IL-36␣ (IL-1F6), IL-36␤ (IL-1F8

SummaryInterleukin (IL)-36a, IL-36b and IL-36g are expressed highly in skin and are involved in the pathogenesis of psoriasis, while the antagonists IL-36Ra or IL-38, another potential IL-36 inhibitor, limit uncontrolled inflammation. The expression and role of IL-36 cytokines in rheumatoid arthritis (RA) and Crohn's disease (CD) is currently debated. Here, we observed that during imiquimod-induced mouse skin inflammation and in human psoriasis, expression of IL-36a, g and IL-36Ra, but not IL-36b and IL-38 mRNA, was induced and correlated with IL-1b and T helper type 17 (Th17) cytokines (IL-17A, IL-22, IL-23, CCL20). In mice with collageninduced arthritis and in the synovium of patients with RA, IL-36a, b, g, IL36Ra and IL-38 were all elevated and correlated with IL-1b, CCL3, CCL4 and macrophage colony-stimulating factor (M-CSF), but not with Th17 cytokines. In the colon of mice with dextran sulphate sodium-induced colitis and in patients with CD, only IL-36a, g and IL-38 were induced at relatively low levels and correlated with IL-1b and IL-17A. We suggest that only a minor subgroup of patients with RA (17-29%) or CD (25%) had an elevated IL-36 agonists/antagonists ratio, versus 93% of patients with psoriasis. By immunohistochemistry, IL-36 cytokines were produced by various cell types in skin, synovium and colonic mucosa such as keratinocytes, CD68 1 macrophages, dendritic/Langerhans cells and CD79a 1 plasma cells. In primary cultures of monocytes or inflammatory macrophages (M1), IL-36b and IL-36Ra were produced constitutively, but IL-36a, g and IL-38 were produced after lipopolysaccharide stimulation. These distinct expression profiles may help to explain why only subgroups of RA and CD patients have a potentially elevated IL-36 agonists/ antagonists ratio.

IntroductionThe interleukin-1 (IL-1) family of cytokines comprises 11 members, including IL-1␣, IL-1␤, IL-18, IL-33, and the recently renamed IL-36␣, ␤, ␥ (previously known as IL-1F6, IL-1F8, and IL-1F9). 1 All these cytokines use heterodimeric receptors for signaling. IL-1, IL-33, and IL-36 bind to specific receptor ␣-chains, which are IL-1RI for IL-1␣ and IL-1␤, T1/ST2 (also known as IL-33R) for IL-33, and IL-36R (previously termed IL-1Rrp2) for IL-36, and then recruit the same coreceptor IL-1R accessory protein (IL-1RAcP). IL-18 uses IL-18R␣ and the coreceptor IL-18R␤. On receptor binding, all IL-1 family cytokines activate similar intracellular signals, including NF-B and mitogenactivated protein kinase (MAPK) pathways. IL-1 receptor antagonist (IL-1Ra) and IL-36Ra (previously termed IL-1F5), 2 additional members of the IL-1 family, act as natural inhibitors for the biologic activities of IL-1 and IL-36, respectively. [2][3][4] IL-1␣, IL-1␤, IL-18, and IL-33 are produced by activated innate immune cells (neutrophils, monocytes, macrophages, and dendritic cells) and epithelial cells, and stimulate proinflammatory innate and adaptive immune responses. More specifically, these cytokines influence CD4 ϩ T-cell responses and their polarization into the different T helper (Th) subsets Th1, Th2, and Th17. IL-1 promotes the proliferation and survival of naive CD4 ϩ T cells and plays a critical role in Th17 differentiation. 5-9 IL-18 and IL-33 stimulate the polarization of CD4 ϩ T cells into Th1 and Th2, respectively,10 although the selectivity of these responses may be modulated by the cytokine environment. Consistently, Th1, Th2, and Th17 cells selectively express the IL-1 family cytokine receptors IL-18R␣, T1/ST2, and IL-1RI, respectively. 10 IL-36 cytokines and IL-36R are abundantly expressed by keratinocytes and other epithelial cell types. 4,[11][12][13] IL-36 plays a major role in mouse experimental skin inflammation and in human psoriasis both in the initiation and regulation of inflammatory responses. [14][15][16][17][18][19] Furthermore, the association of a form of generalized pustular psoriasis with genetic IL-36Ra deficiency in humans argues in favor of a significant role of IL-36 in inflammatory skin diseases. 20,21 Recently, we have shown that dendritic cells (DCs) express IL-36R and that IL-36 stimulates the production of several cytokines and enhances the expression of costimulatory molecules in bone marrow-derived DCs (BMDCs). The stimulatory effects of IL-36 were more robust than those of the other members of the IL-1 family. In addition, IL-36 stimulated the production of interferon ␥ (IFN-␥), IL-4, and to a lesser extent IL-17 by cultured splenocytes and activated CD4 ϩ T cells, and IL-36 was able to act as an adjuvant to stimulate Th1 responses in vivo. 22 In the results described herein we show that, among CD4 ϩ T-cell subsets, IL-36R is predominantly expressed by naive CD4 ϩ T (referred to also as naive Th) cells and that IL-36 stimulates activated naive CD4 ϩ T (referred to also as Th0) cell ...

The proinflammatory activities of IL-1 are tightly controlled at different levels. IL-1R2 acts as a decoy receptor and has been shown to regulate the biological effects of IL-1 in vitro and in vivo. However, little is known about its natural expression in the mouse in physiologic and pathologic conditions. In this study, we examined IL-1R2 mRNA and protein expression in isolated cells and tissues in response to different stimulatory conditions. Data obtained using ex vivo CD11b(+)Ly6G(+) peripheral blood cells and in vitro-differentiated CD11b(+)Ly6G(+) BMG indicated that neutrophils are the major source of constitutively expressed IL-1R2 in the mouse. The expression of IL-1R2 on BMG and ex vivo Ly6G(+) peripheral blood cells was highly up-regulated by HC. IL-1R2 pull-down experiments showed that mouse rIL-1β binds to BMG IL-1R2, whereas binding of IL-1Ra could not be detected. Furthermore, LPS treatment induced shedding of IL-1R2 from the neutrophil membrane in vitro and in vivo, executed mainly by ADAM17. Finally, in in vivo models of inflammation, including thioglycolate-induced acute peritonitis and acute lung injury, infiltrating Ly6G(+) neutrophils, expressed IL-1R2. Our data show that in the mouse, neutrophils mainly express the decoy receptor IL-1R2 under naïve and inflammatory conditions. These data suggest that neutrophils may contribute to the resolution of acute inflammation.

GenBank entries for mouse Il33 reveal the existence of two transcripts, Il33a and Il33b, with different 5'UTRs but coding for the same protein. We investigated expression of these transcripts in different mouse organs and cell types in basal and inflammatory conditions. Il33a and Il33b mRNAs start with different noncoding first exons, transcribed from different promoter regions, which both contain a consensus TATA-like sequence. Constitutive Il33a mRNA expression was detected in mouse stomach, lung, spleen, and brain, whereas basal Il33b mRNA expression was observed only in the stomach. Expression of both transcripts increased after systemic LPS administration. In vitro, we observed high constitutive expression of Il33 transcripts in MEFs. Constitutive Il33a mRNA expression was observed also in BMDCs, where it was preferentially increased in response to poly(I:C), whereas LPS increased levels of Il33a and Il33b mRNA. In contrast, BMMs and Raw 264.7 cells did not express Il33 mRNA constitutively, and LPS stimulation selectively induced expression of Il33b mRNA in these cells. Our data indicate that the Il33 gene is expressed from two alternative promoters in the mouse and that the relative expression of Il33a and Il33b transcripts is cell type- and stimulus-dependent.