The Role Of Short Chain Fatty Acids In Pulmonlary Infection Caused By Porcine Circovirus Type 2

Porcine circovirus type 2(PCV2)is an important pathogen that poses a threat to the world’s pig industry.PCV2 infection with monocytes/macrophages can lead to immune suppression in pigs,leading to secondary infections with other pathogens and the formation of porcine respiratory disease syndrome,which seriously affects the growth and development of pigs.As the largest immune organ in the body,the gut contains a large number of bacteria.The metabolites produced by the bacteria can be absorbed and transported to distant organs through the intestinal epithelium or affect the function of immune cells in the gut,thereby affecting other organs,such as the lungs.Therefore,this experiment first selected C57BL/6J mice to construct a PCV2 infection model with pulmonary inflammation as the main factor.Based on this,the effect of infection on short-chain fatty acids(SCFAs)in the cecum was detected,and the effects of different short-chain fatty acids on the lungs of PCV2-infected mice were studied by supplementing short-chain fatty acid salts;On this basis,macrophage(RAW264.7)infected with PCV2 were incubated with different types of short-chain fatty acid salts to explore the mechanism of action of short-chain fatty acids on lung immune damage caused by PCV2.Key short-chain fatty acids that resist PCV2 function were screened through RNA-seq sequencing analysis.The main content and results are as follows:1.Construction and identification of a mouse pulmonary infection model infected with PCV2This experiment first administered PCV2 to mice through the nasal drip,successfully constructing a model with pulmonary infection as the main symptom.On this basis,we studied the effect of PCV2 infection on the gut microbiota metabolite SCFAs.The HE staining results showed that the infected mice had ruptured alveoli,bleeding in the pulmonary interstitium,significantly thickened alveolar septa,fibrosis around the bronchi,and some individuals had inflammatory cells filled in the bronchial air.Immunohistochemical results showed that the virus was mainly distributed in macrophages in the lungs.The above results indicate that infection with PCV2 has a significantly destructive effect on the lung tissue structure of mice,and nasal instillation can create a lung infection model characterized by inflammatory cell infiltration.2.Effects of PCV2 infection on the gut and gut microbiota metabolites of short chain fatty acids in miceIn the model of PCV2-induced lung infection in mice,severe intestinal inflammation was observed.Inflammatory cell infiltration was found in the ileum,cecum,and colon of mice in the infection group.The villi of the ileum and colon became shorter,the number of goblet cells in the cecum decreased,and the upper edge of the villi was defective.Gas chromatography was used to detect the short-chain fatty acids in the cecum content.The results showed that the content of acetic acid,propionic acid,and butyric acid in the cecum of mice infected with PCV2 decreased.This result showed that the lung infection of mice caused by PCV2 would lead to the imbalance of the production of short-chain fatty acids acetate,propionate,and butyrate.3.The effect of short chain fatty acids on PCV2 induced lung infection in miceIn order to explore whether the disordered short-chain fatty acids have an effect on the lung inflammation caused by PCV2,mice were infected with PCV2 after one week of gavage of acetate,propionate,and butyrate respectively.The HE staining results showed that compared to mice infected only with PCV2,the three short-chain fatty acid treatment groups showed reduced lung bleeding,weakened pulmonary septal hyperplasia,and reduced inflammatory cells in the lungs.However,the sodium butyrate treatment group still had more severe lung inflammation.The expression level of lung-related inflammatory factors was detected by qPCR,and the results showed that IL-1 was present in the three short-chain fatty acid salt-treated groups α The expression was downregulated,indicating that supplementation of short-chain fatty acid salts could reduce the damage of lung inflammation induced by PCV2 in mice,especially acetate and propionate.In addition,to investigate the effects of different short-chain fatty acid salts on the function of PCV2-infected macrophages.4.The effect of short chain fatty acids on the function of monocytes/macrophages infected with PCV2To investigate the effect of different short-chain fatty acid salts on macrophage proliferation,CCK8,and Edu staining methods were first used to detect the effect of gradient concentrations of different types of short-chain fatty acids on RAW264.7 cell proliferation.The results showed that low concentrations(0.25 m M-1 m M)of short-chain fatty acids can promote the proliferation of RAW264.7 cells,while high concentrations(4m M-16 m M)of short-chain fatty acids can actually inhibit the proliferation of RAW264.7cells.To investigate the effect of short-chain fatty acids on the replication of PCV2 in macrophages,an absolute quantitative method was used to detect the content of PCV2 in RAW264.7 cells.The results showed that all three short-chain fatty acid salts could inhibit the replication of PCV2 in macrophages.Using the qPCR method to detect the expression levels of inflammatory factors,chemokines,and macrophage polarization-related genes in macrophages of different treatment groups.The results showed that acetate could increase the expression of CCL5 in RAW264.7(P<0.001)and promote the expression of MRC1 and iNOS,the polarization marker genes of M2macrophages(P<0.05);Propionate can upregulate the expression of IL-27 and CCL5(P<0.05);Butyrate makes IL-1α,IL-27 expression was upregulated,while CXCL10 and CXCL11 expression were downregulated(P<0.01),promoting the upregulation of M1 macrophage polarization marker gene CD86 expression(P<0.01).The above results show that three kinds of short-chain fatty acids have different emphases on the regulation of RAW264.7 cells.Among them,acetate promotes the differentiation of monocytes to inhibit inflammation,propionate regulates the anti-virus of macrophages themselves and chemotaxis of other anti-virus immune cells,and butyrate promotes the anti-inflammatory effect of monocytes.5.Screening of key short chain fatty acids for regulating the immune function of monocytes/macrophages infected with PCV2On the basis of previous studies,in order to further explore the key short-chain fatty acids that play an anti-PCV2 role in acetate and propionate,RNA seq sequencing was performed on the cells infected with PCV2 after acetate and propionate treatment.The results of differential gene analysis showed that the functions of acetate mainly focused on the functions related to the deformation and migration of macrophages;The effect of propionate on RAW cell function is mainly focused on genes that can affect inflammation,including the cell’s response to interferon β Response to cytokines,innate immune response.Under the action of sodium propionate,the signal pathways of biological functions related to viral infection are enriched in: the TNF signal pathway,Toll-like receptor signal pathway,NF-kappa B signal pathway,cytokine receptor interaction,the interaction of the viral protein with cytokines and cytokine receptors and so on.Based on the above experimental results,propionate is a key short-chain fatty acid that promotes monocyte resistance to PCV2 infection.The upregulated genes include Map2k6,H2-DMb2,H2-Ob,Tnfsf14,Il4 ra,and downregulated genes such as CXCL2,Traf1,Tnfsf18.Based on the above research results,it can be concluded that nasal administration of PCV2 can cause severe lung and intestinal inflammation in mice,and lead to a decrease in the production of acetic acid,propionic acid,and butyric acid in short-chain fatty acids;Respectively supplement acetate,propionate and butyrate,of which acetate and propionate can significantly alleviate lung inflammation in mice.The possible mechanism is that acetate induces macrophages to polarize toward M2 macrophages that inhibit inflammation and inhibit the production of inflammatory factors;Propionate promotes the secretion of regulatory inflammatory factors by macrophages,thereby inhibiting the development of inflammation;In vivo and in vitro experiments combined with transcriptome sequencing results suggest that propionate is the key short-chain fatty acid to promote the anti PCV2 infection of macrophages..