TLR4 regulates IFN-γ and IL-17 production by both thymic and induced Foxp3+ Tregs during 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. Treg production of the proinflammatory cytokines IFN-γ and/or IL-17 may arguably explain their ineffectiveness in suppressing intestinal inflammation. However, it remains unknown whether iTreg and tTreg produce proinflammatory cytokines and how TLR signaling regulates this process. Here, we found that Foxp3(+)Tregs 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 Tregs within inflamed intestines. The majority of Foxp3(+) Tregs in the spleen was Helios(+)Nrp1(+), whereas most Foxp3(+) Tregs in the intestinal LP were Helios(-)Nrp1(-). More Helios(+)Nrp1(+) Tregs expressed IFN-γ and/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(+) tTregs and Helios(-)Nrp1(-) iTregs produce proinflammatory cytokines in the intestines during inflammation, which was regulated by TLR4 signaling.