STAT3 regulates inflammatory cytokine production downstream of TNFR1 by inducing expression of TNFAIP3/A20

Tumour Necrosis Factor (TNF) potently induces a transient inflammatory response that must be downregulated once any invasive stimulus has resolved. Yet, how TNF-induced inflammation is shut down in normal cells is incompletely understood. The present study shows that STAT3 was activated in mouse embryo fibroblasts (MEFs) by treatment with TNF or an agonist antibody to TNFR1. STAT3 activation was inhibited by pharmacological inhibition of the Jak2 tyrosine kinase that associates with TNFR1. To identify STAT3 target genes, global transcriptome analysis by RNA sequencing was performed in wild-type MEFs and MEFs from STAT3 knockout (STAT3(KO)) mice that were stimulated with TNF, and the results were validated at the protein level by using multiplex cytokine assays and immunoblotting. After TNF stimulation, STAT3(KO) MEFs showed greater gene and protein induction of the inflammatory chemokines Ccl2, Cxcl1 and Cxcl10 than WT MEFs. These observations show that, by activating STAT3, TNF selectively modulates expression of a cohort of chemokines that promote inflammation. The greater induction by TNF of chemokines in STAT3(KO) than WT MEFs suggested that TNF induced an inhibitory protein in WT MEFs. Consistent with this possibility, STAT3 activation by TNFR1 increased the expression of Tnfaip3/A20, a ubiquitin modifying enzyme that inhibits inflammation, in WT MEFs but not in STAT3(KO) MEFs. Moreover, enforced expression of Tnfaip3/A20 in STAT3(KO) MEFs suppressed proinflammatory chemokine expression induced by TNF. Our observations identify Tnfaip3/A20 as a new downstream target for STAT3 which limits the induction of Ccl2, Cxcl1 and Cxcl10 and inflammation induced by TNF.