Human fibroblasts support the expansion of IL-17–producing T cells via up-regulation of IL-23 production by dendritic cells by Christine Schirmer, Claudia Klein, Martin von Bergen, Jan C. Simon, and Anja Saalbach Blood Volume 116(10):1715-1725 September 9, 2010 ©2010 by American Society of Hematology

Fibroblasts support IL-23 release from DCs Fibroblasts support IL-23 release from DCs. (A) Monocyte-derived DCs were preactivated with LPS for 3 hours. Fibroblasts support IL-23 release from DCs. (A) Monocyte-derived DCs were preactivated with LPS for 3 hours. Subsequently, LPS-stimulated DCs (termed DCact in the entire manuscript) were washed extensively and then cocultured with fibroblasts (DCact + Fb) overnight. As control, LPS-stimulated DCs were cultured alone (DCact). Furthermore, we cultured immature DCs without (DC) and with fibroblasts (DC + Fb) or fibroblasts (Fb) alone. IL-23 protein levels were measured by ELISA. *P < .001 compared with DCact (n = 5 independent experiments). (B) DCact and fibroblasts were cocultured for 3 hours; afterward, DCs (DCact separated after coculture) and fibroblasts (Fb separated after coculture) were separated from the coculture by anti–Thy-1–coupled magnetic beads. For comparison, DCact and fibroblasts (Fb) were cultured alone. Subsequently, RNA preparation and real-time PCR of IL-23p19 mRNA were performed. IL-23p19 mRNA expression values were normalized to the unregulated housekeeping gene RPS26 and are given as percentage of IL-23p19 mRNA expression in DCact. *P < .001 compared with DCact (n = 3 independent experiments). Christine Schirmer et al. Blood 2010;116:1715-1725 ©2010 by American Society of Hematology

DC-derived TNF-α and IL-1β are involved in a feedback loop mechanism that causes increased IL-23 production in cocultures of DCs and fibroblasts. DC-derived TNF-α and IL-1β are involved in a feedback loop mechanism that causes increased IL-23 production in cocultures of DCs and fibroblasts. (A) DCact were cultured in the presence of fibroblast supernatants (DCact + [Fb-sn]) or PFA-fixated fibroblasts (DCact + Fb-fix). As controls, DCact either cultured alone (DCact) or cocultured with fibroblasts (DCact + Fb) were used. IL-23 was detected by ELISA. *P < .001 compared with DCact (n = 3 independent experiments). (B) Neutralizing anti–TNF-α or anti–IL-1β antibodies were added to coculture of DCact and fibroblasts separately (DCact + Fb + aTNF-α and DCact + Fb +aIL-1beta) or in combination (DCact + Fb + aTNFalpha/aIL-1beta). IL-23 levels were compared with coculture with isotype control (DCact + Fb + iso) and preactivated DCs cultured alone (DCact). #,*P < .05 (n = 4 independent experiments). (C-D) DCact and fibroblasts were cocultured for 3 hours and then separated (DCact separated after coculture and Fb separated after coculture). For comparison, preactivated DCs (DCact) and fibroblasts (Fb) were cultured alone. Subsequently, RNA preparations and real-time PCR were performed. (C) Relative level of TNF-α mRNA was calculated on the basis of ΔCt values. mRNA expression was normalized to the unregulated housekeeping gene RPS26 and computed as percentage of TNF-α mRNA expression in DCact. *P < .01 compared with DCact (n = 3 independent experiments). (D) IL-1β mRNA was quantified through a standard curve, normalized to the unregulated housekeeping gene RPS26, and computed as percentage of mRNA expression in DCact (n = 3 independent experiments). Christine Schirmer et al. Blood 2010;116:1715-1725 ©2010 by American Society of Hematology

PGE2, which is produced because of coculturing of activated DCs and fibroblasts, is also involved in IL-23 up-regulation. PGE2, which is produced because of coculturing of activated DCs and fibroblasts, is also involved in IL-23 up-regulation. (A) Indomethacin's PGE2 inhibiting efficiency was verified by detection of PGE2 levels in DCact cultured alone (DCact) and in coculture of DCact and fibroblasts with and without 2μM indomethacin (DCact + Fb and DCact + Fb + indomethacin, respectively). *P < .01 (n = 7 independent experiments). (B) DCs were preactivated with LPS and subsequently cocultured with fibroblasts (DCact + Fb) or without fibroblasts (DCact). Indomethacin (2μM) was added to the cocultures (DCact + Fb + indomethacin) and IL-23 was measured by ELISA. *P < .001 (n = 7 independent experiments). (C) DCs and fibroblasts were separated using anti–Thy-1 coated microbeads after a coculture time of 3 hours (DCact separated after coculture and Fb separated after coculture). Controls were again LPS-stimulated DC (DCact) and fibroblasts (Fb) cultured alone. Then, RNA was prepared and quantitative RT-PCR was performed. Cox-2 mRNA expression values were normalized to the unregulated housekeeping gene RPS26 and are given as percentage of Cox-2 mRNA expression in DCact. *P < .01 (n = 3 independent experiments). Christine Schirmer et al. Blood 2010;116:1715-1725 ©2010 by American Society of Hematology

Fibroblast-derived PGE2 is the critical PGE2 for stimulation of IL-23 secretion from DCact. Fibroblast-derived PGE2 is the critical PGE2 for stimulation of IL-23 secretion from DCact. (A) Fibroblasts or DCs were transfected with Cox-2 siRNA or scrambled siRNA. To induce PGE2 expression, Cox-2– and scrambled-transfected fibroblasts were stimulated with TNF-α/IL-1β (Fb[Cox-2si]+TNFalpha/IL-1beta; Fb[scr] + TNFalpha/IL-1beta). Cox-2– and scrambled-transfected DCs (DC[Cox-2si] + LPS; DC[scr] + LPS) were activated with LPS. PGE2 release was measured by ELISA. *P < .05 compared with scrambled-transfected cells (n = 3 independent experiments). (B) LPS-preactivated DCs (DCact) were cocultured with scrambled-siRNA-transfected fibroblasts (DCact + Fb[scr]) or with Cox-2-siRNA transfected fibroblasts (DCact + Fb[Cox-2si]). As control, DCact were cultured alone. IL-23 production was measured by ELISA. *P < .001 (n = 5 independent experiments). (C) Cox-2– or scrambled siRNA–transfected DCs were preactivated with LPS and subsequently cocultured with fibroblasts (DCact[scr] + Fb; DCact[Cox-2si] + Fb). As control, transfected DCact were cultured alone (DCact[scr]; DCact[Cox-2si]). *P < .001. Christine Schirmer et al. Blood 2010;116:1715-1725 ©2010 by American Society of Hematology

Fibroblasts promote Th17 development from TCs via the stimulation of IL-23 from activated DCs. LPS-preactivated DCs were cocultured with fibroblasts. Fibroblasts promote Th17 development from TCs via the stimulation of IL-23 from activated DCs. LPS-preactivated DCs were cocultured with fibroblasts. After 18 hours, fibroblasts were removed from the coculture using anti–Thy-1–coupled magnetic beads. As control, LPS-preactivated DCs (DCact) were cultured alone. Subsequently, CD3+ TCs or CD4+ were cultured alone (TC) or with either LPS-preactivated DCs (TC + DCact) or LPS-preactivated DCs isolated from the coculture with fibroblasts (TC + [DCact + Fb]) in the presence of respective coculture supernatants. (A,D-E) After 6 days of coculture, IL-17A (A), IL-22 (D), and IFN-γ (E) production was measured by ELISA. *P < .01, compared with TC + DCact (n = 5 independent experiments). (B-C) After 3 days of coculture, TCs were restimulated with phorbol myristate acetate/ionomycin in the presence of brefeldin A and IL-17A expression (B) and transcription factor RORγt expression (C) were measured by intracellular flow cytometric staining. One representative experiment of 3 is shown. (F) Coculture of CD3+ TCs and DCact (TC + DCact) or with fibroblast-stimulated DCact (TC + [DCact + Fb]) were performed without antibody (TC + [DCact + Fb]) in the presence of a control antibody (TC + [DCact + Fb + ctr ab]), a neutralizing anti–IL-6 (TC + [DCact + Fb + aIL-6]), or anti–IL-23 antibody (TC + [DCact + Fb + aIL-23]). After 5 days, IL-17A was quantified by ELISA. *#P < .05 (n = 7 independent experiments). (G) IL-17A secretion of CD3+ Pan TCs, CD4+ TCs, CD4+CD45RO+ memory TCs, and CD4+CD45RA+ naive TCs was compared by culturing the different TC types with medium (aTC), supernatants of LPS-preactivated DC (aTC + [DCact]sn), or supernatants of DCact-fibroblast coculture (aTC + [DCact + Fb]sn) in the presence of CD3/CD28 beads for 5 days. *P < .05, compared with aTC. #P < .01, compared with aTC[DCact]sn (n = 4 independent experiments). Christine Schirmer et al. Blood 2010;116:1715-1725 ©2010 by American Society of Hematology

Cox-2 expression is enhanced in lesional psoriatic skin. Cox-2 expression is enhanced in lesional psoriatic skin. Cox-2 expression (red) was detected by immunohistochemistry in (A) healthy skin and (C-D) in psoriatic skin. Nuclei were stained by hematoxylin (blue). (B) An isotype control antibody in lesional psoriatic skin served as negative control. (E) For quantification of Cox-2 expression, morphometric image analysis was performed by scanning 5 images per sections. The sum of pixels was evaluated, and the mean plus or minus SD of the number of pixels from 6 different psoriatic skin samples and 6 healthy skin samples is shown. (F-G) In lesional psoriatic skin, fibroblasts were detected by an anti–Thy-1 antibody (blue-gray) and Cox-2 expression by the anti–Cox-2 antibody (red). Arrows indicate Thy-1/Cox-2 double-positive cells. Christine Schirmer et al. Blood 2010;116:1715-1725 ©2010 by American Society of Hematology

Model how fibroblasts could stimulate IL-23 secretion from preactivated DCs resulting in expansion of Th17 cells. Model how fibroblasts could stimulate IL-23 secretion from preactivated DCs resulting in expansion of Th17 cells. (1) Preactivation of DC (LPS). (2) Then, DCs secrete IL-1β and TNF-α, which stimulate fibroblasts. (3) Stimulated fibroblasts secrete PGE2, which acts on DCs and therewith increases their IL-23 production. (4) Through the action of elevated IL-23, CD3+ (Pan), CD4+, and CD4+CD45RO+ (memory) TCs are supported to produce IL-17A, IL-22, and to express RORγt as well as to secrete IFN-γ. Christine Schirmer et al. Blood 2010;116:1715-1725 ©2010 by American Society of Hematology