The Mechanism Of Restraint Stress Increases Mice Influenza Susceptibility And The Improvement Of Carnosine

The susceptibility of host plays important role in affecting thegeneration and development of infection diseases. During pathogens infection, hosts through direct anti-infection or increasing disease tolerance offer protection. When the host is unable to keep fitness by using these two ways, it will make host more easily to be destroyed, and we call this “susceptible host”. It’s reported that newborns, elderly, the sick and individuals presenting fatigue or stress are “susceptible population” of influenza. These “susceptible population” infected with influenza have higher mortality, but the underlying mechanisms that how these factors increased influenza “susceptibility” are still unknown. Therefore, exploring the mechanisms of influenza “susceptibility” and how to regulating host “susceptibility” paly important roles in the prevention of influenza infection. Restraint stress can activate hypothalamic pituitaryadrenal(HPA) axis to induce high level of corticostone(CORT), and CORT can regulate the innate immunity to prevent pathogens infection. Our previous studies found that restraint stress increased the “susceptibility” of influenzain virus fected mice. Therefore, the current study established “susceptibile” animal and cell models with restraint stress and stress hormone CORT to investigate the important roles and mechanisms of CORT on restraint stress increased influenza “susceptibility”.Firstly, we studied the effects of stress hormone CORT induced byrestraint stress on influencing the “susceptibility” of influenza virus infected mice. Mice were restrained stress within a 50 m L centrifuge tube for 18 h. Then, mice were infected with 2×LD50 H1N1 virus on the 3rd day after restraint stress. After stress,RU486(25 mg/kg) or CORT(1 mg/kg) were injected intraperitoneally(i.p.) to mice for 3 consecutive days. The survivals and weight of mice were measured for 21 days or mice were sacrificed on the 4th dayafter virus infection.The morbidity and mortality, NP protein expression and lung histopathologic changes of mice were measured. Results found that CORT treated mice had higher morbidity and mortality, increased viral replication(NP protein expression levels) and enhanced lung histopathologic damages after virus infection. RU486 obviously alleviated those symptoms, which suggestedthat CORT treated mice hadhigher “susceptibility”. These results suggested that CORT increased the “susceptibility” of influenza virus infected mice.Secondly, we studied the effects of CORT on mitochondrial antiviral signaling(MAVS) pathway. Virus infection can induce MAVS activation to form large aggeregates, which will activate IRF3 and NF-κB to produce type I interferons(IFN-I)andantiviral proteins(AVP) so as to inhibit influenza infection synergistically. Therefore, this study investigated whether restraint stress increased influenza “susceptibility” was associated with MAVS signaling or not. KM mice were randomly divided into 4 groups, including controlgroup, virus group, stress+virus(model) group, and RU486(RU486 25mg/kg+stress+virus)group. Mice were infected with 2×LD50 H1N1 virus on the 3rd day after restraint stress. After stress, RU486(25mg/kg)were injected intraperitoneally(i.p.) to mice for 3 consecutive days. All mice were sacrificed on the 4thdayafter virus infection. MAVSand MAVS aggeregates protein levels were measured by western blot and SDD-AGE. Compared with virus group, stress significantly downregulated protein levelsof MAVS and MAVS aggeregates, and RU486 increased those two protein expressions. These results suggested that stress hormone CORT couldinhibit MAVS protein and activity to decrease antiviral signaling. In order to further confirm wether CORT decreased IFN-β response to increase mice influenza “susceptibility” was regulated by NF-κB or not, we used NF-κB inhibitor BAY11-7082 to examine the regulating effects of NF-κB signaling pathway on IFN-β response in A549 cells with CORT and virus exposure. Results found thatcompared with CORT treated alone, the protein expression of NF-κB in A549 cells treated with CORT and virus exposurewas significantlyincreased, andpro-inflammatory factors(TNF-α and IL-6) were also promoted. Compared with CORT and virus group cells,BAY11-7082 significantly lowered the protein expressions of NF-κB, TNF-α and IL-6, suggesting that CORT could directly activate NF-κB signaling to induce inflammatory response.However, BAY11-7082 VII administration had no effectson NP or IFN-βprotein expressions in A549 cells with CORT and virus exposure. These results proposed that IFN-βresponse were regulated by MAVS signaling pathway but not NF-κBpathway. The aboveresults indicated that stress hormone CORT could inhibit the MAVS anti-viral signaling pathway and also directly activate NF-κBsignaling pathway to induce inflammatory factors, which ultimately promoted the influenza “susceptibility”.Thirdly, weakened MAVS anti-viral signaling pathway is the pivitol mechanism of restraint stress enhanced influenza “susceptibility”. Influenza virus could through ubiquitin-mediated protesome pathway to regulatesignal transduction of host innate immunity, and mitofusin-2(Mfn2) could inhibit MAVSanti-viral signaling pathway to reduce IFN response. So, in this part from effects of restraint stress and CORT on genome expression sequencing of mice, and the ubiquitination of MAVS to investigate the role of Mfn2 in CORT regulating MAVSubiquitination.(1) Using genome expression sequencing of mice exposured with CORT or restraint stress to measure innate immunity and MAVS anti-viral signaling genelevels. Results showed that CORT significantly enhanced Vdac1, Irf3, Mfn2 and Jungene levels, especially Mfn2, but had noinfluence on MAVS gene levels. Next, we used RT-PCR and western blot to measure the gene and protein expression levels of Mfn2 and MAVS in mice and A549 cells with restraint stress or CORT. Vitro and vivo results demonstrated that CORT enhanced the gene and protein expression levels of Mfn2 andhad no influence on MAVS gene expression, but significantly inhibited MAVS protein expression. These results suggested that Mfn2 may paly a role in CORT-enhanced viral“susceptibility”.(2) Using p IRES2-EGFP-Mfn2 plasmid transfected A549 cells to explore the effects of Mfn2 on the“susceptibility” of virus infection. PIRES2-EGFP-Mfn2 plasmid transfected A549 cells treated with CORT and virus exposure, the protein expressions of NP, IFN-β and IFITM3 were measured.Resultsdemonstrated that overexpression of Mfn2 increased NP expression, but inhibited protein expressions of IFN-β and IFITM3 in A549 cells treated with CORT and virus.These results suggested that CORT increased Mfn2 to inhibit IFN-β responses.(3) In order to examine the role of Mfn2 in CORT regulating MAVS VIII pathway to inhibit IFN-β responses, we used Co-IP to confirm the interaction of Mfn2 and MAVS. Result found that Mfn2 interacted with MAVS in mice. Then,using Co-IP and western blot assays to measure the effects of stress hormone CORT on the lung ubiquitination of MAVS in virus infected mice. Resultsfound that compared with virus goup mice, restraint stress and CORT increased the ubiquitination of MAVS in lung of mice infected by H1N1 virus. These results indicated that Mfn2 may through promotting the ubiquitination of MAVS to inhibit MAVS anti-viral signaling pathway.(4) Using p IRES2-EGFP-Mfn2 plasmid transfected A549 cells andexamingthe effects of Mfn2 on the ubiquitination of MAVS in cells with CORT and virus exposure.Result demonstrated that overexpression of Mfn2 promotedthe ubiquitination of MAVS in A549 cells treated with CORT and virus, suggestingthat CORT elevated Mfn2 to promote Mfn2-mediated ubiquitination of MAVS.Finally, wemeasured the effects and mechanisms of Mfn2 regulated MAVS signaling pathway in carnosine, a natural small molecule active peptide, protecting influenza infected mice treated with restraint stress. Our previous studies have established the viral “susceptibile” mice modelwith restraint stress, and using this model we have evaluated the protective effects of many natural products on influenza infection. We have found that carnosine had anti-stress and anti-oxidation effects, and it alsocould increase the survivals of influzenza virus infected-mice. But carnosine decreases influenza “susceptibility”is related with Mfn2 regulating MAVS pathway or not is still unkown.(1) The lung index, virus replication and lung histopathologic changes of mice treated with restraint stress and virus exposure were measured. Results showed that carnosine significantly decreased lung index and virus replication, and alleviated the lung histopathologic damages. These results suggested that carnosinehad protective effects on influenza infected mice with restraint stress.(2) Examiningcarnosine decreased influenza susceptibility was related with MAVS pathway.Lung protein expressions of IFN-β, IFITM3, MAVS, p-IRF3,NF-κB and p-IκB were measured in mice treated withrestraint stress and virus exposure. Results found that carnosine enhanced IFN-β and IFITM3 protein expression, increased MAVS and p-IRF3 protein levels, meanwhile decreased NF-κB and p-IκB protein expressions. These results demonstrated that carnosine activated MAVS pathway to promote IFN-β response so as to decrease influenza “susceptibility”.(3) Exploring the effects of Mfn2 regulating MAVS pathway on carnosine decreasing host influenza susceptibility. Lung protein expressions of MAVS, MAVS aggregates and Mfn2 were measured in mice treated withrestraint stress and virus exposure. Results found that compared with model group mice, carnosine obviously enhanced lung protein expressions of MAVS and MAVS aggregates, and decreased Mfn2 protein level in mice. These results suggested that carnosine through Mfn2 could enhance MAVS anti-viral signaling pathway so as to decrease influenza “susceptibility”.In summary, restraint stress enhances the influenza “susceptibility” by CORT up-regulating Mfn2 mediated MAVSubiquitination to inhibit IFN response.Carnosine can downregulate Mfn2 to improve MAVS mediated IFN responses ininfluenza infected mice treated with restraint stress.