Mechanism Of RAD50-Mediated DNA Damage Response In Particulate Matter-Induced Airway Inflammation

The DNA damage response(DDR)is an important pathophysiological process for the multi-celluar organism to cope with various exogenous stimuli such as ultraviolet rays,ionizing radiation and chemotherapeutic drugs or byproducts of endogenous physiological processes like DNA replication and transcription.DDR is a multi-level signal transduction pathway that includes sensors,mediators,transducers,and effectors.The result of DDR may be the repair of damaged DNA molecules,cell cycle arrest,apoptosis,or the entry of damaged DNA into daughter cells to promote tumourigenesis.A large body of evidence suggest that DNA damage repair responses are widely involved in the pathophysiological processes of lung diseases including asthma,chronic obstructive pulmonary disease,lung cancer,and acute lung injury.Inflammation is the ultimate response to the constant challenges of the immune system by microbes,irritants or injury.The pathological characters of respiratory diseases is acute or chronic airway inflammation.Asthma,an allergic disease,is a kind of chronic airway inflammation involving various effector cells such as eosinophils,T lymphocytes,macrophages,and neutrophils,and regulated by complex network of cytokines,chemokines,histamine,the leukotrienes and other components of the Th2 type immune response.The basic pathological feature of chronic obstructive pulmonary disease is pulmonary emphysema and chronic bronchitis with neutrophils,alveolar macrophages and corresponding cytokines such as IL8,LTB4,and proteases such as matrix metalloproteinases,elastase,etc.The acute lung injury caused by various pathogenic microorganisms(such as LPS,bacteria),atmospheric particulate matter(PM),and physical damage is Thl-type inflammation as well as neutrophil-based innate immune response.As a DDR sensor,the MRE11-RAD50-NBS1(MRN)protein complex play multiple roles in maintaining genomic stability,including repair of DNA double-strand breaks(DSBs),checkpoint activation,meiotic chromosomal rearrangements,and telomere length maintain.Previous studies have confirmed that the MRN complex play an important role in tumor development.The hypomorphic mutation of RAD50 in the MRN complex facilitate the formation of tumors.Targeted therapy with RAD50 enhances platinum-based chemosensitivity in lung squamous cell carcinoma,making the study of the MRN complex shift from previous structural and functional exploration to clinical application.Recent studies have confirmed that the MRN complex also plays an important role in the regulation of inflammation.MRE11 and RAD50 induce the expression of type I interferons by regulating STING trafficking.After transfection of DNA viruses in dendritic cells(DCs),the MRN complex translocate from the nucleus to the cytoplasm,forming the RAD50-dependent Rad50-CARD9-dsDNA complex,which then activates NF-?B and promotes IL-lb production,while in rad50 conditions knock-out DCs,type I interferon production was significantly inhibited after DNA transfection.NBS1,as a subunit of the MRN complex,plays an important role in macrophage function during normal aging.The inflammatory response is markedly enhanced in 2,4-dinitrobenzene(DNFB)-induced inflammation model following NBS1 deletion in macrophages.DNA damage repair response-related signaling pathways and inflammatory immune responses are two important mechanisms that regulate each other during the disease process,affecting the final outcome of the disease.However,it occurs in response to classical stimulation by exogenous insults such as viral or bacterial infections and endogenous byproducts of normal physiologic processes.Previous research has shown that airborne particulate matter,a complex multi-component system threatening public health and safety,could cause relatively moderate damage to DNA in both in-vitro cell lines and in occupational groups.In this case,is there any connetion between the DDR and inflammatory response induced by particulate matter?Notably,airway epitheliums and alveolar macrophages(AMs)are the first responders to foreign particles in the lung could phagocytose inhaled particles and produce a striking proinflammatory response upon exposure to PM[25,26].Nevertheless,little is known about the possible role of DDR signaling in proinflammatory function of macrophages following PM exposure.Can PM cause direct DNA damage in macrophages?Is DDR signaling involved in PM-treated macrophages?Does DDR signaling impact the function of macrophages in PM-induced inflammation?Therefore,we investigated the function and mechanisms of RAD50-mediated DNA damage response in PM2.5-induced acute inflammation both in in vivo and in vitro model in airway epithelium and macrophages and thus suggest a new potential target for interventions of particulate matter-related pulmonary disorders.Part I Mechanism Of RAD50-Mediated DNA Damage Response In Macrophages In Particulate Matter-Induced Airway InflammationObjective:To establish both in vitro and in vivo model with PM2.5 treatment and study the particulate matter-induced DNA damage response in macrophages and its function in regulation of airway inflammation.Methods:Both bone marrow-derived macrophages and peritoneal-derived macrophages were treated with PM in vitro.Comet assay was used to detect DNA breaks.Q-PCR and ELISA were used to detect the expression of cytokines in macrophages and the culture supernatants,respectively.Western Blot and immunofluorescence were used to detect the expression of specific proteins involved in DNA damage response,autophagy,cGAS-STING and NF-?B signaling pathway.In vivo,RAD50 myeloid specific knockout(RAD50f/fLysmcre)mice of 8 weeks were used as the KO group,and the LysMCre-negative,RAD50flox/flox littermates served as controls.All mice were divided into 4 groups:the control-NS group,the control-PM group,KO-NS group and KO-PM group.The control-NS group and the KO-NS group were instilled intratracheally with NS(50?l),and the corresponding model group with PM2.5(100?g/50?l).The total number of inflammatory cells was quantified and the number of neutrophils was calculated Expressions of cytokine levels in the lung tissue and BALF were determined using real-time PCR and ELISA,respectively.Lung sections stained with hematoxylin and eosin(H&E).were used to detect pathological changes.Results:We found that particulate matter induced significant DNA damage both in vitro and in vivo and simultaneously triggered a rapid DNA damage response,represented by nuclear RPA,53BP1 and ?H2AX foci formation.Genetic and chemical inhibition of the DNA damage response sensor amplified the production of cytokines including Cxcll,Cxcl2 and Ifn-? after particulate matter stimulation in bone marrow-derived macrophages.Similar to that seen in vitro,mice with myeloid-specific deletion of RAD50 showed higher levels of airway inflammation in response to the particulate matter challenge,suggesting a protective role of DNA damage response during inflammation.Conclusions:1.PM2.5 can induce DNA strand breaks and corresponding DNA damage responses in macrophages both in vitro and in vivo models;2.Unrepaired and persistent DNA damage resulted in disruption of DNA damage response lead to elevated inflammatory response through the NF-?B signaling pathway;3.DDR might be a new potential target for interventions of particulate matter-related pulmonary disorders.Part ? RAD50-mediated DNA Damage Response in airway epithelials Particulate Matter-Induced Airway InflammationObjective:To establish both in vitro and in vivo model with PM2.5 treatment in airway epithelials and mice and explore the role of DNA damage response in airway epithelial cells in PM2.5-induced pulmonary inflammation.Methods:Both HBE and Beas2B cells were treated with PM in vitro.Comet assay was used to detect DNA breaks.Q-PCR and ELISA were used to detect the expression of cytokines in macrophages and the culture supernatants,respectively.Western Blot and immunofluorescence were used to detect the expression of specific proteins involved in DNA damage response pathway.In vivo,C57 male mice,aged 6-8 weeks,were instilled intratracheally with PM following the mirin treatment.The total number of inflammatory cells and the number of neutrophils were calculated Expressions of cytokine levels in the lung tissue were determined using real-time PCR.Lung sections stained with hematoxylin and eosin(H&E).were used to detect pathological changes.Results:We found that PM2.5 can induce significant DNA damage in airway epithelial cells in vitro,and initiate a rapid DNA damage response represented by nuclear yH2AX Foci formation.Inhibition of DNA damage response sensor MRN complexes with mirin can significantly inhibit PM-induced elevation of IL6,IL8 and MUC5AC in airway epithelial cells.Similar to the results observed in vitro,mirin treatment in vivo has a significant protective effect on PM-induced airway inflammation.Conclusions:1.PM can induce DNA damage in airway epithelial cells and initiate DNA damage response;2.The inhibition of DDR sensor function in airway epithelial cells can alleviate PM-induced inflammation;...