Interleukin 17 (IL-17) plays a critical role in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for IL-17R, forms a complex with IKKi upon IL-17 stimulation. Using IKKi-deficient mice, we show that IKKi was required for IL-17-induced inflammatory gene expression in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced Act1-TRAF2/5 complex formation, MAPK activation and mRNA stability, whereas the Act1-TRAF6-NFκB axis was retained. IKKi was required for IL-17-induced Act1 phosphorylation on serine 311, adjacent to a putative TRAF binding motif. S311A mutation impaired IL-17-mediated Act1-TRAF2/5 interaction and gene expression. Thus, IKKi is a novel kinase modulating IL-17 signaling through its impact on Act1 phosphorylation and consequent function.

The cellular and molecular mechanisms driven by IL-25 and its cognate receptor IL-17RB necessary for the promotion of T helper type 2 (Th2) mediating pathogenic pulmonary inflammation remain to be defined. We have previously reported the critical role of the U-box-type E3 ubiquitin ligase Act1 (1), for the downstream signaling of the IL-17 cytokine family including the Th2 promoting cytokine IL-25 (IL-17E) (2). In this study, we report that IL-25 driven but not conventional IL-4 driven Th2 polarization and cytokine production is impaired in Act1-deficient T cells. Also, Act1 deficiency in the T cell compartment results in the abrogation of eosinophilic airway infiltration as well as airway hyperresponsiveness (AHR) in a mouse model of antigen induced airway inflammation. The in vivo generation of antigen specific Th2 cytokine producing cells is defective in the absence of Act1 expression in T cells following ovalbumin/Alum immunization. Interestingly, the production of ovalbumin specific IgG1 but not IgG2a or IgE is also impaired. At the molecular level, we report that IL-25-mediated induction of Th2 master regulator GATA-3, and the transcription factor GFI-1 is attenuated in Act1-deficient T cells. Taken together, our findings indicated that Act1 expression in T cells is required for cellular and humoral Th2-mediated allergic responses and the development of AHR, in part, through its function in IL-25 induced development of Th2 T cells.

Monocytes are rapidly recruited to sites of diabetic complications and differentiate into macrophages. Previously, we showed that rat kidney mesangial cells dividing during hyperglycemic stress abnormally synthesize hyaluronan (HA) in intracellular compartments. This initiates a stress response, resulting in an extracellular HA matrix after division that recruits inflammatory cells. Cell–cell communication among macrophages that are recruited into the glomeruli and the damaged rat mesangial cells leads to diabetic nephropathy, fibrosis, and proteinurea, which are inhibited in heparin-treated diabetic rats. In this study, we found that murine bone marrow–derived macrophages (BMDMs) and a human leukemic cell line, U937 cells, dividing in hyperglycemia also accumulate intracellular HA and that heparin inhibits the HA accumulation. Both cell types expressed increased levels of proinflammatory markers: inducible nitric-oxide synthase and tumor necrosis factor-α, when cultured under hyperglycemic stress, which was inhibited by heparin. Furthermore, the abnormal intracellular HA was also observed in peripheral blood monocytes derived from three different hyperglycemic diabetic mouse models: streptozotocin-treated, high-fat fed, and Ins2Akita. Moreover, peripheral blood monocytes in humans with type 2 diabetes and poorly controlled blood glucose levels (hemoglobin A1c (HbA1c) levels of >7) also had intracellular HA, whereas those with HbA1c of <7, did not. Of note, heparin increased the anti-inflammatory markers arginase 1 and interleukin-10 in murine BMDMs. We conclude that heparin treatment of high glucose–exposed dividing BMDMs promotes an anti-inflammatory tissue-repair phenotype in these cells.

Background: Heparin prevents intracellular hyaluronan synthesis and subsequent autophagy in hyperglycemic dividing mesangial cells. Results: The non-reducing terminal trisaccharide of heparin is sufficient for this response. Conclusion: This heparin trisaccharide motif is exposed by the mammalian heparanase and is recognized by a receptor on dividing cells. Significance: The trisaccharide does not have the anti-coagulant properties of heparin.

Many cells, including murine airway epithelial cells, respond to a variety of inflammatory stimuli by synthesizing leukocyteadhesive hyaluronan (HA) cables that remain attached to their cell surfaces. This study shows that air-liquid interface cultures of murine airway epithelial cells (AECs) also actively synthesize and release a majority of their HA onto their ciliated apical surfaces to form a heavy chain hyaluronan (HC-HA) matrix in the absence of inflammatory stimuli. These matrices do not resemble the rope-like HA cables but occur in distinct sheets or rafts that can capture and embed leukocytes from cell suspensions. The HC-HA modification involves the transfer of heavy chains from the inter-␣-inhibitor (I␣I) proteoglycan, which has two heavy chains (HC1 and HC2) on its chondroitin sulfate chain. The transesterification transfer of HCs from chondroitin sulfate to HA is mediated by tumor necrosis factor-induced gene 6 (TSG-6), which is up-regulated in inflammatory reactions. Because the AEC cultures do not have TSG-6 nor serum, the source of I␣I, assays for HCs and TSG-6 were done. The results show that AECs synthesize TSG-6 and their own heavy chain donor (pre-I␣I) with a single heavy chain 3 (HC3), which are also constitutively expressed by human renal proximal tubular epithelial cells. These leukocyte adhesive HC3-HA structures were also found in the bronchoalveolar lavage of naïve mice and were observed on their apical ciliated surfaces. Thus, these leukocyteadhesive HA rafts are now identified as HC3-HA complexes that could be part of a host defense mechanism filling some important gaps in our current understanding of murine airway epithelial biology and secretions.