The Effect And Mechanism Of Heme Oxygenase-1 In Regulating Th17/Treg Cell Balance Of Asthmatic Airway Inflammation

BackgroundTh17 cells play an important role in the pathogenesis of non-eosinophilic asthma(NEA), resulting in steroid-resistant neutrophilic airway inflammation. Heme oxygenase(HO)-1 is a rate-limiting enzyme for heme metabolism, which exerts anti-inflammatory and immunoregulatory functions in several Th cell-mediated autoimmune diseases. However, the role of HO-1 in NEA remains unclear. Our previous studies show that induction of HO-1 can suppress Th2 cell-mediated eosinophilic airway inflammation in a mouse model of eosinophilic asthma(EA), indicating a protective role of HO-1 in asthmatic airway inflammation. The current studies further explore the effect of HO-1 on Th17/Treg cell balance in the NEA mouse model and investigate the underlying mechanism by which HO-1 regulates Th17 immunity. ObjectiveThis study aims to determine the role of Th17/Treg cells and the related cytokines in the NEA mouse model, and to elucidate the effect and mechanism of HO-1 in regulating Th17/Treg cell balance, thus providing a novel target and experimental evidences in clinical understanding and managing severe/steroid-resistant asthma. Methods1. The NEA model was established by intranasally challenge with OVA in transgenic DO11.10 mice. HO-1 inducer hemin or enzymatic inhibitor Sn PP was injected intraperitoneally into OVA-challanged DO11.10 mice. The expression of HO-1 in lung tissues was detected by western blot. The effect of HO-1 on neutrophilic airway inflammation was evaluated in the NEA model by differentiatial cell counts of BALF and lung tissue pathology.2. In vivo experiments, we assessed the role of HO-1 in regulating Th17/Treg cell balance of mediastinal lympho nodes(MLN) and spleen by flow cytometry analysis, RORγt/Foxp3 expression in lung tissues by western blot, and IL-17A/IL-10 level in BALF using ELISA. Meanwhile, we observed whether HO-1 affected Th1/Th2 subsets of MLN and spleen by flow cytometry analysis, T-bet/GATA-3 m RNA expression in lung tissues by real-time PCR, IFN-γ/IL-4 level in BALF with ELISA.3. HO-1 si RNA was injected through tail vein to specifically suppress HO-1 expression followed by intraperitoneal injection of hemin in the NEA mouse model. We observed the pathology of lung tissues and determined the expression of HO-1 and RORγt in lung tissues, to confirm that HO-1 si RNA could reverse the hemin effect in vivo.4. Mouse splenic na?ve T cells were purified and cultured in Th17 or Treg condition in vitro in the presence of hemin or Sn PP. We assess the effect of HO-1 on the differentiation of na?ve T cells towards Th17/Treg cells in vitro by flow cytometry analysis, m RNA level of RORγt, IL-17 A and IL-10 by real-time PCR, as well as IL-17 A production in supernatants using ELISA.5. Mice were intraperitoneally treated with hemin or Sn PP to induce or inhibit HO-1 activity followed by isolation of CD4+ T cells from mouse spleen. Purified CD4+ T cells were culured in vitro with IL-6 or IL-2 for 1 hour to phosphorylate transcription factors STAT3 or STAT5, respectively. Cell lysate was assessed for the expression of p-STAT3/STAT3 or p-STAT5/STAT5 by western blot to further elucidate the mechanism on HO-1regulating Th17/Treg cell differentiation. Results1. OVA induced Th17-mediated antigen-specific airway inflammation in DO11.10 mice, characterized by PMN infiltration in lung. Induction of HO-1 by hemin could significantly alleviate airway inflammation, reduce the total number of cells, especially the number of PMN in BALF.2. Induction of HO-1 by hemin lowered the percentages of Th17 cells in spleen and MLN, down-regulated RORγt expression in lung and decreased IL-17 A production in BALF. Meanwhile, HO-1 induction promoted Treg and IL-10 production, thus suppressing Th17 response.3. HO-1 si RNA transfection reversed the anti-inflammatory effect of hemin in vivo. Moreover, the inhibition of Th17 cells by hemin was also been neutralized by HO-1 si RNA.4. HO-1 induction by hemin could switch na?ve T cell differentiation from Th17 to Treg cells through inhibiting IL-6-induced STAT3 phosphorylation and down-regulating RORγt expression. Therefore, HO-1 manipulated Th17/Treg cell differentiation balance in vitro. Conclusions1. HO-1 can suppress Th17 response in OVA-induced NEA model, as well as promotes Treg cells and reconstruct Th17/Treg cell balance in vivo, thus inhibiting OVA-induced NEA airway inflammation.2. HO-1 switches na?ve T cell differentiation in vitro from Th17 to Treg cells through inhibiting IL-6-induced STAT3 phosphorylation and down-regulating RORγt expression, which negatively regulates Th17 response in vivo.3. HO-1 may provide a novel therapeutic target in managing severe/steroid-restant asthma and other Th17-mediated autoimmune diseases.