NOD2 Deficiency Promotes Intestinal CD4+ T Lymphocyte Imbalance, Metainflammation, and Aggravates Type 2 Diabetes in Murine Model.

Type 2 diabetes (T2D) is a metabolic disease characterized by increased inflammation, NOD-like receptors (NLRs) activation and gut dysbiosis. Our research group has recently reported that intestinal Th17 response limits gut dysbiosis and LPS translocation to visceral adipose tissue (VAT), protecting against metabolic syndrome. However, whether NOD2 receptor contributes intestinal Th17 immunity, modulates dysbiosis-driven metabolic tissue inflammation, and obesity-induced T2D remain poorly understood. In this context, we observed that mice lacking NOD2 fed a high-fat diet (HFD) display severe obesity, exhibit greater adiposity, and more hepatic steatosis compared to HFD-fed wild-type (WT) mice. In addition, they develop increased hyperglycemia, worsening of glucose intolerance, and insulin resistance. Notably, the deficiency of NOD2 causes a deviation from M2 macrophage and regulatory T cells (Treg) to M1 macrophage and mast cells into VAT compared to WT mice fed HFD. An imbalance was also observed in Th17/Th1 cell populations, with reduced IL-17 and IL-22 gene expression in the mesenteric lymph nodes (MLNs) and ileum, respectively, of NOD2-deficient mice fed HFD. 16S rRNA sequencing indicates lower richness, alpha diversity, and a depletion of Allobaculum, Lactobacillus, and enrichment with Bacteroides genera in these mice compared to HFD-fed WT mice. These alterations were associated with disrupted tight-junctions expression, augmented serum LPS, and bacterial translocation into VAT. Overall, NOD2 activation is required for a protective Th17 over Th1 immunity in the gut, which seems to decrease gram-negative bacteria outgrowth in gut microbiota, attenuating the endotoxemia, metainflammation, and protecting against obesity-induced T2D.

Bone Marrow Monocytes and Derived Dendritic Cells from Myelodysplastic Patients Have Functional Abnormalities Associated with Defective Response to Bacterial Infection.

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell diseases characterized by dysplasia of one or more hematologic lineages and a high risk of developing into acute myeloid leukemia. MDS patients have recurrent bacterial infections and abnormal expression of CD56 by monocytes. We investigated MDS patients' bone marrow CD56+/CD56- monocytes and their in vitro-derived dendritic cell populations in comparison with cells obtained from disease-free subjects. We found that monocytes from MDS patients, irrespective of CD56 expression, have reduced phagocytosis activity and low expression of genes involved in triggering immune responses, regulation of immune and inflammatory response signaling pathways, and in the response to LPS. Dendritic cells derived in vitro from MDS monocytes failed to develop dendritic projections and had reduced expression of HLA-DR and CD86, suggesting that Ag processing and T cell activation capabilities are impaired. In conclusion, we identified, in both CD56+ and CD56- monocytes from MDS patients, several abnormalities that may be related to the increased susceptibility to infections observed in these patients.

Mitochondrial DNA Activates the NLRP3 Inflammasome and Predisposes to Type 1 Diabetes in Murine Model.

Although a correlation between polymorphisms of NOD-like receptor family-pyrin domain containing 3 (NLRP3) and predisposition to type 1 diabetes (T1D) has been identified, the potential function and activation of the NLRP3 inflammasome in T1D have not been clarified. The present study shows that non-obese diabetic mice exhibited increased NLRP3, and pro-IL-1ß gene expression in pancreatic lymph nodes (PLNs). Similar increases in gene expression of NLRP3, apoptosis associated speck like protein (ASC) and pro-IL-1ß were induced by multiple low doses of streptozotocin (STZ) in C57BL/6 mice. In addition, diabetic C57BL/6 mice also exhibited increased IL-1ß protein expression in the pancreatic tissue at day 7, which remained elevated until day 15. Diabetic mice also showed increased positive caspase-1 macrophages in the PLNs, which were decreased in NLRP3-/- mice, but not in ASC-/- mice, after STZ treatment. NLRP3- and IL-1R-deficient mice, but not ASC-deficient mice, showed reduced incidence of diabetes, less insulitis, lower hyperglycemia, and normal insulin levels compared to wild-type (WT) diabetic mice. Notably, these mice also displayed a decrease in IL-17-producing CD4 and CD8 T cells (Th17 and Tc17) and IFN-γ-producing CD4 and CD8 T cells (Th1 and Tc1) in the PLNs. Following STZ treatment to induce T1D, NLRP3-deficient mice also exhibited an increase in myeloid-derived suppressor cell and mast cell numbers in the PLNs along with a significant increase in IL-6, IL-10, and IL-4 expression in the pancreatic tissue. Interestingly, diabetic mice revealed increased circulating expression of genes related to mitochondrial DNA, such as cytochrome b and cytochrome c, but not NADH dehydrogenase subunit 6 (NADH). Mitochondrial DNA (mDNA) from diabetic mice, but not from non-diabetic mice, induced significant IL-1ß production and caspase-1 activation by WT macrophages, which was reduced in NLRP3-/- macrophages. Finally, mDNA administration in vivo increased Th17/Tc17/Th1/Tc1 cells in the PLNs and precipitated T1D onset, which was abolished in NLRP3-/- mice. Overall, our results demonstrate that mDNA-mediated NLRP3 activation triggers caspase-1-dependent IL-1ß production and contributes to pathogenic cellular responses during the development of STZ-induced T1D.

Mast cells control insulitis and increase Treg cells to confer protection against STZ-induced type 1 diabetes in mice.

Quantitative alterations in mast cell numbers in pancreatic lymph nodes (PLNs) have been reported to be associated with type 1 diabetes (T1D) progression, but their potential role during T1D remains unclear. In this study, we evaluated the role of mast cells in T1D induced by multiple low-dose streptozotocin (MLD-STZ) treatments, using two strains of mast cell-deficient mice (W/W(v) or Wsh/Wsh) and the adoptive transfer of mast cells. Mast cell deficient mice developed severe insulitis and accelerated hyperglycemia, with 100% of mice becoming diabetic compared to their littermates. In parallel, these diabetic mice had decreased numbers of T regulatory (Treg) cells in the PLNs. Additionally, mast cell deficiency caused a significant reduction in IL-10, TGF-ß, and IL-6 expression in the pancreatic tissue. Interestingly, IL-6-deficient mice are more susceptible to T1D associated with reduced Treg-cell numbers in the PLNs, but mast cell transfer from wild-type mice induced protection to T1D in these mice. Finally, mast cell adoptive transfer prior to MLD-STZ administration conferred resistance to T1D, promoted increased Treg cells, and decreased IL-17-producing T cells in the PLNs. Taken together, our results indicate that mast cells are implicated in resistance to STZ-induced T1D via an immunological tolerance mechanism mediated by Treg cells.

Effects of antimicrobial photodynamic therapy on transforming growth factor-ß1 levels in the gingival crevicular fluid.

BACKGROUND AND OBJECTIVE: Antimicrobial photodynamic therapy (aPDT) has been proposed as an adjunctive therapy to scaling and root planing (SRP). The transforming growth factor-ß1 (TGF-ß) has been considered as an anti-inflammatory cytokine, and its levels in the gingival crevicular fluid (GCF) could monitor the periodontal repair. This study evaluated the adjunct effect of aPDT compared with SRP, analyzing the TGF-ß levels in GCF after nonsurgical and surgical therapy in chronic periodontitis patients. METHODS: Fifteen patients, presenting bilaterally lower molars with class III furcation lesions, were selected. Each pair of teeth was randomly assigned to a control group (CG) or test group (TG). Initially, SRP was performed in the CG, and SRP + aPDT in the TG. Forty-five days later, flap surgery plus SRP, and flap surgery plus SRP + aPDT were performed in CG and TG, respectively. GCF was collected and an enzyme-linked immunosorbent assay (ELISA) test was conducted to determine the amount and concentration of TGF-ß in the GCF at baseline, 45 days post-initial therapy, and 21 days after surgery. RESULTS: Statistically significant differences between groups were found in relation to GCF volume 21 days after the surgical procedures (p=0.03) and TGF-ß concentration in GCF 45 days post-initial therapy (p=0.04), favoring the TG. CONCLUSIONS: There was an additional effect of the aPDT protocol compared with SRP for the TGF-ß concentration in GCF 45 days after nonsurgical therapy, and for the GCF volume 21 days after surgical therapy.

Cell-free antigens from Paracoccidioides brasiliensis drive IL-4 production and increase the severity of paracoccidioidomycosis.

The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.