IL-36 promotes myeloid cell infiltration, activation, and inflammatory activity in skin

Abstract

The IL-1 family members IL-36α (IL-1F6), IL-36β (IL-1F8), and IL-36γ (IL-1F9) and the receptor antagonist IL-36Ra (IL-1F5) constitute a novel signaling system that is poorly understood. We now show that these cytokines have profound effects on the skin immune system. Treatment of human keratinocytes with IL-36 cytokines significantly increased the expression of CXCL1, CXCL8, CCL3, CCL5, and CCL20, potent chemotactic agents for activated leukocytes, and IL-36α injected intradermally resulted in chemokine expression, leukocyte infiltration, and acanthosis of mouse skin. Blood monocytes, myeloid dendritic cells (mDC), and monocyte-derived DC (MO-DC) expressed IL-36R and responded to IL-36. In contrast, no direct effects of IL-36 on resting or activated human CD4(+) or CD8(+) T cells, or blood neutrophils, could be demonstrated. Monocytes expressed IL-1A, IL-1B, and IL-6 mRNA and IL-1β and IL-6 protein, and mDC upregulated surface expression of CD83, CD86, and HLA-DR and secretion of IL-1β and IL-6 after treatment with IL-36. Furthermore, IL-36α-treated MO-DC enhanced allogeneic CD4(+) T cell proliferation, demonstrating that IL-36 can stimulate the maturation and function of DC and drive T cell proliferation. These data indicate that IL-36 cytokines actively propagate skin inflammation via the activation of keratinocytes, APC, and, indirectly, T cells.

Figure 1. IL-36 cytokines induce chemokine expression by keratinocytes

4-day post-confluent normal human keratinocytes (NHK) were stimulated for 24h with recombinant truncated IL-36R ligands or IL-1β. Total RNA was extracted and mRNA transcripts quantified by qRT-PCR relative to the housekeeping gene RPL-P0 and conditioned medium assayed by ELISA. 100ng/ml IL-36α, IL-36β and IL-36γ significantly induced T cell chemokine mRNA expression compared with untreated cells, mean ± S.D. (n=3) (a). IL-36α, IL-36β, IL-36γ and IL-1β but not IL-36Ra dose-dependently induced CXCL1, CCL5, CXCL8 and CCL20 mRNA expression (b-e) and CXCL8 and CCL20 protein secretion (f and g) by keratinocytes. Mean ± S.D. (n=3). Statistical significance indicated by * p<0.05, t** p<0.01 or *** p<0.001, Student’s t-test.

Figure 2. IL-36 induces myeloid cell infiltration of skin concomitant with chemokine and growth factor induction

5μg rmIL-36α or BSA was injected intradermally into CD1 mice every other day for 10 days. Back skin was harvested, snap frozen, processed for RNA and histochemistry. IL-36α treatment lead to acanthosis and an increase in eosinophilic dermal collagen (a) and striking infiltration of granulocytes (CD11b, b), macrophages (F4/80, c), dendritic cells (CD11c, d) CD4+ cells (e) but not CD8+ cells (f). These changes were accompanied by increases in chemokines (g) and cytokines/growth factors (h). Mean ± SEM (n=4 mice). Statistical significance indicated by * p<0.05 (2-tailed t-test). Scale bar = 100μm.

Figure 3. Human APC but not T cells express the IL-36 receptor

Keratinocytes (KC), monocytes and myeloid DC (mDC) express IL-1R1, IL-1RAcP and IL-36R mRNA transcripts, however, CD4+ and CD8+ T cells were found not to express IL-36R as determined by qRT-PCR (a-c, n=4 donors). Flow cytometric analysis reveals that in contrast to mDC (d and g) and monocytes (e, f and h), T cells (e, f and i) did not express surface IL-36R. Filled histogram: anti-IL36R, dotted histogram, isotype control antibody. Flow cytometry gating shown in panels 3d-f; Flow cytometry data are representative of 6 donors.

Figure 4. IL-36 cytokines induce monocyte expression of inflammatory cytokines

Monocytes treated with 100ng/ml IL-36 cytokines for 12h upregulate IL-1A, IL-1B and IL-6, cytokine transcripts (n=9 donors, a-c). Significantly elevated levels of IL-1β and IL-6 were detected in the conditioned culture media after 12h (n=6 donors, e and f). Bars, mean ± SEM. Statistical significance indicated by * p<0.05, ** p<0.01, *** p<0.001 (2-tailed t-test).

Figure 5. IL-36 cytokines facilitate myeloid DC activation and cytokine secretion

Ex vivo blood myeloid DC treated with 100ng/ml IL-36 cytokines for 12h up-regulate CD83 (a), CD86 (b) and HLA-DR (c) expression (FACS, n=6 donors) and secretion of IL-1B (d) and IL-6 (e) (ELISA, n=9). Bars, mean ± SD. Statistical significance indicated by * p<0.05, ** p<0.01, *** p<0.001 (2-tailed t-test).

Figure 6. Monocyte-derived dendritic cells (MO-DC) have enhanced IL-36R expression and IL-36 cytokines facilitate DC maturation, driving T cell proliferation

MO-DC expressed 6-fold more IL-36R mRNA than their monocyte precursors (qRT-PCR, n=6, a). When cultured with 10ng/ml IL-6+0.1μM PGE₂ and either 10ng/ml IL-1β or 100ng/ml IL-36α, MODC significantly increased their surface expression of CD86 (b) and HLA-DR (c), indicative of DC maturation (48hr, bars, mean ± SEM, n=8 donors). MO-DC matured with 10ng/ml IL-6 + 0.1μM PGE₂ and 10ng/ml TNF-α, 10ng/ml IL-1β or 100ng/ml IL-36α drove allogeneic T cell proliferation as determined by CFSE dye dilution (d and e) or DAPI labeling of DNA (f). No DC (T cells only), 1:100 DC:T cell ratio, 1:20 DC:T cell ratio showing mean ± SEM (n=4 donors). Statistical significance indicated by * p<0.05, ** p<0.01, *** p<0.001 compared with IL-6+PGE₂ (2-tailed t-test).