Excessive cytokine activity underlies many autoimmune conditions, particularly through the interleukin-17 (IL-17) and tumor necrosis factor–α (TNFα) signaling axis. Both cytokines activate nuclear factor κB, but appropriate induction of downstream effector genes requires coordinated activation of other transcription factors, notably, CCAAT/enhancer binding proteins (C/EBPs). Here, we demonstrate the unexpected involvement of a posttranscriptional “epitranscriptomic” mRNA modification [N6-methyladenosine (m6A)] in regulating C/EBPβ and C/EBPδ in response to IL-17A, as well as IL-17F and TNFα. Prompted by the observation that C/EBPβ/δ-encoding transcripts contain m6A consensus sites, we show that Cebpd and Cebpb mRNAs are subject to m6A modification. Induction of C/EBPs is enhanced by an m6A methylase “writer” and suppressed by a demethylase “eraser.” The only m6A “reader” found to be involved in this pathway was IGF2BP2 (IMP2), and IMP2 occupancy of Cebpd and Cebpb mRNA was enhanced by m6A modification. IMP2 facilitated IL-17–mediated Cebpd mRNA stabilization and promoted translation of C/EBPβ/δ in response to IL-17A, IL-17F, and TNFα. RNA sequencing revealed transcriptome-wide IL-17–induced transcripts that are IMP2 influenced, and RNA immunoprecipitation sequencing identified the subset of mRNAs that are directly occupied by IMP2, which included Cebpb and Cebpd. Lipocalin-2 (Lcn2), a hallmark of autoimmune kidney injury, was strongly dependent on IL-17, IMP2, and C/EBPβ/δ. Imp2−/− mice were resistant to autoantibody-induced glomerulonephritis (AGN), showing impaired renal expression of C/EBPs and Lcn2. Moreover, IMP2 deletion initiated only after AGN onset ameliorated disease. Thus, posttranscriptional regulation of C/EBPs through m6A/IMP2 represents a previously unidentified paradigm of cytokine-driven autoimmune inflammation.
SARS-CoV-2 has caused an estimated 7 million deaths worldwide to date. A secreted SARS-CoV-2 accessory protein, known as open reading frame 8 (ORF8), elicits inflammatory pulmonary cytokine responses and is associated with disease severity in COVID-19 patients. Recent reports proposed that ORF8 mediates downstream signals in macrophages and monocytes through the IL-17 receptor complex (IL-17RA, IL-17RC). However, generally IL-17 signals are found to be restricted to the nonhematopoietic compartment, thought to be due to rate-limiting expression of IL-17RC. Accordingly, we revisited the capacity of IL-17 and ORF8 to induce cytokine gene expression in mouse and human macrophages and monocytes. In SARS-CoV-2–infected human and mouse lungs, IL17RC mRNA was undetectable in monocyte/macrophage populations. In cultured mouse and human monocytes and macrophages, ORF8 but not IL-17 led to elevated expression of target cytokines. ORF8-induced signaling was fully preserved in the presence of anti–IL-17RA/RC neutralizing Abs and in Il17ra−/− cells. ORF8 signaling was also operative in Il1r1−/− bone marrow–derived macrophages. However, the TLR/IL-1R family adaptor MyD88, which is dispensable for IL-17R signaling, was required for ORF8 activity yet MyD88 is not required for IL-17 signaling. Thus, we conclude that ORF8 transduces inflammatory signaling in monocytes and macrophages via MyD88 independently of the IL-17R.
Interleukin 17 is a proinflammatory cytokine required for host defense against extracellular microorganisms but also contributes to the pathogenesis of certain autoimmune diseases. Candida albicans is a commensal fungus that colonizes mucosal tissues. During immunosuppression, C. albicans can cause superficial infections, such as oropharyngeal candidiasis (OPC). Defects in IL-17 signaling increase susceptibility to OPC in both humans and mice. IL-17 receptor signaling involves RNA binding proteins (RBP) that regulate translation and stability of target mRNA transcripts. During OPC, IL-17 induces several RBPs including Arid5a. This RBP interacts with AU-rich elements located in the 3′ UTR of many inflammatory mRNAs. In T cells, loss of Arid5a reduces expression of IL-6 and STAT3 and leads to decreased frequency of Th17 cells. Consequently, Arid5a−/− mice are resistant to experimental autoimmune encephalomyelitis (EAE), an IL-17-driven model of multiple sclerosis. Although made by T cells, IL-17 signals on non-hematopoietic cell types. We reported an in vitro role for Arid5a in the downstream IL-17 receptor pathway. Specifically, Arid5a regulates stability and/or translation of several IL-17-dependent mRNAs including Il6. Accordingly, we postulated that Arid5a plays a critical role in protection against OPC by stabilizing and increasing expression of IL-17-responsive genes. To test this hypothesis, CRISPR/Cas9 was used to generate an Arid5a knockout mouse. We confirmed that Arid5a−/− mice are resistant to EAE. To ascertain the effects of Arid5a in OPC, we infected mice orally with C. albicans and assessed fungal loads and weight loss. Surprisingly, Arid5a−/− mice were resistant to OPC, in contrast to Il17ra−/− mice.
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