Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>The complement system is thought to be involved in the pathogenesis of numerous neurological diseases. We previously reported that pre-treatment of murine cortico-hippocampal neuronal cultures with the complement derived anaphylatoxin C5a, protects against glutamate mediated apoptosis. Our present study with C5a receptor knock out (C5aRKO) mice corroborates that the deficiency of C5a renders C5aRKO mouse more susceptible to apoptotic injury <it>in vivo</it>. In this study we explored potential upstream mechanisms involved in C5a mediated neuroprotection <it>in vivo </it>and <it>in vitro</it>.</p> <p>Methods</p> <p>Based on evidence suggesting that reduced expression of glutamate receptor subunit 2 (GluR2) may influence apoptosis in neurons, we studied the effect of human recombinant C5a on GluR2 expression in response to glutamate neurotoxicity. Glutamate analogs were injected into C5aRKO mice or used to treat <it>in vitro </it>neuronal culture and GluR2 expression were assessed in respect with cell death.</p> <p>Results</p> <p>In C5aRKO mice we found that the neurons are more susceptible to excitotoxicity resulting in apoptotic injury in the absence of the C5a receptor compared to WT control mice. Our results suggest that C5a protects against apoptotic pathways in neurons <it>in vitro </it>and <it>in vivo </it>through regulation of GluR2 receptor expression.</p> <p>Conclusion</p> <p>Complement C5a neuroprotects through regulation of GluR2 receptor subunit.</p

Comprehensive immunophenotyping of solid tumor-infiltrating immune cells reveals the expression characteristics of LAG-3 and its ligands

BackgroundImmune cell expression profiling from patient samples is critical for the successful development of immuno-oncology agents and is useful to understand mechanism-of-action, to identify exploratory biomarkers predictive of response, and to guide treatment selection and combination therapy strategies. LAG-3 is an inhibitory immune checkpoint that can suppress antitumor T-cell responses and targeting LAG-3, in combination with PD-1, is a rational approach to enhance antitumor immunity that has recently demonstrated clinical success. Here, we sought to identify human immune cell subsets that express LAG-3 and its ligands, to characterize the marker expression profile of these subsets, and to investigate the potential relationship between LAG-3 expressing subsets and clinical outcomes to immuno-oncology therapies.MethodsComprehensive high-parameter immunophenotyping was performed using mass and flow cytometry of tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) from two independent cohorts of samples from patients with various solid tumor types. Profiling of circulating immune cells by single cell RNA-seq was conducted on samples from a clinical trial cohort of melanoma patients treated with immunotherapy.ResultsLAG-3 was most highly expressed by subsets of tumor-infiltrating CD8 T central memory (TCM) and effector memory (TEM) cells and was frequently co-expressed with PD-1. We determined that these PD-1+ LAG-3+ CD8 memory T cells exhibited a unique marker profile, with greater expression of activation (CD69, HLA-DR), inhibitory (TIM-3, TIGIT, CTLA-4) and stimulatory (4-1BB, ICOS) markers compared to cells that expressed only PD-1 or LAG-3, or that were negative for both checkpoints. In contrast to tumors, LAG-3 expression was more limited in circulating immune cells from healthy donors and solid tumor patients. Additionally, we found abundant expression of the LAG-3 ligands MHC-II and galectin-3 in diverse immune cell types, whereas FGL1 and LSECtin were minimally expressed by immune cells in the tumor microenvironment (TME). Lastly, we found an inverse relationship between baseline and on-treatment levels of circulating LAG3 transcript-expressing CD8 memory T cells and response to combination PD-1 and CTLA-4 blockade in a clinical trial cohort of melanoma patients profiled by scRNAseq.ConclusionsThese results provide insights into the nature of LAG-3- and ligand-expressing immune cells within the TME, and suggest a biological basis for informing mechanistic hypotheses, treatment selection strategies, and combination immunotherapy approaches to support continued development of dual PD-1 and LAG-3 blockade

Effects of tobacco smoke on gene expression and cellular pathways in a cellular model of oral leukoplakia

Abstract In addition to being causally linked to the formation of multiple tumor types, tobacco use has been associated with decreased efficacy of anticancer treatment and reduced survival time. A detailed understanding of the cellular mechanisms that are affected by tobacco smoke (TS) should facilitate the development of improved preventive and therapeutic strategies. We have investigated the effects of a TS extract on the transcriptome of MSK-Leuk1 cells, a cellular model of oral leukoplakia. Using Affymetrix HGU133 Plus 2 arrays, 411 differentially expressed probe sets were identified. The observed transcriptome changes were grouped according to functional information and translated into molecular interaction network maps and signaling pathways. Pathways related to cellular proliferation, inflammation, apoptosis, and tissue injury seemed to be perturbed. Analysis of networks connecting the affected genes identified specific modulated molecular interactions, hubs, and key transcription regulators. Thus, TS was found to induce several epidermal growth factor receptor (EGFR) ligands forming an EGFR-centered molecular interaction network, as well as several aryl hydrocarbon receptor-dependent genes, including the xenobiotic metabolizing enzymes CYP1A1 and CYP1B1. Notably, the latter findings in vitro are consistent with our parallel finding that CYP1A1 and CYP1B1 levels were increased in oral mucosa of smokers. Collectively, these results offer insights into the mechanisms underlying the procarcinogenic effects of TS and raise the possibility that inhibitors of EGFR or aryl hydrocarbon receptor signaling will prevent or delay the development of TS-related tumors. Moreover, the inductive effects of TS on xenobiotic metabolizing enzymes may help explain the reduced efficacy of chemotherapy, and suggest targets for chemopreventive agents in smokers

Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 and -1

<p><b>Copyright information:</b></p><p>Taken from "Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 and "</p><p>http://www.jneuroinflammation.com/content/5/1/5</p><p>Journal of Neuroinflammation 2008;5():5-5.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2246107.</p><p></p>termates (WT) (n = 4). Frozen hippocampal brain sections from these mice were then probed immunocytochemically for GluR2 expression using GluR2 antibody. GluR2 immunoreactivity was quantitated from 8–10 randomly selected fields and results were expressed as a percent of vehicle treated WT controls. KA significantly decreased the expression of GluR2 immunoreactivity in the mossy fiber region of C5aRKO mice (Fig. A, B). The arrow points towards the region in the inset which shows the GluR2 immunoreactivity in the mossy fibers of the CA3 region. Hippocampal GluR2 immunoreactivity was reduced in C5aRKO mice treated with KA compared to C5aRKO mice treated with vehicle. All results are expressed as mean ± SD. Difference between groups was assessed by t-test, *< 0.05. SO = stratum oriens; SR = stratum radiatum; SLM = stratum lacunosum moleculare