Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7-dependent lupus.

The peptidylarginine deiminases PAD2 and PAD4 are implicated in the pathogenesis of several autoimmune diseases. PAD4 may be pathogenic in systemic lupus erythematosus (SLE) through its role in neutrophil extracellular trap (NET) formation that promotes autoantigen externalization, immune dysregulation, and organ damage. The role of this enzyme in mouse models of autoimmunity remains unclear, as pan-PAD chemical inhibitors improve clinical phenotype, whereas PAD4-KO models have given conflicting results. The role of PAD2 in SLE has not been investigated. The differential roles of PAD2 and PAD4 in TLR-7-dependent lupus autoimmunity were examined. Padi4-/- displayed decreased autoantibodies, type I IFN responses, immune cell activation, vascular dysfunction, and NET immunogenicity. Padi2-/- mice showed abrogation of Th subset polarization, with some disease manifestations reduced compared with WT but to a lesser extent than Padi4-/- mice. RNA sequencing analysis revealed distinct modulation of immune-related pathways in PAD-KO lymphoid organs. Human T cells express both PADs and, when exposed to either PAD2 or PAD4 inhibitors, displayed abrogation of Th1 polarization. These results suggest that targeting PAD2 and/or PAD4 activity modulates dysregulated TLR-7-dependent immune responses in lupus through differential effects of innate and adaptive immunity. Compounds that target PADs may have potential therapeutic roles in T cell-mediated diseases

Epithelial CaSR deficiency alters intestinal integrity and promotes proinflammatory immune responses

AbstractThe intestinal epithelium is equipped with sensing receptor mechanisms that interact with luminal microorganisms and nutrients to regulate barrier function and gut immune responses, thereby maintaining intestinal homeostasis. Herein, we clarify the role of the extracellular calcium-sensing receptor (CaSR) using intestinal epithelium-specific Casr−/− mice. Epithelial CaSR deficiency diminished intestinal barrier function, altered microbiota composition, and skewed immune responses towards proinflammatory. Consequently, Casr−/− mice were significantly more prone to chemically induced intestinal inflammation resulting in colitis. Accordingly, CaSR represents a potential therapeutic target for autoinflammatory disorders, including inflammatory bowel diseases

Role of the Mitochondria in Immune-Mediated Apoptotic Death of the Human Pancreatic β Cell Line βLox5

Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA) depleted βLox5 cells, or βLox5 ρ0 cells. βLox5 ρ0 cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ0 cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways

Colonic Immune Stimulation by Targeted Oral Vaccine

<div><h3>Background</h3><p>Currently, sufficient data exist to support the use of lactobacilli as candidates for the development of new oral targeted vaccines. To this end, we have previously shown that <em>Lactobacillus gasseri</em> expressing the protective antigen (PA) component of anthrax toxin genetically fused to a dendritic cell (DC)-binding peptide (DCpep) induced efficacious humoral and T cell-mediated immune responses against <em>Bacillus anthracis</em> Sterne challenge.</p> <h3>Methodology/Principal Finding</h3><p>In the present study, we investigated the effects of a dose dependent treatment of mice with <em>L. gasseri</em> expressing the PA-DCpep fusion protein on intestinal and systemic immune responses and confirmed its safety. Treatment of mice with different doses of <em>L. gasseri</em> expressing PA-DCpep stimulated colonic immune responses, resulting in the activation of innate immune cells, including dendritic cells, which induced robust Th1, Th17, CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ T cell immune responses. Notably, high doses of <em>L. gasseri</em> expressing PA-DCpep (10¹² CFU) were not toxic to the mice. Treatment of mice with <em>L. gasseri</em> expressing PA-DCpep triggered phenotypic maturation and the release of proinflammatory cytokines by dendritic cells and macrophages. Moreover, treatment of mice with <em>L. gasseri</em> expressing PA-DCpep enhanced antibody immune responses, including IgA, IgG₁, IgG_2b, IgG_2c and IgG₃. <em>L. gasseri</em> expressing PA-DCpep also increased the gene expression of numerous pattern recognition receptors, including Toll-like receptors, C-type lectin receptors and NOD-like receptors.</p> <h3>Conclusion/Significance</h3><p>These findings suggest that <em>L. gasseri</em> expressing PA-DCpep has substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration and may be used as a safe oral vaccine against anthrax challenge.</p> </div

<i>L. gasseri</i> expressing PA-DCpep promotes Th17 responses.

<p>C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS; LPLs were harvested by collagenase digestion after days 1, 3, 7 and 14, and stained with antibodies against CD4, CD8, RORγT, IL-17 and IL-22 (A&B) before analysis by flow cytometry. Data are representative of two independent experiments. Error bars represent ±SEM. *P<0.05 and **P<0.01 compared with PBS. (C) C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS; colonic sections were stained with RORγT (red) and CD4 (green) antibodies, and visualized using confocal microscopy.</p

Colonic Immune Suppression, Barrier Dysfunction, and Dysbiosis by Gastrointestinal <i>Bacillus anthracis</i> Infection

<div><p>Gastrointestinal (GI) anthrax results from the ingestion of <i>Bacillus anthracis</i>. Herein, we investigated the pathogenesis of GI anthrax in animals orally infected with toxigenic non-encapsulated <i>B. anthracis</i> Sterne strain (pXO1⁺ pXO2⁻) spores that resulted in rapid animal death. <i>B. anthracis</i> Sterne induced significant breakdown of intestinal barrier function and led to gut dysbiosis, resulting in systemic dissemination of not only <i>B. anthracis</i>, but also of commensals. Disease progression significantly correlated with the deterioration of innate and T cell functions. Our studies provide critical immunologic and physiologic insights into the pathogenesis of GI anthrax infection, whereupon cleavage of mitogen-activated protein kinases (MAPKs) in immune cells may play a central role in promoting dysfunctional immune responses against this deadly pathogen.</p></div

Activation of PRR-genes by <i>L. gasseri</i> expressing PA-DCpep.

<p>C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU), or PBS; RNA was isolated from colon tissue after days 1, 3 and 7 of treatment. Quantitative real-time PCR was performed to measure changes in gene expression. Hierarchical matrix cluster analysis (A) and comparative gene analysis presented as a heat map (B). Data are representative of two independent experiments with duplicates.</p

<i>L. gasseri</i> expressing PA-DCpep enhances the induction of regulatory T cells.

<p>C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS; LPLs were harvested by collagenase digestion after days 1, 3, 7 and 14, stained with antibodies against CD4, CD8, IFNγ (A&B), TGFβ and FoxP3 (C&D), and analyzed by flow cytometry. Data are representative of two independent experiments. Error bars represent ±SEM. *P<0.05 and **P<0.01 compared with PBS.</p