H1N1 Influenza Virus Infection Induces Ferroptosis In Nasal Mucosal Epithelial Cells

IntroductionAs a major social health issue,influenza virus infection has received more and more attention in recent years with the epidemic of the new coronavirus.The mucosa of the nasal cavity is the first barrier against the influenza A virus;however,a basic understanding of its pathogenesis is yet to be clarified.Ferroptosis is suggested to be at the root of viral infectioninduced inflammation.However,the mechanisms of H1N1-infection-induced death of human nasal epithelial cells(hNECs)remain unclear.For instance,the NRF2 gene regulates the expression of viral mediators and the level of virus entry by regulating cellular antioxidant levels.Although oxidative stress has been associated with the severity of viral infections,the precise mechanisms,including the role of NRF2 in these responses,lack clarity.Ferroptosisdriven diseases can benefit from targeting NRF2 and its downstream targets because of their importance in preventing ferroptosis.L-glutamine(Gln)has been found to induce ferroptosis via the glutaminolysis pathway under amino acid starvation conditions.Because glutaminase plays a key role in ferroptosis driven disease,it is considered a potential therapeutic target.Therefore,through the study of its mechanism,we provide new and attractive therapeutic targets for virus-induced inflammation.Methods1.Culture of nasal mucosal epithelial primary cells,nasal mucosal epithelial stem cells cultured on trophoblast cells NIH3T3 after nasal septal mucosal pancreatic digestion,and then differentiated nasal mucosal epithelial cells were obtained in B-ALITM growth and differentiation medium.2.The establishment of a model of nasal mucosal epithelial cells infected by H1N1 virus was established,and the expanded H1N1 influenza virus was incubated with nasal mucosal epithelial cells at 37℃ for 2 h.It is then cultured at 37℃ after removal of the viral inoculum and divided into groups 0 h,8 h,24 h,and 48 h according to subsequent incubation time.3.Flow cytometry and MTT were used to observe the activity and death of cells after virus stimulation.We used the following to act on hNECs stimulated by the H1N1 virus:ferroptosis inhibitors,such as ferrostatin-1,deferoxamine(DFO),and α-Tocopherol;apoptosis inhibitors,such as Z-VAD-FMK;necroptosis inhibitors,such as necrostatin-1.4.We stained hNECs with 10 μM BODIPY C11 581/591(Cat.No.GC40165,GLPBIO)for 20 min at 37℃ to detect lipid peroxidation after the incubation medium was removed.The cells were washed with DMEM more than twice,digested with trypsin,and resuspended in PBS containing 5%FBS.We ultimately used a flow cytometer to analyze the oxidation of the polyunsaturated butadienyl portion of C11-BODIPY,which caused the fluorescence emission peak to shift from～590 nm to～510 nm.We used the human 4hydroxynonenal(4-HNE)assay kit,the malondialdehyde(MDA)assay kit and reactive oxygen species(ROS)assay kit to measure the lipid peroxidation level in hNECs.5.RNA sequencing.A NanoDrop spectrophotometer(Thermo Scientific)was used to test RNA concentration and integrity in hNECs,and total RNA was isolated from hNECs by using TRIzol Reagent(Invitrogen Life Technologies).The entire library preparation is then completed by purification,lysis,and amplification.The raw sequencing data is filtered and assembled.We performed expression difference analysis,enrichment analysis and cluster analysis to screen out the genes NRF2,KEAP1 and GCLC that were differentially expressed under viral stimulation.Ultrahigh performance liquid chromatography tandem time-of-flight mass spectrometry combined with ultrahigh performance liquid chromatography was employed to analyze the samples for untargeted metabolomics analysis.To identify metabolites,we compared the retention time,molecular weight error by 10 ppm,secondary fragmentation spectrum,collision energy,and other information.The metabolites were identified based on the literature,followed by secondary confirmation.6.RNA interference and transfection technology,we used lentiviral transfection technology to co-culture GCLC overexpression lentivirus,GCLC knockdown lentivirus,KEAP1 knockdown lentivirus,SLC1A5 knockdown lentivirus and corresponding negative control lentivirus nasal mucosal epithelial cells,and observed the interference efficiency with WB.7.We randomly divided 40 mice into 4 groups,nasal drops with virus solution were administered to the experimental group.A normal saline solution was given to the control group.And we administered JHU-083 to mice infected with the H1N1 virus via intraperitoneal injection.Results1.H1N1 Infection Induced Ferroptosis in hNECs.We found that hNECs declined in activity and proliferative capacity with prolonged stimulation and increased viral titers of H1N1.We also found that compared with other cell death inhibitors,ferrostatin-1 significantly attenuated cell death induced by the influenza A virus.The effect of ferrostatin-1 on the viral stimulation of hNECs verified that H1N1 infection induced ferroptosis.2.NRF2-KEAP1 Pathway May Regulate Ferroptosis in hNECs.Sequencing analysis results indicated that genes associated with ferroptosis showed significant differences before and after viral stimulation.We further analyzed the obtained differential genes,screening three genes:NRF2,KEAP1 and GCLC.We detected the expression of ferroptosis-related genes by qPCR after KEAP1 knockdown.We found that the transcription of prostaglandin-endoperoxide synthase 2(Ptgs2),TfR1,ChaC glutathione specific gamma-glutamylcyclotransferase 1(CHAC1),and ACSL4 decreased,whereas the transcription of GPX4 and SLC7A11 increased.3.H1N1-Infected hNECs Induced L-Glutamine Metabolic Reprogramming.After viral stimulation,glutamate and glutamine levels in hNECs increased,as determined by metabolic analysis.After ferrostatin-1 application,glutamine and its metabolites were reduced,except for GSH.4.GCLC May be Involved in the Regulation of H1N1-Induced Ferroptosis in hNECs.MTT assay detected a significant reduction in cell death of hNECs stimulated by the H1N1 virus after GCLC overexpression.We detected the expression of ferroptosis-related genes by qPCR after GCLC overexpression in viral stimulation.We found that the transcription of Ptgs2,TfR1,CHAC1,and ACSL4 decreased,whereas the transcription of GPX4 and SLC7A11 increased.These changes confirmed the involvement of the NRF2-KEAP1-GCLC signaling pathway in ferroptosis in hNECs.5.JHU-083 Attenuated H1N1 Infection-Induced Damage to Mouse Immune System.We administered JHU-083 to mice infected with the H1N1 virus via intraperitoneal injection to find that ferroptosis in the mouse nasal mucosa was significantly reduced,and JHU-083 could reduce the damage caused by the H1N1 virus to the body via the glutamine metabolic pathway.ConclusionThe H1N1 influenza virus can induce ferroptosis via the NRF2-KEAP1-GCLC signaling pathway in hNECs.Besides,the H1N1 influenza virus promotes ferroptosis by causing glutaminolysis in hNECs.By inhibiting ferroptosis caused by H1N1,JHU-083 can reduce the severity of the inflammation.