Influenza Virus-Specific Immunological Memory Is Enhanced by Repeated Social Defeat

Immunological memory (MEM) development is affected by stress-induced neuroendocrine mediators. Current knowledge about how a behavioral interaction, such as social defeat, alters the development of adaptive immunity, and MEM is incomplete. In this study, the experience of social disruption stress (SDR) prior to a primary influenza viral infection enhanced the frequency and function of the T cell memory pool. Socially stressed mice had a significantly enlarged population of CD8+ T cells specific for the immunodominant NP366–74 epitope of A/PR/8/34 virus in lung and spleen tissues at 6–12 wk after primary infection (resting memory). Moreover, during resting memory, SDR-MEM mice responded with an enhanced footpad delayed-type hypersensitivity response, and more IFN-γ–producing CD4+ T cells were detected after ex vivo stimulation. When mice were rechallenged with A/PR/8/34 virus, SDR-MEM mice terminated viral gene expression significantly earlier than MEM mice and generated a greater DbNP366–74CD8+ T cell response in the lung parenchyma and airways. This enhancement was specific to the T cell response. SDR-MEM mice had significantly attenuated anti-influenza IgG titers during resting memory. Similar experiments in which mice were primed with X-31 influenza and challenged with A/PR/8/34 virus elicited similar enhancements in the splenic and lung airway Db NP366–74CD8+ T cell populations in SDR-MEM mice. This study demonstrates that the experience of repeated social defeat prior to a primary viral infection significantly enhances virus-specific memory via augmentation of memory T cell populations and suggests that social stressors should be carefully considered in the design and analysis of future studies on antiviral immunity

Identification of myeloid derived suppressor cells in the peripheral blood of tumor bearing dogs

<p>Abstract</p> <p>Background</p> <p>Myeloid derived suppressor cells (MDSCs) are a recently described population of immune cells that significantly contribute to the immunosuppression seen in cancer patients. MDSCs are one of the most important factors that limit the efficacy of cancer immunotherapy (e.g. cancer vaccines) and MDSC levels are increased in cancer in multiple species. Identifying and targeting MDSCs is actively being investigated in the field of human oncology and is increasingly being investigated in veterinary oncology. The treatment of canine cancer not only benefits dogs, but is being used for translational studies evaluating and modifcying candidate therapies for use in humans. Thus, it is necessary to understand the immune alterations seen in canine cancer patients which, to date, have been relatively limited. This study investigates the use of commercially available canine antibodies to detect an immunosuppressive (CD11b^low/CADO48^low) cell population that is increased in the peripheral blood of tumor-bearing dogs.</p> <p>Results</p> <p>Commercially available canine antibodies CD11b and CADO48A were used to evaluate white blood cells from the peripheral blood cells of forty healthy control dogs and forty untreated, tumor-bearing dogs. Tumor-bearing dogs had a statistically significant increase in CD11b^low/CADO48A^low cells (7.9%) as compared to the control dogs (3.6%). Additionally, sorted CD11b^low/CADO48A^low generated <it>in vitro</it> suppressed the proliferation of canine lymphocytes.</p> <p>Conclusions</p> <p>The purpose of this study was aimed at identifying potential canine specific markers for identifying MDSCs in the peripheral blood circulation of dogs. This study demonstrates an increase in a unique CD11b^low/CADO48A^low cell population in tumor-bearing dogs. This immunophenotype is consistent with described phenotypes of MDSCs in other species (i.e. mice) and utilizes commercially available canine-specific antibodies. Importantly, CD11b^low/CADO48A^low from a tumor environment suppress the proliferation of lymphocytes. These results provide a useful phenotype of cells increased in canine cancer patients that may serve as a useful prognostic marker for assessing immune status and functional response to cancer immunotherapies in dogs. Understanding MDSCs in dogs will allow for increased effectiveness of cancer immunotherapy in both dogs and humans.</p

Mitochondrial transcription factor A, an endogenous danger signal, promotes TNFα release via RAGE- and TLR9-responsive plasmacytoid dendritic cells.

Mitochondrial transcription factor A (TFAM) is normally bound to and remains associated with mitochondrial DNA (mtDNA) when released from damaged cells. We hypothesized that TFAM, bound to mtDNA (or equivalent CpG-enriched DNA), amplifies TNFα release from TLR9-expressing plasmacytoid dendritic cells (pDCs) by engaging RAGE.Murine Flt3 ligand-expanded splenocytes obtained from C57BL/6 mice were treated with recombinant human TFAM, alone or in combination with CpG-enriched DNA with subsequent TNFα release measured by ELISA. The role of RAGE was determined by pre-treatment with soluble RAGE or heparin or by employing matching RAGE (-/-) splenocytes. TLR9 signaling was evaluated using a specific TLR9-blocking oligonucleotide and by inhibiting endosomal processing, PI3K and NF-κB. Additional studies examined whether heparin sulfate moieties or endothelin converting enzyme-1 (ECE-1)-dependent recycling of endosomal receptors were required for TFAM and CpG DNA recognition.TFAM augmented splenocyte TNFα release in response to CpGA DNA, which was strongly dependent upon pDCs and regulated by RAGE and TLR9 receptors. Putative TLR9 signaling pathways, including endosomal acidification and signaling through PI3K and NF-κB, were essential for splenocyte TNFα release in response to TFAM+CpGA DNA. Interestingly, TNFα release depended upon endothelin converting enzyme (ECE)-1, which cleaves and presumably activates TLR9 within endosomes. Recognition of the TFAM-CpGA DNA complex was dependent upon heparin sulfate moieties, and recombinant TFAM Box 1 and Box 2 proteins were equivalent in terms of augmenting TNFα release.TFAM promoted TNFα release in a splenocyte culture model representing complex cell-cell interactions in vivo with pDCs playing a critical role. To our knowledge, this study is the first to incriminate ECE-1-dependent endosomal cleavage of TLR9 as a critical step in the signaling pathway leading to TNFα release. These findings, and others reported herein, significantly advance our understanding of sterile immune responses triggered by mitochondrial danger signals

Physiologic Doses of Bilirubin Contribute to Tolerance of Islet Transplants by Suppressing the Innate Immune Response

Bilirubin has been recognized as a powerful cytoprotectant when used at physiologic doses and was recently shown to have immunomodulatory effects in islet allograft transplantation, conveying donor-specific tolerance in a murine model. We hypothesized that bilirubin, an antioxidant, acts to suppress the innate immune response to islet allografts through two mechanisms: 1) by suppressing graft release of damage-associated molecular patterns (DAMPs) and inflammatory cytokines, and 2) by producing a tolerogenic phenotype in antigen-presenting cells. Bilirubin was administered intraperitoneally before pancreatic procurement or was added to culture media after islet isolation in AJ mice. Islets were exposed to transplant-associated nutrient deprivation and hypoxia. Bilirubin significantly decreased islet cell death after isolation and hypoxic stress. Bilirubin supplementation of islet media also decreased the release of DAMPs (HMGB1), inflammatory cytokines (IL-1β and IL-6), and chemokines (MCP-1). Cytoprotection was mediated by the antioxidant effects of bilirubin. Treatment of macrophages with bilirubin induced a regulatory phenotype, with increased expression of PD-L1. Coculture of these macrophages with splenocytes led to expansion of Foxp3+ Tregs. In conclusion, exogenous bilirubin supplementation showed cytoprotective and antioxidant effects in a relevant model of islet isolation and hypoxic stress. Suppression of DAMP release, alterations in cytokine profiles, and tolerogenic effects on macrophages suggest that the use of this natural antioxidant may provide a method of preconditioning to improve outcomes after allograft transplantation

TFAM and CpGA DNA Induce a Splenocyte Proinflammatory Immune Response through PI3K and NF-κB Signaling.

<p>The presented data was derived from at least 5 independent experiments. TFAM (5 µg/ml) + CpGA DNA (0.3 µM)-induced Flt3L-expanded splenocyte (1 × 10⁶ cells/ml) TNFα release was completely blocked 24 hours post-treatment following pre-treatment (30 minutes) with inhibitors of PI3K (LY294002, 5 µM) and NF-κB (BAY 11-7085, 5 µM) signaling (*<i>p</i> < 0.01, relative to no treatment; † <i>p</i> < 0.01, compared to the TFAM + CpGA treatment group).</p

Schematic Representation of Splenic Dendritic Cell Activation by Necrotic Cells.

<p>Mitochondrial DNA (mtDNA), when released from necrotic cells, remains associated with TFAM. As in Type I interferon production, TFAM augments the proinflammatory immune response to mtDNA by associating with RAGE (and related heparin sulfate moieties) and TLR9, respectively, to promote endosomal processing, including endosomal receptor (TLR9) recycling by ECE-1, and signal transduction via PI3K/Akt and ERK pathway activation to induce splenic dendritic cell TNFα gene transcription and protein translation and release.</p

Amplification of CpGA DNA-provoked Splenocyte TNFα Release by TFAM Involves RAGE and TLR9 but Not FPR.

<p>The presented data was derived from at least 5 independent experiments. (A) 24 hours post-treatment, TFAM (5 µg/ml) increased CpGA DNA (0.3 µM)-induced Flt3L-expanded mouse splenocyte (1 × 10⁶ cells/ml) TNFα release. This effect was dramatically attenuated through RAGE (sRAGE, 20 µg/ml) or TLR9 (G-ODN, 10 µM) inhibition 30 minutes pre-treatment. In addition, splenocytes from RAGE -/- mice demonstrated significantly diminished TNFα release in response to TFAM ± CpGA DNA treatment. (B) Similarly, exposure of Flt3L-expanded splenocytes (1 × 10⁶ cells/ml) to immunoprecipitated native TFAM complexed with mtDNA (IP TFAM, 0.5 µg/ml) derived from necrotic cells yielded marked release of TNFα by 24 hours post-treatment which was significantly reduced when treating corresponding splenocytes from RAGE -/- mice. (C) Pre-treatment (30 minutes) with FPR antagonists [CsH (1 µM), BOC (10 µM) or WRW4 (20 µg/ml)] had no effect upon TFAM (5 µg/ml) + CpGA DNA (0.3 µM)-induced splenocyte TNFα release 24 hours post-treatment. Likewise, 24 hours after treatment, FPR agonists [fMLP (5 µg/ml), ND6 (5 µg/ml)] did not stimulate TNFα release (*<i>p</i> < 0.01, compared to no treatment; ^†<i>p</i> < 0.01, relative to the wild type TFAM + CpGA treatment group).</p

TFAM Augments CpGA DNA-induced Splenocyte TNFα Release which Is Highly Dependent upon the Presence of Plasmacytoid Dendritic Cells.

<p>The presented data was derived from at least 5 independent experiments. (A) As previously observed for other cytokines in different cell preparations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072354#B2" target="_blank">2</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072354#B3" target="_blank">3</a>], native mitochondrial protein (5 µg/ml) induced significant TNFα release from cultured mouse Flt3L-expanded splenocytes (1 × 10⁶ cells/ml) 24 hours post-treatment. CpGA DNA (0.3 µM), which is akin to that naturally associated with mitochondrial protein, promoted splenocyte TNFα release which was significantly amplified by co-incubation with recombinant human TFAM (5 µg/ml) despite that having no effect when treated alone. Compared to splenocytes obtained from a normal spleen, TNFα release was markedly increased following all treatments when the constituent pDC population was expanded by Flt3L over-expression as employed in the present study. (B) Selective removal of pDCs from matching Flt3L-expanded splenocyte preparations notably reduced the TNFα release induced by CpGA DNA (0.3 µM) ± TFAM (5 µg/ml) 24 hours post-treatment. (C) pDCs (1 × 10⁶ cells/ml) isolated from similar mouse splenocyte preparations demonstrated comparably minimal yet significant TNFα release 24 hours after CpGA DNA (0.3 µM) ± TFAM (5 µg/ml) treatment (*<i>p</i> < 0.01, relative to no treatment; ^†<i>p</i> < 0.01, compared to CpGA DNA treatment alone, and ^‡<i>p</i> < 0.01, relative to matching normal splenocyte treatments).</p

Enhancement by TFAM of the CpGA DNA-induced Splenocyte Proinflammatory Immune Response Results from Charge-Dependent Recognition of TFAM and Is Equipotent for Both TFAM DNA-binding Subunit Proteins, Box 1 and Box 2.

<p>The presented data was derived from at least 5 independent experiments. (A) Pre-treatment (30 minutes) with heparin sodium (1-100 U/ml) or heparin lyase (heparinase) II or III (5 mU/ml) dramatically suppressed TFAM (5 µg/ml) + CpGA DNA (0.3 µM)-induced Flt3L-expanded splenocyte (1 × 10⁶ cells/ml) TNFα release 24 hours post-treatment. Heparin demonstrated significant inhibition in a dose-dependent manner (*<i>p</i> < 0.01, compared to no treatment; † <i>p</i> < 0.01, relative to the TFAM + CpGA treatment group). (B) Though not as potent at augmenting CpGA DNA (0.3 µM)-induced splenocyte (1 × 10⁶ cells/ml) TNFα release as full-length recombinant TFAM (5 µg/ml) 24 hours post-treatment, recombinant TFAM Box 1 and Box 2 (5 µg/ml) each demonstrated significant amplification of CpGA DNA-induced TNFα release (*<i>p</i> < 0.01, compared to no treatment; † <i>p</i> < 0.01, relative to CpGA DNA treatment alone, and ‡<i>p</i> < 0.01, compared to CpGA DNA alone and the TFAM + CpGA treatment group).</p