| Background Th17 cell-derived IL-17 plays a crucial role in mediating the neutrophilic airway inflammation in non-eosinophilic asthma(NEA).IL-6 is a key factor in the priming na?ve T cells to Th17 cells through triggering the phosphorylation of STAT3.Suppressor of cytokine signaling 3(SOCS3)is a negative feedback regulator of Th17 cells differentiation.Our previous studies have shown that heme oxygenase-1(HO-1)suppresses Th17 cells differentiation and subsequently attenuates neutrophilic airway inflammation.However,the molecular mechanisms and signal transduction pathways by which HO-1 regulates Th17 cells differentiation in NEA remain incompletely understood.Objective The effects of HO-1 on airway inflammation and Th17 cells differentiation were explored using the NEA mouse model,and the molecules associated with signaling pathway of Th17 cells differentiation were performed in vitro to elucidate the mechanisms involved in the inhibition of Th17 cells differentiation by HO-1.Methods 1.Female DO11.10 mice were challenged intranasally with OVA for 3 continuous days to establish the NEA model.Hemin and Sn PP were administered by intraperitoneal injection to regulate the expression of HO-1.Total cells and Gr-1+ cells were counted.The levels of IL-17 A,IL-6 and IL-10 in lung homogenate(LH)or BALF were measured by ELISA.And the airway inflammation was observed.2.Mice were challenged intranasally with OVA in the process of establishing the NEA model.The expressions of HO-1,ROR?t,SOCS1 and SOCS3 in lung or spleen were detected by Western blot analysis,the levels of IL-17 A and IL-10 in LH or BALF were measured by ELISA,after 24 hours each time of OVA-challenged.3.Proteins expression associated with Th17-differentiation signaling pathway in splenic na?ve T cells purified by MACS from NEA mouse model,following stimulated by OVA323-339,in vitro,were detected by Western blot analysis.4.Na?ve T cells were isolated from BALB/c mice.The expression of HO-1 in the na?ve T cells treated with Hemin or Sn PP were detected at 24 h,48 h,72 h and 96 h.Na?ve T cells were cultured under the Th17-skewing differentiation and the proportions of CD4+IL-17A+ cells(Th17 cells)were analyzed by FCM.Moreover,the expressions levels of STAT3,p-STAT3 and SOCS3 in the cell lysates were detected by Western blot.The levels of IL-17 A in the supernatant at different time points were measured by ELISA.5.Hemin was administered by intraperitoneal injection in BALB/c mice to induce HO-1 expression for 2 consecutive days.(1)Spleen na?ve T cells sorted by MACS were cultured under the condition of Th17-skewing differentiation in vitro,and proteins associated with the Th17 cells pathway at 1 h,4 h,24 h,5 d were detected by Western blot analysis.(2)The frequency of CD4+IL-6R+ or CD4+IL-23R+ cells from spleen was analyzed by FCM.(3)STAT3,p-STAT3 and HO-1 in na?ve T cells at different time points treated with IL-6 were detected by Western blot.6.To detect the proteins interacted with HO-1 in the associated signaling pathway,co-Immunoprecipitation(Co-IP)and Western blot were uesd.The HO-1 and STAT3 domain expression plasmids were co-transfected into 293 T cells to identify the interaction sites of the STAT3 domain with HO-1.7.Mice transfected with HO-1 si RNA and then treated by Hemin via intraperitoneal injection,were used to establish the NEA model.Expression of FAM si RNA in lung tissues was detected by immunofluorescence staining.The frequency of Gr-1+ cells in BALF was analyzed by FCM.H&E and PAS staining were used to assess the pathological change of lung tissues.Western blot was used to detect the expression levels of associated protein in lung tissues and na?ve T cells.The levels of IL-17 A and IL-10 in the supernatant of na?ve T cells,in vitro,were measured by ELISA.8.Mice were transfected with HO-1 si RNA via tail vein injection and then Hemin was administered via intraperitoneal injection for 2 days.Na?ve T cells purified and cultured for 5 days under the condition of Th17-skewing differentiation,proteins associated Th17 cells signaling pathway were observed by Western blot analysis.Results 1.Compared to control mice,total cell numbers and Gr-1+ cells in BALF from NEA mice remarkably increased,IL-17 A,IL-6 and IL-10 in lung tissues were enhanced,and more inflammatory cells infiltrated in lung tissues.However,total cell numbers and Gr-1+ cells in BALF,levels of IL-17 A and IL-6 in lung tissues were decreased in NEA+Hemin group upon HO-1 up-regulation.Additionally,the extent of inflammation in the lung was alleviated,and the production of IL-10 was increased.In contrast,the above changes were reserved in the NEA+Sn PP group.2.In the process of establishing the NEA model,with the number of OVA-challenged increased:(1)IL-17 A production was gradually increased in all groups.After OVA-challenged,IL-10 production was first increased,and then gradually reduced.HO-1 overexpression inhibited the amplitude of IL-17 A production but maintained the high level of IL-10.(2)In the OVA group,the expression levels of ROR?t,SOCS3 and HO-1 in lung tissues increased gradually.Furthermore,HO-1 induced by Hemin inhibited the expression of ROR?t and SOCS3,but this change could be reversed by Sn PP.(3)The expression of SOCS1 in lung tissues was weak and HO-1 had no effect on its level in the NEA mouse model.3.HO-1 down-regulated the levels of ROR?t and SOCS3 expression under Th17 cells differentiation through inhibition the phosphorylation STAT3 of na?ve T cells in the NEA mouse model.In contrast,there was no change of activation of JAK2. 4.HO-1 expression induced by Hemin was highest at 48~72 hours,but Sn PP did not induce HO-1 expression.HO-1 suppressed the frequency of Th17 cells and the level of IL-17 A under Th17-skewing differentiation cultured for 5 days.The inhibitory effect of HO-1 on IL-17 A production was most obvious at 72 hours.HO-1 also inhibited the phosphorylation of STAT3 and SOCS3 expression under the Th17-skewing differentiation.5.We examined the dynamic role of HO-1 in the regulation of Th17 cells differentiation,and found that HO-1 played its role for inhibiting the expression of ROR?t and SOCS3 just after the phosphorylation of STAT3 activation.Results also confirmed that HO-1 suppressed the downstream signaling pathway mainly through inhibiting the IL-6-STAT3 pathway.6.Endogenous HO-1 was mainly interacted with STAT3.However,there was no interaction between JAK1,JAK2,SOCS3 and HO-1.Furthermore,the results showed that HO-1 had interaction with the whole STAT3 domain except for the coiled-coil domain.7.The protective effect of HO-1 was reversed by HO-1 si RNA in the NEA model,which was characterized by the increased frequency of Gr-1+ cells in BALF,the aggravated airway inflammation,and the promotion of activation of STAT3-ROR?t signaling pathway.8.In the in-vitro study,HO-1 si RNA reversed the effect of HO-1 that suppressed the phosphorylation of STAT3 under Th17-skewing differentiation.However,HO-1 did not regulate the activation of either JAK1 or JAK2.Conclusions 1.HO-1 suppresses Th17 response and increases IL-10 production,thereby alleviating Th17 cell-mediated neutrophilic airway inflammation.Interfering HO-1 expression can reverse the role of HO-1 for anti-inflammatory effect in neutrophilic airway inflammation and inhibition for Th17 cells differentiation related signaling pathway.2.HO-1 inhibits downstream nuclear transcription factor ROR?t and negative feedback regulator SOCS3 through blocking IL-6-STAT3-ROR?t/SOCS3 signaling pathway,which reduces Th17 cells differentiation,thereby alleviating Th17 cell-mediated neutrophilic airway inflammation.However,SOCS1 is not involved in this inflammatory response.3.The regulation of HO-1 in Th17 cells differentiation is mainly mediated through combination to STAT3 and further inhibiting the phosphorylation of STAT3,suggesting that STAT3 is the target for HO-1 regulating Th17 cells differentiation. |
PDF Full Text Request