TLR4 regulates IFN-γ and IL-17 production by both thymic and induced Foxp3+ Tregs during intestinal inflammation

T-regs play a crucial role in the maintenance of intestinal immune homeostasis. However, significant numbers of Foxp3(+) T-regs accumulate in the inflamed lesions in experimental colitis and in IBD patients. T-reg production of the proinflammatory cytokines IFN-gamma and/or IL-17 may arguably explain their ineffectiveness in suppressing intestinal inflammation. However, it remains unknown whether iT(reg) and tT(reg) produce proinflammatory cytokines and how TLR signaling regulates this process. Here, we found that Foxp3(+) T-regs were 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-regs within inflamed intestines. The majority of Foxp3(+) T-regs in the spleen was Helios(+)Nrp1(+), whereas most Foxp3(+) T-regs in the intestinal LP were Helios(-)Nrp1(-). More Helios(+)Nrp1(+) T-regs expressed IFN-gamma and/or IL-17 than did Helios(-)Nrp1(-) T-regs 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(regs) and Helios(-)Nrp1(-) iT(regs) produce proinflammatory cytokines in the intestines during inflammation, which was regulated by TLR4 signaling.