Research ArticleImmunology

Noncanonical STAT1 phosphorylation expands its transcriptional activity into promoting LPS-induced IL-6 and IL-12p40 production

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Science Signaling  24 Mar 2020:
Vol. 13, Issue 624, eaay0574
DOI: 10.1126/scisignal.aay0574

Inflammatory internalization

When bound to its ligand LPS, the cell surface pattern recognition receptor TLR4 stimulates the production of proinflammatory cytokines. However, endocytosed TLR4 signals through different effectors to drive production of interferon-β (IFN-β), an antiviral cytokine. Metwally et al. found an alternative signaling pathway activated by endocytosed TLR4 in mouse and human macrophages that contributed to the production of the proinflammatory cytokines IL-6 and IL-12p40 independently of IFN-β. Endocytosis of TLR4 led to the noncanonical phosphorylation of the transcriptional regulator STAT1, which altered its target DNA motif. STAT1 stimulated the expression of the genes encoding IL-12p40 and the protein ARID5A, which increased the stability of IL6 mRNA. Together, these findings suggest how endocytosed TLR4 contributes to proinflammatory cytokine production, which may have implications for the use of vaccine adjuvants that target this receptor.


The lipopolysaccharide (LPS)–induced endocytosis of Toll-like receptor 4 (TLR4) is an essential step in the production of interferon-β (IFN-β), which activates the transcription of antiviral response genes by STAT1 phosphorylated at Tyr701. Here, we showed that STAT1 regulated proinflammatory cytokine production downstream of TLR4 endocytosis independently of IFN-β signaling and the key proinflammatory regulator NF-κB. In human macrophages, TLR4 endocytosis activated a noncanonical phosphorylation of STAT1 at Thr749, which subsequently promoted the production of interleukin-6 (IL-6) and IL-12p40 through distinct mechanisms. STAT1 phosphorylated at Thr749 activated the expression of the gene encoding ARID5A, which stabilizes IL6 mRNA. Moreover, STAT1 phosphorylated at Thr749 directly enhanced transcription of the gene encoding IL-12p40 (IL12B). Instead of affecting STAT1 nuclear translocation, phosphorylation of Thr749 facilitated the binding of STAT1 to a noncanonical DNA motif (5′-TTTGANNC-3′) in the promoter regions of ARID5A and IL12B. The endocytosis of TLR4 induced the formation of a complex between the kinases TBK1 and IKKβ, which mediated the phosphorylation of STAT1 at Thr749. Our data suggest that noncanonical phosphorylation in response to LPS confers STAT1 with distinct DNA binding and gene-regulatory properties that promote both IL12B expression and IL6 mRNA stabilization. Thus, our study provides a potential mechanism for how TLR4 endocytosis might regulate proinflammatory cytokine production.

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