Ochronna rola chemeryny w fizjologii naskórka

Uniwersytet Jagielloński Wydział Biochemii, Biofizyki i Biotechnologii Ochronna rola chemeryny w fizjologii naskórka mgr Magdalena Banaś Praca wykonana pod kierunkiem prof. dr hab. Joanny Cichy w Zakładzie Immunologii Wydziału Biochemii, Biofizyki i Biotechnologii Uniwersytetu Jagiellońskiego Kraków 2015

Składam serdeczne podziękowania mojemu promotorowi Pani Profesor Joannie Cichy za owocną współpracę, pomoc, cenne rady oraz wyrozumiałość okazaną podczas przygotowywania niniejszej pracy. Szczególne podziękowania kieruję do Asi Marczyńskiej oraz Mateusza Kwitniewskiego, bez których niniejsza praca nie mogłaby powstać. Dziękuję również Anecie Zegar i Kasi Zabiegło za współpracę, oraz całemu Zespołowi za wspaniałą atmosferę w laboratorium. 1

Dla Moich Rodziców. Sprawiacie, że wszystko jest możliwe. 2

Spis treści 1. Streszczenie... 4 2. Abstract... 6 3. Cele pracy... 8 4. Regulacja ekspresji chemeryny w naskórku... 10 5. Potranslacyjne modyfikacje chemeryny w ludzkim naskórku... 17 6. Funkcja chemeryny w naskórku... 22 7. Bibliografia... 26 8. Załączniki:... 31 3

1. Streszczenie Chemeryna, białkowy produkt genu TIG2 (ang. tazarotene induced gene 2), to niedawno odkryty czynnik chemotaktyczny regulujący migrację komórek układu immunologicznego, posiadających na swej powierzchni receptor CMKLR1. Chemeryna kontroluje również rozwój i fizjologię komórek tłuszczowych, osteoblastów, miocytów oraz reguluje metabolizm glukozy, co czyni ją łącznikiem pomiędzy procesami metabolicznymi, a układem odporności. Chociaż za głównych producentów chemeryny uważa się wątrobę i tkankę tłuszczową, chemeryna obecna jest również w nabłonkach, np. w naskórku, jednak jej funkcja i forma, w jakiej występuje oraz mechanizmy jej regulacji w tkankach nabłonkowych pozostają niewyjaśnione. Co ciekawe, ekspresja chemeryny w naskórku zanika w łuszczycy, chorobie o podłożu autoimmunizacyjnym, co sugeruje ochronne funkcje chemeryny w tej tkance. Aby określić mechanizmy regulacji ekspresji chemeryny w naskórku, przygotowano trójwymiarowe (3D), organotypowe hodowle pierwotnych ludzkich keratynocytów, które naśladują morfologicznie i funkcjonalnie wielowarstwowy, ludzki naskórek. Naskórek stanowi naturalną barierę ciała, w związku z tym oddziałują na tą tkankę zarówno obecne w środowisku mikroorganizmy, jak i czynniki uwalnianie przez komórki układu odporności naciekające skórę w stanach zapalnych. Założono zatem, że wśród czynników wpływających na poziom ekspresji chemeryny w naskórku znajdują się zarówno bakterie, jak również cytokiny o kluczowym znaczeniu w indukcji łuszczycy, a także cytokiny ostrej fazy, których zadanie polega na przywróceniu naturalnej homeostazy organizmu naruszonej w wyniku stanu zapalnego. Wykazano, że chemeryna w hodowlach naskórka, jest różnicowo regulowana przez bakterie. Podczas gdy cytokiny ostrej fazy tj. onkostatyna M i/lub IL-1 stymulują ekspresję chemeryny w keratynocytach, cytokiny pełniące kluczową rolę w patogenezie łuszczycy, IL-17 i/lub IL-22 hamują syntezę tego białka w naskórku. Zgadza się to z obrazem klinicznym łuszczycy, gdzie ekspresja chemeryny w zmienionym chorobowo naskórku jest zahamowana. Chemeryna wydzielana jest poza komórkę jako nieaktywna biologicznie cząsteczka, która ulega aktywacji w wyniku usunięcia hamującej sekwencji zlokalizowanej na końcu C przez proteinazy serynowe i cysteinowe. Chemeryna występuje w różnych izoformach, które różnią się długością i stopniem aktywności. Podjęto próbę 4

zidentyfikowania izoformy w jakiej chemeryna występuje w naskórku. Wykazano, iż chemeryna ulega potranslacyjnym modyfikacjom in vitro w wyniku reakcji transglutaminacji katalizowanej przez transglutaminazy naskórkowe. Tego rodzaju modyfikacje mogą być potencjalnie odpowiedzialne za tworzenie wysokocząsteczkowych form chemeryny wykrywanych w naskórku. Chemeryna pod względem budowy zbliżona jest do katelicydyn, czynników o działaniu antybakteryjnym. Postawiono zatem hipotezę, iż chemeryna może posiadać własności antybakteryjne. Pokazano po raz pierwszy, że zarówno rekombinowana ludzka chemeryna jak i chemeryna wydzielana przez keratynocyty w hodowlach organotypowych hamuje wzrost bakterii. Ponadto, wykazano, że myszy z genetycznym deficytem chemeryny charakteryzują się ograniczoną zdolnością kontroli wzrostu bakterii w modelu infekcji skórnej. Odkryto tym samym zupełnie nową funkcję tego białka. Ponadto zidentyfikowano region Val 66 -Pro 85 (peptyd 4) zlokalizowany w środkowej części chemeryny, jako fragment głównie odpowiedzialny za własności antybakteryjne całego białka. Syntetyczny peptyd Val 66 -Pro 85 charakteryzuje się dużym potencjałem bakteriobójczym w porównaniu do znanych czynników przeciwbakteryjnych oraz szerokim spektrum działania. Podsumowując, w niniejszej pracy zidentyfikowano po raz pierwszy czynniki mogące regulować poziom ekspresji chemeryny w naskórku. Pokazano niebadane do tej pory modyfikacje chemeryny, które mogą wpływać na jej stabilność i funkcję w naskórku. Ponadto zaprezentowano nową, przeciwbakteryjną funkcję tego białka. Sugeruje to, iż chemeryna jest istotnym czynnikiem biorącym udział w utrzymaniu homeostazy naskórka. 5

2. Abstract Chemerin also known as tazarotene induced gene 2 (TIG2) is a ligand for CMKLR1 receptor and is responsible for regulating CMKLR1+ immune cells influx during inflammation. Chemerin is also involved in adiopogenesis, osteoblastogenesis and myogenesis. Moreover, it regulates glucose intake and can serve as a link between metabolism and immunity. Although, liver and fat tissue are the main producers of chemerin, this protein is also present in epithelium, for example in epidermis, where its function, form, and regulation of expression are yet to be determined. Interestingly, chemerin expression in epidermis is strongly downregulated in skin of individuals suffering from psoriasis, an autoinflammatory disease, suggesting that chemerin may play a protective role in epidermis. In order to determine mechanism(s) that underlay chemerin expression in human epidermis, we developed a model of human epidermis, that consist of primary, human keratinocytes growing in three-dimensional (3D) cell cultures. Because epidermis is a natural body barrier, it has a contact with both, numerous microorganisms that are present in the skin environment, and factors released by immune cells that infiltrate the tissue. Therefore, we hypothesized chemerin expression may be regulated on the one hand, by bacteria, and on the other, by cytokines released during skin inflammation. The latter involves IL-17 and IL-22 that drive keratinocyte pathology in psoriasis, and acute phase mediators oncostatin M (OSM) and IL-1, which mobilize protective responses. We showed that specific strains of bacteria have different potential to induce chemerin expression in epidermis. Acute phase mediators such as OSM and IL-1 also upregulated, whereas IL-17 and IL-22 downregulated chemerin expression in human epidermis. This is consistent with clinical manifestation of psoriasis, where chemerin levels are significantly reduced in epidermis. Chemerin is produced and secreted in its biologically inactive form, and requires C terminal proteolytic processing in order to gain chemotactic activity. Serine and cysteine proteases are responsible for generation of chemotactively active chemerin isoform(s). Therefore, we asked which chemerin isoform is present in human epidermis. We determined that chemerin undergoes posttranslational modifications in vitro and epidermal transglutaminases might be responsible for these modifications, leading to generation of high-molecular weight chemerin isoforms detected in epidermis. 6

Given that chemerin and antibacterial cathelicidins share similar structure, and that chemerin is present in natural body barriers, we hypothesized that chemerin may posses antimicrobial properties. Indeed, both recombinant chemerin and chemerin released by human primary keratinocytes growing in 3D cultures, was able to inhibit bacteria growth in vitro. Moreover, chemerin deficient mice in cutaneous infection model, were more prone to bacterial infections in comparison to wild type animals. In addition, we identified internal chemerin fragment Val 66 -Pro 85 (peptide 4) as chemerin domain primarily responsible for chemerin antibacterial activity. Synthetic peptide 4 alone was proven to be a strong antimicrobial agent with wide spectrum of activity toward different strains of bacteria. In conclusion, we identified for the first time agents that may regulate chemerin expression in epidermis, and characterized new posttranslational modifications of chemerin that may influence chemerin stability, activity and bioavailability in epidermis. Finally, we identified novel antimicrobial function of chemerin. Together, these findings suggest that chemerin may play an important role in maintaining skin homeostasis. 7

3. Cele pracy Chemeryna jest białkowym chemoatraktantem i adipokiną o istotnej funkcji w procesach zapalnych i metabolicznych. Wydzielana jest w postaci nieaktywnej biologicznie proformy i do uzyskania pełnej aktywności chemotaktycznej wymaga proteolitycznej modyfikacji końca C. Chociaż chemeryna jest wykrywana w naskórku osób zdrowych, a jej stężenie w naskórku drastycznie maleje w niektórych chorobach skóry, to jej funkcja, mechanizmy regulacji ekspresji oraz forma w jakiej występuje w tej tkance pozostawała niewyjaśniona. W niniejszej pracy postawiono następujące cele badawcze: 1. Określenie poziomu ekspresji chemeryny w naskórku w porównaniu do innych tkanek oraz identyfikacja czynników potencjalnie regulujących ekspresję chemeryny w tej tkance. Opis tych badań zawarto w pracy: Banas M, Zegar A, Kwitniewski M, Zabieglo K, Marczynska J, Kapinska- Mrowiecka M, LaJevic M, Zabel BA, Cichy J. (2015) The expression and regulation of chemerin in the epidermis. Plos ONE 10(2): e0117830 (Załącznik 1) 2. Określenie formy w jakiej chemeryna występuje w ludzkim naskórku oraz opis potencjalnych modyfikacji jakim chemeryna może być poddawana w tej tkance. Uzyskane wyniki badań przedstawione w niniejszej pracy nie zostały opublikowane. 3. Określenie roli chemeryny w naskórku. Założono, że ze względu na strategiczną pozycję chemeryny w naskórku reprezentującym naturalną barierę ciała, oraz podobieństwo strukturalne do przeciwbakteryjnych katelicydyn, funkcja chemeryny w naskórku będzie polegała na kontroli wzrostu bakterii mających kontakt z tą tkanką. Weryfikacja tej hipotezy zawierała trzy etapy: a) Zbadanie funkcji rekombinowanej chemeryny jako czynnika przeciwbakteryjnego oraz identyfikację potencjalnej domeny odpowiedzianej za przeciwbakteryjną funkcję tego białka. Opis tych badań zawarto w pracach: 8

Kulig P, Kantyka T, Zabel BA, Banas M, Chyra A, Stefanska A, Tu H, Allen SJ, Handel TM, Kozik A, Potempa J, Butcher EC, Cichy J. (2011) Regulation of chemerin chemoattractant and antibacterial activity by human cysteine cathepsins. J Immunol 187: 1403-1410 (Załącznik 2) Banas M, Zabieglo K, Kasetty G, Kapinska-Mrowiecka M, Borowczyk J, Drukala J, Murzyn K, Zabel BA, Butcher EC, Schroeder JM, Schmidtchen A, Cichy J. (2013) Chemerin is an antimicrobial agent in human epidermis. Plos ONE 8(3): e58709 (Załącznik 3) b) Zbadanie czy chemeryna wydzielana przez pierwotne, ludzkie keratynocyty posiada własności bakteriobójcze. Opis tych badań zawarto w pracy: Banas M, Zabieglo K, Kasetty G, Kapinska-Mrowiecka M, Borowczyk J, Drukala J, Murzyn K, Zabel BA, Butcher EC, Schroeder JM, Schmidtchen A, Cichy J. (2013) Chemerin is an antimicrobial agent in human epidermis. Plos ONE 8(3): e58709 (Załącznik 3) c) Analizę porównawczą podatności na infekcje bakteryjne skóry zwierząt produkujących funkcjonalną chemerynę i zwierząt z deficytem chemeryny. Opis tych badań zawarto w pracy: Banas M, Zegar A, Kwitniewski M, Zabieglo K, Marczynska J, Kapinska- Mrowiecka M, LaJevic M, Zabel BA, Cichy J. (2015) The expression and regulation of chemerin in the epidermis. Plos ONE 10(2): e0117830 (Załącznik 1) 9

4. Regulacja ekspresji chemeryny w naskórku Chemeryna jest wielofunkcyjnym białkiem głównie znanym ze względu na swoje własności chemotaktyczne oraz regulację różnicowania komórek tłuszczowych. Gen kodujący chemerynę po raz pierwszy został odkryty w skórze stymulowanej tazarotenem, pochodną kwasu retinowego i nazwany TIG2 (ang. tazarotene induced gene 2) [1]. Chemeryna jest ligandem dla metabotropowego receptora związanego z białkiem G, CMKLR1 (ang. chemokine-like receptor 1) [2, 3, 4] i ma zdolność regulacji napływu komórek, które posiadają na swojej powierzchni receptor CMKLR1. Należą do nich komórki układu odporności takie jak plazmacytoidalne komórki dendrytyczne (pdc), makrofagi oraz komórki NK [4, 5, 6, 7]. Czyni to chemerynę istotnym czynnikiem biorącym udział w rozwoju odpowiedzi immunologicznej. Oprócz CMKLR1, z podobnym powinowactwem chemeryna wiąże się z dwoma innymi receptorami, GPR1 (ang. G protein-coupled receptor 1) oraz CCLR2 (ang. CCmotif chemokie like receptor 2). [8, 9]. Wśród wymienionych receptorów związanie chemeryny tylko z CMKLR1 skutkuje internalizacją receptora, mobilizacją jonów wapnia w komórce oraz migracją komórki zgodnie z gradientem chemeryny [3, 10]. Funkcje dwóch pozostałych receptorów pozostają słabo poznane, chociaż istnieją doniesienia, iż GPR1 może brać udział w utrzymywaniu prawidłowego stężenia glukozy we krwi [11], a CCLR2 reguluje lokalny poziom chemeryny poprzez wiązanie i prezentację chemeryny innym komórkom wyposażonym w receptor CMKLR1 [9, 12, 13]. Za głównych producentów chemeryny w organizmie, odpowiedzialnych za utrzymywanie stosunkowo wysokiego, nanomolowego (~4 nm) stężenia chemeryny w surowicy [14] uznaje się wątrobę oraz tkankę tłuszczową [10]. Badania ostatnich lat pokazują, że chemeryna ulega znaczącej ekspresji również w nabłonkach tworzących naturalne bariery organizmu, w płucach, jelitach oraz skórze [1, 16, 17, 18]. Jednakże poziom ekspresji w tych barierach na tle innych rezerwuarów chemeryny pozostawał nieznany. Dlatego w pierwszym etapie tej części badań wykonano analizę porównawczą mysich tkanek, która ujawniła, że ekspresja genu TIG2 w skórze jest w przybliżeniu dziesięć i sześć razy niższa w porównaniu do wątroby i tłuszczu (Załącznik 1, Ryc. 1A), a na poziomie białka stężenie chemeryny w lizatach z tkanek było około dwa i trzy razy niższe w skórze w odniesieniu do wątroby i tłuszczu (Załącznik 1, Ryc. 1B). 10

Rozwarstwienie skóry na naskórek i skórę właściwą ujawniło, iż to frakcja epidermalna jest głównie odpowiedzialna za produkcję chemeryny w zdrowej skórze, a poziom ekspresji chemeryny w tej tkance zbliżony jest do tego obserwowanego w wątrobie (Załącznik 1, Ryc. 1B). Zgadza się to z danymi literaturowymi, uzyskanymi w oparciu o badania immunohistochemiczne preparatów skórnych, iż chemeryna w zdrowej skórze lokalizuje się głównie w naskórku [16]. Jak opisano to powyżej, chemeryna może się wiązać do trzech receptorów, dlatego też aby zbadać czy poziom chemeryny jest regulowany lokalnie w tkankach poprzez wiązanie się chemeryny do tych receptorów przeprowadzono analizę ekspresji CMLR1, CCLR2 oraz GPR1 w wątrobie, tłuszczu trzewnym oraz skórze i jej frakcjach. Transkrypt dla wszystkich receptorów był obecny w każdej z badanych tkanek. Najwyższą ekspresję CMKLR1 zanotowano w tłuszczu, natomiast poziom mrna dla CCLR2 i GPR1 najwyższy był w skórze, w szczególności w skórze właściwej (Załącznik 1, Ryc. 1C-1E). Uzyskane wyniki sugerują, że lokalne stężenie chemeryny w skórze może być regulowane poprzez wiązanie się chemeryny do wszystkich receptorów. Potwierdzają to wyniki uzyskane przez innych członków zespołu na zwierzętach z wyciszonymi genami kodującymi CMKLR1 i CCLR2 (Załącznik 1, Ryc. 1F-1G). Myszy z deficytem GPR1 nie były dostępne do wykonania podobnych badań. Analiza porównawcza ekspresji chemeryny i jej receptorów w ludzkich tkankach dała zbliżone wyniki do tych obserwowanych w mysich tkankach, za wyjątkiem braku znaczących różnic w ekspresji CMKLR1, CCLR2 oraz GPR1 pomiędzy naskórkiem, a skórą właściwą (Załącznik 1, Ryc. 2A-2E). Profil ekspresji chemeryny jest inny w skórze zdrowej niż tej dotkniętej zmianami chorobowymi o podłożu autoimmunizacyjnym, takich jak łuszczyca. Jak wspomniano wyżej, w zdrowej skórze głównymi producentami chemeryny są komórki budulcowe naskórka keratynocyty, natomiast chemeryna nie jest wykrywana w skórze właściwej. U pacjentów cierpiących na łuszczycę ekspresja chemeryny w naskórku jest znacząco obniżona, przy jednoczesnej produkcji chemeryny przez komórki tworzące skórę właściwą, gdzie pełni ona rolę chemoatraktanta dla komórek układu odporności [1, 15, 16]. Korelacja pomiędzy profilem ekspresji chemeryny w naskórku i skórze właściwej, a wystąpieniem łuszczycy sugeruje, iż czynniki odpowiedzialne za patofizjologię tego schorzenia mogą wpływać na regulację produkcji chemeryny w skórze. Aby to zbadać zastosowano model badawczy ludzkiego naskórka, który opierał się na hodowlach pierwotnych, ludzkich keratynocytów, tworzących 11

wielowarstwową tkankę, tzw. hodowlach organotypowych lub inaczej hodowlach 3D (Ryc. 1). Ryc. 1 Schemat organotypowej hodowli pierwotnych, ludzkich keratynocytów (hodowla 3D). Keratynocyty ściśle porastają dno koszyka, który stanowi filtr o porach wielkości 0,4 m. Koszyk umieszczany jest w naczyniu wypełnionym pożywką hodowlaną, która przedostaje się przez pory i odżywia komórki warstwy podstawnej. Górna powierzchnia hodowli ma kontakt z powietrzem co wraz z odpowiednio dobranym składem pożywki hodowlanej umożliwia różnicowanie keratynocytów i tworzenie wielowarstwowego modelu naskórka. Obraz histologiczny takiej hodowli przedstawiony jest w Załączniku 1, Ryc. 4E. Cytokiny uwalniane przez komórki układu odporności naciekające zmienioną chorobowo skórę oddziałują w pierwszej kolejności na keratynocyty budujące warstwę podstawną naskórka, która znajduje się tuż nad skórą właściwą. Aby naśladować rzeczywisty układ, w którym cytokiny działają na naskórek od spodu, badane czynniki podawane były do medium hodowlanego, w którym następnie zanurzone były filtry porośnięte przez organotypowe hodowle keratynocytów. Badanymi czynnikami była IL-17 odgrywająca kluczową rolę w rozwoju stanu zapalnego w zmienionej łuszczycowo skórze [19, 20] oraz IL-22 odpowiedzialna za nadmierne podziały keratynocytów, co jest charakterystyczne dla tego schorzenia [21, 22]. Podanie cytokin skutkowało zahamowaniem ekspresji chemeryny na poziomie transkryptu po 48 godzinach (Załącznik 1, Ryc. 3A i 3C). Ilość chemeryny wydzielonej do medium była nieznacznie obniżona, jednak uzyskany wynik nie był istotny statystycznie (Załącznik 1, Ryc. 3B i 3D). W przypadku jednoczesnego podania cytokinin obserwowany efekt nasilał się. Sugeruje to uzupełniające się nawzajem działanie tych cytokin. Te wyniki uprawdopodabniają tezę, że IL-17 oraz IL-22 mogą być czynnikami bezpośrednio 12

odpowiedzialnymi za zmniejszoną ekspresję chemeryny w zmienionym łuszczycowo naskórku. Przerwanie integralności skóry może prowadzić do infekcji. Towarzyszące temu powstanie stanu zapalnego ma na celu przeciwdziałać rozwojowi infekcji poprzez napływ komórek układu odporności, aktywować proces gojenia się rany oraz regenerację uszkodzonej tkanki. Komórki w miejscu zapalenia wydzielają tzw. czynniki ostrej fazy mające na celu przywrócenie homeostazy organizmu, zaburzonej w wyniku procesu zapalnego [23]. Jedną z głównych cytokin ostrej fazy jest onkostatyna M (OSM), a jej działanie jest często wspomagane przez IL-1. Co ciekawe, zwiększona ekspresja zarówno OSM jak i IL-1 jest obserwowana w skórze objętej zmianami łuszczycowymi [24, 25, 26]. Zadano sobie pytanie, czy czynniki ostrej fazy będą wpływały na profil ekspresji chemeryny w skórze. Stymulacja keratynocytów za pomocą OSM i IL-1 ujawniła odmienny od IL-17 i IL-22 mechanizm regulacji ekspresji chemeryny w naskórku. OSM samodzielnie oraz w kombinacji z IL-1, silnie stymulowała ekspresję chemeryny w keratynocytach na poziomie mrna (Załącznik 1, Ryc. 3A i 3C). Na poziomie białka zwiększoną ekspresję chemeryny obserwowano po 24 godzinach od podania OSM, a także OSM z IL- (Załącznik 1, Ryc. 3B). Po upływie 48 godzin różnica pomiędzy próbkami kontrolnymi, a stymulowanymi zmniejszała się (Załącznik 1, Ryc. 3D), choć wykazywała podobną tendencję do tej obserwowanej po 24 godzinnej stymulacji. Celem sprawdzenia czy chemeryna uwalniana do mediów jest wiązana przez receptory obecne na powierzchni keratynocytów zbadano poziom ekspresji CMKLR1, CCLR2 i GPR1, po stymulacji OSM i IL-1 (Załącznik 1. Ryc. 5). 24 godzinna stymulacja IL-1 oraz IL-1 w kombinacji z OSM skutkowała zwiększeniem ekspresji wszystkich receptorów. W przypadku 48 godzinnej stymulacji tylko IL-1 zwiększała poziom ekspresji CMKLR1 i CCLR2, natomiast poziom GPR1 pozostawał niezmieniony. Przedstawione wyniki pokazują, iż wydzielana przez keratynocyty chemeryna może być wiązana przez receptory obecne na powierzchni komórek i autokrynnie wpływać na ich fizjologię. Uzyskane wyniki sugerują, że prawdopodobnie istnieją różne ścieżki regulacji ekspresji chemeryny przez cytokiny zaangażowane w rozwój łuszczycy. IL-17 i IL-22 hamują, natomiast IL-1 i OSM zwiększają ekspresję chemeryny w keratynocytach, pomimo, iż rozwojowi łuszczycy towarzyszy zwiększona ekspresja wszystkich tych cytokin [20, 21, 24, 25, 26]. Dodatkowo, istnieją doniesienia, że IL-1 podtrzymuje przewlekły stan 13

zapalny poprzez regulację dojrzewania limfocytów T produkujących IL-17 [27, 28, 29], a OSM hamuje różnicowanie keratynocytów [24], co najprawdopodobniej sprzyja rozwojowi łuszczycy. Z drugiej jednak strony OSM na wczesnych etapach łuszczycy hamuje ekspresję genów zaangażowanych w rozwój stanu zapalnego [26]. Ponadto IL-1, a w mniejszym stopniu OSM uwrażliwiają naskórek na autokrynne działanie chemeryny poprzez zwiększenie poziomu ekspresji receptorów, co nie było obserwowane w przypadku stymulacji IL-17 i/lub IL-22 (Załącznik 1, Ryc. 5). OSM i IL-1 jako czynniki ostrej fazy regulują procesy naprawcze w uszkodzonej tkance, a fakt, że zwiększają również ekspresję chemeryny i jej receptorów sugeruje, iż chemeryna i jej receptory mogą być zaangażowane w poprawę stanu zmienionego łuszczycowo naskórka. Jedno z głównych zadań naskórka jako zewnętrznej bariery organizmu, polega na ochronie przed patogennymi czynnikami obecnymi w środowisku zewnętrznym, takimi jak bakterie. Są wśród nich Staphylococcus aureus główny czynnik bakteryjnych infekcji skórnych [30] oraz Escherichia coli bakteria odpowiedzialna za infekcje wynikające z kontaktu skóry z treścią jelitową [31]. Aby uzyskać odpowiedź na pytanie czy mikroorganizmy mają wpływ na ekspresję chemeryny w naskórku, infekowano pierwotne, ludzkie keratynocyty rosnące w trójwymiarowej hodowli organotypowej (model ludzkiego naskórka) oraz mysią skórę za pomocą S. aureus i E. coli. Ponieważ w rzeczywistości bakterie oddziałują na najbardziej zewnętrzne warstwy naskórka (w odróżnieniu od cytokin głównie pochodzących z komórek naciekających skórę), badane czynniki podawane były od góry na keratynocyty mające kontakt z powietrzem. Uzyskane wyniki pokazują, iż bakterie indukują produkcję chemeryny przez keratynocyty (Załącznik 1. Ryc. 4A-4D, 7A-7B). Bakterie szczepu S. aureus silniej niż E. coli zwiększały ekspresję chemeryny, pomimo zastosowania takiego samego inokulum (10 7 CFU). Mogło to wynikać z lepszej przeżywalności tych bakterii na skórze lub stanowić specyficzną cechę S. aureus. Celem sprawdzenia niniejszej hipotezy keratynocyty stymulowano za pomocą bakterii, których wzrost był ograniczony przez obecność antybiotyku. Stężenie ampicyliny zostało tak dobrane aby bakterie nie ulegały podziałom komórkowym, ale nadal pozostawały żywe, a liczba bakterii S. aureus hodowanych z antybiotykiem i liczba E. coli po 24 godzinach były porównywalne. Zarówno S. aureus jak i S. aureus traktowane ampicyliną charakteryzowały się podobnym potencjałem stymulacji 14

produkcji chemeryny (Załącznik1, Ryc. 4A-4D). Sugeruje to, iż bakterie S. aureus produkują specyficzne dla swojego szczepu czynniki, które silniej niż czynniki produkowane przez E. coli wpływając na ekspresję chemeryny. Aby zbadać czy keratynocyty reagują zwiększoną produkcją chemeryny w odpowiedzi na obecność żywych bakterii, hodowle keratynocytów stymulowano przy pomocy komórek bakteryjnych, które uprzednio zostały zabite pod wpływem inkubacji w wysokiej temperaturze. W istocie, żywe bakterie mocniej niż zabijane termicznie stymulowały produkcję chemeryny (Załącznik 1, Ryc. 4A-4D) co sugeruje, że czynniki specyficzne dla żywych mikroorganizmów mogą brać udział w tym procesie. Na przestrzeni ostatnich lat pojawiły się teorie dotyczące tzw. vita-pamps (ang. viability associated pathogen-associated mlecular patterns), czyli czynników obecnych tylko u żywych baterii np. mrna [32]. Prawdopodobnym wydaje się, że czynniki te mogą brać udział w regulacji ekspresji chemeryny w naskórku. Dodatkowo hodowle keratynocytów były stymulowane płynami pohodowlanymi uzyskanymi po odwirowaniu komórek z 24 godzinnej hodowli bakteryjnej. Zaobserwowano, że nie tylko zawiesiny bakterii, ale i w niektórych przypadkach również czynniki uwalniane do płynu hodowlanego przez bakterie wykazują tendencję do stymulacji ekspresji chemeryny (Załącznik1, Ryc. 4A- 4D). Podsumowując, uzyskane wyniki sugerują, że patogeny obecne w środowisku mogą, angażując różne mechanizmy stymulować keratynocyty do produkcji chemeryny. Wykonano również analizę immunohistochemiczną organotypowych hodowli keratynocytów traktowanych za pomocą bakterii (Załącznik 1, Ryc. 4E). Naskórek mający kontakt z żywymi bakteriami produkował więcej chemeryny niż hodowla kontrolna. Najwięcej chemeryny wydzielały keratynocyty tworzące warstwę podstawną, co pozwala przypuszczać, że dzielące się keratynocyty w największym stopniu odpowiadają za poziom chemeryny uwolnionej do medium hodowlanego. Podczas gdy większość badanych czynników bakteryjnych zwiększała ekspresję chemeryny w naskórku, taki efekt nie był obserwowany w przypadku receptorów chemerynowych. 24 godzinna stymulacja hodowli keratynocytów za pomocą bakterii E. coli oraz w mniejszym stopniu S. aureus skutkowała zmniejszeniem ekspresji CCLR2 oraz GPR1, natomiast poziom mrna dla CMKLR1 pozostawał niezmieniony (Załącznik 1, Ryc. 6A). Co ciekawe, ekspresja CMKLR1 oraz CCLR2 wydawała się być indukowana pod wpływem dłuższego kontaktu keratynocytów - 48 godzin z żywymi bakteriami S. aureus (Załącznik 1, Ryc. 6B). Tego efektu nie obserwowano 15

w przypadku zastosowania bakterii E. coli ani czynników uwalnianych przez komórki bakteryjne do płynu hodowlanego. Na podstawie uzyskanych wyników można zaproponować istnienie odrębnych mechanizmów, które aktywowane są w naskórku pod wpływem kontaktu z bakteriami. Bakterie S. aureus stymulują wyłapywanie chemeryny przez receptory i prezentację chemeryny na powierzchni komórek tworząc tarczę ochronną, natomiast bakterie E. coli generują pulę wolnej chemeryny, działającej jak rozpuszczalny czynnik np. chemotaktyczny lub bakteriobójczy. Analiza ekspresji receptorów dla chemeryny w mysiej skórze pod wpływem kontaktu z bakteriami pokazała, że bakterie S. aureus zwiększają ekspresję CMKLR1 oraz GPR1 (Załącznik 1, Ryc. 7C i 7D). Bakterie E. coli natomiast zwiększają ekspresję CCLR2 i GPR1 (Załącznik1, Ryc. 7D i 7E). Współgra to z obserwacjami poczynionymi przy użyciu modelu ludzkiego naskórka, iż keratynocyty w odmienny sposób regulują ekspresję chemeryny i jej receptorów w odpowiedzi na E. coli i S. aureus. 16

5. Potranslacyjne modyfikacje chemeryny w ludzkim naskórku Ludzka chemeryna syntetyzowana jest jako prekursor zbudowany ze 163 aminokwasów, a po usunięciu 20 aminokwasowego peptydu sygnałowego, wydzielana jest poza komórkę jako chemeryna 163S (gdzie 163S oznacza terminalną serynę w pozycji 163). Ta obecna w surowicy forma jest nieaktywna biologicznie [33]. Do uzyskania pełnej aktywności niezbędna jest modyfikacja proteolityczna jej końca karboksylowego. Enzymami zaangażowanymi w aktywację chemeryny są białka z rodziny proteaz serynowych biorących udział w reakcjach zapalnych [33, 7] oraz proteaz cysteinowych pochodzących zarówno z własnych komórek [34, 35] jak i niektórych drobnoustrojów [36]. Ilość aminokwasów usuniętych z końca karboksylowego chemeryny bezpośrednio wpływa na jej aktywność chemotaktyczną. Do najbardziej aktywnych chemotaktycznie form chemeryny należy forma Chem157S, która kończy się na serynie w pozycji 157 i jest krótsza od formy 163S o sześć aminokwasów na końcu C [10]. Ryc. 2 Sekwencja chemeryny. Kolorem żółtym podkreślono peptyd sygnałowy, kolorem czerwonym i niebieskim zaznaczony jest region, który podlega proteolizie w różnych miejscach chemeryny obecnej w płynach zapalnych. Kolorem zielonym zaznaczone są reszty lizyny i glutaminy. Liczbami oznaczono koniec-c form chemeryny, odpowiednio Chem149Y, Chem154F, Chem157S i Chem163S. Forma w jakiej chemeryna występuje w ludzkim naskórku pozostaje nieznana. Ponieważ ilość aminokwasów usuniętych z karboksylowego końca chemeryny może rzutować na jej aktywność biologiczną, istotnym wydawało się zidentyfikowanie formy w jakiej chemeryna występuje w naskórku. Próbując odpowiedzieć na to pytanie wykonano analizę Western Blot naskórków pobranych od piętnastu różniących się wiekiem pacjentów obojga płci. Biorąc pod uwagę, iż forma chemeryny może zależeć od miejsca pobrania, badano wycinki skórne pochodzące z różnych rejonów ciała. 17

Ponieważ nie zaobserwowano znaczących różnic pomiędzy dawcami materiału ani miejscami pobrania wycinków skóry, wyniki przedstawiono dla trzech reprezentatywnych próbek (Ryc. 3A). Analiza Western Blot prowadzona w warunkach redukujących, chemeryny obecnej w ludzkim naskórku ujawniła dominujący produkt o masie cząsteczkowej (>70 kda) przekraczającej znacznie masę cząsteczkową rekombinowanego białka Chem157S (~18 kda). Zaobserwowano również szereg innych produktów, wszystkie o masie przekraczającej masę cząsteczkową rekombinowanej chemeryny. Aby precyzyjniej określić formę w jakiej produkowana jest chemeryna przez keratynocyty, przeprowadzono również analizę Western Blot mediów pochodzących z hodowli organotypowych pierwotnych, ludzkich keratynocytów (Ryc. 3B). Tak jak w przypadku lizatów naskórka, białko uwalniane do medium przez keratynocyty i najsilniej wykrywane przez przeciwciała przeciwko ludzkiej chemerynie miało masę >70 kda. Ryc. 3 Chemeryna produkowana przez keratynocyty występuje w wysokocząsteczkowej formie. Analiza Western Blot: A chemeryny obecnej w ludzkim naskórku (M37 skroń skóra pobrana ze skroni 37 letniego mężczyzny, K61 udo skóra pobrana z uda 61 letniej kobiety, K33 ramię skóra pobrana z ramienia 33 letniej kobiety), wyniki reprezentatywne dla 15 dawców i 3 powtórzeń, B - chemeryny uwolnionej do medium przez keratynocyty wyizolowane z ucha 15 letniego mężczyzny (medium), wyniki reprezentatywne dla 5 powtórzeń. Jako kontrolę wykorzystano rekombinowaną chemerynę, której sekwencja kończy się na serynie w pozycji 157-157S (chemeryna). Do detekcji użyto biotynylowanych kozich przeciwciał przeciw ludzkiej chemerynie oraz streptawidyny sprzęgniętej z peroksydazą chrzanową. 18

Uzyskany wynik sugeruje, iż chemeryna produkowana przez keratynocyty łączy się poprzez wiązanie kowalencyjne z innymi białkami, tworząc białkowe kompleksy o dużej masie cząsteczkowej. Odpowiedź na pytanie jaki rodzaj modyfikacji może leżeć u podstaw tworzenia wielkocząsteczkowych produktów chemerynowych pochodzi z wnętrza sekwencji chemeryny (Ryc. 2), w której skład wchodzą liczne reszty lizyny i glutaminy (zaznaczone kolorem zielonym) oraz ko-lokalizacja w naskórku chemeryny i enzymów mających zdolność do tworzenia wiązań pomiędzy resztami lizyny i glutaminy tzw. transglutaminaz [37]. Transglutaminazy (TG), enzymy licznie występujące w tkankach, odpowiedzialne są za potranslacyjne modyfikacje białek - katalizują tworzenie się wiązań peptydowych pomiędzy lizyną, a glutaminą. TG tworzą pewnego rodzaju tarczę ochronną w warstwie rogowej naskórka poprzez wiązanie białek podatnych na proteolizę z inhibitorami proteaz bądź proteoglikanami lub sfingolipidami macierzy zewnątrzkomórkowej. Produkty reakcji transglutaminacji, nierozpuszczalne białka o duże masie cząsteczkowej, charakteryzują się znaczną odpornością na mechaniczne uszkodzenia jak i proteolityczną degradację [38]. Do tej pory zidentyfikowano 8 białek należących do rodziny transglutaminaz [39]. Enzymami ulegającymi ekspresji w skórze są TG1 i 3 (obecne we wszystkich warstwach naskórka) [37], oraz TG5 produkowana głównie przez keratynocyty warstwy ziarnistej [40]. Enzymy proteolityczne obecne w naskórku mają zdolność kształtowania lokalnego środowiska zarówno poprzez degradację białek macierzy zewnątrzkomórkowej jak i regulację procesów zapalnych aktywując prekursory cytokin i chemokin [41]. W stanach chorobowych skóry takich jak łuszczyca czy atopowe zapalenie skóry obserwowana jest zwiększona ilość enzymów proteolitycznych w naskórku [37, 41, 42]. Nadmierna lub nieprawidłowa proteoliza białek może prowadzić to rozwoju stanu zapalnego, którego efektem będą zmiany chorobowe w obrębie tkanki objętej procesem zapalnym. Proces transglutaminacji wydaje się być mechanizmem zaradczym, przeciwdziałającym wzmożonej proteolizie białek [38]. Zwiększona masa cząsteczkowa chemeryny wydzielanej przez keratynocyty, obecność licznych reszt lizyny i glutaminy w jej sekwencji, ko-lokalizacja chemeryny i transglutaminaz oraz zbieżność w strukturze chemeryny i cystatyn [14, 43], jednego ze znanych substratów dla transglutaminaz [44] sugerują, iż chemeryna może być potencjalnym substratem dla tych enzymów. Celem potwierdzenia tej tezy, 19

rekombinowaną chemerynę inkubowano w obecności medium z hodowli ludzkich keratynocytów, transglutaminaz oraz inhibitorów transglutaminaz. Ponieważ TG1 i TG3, tak jak chemeryna, obecne są we wszystkich warstwach naskórka, i ze względu na wspólną ko-lokalizację z dużym prawdopodobieństwem mogą być zaangażowane w modyfikacje chemeryny w naskórku, w badaniach wykorzystano TG1 i TG3. Wyniki analizy Western Blot produktów transglutaminacji chemeryny zostały przedstawione na Ryc. 4A oraz 4B. Ryc. 4 Chemeryna jest substratem dla transglutaminaz naskórkowych. Analiza Western Blot produktów reakcji transglutaminacji chemeryny. Badany materiał: A rekombinowana chemeryna inkubowana z medium z organotypowej hodowli ludzkich keratynocytów w obecności transglutaminaz 1 lub 3 oraz inhibitorów transglutaminaz, B rekombinowana chemeryna inkubowana w obecności transglutaminaz 1 i 3 oraz inhibitorów. Do detekcji chemeryny użyto biotynylowanych kozich przeciwciał przeciw ludzkiej chemerynie oraz streptawidyny sprzęgniętej z peroksydazą chrzanową. Wyniki reprezentatywne dla pięciu powtórzeń. Inkubacja rekombinowanej chemeryny w obecności medium z hodowli keratynocytów skutkowała pojawieniem się dominującego produktu o masie >70 kda oraz dwóch dodatkowych produktów o masie ~40 kda i ~55 kda (Ryc. 4A). Uzyskane wyniki wskazują, iż w medium mogą być obecne transglutaminazy mające zdolność tworzenia wiązań pomiędzy chemeryną, a innymi białkami wydzielanymi do medium. Dodanie rekombinowanych transglutaminaz powodowało, że przeciwciała skierowane przeciwko ludzkiej chemerynie silniej wykrywały produkty o masie ~40 kda i ~55 kda oraz wykrywały produkty o masie >100 kda. Tworzenie się produktów o zwiększonej 20

masie cząsteczkowej hamowane było poprzez dodanie inhibitorów. Obecność produktów o masie ~40 kda i ~55 kda, co w przybliżeniu odpowiada kolejno masie dwóch i trzech cząsteczek chemeryny, sugeruje, iż chemeryna potencjalnie może tworzyć produkty transglutaminacji sama ze sobą. Istotnie, wykrywane produkty inkubacji rekombinowanej chemeryny w obecności obu transglutaminaz również miały masę ~40 kda, ~55 kda i ~70 kda (Ryc. 4B). Odpowiada to masie dwóch, trzech i czterech cząsteczek chemeryny. Produkty przekraczające swoją masą 100kDa mogły być efektem powstania wielocząsteczkowych konglomeratów chemeryny lub chemeryny związanej z enzymem (co stanowi produkt pośredni w procesie transglutaminacji). Uzyskane wyniki pokazują, że chemeryna poprzez wiązania kowalencyjne ma zdolność łączenia się sama ze sobą oraz z innymi białkami wydzielanymi przez keratynocyty, a enzymami katalizującymi powstawanie tych połączeń są transglutaminazy naskórkowe. Znaczeniem fizjologicznym obserwowanego procesu może być konieczność ochrony chemeryny przed wzmożoną proteolizą i generowaniem chemotaktycznie aktywnych from. To z kolei może przeciwdziałać nadmiernemu napływowi komórek układu odporności i hamowaniu powstawania stanu zapalnego. Tworzenie agregatów chemerynowych może również wpływać na lokalne zagęszczenie chemeryny, co będzie wspomagało inne niż napływ komórek immunologicznych, funkcje chemeryny w naskórku. W niniejszej pracy po raz pierwszy podjęto próbę określenia formy w jakiej chemeryna występuje w naskórku. Pokazano, iż chemeryna ma zdolność wiązania się zarówno z innymi białkami macierzy zewnątrzkomórkowej naskórka jak i tworzenia chemerynowych super-cząsteczek oraz zidentyfikowano enzymy potencjalnie zaangażowanie w te procesy. Jednakże, uzyskane wyniki są punktem wyjścia do dalszych badań nad modyfikacjami chemeryny w naskórku, gdyż niezbędnym jest określenie składu super-cząsteczek chemerynowych przy wykorzystaniu np. spektroskopii masowej. 21

6. Funkcja chemeryny w naskórku Chemeryna jest wielofunkcyjnym białkiem zaangażowanym w kierowanie napływu komórek układu odporności do miejsca zapalenia, regulację różnicowania i metabolizmu komórek tłuszczowych [10, 45, 46], ponadto bierze udział w procesach takich jak angiogeneza [47], osteoblastogeneza [48] oraz rozwój komórek mięśniowych [49]. Od wielu lat wiadomo, że chemeryna obecna jest w naskórku [1], jednak funkcja chemeryny w tej tkance pozostawała niewyjaśniona. Ryc. 5 Izoformy chemeryny i peptydy chemerynowe badane pod kątem własności przeciwbakteryjnych. W sekwencji chemeryny zaznaczono pozycje syntetycznych peptydów (p1 - p14) użytych do zbadania własności antybakteryjnych. Kolorem czerwonym oznaczony został peptyd 4, głównie odpowiedzialny za przeciwbakteryjne własności chemeryny. Liczby nad aminokwasami oznaczają koniec C testowanych izoform chemeryny, odpowiednio Chem125R, Chem157S i Chem163S. Podobieństwo strukturalne chemeryny i przeciwbakteryjnych katelicytydyn [10, 50] oraz fakt, że chemeryna obecna jest w naturalnych barierach ciała, nie tylko w naskórku ale również w nabłonkach wyściełających płuca oraz jelita [10, 18] wskazuje, iż chemeryna może pełnić ochronną, niezależną od chemotaksji rolę w tych tkankach. Analiza form chemeryny o rożnym potencjale chemotaktycznym ujawniła, że wszystkie te formy mają zdolność hamowania wzrostu bakterii tj. E. coli oraz K. pneumoniae (Załącznik 2, Fig. 5A). Najniższy potencjał przeciwbakteryjny wykazywała pro-forma Chem163S. Usunięcie sześciu aminokwasów z końca karboksylowego, co generuje formę Chem157S o największej aktywności chemotaktycznej skutkowało również zwiększeniem aktywności bakteriobójczej. 22

Wyniki te sugerują, że tak jak w przypadku funkcji chemotaktycznej, usunięcie hamującego, C-końcowego fragmentu tego białka, jest konieczne do ujawnienia pełnej funkcji antybakteryjnej chemeryny. Co ciekawe, zarówno chemotaktycznie aktywna Chem157S, jak i chemotaktycznie nieaktywna forma Chem125R forma kończąca się na argininie w pozycji 125, krótsza od form 157S o 22 aminokwasy (Ryc. 5), w równym stopniu hamowały wzrost bakterii (Załącznik 2, Fig. 5D). Sugeruje to, że za funkcje chemotaktyczną i bakteriobójczą chemeryny odpowiedzialne są różne fragmenty tego białka. Testy przeciwbakteryjne z wykorzystaniem zsyntetyzowanych chemicznie 20-aminokwasowych peptydów pokrywających całą sekwencję chemeryny (Ryc. 5) pozwoliły zidentyfikować peptyd 4 (p4) reprezentujący sekwencję Val 66 -Pro 85, jako region głównie odpowiedzialny za zabijanie komórek E. coli przez chemerynę (Załącznik 3, Fig. 3). W kolejnym etapie zbadano czy aktywność przeciwbakteryjna peptydu 4 jest specyficzna dla określonych szczepów mikroorganizmów. Prace innych członków zespołu ujawniły, że peptyd 4 charakteryzuje się szerokim spektrum działania (Załącznik 3, Fig. 4 oraz Tab. 3). Dodatkowo wykonano testy przeciwbakteryjne z użyciem bakterii szczepów kolonizujących skórę [51] tj. Pseudomonas, Staphylococcus, Dermabacter, Streptococcus, Corynebacterium i Deinococcus (Ryc. 6). Wszystkie badane szczepy charakteryzowały się wrażliwością na peptyd 4. Rozlokowanie chemeryny we wszystkich warstwach naskórka (Załącznik 1, Fig. 4E) oraz szerokie spektrum działania peptydu 4 (Ryc. 6, Załączniki 3, Fig. 4,) sugerują, że chemeryna może pełnić ochronę funkcję w naskórku zapobiegając infekcjom bakteryjnym. Badania nad peptydem 4 ujawniły również, iż najwyższą aktywność bakteriobójczą wykazuje on w ph zbliżonym do neutralnego oraz w środowisku o niskiej zawartości soli (Załącznik 2, Fig. 5). ph zdrowej, ludzkiej skóry waha się od 5 w wyższej warstwie rogowej do 7 w niższej warstwie granularnej [52], natomiast stężenie soli wynosi około 40mM [53] i zależy od ilości wody traconej w wyniku pocenia się. Oznacza to więc, że peptyd pochodzenia chemerynowego będzie w pełni aktywny w środowisku zdrowego naskórka. Ze względu na efektywność i wszechstronność peptydu chemerynowego p4, zaproponowano użycie tego peptydu do wytwarzania leku o właściwościach ochronnych względem komórek nabłonkowych, w tym w szczególności do stosowania przeciw zakażeniom bakteryjnym i grzybiczym, oraz do leczenia chorób związanych ze stanem zapalnym skóry, płuc czy przewodu pokarmowego. Peptydy chemerynowe, kompozycja farmaceutyczna zawierająca te peptydy oraz ich zastosowanie stały się przedmiotem zgłoszeń patentowych do 23

Polskiego oraz Europejskiego Biura Patentowego (zgłoszenie numer P.402070 oraz PCT/IB2013/061001). Strefa zahamowania wzrostu [mm] 15 10 5 0 P. aeruginosa S. capitis D. hominis S. agalactiae C. simulans D. indicus p4 Lizozym Ryc. 6 Chemerynowy peptyd 4 jest czynnikiem przeciwbakteryjnym o szerokim spektrum działania. Przedstawione na wykresie szczepy bakterii (P. aeruginsa szczep kliniczny, S. capitis ATCC27840, D. hominis ATCC49369, S. agalactiae ATCC13813, C. simulans DSM44392, D. indicus DSM15307) wykorzystano w teście RDA badającym potencjał przeciwbakteryjny peptydu 4 (100 M) oraz lizozymu (75 M). Badane czynniki przez 3 godziny ulegały dyfuzji w żelu agarozowym z zawiesiną bakterii. Po upływie 24 godzin mierzono średnice stref zahamowania wzrostu bakterii, a wyniki podawano pomniejszone o średnicę otworów do których nakładane były czynniki. Przestawione dane są średnią ± odchylenie standardowe z 4 niezależnych eksperymentów. W opisanych powyżej badaniach wykazano przeciwbakteryjną funkcję rekombinowanej chemeryny oraz peptydu chemerynowego p4. Kolejnym etapem było określenie czy tak jak forma rekombinowana, chemeryna wydzielana przez ludzkie keratynocyty posiada własności bakteriobójcze. Dodanie medium z hodowli keratynocytów do hodowli bakterii E. coli znacząco hamowało wzrost bakterii. Aby zbadać jaką rolę pełni w tym procesie chemeryna zastosowano medium, z którego usunięto chemerynę poprzez immunoprecypitację (wykorzystano do tego celu królicze przeciwciało przeciwko ludzkiej chemerynie sprzężone z sefarozą). Kontrolę stanowiło medium z hodowli keratynocytów traktowane przeciwciałami pochodzącymi z nieimmunizowanego królika. Użycie medium bez chemeryny w mniejszym stopniu hamowało wzrost bakterii niż medium kontrolne. Dodanie rekombinowanego białka do mediów pozbawionych chemeryny przywracało pierwotny efekt bakteriobójczy (Załącznik 2, Fig. 1C). Uzyskane wyniki pokazują, że ludzkie keratynocyty wydzielają 24

czynniki bakteriobójcze, a chemeryna jest jednym z nich i w znaczący sposób odpowiada za hamowanie wzrostu bakterii w naskórku. Potwierdzeniem tej hipotezy były porównawcze eksperymenty przeprowadzone na myszach z wyciszonym genem kodującym chemerynę oraz zwierzętach typu dzikiego (Załącznik 1, Ryc. 8). W badaniach wykorzystano bakterie szczepu S. aureus ponieważ są bakteriami zasiedlającymi skórę oraz odpowiedzialne są za liczne infekcje skóry, przy czym są wrażliwe na działanie peptydu 4 (dane nieprezentowane). Na skórę myszy nanoszono zawiesinę bakterii szczepu S. aureus. Po 24 godzinach zawiesina bakterii była pobierana i wyznaczano CFU/ml oraz określano procent przeżycia komórek bakteryjnych, względem wyjściowego inokulum. W przypadku zwierząt dzikich obserwowano prawie całkowite zahamowanie wzrostu bakterii na skórze, natomiast u zwierząt z deficytem chemeryny nie obserwowano tego efektu. Uzyskane wyniki jednoznacznie potwierdzają, że chemeryna obecna w naskórku pełni funkcję ochronną hamując wzrost bakterii. 25

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8. Załączniki: 1. Banas M, Zegar A, Kwitniewski M, Zabieglo K, Marczynska J, Kapinska- Mrowiecka M, LaJevic M, Zabel BA, Cichy J. (2015) The expression and regulation of chemerin in the epidermis. Plos ONE 10(2): e0117830. 2. Kulig P, Kantyka T, Zabel BA, Banas M, Chyra A, Stefanska A, Tu H, Allen SJ, Handel TM, Kozik A, Potempa J, Butcher EC, Cichy J. (2011) Regulation of chemerin chemoattractant and antibacterial activity by human cysteine cathepsins. J Immunol 187: 1403-1410. 3. Banas M, Zabieglo K, Kasetty G, Kapinska-Mrowiecka M, Borowczyk J, Drukala J, Murzyn K, Zabel BA, Butcher EC, Schroeder JM, Schmidtchen A, Cichy J. (2013) Chemerin is an antimicrobial agent in human epidermis. Plos ONE 8(3): e58709. 31

RESEARCH ARTICLE The Expression and Regulation of Chemerin in the Epidermis Magdalena Banas 1, Aneta Zegar 1, Mateusz Kwitniewski 1, Katarzyna Zabieglo 1, Joanna Marczynska 1, Monika Kapinska-Mrowiecka 2, Melissa LaJevic 3,4, Brian A. Zabel 4, Joanna Cichy 1 * 1 Department of Immunology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland, 2 Department of Dermatology, Zeromski Hospital, Kraków, Poland, 3 Stanford University School of Medicine, Department of Pathology, Stanford, California, United States of America, 4 Palo Alto Veterans Institute for Research, VA Palo Alto Health Care System, Palo Alto, California, United States of America * Joanna.Cichy@uj.edu.pl Abstract OPEN ACCESS Citation: Banas M, Zegar A, Kwitniewski M, Zabieglo K, Marczynska J, Kapinska-Mrowiecka M, et al. (2015) The Expression and Regulation of Chemerin in the Epidermis. PLoS ONE 10(2): e0117830. doi:10.1371/journal.pone.0117830 Academic Editor: Bernhard Ryffel, French National Centre for Scientific Research, FRANCE Received: October 1, 2014 Accepted: December 31, 2014 Published: February 6, 2015 Copyright: 2015 Banas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chemerin is a protein ligand for the G protein-coupled receptor CMKLR1 and also binds to two atypical heptahelical receptors, CCRL2 and GPR1. Chemerin is a leukocyte attractant, adipokine, and antimicrobial protein. Although chemerin was initially identified as a highly expressed gene in healthy skin keratinocytes that was downregulated during psoriasis, the regulation of chemerin and its receptors in the skin by specific cytokines and microbial factors remains unexplored. Here we show that chemerin, CMKLR1, CCRL2 and GPR1 are expressed in human and mouse epidermis, suggesting that this tissue may be both a source and target for chemerin mediated effects. In human skin cultures, chemerin is significantly downregulated by IL-17 and IL-22, key cytokines implicated in psoriasis, whereas it is upregulated by acute phase cytokines oncostatin M and IL-1β. Moreover, we show that human keratinocytes in vitroand mouse skin in vivorespond to specific microbial signals to regulate expression levels of chemerin and its receptors. Furthermore, in a cutaneous infection model, chemerin is required for maximal bactericidal effects in vivo. Together, our findings reveal previously uncharacterized regulators of chemerin expression in skin and identify a physiologic role for chemerin in skin barrier defense against microbial pathogens. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported in part by grants from Polish National Science Center 0724/B/P01/ 2011/40, UMO-2014/12/W/NZ6/00454 and the Foundation for Polish Science TEAM/2010-5/1, cofinanced by the European Union within European Regional Development Fund (to JC); The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University is a beneficiary of the structural funds from the European Union (grant No: POIG.02.01.00-12-064/08). This work was also supported by DoD grant W81XWH-11-1-0512 and Introduction Chemerin, also known as tazarotene induced gene 2 (Tig2) or retinoic acid receptor responder protein 2 (RARRES2), is a broadly expressed leukocyte attractant ligand for serpentine, G protein-associated receptor CMKLR1 (chemokine-like receptor 1) [1,2,3]. CMKLR1+ plasmacytoid dendritic cells (pdcs), macrophages and NK cells are critical in bridging the innate and adaptive immune responses [3,4,5,6]. Chemerin is secreted as an inactive precursor protein (Chem163S, with number and capital letter referring to the terminal amino acid position and single amino acid code, respectively). Chem163S can be converted to chemotactically active PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 1/ 19

Chemerin Regulation in Epidermis NIH grant AI-079320 (to BAZ). ML was a recipient of fellowship support under National Institutes of Health Training Grants 5 T32AI07290, T32CA09151 and F32CA180415. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. isoforms, such as Chem157S, through posttranslational carboxyl-terminal processing by a variety of proteinases [2,7,8,9]. Interest in chemerin has surged in the last few years as chemerin, in addition to its chemotactic function, was reported to regulate adipocyte differentiation [10], angiogenesis [11], osteoblastogenesis [12], myogenesis [13], and glucose homeostasis [14,15,16]. In addition to CMKLR1, two additional heptahelical receptors, GPR1 and CC-motif chemokine receptor-like 2 (CCRL2), bind chemerin with low nanomolar affinities similar to CMKLR1 [17,18]. However, among these receptors only chemerin binding to CMKLR1 triggers cell migration, intracellular calcium mobilization, and β-arrestin2 association and receptor internalization, all features common to classical G protein-coupled receptors. In contrast, chemerin binding to GPR1 triggers β-arrestin2 association and receptor internalization: whether it triggers intracellular calcium signaling is unclear [2,17]. The in vivo function of GPR1 remains relatively obscure, although recent studies using GPR1-deficient mice implicate the receptor in regulating glucose homeostasis during obesity [19]. CCRL2 regulates chemerin concentrations by sequestering secreted chemerin, concentrating it on the cell surface and presenting it to adjacent CMKLR1+ cells [18,20,21]. Although fat tissue and liver have been confirmed by multiple groups as key sites of chemerin production [22] and possibly responsible for the high nanomolar chemerin levels found circulating in plasma [23], chemerin is also expressed at epithelial barriers, including skin epidermis [24,25,26,27]. There is regional variation in the distribution of chemerin in healthy and diseased skin. Whereas chemerin is produced by keratinocytes in healthy skin, it is markedly downregulated in the epidermis of patients suffering from the autoinflammatory skin disease psoriasis. In contrast, normal dermis contains little chemerin, but affected psoriatic dermis is a significant source of chemerin as determined by immunohistochemistry [24,27]. These findings suggest an association between skin dysfunction and altered chemerin levels. We and others have previously reported that chemerin likely contributes to pdc recruitment to lesional psoriatic skin [5,24,28]. In addition, in normal skin, specifically the epidermis, chemerin functions as a potent antimicrobial protein, where it embodies a quantitatively significant fraction of the anti-bacterial activity of cultured keratinocytes [25]. Despite its roles in host defense and the pathogenesis of skin disease, the mechanisms underlying chemerin expression in skin remain poorly defined. The only known regulator of chemerin expression in epidermis is the anti-psoriatic synthetic retinoid-tazarotene, which upregulates chemerin level in skin raft cultures [27]. Here we show that epidermal chemerin represents an important source of this protein in the skin under steady-state conditions and is significantly downregulated by cytokines implicated in psoriasis, whereas it is markedly upregulated by bacteria and acute phase mediators. Materials and Methods Materials Human recombinant OSM, IL-1β, IL17 and IL22 were purchased from R&D Systems, whereas E. coli-derived LPS from Sigma-Aldrich. S. aureus ATCC 35556 and E. coli HB101 were obtained from DSMZ. Bacteria were grown in tryptic soy broth (TSB) (Sigma) to mid-logarithmic phase and used for subsequent experiments at 1x10 7 colony-forming units (CFU). When indicated bacteria were heat-killed by incubation of 10 7 CFU bacteria/100 microl PBS at 85 C for 20 min., or were incubated with bacteriocidic concentration of ampicilin (1 μg/ml) for 24h/ 48h. PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 2/ 19

Chemerin Regulation in Epidermis Mice Female or male 8 12 weeks old C57BL6 mice and chemerin-deficient mice on C57BL6 background, as well as WT Balb/C mice, CMKLR1KO [29], CCRL2KO [18] or double CMKLR1/ CCRL2KO mice on Balb/C background were used in these studies. The chemerin KO mice used for this research project were generated by the trans-nih Knock-Out Mouse Project (KOMP) and obtained from the KOMP Repository (www.komp.org). NIH grants to Velocigene at Regeneron Inc (U01HG004085) and the CSD Consortium (U01HG004080) funded the generation of gene-targeted ES cells for 8500 genes in the KOMP Program and archived and distributed by the KOMP Repository at UC Davis and CHORI (U42RR024244). Mice were housed under pathogen-free conditions in the animal facility at the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University or the Veterans Affairs Palo Alto Health Care System. Liver, white adipose tissue (WAT) and skin were harvested and subjected to RT-QPCR or ELISA analysis. Blood was collected in EDTA coated tubes and centrifuged at 2000g for 6 min. Collected plasma was then subjected to ELISA analysis. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols 119/2010, 149/2013 and A3088-01 were approved by the First Local Ethical Committee on Animal Testing at the Jagiellonian University in Krakow or the Institutional Animal Use and Care Committee at the Veterans Affairs Palo Alto Health Care System (AAALAC-accredited facility). All surgery was performed under ketamine/xylazine anesthesia, and all efforts were made to minimize suffering. Mice were sacrificed by inhalation of CO2. Topical skin infection Mice were anesthetized and a small dorsal area of the skin was shaved and sterilized with ethanol. The shaved area was punctured six times at two places using a syringe needle (BD Microlance, 0.3 19 mm) (MidMeds). Two rubber rings both with 8-mm inner diameter were subsequently attached using an ethylcyanoacrylate-based adhesive and the rings were covered with OpSite (Medisave). 1 10 7 CFU of S. aureus or E. coli in a volume of 50μl (PBS) was thereafter injected through the OpSite into the cavity formed by the rubber rings. The ring injected with sterile PBS was used as control. Mice were killed after 24h and the skin within the side of the rings was retrieved for RT-QPCR and ELISA analysis as well as enumeration of CFU. Cell culture All human studies were performed in compliance with ethical protocols KBET/44/B/2011 and KBET/87/B/2014 approved by the Jagiellonian University Institutional Bioethics Committee. Declaration of Helsinki protocols were followed. All participants provided their written informed consent to participate in these studies as recommended by the ethical board. Normal human keratinocytes were isolated from excess skin from donors obtained at the time of cosmetic surgery for mole removal or during plastic surgery. Donors included 23 healthy individuals (age 36±18 years; F:M, 10:13). Skin biopsies were rinsed three times in calcium- and magnesium-free PBS supplemented with penicillin (5000U/ml) streptomycin (5mg/ml) (all from Sigma). After washing, the biopsy was placed in PBS containing dispase (12U/mL, Gibco) for 16h at 4 C. Next, the epidermis was separated from the dermis with forceps followed by treatment with 0.05% trypsin with 2 mm EDTA (Sigma) to isolate epidermal cells. Cells were cultured in serum free KGM-Gold medium (Lonza Group Ltd.) to generate passage 1 cells. The keratinocytes were then plated at density of 1 10 5 cells per well on permeable inserts (12-mmdiameter, 0.4μm pore size; Millipore, Millicell culture inserts) in PCT Epidermal Keratinocyte PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 3/ 19

Chemerin Regulation in Epidermis Medium (CellnTec). Cells were cultured at 37 C in presence of 5% CO 2 until confluence. Polarized skin structures that resemble in vivo stratified epidermis were generated by air-liquid interface cultures grown in 3D Prime Medium (CellnTec) for 11 days. Cells were than treated with the indicated factors for 24h or 48h. The final concentration of stimulating factors were based on previous publications [30,31,32], or for LPS optimized experimentally [from 10ng to 10 μg/ml], and were as follows; OSM 50ng/ml, IL1 10ng/ml, IL17 200ng/ml, IL22 200ng/ml and LPS 1μg/ml. Preparation of skin homogenates and epidermis lysates Skin was homogenized at 100 mg/ml in water containing protease inhibitor (Complete, Roche) or lysed as described for the epidermis. The epidermis was separated from the dermis as described above. Epidermis was then lysed in a RIPA buffer (25mM Tris-HCl, ph 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibitors, passed through a 40 μm cell strainer and incubated o/n at 4 C. Extracts were centrifuged at 10,000g for 30 min to remove cellular debris and then normalized based on protein concentration as determined by BCA assay (Sigma). Lysates were stored at -20 C until used. RT-QPCR Total RNA was extracted as previously described [33] and converted to cdna using NxGen M-MulV reverse transcriptase (Lucigen) with random primers (Promega). Real time PCR was performed on the 7500Fast (Applied Biosystems) using SYBR Green I containing universal PC R master mix (A&A Biotechnology) and primers specific for; human chemerin (5 TGGAA GAAACCCGAGTGCAAA-3, 5 -AGAACTTGGGTCTCTATGGGG), CMKLR1 (5 - ATGGA CTACCACTGGGTTTTCGGG-3,5 -GAAGACGAGAGATGGGGAAACTCAAG-3 ), CCLR2 (5 -CCGTTTCTTAAAAGGCAGTCTGAA-3,5 -GTCATACTTGTCACA TTGCTCTGC-3 ), GPR1 (5 -AATGCCATCGTCATTTGGTT-3, 5 -CAACTGGGCAGTGAA GGAAT-3 ), GAPDH (5 - GAGTCAACGGTTTGGTCGTATTG-3, 5 - ATGTAGTTGA GGTCAATGAAGGGG -3 ) and beta-2-microglobulin (B2M) (5 - TCAGCAAGGA CTGGTCTTTCTATC-3,5 - GCTTACATGTCTCGATCCCACTTA-3 ), as well as mouse chemerin (5 - CTTCTCCCGTTTGGTTTGATT-3,5 - TACAGGTGGCTCCTCTGGAGGA GT-3 ), CMKLR1 (5 -CAAGCAAACAGCCACTACCA-3,5 -TAGATGCCGGAGTCGTTG TAA- 3 ), CCLR2 (5 - TTCCAACATCCTCCTCCTTG -3,5 - GATGCACGCAACAATACC AC -3 ), GPR1 (5 - AAAAGCTGTTTGAGGCTAGAAAGG -3,5 - AGGAAATCTGTTAA TGTTCTGTGCG -3 ), cyclophilin (5 - AGCATACAGGTCCTGGCATCTTGT -3,5 - CAAA GACCACATGCTTGCCATCCA -3 ) and ribosomal protein L13a (RPL13A) (5 -CCTCAA GGTGTTGGATGGGAT-3, 5 - GTAAGCAAACTTTCTGGTAGGCTTC-3 ). The Excel based application Best-Keeper was used to analyze the expression stabilities of the commonly used reference genes [34]. Based on this analysis, murine cyclophylin A and RPL13A, or human B2M and GAPDH were selected as housekeeping genes for normalizing RNA expression in RT-QPCR. Relative gene expression normalized to geometric mean of these housekeeping genes was calculated using the 2 -ΔΔCT method [35,36]. Whenever possible, specificity of PCR products was verified using KO mice. ELISA Chemerin levels in conditioned media or in epidermis lysates was quantified by human or mouse-specific ELISA. High-binding ELISA strips (Nunc) were coated with mouse-antihuman chemerin mabs (MAB23241), or goat-anti-mouse Abs (AF2325) (both from R&D Systems) in Tris-buffered saline (50 mm Tris-HCl ph 9.5, 150 mm NaCl). The plates were then PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 4/ 19

Chemerin Regulation in Epidermis washed with PBS containing 0.1% Tween 20, and nonspecific protein-binding sites were blocked with 3% BSA in PBS. Human or mouse recombinant chemerin was used as a standard. Chemerin was detected using biotin-conjugated goat anti-human chemerin Abs (BAF2324) or biotin-conjugated rat-anti mouse chemerin mabs (BAM2325) followed by streptavidin-hrp (BD Science). The reaction was developed with TMB substrate (BD Science).The ELISA detects both the 163S and 157S chemerin. Alternatively, chemerin in mouse skin homogenates (100%) and plasma samples (diluted 1/200) was detected by comercially available ELISA (R&D Systems), according to the manufacturer s instructions. The levels of chemerin in plasma or skin homogenates and epidermis extracts were undetectable in chemerin KO mice. Immunohistochemistry Epidermal tissues were fixed in 4% formaldehyde and embedded in paraffin. Paraffin 6-μm sections were then prepared from keratinocyte cultures. Sections were blocked with goat IgG and stained with the rabbit anti-human chemerin (H-002-52 Phoenix Pharmaceuticals) or control IgG (normal rabbit IgG, Jackson Immunoresearch) followed by APC-goat anti-rabbit IgG F(ab)2 (Jackson Immunoresearch). Blocking and staining were performed in the presence of 0.1% saponin. The sections were counterstained with Hoechst 33258 (Invitrogen). Images were captured with a fluorescence microscope (NIKON, Eclipse) and analyzed by NIS elements software (Nikon). Statistical analysis For statistical evaluation, one way ANOVA followed by a Bonferroni post hoc test, or twotailed Student s t test was performed. Results Expression of chemerin and its receptors in normal skin Under normal conditions, expression of chemerin mrna in skin was approximately ten-fold and six-fold lower compared to liver and white adipose tissue (WAT), respectively (Fig. 1A). On the other hand, chemerin protein levels in tissue lysates were only two-fold and three-fold lower compared to liver and WAT, respectively (liver: 190±40 ng/mg total protein; WAT: 267±37 ng/mg; skin: 86±17 ng/mg) (Fig. 1B). When the skin was split into epidermal and dermal sheets, chemerin was found primarily in the epidermis (Fig. 1A and B), in agreement with previous immunohistochemistry results [26], suggesting that chemerin mrna and protein levels in total skin might be diluted by low expression of chemerin in dermis. Notably, chemerin protein levels in epidermal isolations (133±41 ng/mg of total protein) were similar to the levels detected in the liver. Since chemerin protein levels in tissue lysates might be affected by binding of secreted chemerin to chemerin receptors [22], we next analyzed expression of CMKLR1, CCRL2, and GPR1. Although mrna for all three receptors was present in liver, WAT and skin, CMKLR1 was expressed most highly in WAT, whereas CCRL2 and GPR1 were expressed most highly in skin (Fig. 1C-E). CMKLR1and GPR1 expression tended to be higher in the dermal compartment compared with epidermal layers, and was significantly higher for CCRL2. If CMKLR1 and CCRL2 serve as chemerin receptors in skin, then skin chemerin levels may be diminished in the absence of these receptors. As demonstrated in Fig. 1F, skin chemerin levels tended to be lower in CMKLR1 KO and CCRL2 KO mice and were the lowest in mice with a combined deletion of CMKLR1 and CCRL2 (CMKLR1/CCRL2 KO) compared to WT mice. On the other hand, plasma chemerin levels showed the opposite trend and were highest in CMKLR1/CCRL2 KO mice (Fig. 1G). This is consistent with a previous report indicating elevated chemerin levels in CCRL2 PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 5/ 19

Chemerin Regulation in Epidermis Fig 1. Chemerin and chemerin receptor expression in mouse skin. Chemerin mrna expression (A), chemerin protein expression in tissue lysates (B), chemerin receptor (CMKLR1, CCRL2, GPR1) mrna expression (C-E), or chemerin protein expression in skin homogenates (F) and chemerin expression in plasma (G) was measured in the indicated tissues isolated from C57BL/6 mice (A-E) or the indicated mice on BalbC backround (F-G) by RT-QPCR and ELISA. The expression data of the indicated genes was normalized to cyclophilin A and RPL13A, and presented relative to liver as the mean ± SEM, n = 4 5 different mice (A, C-E). The amount of chemerin protein in plasma, or skin lysates and homogenates normalized to total protein is shown as the mean ± SEM, n = 4 (B), or n6 mice (F-G). Statistical significance is indicated by asterisk(s); *p<0.05, **p<0.01, by ANOVA followed by a Bonferroni post hoc test. doi:10.1371/journal.pone.0117830.g001 KO mice [21] and is a general phenomenon common to cognate receptor-deficient mice [37]. Together, these data suggest that chemerin is sequestered in skin by CMKLR1 and CCRL2. We next evaluated chemerin and chemerin receptor levels in human skin. Similar to mouse skin, chemerin levels were significantly higher in human epidermis compared with dermis (Fig. 2A and B). On the other hand, there were no significant differences in human epidermal vs. dermal expression of CMKLR1, CCRL2 and GPR1 (Fig. 2C-E). Regulation of chemerin expression in keratinocytes by acute phase mediators To facilitate our studies investigating the mechanisms regulating chemerin expression in skin we generated pseudo-stratified, highly differentiated human epidermal tissue in vitro.in PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 6/ 19

Chemerin Regulation in Epidermis Fig 2. Chemerin in healthy human skin is primarily expressed in the epidermis. Chemerin mrna expression (A), chemerin protein expression in tissue lysates (B), and chemerin receptor (CMKLR1, CCRL2, GPR1) mrna expression (C-E), was measured in the indicated tissues isolated from healthy human donors. Total RNA was subjected to RT-QPCR. The expression data of the indicated genes was normalized to B2M and GAPDH, and presented relative to skin (A, C-E). The amount of chemerin in skin lysates, normalized to total protein was determined by ELISA (B). The mean of n = 6 (A, C-E) or n = 8 (B) different donors ± SEM is shown. Statistical significance between epidermis and dermis is shown by asterisk(s); *p<0.05, **p<0.01, by ANOVA followed by a Bonferroni post hoc test. doi:10.1371/journal.pone.0117830.g002 PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 7/ 19

Chemerin Regulation in Epidermis Fig 3. Psoriasis-associated cytokines downregulate chemerin and acute phase cytokines upregulate chemerin expression in epidermis. Normal keratinocytes grown in 3D culture were treated with the indicated factors for 24 (A-B) or 48h (C-D). Total RNA was subjected to RT-QPCR. Relative expression of stimulated cells over control is shown as the mean ±SD from five-nine independent experiments (A, C). Levels of secreted chemerin were determined in parallel in conditioned media by ELISA. Data show the mean ±SD from five-nine independent experiments (B, D). Statistical significance between control and the treated cells is shown by asterisk; *p<0.05 by ANOVA followed by a Bonferroni post hoc test. doi:10.1371/journal.pone.0117830.g003 contrast to keratinocyte monolayers that do not fully recapitulate the multilayered differentiation of epidermis and express little-to-no chemerin ([27] and data not shown), this 3D tissue closely resembles the epidermis, and keratinocytes in these 3D cultures express high levels of chemerin Figs. 3 & 4. Importantly, the polarized nature of skin keratinocytes in this model allows for the anatomical segregation of epidermal responses. We applied cytokines to the basolateral compartment to mimic epidermal cytokine exposure resulting from immune cells infiltrating the skin [38,39,40,41]. We first asked if local chemerin synthesis in the skin was induced by acute phase mediators such as oncostatin M (OSM) and IL-1β, which mobilize protective acute phase reactants. Cells and conditioned media from cultured human skin equivalents were collected 24 48h after basolateral treatment, since the effect of OSM on gene expression is typically most profound at these time points [31,42,43,44]. Treatment with OSM, IL-1β, and the combination resulted in either a tendency to higher chemerin levels or statistically significant upregulation of chemerin mrna and protein at both time points (Fig. 3). Chemerin production was the highest in response to OSM + IL-1β at 24h (7.3-fold increase over control by RNA analysis, and 2-fold by secreted protein analysis), suggesting additive effects (Fig. 3). PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 8/ 19

Chemerin Regulation in Epidermis Regulation of chemerin expression in keratinocytes by psoriatic cytokines IL-17 and IL-22 drive keratinocyte pathology in psoriasis [39,40,41]. We next asked if IL-17 and Il-22 applied to the basolateral compartment affected chemerin expression/secretion in the epidermis model. IL-17 and IL-22 were equally efficacious in downregulating chemerin expression at 48h (on average 2.5-fold compared with untreated controls), and when used together exhibited an additive effect (4.3-fold reduction). Consistent with IL-17- and IL-22-mediated inhibition of chemerin RNA expression, secreted protein tend to be diminished (Fig. 3C and D). Together, these data suggest that chemerin is a regulatory target of IL-17 and IL-22 in epidermal tissue. Regulation of chemerin expression in human keratinocytes and mouse skin by bacteria Since chemerin has antimicrobial activity in normal human skin, we next asked if its expression was modulated by bacteria exposure in the epidermal model (apical side treatment). We selected two bacteria strains, E. coli and S. aureus, both of which are susceptible to chemerin-dependent killing, although with different potencies (MIC = 3.1 6.3μg/ml vs. 12.5μg/ml for E. coli and S. aureus, respectively) [25]. E. coli markedly upregulated chemerin RNA expression (~7- fold), (Fig. 4A) and secreted protein (75±20 ng/ml versus 43±12 ng/ml in untreated cultures) at 24h (Fig. 4B). The effect of E. coli remained significant although somewhat diminished by 48h (Fig. 4C and D). Interestingly, compared with live bacteria, heat-killed counterparts triggered no significant effects on chemerin expression or secretion. This may be attributed to the ability of live microorganisms to replicate and/or express specialized stimulating factors. At least part of the stimulatory effect of E. coli was mediated by soluble factors, most likely LPS, as LPS alone significantly increased chemerin mrna at 24h. Compared with E. coli, S. aureus was more effective in boosting chemerin expression, resulting in 10-fold and 8-fold induction of chemerin mrna levels at 24h and 48h, respectively (Fig. 4A and C). The effect of S. aureus on chemerin gene expression was reflected in secreted chemerin protein levels, which increased from 43±12 to 123±24 ng/ml following 24h co-incubation, and from 53±17 to 185±47 ng/ml after 48h incubation with the bacteria (Fig. 4B and D). We hypothesized that since chemerin is more potent in killing E. coli compared with S. aureus, the keratinocytes in the in vitro culture may have been exposed to lower doses of E. coli than S. aureus. This may in turn result in the appearance of a more robust induction of chemerin by S. aureus. To address this, we treated S. aureus with bacteriostatic doses of ampicilin. This treatment resulted in comparable numbers of CFU for E. coli and S. aureus during their incubation with keratinocytes, and did not significantly change the chemerin levels in control keratinocytes (data not shown). There was no significant difference in epidermal chemerin induction by S. aureus vs. S. aureus treated with ampicillin, both of which were more effective than E. coli (Fig. 4A-D). These data imply that a S. aureus-intrinsic component stimulates higher levels of chemerin expression than E. coli, and that the effect is unrelated to differential microbe-specific killing potencies of chemerin. In addition, live S. aureus was ~3-fold more effective than heat-killed counterparts in inducing chemerin (Fig. 4A and C). Since the effect of live S. aureus on chemerin expression dominated over other factors, we investigated this effect more closely by immunohistochemistry. S. aureus-treated and untreated skin equivalents appeared similar by microscopic analysis of H&E stained sections (Fig. 4E). Chemerin was present in all strata, with the exception of stratum granulosum. However, chemerin staining was more intense in all epidermal strata in S. aureus-treated skin vs. control, most notably in the stratum basale, suggesting that the elevated chemerin protein levels PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 9/ 19

Chemerin Regulation in Epidermis Fig 4. Bacteria upregulate chemerin expression in epidermis. Keratinocytes were treated with the indicated factors for 24 (A, B, E) or 48h (C, D). Total RNA was subjected to RT-QPCR. Relative expression of stimulated cells over control is shown as the mean ±SD from five-nine independent experiments (A, C). Levels of secreted chemerin were determined in parallel in conditioned media by ELISA. Data show the mean ±SD from five-nine independent experiments (B, D). Statistical significance between control and the treated cells is shown by asterisk; *p<0.05 by ANOVA followed by a Bonferroni post hoc test. E. c., E. coli; HK, heat-killed; SN, supernatant; S. a., S. aureus; A, ampicillin-treated. Microscope images of keratinocytes stained with hematoxilin and eosin (H&E) and fluorescence microscope images of keratinocytes stained for chemerin (chem) or control rabbit Abs (cab) (red), with Hoechst counterstain to detect cell nuclei (blue). Scale bar = 10 μm. Data are representative of three different donors. SC, stratum corneum, SG, stratum granulosum, SS, stratum spinosum, SB stratum basale, TM transwell membrane (E). doi:10.1371/journal.pone.0117830.g004 detected in conditioned media likely result from its secretion primarily by proliferating keratinocytes (Fig. 4E). Regulation of CMKLR1, CCRL2 and GPR1 expression in human epidermis equivalents We next evaluated the expression of chemerin receptors in skin equivalents in response to cytokines and bacteria. CMKLR1 mrna levels were significantly upregulated following 24h treatment with IL-1β and the combination of IL-1β and OSM, while IL-17 and/or IL22 had no effect (Fig. 5A). Of the cytokines tested, only IL-1β had a significant effect on CMKLR1 levels at 48h (Fig. 5B) Likewise, CCRL2 and GPR1 were significantly upregulated only by IL1β or IL1β+OSM at the 24h time point, and by IL1β at the 48h time point in the case of CCRL2 PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 10 / 19

Chemerin Regulation in Epidermis Fig 5. Expression of chemerin receptors in human skin equivalents treated with cytokines. Keratinocytes were treated with the indicated factors for 24h (A) or 48 h (B). RT-QPCR was performed and the expression data were normalized to cyclophilin A and expressed relative to unstimulated cells. Mean ± SD of 5 8 independent experiments is shown. Statistical significance comparing cytokine-treated cells vs. untreated cells (* p<0.05) was determined by ANOVA followed by a Bonferroni post hoc test. doi:10.1371/journal.pone.0117830.g005 (Fig. 5). CCRL2 was also significantly dowregulated by IL22 at 48h, whereas GPR1 expression was not altered (Fig. 5B). CCRL2 and GPR1 RNA expression was significantly downregulated by 24h-treatment with E. coli and E. coli products, and to a lesser extent by S. aureus, whereas levels of CMKLR1 were unaffected (Fig. 6A). Interestingly, at 48h, CCRL2 expression was significantly induced by live S. aureus but not by E. coli and its derivatives (Fig. 6B). A similar trend was noted for CMKLR1. Taken together, these data suggest that different regulatory mechanisms underlie the expression of each of the chemerin receptors in human epidermis. PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 11 / 19

Chemerin Regulation in Epidermis Fig 6. Expression of chemerin receptors in human skin equivalents treated with bacteria. Keratinocytes were treated with the indicated factors for 24h (A) or 48 h (B). RT-QPCR was performed and the expression data were normalized to cyclophilin A and expressed relative to unstimulated cells. Mean ± SD of 5 7 independent experiments is shown. Statistical significance comparing cytokine-treated cells vs. untreated cells (* p<0.05) was determined by ANOVA followed by a Bonferroni post hoc test. doi:10.1371/journal.pone.0117830.g006 PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 12 / 19

Chemerin Regulation in Epidermis Regulation of chemerin and its receptors by bacteria in mouse skin in vivo Due to the pronounced elevation of chemerin levels by bacteria and the differential effects of E. coli and S. aureus on chemerin receptor expression in human skin equivalents, we next asked if these responses occur in vivo. Mice were ectopically treated with E. coli or S. aureus, and the skin analyzed for chemerin and chemerin receptor expression 24h later. Both E. coli or S. aureus significantly upregulated chemerin mrna and chemerin protein levels in skin lysates (Fig. 7A and B). However, similar to human skin equivalents, S. aureus seemed to be more potent in inducing chemerin expression compared with E. coli, although this trend did not reach statistical significance. S. aureus significantly increased CMKLR1 and GPR1 RNA expression in the skin (Fig. 7C and E), while E. coli significantly increased the expression of CCRL2 and GPR1 (Fig. 7D &E). Together, these data suggest that the expression of chemerin and its receptors are influenced in distinct fashion by cutaneous microbes. Chemerin is required for maximal bactericidal effects in vivo Given the significant local induction of chemerin in the skin in response to bacterial challenge, we next asked if chemerin controls bacterial burden in skin. Chemerin-deficient mice and wild type controls were topically infected with S. aureus, and the bacterial load recovered from the skin surface 24h later was measured by colony-forming assay. Chemerin-deficient mice harbored at least 10-fold higher bacterial levels compared to WT (Fig. 8). These data suggest that chemerin plays a key role in restricting bacteria growth in skin. Discussion Here we report on previously unappreciated regulators of chemerin synthesis in the epidermis that link chemerin expression to both clinical findings in psoriasis and antimicrobial functions of chemerin in skin. First, treatment of model epidermis with IL-17 and IL-22 recapitulate the reduction in chemerin levels reported in affected skin from psoriasis patients. Although the nature and significance of chemerin downregulation in lesional psoriatic skin remains obscure, we reasoned that chemerin expression might be affected by the same mediators that drive the disease processes. Genetic studies, usage of therapeutic antagonists, as well as recently developed imiquimodbased mouse model of psoriasis, established a pivotal role for the IL-17 as a driver in skin inflammation in psoriasis [39,45]. In addition, IL-22 has emerged as a key regulator of keratinocyte hyperplasia in this disorder [40,46,47]. Deficiencies in either, IL-17 or IL-22 result in partial protection, whereas absence of both IL-17- and IL-22-mediated responses confers almost total protection against the disease, suggesting additive or synergistic effects of these cytokines in the development of skin changes. Keratinocytes appear to be one of the main targets of IL-17 and IL-22 in psoriatic skin [39,40]. This is supported by the finding that the absence of IL-17 or IL-22 correlates with marked reduction in epidermal thickening along with diminished numbers of skin infiltrating immune cells in vivo. Moreover, keratinocytes respond to these cytokines in vitro with a psoriatic-like gene expression signature that includes production of proinflammatory cytokines, chemokines, complement components and antimicrobial peptides [39,40,47]. Our work indicates that chemerin may be a regulatory target of IL-17 and IL- 22 in epidermis, potentially influencing skin cell responses in psoriasis. Second, we identified two different chemerin regulation patterns in response to cytokines that are elevated or induced in psoriatic skin. In contrast to IL-17 and IL-22, which suppressed chemerin expression, OSM and IL-1β significantly increased chemerin production, despite the PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 13 / 19

Chemerin Regulation in Epidermis PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 14 / 19

Chemerin Regulation in Epidermis Fig 7. Bacteria controls the expression of chemerin and its receptors in vivo. Mice were ectopically treated with S. aureus, E coli or PBS (control) for 24h. The skin exposed to the treatment was then collected for RNA and protein isolation. Chemerin and chemerin receptor message was determined by RT-QPCR. The expression data was normalized to cyclophilin A and presented relative to PBS-treated skin (A, C-E). The amount of chemerin in skin lysates, normalized to total protein was determined by ELISA (B). Data are shown as the mean ±SEM from six mice in each group. Statistically significant differences between PBS-treated and bacteria-treated mice is indicated by an asterisk (*, p<0.05; ANOVA followed by a Bonferroni post hoc test). doi:10.1371/journal.pone.0117830.g007 fact that all four cytokines are potent keratinocyte activators with potential roles in the pathology of psoriasis [38,43,48]. IL-1β has been assigned a prominent function in various aspects of cutaneous inflammation, for example, as a key contributing factor to the development and maturation of IL-17 secreting T cells, or in the recruitment of neutrophils to psoriatic skin [49,50,51]. On the other hand, OSM was linked to the pathology of psoriasis through its ability to inhibit expression of keratinocyte differentiation markers, including filaggrin and loricrin, which are decreased in the skin of psoriatic patients, or through inducing AMPs in reconstituted epidermis, such as psoriasin (S100A7), calgranulin C (S100A12) and β-defensin 2, which are strongly associated with psoriasis [38,43,52]. Although these OSM-mediated skin alterations suggest a pathogenic role of OSM in the disease, this cytokine may also contribute to attenuating the pathology, depending, for example, on the phase of the disease. This is supported by its well-defined role as an acute phase mediator as well as the observation that in reconstituted epidermis, OSM also downregulated sets of genes regarded as pro-inflammatory in psoriasis, such as Th1-type signaling molecules [43]. The opposing effects of OSM and IL-1β compared with IL-17 and IL-22 on chemerin production in keratinocytes suggests different roles for the former in regulating chemerin-mediated skin changes. Notably, in contrast to IL- 17 and IL-22, which had no effect or downregulated the chemerin receptors, IL-1β and to the lesser extend OSM increased expression of the receptors, suggesting that chemerin might have a particularly strong impact on skin pathophysiology when IL-1β and/or OSM are present. Since the epidermal disruption that occurs in psoriasis may lead to a compensatory engagement of cytokines involved in restoration of homeostasis, such as acute phase mediators-osm and IL-1, chemerin and chemerin receptor levels that rise in response to OSM and IL-1β may serve to improve skin conditions. Fig 8. Chemerin is bactericidal in vivo. Chemerin deficient (ChemKO) and WT mice were ectopically treated with S. aureus. Bacteria were retrieved from skin 24h later, and presented as a % of input inoculum. Each data point represents one experiment and a horizontal line indicate the mean value in each group. *p<0.05, by t test. doi:10.1371/journal.pone.0117830.g008 PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 15 / 19

Chemerin Regulation in Epidermis Third, our findings indicate that the epidermis is a functional bacteria-responsive anatomic site for chemerin production. The major function of the epidermis is to provide a barrier against the external environment that includes a variety of pathogenic microorganisms. Our data suggest that keratinocytes respond to microbial stimuli with chemerin synthesis. They also indicate that the epidermis, through upregulation of CCRL2 or CMKLR1, is likely to respond to chemerin in an autocrine manner when challenged by specific bacteria strains. Whereas E. coli and S. aureus both increased chemerin expression in human skin equivalents in vitro as well as mouse skin in vivo, chemerin receptor expression appeared to be differentially regulated by these bacteria strains. Most striking was a stimulatory role of S. aureus but not E. coli on CCRL2 expression in human skin equivalents. Restricting keratinocyte response to upregulation of chemerin but not CMKLR1 or CCRL2, as was the case for E. coli-mediated stimulation, may be a mechanism that diminishes CCRL2-mediated accumulation of chemerin on keratinocyte surfaces or CMKLR1-mediated signaling in keratinocytes, allowing free chemerin to act as an AMP. In contrast, S. aureus has the potential to contribute to epidermal biology by virtue of its reciprocal induction of chemerin and chemerin receptor expression. Whereas the secretion of chemerin by S. aureus-stimulated keratinocytes may contribute to establishing a biochemical shield to microbial colonization of skin by other bacteria, upregulation of chemerin receptors might foster chemerin-mediated, yet-to- be-identified functional changes in mammalian skin. S. aureus and E. coli are likely to deploy various mechanisms to affect production of chemerin and chemerin receptors in keratinocytes. These may include soluble factors and/or nonsecreted bacterial components, such as structures of the bacterial wall that differ substantially between these two microorganisms. Killing of either bacteria with heat, diminished chemerin production in keratinocytes, suggesting that bacteria viability is an important determinant associated with chemerin synthesis. A new concept has emerged that the recognition of so-called vita-pamps (viability associated pathogen-associated molecular patterns) that are present only in viable bacteria elicits unique responses [53]. These include bacterial messenger RNA. The stimulation of chemerin production by vita-pamps may explain the differential potency of live and dead bacteria to regulate chemerin expression in keratinocytes. Since chemerin synthesis in reconstituted human epidermis is also triggered to some extent by bacterial supernatants, soluble factors may also be involved in promoting chemerin synthesis in keratinocytes. Together, our findings reveal an inherent ability of human and mouse epidermis to express high levels of chemerin. Our previous work demonstrated the potent antimicrobial activity of human keratinocyte-derived chemerin [25], and our present study shows substantially diminished antimicrobial activity in chemerin-deficient mice. Thus, elevation of chemerin levels by acute phase cytokines and specific bacteria strains, and downregulation by cytokines associated with psoriasis may reflect a programmed response to skin challenge that regulates defensive functions of this organ. Acknowledgments We thank J. Borowczyk and Dr J. Drukala for help with keratinocyte cultures. Author Contributions Conceived and designed the experiments: JC. Performed the experiments: MB AZ MK KZ JM ML. Analyzed the data: JC BAZ MK. Contributed reagents/materials/analysis tools: MKM. Wrote the paper: JC BAZ. PLOS ONE DOI:10.1371/journal.pone.0117830 February 6, 2015 16 / 19

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The Journal of Immunology Regulation of Chemerin Chemoattractant and Antibacterial Activity by Human Cysteine Cathepsins Paulina Kulig,*,1 Tomasz Kantyka,,1 Brian A. Zabel,,1 Magdalena Banaś,* Agnieszka Chyra,* Anna Stefańska,* Hua Tu, x Samantha J. Allen, { Tracy M. Handel, { Andrzej Kozik, Jan Potempa,,# Eugene C. Butcher,, ** and Joanna Cichy* Chemerin, a ligand for the G-protein coupled receptor chemokine-like receptor 1, requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, including chemokine-like receptor 1-positive immature plasmacytoid dendritic cells (pdc). Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins composed of antibacterial polypeptide cathelicidins and inhibitors of cysteine proteinases (cystatins). We therefore hypothesized that chemerin may interact directly with cysteine proteases, and that it might also function as an antibacterial agent. In this article, we show that chemerin does not inhibit human cysteine proteases, but rather is a new substrate for cathepsin (cat) K and L. cat K- and L-cleaved chemerin triggered robust migration of human bloodderived pdc ex vivo. Furthermore, cat K- and L-truncated chemerin also displayed antibacterial activity against Enterobacteriaceae. Cathepsins may therefore contribute to host defense by activating chemerin to directly inhibit bacterial growth and to recruit pdc to sites of infection. The Journal of Immunology, 2011, 187: 1403 1410. *Department of Immunology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland; Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland; Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304; x LakePharma, Inc., Belmont, CA 94002; { Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093; Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland; # Center for Oral Health and Systemic Diseases, University of Louisville School of Dentistry, Louisville, KY 40202; and **Stanford University Medical School, Stanford, CA 94305 1 P.K., T.K., and B.A.Z. contributed equally to this work. Received for publication July 13, 2010. Accepted for publication May 29, 2011. This work was supported by the Ministry of Scientific Research, Poland (Grants SPUB3088 and 0724/B/P01/2011/40 to J.C.), a Fogarty International Research Collaborative award (R03TW007174-01 to E.C.B. and J.C.), a grant from the European Union 6th FP project (SP6MTKD-CT-2006-042586 to J.C.), a Team award from the Foundation for Polish Science (TEAM/2010-5/1 to J.C.), and the National Institutes of Health (Grant AI079320 to B.A.Z. and National Institutes of Health grants to E.C.B.). T.H. is supported by National Institutes of Health Grant R01-AI37113. J.P. is supported by the European Community Gums & Joints project (7FP-HEALTH-2010-261460), the Ministry of Scientific Research, Poland (Warsaw, Poland; Grant 1642/B/ P01/2008/35), and the Foundation for Polish Science (TEAM Project DPS/424-329/ 10). The Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University is a beneficiary of structural funds from the European Union (Grant POIG.02.01.00-12-064/08). Address correspondence and reprint requests to Dr. Joanna Cichy, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7 Street, 30-387 Krakow, Poland. E-mail address: joanna.cichy@uj.edu.pl Abbreviations used in this article: cat, cathepsin; chem, chemerin truncated at indicated residue position; chems, chemerin serum form; chem/sspb, staphopain B- truncated chemerin; CID, collision-induced dissociation; CMKLR1, chemokine-like receptor 1; hcap18, human cationic antimicrobial peptide of 18 kda; MHB, Mueller Hinton broth; MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; pdc, plasmacytoid dendritic cell; SspB, staphopain B. Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 Chemerin is a recently characterized chemoattractant protein that serves as a ligand for the seven-pass G-protein coupled receptor chemokine-like receptor 1 (CMKLR1) (1, 2). Two additional heptahelical receptors, GPR1 and CCRL2, have been reported to bind chemerin, although they do not appear to directly support chemotaxis (3, 4). Chemerin circulates as an inactive precursor (prochemerin) in blood and undergoes protease-mediated, C-terminal truncation to acquire chemotactic activity. Truncated, bioactive chemerin lacking 6 (chems157), 8 (chema155), or 9 (chemf154) amino acids in the C terminus have been isolated from several biofluids, including ascites, serum, and hemofiltrate, respectively (reviewed in Ref. 5). Serine proteases of the coagulation, fibrinolytic, and inflammatory cascades, including neutrophil elastase and cathepsin (cat) G, generate bioactive chemerin (6, 7). In addition, chemerin can be activated in a sequential manner by plasma carboxypeptidases after initial cleavage by plasmin (8). Cells that are critical in linking the innate and adaptive immune responses, such as plasmacytoid dendritic cells (pdc), NK cells, and macrophages, express CMKLR1 and respond to chemerin through chemotaxis (1, 2, 9 12). Although the structure of chemerin has not yet been solved, the predicted structural homology between chemerin and inhibitors of cysteine proteinases (cystatins) and antimicrobial cathelicidins (1, 5) suggests that chemerin may inhibit endogenous human cysteine proteases and possibly exhibit antibacterial activity. Alternatively, host cysteine proteases may bind and proteolytically process chemerin. In support of the latter, we recently reported that the cysteine protease staphopain B (SspB) secreted by the human pathogen Staphylococcus aureus selectively cleaves and activates chemerin (13). The papain-like cysteine proteases, including cat B, cat L, and cat K are well-known degradative enzymes of mammalian cells, participating primarily in intracellular proteolytic pathways (such as Ag processing and presentation), but also extracellular protein turnover. Recent studies show that lysosomal cathepsins can exert their proteolytic activity at extracellular sites (14, 15), where they contribute to a variety of pathophysiological processes, including chronic inflammation associated with obesity (16 18). Cathelicidins consist of two distinct domains: a highly conserved N-terminal cathelin-like domain with homology to the cystatins and a divergent C-terminal antimicrobial region that varies among species. Only one cathelicidin has been described in humans: human cationic antimicrobial peptide of 18 kda (hcap18). hcap18 is cleaved by neutrophil serine proteases such as pro- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002352

1404 CHEMERIN ACTIVATION BY HOST CYSTEINE CATHEPSINS teinase 3 to generate a 37-aa antimicrobial peptide LL-37 and a 103-aa cathelin-like domain (19, 20). In this study, we found that although chemerin does not inhibit the proteolytic activities of cat L or K, these cysteine proteases are potent activators of chemerin. cat L and K initially and efficiently cleave prochemerin to release a 6-aa peptide from the carboxyl terminus, generating chems157; the enzymes can also cleave chemerin to release a 38-residue C-terminal peptide, generating chemr125. The activated chemerin S157 is a potent attractant for CMKLR1 + cells, including human blood pdc. In addition, we demonstrate that although the smaller chemerin fragment generated by both cathepsins (chemerin R125) does not support chemotaxis of CMKLR1 + cells, both chems157 and chemr125 display comparable antimicrobial activity against Enterobacteriaceae. Materials and Methods Materials Recombinant cat B, L, and K, and chems157 were purchased from R&D Systems. Anti-CD3, -CD14, -CD16, -CD19, -CD20, -CD56 biotin-linked mabs, as well as FITC-labeled CD123 and allophycocyanin-labeled BDCA-2 were obtained from BD Pharmingen, Miltenyi Biotec, ebioscience, and BioLegend. Recombinant chemerin isoforms, full-length prochemerin, chemerin serum form (chema155), and SspB-truncated chemerin (chem/sspb; chems157) were produced as previously described (2, 13, 21). Recombinant Fc-chemerin proteins were produced and purified from Chinese hamster ovary cells via transient transfection and protein A purification. DNA fragments corresponding to the desired chemerin proteins were amplified by PCR and cloned in-frame downstream of human IgG1 Fc domain, which is downstream of a secretion signal peptide in mammalian expression vector plev113 (LakePharma). There is a 9-aa glycine-rich linker between the Fc and chemerin domains. Plasmid DNA was transfected into Chinese hamster ovary cells using Lafectine transfection reagent (LakePharma), and cell culture supernatant was collected 3 d posttransfection. Fc fusion proteins were purified with Protein A resins (Mab Select SuRe GE Healthcare), and final proteins were formulated in 100 mm Tris, 150 mm NaCl, and 0.45% NaOAc. Inhibition assays Inhibitory activity against cat B, K, and L was assayed fluorometrically using a Molecular Devices Gemini XS system, with an excitation of 350 nm and emission wavelength of 460 nm. Cathepsins were preactivated in a buffer containing 100 mm sodium acetate, ph 5.5, 100 mm NaCl, 10 mm DTT, 1 mm EDTA, and 0.01% Tween 20 for 10 min at 25 C. Cathepsins (0.2 nm) were then incubated with 10 or 100 molar excess of recombinant prochemerin or the nonspecific cysteine proteinase inhibitor E64 for 30 min at 37 C in the same buffer containing the fluorogenic substrates Z-Phe-Arg-AMC (10 mm; Sigma) for cat B and L or Boc-VLK- AMC (10 mm; Peptides International) for cat K, in a total volume of 100 ml. The progress of the reaction was monitored by fluorescence spectroscopy, and the data were plotted versus time. Maximum velocity values were calculated by linear fit to the time-dependent curve. The residual cathepsin activity present in each treatment group is presented as a percentage of the maximum velocity determined in the absence of inhibitor (cathepsin + substrate only). Mass spectrometry Matrix-assisted laser desorption ionization time-of-flight spectrometry (MS) was performed by the Stanford Protein and Nucleic Acid Biotechnology Facility (Stanford University, Stanford, CA). After trypsin digest of the cat K and L truncated-chemerin band, MS was performed and the mass values were used in a Mascot search (http://www.matrixscience.com) of public peptide databases. PeptideCutter was used to predict the mass values of tryptic chemerin fragments (http://www.expasy.org). Mass values obtained after collision-induced dissociation (CID) of the predicted C-terminal peptide (f) were compared with the predicted CID mass values for the peptide corresponding to residues 141 157 of chemerin (sequence: AGEDPHSFYFPGQFAFS), using Mascot software (Matrix Science). Purification of pdc The Institutional Review Board at Jagiellonian University approved all human subject protocols. Human blood was collected and PBMCs were harvested after LSM1077 (PAA Laboratories) gradient separation, as described by the manufacturer. pdc were enriched from PBMC using negative selection with biotinylated mabs directed against CD3, CD14, CD16, CD19, CD20, CD56, and anti-biotin MACS Microbeads (Miltenyi Biotec), according to the manufacturer s recommendations. Cells were blocked with 50 80% autologous plasma and then stained for flow cytometry analysis using mabs against CD123 and BDCA-2 to identify pdc. Stained cells were analyzed on an LSRII flow cytometer (Becton Dickinson). Chemotaxis assay Cathepsins were incubated with recombinant human prochemerin for 10 min at 37 C and then tested in an in vitro chemotaxis assay using murine pre-b lymphoma L1.2 cells stably transfected with human recombinant CMKLR1 (CMKLR1/L1.2) or purified human blood pdc. In each case, enzymatic digestion was stopped by placing samples on ice and diluting with chemotaxis medium (RPMI 1640 containing 10% FBS). Where indicated, recombinant chemerin isoforms, Fc-chemerin fusion proteins, or Fc alone were used. A total of 100 ml cells (2.5 3 10 5 cells/well) was added to the top well of 5-mm pore transwell inserts (Costar), and test samples were added to the bottom well in a 600-ml volume. Migration was assayed for 2 h at 37 C. The inserts were then removed, and cells that had migrated through the filter to the lower chamber were collected and counted by flow cytometry (FACSCalibur; BD Biosciences). The results are presented as percentage input migration. CXCL12 served as a positive control. Antimicrobial assay Escherichia coli (HB101, a conventional laboratory strain) and Klebsiella pneumoniae (a clinical isolate from human bronchoalveolar lavage fluid) were used in this study. The antimicrobial activity of the indicated chemerin forms was estimated using a microtiter broth dilution assay (22). A single colony of bacteria was inoculated into 20 ml Mueller Hinton broth (MHB; Difco) and incubated overnight at 37 C, subcultured once at 1:100 dilution in MHB, and then grown for 2 3 h to midlogarithmic phase. Cell numbers were calculated using previously determined standard curves, and for subsequent experiments, bacteria were used at 2 7 3 10 5 CFU/ml. Bacterial suspensions (90 ml) in MHB were mixed with 10 ml diluent (10 mm HEPES, 100 mm Tris, 150 mm NaCl, and 0.45% NaOAc, or MHB) (control) or 10 ml different concentrations of chemerin, Fcchemerin fusion proteins, Fc alone, or synthetic LL-37 (Emory Microchemical Facility) and incubated at 37 C for the indicated times. After serial dilutions with MHB, the diluted mixture was plated on MHB agar plates and incubated at 37 C overnight for enumeration of CFU. In selected experiments, samples of the bacteria/peptide mixtures were also analyzed by flow cytometry and by spectrophotometry. These methods produced comparable results to the colony-forming assay (data not shown). Results cat L and K activate chemerin chemoattractant activity We initially tested chemerin for cystatin-like activity. Substrate hydrolysis by cat B, L, or K was not significantly inhibited by prochemerin, serum-form bioactive chemerin (chema155), or SspB-activated chemerin (chems157), even at 10:1 and 100:1 molar ratios of chemerin-to-cathepsin (Fig. 1, Table I). The general cysteine proteinase inhibitor E64 efficiently abolished the activity of all cathepsins examined (Fig. 1). Thus, chemerin does not appear to function as a cystatin. Prochemerin was incubated with purified cat B, L, or K and tested for attractant activity to test whether prochemerin is a substrate for human cysteine proteases. A controlled digest of prochemerin, using low concentrations of either cat L or K (1000-fold less than prochemerin) generated a single primary proteolytic product (Fig. 2A). Two apparent cleavage products were generated when cat L and K concentrations were increased 10 50 times, suggesting further digestion of prochemerin. Under similar conditions, cat B did not cleave prochemerin (data not shown). Interestingly, the two specific.10-kda chemerin cleavage products generated by cat L and K can be distinguished by PAGE only under reducing conditions (Fig. 2A). Because prochemerin contains three disulfide bonds, these data suggest that under nonreducing conditions, the dual-cleaved chemerin products remain associated with the holo-molecule through S S bond(s).

The Journal of Immunology 1405 FIGURE 1. Chemerin does not inhibit the activity of the cysteine cathepsins. Cathepsins were incubated with 10 (103) or 100 molar excess (1003) of recombinant prochemerin (pro-chem), serum chemerin (chema155), or chem/sspb (chems157), or 100 molar excess of the general cysteine proteinase inhibitor E64 for 30 min at 37 C with fluorogenic substrates. *p, 0.05, Student t test. Data are shown as mean 6 SD of three independent measurements done in duplicate. Control contained no inhibitor. CMKLR1-transfected L1.2 cells migrated significantly to cat L- or K-treated prochemerin (Fig. 2B). The chemotactic response of CMKLR1/L1.2 transfectants was dependent on cathepsin concentration, with cat L and K eliciting maximal effects on chemerin-mediated migration at a 1:100 prochemerin/cathepsin ratio (Fig. 2B). Although chemerin treated with cat L appeared to elicit slightly higher chemotactic response compared with cat K-treated chemerin (Fig. 2B), the difference was not statistically significant. Freshly isolated CMKLR1 + human blood pdc also migrated in response to cathepsin-cleaved chemerin, suggesting that these enzymes may be involved in pdc recruitment (Fig. 2C). No cell migration was detected in the absence of chemerin, or when prochemerin or the cathepsins were tested alone (Fig. 2). Compared with cat L and K, cat B had negligible effects on chemerin chemoattractant activity (data not shown). Taken together, these data suggest that incubation of cat L and K with prochemerin results in generation of bioactive chemerin chemoattractant. Identification of the cathepsin chemerin cleavage sites We used MS to determine the cathepsin chemerin cleavage sites. The mass value for the larger chemerin cleavage product initially and efficiently generated by cat L and K (experimental mass [M+H] +, 16,152 Da; calculated mass [M+H] +, 16,155 Da; D mass, 3 Da) corresponds to chemerin residues 18 157 (ADPELT GQFAFS, chem157s). The larger protein band (indicated by arrowheads in Fig. 2A) was isolated, digested with trypsin, and analyzed by mass spectrometry to confirm the processing site. A peptide fragment with a mass value of 1903.8 Da was identified, Table I. Comparison of chemerin isoforms used in this study Chemerin Form Abbreviations C-Terminal Amino Acid Sequence a References Prochemerin (full-length chemerin) Prochem 111 VLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQFAFSKALPRS 163 7 Serum form chema155 111 VLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQFAPH 155 7, 21 SspB-treated chemerin chem/sspb; chems157 111 VLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQFAFS 157 13 Minor form chems148 111 VLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHS 148 13 Cat L-treated chemerin, Cat K-treated chemerin chems157 111 VLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQFAFS 157 This study Cat L-treated chemerin, Cat K-treated chemerin chemr125 111 VLGRLVHCPIETQVLR 125 This study a Residue position is italic. Nonnative amino acids are shown in bold.

1406 CHEMERIN ACTIVATION BY HOST CYSTEINE CATHEPSINS FIGURE 2. cat L- and K-activated chemerin triggers CMKLR1/L1.2 cell and human blood pdc chemotaxis. A, Various concentrations of cat L or K were incubated with 2.9 mm chemerin, and the resulting products were separated by SDS-PAGE under either reducing (left panel) or nonreducing (right panel) conditions. Arrowheads indicate the two predominant chemerin cleavage products (two-pointed arrowheads signify the larger, three-pointed arrowheads signify the smaller cleavage products). Representative of n = 3 performed with similar results. B and C, cat K- or L- activated chemerin was tested in in vitro transwell chemotaxis using CMKLR1/L1.2 transfectants (B) or human blood pdc (C). The chemerin samples described in A were diluted to a final concentration of 1 nm and tested in chemotaxis assays. CXCL12 (10 nm) was used as a positive control for pdc chemotaxis (C). The mean 6 SD from duplicate wells of three or four experiments is shown. *p, 0.05 by Student t test comparing cathepsin-treated chemerin versus prochemerin, or CXCL12 versus (2) control. corresponding to a nontryptic peptide comprising amino acids 141 157 from the C terminus of chemerin (Fig. 3A, 3B). This peptide confirmed the initial and predominant cat K- and L-mediated chemerin cleavage site as NH 2..AFS KAL..COOH. Further microsequencing of this peptide by CID (tandem MS/MS) confirmed the sequence (Fig. 3C). Interestingly, this cleavage site is identical to a previously identified endogenous active human chemerin isoform isolated from ascites fluid (1), and to a main chemerin isoform generated by the S. aureus-secreted cysteine protease SspB (chem/sspb; Table I). The mass value for the smaller chemerin cleavage product generated by cat L and K (experimental mass [M+H] +, 12,442 Da; calculated mass [M+H] +, 12,437 Da; D mass, 5 Da) corresponds to chemerin residues 18 125 (ADPELT ETQVLR, chemr125). MS and CID analysis of the tryptic digests confirmed that the most distal tryptic or nontryptic fragment was LVHCPIETQVLR (fragment d in Fig. 3B, data not shown). Thus, prolonged incubation (data not shown) or incubation with 10 50 times higher concentrations of cat K or L (Fig. 2A) cleaves chemerin at position NH 2..QVLR EAEE..COOH, and the released C-terminal peptide likely remains linked to the holo-molecule through a disulfide bond, as indicated by Fig. 2A. Chemerin S157, but not chemerin R125, is chemotactively active Because separation of the smaller cleavage product by HPLC required reducing the S-S bonds, which would likely alter its secondary structure and possibly bioactivity, to determine the relative biological activities of the two cathepsin-generated chemerin products, we generated recombinant Fc-chemerins, abbreviated further as Fc-chemS157 (the larger cleavage product) and Fc-chemR125 (the smaller cleavage product). The Fc-chemerins have a glycine linker on the N terminus that is connected to the Fc domain of human IgG1; thus, the C terminus of the Fc-chemerin fusion proteins remained native. Fc alone or Fc-chemR125 failed to trigger CMKLR1 + cell migration at every concentration tested (up to 50 nm) (Fig. 4). However, chems157 was the most potent attractant for CMKLR1 + cells, with 1 nm eliciting a maximum 42 6 8% cell migration. Recombinant, commercially available chemerin S157 and HPLC-purified chemerin cleavage product of SspB

The Journal of Immunology 1407 FIGURE 3. Identification of the chemerin cat K and L processing site. Prochemerin was incubated with cat L and K, and the resulting peptide fragments were resolved by SDS-PAGE. The larger chemerin bands (upper bands indicated by two-pointed arrowheads in Fig. 2A) were digested with trypsin and subjected to MS analysis. A, Peptide fragments corresponding to predicted tryptic chemerin mass values are labeled with their mass values (a f). Asterisk indicates carbamidomethyl modification of cysteine. B, The peptides corresponding to the chemerin fragments identified by MS are underlined and lettered (a f). Vertical bars indicate predicted trypsin cleavage sites. The N-terminal signal peptide is indicated by italics, as is the C-terminal peptide released on cathepsin cleavage. The carboxy terminus of truncated chemerin is nontryptic C-terminal peptide (f). C, CID mass spectra (tandem MS/MS analysis) of peptide (f) confirms the sequence identity of the nontryptic C-terminal peptide. The major intensity peaks are predicted b or y series ions. (13) were used interchangeably as chems157, because both gave similar results in chemotaxis assays (data not shown). In previous work, we and others have shown that chems157 is a chemoattractant for pdc, NK cells, and monocyte-derived dendritic cells and macrophages (1, 11, 13). FIGURE 4. Chemerin S157 is the most active chemoattractant chemerin. CMKLR1/L1.2 cell migration to the indicated doses of the indicated chemerin isoforms was assessed by in vitro transwell chemotaxis. Migration to media only or to Fc only (tested at molar concentrations equivalent to the Fc-chemerin fusion proteins) are shown as negative controls. The mean 6 SD from three experiments is shown. Statistically significant differences between media or Fc-control and the chemerin isoforms are indicated by asterisks. *p, 0.05, Student t test. The Fc-chemS157 fusion protein also triggered statistically significant CMKLR1/L1.2 cell migration at concentrations of 5, 10, and 50 nm, although the response was not as robust as the unmodified form. ChemA155 also triggered CMKLR1 + cell migration, although it should be noted that this recombinant protein has a nonnative PH C-terminal dipeptide following the native terminal alanine (residue 155; Table I), which may alter its activity. Chemerin displays antibacterial activity The predicted homology between chemerin and the antimicrobial cathelicidins, coupled with the expression of chemerin in the skin and other epithelial cell surfaces continually exposed to bacterial challenge (9), led us to evaluate possible antibacterial activities of chemerin. Different chemerin isoforms were first tested for antibacterial activity against a laboratory strain of E. coli. Human cathelicidin LL-37 (3 mm) was used as a positive control peptide inhibitor of bacterial growth (20). As demonstrated in Fig. 5A, full-length chemerin (prochemerin, 2 mm) significantly inhibited the growth of E. coli when incubated for 24 h, leading to survival of 59 6 13% of bacteria compared with vehicle-treated E. coli set as 100%. Notably, truncated chemerin was an even more effective inhibitor of bacterial growth. The primary chemerin cleavage product generated by cat K and L (chems157; Table I) was significantly more effective than prochemerin in inhibiting bacterial

1408 CHEMERIN ACTIVATION BY HOST CYSTEINE CATHEPSINS FIGURE 5. Chemerin exhibits antibacterial activity. The antimicrobial activity of full-length (pro-chem), chema155, chems157 (HPLC-purified chem/sspb; Table I), Fc-chemS157, Fc-chemR125, and Fc alone were tested against E. coli (A D) and K. pneumoniae (A) using the microtiter broth dilution assay. Chemerin isoforms were tested at 2 mm (A, B), 0.5 mm (D), or at the indicated concentrations (C). Synthetic LL-37 (3 mm) was used as a positive control (A). Bacteria were incubated with the peptides for 24 h (A, C, D) or for indicated times (B). The results are expressed as the mean 6 SD of nine (A, E. coli), four (A, K. pneumoniae, B, C), or two (D) independent experiments done in duplicate. A, Statistically significant differences between prochemerin and the truncated chemerin isoforms are indicated by asterisks and p values (Student t test). B D, Asterisks indicate statistically significant differences comparing the indicated protein preparations with vehicle- or Fc alone-treated E. coli. *p, 0.05, **p, 0.001, Student t test. growth, resulting in 33 6 15% E. coli survival (Fig. 5A). Truncated chema155 also significantly reduced E. coli survival (39 6 19%) versus prochemerin (Fig. 5A). We also tested chemerin (2 mm) for antibacterial activity against a clinical isolate of another genus within the family of Enterobacteriaceae, K. pneumoniae. As demonstrated in Fig. 5A, incubation of K. pneumoniae with prochemerin resulted in 58 6 14% viable bacteria compared with control, whereas chema155 and chems157 reduced K. pneumoniae survival to 32 6 11 and 46 6 13%, respectively. Thus, similar to E. coli, the truncated form of chemerin was more effective at inhibiting K. pneumoniae growth than prochemerin. However, it should be noted that 24-h incubation of prochemerin with either E. coli or K. pneumoniae also resulted in some truncation of prochemerin (data not shown), suggesting that native protein requires removal of inhibitory C-terminal sequence to display full antibacterial activity. Taken together, these data suggest that proteolytic cleavage increases the antimicrobial activity of chemerin. The inhibition of E. coli growth by chemerin was detectable after just 8 h (Fig. 5B). Treatment with prochemerin, chema155, or chems157 diminished the survival of E. coli to 65 6 7, 53 6 10, and 60 6 5% of control, respectively (Fig. 5B). The chemerinmediated decrease in bacterial viability grew even more pronounced at prolonged incubation times; after 24 h, for example, the percentage of viable bacteria decreased to 53 6 9, 35 6 9, and 35 6 4%, respectively (Fig. 5B). With the exception of 4 h, when prochemerin seemed to be more effective compared with chema155 and chems157 (87 6 5, 95 6 3, 94 6 4), the truncated chemerin forms demonstrated stronger antibacterial activity against E. coli (Fig. 5B). Interestingly, chemerin-mediated inhibition of E. coli survival was detectable over a relatively wide range of concentrations, from 2 mm (the highest concentration tested leading to statistically significant growth inhibition) to as little as 0.125 to 0.0625 mm for chema155 and chems157 (Fig. 5C). This was in contrast with LL-37, which was highly effective at inhibiting E. coli growth when used at 3 mm (Fig. 5A) but had almost no effect when tested at 1.5 mm (data not shown). The enhanced antibacterial activity of the truncated chemerin isoforms compared with prochemerin suggests an inhibitory role for the prochemerin C-terminal peptide. Addition of chemically synthesized C-terminal peptide KALPRS that is released from prochemerin by SspB, cat K, and cat L, however, did not reduce the antibacterial activity of the larger truncated chemerin forms (data not shown). These data suggest that after release from the core protein, the C-terminal peptide no longer plays an inhibitory role. Interestingly, both chemerin Fc fusion proteins, Fc-chemS157 and Fc-chemR125, displayed comparable antibacterial activity against E. coli (Fig. 5D). These data suggest that in contrast with chemotactic activity, the antibacterial properties of chemerin appear to be localized closer to the N terminus, because removal of 38 residues from the C terminus did not abrogate its antibacterial activity. Discussion In this article, we identify a novel antimicrobial activity associated with chemerin, and show that host-derived cat L and K can cleave and activate the leukocyte attractant activity of chemerin, as well as enhance its antibacterial effects. Various serine proteases have been reported to effectively convert chemerin to a potent chemoattractant in vitro. There is also a single example of a cysteine protease, S. aureus-derived SspB that can efficiently activate human chemerin. In addition, hostoriginating cathepsin S and calpains have been reported to process mouse chemerin, although in this case, the proteolysis of the C terminus generates chemerin variants equipped primarily with anti-inflammatory properties (13, 23). Cysteine cathepsins of the papain-like family are normally confined to the endosomal/ lysosomal network. However, there is evidence that certain cathepsins are also active extracellularly, either in association with the cell surface or in soluble form (14). Some cells such as macrophages and fibroblasts constitutively secrete cysteine cathepsins as zymogens (14). Moreover, macrophages have been reported to deploy enzymatically active cat B, L, and S, and exhibit an elastindegrading phenotype, indicating that macrophages can mobilize

The Journal of Immunology 1409 cysteine cathepsins to participate in the pathophysiologic remodeling of the extracellular matrix (17). Massive amounts of extracellular cathepsins, probably released from macrophages, are found in the bronchial tree of patients suffering from acute pulmonary inflammation (24). In addition, cat K is strongly implicated in maintaining the homeostasis of the extracellular matrix in the lung (25). Because chemerin mrna is abundantly produced in lung (1, 2), collectively, these data suggest that either cat L or K may be involved in chemerin processing in this organ. Alternatively, significant expression and/or activity of cat K and L in the joints of patients with rheumatoid arthritis and skin dermatoses, respectively (26, 27), together with reported chemerin immunoreactivity or bioactivity, or both, in psoriasis skin and inflamed synovial fluid (1, 9, 28), suggest that these cathepsins may play a role in chemerin cleavage in joints and skin. Because cathepsinmediated processing releases chemerin attractant activity, these enzymes may have an important regulatory role in immune cell migration. Notably, the presence of pdc in lung, as well as the inflamed joints and psoriatic skin (1, 9, 28, 29), supports the notion that cat K and L, through the generation of active chemerin, may contribute to pdc recruitment to these sites. Our data also uncover a novel role for chemerin as a hostexpressed antibacterial agent in host defense. Despite low primary sequence homology between chemerin and antibacterial cathelicidins, the conserved positioning of key cysteine residues leads to a predicted shared similar tertiary structure, although recent NMR assignment of human chemerin does not exclude a different fold (30). LL-37, the 37-aa C-terminal derivative of human cathelicidin hcap18, is well known for its potent and broad-spectrum bacterial killing activity. However, chemerin is structurally similar to the cathelin-like N-terminal region. Interestingly, the cathelin-like domain of hcap18 has been reported to possess antimicrobial activity, although the mechanism by which it inhibits bacterial growth is not known (20). Chemerin may exert antimicrobial activity on the surface of skin and/or lung where it is locally expressed (1, 9). For example, the respiratory surface is continually exposed to pathogenic organisms, such as K. pneumoniae, which, as shown in this report, might be a direct chemerin target. Although either prochemerin or the C-terminal truncated chemerin forms displayed antibacterial activity, C-terminal processing augmented the inhibitory effect of chemerin on the growth of Enterobacteriaceae. However, our data suggest that prochemerin is also processed by bacterial proteases during incubation, although the protease(s) responsible remain to be identified. It will be interesting to map the specific chemerin domains/regions responsible for its antimicrobial activity. Our preliminary data suggest that most of the antibacterial activity is associated with the chemerin region(s) located within 65 115 aa (data not shown). This is consistent with our data showing that FcchemS157 and Fc-chemR125 have similar antibacterial activity, although the inhibitory C-terminal peptide must be removed for full antibacterial effects. Although the antimicrobial effects of chemerin on E. coli and K. pneumoniae were less potent compared with the classical antibacterial peptide LL-37, chemerin showed bactericidal properties at much lower concentrations. In general, pore-forming antimicrobial peptides, such as LL-37, require micromolar concentrations for activity. However, some antibacterial peptides, such as Lactococcus-derived nisin, operate in the nanomolar range (31). This ability is attributed to docking to a specific component on the bacteria cell wall for subsequent pore formation, or to the dualkilling mechanisms of the peptide, which in addition inhibits bacterial cell wall biosynthesis (32). Chemerin might use a similar strategy to exert its antimicrobial activity in the nanomolar range. However, because antimicrobial properties can be sensitive to ph and ionic composition of the peptide environment (31), it will be important to determine whether chemerin operates in conditions similar to those found in the skin, bronchial tree, or both. Thus, our work uncovers a novel antibacterial property of chemerin and characterizes the activation of chemerin by hostderived cysteine proteases of the cathepsin family, and adds a new dimension to the ways chemerin may modulate and augment immunity. Acknowledgments We are grateful to Dr. M. Bulanda and K. Palaga for help with bacteria collection. 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1410 CHEMERIN ACTIVATION BY HOST CYSTEINE CATHEPSINS 19. Sørensen, O. E., P. Follin, A. H. Johnsen, J. Calafat, G. S. Tjabringa, P. S. Hiemstra, and N. Borregaard. 2001. Human cathelicidin, hcap-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood 97: 3951 3959. 20. Zaiou, M., V. Nizet, and R. L. Gallo. 2003. Antimicrobial and protease inhibitory functions of the human cathelicidin (hcap18/ll-37) prosequence. J. Invest. Dermatol. 120: 810 816. 21. Zabel, B. A., T. Ohyama, L. Zuniga, J. Y. Kim, B. Johnston, S. J. Allen, D. G. Guido, T. M. Handel, and E. C. Butcher. 2006. Chemokine-like receptor 1 expression by macrophages in vivo: regulation by TGF-beta and TLR ligands. Exp. Hematol. 34: 1106 1114. 22. Radetsky, M., R. C. Wheeler, M. H. Roe, and J. K. Todd. 1986. Microtiter broth dilution method for yeast susceptibility testing with validation by clinical outcome. J. Clin. Microbiol. 24: 600 606. 23. Cash, J. L., R. Hart, A. Russ, J. P. Dixon, W. H. Colledge, J. Doran, A. G. Hendrick, M. B. Carlton, and D. R. Greaves. 2008. Synthetic chemerinderived peptides suppress inflammation through ChemR23. J. Exp. Med. 205: 767 775. 24. Serveau-Avesque, C., M. F. Martino, V. Hervé-Grépinet, E. Hazouard, F. Gauthier, E. Diot, and G. Lalmanach. 2006. Active cathepsins B, H, K, L and S in human inflammatory bronchoalveolar lavage fluids. Biol. Cell 98: 15 22. 25. Bühling, F., C. Röcken, F. Brasch, R. Hartig, Y. Yasuda, P. Saftig, D. Brömme, and T. Welte. 2004. Pivotal role of cathepsin K in lung fibrosis. Am. J. Pathol. 164: 2203 2216. 26. Bylaite, M., H. Moussali, I. Marciukaitiene, T. Ruzicka, and M. Walz. 2006. Expression of cathepsin L and its inhibitor hurpin in inflammatory and neoplastic skin diseases. Exp. Dermatol. 15: 110 118. 27. Hou, W. S., Z. Li, R. E. Gordon, K. Chan, M. J. Klein, R. Levy, M. Keysser, G. Keyszer, and D. Brömme. 2001. Cathepsin k is a critical protease in synovial fibroblast-mediated collagen degradation. Am. J. Pathol. 159: 2167 2177. 28. Skrzeczyńska-Moncznik, J., K. Wawro, A. Stefańska, E. Oleszycka, P. Kulig, B. A. Zabel, M. Sulkowski, M. Kapińska-Mrowiecka, M. Czubak-Macugowska, E. C. Butcher, and J. Cichy. 2009. Potential role of chemerin in recruitment of plasmacytoid dendritic cells to diseased skin. Biochem. Biophys. Res. Commun. 380: 323 327. 29. de Heer, H. J., H. Hammad, T. Soullié, D. Hijdra, N. Vos, M. A. Willart, H. C. Hoogsteden, and B. N. Lambrecht. 2004. Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen. J. Exp. Med. 200: 89 98. 30. Allen, S. J., B. A. Zabel, J. Kirkpatrick, E. C. Butcher, D. Nietlispach, and T. M. Handel. 2007. NMR assignment of human chemerin, a novel chemoattractant. Biomol. NMR Assign. 1: 171 173. 31. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415: 389 395. 32. Wiedemann, I., E. Breukink, C. van Kraaij, O. P. Kuipers, G. Bierbaum, B. de Kruijff, and H. G. Sahl. 2001. Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J. Biol. Chem. 276: 1772 1779.

Chemerin Is an Antimicrobial Agent in Human Epidermis Magdalena Banas 1, Katarzyna Zabieglo 1, Gopinath Kasetty 4, Monika Kapinska-Mrowiecka 5, Julia Borowczyk 2, Justyna Drukala 2, Krzysztof Murzyn 3, Brian A. Zabel 6, Eugene C. Butcher 7, Jens M. Schroeder 8, Artur Schmidtchen 4, Joanna Cichy 1 * 1 Department of Immunology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland, 2 Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland, 3 Department of Computational Biophysics and Bioinformatics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland, 4 Division of Dermatology and Venerology, Department of Clinical Sciences, Lund University, Lund, Sweden, 5 Department of Dermatology, Zeromski Hospital, Kraków, Poland, 6 Palo Alto Institute for Research and Education, Veterans Affairs Palo Alto Health Care System, Palo Alto, California, United States of America, 7 Stanford University School of Medicine, Stanford, California, United States of America, 8 Department of Dermatology, University Hospital Schleswig-Holstein, Kiel, Germany Abstract Chemerin, a chemoattractant ligand for chemokine-like receptor 1 (CMKLR1) is predicted to share similar tertiary structure with antibacterial cathelicidins. Recombinant chemerin has antimicrobial activity. Here we show that endogenous chemerin is abundant in human epidermis, and that inhibition of bacteria growth by exudates from organ cultures of primary human skin keratinocytes is largely chemerin-dependent. Using a panel of overlapping chemerin-derived synthetic peptides, we demonstrate that the antibacterial activity of chemerin is primarily mediated by Val 66 -Pro 85, which causes direct bacterial lysis. Therefore, chemerin is an antimicrobial agent in human skin. Citation: Banas M, Zabieglo K, Kasetty G, Kapinska-Mrowiecka M, Borowczyk J, et al. (2013) Chemerin Is an Antimicrobial Agent in Human Epidermis. PLoS ONE 8(3): e58709. doi:10.1371/journal.pone.0058709 Editor: Markus M. Heimesaat, Charité, Campus Benjamin Franklin, Germany Received November 16, 2012; Accepted February 8, 2013; Published March 20, 2013 Copyright: ß 2013 Banas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by the Team Award and Polish National Science Center grant 2011/02/A/NZ5/00337 (to JC). The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University is a beneficiary of the structural funds from the European Union (grant No: POIG.02.01.00-12-064/08). ECB is supported by grants from the National Institutes of Health (NIH) and by an endowed chair in Experimental Pathology. BAZ is supported by NIH grant AI-079320. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Joanna.Cichy@uj.edu.pl Introduction Chemerin is a multifunctional protein implicated in chemotaxis of immune cells, regulation of differentiation and metabolic function of adipocytes, and glucose homeostasis [1,2,3]. It binds with high affinity to three receptors, chemokine-like receptor 1 (CMKLR1) and atypical chemokine CC motif receptor-like 2 (CCRL2) as well as G protein-coupled receptor 1 (GPR1). However, among these receptors, only CMKLR1 is responsible for direct chemerin-mediated chemotactic effects [4,5]. Chemerin mrna is present in many tissues, including liver, fat, placenta, pancreas, lung and skin [6,7]. Chemerin is also present in plasma in the nanomolar range. Like other serum proteins, the liver may be a primary source for circulating blood chemerin [3]. However, chemerin is also expressed by epithelial cells, including kertinocytes [8], although the biological significance of chemerin in skin remains unknown. Human chemerin is secreted as a 143-amino acid precursor, pro-chem163s. Proteolytic processing of the C-terminus of prochem163s is required for this protein to become an active chemoattractant. Chemerin lacking 6 amino acids from the C- terminus, thus ending at serine 157 (chem157s), appears to be the most effective form in controlling chemotaxis of several types of immune cells. Among cells responsive to chemerin gradients are plasmacytoid dendritic cells (pdcs), macrophages and NK cells [7,9,10,11,12]. Serine proteases of the inflammatory cascade, such as neutrophil elastase and cathepsin G, as well as host cysteine proteases including cathepsin L and K or pathogen-derived staphopain B, are potent activators of chemerin chemotactic activity [13,14,15]. These enzymes can process chemerin in vitro to generate bioactive chemerin isoforms identical to the endogenous isoforms isolated from body fluids [16]. However, extensive cleavage of this protein that has been reported to occur either in vitro or in vivo, also results in generating chemerin isoforms that lack chemotactic activity [3,17,18]. These data suggest that at least some chemerin fragments may play other, not chemotaxis-related functions. Chemerin expression in the skin is not uniform and varies based on anatomic position as well as disease state. Chemotactically active chemerin was detected in lesional skin of psoriasis patients, where it may contribute to selective pdc recruitment [11,19]. However, psoriatic lesions show much lower chemerin levels in the epidermis compared to the healthy skin, but strong chemerin immunoreactivity in the dermis. This is in contrast to normal skin where chemerin is primarily expressed by epidermal keratinocytes, but rarely, if at all, in the dermis [19,20]. Therefore, chemerin reactivity in the epidermis suggests an additional, non-pdcrecruitment-related role for this protein in skin biology. The predicted structural homology between chemerin and antimicrobial cathelicidins such as cathelin-like N-terminal region of human hcap18 [6,13,21,22], led us to hypothesize that chemerin may confer some protection against invading microbes. This was supported by our previous studies demonstrating antimicrobial activity of two chemerin isoforms (chems157 and PLOS ONE www.plosone.org 1 March 2013 Volume 8 Issue 3 e58709

Chemerin in Epidermis chemr125) against E. coli and K. pneumoniae [13]. These recombinant chemerin isoforms lack 6aa and 38 aa, and terminate at Ser 157 and Arg 125, respectively. Although both isoforms differed significantly in supporting chemotaxis, they were equally effective in reducing the growth of E. coli [13]. These data suggest that different chemerin domains are responsible for chemotactic and antimicrobial properties of this protein. Since recombinant chemerin was previously used in order to demonstrate its antibacterial properties, it was important to determine whether chemerin exhibits antimicrobial functions in the skin environment, and whether its activity comprises a significant component of the secreted antibacterial products of skin. In this work we show that chemerin originating from exudates from organ cultures of human skin keratinocytes displays antimicrobial activity. Moreover, using chemically-synthesized chemerin-derived peptides we provide mechanistic information on the action of chemerin as well as insights into the domains that mediate its antimicrobial activity. Materials and Methods Peptides Peptides were generated by a peptide synthesis platform (PEPscreenH, Custom Peptide Libraries, Sigma Genosys). MALDI-ToF Mass Spectrometry was performed on these peptides, and average Crude Purity of the peptides was found to be 60 70%. In addition, peptide 4 was synthesized and purified.98% by thinkpeptides, UK. Peptide selection Mean hydrophobicity (H), and relative mean hydrophobic moments (rhm) were calculated using Combined Consensus Scale (CCS) of hydrophobicity [Tossi-2002] according to definitions given by Eisenberg et al. [23] with periodicity angle set to 100u and 160u, for a-helical and twisted b-strand conformations, respectively. All calculations were performed using an in-house program (hm.py). rhm by definition gives the value of mean hydrophobic moment relative to a perfectly amphipathic peptide of certain length, i.e. the amino acid sequence which maximizes rhm when adopting a given conformation. For CCS and 20 amino acid peptides, perfectly amphipathic peptides have the following sequence: RFFRRFFRRFRRFFRRFFRF (a-helix) and RFRFRRFRFRFRFRRFRFRF (twisted b strand). Cell culture All human studies were performed in compliance with ethical protocols KBET/72/B/2008 and KBET/44/B/2011 approved by Jagiellonian University Institutional Bioethics Committee. Declaration of Helsinki protocols were followed. All participants provided their written informed consent to participate in these studies as recommended by the ethical board. Normal human keratinocytes were isolated from excess skin from donors obtained at the time of cosmetic surgery for mole removal or during plastic surgery. Skin biopsies were rinsed 3 times in calcium- and magnesium-free PBS supplemented with penicillin (5000 U/ml) streptomycin (5 mg/ml) (all from Sigma). After washing, the biopsy was placed in PBS containing dispase (12 U/mL, Gibco) for 16 h in 4uC. Next, the epidermis was separated from the dermis with forceps followed by treatment with 0.05% trypsin with 2 mm EDTA (Sigma) to isolate epidermal cells. Cells were cultured in serum free KGM-Gold medium (Lonza Group Ltd.) to generate passage 1 cells. The keratinocytes were then plated at density of 5610 4 cells per well on permeable Transwell inserts (6.5-mm-diameter, 0.4 mm pore size; Falcon Transwell-Clear supports) in KGM-Gold medium. Cells were cultured at 37uC in presence of 5% CO 2 until confluence. Polarized skin structures that resemble in vivo stratified epidermis were generated by airliquid interface cultures for 1 to 3 weeks. Conditioned media were collected two days after the cells were exposed to the air-liquid interface and then every 48 h. The pulled conditioned media was analyzed. Preparation of epidermis lysate The epidermis was separated from the dermis as described above. Epidermis was then homogenized in a RIPA buffer (25 mm Tris-HCl, ph 7.6, 150 mm NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibitors (Complete, Roche), passed through a 40 mm cell strainer and incubated o/n at 4uC. Extracts were centrifuged at 10,000 g for 30 min to remove cellular debris and then normalized based on protein concentration as determined by BCA assay (Sigma). Lysates were stored at 220uC until used. Immunohistochemistry Paraffin 6-mm sections were prepared from skin biopsies or keratinocyte cultures. Sections were blocked with goat IgG and stained with the rabbit anti-human chemerin (H-002-52 Phoenix Pharmaceuticals) or control IgG (normal rabbit IgG, Jackson Immunoresearch) followed by APC-goat anti-rabbit IgG F(ab)2 (Jackson Immunoresearch). The sections were counterstained with Hoechst 33258 (Invitrogen). Images were captured with a fluorescence microscope (NIKON, Eclipse) and analyzed by NIS elements software (Nikon). ELISA Chemerin in conditioned media or in epidermis lysates was quantified by ELISA. Monoclonal mouse-anti-human chemerin (R&D System) Abs were used to capture chemerin and biotinlabeled polyclonal goat anti human chemerin (R&D System) followed by HRP-labeled streptavidin (BD PharMingen) were used to quantitate chemerin. The reaction was developed with TMB substrate (BD Science). Chemerin depletion Chemerin was removed form keratinocyte conditioned media by immunoprecipitation with sepharose-conjugated anti-chemerin Abs. The conjugation of anti-chemerin IgG (G-002-52 rabbit antihuman chemerin, Phoenix Pharmaceuticals) or control IgG (normal rabbit IgG, Jackson Immunoresearch) to Sepharose4B (Pharmacia) was performed according to the manufacturer s recommendations. Microtitre broth dilution (MBD) assay The antimicrobial activity of the indicated chemerin peptides or keratinocyte conditioned media against Escherichia coli (HB101, a conventional laboratory strain) was estimated as previously described [24]. Briefly, bacteria were grown in Mueller Hinton Broth (MHB) (Difco) to mid-logarithmic phase and used for subsequent experiments at 2 5610 5 or 2610 4 colony-forming units (CFU)/ml. Chemerin levels in the keratinocyte conditioned media did not exceed 20 ng/ml. Therefore, to investigate the antimicrobial effect of these media, we used 10 times less bacteria (2610 4 CFU/ml) compared to the standard MBD assay. Bacterial suspensions in MHB were mixed with diluent (90%:10% 10 mm HEPES or 50%:50% keratinocyte growth media) (control), chemerin peptides or keratinocyte conditioned media and incubated at 37uC for 18 24 h. After serial dilutions with MHB, PLOS ONE www.plosone.org 2 March 2013 Volume 8 Issue 3 e58709

Chemerin in Epidermis Figure 1. Keratinocyte-derived chemerin displays anti-bacterial activity. Paraffin sections of normal, shoulder skin biopsies (A) or chest keratinocytes grown in 3D culture for 1 week (B) were stained for chemerin or control rabbit Abs (red), with Hoechst counterstain to detect cell nuclei (blue). The slides were examined by fluoresce microscopy. Dotted lines in A indicate location of epidermis. Scale bar = 10 mm. Data are representative of three different donors. The antimicrobial activity of conditioned media from 3D cultures of keratinocytes (conditioned media) was tested against E. coli using the microtitre broth dilution assay (C). Where indicated, the conditioned media were first treated with sepharose-conjugated anti-chemerin Ab (anti-chemerin IgG), sepharose-conjugated control IgG (control IgG), or anti-chemerin Ab followed by recombinant chemerins157 (chems157) at 20 ng/ml. The results are expressed as the mean 6 SD of four independent experiments. Statistically significant differences are indicated by asterisks (p#0.01, Student s t test). doi:10.1371/journal.pone.0058709.g001 the diluted mixture was plated on MHB agar plates and incubated at 37uC overnight for enumeration of CFU. In selected experiments, samples of the bacteria/peptide mixtures were also analyzed by spectrophotometry. These methods produced comparable results to the colony-forming assay (data not shown). Microdilution assay Test microorganisms were incubated with various concentrations of chemerin-derived peptide 4 (.98% pure) in 10 mm sodium phosphate buffer ph 7.4 containing 1% (v/v) trypticase soy broth (TSB) for 2 h at 37uC. The antimicrobial activity was then analyzed by plating serial dilutions of the incubation mixtures and determining the number of CFU the following day. Radial diffusion assay The indicated bacteria were grown to mid-logarithmic phase in 10 ml of full-strength (3% w/v) TSB as previously described [25]. The microorganisms were then washed once with 10 mm Tris, ph 7.4. 4610 6 bacterial CFUs were then added to 15 ml of the underlay agarose gel, consisting of 0.03% (w/v) TSB, 1% (w/v) low electroendosmosis type (EEO) agarose (Sigma) and 0.02% (v/ v) Tween 20 (Sigma), with or without 0.15 M NaCl. The underlay was poured into a Ø 144 mm Petri dish. After agarose solidification, 4 mm-diameter wells were punched and 6 ml of chemerin-derived peptides or LL37 (Innovagen AB) was added to each well. Plates were incubated at 37uC for 3 hours to allow diffusion of the peptides. The underlay gel was then covered with 15 ml of molten overlay (6% TSB and 1% Low-EEO agarose in PLOS ONE www.plosone.org 3 March 2013 Volume 8 Issue 3 e58709

Chemerin in Epidermis Table 1. Chemerin levels in lysates isolated from epidermis of healthy donors. Patient number Gender Age Anatomic location Body mass index Chemerin ng/mg of total protein 1 F 50 thigh 25.15 441.4 2 F 28 back 21.18 1293.2 3 F 26 nape of the neck 18.67 734 4 M 55 temple 27.15 417.8 5 M 57 the inside of elbow 26.51 1398.8 6 M 33 neck 22.09 655.5 Mean ± SD 823±424 doi:10.1371/journal.pone.0058709.t001 dh 2 O). Antimicrobial activity of a peptide was visualized as a zone of clearing around each well after 18 24 hours of incubation at 37uC. Lytic assay E. coli JM83 strain containing plasmid pch110 (Pharmacia- Amersham) encoding beta-galactosidase and ampicillin-resistance genes was grown in Luria-Bertani medium (LB) (Difco) containing 1.25 mg/ml ampicillin. All assays were performed using midlogarithmic phase bacteria inoculated from overnight culture. Triton-lysed bacteria were served as 100% control. The phdependence was determined in 20 mm citrate-phosphate buffers of indicated ph (no NaCl), whereas the salt-dependence was assayed in 20 mm phosphate buffer with a constant ph = 7.2 containing the indicated amount of NaCl. To detect b-galactosidase activity, p-nitrophenyl-b-d-galactopyranoside, was used as a substrate. Results To investigate the role of chemerin in antibacterial defense of epithelial tissue, we first determined chemerin levels in lysates obtained from epidermis of healthy individuals. Since previous studies showed strong chemerin RNA expression in the epidermis from healthy individuals, which we confirmed by RT-QPCR (data not shown), we focused on quantifying chemerin protein in the skin. Indeed, chemerin protein was abundant in epidermis samples from multiple anatomic positions (8246424 ng per mg of total protein, n = 6) by ELISA (Table 1). Immunohistochemistry of paraffin embedded healthy skin derived from a shoulder biopsy revealed that chemerin is primarily expressed in the basal and suprabasal layers of epidermis, although its expression can also be detected in upper layers (Fig. 1A and data not shown). Based on the expression level and location of chemerin in healthy skin, chemerin is well-positioned to provide protection against skincolonizing bacteria. Unlike standard cultures of normal human keratinocytes, tissuelike 3-dimensional structures express high levels of chemerin [20]. Therefore, to determine whether keratinocyte-derived chemerin is equipped with antimicrobial activity we established polarized cultures of keratinocytes isolated from healthy human skin derived from a chest biopsy. Interestingly, chemerin levels were the highest in the most matured 3D cultures, suggesting that differentiation status influence the chemerin expression (data not shown). In vitro cultured skin expressed chemerin in the basal- and suprabasal-like epithelial layers, correlating with its localization in situ in normal skin (Fig. 1B). To determine whether chemerin is a relevant antimicrobial agent in human keratinocytes, we tested conditioned media from these 3D cultures for antibacterial chemerin activity using MBD assays. We used E. coli for these studies, since human skin is frequently exposed to this bacteria. As demonstrated in Fig. 1C, the keratinocyte conditioned media (chemerin level,20 ng/ml) significantly inhibited the growth of E. coli strain H101, leading to survival of 47612% of bacteria compared to vehicle-treated E. coli set as 100%. We previously used this strain to show inhibition of the bacterial growth by recombinant chemerin isoforms chems157 and chemr125 [13]. To define the contribution of chemerin to the bacterial killing, we depleted chemerin from the conditioned media using sepharose-bound antichemerin Abs. Treatment of the supernatants with sepharose- Figure 2. Overlapping peptides (p1-p14) are underlined in the chemerin sequence. The N-terminal signal peptide is indicated by italics. Peptide 4 is shown in bold. doi:10.1371/journal.pone.0058709.g002 PLOS ONE www.plosone.org 4 March 2013 Volume 8 Issue 3 e58709

Chemerin in Epidermis bound anti-chemerin Abs reduced chemerin levels from 17 18 ng/ml to,10 pg/ml (below the limit of ELISA detection); sepharose-bound control Abs had no major effect on chemerin levels (not shown). The depletion of chemerin from the conditioned media significantly increased the survival of bacteria from 47612% to 75613%, whereas the conditioned media treated with sepharose-bound control Abs had no effect (Fig. 1C). Moreover, reconstitution of the conditioned media devoid of endogenous chemerin with recombinant human chems157 (20 ng/ml) restored the killing activity of the conditioned media (bacterial viability significantly decreased to 22610%) (Fig. 1C). Taken together, these data suggest that chemerin significantly contributes to the antibacterial properties of keratinocyte secretions. To define the potential antimicrobial epitopes of chemerin, we selected and chemically synthesized 14 partially overlapping peptides covering the entire chemerin sequence (Fig. 2 and Table 2). These peptides, each,20 residue long, were selected to cover a wide range of net charge, mean hydrophobicity, and relative mean hydrophobic moment (rhm) values, allowing us to evaluate different determinants that might constitute the antibacterial activity of chemerin. The amphipathicity of chemerin peptides was analyzed by comparison of the rhm values, assuming for each of the peptide two distinct conformations: the a-helical and a b-structure. Owing to the presence of hydrophobicity patterns in native proteins [23], a substantially higher value of the calculated rhm for one of the alternative peptide structures (rhma for a-helical and rhmb for a b-structure) indicates the Table 2. Synthetic chemerin peptides 1. Name sequence H rhmb rhma ratio netchg more probable conformation of the peptide. The analysis of the rhmb/rhma ratio for different reference peptides (Table 2 and data not shown), allowed us to classify p1 p14 peptides with the ratio.1.4 as showing propensity to adopt b-structures and those with the ratio,0.7 to adopt the a-helical conformation. Interestingly, all 20 residue long chemerin peptides with the net-charge higher than +2 clearly prefer a b-structure rather than the a- helical structure (Table 2), suggesting that the peptide conformation may be non-helical in the intact structure. The selected chemerin-derived peptides (100 mm) were tested for antibacterial activity against E. coli strains HB101 and ATCC 25922 using the MBD and RDA assays, respectively. Several peptides inhibited growth of E. coli to some degree. Among them, peptide 4 (Fig. 2 and Table 2) corresponding to internal Val 66 - Pro 85 region of human chemerin exhibited the strongest antimi- p1 ELTEAQRRGLQVALEEFHKH 22.40 0.124 0.466 0.27 0.1 p2 EFHKHPPVQWAFQETSVESA 21.58 0.169 0.204 0.83 20.9 p3 SVESAVDTPFPAGIFVRLEF 0.42 0.247 0.109 2.27 22.0 p4 VRLEFKLQQTSCRKRDWKKP 23.28 0.375 0.157 2.40 5.0 p5 DWKKPECKVRPNGRKRKCLA 24.41 0.295 0.205 1.44 6.0 p6 RKCLACIKLGSEDKVLGRLV 21.27 0.170 0.119 1.43 3.0 p7 LGRLVHCPIETQVLREAEEH 21.53 0.095 0.080 1.18 20.9 p8 EAEEHQETQCLRVQRAGEDP 24.44 0.244 0.177 1.38 23.4 p9 DPHSFYFPGQFAFSKELPRS 20.56 0.205 0.250 0.82 0.5 p10 VQRAGEDPHSFYFPGQFAFS 20.59 0.223 0.186 1.20 20.5 p11 QVLREAEEHQETQCLRVQRA 23.58 0.141 0.278 0.51 20.4 p12 NGRKRKCLACIKLGSEDKVL 22.81 0.284 0.046 6.20 4.0 p13 NGRKRKCLACIKLGSEDKVLGRLVH 22.34 0.168 0.167 1.00 5.5 p14 KALPRS 22.63 0.100 0.609 0.16 2.0 pg-1 RGGRLCYCRRRFCVCVGR 22.56 0.376 0.227 1.66 6.0 mag- GIGKFLHSAKKFGKAFVGEIMNS 20.58 0.104 0.505 0.21 3.5 2 Peptide 4 is shown in bold. The net charge at ph 6 (netchg), mean hydrophobicity (H), relative mean hydrophobic moment assuming a b structure and a-helix, (rhmb) and (rhma) respectively, and rhmb/rhma ratio are indicated for each peptide. Data for the antibacterial peptide protegrin-1 (pg-1) and magainin-2 (Mag-2) known to adopt b structure and a-helical conformation, respectively when bound to the lipid membrane [36,37], are shown for comparison. The rhm values and rhmb/rhma ratio for preferred peptide conformation are shown in bold. 1 chemerin peptides-patent pending. doi:10.1371/journal.pone.0058709.t002 Figure 3. The chemerin-derived peptide 4 (Val 66 -Pro 85 ) strongly inhibits growth of E. coli. Chemically synthesized chemerin peptides (p1-p14) were tested against E. coli HB101 using the microtitre broth dilution assay (A) or against E. coli ATCC 25922 using radial diffusion assay (RDA) in physiological 0.15 M NaCl (B) or low salt concentration (C). Bacteria were incubated with the peptides at 100 mm. The results are expressed as the mean 6 SD of three independent experiments. doi:10.1371/journal.pone.0058709.g003 PLOS ONE www.plosone.org 5 March 2013 Volume 8 Issue 3 e58709

Chemerin in Epidermis Figure 4. Chemerin peptide 4 exhibits anti-microbial activity against variety of microbial species. The indicated microorganisms (E. coli ATCC 25922, S. aureus ATCC 29213, P. aeruginosa ATCC 27853 and C. albicans ATCC 90028) were tested for antimicrobial activity of chemerin peptide 4 or LL-37 (both at 100 mm), using RDA assay. The results are expressed as the mean 6 SD of three independent experiments. * p,0.005 (Student s t test). doi:10.1371/journal.pone.0058709.g004 crobial potency, resulting in almost complete inhibition of viable counts of E. coli H101 following 24 h treatment (Fig. 3A). P4 also significantly inhibited growth zones of E. coli ATCC 25922 in RDA under physiological salt conditions (0.15 M NaCl) (Fig. 3B). Other peptides such as p5 and p6 inhibited growth zones in low salt conditions, however, their inhibitory effects were less robust than p4 (Fig. 3C). For comparison, using a similar MBD assay, we previously demonstrated that pro-chem163s and chems157 (evaluated at 2 mm) significantly inhibited bacterial growth, resulting in 59613% and 33615% E. coli survival, respectively [13]. Thus, the analysis of overlapping chemerin-derived peptides demonstrate that the region Val 66 -Pro 85 of chemerin mediates the majority of the antibacterial activity of the full-length or chemotactically active chemerin, although cationic regions further C-terminal of p4 may also contribute to the resulting antibacterial activity of the intact holomolecule. We next examined a collection of clinically relevant human pathogens known to colonize the skin for sensitivity to peptide 4. The peptide was purified by HPLC to.98% and tested for antimicrobial activity using E. coli ATCC 25922, S. aureus ATCC 29213, P. aeruginosa ATCC 27853, as well as C. albicans ATCC 90028 by RDA assay. As demonstrated in Fig. 4, p4 at 100 mm inhibited the growth of all microorganisms, although it was particularly effective against Gram-negative bacteria, especially E. coli, but also the fungus Candida. Moreover, at 100 mm p4 was more potent in inhibiting growth of E. coli and C. albicans than the well-known keratinocyte-expressed antimicrobial agent LL-37 (Fig. 4). Similar results were generated with p4 against alternative strains of each and when p4 was tested at 40 mm (data not shown). The strong anti-microbial activity of p4 was further demonstrated by minimal inhibitory concentration (MIC) values which were in the range of 3.1 6.3 mg/ml (1.2 2.4 mm) for the most susceptible E. coli, to 12.5 mg/ml (4.8 mm) for the least susceptible S. aureus (Table 3). P4 also effectively inhibited the growth of two strains of Staphylococcus epidermidis, a common commensal skin bacteria (MIC = 12.5 mg/ml, Table 3). The MIC values were within the concentration range of most well-known anti-microbial agents [26]. Collectively, these data demonstrate that chemerin-derived peptide 4 is a potent anti-microbial agent. Like other potent anti-microbial peptides, we hypothesize that the highly positively- charged p4 (Table 2) interacts with negatively-charged bacterial surfaces to disrupt membrane integrity. To ask if p4 causes direct bacterial lysis, we used a b- galactosidase reporter E. coli strain, where cytoplasmic b-galactosidase is released into the supernatant following effective lysis [27]. Indeed, incubation with 10 mm of p4 released b-galactosidase suggesting a direct lytic effect. P4 was most active at neutral physiological ph and in low salt, although it seemed to retain activity in physiological (0.15 M) salt concentration (Fig. 5). These data suggest that although charge mostly governs the antimicrobial activity of p4, other mechanisms, such as those based on hydrophobic interaction also play a role in its activity. Table 3. MIC values for indicated microorganisms as determined by microdilution assay. p4 (mg/ml) E. coli ATCC 11775 S. aureus ATCC 6538 P. aerugin. ATCC 10145 C. albicans ATCC 24433 S. epiderm. ATCC 12228 S. epiderm. ATCC 14990 100 100 100 100 100 100 100 50 100 100 100 100 100 100 25 100 100 100 100 100 100 12.5 100 100 100 100 100 100 6.3 100 98.9 100 100 99.7 99.1 3.1 100/98.6 72 99.4 80 96.8 97 1.6 92.1 57 96.7 39 83 84 0.8 82 23 71 18 61 38 0.4 57 11 23 7 16 34 0.2 16 0 6 14 17 17 0.1 20 0 17 0 8 8 0.05 7 0 0 0 0 0 0.02 26 0 0 0 0 0 0.01 0 0 0 0 0 0 MIC (mg/ml) 3.1 6.3 12.5 6.3 6.3 12.5 12.5 Data in columns indicate % of killing for each strain. The MIC was defined as the lowest concentration of p4 showing no visible growth (100% of killing). Mean of at least 3 measurements is shown. doi:10.1371/journal.pone.0058709.t003 PLOS ONE www.plosone.org 6 March 2013 Volume 8 Issue 3 e58709