Deletion of P2Y2 receptor reveals a role for lymphotoxin-α in fatty streak formation

Background Lymphotoxin alpha (LTα) is expressed in human atherosclerotic lesions and genetic variations in the LTα pathway have been linked to myocardial infarction. Activation of the P2Y2 nucleotide receptor (P2Y2R) regulates the production of LTα. in vitro. We aimed to uncover a potential pathway linking purinergic receptor to LTα-mediated inflammatory processes pivotal to the early stages of atherosclerosis in apolipoprotein E (ApoE−/−) deficient mice. Methods and results En face immunostaining revealed that P2Y2R and VCAM-1 are preferentially expressed in the atherosclerosis prone site of the mouse aortic sinus. Deletion of the P2Y2R gene suppresses VCAM-1 expression. Compared with ApoE−/− mice, ApoE−/− mice lacking the P2Y2R gene (ApoE−/−/P2Y2R−/−) did not develop fatty streak lesions when fed a standard chow diet for 15 weeks. Systemic and CD4+ T cell production of the pro-inflammatory cytokine lymphotoxin-alpha (LTα) were specifically inhibited in ApoE−/−/P2Y2R−/−mice. Anti-LTα preventive treatment was initiated in ApoE−/− mice with intraperitoneal administration of recombinant human tumor necrosis factor receptor 1 fusion protein (TNFR1-Fc) on 5 consecutive days before the disease onset. Remarkably, none of the TNFR1:Fc-treated ApoE−/− mice exhibited atherosclerotic lesions at any developmental stage. Significance ApoE−/− mice deficient in P2Y2R exhibit low endothelial cell VCAM-1 levels, decreased production of LTα and delayed onset of atherosclerosis. These data suggest that targeting this nucleotide receptor could be an effective therapeutic approach in atherosclerosis

LAB/NTAL Facilitates Fungal/PAMP-induced IL-12 and IFN-γ Production by Repressing β-Catenin Activation in Dendritic Cells.

Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2−/− mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear β-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted inLat2−/− DCs. Accordingly, Lat2−/− DCs directed reduced Th1 polarization in vitro and Lat2−/−mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and β-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance

Ablation of Tumor Necrosis Factor Is Associated with Decreased Inflammation and Alterations of the Microbiota in a Mouse Model of Inflammatory Bowel Disease

<div><p>Inflammatory bowel disease (IBD) is associated with prolonged, excess secretions of Tumor Necrosis Factor (TNF). Many patients with IBD have successful management of IBD symptoms by blocking TNF secretion or signaling. However, some patients are non-responsive to this therapy, eventually become refractory to therapy, or Alterations in the microbiota that are associated with the lack of TNF could be a contributing cause of this therapeutic insufficiency seen in some patients. Here we use wildtype (WT) and mice lacking <i>Tnf</i> (<i>Tnf</i>^-/-) in an acute TNBS colitis model to investigate the role of TNF in colitis and how its presence or absence affects the colonic microbiota. As expected, <i>Tnf</i>^-/- had less severe inflammation than WT mice. Microbiome analysis revealed significant <i>Tnf</i> dependent-differences in alpha and beta diversity. There were also notable differences in many species that were also primarily <i>Tnf</i> dependent. Taken together, our data indicates that TNF contributes significantly to the inflammation and microbiotal alterations in that occur in IBD.</p></div

Relative proportions of significantly different bacterial genera in WT mice.

<p>Superscript letters across rows denote significant difference at P<0.05 determined using Mann Whitney U test.</p><p>“Unclassified” are taxa in the families listed that are yet to be classified with a genus name. “Other” are taxa that cannot be clearly assigned to a reference group in the Greengenes data set (version 13_5).</p><p>Relative proportions of significantly different bacterial genera in WT mice.</p

Canonical correspondence analysis (CCA) of fecal bacterial composition, mouse genotype, treatment, and histopathology criterion of mice on day 10 revealed more close associations between genotype and erosions in TNBS treated WT mice.

<p>Variation explained in horizontal axis is 7.9% and the vertical axis is 6.8%.</p

Bacteroidetes and Firmicutes are the predominant relative proportions of phyla in the feces of WT and <i>Tnf</i>^<i>-/-</i> mice prior to colitis (F0) and post colitis (F10) in SHAM and TNBS treated mice.

<p>ANOVA indicates significant differences between Bacteroidetes and Firmicutes.</p

Canonical correspondence analysis (CCA) of fecal bacterial composition, mouse genotype, treatment, sampling date and weight of mice.

<p>Fecal sample collected from WT and <i>Tnf</i>^<i>-/-</i> mice treated with TNBS or SHAM at the beginning (F0) versus end (F10) of acute treatment. Variation explained in horizontal axis is 7.7% and the vertical axis is 2.7%.</p

Relative proportion (%) of bacterial genera that significantly differed between the two mouse genotypes on day 0.

<p>n = 20 for each genotype. Significance tested using Mann Whitney U with Bonferroni correction.</p><p>“Unclassified” are taxa in the families listed that are yet to be classified with a genus name. “Other” is taxa that cannot be clearly assigned to a reference group in the Greengenes data set (version 13_5).</p><p>Relative proportion (%) of bacterial genera that significantly differed between the two mouse genotypes on day 0.</p