TLR2-HIF-1α Mediated Polarization Of Macrophages And Oxidative Stress In Allergic Airway Inflammation

BackgroundBronchial asthma（asthma）is a heterogeneous disease characterized by chronic inflammation of the respiratory tract,hyperresponsiveness,increased mucus secretion,and structural remodeling of the respiratory tract,regulated by innate immunity and adaptive immunity.Toll-like receptors（TLRs）play an important role in innate immunity and serve as the first line of defense to recognize and process invading pathogens and aeroallergens.Toll-like receptor 2（TLR2）is the most widely distributed member of Toll-like receptor family and plays an important role in the pathogenesis of asthma.Similarly,the anoxia response and the increase of Hypoxia-inducible factor-1α（HIF-1α）are involved in respiratory inflammation and airway remodeling.Previous studies have suggested a direct link between TLRs signaling pathways and HIF-1α.For example,the promoter of TLR2 contains binding sites for HIF-1α.TLRs signal can induce the expression of HIF-1αand increase the stability of HIF-1αIn case of hypoxia.Previous studies have shown that macrophage polarization is also significantly involved in the pathogenesis of asthma,macrophage increase and activation of the polarization are closely related to the pathogenesis of asthma.Oxidative stress is caused by an imbalance between the production of oxides such as Reactive Oxygen Species（ROS）and Reactivenitrogen species（RNS）and antioxidant defenses,and is closely related to the inflammatory response of asthma.But does TLR2 participate in asthmatic airway inflammation through HIF-1α-mediated macrophage polarization and oxidative stress?And how the polarization of macrophages and oxidative stress mediated by TLR2-HIF-1αparticipate in the inflammatory waterfall effect during asthma attack remains to be further investigated.ObjectiveIn this paper,we used ovalbumin（OVA）to sensitize and stimulate the animal model of allergic airway inflammation in mice to explore the induction of allergic airway inflammation by TLR2 and its specific molecular regulatory mechanism.Methods6-8-week-old female C57BL/6J mice were randomly divided into control group,OVA model group and TLR2^-/-OVA group.PCR and Western blotting assay were used to identify the homozygotes of TLR2 absent mice;hematoxylin-eosin（H&E）staining was used to assess the inflammatory cell infiltration around the airway;glycogen（PAS）staining was used to detect the goblet cell proliferation and mucus secretion in the airway;the total number of white blood cells in bronchoalveolar lavage fluid（BALF）was calculated and classified;Western blotting assay was used to detect Th2 cytokines（IL-4,IL-5 and IL-13）;ELISA was used to detect serum Ig E level;Western blotting and immunohistochemistry were used to detect mitochondrial mitochondrial fusion fusion and oxidative stress associated proteins;RNA sequencing was used to analyze polarization-related indicators of macrophages;fluorescence stained TLR2 expression location and relationship with macrophages;Western blotting and immunohistochemistry were used to assess the polarization level of pulmonary and macrophages.Female C57BL/6J mice were randomly divided into OVA group,TLR2-/-OVA group,TLR2-/-OVA+EDHB group.The levels of HIF-1αafter OVA stimulation were detected by western blotting and immunohistochemistry.Hematoxylin-Eosin（H&E）staining was used to assess inflammatory cell infiltration around airways;goblet cell proliferation and mucus secretion in airways were detected by glycogen（PAS）staining;the total number of white blood cells in bronchoalveolar lavage fluid（BALF）was calculated and classified;Th2 cytokines（IL-4,IL-5 and IL-13）were detected by immunoblotting;serum Ig E level was detected by ELISA;and polarization of macrophages was detected by immunoblotting assay and immunohistochemical/fluorescence assay.In vivo AMs from WT and TLR2-/-mice challenged with or without OVA were isolated,then the expression levels of TLR2,HIF-1α,CD206,Arg1,and i NOS were detected by WB and immunofluorescence was used to detected CD206 and i NOS.WT and TLR2-/-mice alveolar macrophages were isolated.We then stimulated AMs with FITC-OVA for24h and confirmed that OVA can easily enter AMs.Then,the obtained AMs were stimulated with or without OVA for 24h.The detection contents were consistent with the above.ResultsIn OVA-induced allergic airway inflammation,the airway inflammatory response increased significantly in mice,while TLR2 deficiency reduced allergic airway inflammation.OVA exposure strongly increased Nitrotyrosine,TXNIP,8-OHd G,MDA,HO-1,GPX1/2,TRX1,OPA1,Mfn2,DRP1,Fis1,i NOS,CD86,CD206,Fizz1,Arg1.When TLR2 was knocked out,oxidative stress,mitochondrial mitotic fusion,and macrophage polarization decreased.However,EDHB restores oxidative stress and macrophage polarization in TLR2^-/-mice following OVA challenge.Further studies showed that the expression of TLR2,HIF-1αand macrophage polarization were also increased in in vivo AMs from OVA-challenged mice or ex vivo cultured resident AMs,and these were inhibited in TLR2^-/-AMs.ConclusionTLR2-HIF-1α-mediated macrophage polarization and oxidative stress are involved in allergic airway inflammation.This will provide a theoretical basis for the treatment of allergic airway inflammation based on the TLR2-HIF-1αsignaling pathway of alveolar macrophages.