Nod2 Suppresses Borrelia burgdorferi Mediated Murine Lyme Arthritis and Carditis through the Induction of Tolerance

The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance

Nod2 Suppresses Borrelia burgdorferi Mediated Murine Lyme Arthritis and Carditis through the Induction of Tolerance

The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance

Human Integrin α3β1 Regulates TLR2 Recognition of Lipopeptides from Endosomal Compartments

Toll-like receptor (TLR)-2/TLR1 heterodimers recognize bacterial lipopeptides and initiate the production of inflammatory mediators. Adaptors and co-receptors that mediate this process, as well as the mechanisms by which these adaptors and co-receptors function, are still being discovered.Using shRNA, blocking antibodies, and fluorescent microscopy, we show that U937 macrophage responses to the TLR2/1 ligand, Pam(3)CSK(4), are dependent upon an integrin, α(3)β(1). The mechanism for integrin α(3)β(1) involvement in TLR2/1 signaling is through its role in endocytosis of lipopeptides. Using inhibitors of endosomal acidification/maturation and physical tethering of the ligand, we show that the endocytosis of Pam(3)CSK(4) is necessary for the complete TLR2/1-mediated pro-inflammatory cytokine response. We also show that TLR2/1 signaling from the endosome results in the induction of different inflammatory mediators than TLR2/1 signaling from the plasma membrane.Here we identify integrin α(3)β(1) as a novel regulator for the recognition of bacterial lipopeptides. We demonstrate that induction of a specific subset of cytokines is dependent upon integrin α(3)β(1)-mediated endocytosis of the ligand. In addition, we address an ongoing controversy regarding endosomal recognition of bacterial lipopeptides by demonstrating that TLR2/1 signals from within endosomal compartments as well as the plasma membrane, and that downstream responses may differ depending upon receptor localization. We propose that the regulation of endosomal TLR2/1 signaling by integrin α(3)β(1) serves as a mechanism for modulating inflammatory responses

Mammal responses to global changes in human activity vary by trophic group and landscape

Wildlife must adapt to human presence to survive in the Anthropocene, so it is critical to understand species responses to humans in different contexts. We used camera trapping as a lens to view mammal responses to changes in human activity during the COVID-19 pandemic. Across 163 species sampled in 102 projects around the world, changes in the amount and timing of animal activity varied widely. Under higher human activity, mammals were less active in undeveloped areas but unexpectedly more active in developed areas while exhibiting greater nocturnality. Carnivores were most sensitive, showing the strongest decreases in activity and greatest increases in nocturnality. Wildlife managers must consider how habituation and uneven sensitivity across species may cause fundamental differences in human–wildlife interactions along gradients of human influence.Peer reviewe

CCNE1 and survival of patients with tubo-ovarian high-grade serous carcinoma: An Ovarian Tumor Tissue Analysis consortium study

BACKGROUND: Cyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo-ovarian high-grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large-scale, histotype-specific validation has been performed. The hypothesis was that high-level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC. METHODS: Within the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated. RESULTS: High-level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high-level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08-1.47, p = .034, and HR, 1.18; 95% CI, 1.05-1.32, p = .015, respectively). This was also true for cases with combined high-level amplification/overexpression (HR, 1.26; 95% CI, 1.09-1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1-SD increase; 95% CI, 0.94-1.06; p = .58). CCNE1 high-level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss. CONCLUSION: This study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC

Role of Adrenomedullin in Lyme Disease▿

Borrelia burgdorferi stimulates a strong inflammatory response during infection of a mammalian host. To understand the mechanisms of immune regulation employed by the host to control this inflammatory response, we focused our studies on adrenomedullin, a peptide produced in response to bacterial stimuli that exhibits antimicrobial activity and regulates inflammatory responses by modulating the expression of inflammatory cytokines. Specifically, we investigated the effect of B. burgdorferi on the expression of adrenomedullin as well as the ability of adrenomedullin to dampen host inflammatory responses to the spirochete. The concentration of adrenomedullin in the synovial fluid of untreated Lyme arthritis patients was elevated compared with that in control osteoarthritis patient samples. In addition, coculture with B. burgdorferi significantly increased the expression of adrenomedullin in RAW264.7 macrophages through MyD88-, phosphatidylinositol 3-kinase (PI3-K)-, and p38-dependent signaling cascades. Furthermore, the addition of exogenous adrenomedullin to B. burgdorferi-stimulated RAW264.7 macrophages resulted in a significant decrease in the induction of proinflammatory cytokines. Taken together, these results suggest that B. burgdorferi increases the production of adrenomedullin, which in turn negatively regulates the B. burgdorferi-stimulated inflammatory response

IL-10 and beta-defensin deficiency are not responsible for increased inflammation in joints and hearts of <i>B. burgdorferi</i> infected Nod2 deficient mice.

<p><b>A</b>) BMDM from wild type (B6) and Nod2 deficient mice were stimulated with <i>B. burgdorferi</i> at MOI 10 for 6 hours. mRNA expression for <i>il-10</i> was measured by RT-qPCR. Values represent relative expression ± s.e.m. of three independent experiments, * p<0.05. <b>B</b>) IL-10 levels in the culture supernatant were measured by ELISA. For each experiment, the control value was set at 100%. Bars represent mean percent secretion relative to control cells ± s.e.m. of three independent experiments. Values for wild type (B6) cells for each experiment were normalized to 100% and other values are shown relative to B6. Because of the normalization, the s.e.m. for B6 is zero and no error bars are visible, * p<0.05. Wild type cells secreted a mean of 80 pg/ml and Nod2 deficient cells a mean of 310 pg/ml. <b>C</b>) Three or four day old wild type (B6) and Nod2 deficient mice were subcutaneously injected with <i>B. burgdorferi</i> at 1×10⁴ cells. Mice were sacrificed at 4 weeks post-infection and joints were collected and frozen in RNAlater. RNA from joints was isolated by grinding in TRIzol. Transcripts from joints were assessed by RT-qPCR for <i>il-10</i> and <i>mBD14</i> (<b>D</b>). Horizontal lines represent the median values.</p

Nod2 deficient mice exhibit increased arthritis and carditis with <i>B. burgdorferi</i> infection.

<p><b>A</b>) Three or four day old wild type (B6) and Nod2 deficient mice were injected subcutaneously with <i>B. burgdorferi</i> at 1×10⁴ cells. Mice were sacrificed at 4 weeks post-infection. Shown are caliper measurements of joint thickness in millimeters at time of sacrifice. <b>B</b>) Joints from mice infected in 3 independent experiments were sectioned and stained with H&E and scored blindly by a pathologist for inflammation using a scale of 0-normal, 1- mild, 2-moderate, or 3-severe inflammation. Horizontal lines on the graphs represent median values for the group. <b>C</b>) Pictured are representative images of the tibiotarsal joints from an infected wild type and a Nod2 deficient mouse infected with <i>B. burgdorferi</i>. In the Nod2 deficient mice, there is severe inflammation with destruction of the cartilage and thickened synovium. The wild type mouse shows minimal inflammation. <b>D</b>) Hearts from infected mice were sectioned and stained with H&E and scored blindly by a pathologist with a score of 0-normal, 1- mild, 2-moderate, or 3-severe inflammation. Horizontal lines on the graphs represent median values for the group. Significance was calculated using Fisher's exact test. <b>E</b>) Levels of <i>il-6</i> transcript were measured by RT-qPCR from joint tissue in wild type and Nod2 deficient mice. The lowest value in the group was arbitrarily set to a value of 1 and all other values are shown in reference to that value using the 2^-ΔΔCt method after normalization for the value of <i>nidogen</i>.</p

Nod2-mediated tolerance affects <i>ifn-α</i>, <i>cxcl10</i>, but not <i>ifit1</i> and <i>igtp</i> mRNA.

<p>Activated BMDM from wild type (B6) and Nod2 deficient mice were treated with MDP at 100 µg/ml 24 hours prior to stimulation to <i>B. burgdorferi</i> at an MOI 10. Just prior to infection, cells were washed in DMEM containing 10% FBS and fresh media was added. Cells were collected 16 hours post-infection for <i>inf-α</i> (<b>A</b>) and 6 hours post-infection for <i>cxcl10</i> (<b>B</b>), <i>igtp</i> (<b>C</b>) and <i>ifit1</i> (<b>D</b>)<b>.</b> mRNA expression was measured by RT-qPCR. Bars represent mean percent change ± s.e.m. of three independent experiments, * p<0.05.</p