SummaryWhile much is known about tolerogenic dendritic cell effects on forkhead box protein 3 (FoxP3) + regulatory T cells, virtually nothing is known about their effects on another arm of immunoregulation that is mediated by a subpopulation of immunosuppressive B cells. These cells suppress rheumatoid arthritis, lupus and inflammatory bowel disease in mice, and functional defects have been reported in human lupus. We show that co-stimulationimpaired tolerogenic dendritic cells that prevent and reverse type 1 diabetes mellitus induce the proliferation of human immunosuppressive B cells in vitro. We also show that the suppressive properties of these B cells concentrate inside the CD19 + CD24 + B cell population and more specifically inside the CD19 + CD24 + CD38 + regulatory B cell population. We discovered that B cell conversion into suppressive cells in vitro is partially dependent on dendritic cell production of retinoic acid and also that CD19 + CD24 + CD38 + B regulatory cells express retinoic acid receptors. Taken together, our data suggest a model whereby part of the immunosuppressive properties of human tolerogenic dendritic cells could be mediated by retinoic acid which, in addition to its known role in favouring T cell differentiation to FoxP3 + regulatory T cells, acts to convert B cells into immunosuppressive cells.

Objectives Links between microbial alterations and systemic inflammation have been demonstrated in chronic disease, but little is known about these interactions during acute inflammation. This study investigates the effect of dietary supplementation with cellulose, a nonfermentable fiber, on the gut microbiota, inflammatory markers, and survival in two murine models of sepsis. Design Prospective experimental study. Setting University laboratory. Subjects: 6 week old male C57BL/6 wild-type mice. Interventions Mice were assigned to low-fiber (LF), normal-fiber (NF), or high-fiber (HF) diets with or without antibiotics for two weeks and then subjected to sepsis by cecal ligation and puncture (CLP) or endotoxin injection. Fecal samples were collected for microbiota analyses before and after dietary interventions. Measurements and Main Results Mice that received a HF diet demonstrated increased survival after CLP relative to mice receiving LF or NF diets. The survival benefit was associated with decreased serum concentration of pro-inflammatory cytokines, reduced neutrophil infiltration in the lungs, and diminished hepatic inflammation. The HF diet also increased survival after endotoxin injection. Bacterial 16S rRNA gene sequences from each sample were amplified, sequenced, and analyzed. Fiber supplementation yielded an increase in relative abundance of the genera Akkermansia and Lachnospiraceae, taxa commonly associated with metabolic health. Administration of antibiotics to mice on the HF diet negated the enrichment of Akkermansia species and the survival benefit after CLP. Conclusion Dietary supplementation with cellulose offers a microbe-mediated survival advantage in murine models of sepsis. Improved understanding of the link between diet, the microbiota, and systemic illness may yield new therapeutic strategies for patients with sepsis.

The objective of the study was to identify immune cell populations, in addition to Foxp3+ T-regulatory cells, that participate in the mechanisms of action of tolerogenic dendritic cells shown to prevent and reverse type 1 diabetes in the Non-Obese Diabetic (NOD) mouse strain. Co-culture experiments using tolerogenic dendritic cells and B-cells from NOD as well as transgenic interleukin-10 promoter-reporter mice along with transfer of tolerogenic dendritic cells and CD19+ B-cells into NOD and transgenic mice, showed that these dendritic cells increased the frequency and numbers of interleukin-10-expressing B-cells in vitro and in vivo. The expansion of these cells was a consequence of both the proliferation of pre-existing interleukin-10-expressing B-lymphocytes and the conversion of CD19+ B-lymphcytes into interleukin-10-expressing cells. The tolerogenic dendritic cells did not affect the suppressive activity of these B-cells. Furthermore, we discovered that the suppressive murine B-lymphocytes expressed receptors for retinoic acid which is produced by the tolerogenic dendritic cells. These data assist in identifying the nature of the B-cell population increased in response to the tolerogenic dendritic cells in a clinical trial and also validate very recent findings demonstrating a mechanistic link between human tolerogenic dendritic cells and immunosuppressive regulatory B-cells.

Mitochondrial DNA (mtDNA) is a novel danger-associated molecular pattern that on its release into the extracellular milieu acts via toll-like receptor-9, a pattern recognition receptor of the immune system. We hypothesized that plasma mtDNA concentrations will be elevated in septic children, and these elevations are associated with an increase in the severity of illness. In a separate set of in vitro experiments, we test the hypothesis that exposing peripheral blood mononuclear cells (PBMC) to mtDNA activates the immune response and induces tumor necrosis factor (TNF) release. Children with sepsis/systemic inflammatory response syndrome or control groups were enrolled within 24 h of admission to the pediatric intensive care unit. Mitochondrial gene cytochrome c oxidase 1 (COX1) concentrations were measured by realtime quantitative PCR in the DNA extracted from plasma. PBMCs were treated with mtDNA (10μg/mL) and supernatant TNF levels were measured. The median plasma mtDNA concentrations were significantly elevated in the septic patients as compared with the critically ill non-septic and healthy control patients [1.75E+05 (IQR 6.64E+04-3.67E+05) versus 5.73E+03 (IQR 3.90E+03-1.28E+04) and 6.64E+03 (IQR 5.22E+03-1.63E+04) copies/μL respectively]. The median concentrations of plasma mtDNA were significantly greater in patients with MOF as compared with patients without MOF (3.2E+05 (IQR 1.41E+05-1.08E+06) vs. 2.9E+04 (IQR 2.47E+04-5.43E+04) copies/μL). PBMCs treated with mtDNA demonstrated higher supernatant TNF levels as compared with control cells (6.5 ±1.8 vs. 3.5±0.5 pg/mL, P>0.05). Our data suggest that plasma mtDNA is a novel danger-associated molecular pattern in pediatric sepsis and appears to be associated with MOF.