Characterization Of Immune Response To Inhalational Infection Of Methicillin-resistant Staphylococcus Aureus In Mice

Objective: Methicillin-resistant Staphylococcus aureus(MRSA)pneumonia causes a high mortality rate and is associated with early excessive inflammation of unknown mechanism.MRSA has a powerful virulent secretory system to evade the host’s immune response and may even promote excessive inflammatory responses.Hence,the regulation of host immune response,especially the key mechanism of controlling inflammation,is crucial to successfully resist MRSA pneumonia.However,compared with the studies focusing on the mechanism of bacterial virulence,the studies focusing on the mechanism of host immune response are limited.Therefore,this study focused on the immune response characteristics of mice after infected with MRSA via aerosolized intratracheal(i.t.)inoculation,aiming to discover the key targets of host regulation of immunity and inflammation,and provide a theoretical basis for the development of non-antibiotic therapies.Methods: 1.PCR and second-generation sequencing were used to screen and confirm the strains.2.A hand-held liquid aerosol delivery device was used to convert bacterial liquids of different concentrations into aerosols and deliver them directly from the trachea to the lungs to construct a mouse infection model.Survival curve,bacterial load and histopathological changes were detected to evaluate the model.3.Luminex 200 liquid suspension chip system was used to detect the changes of 18 cytokines in serum and bronchoalveolar lavage fluid(BALF)of mice at different time points after infection.Flow cytometry was used to detect the change trend of main cell groups in lung tissue of mice at different time points after infection.4.RNA seq was used to sequence the transcriptome of mouse lung tissue at different time points after infection,and sequential analysis was conducted to explore the key regulatory mechanisms: based on the obtained Differential expression gene(DEGs),differential gene expression patterns were analyzed by Mfuzz temporal clustering;Weighted gene co-expression network analysis(WGCNA)was used to obtain gene coexpression modules.On the basis of WGCNA,the core genes and Protein–protein interactions(PPI)in the modules were obtained using Cytoscape software and STRING database,respectively.Biological processes and signaling pathways involved in DEGs were enriched and analyzed using Gene ontology(GO)and Kyotoencyclopedia of genes and genome(KEGG).Results: 1.A mouse pneumonia model infected with MRSA stain ATCC-BAA 1556 was successfully constructed.Three repeated experiments showed that the higher the infection concentration,the more mice died.Mice with infection concentration of 2-4×108 CFU died,and the death time was concentrated within 48 h.Mice infected at 1×108 CFU did not die,but showed disease phenotypes.2.1×108 CFU was selected to construct a stable sublethal model,and the bacterial load in blood and the bacterial load and pathological changes in heart,liver,spleen,lung and kidney were detected: the bacterial load in blood peaked at 24 hpi;while the bacterial load in heart,liver,spleen and kidney peaked at 12 or 24 hpi,and all were cleared by the host at 96 hpi.The bacterial load in the lung showed an exponential decrease from 0 to 96 hpi and was cleared by the host at 168 hpi.The lung tissues showed obvious lesions including alveolar wall thickening,inflammatory cell infiltration,and hemorrhagic edema after infection,which were most severe at 24-72 hpi.The liver and spleen tissues showed a small amount of pathological changes,while the heart and kidney tissues did not show obvious pathological changes throughout the infection.3.Cytokine secretion in BALF of infected mice increased,and mostly reached the peak growth at 12-24 hpi,and began to decline significantly at 96 hpi.The expression pattern of cytokines in serum was similar to that in BALF,but the number and peak value of cytokines were significantly smaller than those in BALF.4.After infection,a large number of neutrophils and inflammatory monocytes were recruited into the lung tissue,and their proportion increased significantly,reaching the peak growth at 24 hpi and 48 hpi,respectively,and the neutrophils had recovered at 96 hpi with no difference from 0 hpi.The percentage of NK cells,dendritic cells,B cells,T cells,and epithelial cells showed a significant decrease,especially at 24 hpi and 48 hpi.The proportion of macrophages only decreased slightly at 12 hpi,and there was no difference at other time points.The proportion of endothelial cells did not change significantly.5.Transcriptome combined phenotypic analysis showed that inflammation was initiated rapidly 12 hpi,during which the IL-17 pathway mainly mediates inflammation response and immune defense.The main stage of host inflammatory response occurs at 24 and 48 hpi,and the regulation of interferon activation and macrophage polarization plays an important role in the control of inflammation balance at this stage.At 96 hpi,cellular proliferation processes associated with host repair,as well as adaptive immune responses and complement system responses involving C1 q molecules,were observed.At the same time,several potential functional genes involved in balancing host anti-inflammatory and pro-inflammatory responses were identified during the above infection and repair processes.Conclusions : In conclusion,this study established a mouse pneumonia model infected with MRSA via aerosolized intratracheal(i.t.)inoculation for the first time,systematically revealing the host response characteristics after inhalation infection from the perspective of protein level,cellular level and gene level respectively,showed the development process of inflammation and the body’s balance regulation between antiinflammatory and pro-inflammatory response.The post-infection inflammation was divided into three stages: 1)0-12 h was the initial stage of inflammation,and IL-17 pathway played an important role in this stage;2)12-48 h is the stage of inflammatory response,and the regulation of interferon response and macrophage function may be the key to the balance of inflammation;3)48-96 h is the stage of inflammation recovery,involving multiple effects of cell proliferation,complement system and adaptive immune response.This study provides new insights into the mechanisms of immune response for successful host defense against MRSA pneumonia,as well as valuable research targets for immune regulation against MRSA pneumonia,which will contribute to the development of new therapeutic strategies for MRSA pneumonia.