Pathogenic Mechanism Of NLRP6 In Brucella And Influenza Virus Co-infection

Objective:To establish coinfection animal models of Brucella-Influenza A virus,explore the synergistic pathogenesis of acute lung injury and cytokine storm caused by Brucella and influenza virus coinfection,screen the characteristic indicators of acute lung injury and cytokine storm,and provide candidate targets for early prevention,diagnosis and clinical intervention.Methods:Influenza A（H1N1）virus strain PR8（A/PR/8/1934）and Brucella suis vaccine strain S2 were selected as the challenge strains,and 4-week-old C57BL/6N mice were selected as the model animals.The model was established by simulating the pulmonary delivery vaccination route of aerosol infection.The establishment effect of the model was evaluated by observing the clinical manifestation,weight change,mortality,q-PCR and tissue TCID₅₀,plate count and ELISA.Results:The coinfection model of Brucella-Influenza A virus with aerosol lung infection was established by using a handheld liquid aerosol lung delivery device.The mortality of infected mice increased（80%）,and the progression of the disease worsened.The count of Brucella S2 in the coinfection group was（7.974±0.2242）CFU/g,which was significantly higher than the single infection group（6.643±0.4695）,and the difference was statistically significant（P<0.05）;The results of tissue TCID₅₀（6.490e±0.8367）and q-PCR（7.673±0.9367）in coinfection group were significantly lower than single infection group TCID₅₀（9.190e±2.074）and q-PCR（13.70±2.589）,and the difference was statistically significant（P<0.05）.Pathological section showed that the injury of lung tissue was aggravated.ELISA results showed a general increase in inflammatory cytokines:Compared with the negative control group,the coinfection group had higher IFN-γ（10682±3088）,IL-6（75891±11518）,IL-1β（731.6±126.1）,IP-10（7953±548.02）,IL-18（294715±124928）,TNF-α（6696±1593）increased significantly,the difference was statistically significant（P<0.05）.Compared with single infection group,IL-18 and TNF-αincreased significantly,the difference was statistically significant（P<0.05）.Compared with the negative control group,IL-10 in the coinfection group decreased significantly（767.2±95.15）,and the difference was statistically significant（P<0.05）.Conclusion:In this study,a mouse model of Brucella-Influenza A virus coinfection with liquid aerosol lung infection was successfully established by using a handheld liquid aerosol lung delivery device.To understand the immunotoxicology reaction of C57BL/6N mice infected with Brucella influenza A virus aerosol.Infected mice had increased mortality,worsened disease progression,more bacteria in the lung,aggravated pathological injury,and stimulated the immune response of high concentrations of inflammatory cytokines.Objective: To explore the functions of various cells in lung tissue in the pathogenic mechanism of Influenza virus-Brucella coinfection.Methods: Using 10×Genomics single cell transcriptome sequencing technology sequenced,to analyze the transcriptome of all cells in the lung tissue of coinfected mice.Results: the lung cell maps of healthy,Influenza virus infection,Brucella infection and coinfection mice were obtained by dimensionality reduction clustering.The analysis of differential gene and pseudo time series showed that compared with the control group,the proportion of neutrophil subtypes increased and the proportion of macrophage subtypes decreased in the two single infection groups;In the coinfection group,the proportion of neutrophil subtypes increased significantly,and the proportion of macrophage subtypes decreased significantly,and the change range was greater than that in the single infection group.Then,the expressions of CCL3,cxcl2,CCl4,S100A8,retnlg and NGP in neutrophils in coinfection group were significantly changed,and the expression of HBB-BS in macrophages in coinfection group was significantly changed.Finally,combined with the characteristics of cytokine spectrum in the first part,differences of molecular expression patterns related to IL-18 and TNF-α between coinfection and single infection were analyzed.Conclusion: After of influenza virus-Brucella coinfection in mouse lung,the proportion of neutrophils and macrophages in lung tissue change significantly,which may be one of the mechanisms of immune imbalance and fatal immune injury caused by coinfection.Screening the significantly changed genes in neutrophils and macrophages,we used new research ideas on the mechanism of coinfection and intervention targets for the pathogenesis of coinfection,and provided new strategies for its treatment.Objective:To clarify the function of NLRP6 in the early stage of coinfection of Influenza A virus and Brucella,for the first time physical vector hypothesis was used to analyze the bi-directional regulation of NLRP6 in the anti-infective immune network.And the potential of NLRP6 was revealed in intervention and diagnosis of infectious acute lung injury.Methods: The inclination of the body to resist different pathogens was evaluated by detecting the viral and bacterial loads;the immune imbalance in the early stage of coinfection was evaluated by detecting the expression of NLRP6;through building NLRP6-/-mice model,to explore the bi-directional regulation of NLRP6 in anti-infective immunity.Results: On the third day of co-infection,WT mice showed obvious symptoms of lung injury,overexpression of NLRP6,excessive increase of cytokines such as TNF-α and IL-18,significantly increased bacterial load and significantly decreased viral load;on the contrary,bacterial load significantly decreased but viral load significantly increased in NLRP6-/-mice.Conclusion: The bi-directional regulation function of NLRP6: the coinfection of Influenza A virus and Brucella enhances the ability of virus clearance by up-regulating NLRP6,but neglects anti-bacterial immunity.And the deficiency of NLRP6 makes the body unable to resist viral infection,although bacteria are significantly inhibited.It is suggested that early detection of the expression of NLRP6 can well reflect the current inclination of immune system to resist different pathogens,and its excessive increase and decrease may induce the severe lung injury and poor prognosis.Therefore,NLRP6 may play a key role in early diagnosis and clinical intervention.