Both TLR2 and TRIF Contribute to Interferon-β Production during Listeria Infection

Synthesis of interferon-β (IFN-β) is an innate response to cytoplasmic infection with bacterial pathogens. Our recent studies showed that Listeria monocytogenes limits immune detection and IFN-β synthesis via deacetylation of its peptidoglycan, which renders the bacterium resistant to lysozyme degradation. Here, we examined signaling requirements for the massive IFN-β production resulting from the infection of murine macrophages with a mutant strain of L. monocytogenes, ΔpgdA, which is unable to modify its peptidoglycan. We report the identification of unconventional signaling pathways to the IFN-β gene, requiring TLR2 and bacterial internalization. Induction of IFN-β was independent of the Mal/TIRAP adaptor protein but required TRIF and the transcription factors IRF3 and IRF7. These pathways were stimulated to a lesser degree by wild-type L. monocytogenes. They operated in both resident and inflammatory macrophages derived from the peritoneal cavity, but not in bone marrow-derived macrophages. The novelty of our findings thus lies in the first description of TLR2 and TRIF as two critical components leading to the induction of the IFN-β gene and in uncovering that individual macrophage populations adopt different strategies to link pathogen recognition signals to IFN-β gene expression

Succinate Dehydrogenase Supports Metabolic Repurposing of Mitochondria to Drive Inflammatory Macrophages.

Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state

Gut Microbial Metabolite-Mediated Regulation of the Intestinal Barrier in the Pathogenesis of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disease. The disease has a multifactorial aetiology, involving genetic, microbial as well as environmental factors. The disease pathogenesis operates at the host–microbe interface in the gut. The intestinal epithelium plays a central role in IBD disease pathogenesis. Apart from being a physical barrier, the epithelium acts as a node that integrates environmental, dietary, and microbial cues to calibrate host immune response and maintain homeostasis in the gut. IBD patients display microbial dysbiosis in the gut, combined with an increased barrier permeability that contributes to disease pathogenesis. Metabolites produced by microbes in the gut are dynamic indicators of diet, host, and microbial interplay in the gut. Microbial metabolites are actively absorbed or diffused across the intestinal lining to affect the host response in the intestine as well as at systemic sites via the engagement of cognate receptors. In this review, we summarize insights from metabolomics studies, uncovering the dynamic changes in gut metabolite profiles in IBD and their importance as potential diagnostic and prognostic biomarkers of disease. We focus on gut microbial metabolites as key regulators of the intestinal barrier and their role in the pathogenesis of IBD

Establishing Boundaries: The Relationship That Exists between Intestinal Epithelial Cells and Gut-Dwelling Bacteria

The human gastrointestinal (GI) tract is a highly complex organ in which various dynamic physiological processes are tightly coordinated while interacting with a complex community of microorganisms. Within the GI tract, intestinal epithelial cells (IECs) create a structural interface that separates the intestinal lumen from the underlying lamina propria. In the lumen, gut-dwelling microbes play an essential role in maintaining gut homeostasis and functionality. Whether commensal or pathogenic, their interaction with IECs is inevitable. IECs and myeloid immune cells express an array of pathogen recognition receptors (PRRs) that define the interaction of both pathogenic and beneficial bacteria with the intestinal mucosa and mount appropriate responses including induction of barrier-related factors which enhance the integrity of the epithelial barrier. Indeed, the integrity of this barrier and induction of appropriate immune responses is critical to health status, with defects in this barrier and over-activation of immune cells by invading microbes contributing to development of a range of inflammatory and infectious diseases. This review describes the complexity of the GI tract and its interactions with gut bacteria

Novel Luciferase Reporter System for In Vitro and Organ-Specific Monitoring of Differential Gene Expression in Listeria monocytogenes

In this paper we describe construction of a luciferase-based vector, pPL2lux, and use of this vector to study gene expression in Listeria monocytogenes. pPL2lux is a derivative of the listerial integration vector pPL2 and harbors a synthetic luxABCDE operon encoding a fatty acid reductase complex (LuxCDE) involved in synthesis of the fatty aldehyde substrate for the bioluminescence reaction catalyzed by the LuxAB luciferase. We constructed pPL2lux derivatives in which the secA and hlyA promoters were translationally fused to luxABCDE and integrated as a single copy into the chromosome of L. monocytogenes EGD-e. Growth experiments revealed that hlyA was expressed predominantly in the stationary phase in LB medium buffered at pH 7.4, whereas secA expression could be detected in the exponential growth phase. Moreover, the correlation between luciferase activity and transcription levels, as determined by reverse transcriptase PCR, was confirmed using conditions known to lead to repression and activation of hemolysin expression (addition of cellobiose and activated charcoal, respectively). Furthermore, hemolysin expression could be monitored in real time during invasion of an intact monolayer of C2Bbe1 (Caco-2-derived) cells. Finally, hemolysin expression could be detected in the livers, spleens, and kidneys of mice 3 days postinfection. These experiments clearly established the effectiveness of pPL2lux as a quantitative reporter system for real-time, noninvasive evaluation of gene expression in L. monocytogenes

MicroRNA-21 Limits Uptake of Listeria monocytogenes by Macrophages to Reduce the Intracellular Niche and Control Infection

MiRNAs are important post-transcriptional regulators of gene expression. MiRNA expression is a crucial part of host responses to bacterial infection, however there is limited knowledge of their impact on the outcome of infections. We investigated the influence of miR-21 on macrophage responses during infection with Listeria monocytogenes, which establishes an intracellular niche within macrophages. MiR-21 is induced following infection of bone marrow-derived macrophages (BMDMs) with Listeria. MiR-21−/− macrophages display an increased bacterial burden with Listeria at 30 min and 2 h post-infection. This phenotype was reversed by the addition of synthetic miR-21 mimics to the system. To assess the immune response of wildtype (WT) and miR-21−/− macrophages, BMDMs were treated with bacterial LPS or infected with Listeria. There was no difference in IL-10 and IL-6 between WT and miR-21−/− BMDMs in response to LPS or Listeria. TNF-α was increased in miR-21−/− BMDMs stimulated with LPS or Listeria compared to WT macrophages. We next assessed the production of nitric oxide (NO), a key bactericidal factor in Listeria infection. There was no significant difference in NO production between WT and miR-21−/− cells, indicating that the increased bacterial burden may not be due to impaired killing. As the increased bacterial load was observed early following infection (30 min), we questioned whether this is due to differences in uptake of Listeria by WT and miR-21−/− macrophages. We show that miR-21-deficiency enhances uptake of FITC-dextran and FITC-Escherichia coli bioparticles by macrophages. The previously observed Listeria burden phenotype was ablated by pre-treatment of cells with the actin polymerization inhibitor cytochalasin-D. From analysis of miR-21 targets, we selected the pro-phagocytic regulators myristoylated alanine-rich C-kinase substrate (MARCKS) and Ras homolog gene family, member B (RhoB) for further investigation. MARCKS and RhoB are increased in miR-21−/− BMDMs, correlating with increased uptake of Listeria. Finally, intra-peritoneal infection of mice with Listeria led to increased bacterial burden in livers of miR-21−/− mice compared to WT mice. These findings suggest a possible role for miR-21 in regulation of phagocytosis during infection, potentially by repression of MARCKS and RhoB, thus serving to limit the availability of the intracellular niche of pathogens like L. monocytogenes

Lactobacillus salivarius UCC118™ Dampens Inflammation and Promotes Microbiota Recovery to Provide Therapeutic Benefit in a DSS-Induced Colitis Model

The use of probiotics such as Lactobacillus and Bifidobacterium spp. as a therapeutic against inflammatory bowel disease (IBD) is of significant interest. Lactobacillus salivarus strain UCC118TM is a commensal that has been shown to possess probiotic properties in vitro and anti-infective properties in vivo. However, the usefulness of UCC118 TM as a therapeutic against colitis remains unclear. This study investigates the probiotic potential of Lactobacillus salivarius, UCC118™ in a mouse model of colitis. DSS-induced colitis was coupled with pre-treatment or post-treatment with UCC118TM by daily oral gavage. In the pre-treatment model of colitis, UCC118TM reduced the severity of the disease in the early stages. Improvement in disease severity was coupled with an upregulation of tissue IL-10 levels and increased expression of macrophage M2 markers. This anti-inflammatory activity of UCC118TM was further confirmed in vitro, using a model of LPS-treated bone marrow-derived macrophages. Taken together, these results suggest that UCC118TM may promote the resolution of inflammation. This was supported in a mouse model of established DSS-induced colitis whereby UCC118TM treatment accelerated recovery, as evidenced by weight, stool, histological markers and the recovery of microbiome-associated dysbiosis with an increased abundance of beneficial commensal species. These results demonstrate the potential of Lactobacillus salivarius UCC118TM as a probiotic-based therapeutic strategy to promote health through the upregulation of anti-inflammatory IL-10 and protect against dysbiosis during IBD

<i>Listeria</i> nucleic acids trigger IFN-β production.

<p>(A) The parental EGDe (black bars) Δ<i>pgdA</i> (grey bars) and complemented Δ<i>pgdA</i> strain (hatched bars) were incubated with lysozyme. The amount of DNA and RNA released after treatment was quantified by spectrophotometry. (B) THP-1 macrophages were transfected with DNA from the Δ<i>pgdA</i> mutant, pretreated or not with DNase, and IFN-β induction was determined using the HEK-blue assay. (C) The parental EGDe strain (black bars) or the Δ<i>pgdA</i> mutant (grey bars) were incubated with lysozyme. PEM were transfected with bacterial lysates, pretreated with DNase or not treated, and IFN-β production was quantified in cells supernatants 24 h after transfection by ELISA. Data are mean ± SD (**, <i>p</i><0.01, n = 3; ***, <i>p</i><0.0001, <i>n</i> = 3).</p

TLR2 is required for PgdA-mediated IFN-β response to <i>Listeria</i> in peritoneal but not bone-marrow macrophages.

<p>(A) PEM from C57BL/6J or <i>tlr2^−/−</i> mice were infected with the parental EGDe strain. After 4 h of infection, IFN-β induction was measured by qRT-PCR. (B) BMM from C57BL/6J or <i>tlr2^−/−</i> mice were infected with the parental EGDe strain. After 4 h of infection, IFN-β induction was measured by qRT-PCR. Data are mean ± SD (NS, non significant; ***, <i>p</i><0.0001, <i>n</i> = 3). PEM from WT C57BL/6J or <i>tlr2</i>^−/− mice were infected with the Δ<i>pgdA</i> mutant (C) or the complemented Δ<i>pgdA</i> strain (D). After 7 h of infection, IFN-β levels were measured in supernatants by ELISA. Data are mean ± SD (**, <i>p</i><0.01, <i>n</i> = 5).</p