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

Background: Previous work demonstrated that the Signal Transducer and Activator of Transcription 3 (STAT3) is activated downstream of the Type 1 TNF Receptor. However, whether and how STAT3 regulates gene expression downstream of TNFR1 has not been elucidated. Methods: Global transcriptome analysis by RNA sequencing was performed in wild type and STAT3 knockout mouse embryonic fibroblasts (MEFs) stimulated with TNF. The fold changes in gene expression were assessed bioinformatically. Results of the RNA sequencing were validated at the protein level by using multiplex cytokine assays and immunoblotting. Results: Stimulation of MEFs with TNF or an agonist antibody to TNFR1 activated STAT3, and this was inhibited by pharmacological inhibition of Jak2 and cSrc. At 4 hours after TNF stimulation, STAT3 knockout MEFs had a greater level than WT MEFs of induction of the chemokines Ccl2, Cxcl1 and Cxcl10 at the RNA and protein levels. Mechanistically, this was due STAT3 promoting the expression of Tnfaip3/A20, a ubiquitin modifying enzyme that inhibits inflammation, in wild-type MEFs at early timepoints after TNFR1 stimulation. In STAT3 knockout MEFs TNF failed to induce the expression of Tnfaip3/A20 or GM-CSF when acting through TNFR1. Expression of A20 into STAT3 knockout MEFs suppressed cytokine expression. Conclusion: STAT3 limits the induction of Ccl2, Cxcl1 and Cxcl10 in response to TNFR1 activation by promoting the expression of Tnfaip3/A20. On the other hand, STAT3 promotes the expression of GM-CSF in response to TNFR1 stimulation. These results show that STAT3 modulates inflammatory signaling by TNF in normal cells. ### Competing Interest Statement R. Antonia is currently employed by NGM Biopharmaceuticals.