Therapeutic Effects Of Disulfiram In Severe Cytomegalovirus Pneumonia In Immunocompromised Mice

BackgroundHuman Cytomegalovirus(HCMV),a member of theβ-herpesvirus subfamily,is an opportunistic pathogen causing disease with an infection rate of over 90%in healthy humans.However,in immunocompromised individuals,CMV infection may lead to high morbidity and mortality,particularly in those patients who undergo lymphocyte depleting therapies,such as with allogeneic hematopoietic cell transplant and solid organ transplant,or HIV infection,despite the availability of antiviral therapies.HCMV is reactivated and enters the lytic replication phase,which cause several diseases in immunocompromised hosts,such as hepatitis,pneumonitis,colitis and neurological disorders.Studies have reported that the lung is a prominent site of CMV infection,no matter acute infection,latency or reactivation.Ganciclovir is currently used for prophylaxis and treatment of CMV infection,but is limited by its drug resistance,bone marrow suppression,and carcinogenesis.Therefore,there is an urgent need to develop new strategies to treat pneumonia caused by CMV.Disulfiram is a FDA approved drug for the treatment of alcoholism,with the advantages of safety,high efficiency and low toxicity.In recent years,it has been found to have some anti-inflammatory and antiviral effects.DSF is effective to inhibit the inflammatory response in several animal models,including sepsis,gouty arthritis,and SARS-Co V 2 infection.The NOD-like receptor 3(NLRP3)inflammasome plays a key role in innate immunity by helping to produce pro-inflammatory cytokines,which are associated with infection,inflammation,and the autoimmune processes.It has been shown that NLRP3 inflammasome is involved in the pathological progression after CMV infection.In addition,DSF can inhibit NLRP3 inflammasome activation and suppress gout inflammation.Therefore,we propose that DSF may have a therapeutic effect on CMV-infected pneumonia and may be involved in inhibiting the activation of NLRP3 inflammasome.To prove this concept,we apply murine CMV(MCMV)as a useful model which shares high similarity to HCMV.ObjectivesThe purpose of this study is to investigate the protections of DSF in mice against lethal MCMV pneumonia as well as the possible underlying mechanism,may throw new light on strategy against HCMV pneumonia.Methods1.The effect of disulfiram on MCMV-infected BMDMs:Mouse bone marrow cells were isolated and induced into mature macrophages with M-CSF.The morphological characteristic were observed and the cells were detected the expressions of macrophage surface markers CD11b and F4/80;Cell proliferation and toxicity assay were estimated by Cell Counting Kit-8 and Calcein/PI cell activity and cytotoxicity test kit.Expression of inflammatory cytokines in BMDMs was detected by RT-PCR and ELISA;Cell supernatants were collected for viral titer assay.2.The effect of disulfiram on MCMV infectious pneumonia:Mice were randomly divided into 5 groups(Control,MCMV,DSF-50,DSF-100 and GCV-60),which were pretreated for 2 days.Mice were anesthetized with isoflurane and inoculated intranasally with 2×10~5 PFU of MCMV in a total volume of 40μL.Control mice were administered with an equal volume of normal saline.After infection,mice were weighed and survival of mice was monitored for up to 30 days post infection or until death.The pathological changes were determined by histological analysis.Inflammatory cytokines were detected by RT-PCR and ELISA.Recruitment of leukocytes into the lung was detected by FACS.Fibrosis-associated proteinsα-SMA and collagen I were revealed by WB and immunofluorescence.3.Molecular mechanism of disulfiram in reducing MCMV infectious pneumonia:NLRP3 and ASC in lung tissues of each group were detected by immunofluorescence;The NLRP3 inflammasome signal pathway related protein in lung tissue and BMDMs was detected by western blotting;DCFH-DA kit was used to detect the production level of ROS in BMDMs.Results1.After 7 days of M-CSF induced treatment of mouse bone marrow cells,most cells adhered to the wall and grew,showing a typical macrophage morphology in vitro.The number of CD11b~+F4/80~+cells in the induced cells was up to 89%,indicating that most cells were successfully induced into macrophages;The results of CCK8 kit and calcein/PI cell activity and cytotoxicity test kit showed that the experimental concentration of DSF had no significant effect on the activity of BMDMs.DSF treatment significantly reduced the level of pro-inflammatory cytokine expression and decreased viral load in MCMV-infected BMDMs.2.After pretreatment of MCMV-infected mice with DSF and GCV,DSF or GCV treatments can significantly prolong the survival and decrease weight loss of mice compared with MCMV infected group;Histological analysis of the lung tissue confirmed infiltration of leukocyte cells,increased alveolar tissue,and decreased alveolar airspace in MCMV-induced pneumonia mice model.Compared to the model group,administration of DSF clearly ameliorated the lung damage induced by MCMV;The virus titer of BALF showed that both DSF and GCV treatment reduced the virus load;Compared with MCMV-infected group,the results of RT-PCR and ELISA in vivo showed that the DSF treatment significantly reduced the levels of pro-inflammatory cytokines(TNF-α,MCP-1,IL-1β)but enhanced anti-inflammatory cytokines(IL-10)expression,especially the high-dose DSF treatment group;The results of flow cytometry showed that DSF treatment could significantly reduce the infiltration of immune cells;Western blotting and immunofluorescence showed that MCMV infection induced high expressions of fibrosis related proteins,such asα-SMA and collagen I,whereas DSF treatment significantly reduced their expressions.3.Similar to the in vivo results,MCMV induced in vitro NF-κB signaling and NLRP3 inflammasome activation,indicated by the upregulated expressions of p NF-κB(pp65),NLRP3,ASC,caspase-1,and IL-1β,which were attenuated by DSF treatment;DCFH-DA kit results showed that DSF treatment significantly reduced ROS production.Conclusions1.Mouse primary macrophages were successfully isolated and cultured from mouse bone marrow cells,which can be used in subsequent experiments;Different concentrations of DSF treatment did not show any cytotoxicity on BMDMs.DSF treatment significantly reduced the level of pro-inflammatory cytokine expression and decreased viral load in MCMV-infected BMDMs.2.DSF treatment significantly prolonged the survival of mice,improved lung lesions and reduced virus load.DSF treatment inhibited lung inflammatory responses,including reducing pro-inflammatory cytokines(TNF-α,MCP-1,IL-1β),upregulating anti-inflammatory cytokines(IL-10)and lowering the accumulation of leukocytes in the lung.It also can reduce pulmonary fibrosis.3.DSF therapeutic mechanism may work by inhibiting the activation of NF-κB/NLRP3 signaling pathway in macrophages,thereby reducing lung inflammation and promoting lung tissue repair in MCMV-infected mice.