By Charlotte O'Donnell PhD
The genes of the IL-36 family members are located in a cluster on chromosome 2 . All three activating ligands, IL-36α, IL-36β and IL-36γ share the classic IL-1 β-trefoil structure, and lack a signal peptide. IL-36α, IL-36β, and IL-36γ lack a signal sequence, and thus cannot be transported to the endoplasmic reticulum. Similar to IL-1β and IL-18, the N-terminus must be cleaved for full agonist or antagonist bioactivity of each of the IL-36 cytokines. The protease responsible for this cleavage has not yet been elucidated. It is not thought to be a caspase as the site does not resemble a caspase cleavage site . These three agonists induce proinflammatory cytokines, chemokines and other stimulatory molecules, thus promoting the infiltration of many immune cells such as DCs and neutrophils, activation of Th1 and IL-17-producing T cells, and keratinocyte proliferation.
Expression and function of IL-36 family members
IL-36R ligands are principally expressed by epithelial cells, keratinocytes, brain tissue and macrophages . However, T cells have also been shown to express IL-36α and IL-36β. Variation in cellular expression of the ligands has also been observed. For instance, IL-36α is constitutively expressed by keratinocytes while IL-36γ is induced upon stimulation with TNFα. In vitro, monocytes were found to specifically express IL-36γ following LPS treatment . IL-36α is secreted by BMDMs upon treatment with LPS, indicating that IL-36α can be externalized in response to a stimulus comparable to IL-1β.
IL-36 cytokines can regulate the immune response by influencing antigen presenting cells (APCs), such as macrophages and DCs. Indeed, the IL-36R is expressed by both monocyte and plasmacytoid derived DCs. Both of these APCs increase expression of CD83 in response to IL-36β and IL-36γ stimulation. In particular, IL-36β can induce secretion of IL-12 and IL-18 production by monocyte derived DCs. These cytokines can then activate IFN-γ producing T-cells . CD11+ cells stimulated with IL-36α produce TNFα, CD40, CXCL1 and CXCL2 which activate proliferation of CD4+ T cells. The IL-36R is also expressed on naïve T-cells, enabling IL-36 agonists to stimulate the proliferation of T-cells and induce IL-2 secretion by naïve T cells. Indeed, IL-36R signalling has also been shown to activate a Th1 and a Th17 response.
To date, few studies have investigated the regulation of IL-36 signalling. The IL-36R and the IL-1RAcP have been shown to be recycled in the absence of agonists. However, in the presence of agonists both receptors accumulated in higher abundance in lysosomes . Toll-interacting protein (Tollip) is a protein that is central to the regulation of Toll-like receptor (TLR) signalling pathways. Tollip also regulates IL-36 trafficking by elevating the levels of IL-36R and IL-1RAcP. In addition, Tollip has been shown to stabilise the IL-36R once the agonist has bound. This is unlike IL-1R signalling where Tollip has been shown to target the ligand-bound IL-1R for degradation. Proteases produced by immune cells such as lymphocytes and neutrophils have been found to enhance the bioactivity of IL-33 and may also have similar effects on IL-36R agonists [ 7].
Unlike IL-33, IL-36 cytokines require proteolytic processing to become activated. Proteases produced by immune cells such as lymphocytes and neutrophils increase the bioactivity of IL-36 cytokines. IL-36α, IL-36β, and IL-36γ are distinctively processed and activated by neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3 . In this manner neutrophil-derived proteases can increase inflammation through the regulation of IL-36 cytokines.
Few studies to date have investigated the role of the IL-36R signalling axis in the intestine. Similar to other IL-1R family members, the co-receptor IL-1RAcP is recruited to the IL-36R upon binding by one of the three agonistic ligands. This stabilizes ligand binding. Conformational changes are induced in the TIR domains of both heterodimers. The IL-36R adaptor protein, MyD88, is subsequently recruited. Similar to other IL-1R family members, IL-36R signalling has been shown to activate MAPK, ERK1/2, JNK (Figure 1) . Indeed, studies investigating IL- 36 signalling in both HT29 and Widr colon cancer cells have shown that stimulation with IL-36α and IL-36γ resulted in the recruitment of MyD88, TRAF6, IRAK1 and TAK1 adaptor proteins.
This adaptor complex induced the activation of NF-κB, the phosphorylation of the MAPKs and AP-1 activation. Similar to IL-33, stimulation of the cells with IL-36 cytokines upregulated CXC chemokines (such as CXCL1, CXCL2, CXCL3) in intestinal epithelial cells . Consistent with a role for the MAPK pathway and NF-kB in IL-36R signalling, activation of the IL-36R by IL-36γ was reduced in the presence of MAPK inhibitors and siRNAs specific for NF-κB and AP-1. In addition to the induction of chemokines, IL-6 was strongly induced in response to IL-36R stimulation in colonic fibroblasts . IL-6 has a broad range of functions in the colon. This cytokine has been implicated in a pro-inflammatory response, but it is also thought to play a role in mucosal healing of mucosal lesions . The antagonist IL-36Ra shares homology with IL-1Ra. It binds to the IL-36R preventing the formation of the heterodimer with IL-1RAcP. However, IL-36Ra can induce IL-4 production in glial cells.
Figure 1: The IL-36 family members and signal transduction. The IL-36R is a member of the IL-1R family. IL-36α, IL-36β and IL-36γ exert their actions by binding to the IL-36R. Ligand binding enables the recruitment of the IL-1RAcP. This leads to signal transduction through MYD88-dependent pathways, and activation of NF-κB and MAPKs. The IL-36R antagonist, IL-36Ra, also binds to the IL-36R, but fails to recruit the IL-1RAcp. Moreover, it also prevents other agonist ligands from binding to the receptor. IL-38 is also thought to act as an antagonist for this receptor.
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