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Transcriptomic profile of murine macrophage RAW264.7 in response to Salmonella flagellin

ABSTRACT: Macrophages are an important component of the innate immune system. Flagellin has been extensively studied for its adjuvant activity owing to its TLR5 and NLRC4 binding sites. However, few studies have comprehensively investigated the effects of flagellin in macrophages using transcriptome sequencing. In this study, RNA sequencing (RNA-seq) was used to analyze the expression patterns of RAW264.7 cells induced by flagellin (FliC) of Salmonella typhimurium compared with unstimulated cells, to identify novel transcriptomic signatures in macrophages.

ORGANISM(S): Mus musculus

PROVIDER: GSE200796 | GEO | 2022/12/31

REPOSITORIES: GEO

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Cytosolic flagellin receptor NLRC4 protects mice against mucosal and systemic challenges

Project description:Bacterial flagellin is a dominant innate immune activator of the intestine. Therefore, we examined the role of the intracellular flagellin receptor, NLRC4, in protecting the gut and/or driving inflammation. In accord with NLRC4 acting via transcription-independent pathways, loss of NLRC4 did not reduce the rapid robust changes in intestinal gene expression induced by flagellin administration. Loss of NLRC4 did not alter basal intestinal homeostasis nor predispose mice to development of colitis upon administration of an anti-IL-10R monoclonal antibody. However, in response to epithelial injury induced by dextran sulfate sodium (DSS), loss of NLRC4 resulted in more severe disease indicating a role for NLRC4 in protecting the gut. Moreover, loss of NLRC4 resulted in increased mortality in response to flagellate, but not aflagellate Salmonella infection. Thus, despite not being involved in rapid intestinal gene remodeling upon detection of flagellin, NLRC4-mediated inflammasome activation protects mice from mucosal and systemic challenges Flagellin (FliC) from WT Salmonella enterica serovar Typhimurium (SL3201, fljB–) was purified through sequential cation and anion-exchange chromatography and purity was verified as previously described 4. WT, T5KO, N4KO and T5/N4-DKO mice (n=6) were given either 0.2 mL PBS or flagellin (10μg/mouse in 0.2 mL PBS) intraperitoneally. After 1h, mice were euthanized and colon was taken and stored in RNAlater (Invitrogen) for 1 day. Total mRNA was isolated from colonic tissues using TRIzol (Invitrogen) and purified using the RNeasy® Plus Mini kit (Qiagen) according to the manufacturer’s instructions

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Cytosolic flagellin receptor NLRC4 protects mice against mucosal and systemic challenges

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Project description:Microarray analysis of IECs from Tlr5+/+ and Tlr5â/â mice stimulated with either medium alone or flagellin (1 Âµg/ml); to elucidate TLR5-mediated immune responses in CD11c+ LPCs Experiment Overall Design: CD11c+ LPCs from Tlr5+/+ and Tlr5â/â mice were treated with or without flagellin (1 ïg/ml) for 4 h. Total RNA was extracted with an RNeasy kit (Qiagen), and purified using an Oligotex mRNA Kit (Pharmacia). Fragmented and biotin-labeled cDNA was synthesized from 100 ng purified mRNA using the Ovation Biotin System (Nugen), according to the manufacturerâs protocol. cDNA was hybridized to Affymetrix Murine Genome 430 2.0 microarray chips (Affymetrix) according to the manufacturerâs instructions. Hybridized chips were stained, washed and scanned using a GeneArray Scanner (Affymetrix). Data analysis was performed with Microarray Suite software (Version 5.0, Affymetrix) and GeneSpring software (Silicon Genetics).

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