Neuronal guidance molecule netrin-1 attenuates inflammatory cell trafficking during acute experimental colitis

Background: Inflammatory bowel diseases, encompassing Crohn’s disease and ulcerative colitis, are characterised by persistent leucocyte tissue infiltration leading to perpetuation of an inappropriate inflammatory cascade. The neuronal guidance molecule netrin-1 has recently been implicated in the orchestration of leucocyte trafficking during acute inflammation. We therefore hypothesised that netrin-1 could modulate leucocyte infiltration and disease activity in a model of inflammatory bowel disease. Design: DSS-colitis was performed in mice with partial genetic netrin-1 deficiency (Ntn-1+/- mice) or wild-type mice treated with exogenous netrin-1 via osmotic pump to examine the role of endogenous and therapeutically administered netrin-1. These studies were supported by in vitro models of transepithelial migration and intestinal epithelial barrier function. Results: Consistent with our hypothesis, we observed induction of netrin-1 during intestinal inflammation in vitro or in mice exposed to experimental colitis. Moreover, mice with partial netrin-1 deficiency demonstrated an exacerbated course of DSS-colitis compared to littermate controls, with enhanced weight loss and colonic shortening. Conversely, mice treated with exogenous mouse netrin-1 experienced attenuated disease severity. Importantly, permeability studies and quantitative assessment of apoptosis reveal that netrin-1 signalling events do not alter mucosal permeability or intestinal epithelial cell apoptosis. In vivo studies of leucocyte transmigration demonstrate suppression of neutrophil trafficking as a key function mediated by endogenous or exogenously administered netrin-1. Finally, genetic studies implicate the A2B adenosine receptor in netrin-1-mediated protection during DSS-colitis. Conclusions: The present study identifies a previously unrecognised role for netrin-1 in attenuating experimental colitis through limitation of neutrophil trafficking

Adora2b Adenosine Receptor Engagement Enhances Regulatory T Cell Abundance during Endotoxin-Induced Pulmonary Inflammation

Anti-inflammatory signals play an essential role in constraining the magnitude of an inflammatory response. Extracellular adenosine is a critical tissue-protective factor, limiting the extent of inflammation. Given the potent anti-inflammatory effects of extracellular adenosine, we sought to investigate how extracellular adenosine regulates T cell activation and differentiation. Adenosine receptor activation by a pan adenosine-receptor agonist enhanced the abundance of murine regulatory T cells (Tregs), a cell type critical in constraining inflammation. Gene expression studies in both naïve CD4 T cells and Tregs revealed that these cells expressed multiple adenosine receptors. Based on recent studies implicating the Adora2b in endogenous anti-inflammatory responses during acute inflammation, we used a pharmacologic approach to specifically activate Adora2b. Indeed, these studies revealed robust enhancement of Treg differentiation in wild-type mice, but not in Adora2b−/− T cells. Finally, when we subjected Adora2b-deficient mice to endotoxin-induced pulmonary inflammation, we found that these mice experienced more severe inflammation, characterized by increased cell recruitment and increased fluid leakage into the airways. Notably, Adora2b-deficient mice failed to induce Tregs after endotoxin-induced inflammation and instead had an enhanced recruitment of pro-inflammatory effector T cells. In total, these data indicate that the Adora2b adenosine receptor serves a potent anti-inflammatory role, functioning at least in part through the enhancement of Tregs, to limit inflammation

DNA Tumor Virus Regulation of Host DNA Methylation and Its Implications for Immune Evasion and Oncogenesis

Viruses have evolved various mechanisms to evade host immunity and ensure efficient viral replication and persistence. Several DNA tumor viruses modulate host DNA methyltransferases for epigenetic dysregulation of immune-related gene expression in host cells. The host immune responses suppressed by virus-induced aberrant DNA methylation are also frequently involved in antitumor immune responses. Here, we describe viral mechanisms and virus–host interactions by which DNA tumor viruses regulate host DNA methylation to evade antiviral immunity, which may contribute to the generation of an immunosuppressive microenvironment during cancer development. Recent trials of immunotherapies have shown promising results to treat multiple cancers; however, a significant number of non-responders necessitate identifying additional targets for cancer immunotherapies. Thus, understanding immune evasion mechanisms of cancer-causing viruses may provide great insights for reversing immune suppression to prevent and treat associated cancers

Roles of APOBEC3A and APOBEC3B in Human Papillomavirus Infection and Disease Progression

The apolipoprotein B messenger RNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family of cytidine deaminases plays an important role in the innate immune response to viral infections by editing viral genomes. However, the cytidine deaminase activity of APOBEC3 enzymes also induces somatic mutations in host genomes, which may drive cancer progression. Recent studies of human papillomavirus (HPV) infection and disease outcome highlight this duality. HPV infection is potently inhibited by one family member, APOBEC3A. Expression of APOBEC3A and APOBEC3B is highly elevated by the HPV oncoproteins E6 and E7 during persistent virus infection and disease progression. Furthermore, there is a high prevalence of APOBEC3A and APOBEC3B mutation signatures in HPV-associated cancers. These findings suggest that induction of an APOBEC3-mediated antiviral response during HPV infection may inadvertently contribute to cancer mutagenesis and virus evolution. Here, we discuss current understanding of APOBEC3A and APOBEC3B biology in HPV restriction, evolution, and associated cancer mutagenesis

An Adora2b-specific agonist enhances Treg abundance in vitro following activation of murine T cells.

<p>Bulk splenocytes from either <i>Adora2b</i>+/+ (C57BL/6J) or <i>Adora2b</i>−/− mice were cultured with soluble anti-CD3 for three days with or without the Bay60-6583 compound, at which time the relative abundance of Tregs was assessed. (A) Fold change in Treg abundance relative to <i>Adora2b</i>+/+ cultures without the Bay compound control, where Tregs were defined as viable, CD4+ FoxP3+ cells by flow cytometry. (B) Flow cytometry plots shown from <i>Adora2b</i>+/+ cultured splenocytes. Bay60-6583, an Adora2b-specific agonist was added to a final concentration of 4 nM. Data from two independent experiments, each containing 1–3 independent replicates. The numbers present on each flow cytometry plot indicate the percentage of cells that are FoxP3+ as defined by the square gate, with FoxP3 expressing cells defined relative to isotype control-stained samples (not shown). Statistically significant differences are indicated and were calculated by one-way ANOVA followed by Bonferroni's post-test correction. ns indicates a comparison that is not statistically significantly different.</p

Relative expression levels of adenosine receptor genes in T cell subsets.

<p>Real-time PCR analysis of mRNA for the four adenosine receptor genes in FACS purified CD4 T cell subsets of naïve CD4 T cells or Tregs, with Tregs isolated from FoxP3GFP or DEREG mice. Values were standardized to bulk spleen mRNA, with each value showing expression relative to actin. High level expression of FoxP3 mRNA is consistent with a highly purified population of Tregs. Data representative of two to three independent experiments, analyzing at least three independently isolated populations for both naïve CD4 T cells and Tregs. Data depict mean ± SEM for each transcript. Statistically significant differences were calculated by unpaired t test comparing expression in naïve CD4 T cells relative to Tregs, as indicated.</p

Adenosine receptor activation enhances the abundance of Tregs following in vitro stimulation of primary mouse T cells.

<p>(A) Adenosine receptor activation enhances the abundance of Tregs following antibody-mediated stimulation of T cells (using anti-CD3 antibody, 1 µg/mL combined with 10 ng/mL of IL-2, in the absence of TGF-β). Samples were either untreated (+Vehicle) or treated with 10 µM of NECA (+NECA), a potent adenosine receptor agonist, analyzed three days post-stimulation for the relative abundance of FoxP3-expressing Tregs. The numbers present in the upper left-hand corner of each flow cytometry plot indicate the percentage of cells that are FoxP3+ as defined by the square gate. Background staining with an isotype control antibody is indicated in the leftmost panel. (B) Quantitation of FoxP3-expressing cells following either control (vehicle treated, white bars) or NECA treated (black bars), with data indicating mean +/− SEM of triplicate cultures done in two separate experiments. (C) Adenosine receptor activation enhances the abundance of Tregs following antibody-mediated stimulation of T cells in the presence of TGF-β, a known inducer of Tregs (using anti-CD3 antibody, 1 µg/mL combined with 10 ng/mL of IL-2, combined with 0.75 ng/mL TGF-β), showing flow cytometric analysis (C) and quantitation (D). The numbers present in the upper left-hand corner of each flow cytometry plot indicate the percentage of cells that are FoxP3+ as defined by the square gate. Relative abundance of Tregs within cultures were defined by flow cytometry, with Tregs defined as live, MHC class II negative, CD8−, CD4+ cells that express the transcription factor FoxP3. Data indicate mean +/− SEM of triplicate cultures, representative of two independent experiments. (E) Tregs generated by TGF-β with NECA (solid black line) relative to Tregs generated by TGF-β treatment alone (indicated in gray) have a comparable cell surface expression of CD25, CD39 and CD73. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032416#s2" target="_blank">Results</a> representative of results from three independent cultures, done in two independent experiments. Statistical analysis was performed using unpaired t test, with statistically significant differences as indicated.</p

Monitoring longitudinal immunological responses to bluetongue virus 17 in experimentally infected sheep

Bluetongue virus (BTV) is an economically important pathogen of ruminant species with worldwide prevalence. While many BTV infections are asymptomatic, animals with symptomatic presentation deteriorate quickly with the sickest succumbing to disease within one week. Animals that survive the infection often require months to recover. The immune response to BTV infection is thought to play a central role in controlling the disease. Key to understanding BTV disease is profiling vertebrate host immunological cellular and cytokine responses. Studies to characterize immune responses in ruminants have been limited by a lack of species-specific reagents and assay technology. Here we assess the longitudinal immunological response to experimental BTV-17-California (CA) infection in sheep using the most up to date assays. We infected a cohort of sheep with BTV-17-CA and longitudinally monitored each animal for clinical disease, viremia and specific immunological parameters (B cells, T cells, monocytes) by RT-qPCR, traditional flow cytometry and/or fluorescent based antibody arrays. BTV-inoculated sheep exhibited clinical signs characteristic of bluetongue virus disease. Circulating virus was demonstrated after 8 days post inoculation (DPI) and remained detectable for the remainder of the time course (24 DPI). A distinct lymphopenia was observed between 7 and 14 DPI that rebounded to mock-inoculated control levels at 17 DPI. In addition, we observed increased expression of pro-inflammatory cytokines after 8 DPI. Taken together, we have established a model of BTV infection in sheep and have successfully monitored the longitudinal vertebrate host immunological response and viral infection progression using a combination of traditional methods and cutting-edge technology

In Vivo and In Vitro Effects of Vortioxetine On Molecules Associated with Neuroplasticity

Neuroplasticity is fundamental for brain functions, abnormal changes of which are associated with mood disorders and cognitive impairment. Neuroplasticity can be affected by neuroactive medications and by aging. Vortioxetine, a multimodal antidepressant, has shown positive effects on cognitive functions in both pre-clinical and clinical studies. In rodent studies, vortioxetine increases glutamate neurotransmission, promotes dendritic branching and spine maturation, and elevates hippocampal expression of the activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) at the transcript level. The present study aims to assess the effects of vortioxetine on several neuroplasticity-related molecules in different experimental systems. Chronic (1 month) vortioxetine increased Arc/Arg3.1 protein levels in the cortical synaptosomes of young and middle-aged mice. In young mice, this was accompanied by an increase in actin-depolymerizing factor (ADF)/cofilin serine 3 phosphorylation without altering the total ADF/cofilin protein level, and an increase in the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor phosphorylation at serine 845 (S845) without altering serine 831 (S831) GluA1 phosphorylation nor the total GluA1 protein level. Similar effects were detected in cultured rat hippocampal neurons: Acute vortioxetine increased S845 GluA1 phosphorylation without changing S831 GluA1 phosphorylation or the total GluA1 protein level. These changes were accompanied by an increase in α subunit of Ca2+/calmodulin-dependent kinase (CaMKIIα) phosphorylation (at threonine 286) without changing the total CaMKIIα protein level in cultured neurons. In addition, chronic (1 month) vortioxetine, but not fluoxetine, restored the age-associated reduction in Arc/Arg3.1 and c-Fos transcripts in the frontal cortex of middle-aged mice. Taken together, these results demonstrated that vortioxetine modulates molecular targets that are related to neuroplasticity