REG3A Induces SHP-1 To Regulate TLR3-dependent Inflammation In Skin Wounds

Wound repair is a critical physiological process for maintaining tissue homeostasis, and inflammation is a key phase in this process. Appropriate inflammatory responses inhibit the growth of bacteria and help to clean up matrix and cell debris, thus promoting tissue repair. However, uncontrolled and excessive production of inflammatory cytokines delays wound healing, tissue necrosis, even leads to multi-organ failure. For long decades the research on the inflammatory responses was focused on the one caused by microbial infection, while the inflammation caused by non-microbial infection was ignored. Therefore, it is urgent to investigate inflammation in wounds in the absence of infection.REG3A is a multi-functional protein which can regulate pancreatitis, enteritis and keratitis. So far, little is known about its functions in skin inflammation. In our study we first demonstrated that the activation of TLR3 induced the expression of REG3A. Compared to WT mice, the expression of REG3A was decreased in skin wounds. TLR3 silencing in keratinocytes disabled poly(I:C) to induce REG3A. Furthermore, REG3A suppressed TLR3-dependent inflammation both in vivo and in vitro, suggesting that activiation of TLR3 promotes inflammatory response as well as increases REG3A, thus preventing excessive inflammation in skin wounds.We next explored the mechanism by which REG3A suppressed TLR3-dependent inflammation. Firstly, we purified different structures of REG3A to stimulate keratinocytes in the presence or absence of poly(I:C), and found that C-type lectin domain (CTLD) was the key fragment of REG3A to inhibit poly(I:C)-induced inflammatory cytokines. We further determined that poly(I:C) induced inflammatory cytokines mainly dependent on JNK pathway by using multiple inhibitors, while REG3A dephosphorylated poly(I:C)-induced JNK2 to inhibit the inflammation. In vivo experiments also supported that REG3A inhibited TLR3-dependent inflammation via dephosphorylation of TLR3-activated p-JNK2. To further dissect how REG3A dephosphorylates p-JNK2, we checked whether REG3A induced negative regulators in keratinocytes, and found that REG3A induced negative regulator SHP-1 both in vivo and in vitro, but had no effects on A20, SARM and TRAF1. Moreover, REG3A bound to the N-terminus of EXTL3(1-141aa) to activate AKT-STAT3 to increase SHP-1 expression, and then SHP-1 silencing in keratinocytes inhibited the inhibitory effect of REG3A on p-JNK2 dephosphorylation, thus inhibiting TLR3-dependent inflammation. These results indicate that REG3A induces SHP-1 to dephosphorylate TLR3-mediated JNK2 to suppress inflammation.Taken together, our findings reveal that the activation of TLR3 induces the expression of REG3A, and REG3A, in turns, suppresses TLR3-denpendent inflammation via the induction of SHP-1 to dephosphorylate TLR3-activated p-JNK2 in keratinocytes. These findings provide new insights into the treatment of non-infectious wound inflammation instead of antibiotic.