Tlr4 regulates IFN-γ and IL-17 production by both thymic and induced Foxp3⁺T_regsduring intestinal inflammation

Tregs play a crucial role in the maintenance of intestinal immune homeostasis. However, significant numbers of Foxp3⁺Tregs accumulate in the inflamed lesions in experimental colitis and in IBD patients. T_regproduction of the proinflammatory cytokines IFN-γ and/or IL-17 may arguably explain their ineffectiveness in suppressing intestinal inflammation. However, it remains unknown whether iT_regand tT_regproduce proinflammatory cytokines and how TLR signaling regulates this process. Here, we found that Foxp3⁺T_regswere increased in the intestines of B6.TLR4^-/-and B6.IL-10^-/-mice when compared with WT B6 mice. TLR4^-/-and IL-10^-/-resulted in more T_regswithin inflamed intestines. The majority of Foxp3⁺T_regsin the spleen was Helios^-Nrp1^-, whereas most Foxp3⁺T_regsin the intestinal LP were Helios⁺Nrp1⁺. More Helios^-Nrp1^-T_regsexpressed IFN-γand/or IL-17 than did Helios⁺Nrp1⁺Tregs in the spleen and intestine, which was increased with TLR4^-/-. TLR4 signaling in T cells and APCs inhibited Foxp3⁺induction via MyD88-dependent, TRIF-independent pathways, which was negatively regulated by SOCS3. Collectively, these data demonstrate Helios⁺Nrp1⁺tT_regsand Helios-^-Nrp1^-iT_regsproduce proinflammatory cytokines in the intestines during inflammation, which was regulated by TLR4 signaling.