The Effect And Mechanism Of The Sumoylation Of NLRP3 Of Alveolar Macrophage On Airway Inflammation In Bronchial Asthma

Research background:Asthma is a heterogeneous chronic inflammatory disease of the lungs with a variety of phenotypes,characterized by airway inflammation,hypersecretion of mucus,and airway hyperresponsiveness.Airway inflammation is characterized by long-term chronic infiltration of various inflammatory cells under the airway epithelium.Airway inflammatory cell infiltration can cause mucus hypersecretion,aggravate airway hyperresponsiveness and airway remodeling,and then lead to fixed airflow limitation and irreversible decline in lung function.Airway inflammation is central to the pathophysiology,clinical features,and risk susceptibility of asthma and is the target of treatment.Macrophages are important innate immune cells with a wide variety of functions related to their role as initial responders to immune stimuli and the subsequent development of innate and adaptive immune responses.These functions include the recognition,response and destruction of invading microorganisms,antigen presentation,maintenance of tissue and immune homeostasis by clearing cell debris,and production of inflammatory regulatory products.Studies have shown that macrophages not only maintain lung homeostasis by performing anti-inflammatory functions,but may also be an integral part of the mechanisms of inflammation and tissue damage associated with asthma.Macrophages can contribute to asthma inflammation in several ways,including altered(anti-inflammatory)cytokine/chemokine production,induction of inflammatory bodies,and altered cellular processes such as phagocytosis deficits.Inflammasome is a multiprotein complex composed of intracytoplasmic pattern-recognition receptors(PRRs)that can be detected in immune and non-immune cells upon stimulation.Among them,NOD-like receptor(NLR)heat protein domain associated protein 3(NLRP3)inflammatories play an important role in innate immunity of human body and have received extensive attention in recent years.NLRP3 inflammasome is highly expressed in patients with rheumatic immune diseases,Alzheimer’s disease,Parkinson’s disease,myocardial infarction,and bronchial asthma and chronic obstructive pulmonary disease.After activation of NLRP3 inflammasome,IL-1β,IL-18 and other inflammatory factors are released to induce immune response,which are closely related to the onset of asthma.In patients with asthma,NLRP3 inflammasome is mainly expressed in alveolar macrophages,and regulating the activation of NLRP3 inflammasome in macrophages is expected to be a potential target for controlling airway inflammation in bronchial asthma.The activation process of NLRP3 inflammasome includes two steps:assembly and activation.Assembly refers to the combination of transcription,translation and oligomerization of inactive parts of NLRP3 inflammasome(NLRP3,ASC,pro-caspase-1),activation refers to the presence of activators,pro-caspase-1 is activated to mature caspase-1 and cleaved to proIL-1β and proIL-18 to form active IL-1β and IL-18,which then participate in the inflammatory process.The activation of NLRP3 inflammasome involves a complex regulatory network,the core of which is the regulation of NLRP3.The regulation of NLRP3 includes transcriptional regulation,post-transcriptional regulation and post-translational modification.The post-translational modification includes NLRP3 phosphorylation/dephosphorylation,sudinization/desudinization,ubiquitination/deubiquitination and other processes.Protein sumoylation modification is a protein modification process mediated by SUMO(small ubiquitin-related modifier)proteins,which can change many characteristics of substrate proteins,such as intracellular sublocalization,enzyme activity,protein structure and stability,and transcriptional activity.In vitro studies,NLRP3 sumoylation modification in peritoneal macrophages and monocyt-induced differentiated macrophages can promote the activation of NLRP3 inflammasome and increase the release of pro-inflammatory factors,but whether it occurs in alveolar macrophages and whether it is involved in the formation of airway inflammation in bronchial asthma remains unclear.Studies at home and abroad have shown that the activation of NLRP3 inflammatories in alveolar macrophages plays a crucial role in the occurrence of airway inflammation in asthma.Sumoylation is an important part of post-translational protein regulation and has been shown to regulate the activation of NLRP3 inflammasome in macrophages.However,whether the sumoylation of NLRP3 in alveolar macrophages can regulate the activation of NLRP3 inflammasome or participate in airway inflammation in bronchial asthma has not been studied.Purpose of research:1.To explore the difference of IL-1β concentration and SUMO1mRNA expression in induced sputum in asthmatic patients and health controls,to explore the relationship between them and the relationship between SUMO1mRNA and lung function of asthmatic patients..2.To confirmed that HDM promoted the activation of NLRP3 inflammasome by enhancing the sumoylation of NLRP3 in alveolar macrophages.3.To explore the effect of inhibition of NLRP3 sumoylation on the activation of NLRP3 inflammasome in HDM induced alveolar macrophages.And explore its possible mechanism.4.In animal experiments,sumoylation inhibition was given to observe whether inhibiting sumoylation inhibition could reduce airway inflammatory cell infiltration,reduce airway inflammatory factor release,and reduce airway mucus hypersecretion in asthmatic mice sensitized by HDM.Research method:1.The induced sputum of asthmatic patients and healthy controls was collected to analyze the differences of IL-1β and SUMO1 mRNA levels in induced sputum between the two groups,and to analyze the correlation between IL-1β and SUMO1 mRNA meanwhile the correlation between SUMO1 mRNA and lung function.2.Western blot was used to analyze the expression of SUMO1,NLRP3/IL-1βpathway related proteins in alveolar macrophages after HDM stimulation,and CO-IP was used to analyze the expression of SUMO-NLRP3 after HDM stimulation.3.After inhibiting the sumoylation of NLRP3,Western blot was used to analyze the expressions of SUMO1,NLRP3/IL-1β pathway related proteins in alveolar macrophages stimulated by HDM,and CO-IP was used to analyze the changes in the SUMO-NLRP3 expression.4.After inhibition of NLRP3 sumoylation,the difference of NLRP3 mRNA expression in alveolar macrophages stimulated by HDM was detected by RT-PCR,and the stability of NLRP3 protein was analyzed by protein degradation assay.5.HE staining and immunohistochemical methods were used to evaluate the effects of sumoylation inhibitor on airway inflammation and mucus secretion in HDM sensitized asthmatic mice.The levels of inflammatory cytokines and the number of inflammatory cells in alveolar lavage fluid were determined by Elisa.The alveolar lavage cell sediment was used to extract the protein for Western blot analysis of SUMO1,NLRP3/IL-1β pathway related protein expression.The changes of SUMO-NLRP3 expression were analyzed by CO-IP method.Research results:1.The levels of IL-1β and SUMO1 mRNA in asthmatic patients were higher than those in healthy controls.and they were positively correlated with the deterioration of lung function in asthmatic patients.2.The expression of SUMO1 and NLRP3/IL-1β pathway related proteins in alveolar macrophages stimulated by HDM was increased,and the sumoylation of NLRP3 was also increased.3.NLRP3/IL-1β pathway related protein expression was decreased in HDM stimulated alveolar macrophages after the inhibition of sumoylation,along with SUMO-NLRP3 reduction.3.1 After the application of si-UBC9 to interfere the UBC9 expression,NLRP3/IL-1β pathway related protein expression in HDM stimulated alveolar macrophages was decreased,accompanied by the decrease of SUMO-NLRP3.3.2 NLRP3/IL-1β pathway related protein expression was decreased in HDM stimulated alveolar macrophages after 2-D08 inhibition of sumoylation,accompanied by a decrease in SUMO-NLRP3 expression.4.Inhibition of sumoylation reduced the stability of NLRP3 protein.5.Inhibition of sumoylation can reduce airway inflammatory infiltration,mucus hypersecretion and release of inflammatory factors in asthmatic mice.Research conclusions:1.HDM promotes the activation of NLRP3 inflammasome by enhancing the sumoylation of NLRP3 in alveolar macrophages.Inhibition of sumoylation can reduce the activation of NLRP3 inflammasome induced by HDM.2.Inhibition of NLRP3 sumoylation reduces inflammasome activation by reducing the stability of NLRP3 protein.3.Inhibition of sumoylation of alveolar macrophage NLRP3 can reduce airway inflammatory cell infiltration,secretion of inflammatory factors and airway mucus hypersecretion in bronchial asthmatic mice.