TLR9 Mediates The Activation Of NLRP3 Inflammasome And Oxidative Stress In Murine Allergic Airway Inflammation

Background:Bronchial asthma is a complicated airway inflammatory disease accompanied by various types of cells and cellular components with its prevalence still being high.NOD-like Receptor with Pyrin Domain Containing 3（NLRP3）,is one of the most characteristic pattern recognition receptors（PRRs）activated by diverse stimuli such as allergens,thus involving the development and progression of asthma.Oxidative stress which strives from the imbalance between the production of oxidants such as reactive oxidative species（ROS）and reactive oxidative species（RNS）and the capacity of antioxidant defense,is closely associated with asthmatic inflammatory response.TLR9 is reported to play a vital role in allergic airway inflammation while the concrete mechanisms remain to be elucidated.Objective:Acute allergic airway inflammatory model induced by ovalbumin（OVA）was established and Raw 264.7 murine macrophage was used in our study so as to investigate the role of TLR9 and its relationship with NLRP3 inflammasome and oxidative stress.Materials and methods:1.In vivo,female wide type and TLR9^-/-mice aged 5 weeks on C57 BL/6 background are randomly divided into four groups（n=6 per group）:Vehicle group（Vehicle）、OVA group（OVA）、TLR9^-/-Vehicle group(TLR9^-/-Vehicle)and TLR9^-/-OVA group(TLR9^-/-OVA).At 0 day,mice were sensitized with 10μg OVA emulsified in alum in a total OVA volume of 500μl intraperitoneally for sensitization and were challenged with aerosolized for 30 min per day for 4 days consecutively from day 14.At day 18,mice were sacrificed to collect lung tissue and bronchial alveolar lavage（BALF）.The whole number of leukocytes in BALF was accessed by a hemocytometer and cellular composition was classified and counted by Swiss-Giemsa staining.Enzyme-linked immunosorbent assay（ELISA）was to qualify the level of IL-1β,IL-18,IL-5 and granulocyte-macrophage colony-stimulating factor（GM-CSF）.Western blot was to analyze the relative protein expression of TLR9,NLRP3,IL-1β,caspase-1（p20）and nitrotyrosine.The degree of inflammatory cell infiltration was observed by H&E and PAS staining was to evaluate goblet cell hyperplasia and mucus production.Immunohistochemistry（IHC）was to detect the expression of 8-OHd G and nitrotyrosine.2.In vitro,murine macrophage line Raw264.7 was randomly divided into three groups:control group,Staphylococcus aureus（S.aureus）group and TLR9 si RNA treatment group.After Raw264.7 macrophages were stimulated by S.aureus for 8 hours,the protein expression of TLR9,NLRP3,IL-1β,caspase-1（p20）,nitrotyrosine in different groups was analyzed by western blot.ELISA was to detect the content of IL-1βand IL-18 in culture medium.Immunofluorescence（IF）was used to examine the expression of 8-OHd G and nitrotyrosine in cells.Mito SOX Red assay was to assess cellular level of ROS..Results:1.In vivo,compared with wild type mice,TLR9 deficiency suppressed markedly inflammatory cell infiltration,goblet cell hyperplasia and mucus production induced by OVA.Simultaneously,TLR9 deficiency deceased the whole inflammatory cells and differential counts as well as the production of IL-1β、IL-18、IL-5、GM-CSF.Moreover,it inhibited the activation of NLRP3 inflammasome and the expression of NLRP3,IL-1β,IL-18,caspase-1,nitrotyrosine and 8-OHd G.2.In vitro,in contrast with vehicle group,after Raw264.7 cells stimulated by S.aureus,the cellular protein level of NLRP3,caspase-1 and IL-1βwas upregulated,and thecytokines production of IL-1βand IL-18 in culture medium were significantly increased.Moreover,the oxidative stress markers like nitrotyrosine and 8-OHd G and the level of ROS were also augmented.However,such increases were all significantly suppressed with TLR9 knock down by si RNA.Conclusions:TLR9 mediates the activation of NLRP3 inflammasome and upregulation of oxidative stress in murine allergic airway inflammation induced by OVA.TLR9 plays a part in regulating the activation of NLRP3 inflammasome and oxidative stress in Raw264.7 cells.TLR9 may be a vital positive mediator in activating NLRP3 inflammasome and oxidative stress in allergic airway inflammation.