RESUMO
Autoimmunity can potentially pre-dispose to, exacerbate or ameliorate pathogenic infections. The current study was designed to compare and understand the infection outcomes with Salmonella enterica serovar Typhimurium ATCC 14028s (S. Typhimurium) wild type (WT) and attenuated ΔrpoS strains, in autoimmune-prone lpr mice. C57BL/6 (B6) and B6/lpr (lpr) 6-8 weeks old mice were orally infected with S. Typhimurium WT and ΔrpoS strains. Disease outcomes were assessed with respect to survival, organ bacterial load, tissue damage and inflammation in infected mice. The acute infection stage (day 4) was examined and compared to the later stages (up to day 12) post ΔrpoS infection. S. Typhimurium WT exhibited an acute and lethal infection in both B6 and lpr mice. However, the ΔrpoS strain exhibited prolonged infection with reduced mortality in B6 mice but complete mortality in lpr mice. During late infection, bacterial load and serum IFNγ levels were higher in the ΔrpoS strain infected lpr mice compared to B6 mice. The ΔrpoS strain infected lpr mice also exhibited greater bacterial faecal shedding and greater tissue histopathological changes. Interestingly, ΔrpoS-infected B6 mice displayed minimal microbial load in the brain; however, sustained brain bacterial load was observed in ΔrpoS-infected lpr mice, corresponding to abnormal gait. Overall, S. Typhimurium ΔrpoS is competent in establishing infection but compromised in sustaining it. Nonetheless, lpr mice are less efficient in controlling this attenuated infection. The findings from the study demonstrate that genetic pre-disposition to autoimmunity is sufficient for greater host susceptibility to infection by attenuated S. Typhimurium strains.
Assuntos
Salmonella enterica , Salmonella typhimurium , Animais , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Salmonella typhimurium/genética , SorogrupoRESUMO
Accurate detection of bacterial antibiotic sensitivity is crucial for theranostics and the containment of antibiotic-resistant infections. However, the intricate task of detecting and quantifying the antibiotic-induced changes in the bacterial cytoplasmic membrane, and their correlation with other metabolic pathways leading to antibiotic resistance, poses significant challenges. Using a novel class of 4-aminophthalimide (4AP)-based fluorescent dyes with precisely tailored alkyl chains, namely 4AP-C9 and 4AP-C13, we quantify stress-mediated alterations in E. coli membranes. Leveraging the unique depth-dependent positioning and environment-sensitive fluorescence properties of these dyes, we detect antibiotic-induced membrane damage through single-cell imaging and monitoring the fluorescence peak maxima difference ratio (PMDR) of the dyes within the bacterial membrane, complemented by other methods. The correlation between the ROS-induced cytoplasmic membrane damage and the PMDR of dyes quantifies sensitivity against bactericidal antibiotics, which correlates to antibiotic-induced lipid peroxidation. Significantly, our findings largely extend to clinical isolates of E. coli and other ESKAPE pathogens like K. pneumoniae and Enterobacter subspecies. Our data reveal that 4AP-Cn probes can potentially act as precise scales to detect antibiotic-induced membrane damage ("thinning") occurring at a subnanometer scale through the quantification of dyes' PMDR, making them promising membrane dyes for rapid detection of bacterial antibiotic resistance, distinguishing sensitive and resistant infections with high specificity in a clinical setup.
Assuntos
Antibacterianos , Membrana Celular , Escherichia coli , Corantes Fluorescentes , Testes de Sensibilidade Microbiana , Corantes Fluorescentes/química , Escherichia coli/efeitos dos fármacos , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana , Humanos , Enterobacter/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacosRESUMO
Introduction: Interferon-gamma (IFN-γ), the sole member of the type-II interferon family, is well known to protect the host from infectious diseases as well as mount anti-tumor responses. The amounts of IFN-γ in the tumor microenvironment determine the host responses against tumors; however, several tumors employ evasive strategies by responding to low IFN-γ signaling. Methods: In this study, the response of various tumor cell lines to IFN-γ was studied in vitro. Results: IFN-γ-activation increases glycolytic flux and reduces mitochondrial function in a nitric oxide (NO)- and reactive oxygen species (ROS)-dependent manner in the H6 hepatoma tumor cell line. The higher glycolysis further fueled NO and ROS production, indicating a reciprocal regulation. These processes are accompanied by Hypoxia inducing factor (HIF)-1α stabilization and HIF-1α-dependent augmentation of the glycolytic flux. The IFN-γ enhancement of lactate production also occurred in other NO-producing cell lines: RAW 264.7 monocyte/macrophage and Renca renal adenocarcinoma. However, two other tumor cell lines, CT26 colon carcinoma and B16F10 melanoma, did not produce NO and lactate upon IFN-γ-activation. HIF-1α stabilization upon IFN-γ-activation led to lower cell growth of B16F10 but not CT26 cells. Importantly, the IFN-γ-activation of both CT26 and B16F10 cells demonstrated significant cellular growth reduction upon metabolic rewiring by exogenous administration of potassium lactate. Discussion: Clinical studies have shown the crucial roles of IFN-γ for successful cancer immunotherapies involving checkpoint inhibitors and chimeric antigen receptor T cells. The positive implications of this study on the metabolic modulation of IFN-γ activation on heterogeneous tumor cells are discussed.
Assuntos
Interferon gama , Neoplasias , Humanos , Interferon gama/metabolismo , Óxido Nítrico/metabolismo , Ácido Láctico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Glicólise , Linhagem Celular Tumoral , Hipóxia/metabolismo , Neoplasias/terapiaRESUMO
Interferon-gamma (IFN-γ) is a type II interferon produced primarily by T cells and natural killer cells. IFN-γ induces the expression of inducible nitric oxide synthase (NOS2) to catalyze Nitric Oxide (NO) production in various immune and non-immune cells. Excessive IFN-γ-activated NO production is implicated in several inflammatory diseases, including peritonitis and inflammatory bowel diseases. In this study, we screened the LOPAC®1280 library in vitro on the H6 mouse hepatoma cell line to identify novel non-steroidal small molecule inhibitors of IFN-γ-induced NO production. Compounds with the highest inhibitory activity were validated, which led to identifying the lead compounds: pentamidine, azithromycin, rolipram, and auranofin. Auranofin was the most potent compound determined based on IC50 and goodness of fit analyses. Mechanistic investigations revealed that majority of the lead compounds suppress the IFN-γ-induced transcription of Nos2 without negatively affecting NO-independent processes, such as the IFN-γ-induced transcription of Irf1, Socs1 and MHC class 1 surface expression. However, all four compounds lower IFN-γ-induced reactive oxygen species amounts. In addition, auranofin significantly reduced IFN-γ-mediated NO and IL6 production in resident as well as thioglycolate-elicited peritoneal macrophages (PMs). Finally, in vivo testing of the lead compounds in the pre-clinical DSS-induced ulcerative colitis mice model revealed pentamidine and auranofin to be the most potent and protective lead compounds. Also, pentamidine and auranofin greatly increase the survival of mice in another inflammatory model: Salmonella Typhimurium-induced sepsis. Overall, this study identifies novel anti-inflammatory compounds targeting IFN-γ-induced NO-dependent processes to alleviate two distinct inflammatory models of disease.
Assuntos
Colite , Sepse , Camundongos , Animais , Interferon gama/metabolismo , Óxido Nítrico/metabolismo , Salmonella typhimurium/fisiologia , Auranofina/farmacologia , Auranofina/uso terapêutico , Pentamidina , Ensaios de Triagem em Larga Escala , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Colite/induzido quimicamente , Colite/tratamento farmacológicoRESUMO
Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice. This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival. Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.
Assuntos
Peritonite , Sepse , Animais , Ceco/patologia , Ceco/cirurgia , Citocinas , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos , Peritonite/microbiologia , Peritonite/patologia , Reprodutibilidade dos Testes , Salmonella typhimurium , Sepse/microbiologiaRESUMO
The hallmarks of the adaptive immune response are specificity and memory. The cellular response is mediated by T cells which express cell surface T cell receptors (TCRs) that recognize peptide antigens in complex with major histocompatibility complex (MHC) molecules on antigen presenting cells (APCs). However, binding of cognate TCRs with MHC-peptide complexes alone (signal 1) does not trigger optimal T cell activation. In addition to signal 1, the binding of positive and negative costimulatory receptors to their ligands modulates T cell activation. This complex signaling network prevents aberrant activation of T cells. CD28 is the main positive costimulatory receptor on nai¨ve T cells; upon activation, CTLA4 is induced but reduces T cell activation. Further studies led to the identification of additional negative costimulatory receptors known as checkpoints, e.g. PD1. This review chronicles the basic studies in T cell costimulation that led to the discovery of checkpoint inhibitors, i.e. antibodies to negative costimulatory receptors (e.g. CTLA4 and PD1) which reduce tumor growth. This discovery has been recognized with the award of the 2018 Nobel prize in Physiology/Medicine. This review highlights the structural and functional roles of costimulatory receptors, the mechanisms by which checkpoint inhibitors work, the challenges encountered and future prospects.