P181 IL-36 signaling amplifies Th1 responses by enhancing proliferation and Th1 polarization of naïve CD4+ T cells

Abstract

Introduction The interleukin (IL)-1 superfamily of cytokines comprises a set of pivotal mediators of inflammation, playing an important role in innate and adaptive immune responses. Since the cloning of IL-1 α and IL-1 β , a number of additional members of this family have been discovered, including IL-18, IL-33, IL-36a, IL-36b, and IL-36g. While IL-1, IL-18 and IL-33 are well recognized for their immunomodulatory effects, the action of IL-36 cytokines in immune responses has remained elusive. We have previously shown that IL-36 cytokines were able to stimulate the production of cytokines by dendritic cells (DC) and that IL-36b acts as an adjuvant to stimulate T helper (Th)1 responses in vivo. In the present study, we investigated more precisely the function of IL-36 in Th1 immune responses. Methods Th0 (CD62L hi CD44 lo CD4 + T) cells were isolated from spleen of WT or IL-36R KO mice by sorting on a FacsVantage and were activated with plate-bound anti-mouse CD3/CD28 antibodies before cytokines stimulation. The expression of receptors and IL-36R ligands were assessed by RT-qPCR. Th0 cell proliferation was determined by 3H thymidine incorporation and FACS (CFSE) analysis. The role of IL-36 in Th1 differentiation was determined using co-culture systems and cytokine combination assays. Cytokine protein levels were monitored by ELISA, Milliplex assay or FACS. The involvement of IL-2 was studied by using anti-IL-2 and anti-IL-2Ra (CD25) neutralizing antibodies. Mycobacterial infection was induced in WT and IL-36R deficient mice by intravenous injection of 10 7 living M. bovis BCG Connaught. Results IL-36R is predominantly expressed on nai¨ve CD4 + T (Th0) cells, while other IL-1R family members are predominantly expressed in other Th subsets. IL-36 cytokines act directly on Th0 cells by enhancing both cell expansion and IL-2 secretion. The specificity of the stimulatory effects of IL-36 cytokines was confirmed by using IL-36R −/− Th0 cells.IL-36R signaling is required both on BMDC and Th0 cells via IL-12 and IL-2 production respectively, to induce IFN- γ production. Like IL-18, IL-36 β acts as a co-polarization factor with IL-12 to promote Th1 response inducing IL-12R β 2, T-bet mRNA expression and IFN- γ production. Using IL-2 neutralizing conditions, we observed that IL-2 is not required for the effects of IL-36 onnai¨ve T cell survival and proliferation, whereas the effect of IL-36 on Th1 differentiation is IL-2 dependent. Moreover, we showed that IL-36 β produced endogenously by Th0 cells acts as an autocrine inducer of IFN- γ production. Finally, the effects of IL-36 on Th1 cell polarization in vitro were confirmed in vivo by using a systemic model of mycobacterial infection. After 4 weeks of BCG infection, there was a significant impairment of IFN- γ , IL-6, TNF- α and nitrite oxide production by ex vivo restimulated splenocytes of IL-36R deficient mice compared to WT mice. Conclusion Our findings point towards a critical function of IL-36 in the initiation and maintenance of Th1 responses in vitro, via IL-12 and IL-2 signaling and in adaptive immunity in a model of mycobacterial infection in vivo.