Propionate has protective and anti-inflammatory effects on the blood–brain barrier

Production of short-chain fatty acids (SCFAs) from dietary substrates by the gut microbiota is associated with health, with these metabolites influencing the host via the ‘gut–brain axis’. Micromolar quantities of microbially derived SCFAs are taken up from the gut and reach systemic circulation, where they can influence host gene expression through a variety of largely unknown mechanisms. The blood–brain barrier (BBB) is the major interface between the circulation and central nervous system, and is critically involved in the pathogenesis of neuroinflammatory disorders such as stroke and vascular dementia. We hypothesized exposure of the BBB to SCFAs influences barrier integrity and function. To test our hypothesis, we investigated the in vitro effects of a physiologically relevant concentration (1 μM) of propionate upon the human immortalised cerebromicrovascular endothelial cell line hCMEC/D3. Propionate is produced by the microbiota from dietary glucans, and is biologically active via the G protein coupled receptors FFAR2 and FFAR3. It is a highly potent FFAR2 agonist (agonist activity 3.99) and has close to optimal ligand efficiency (-ΔG=1.19 kcal mol-1 atom-1) for this receptor. Notably, FFAR3 is expressed on the vascular endothelium and a likely target for propionate in the BBB. After confirming the presence of FFAR3 on hCMEC/D3 cells, we undertook an unbiased transcriptomic analysis of confluent hCMEC/D3 monolayers treated or not for 24 h with 1 μM propionate, supported by in vitro validation of key findings and assessment of functional endothelial permeability barrier properties. Propionate treatment had a significant (PFDR < 0.1) effect on the expression of 1136 genes: 553 upregulated, 583 downregulated. Propionate inhibited several inflammation-associated pathways: namely, TLR-specific signalling, NFkappaB signalling, and cytosolic DNA-sensing. Functional validation of these findings confirmed the down-regulation of TLR signalling by propionate, achieved primarily through down-regulation of endothelial CD14 expression. Accordingly, propionate prevented LPS-induced increases in paracellular permeability to 70 kDa FITC-dextran and loss of transendothelial electrical resistance. Enrichr analysis indicated the activation by propionate of the NFE2L2 (NRF2)-driven protective response against oxidative stress. Confirming these data, propionate limited free reactive oxygen species induction by the mitochondrial respiratory inhibitor rotenone. Together, these data strongly suggest the SCFA propionate contributes to maintaining BBB integrity and protecting against inflammatory challenge by downregulating BBB responsiveness. In addition to its well-described effects on cholesterol metabolism, maintenance of propionate levels in the circulation may be an additional mechanism whereby a glucan-containing diet protects against neurovascular disease

Extracellular vesicles produced by the human commensal gut bacterium Bacteroides thetaiotaomicron affect host immune pathways in a cell-type specific manner that are altered in inflammatory bowel disease

The gastrointestinal (GI) tract harbours a complex microbial community, which contributes to its homeostasis. A disrupted microbiome can cause GI-related diseases, including inflammatory bowel disease (IBD), therefore identifying host-microbe interactions is crucial for better understanding gut health. Bacterial extracellular vesicles (BEVs), released into the gut lumen, can cross the mucus layer and access underlying immune cells. To study BEV-host interactions, we examined the influence of BEVs generated by the gut commensal bacterium, Bacteroides thetaiotaomicron, on host immune cells. Single-cell RNA sequencing data and host-microbe protein-protein interaction networks were used to predict the effect of BEVs on dendritic cells, macrophages and monocytes focusing on the Toll-like receptor (TLR) pathway. We identified biological processes affected in each immune cell type and cell-type specific processes including myeloid cell differentiation. TLR pathway analysis highlighted that BEV targets differ among cells and between the same cells in healthy versus disease (ulcerative colitis) conditions. The in silico findings were validated in BEV-monocyte co-cultures demonstrating the requirement for TLR4 and Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) in BEV-elicited NF-kB activation. This study demonstrates that both cell-type and health status influence BEV-host communication. The results and the pipeline could facilitate BEV-based therapies for the treatment of IBD

Bacteroides thetaiotaomicron-derived outer membrane vesicles promote regulatory dendritic cell responses in health but not in inflammatory bowel disease

Background: Bacteroides thetaiotaomicron (Bt) is a prominent member of the human intestinal microbiota that, like all Gram-negative bacteria, naturally generates nanosized outer membrane vesicles (OMVs) which bud off from the cell surface. Importantly, OMVs can cross the intestinal epithelial barrier to mediate microbe-host cell crosstalk involving both epithelial and immune cells to help maintain intestinal homeostasis. Here we have examined the interaction between Bt OMVs and blood or colonic mucosa-derived dendritic cells (DC) from healthy individuals and patients with Crohn’s disease (CD) or ulcerative colitis (UC). Results: In healthy individuals, Bt OMVs stimulated significant (p<0.05) IL-10 expression by colonic DC, whereas in peripheral blood-derived DC they also stimulated significant (p<0.001 and p<0.01, respectively) expression of IL-6 and the activation marker CD80. Conversely, in UC Bt OMVs were unable to elicit IL-10 expression by colonic DC. There were also reduced numbers of CD103 + DC in the colon of both UC and CD patients compared to controls, supporting a loss of regulatory DC in both diseases. Furthermore, in CD and UC, Bt OMVs elicited a significantly lower proportion of DC which expressed IL-10 (p<0.01 and p<0.001, respectively) in blood compared to controls. These alterations in DC responses to Bt OMVs were seen in patients with inactive disease, and thus are indicative of intrinsic defects in immune responses to this commensal in inflammatory bowel disease (IBD). Conclusions: Overall, our findings suggest a key role for OMVs generated by the commensal gut bacterium Bt in directing a balanced immune response to constituents of the microbiota locally and systemically during health which is altered in IBD patients

The proteome of extracellular vesicles produced by the human gut bacteria bacteroides thetaiotaomicron in vivo Is influenced by environmental and host-derived factors

Bacterial extracellular vesicles (BEVs) released from both Gram-negative and Gram-positive bacteria provide an effective means of communication and trafficking of cell signaling molecules. In the gastrointestinal tract (GIT) BEVs produced by members of the intestinal microbiota can impact host health by mediating microbe-host cell interactions. A major unresolved question, however, is what factors influence the composition of BEV proteins and whether the host influences protein packaging into BEVs and secretion into the GIT. To address this, we have analyzed the proteome of BEVs produced by the major human gut symbiont Bacteroides thetaiotaomicron both in vitro and in vivo in the murine GIT in order to identify proteins specifically enriched in BEVs produced in vivo. We identified 113 proteins enriched in BEVs produced in vivo, the majority (62/113) of which accumulated in BEVs in the absence of any changes in their expression by the parental cells. Among these selectively enriched proteins, we identified dipeptidyl peptidases and an asparaginase and confirmed their increased activity in BEVs produced in vivo. We also showed that intact BEVs are capable of degrading bile acids via a bile salt hydrolase. Collectively these findings provide additional evidence for the dynamic interplay of host-microbe interactions in the GIT and the existence of an active mechanism to drive and enrich a selected group of proteins for secretion into BEVs in the GIT

Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample

We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative) analysis are described here, together with an example from a study on the effects of TGF-β1 in TF-1 cells

Helicobacter pylori Induces Activation of Human Peripheral γδ+ T Lymphocytes

Helicobacter pylori is a Gram-negative bacterium that causes gastric and duodenal diseases in humans. Despite a robust antibody and cellular immune response, H. pylori infection persists chronically. To understand if and how H. pylori could modulate T cell activation, in the present study we investigated in vitro the interaction between H. pylori and human T lymphocytes freshly isolated from peripheral blood of H. pylori-negative donors. A direct interaction of live, but not killed bacteria with purified CD3+ T lymphocytes was observed by microscopy and confirmed by flow cytometry. Live H. pylori activated CD3+ T lymphocytes and predominantly γδ+ T cells bearing the TCR chain Vδ2. Upon interaction with H. pylori, these cells up-regulated the activation molecule CD69 and produced cytokines (such as TNFα, IFNγ) and chemokines (such as MIP-1β, RANTES) in a non-antigen-specific manner. This activation required viable H. pylori and was not exhibited by other Gram-negative bacteria. The cytotoxin-associated antigen-A (CagA), was at least partially responsible of this activation. Our results suggest that H. pylori can directly interact with T cells and modulate the response of γδ+ T cells, thereby favouring an inflammatory environment which can contribute to the chronic persistence of the bacteria and eventually to the gastric pathology

Effects of 15-Deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2) and Rosiglitazone on Human Vδ2+ T Cells

BACKGROUND:Thiazolidinediones (TZD) class of drugs, and 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2) are immune regulators predicted to modulate human autoimmune disease. Their effects on gammadelta T cells, which are involved in animal model and human and animal autoimmune diseases, are unknown. METHODOLOGY/PRINCIPAL FINDINGS:We characterized the activity of rosiglitazone (from the TZD class of drugs) and 15d-PGJ2 in human Vdelta2 T cells. We found that 15d-PGJ2 and rosiglitazone had different effects on Vdelta2 T cell functions. Both 15d-PGJ2 and rosiglitazone suppressed Vdelta2 T cell proliferation in response to IPP and IL2. However, only 15d-PGJ2 suppressed functional responses including cytokine production, degranulation and cytotoxicity against tumor cells. The mechanism for 15d-PGJ2 effects on Vdelta2 T cells acts through inhibiting Erk activation. In contrast, rosiglitazone did not affect Erk activation but the IL2 signaling pathway, which accounts for rosiglitazone suppression of IL2-dependent, Vdelta2 T cell proliferation without affecting TCR-dependent functions. Rosiglitazone and 15d-PGJ2 are designed to be peroxisome proliferator-activated receptor gamma (PPARgamma) ligands and PPARgamma was expressed in Vdelta2 T cell. Surprisingly, when PPARgamma levels were lowered by specific siRNA, 15d-PGJ2 and rosiglitazone were still active, suggesting their target of action induces cellular proteins other than PPARgamma. CONCLUSIONS/SIGNIFICANCE:The current findings expand our understanding of how the immune system is regulated by rosiglitazone and 15d-PGJ2 and will be important to evaluate these compounds as therapeutic agents in human autoimmune disease

cDNA Sequence and Fab Crystal Structure of HL4E10, a Hamster IgG Lambda Light Chain Antibody Stimulatory for γδ T Cells

Hamsters are widely used to generate monoclonal antibodies against mouse, rat, and human antigens, but sequence and structural information for hamster immunoglobulins is sparse. To our knowledge, only three hamster IgG sequences have been published, all of which use kappa light chains, and no three-dimensional structure of a hamster antibody has been reported. We generated antibody HL4E10 as a probe to identify novel costimulatory molecules on the surface of γδ T cells which lack the traditional αβ T cell co-receptors CD4, CD8, and the costimulatory molecule CD28. HL4E10 binding to γδ T cell, surface-expressed, Junctional Adhesion Molecule-Like (JAML) protein leads to potent costimulation via activation of MAP kinase pathways and cytokine production, resulting in cell proliferation. The cDNA sequence of HL4E10 is the first example of a hamster lambda light chain and only the second known complete hamster heavy chain sequence. The crystal structure of the HL4E10 Fab at 2.95 Å resolution reveals a rigid combining site with pockets faceted by solvent-exposed tyrosine residues, which are structurally optimized for JAML binding. The characterization of HL4E10 thus comprises a valuable addition to the spartan database of hamster immunoglobulin genes and structures. As the HL4E10 antibody is uniquely costimulatory for γδ T cells, humanized versions thereof may be of clinical relevance in treating γδ T cell dysfunction-associated diseases, such as chronic non-healing wounds and cancer

A Rapid Crosstalk of Human γδ T Cells and Monocytes Drives the Acute Inflammation in Bacterial Infections

Vγ9/Vδ2 T cells are a minor subset of T cells in human blood and differ from other T cells by their immediate responsiveness to microbes. We previously demonstrated that the primary target for Vγ9/Vδ2 T cells is (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), an essential metabolite produced by a large range of pathogens. Here we wished to study the consequence of this unique responsiveness in microbial infection. The majority of peripheral Vγ9/Vδ2 T cells shares migration properties with circulating monocytes, which explains the presence of these two distinct blood cell types in the inflammatory infiltrate at sites of infection and suggests that they synergize in anti-microbial immune responses. Our present findings demonstrate a rapid and HMB-PP-dependent crosstalk between Vγ9/Vδ2 T cells and autologous monocytes that results in the immediate production of inflammatory mediators including the cytokines interleukin (IL)-6, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and oncostatin M (OSM); the chemokines CCL2, CXCL8, and CXCL10; and TNF-related apoptosis-inducing ligand (TRAIL). Moreover, under these co-culture conditions monocytes differentiate within 18 hours into inflammatory dendritic cells (DCs) with antigen-presenting functions. Addition of further microbial stimuli (lipopolysaccharide, peptidoglycan) induces CCR7 and enables these inflammatory DCs to trigger the generation of CD4+ effector αβ T cells expressing IFN-γ and/or IL-17. Importantly, our in vitro model replicates the responsiveness to microbes of effluent cells from peritoneal dialysis (PD) patients and translates directly to episodes of acute PD-associated bacterial peritonitis, where Vγ9/Vδ2 T cell numbers and soluble inflammatory mediators are elevated in patients infected with HMB-PP-producing pathogens. Collectively, these findings suggest a direct link between invading pathogens, microbe-responsive γδ T cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity