Experimental Study On The Regulation Of NLRP3 By Brucella Ovis Via Caspase-4

Brucellosis is a zoonotic infectious disease widely spread worldwide,and the prevalence of this disease seriously threatens the development of the farming industry and the safety of human public health.Brucella is a gram-negative intracellular parasitic bacterium.After Brucella invades the animal body,it will attack the macrophages in the host and survive and reproduce in its cells.Studies have shown that the genomes of most smooth(S)type Brucella as well as lipopolysaccharide(LPS)are trapped by multiple pattern recognition receptors(PRRs)within host cells to activate intracellular inflammatory responses.Brucella ovis(B.ovis)is a rough(R)type of Brucella whose molecular mechanism for activating the target cell inflammatory response is not well defined.This topic intends to unfold the process of performing transcriptomic sequencing on total cellular RNA extracted from RAW264.7 macrophages infected with B.ovis to bioinformatically analyze the sequencing results,mining out inflammatory response pathways and related differentially expressed genes in B.ovis infected cells,q RT-PCR validation and silencing of selected genes using RNA interference(RNAi),the effect of silencing and the effect on inflammatory pathway related proteins were detected by Western blot,and the changes in inflammatory cytokines were detected by ELISA to explore the molecular regulatory mechanism of inflammation in B.ovis activated RAW264.7 macrophages.The specific research methods are as follows:1.Transcriptomic analysis of expression profiles of B.ovis infected RAW264.7 cells and mining of differentially expressed genes of inflammatory pathwaysIn this experiment,B.ovis infected mice RAW264.7 macrophages according to the MOI value(100:1)for 24 h,and extracted the total RNA of the cells,transcriptomic sequencing was performed,and the sequencing data were subjected to bioinformatics analysis.We utilized Gene Ontology(GO)and Kyoto Encyclopedia of genes and genomes(KEGG)functional enrichment analyses.Go analysis results identified a total of 337 genes that were upregulated and 264 genes that were downregulated in B.ovis infected compared with mock infected;36 differentially expressed genes were included in the cellular inflammatory response(GO: 0006954)(p < 0.01),of which 31 were up-regulated and 5 down regulated;the results of KEGG analysis found that the pathways related to inflammatory response were Toll-like receptor signaling pathway,TNF signaling pathway,NOD-like receptor signaling pathway,NF-κB signaling pathway,IL-17 signaling pathway(p < 0.01).Combined GO and KEGG analysis results,we found that the expression level of Cysteinyl aspartate specific proteinase-4(Caspase-4),which is annotated to NOD-like receptor signaling pathway,was significantly upregulated upon B.ovis stimulation,and this gene affected the expression level of NLRP3 inflammasome by activating the noncanonical inflammasome in cells,which in turn promoted cellular inflammation.We validated 7 transcripts in the Caspase-4 gene by q RT-PCR,all with elevated expression,fold change consistent with the prediction,and highly significant differences(p < 0.01).2.Revealed that B.ovis regulated NLRP3 and thereby mediated IL-1β,TNF-α,and IL-6 secretion in RAW264.7 cells via caspase-4After B.ovis infected mouse RAW264.7 cells,a time gradient was set to extract the total protein of the cell samples,and the expression levels of the corresponding Caspase-4 and NLRP3 proteins in the cells at different time points were detected by Western blot,screening RAW264.7 cells for Caspase-4 as well as the time point with the highest expression level of NLRP3 after B.ovis infection.We set four collection time points of 6 h,12 h,18 h,24 h for total protein extraction in mock infected group and B.ovis infected group in this study.Western blot showed that Caspase-4 and NLRP3 protein expression levels were maximal at the same time point(18h post challenge)and were greatly different from the control at the same time point(p < 0.01),and there was no significant change in Caspase-4 protein expression levels after 18 h and a trend toward a significant decrease in NLRP3 protein expression after 18h(p < 0.01).Using transcripts from Caspase-4 to design and synthesize small interfering RNA(si RNA),RAW264.7 cells were transfected with FAM tagged si RNA for four time points of 6 h,12 h,18 h and 24 h,and the coverage of fluorescence in the cells was observed under an inverted fluorescence microscope to optimize the transfection time.After determining the optimal transfection time,RAW264.7 cells were transfected with si RNA to achieve maximum transfection efficiency time B.ovis(MOI value 100:1)was added and total cellular protein was extracted 18 h after challenge,while cell supernatants were collected.The expression levels of Caspase-4 and NLRP3 proteins in cells were detected by Western blot;The supernatants of different fractions were assayed for the secretion of IL-1β,TNF-α,and IL-6 cytokines by ELISA.The highest percentage of cells with fluorescence coverage was observed 24 h after si RNA transfection by inverted fluorescence microscopy;Western blot showed that the protein expression levels of Caspase-4 and NLRP3 were decreased in the combined Caspase-4-si RNA + B.ovis infected group compared with the B.ovis infected group,and the differences were significant(p < 0.01);ELISA results showed that IL-1β,TNF-α,and IL-6 secretion levels were significantly up-regulated in the B.ovis infected group compared to the Mock infected and NC groups(p < 0.01);The levels of IL-1β,TNF-α,and IL-6 secretion in the combined Caspase-4-si RNA + B.ovis infected group were all significantly downregulated compared with those in the B.ovis infected group(p < 0.01)Conclusions: This study reveals a molecular regulatory mechanism by which B.ovis regulates NLRP3 expression via caspase-4 to mediate the secretion of inflammatory cytokines IL-1β,TNF-α,and IL-6 from RAW264.7 cells.This provides theoretical support for studying the mechanism by which B.ovis affects the inflammatory pathway of macrophages and also provides an important basis for further exploring the pathogenic mechanism of Brucella.