The Functional Mechanism By Which Hyperglycemia-induced MiR-9 Inhibits IL-33 Expression

Inflammatory response is crucial for wound healing. Appropriate inflammatory response can promote normal wound healing, protect from microbial infection and maintain the body homeostasis, while excessive inflammatory response impairs wound healing in some extreme conditions such as diabetes. However, how inflammatory responses are dysregulated in diabetes remains unknown. Our and other groups have reported that IL-33 expression is decreased in diabetic wounds along with the increased expression of TLR3-dependent proinflammatory cytokines such as TNF-a and IL-6. However, how IL-33 is decreased in diabetic wounds is poorly explored, and whether the decreased IL-33 expression is related to excessive inflammation in diabetic wounds remains unclear.Here we firstly treated keratinocytes with high glucose and IL-17 or TLR3 ligand poly(I:C). We showed that the high glucose significantly inhibited IL-17- or poly(I:C)-induced IL-33 expression and increased poly(I:C)-induced proinflammatory cytokines expression such as IL-1β and IL-6. We further found that miR-9 inhibited IL-33 mRNA expression by targeting the 3’UTR of IL-33 mRNA, thus leading to decreased expression of IL-33. Moreover, high glucose significantly induced the expression of miR-9 in keratinocytes, and the inhibitory effect of high glucose on IL-17-induced IL-33 was dependent on miR-9 as the inhition of miR-9 by its miR-9 inhibitor restored the expression of IL-33 in keratinocytes. Furthermore, we found that high glucose up-regulated miR-9 expression dependent on the activation of NF-κB. These results show that high glucose induces miR-9 to inhibit the expression of IL-33 by activating NF-κB.Next we investigated the functional mechanisms by which IL-33 inhibits inflammatory responses in keratinocytes. We treated keratinocytes with recombinant IL-33 and poly(I.C) and found that IL-33 markedly inhibited poly(I:C)-induced proinflammatory cytokines such as TNF-a, IL-1β and IL-6 expression. Further studies demonstrated that IL-33 significantly induced the expression of SHP-1, a negative regulator, in keratinocytes, and SHP-1 dephosphorylated TLR3-activated JNK2 via binding to JNK2. Thus, SHP-1 mediates IL-33 dephosphorylation of JNK2 and then inhibits TLR3-dependent inflammation.In summary, our studies show for the first time that high glucose in diabetes induces miR-9 to decrease IL-33 mRNA expression, thus leading to decrease IL-33 but increased TLR3-dependent proinflammatory cytokines in skin wounds. These findings also provide new insights into pathways contributing to wound healing and therapeutic targets in treatment with non-healed wounds due to excessive inflammation in diabetes.