Deciphering the role of gammaherpesvirus-imprinted monocytes in preventing deleterious inflammation

Les gammaherpèsvirus (γHVs) sont des pathogènes très prévalents au sein de la population mondiale. Ces virus modulent le système immunitaire de l’hôte pour l’infecter de façon persistante et dans la plupart des cas, de façon asymptomatique. Dans certains cas, l’infection par les γHVs peut mener au développement d’immunopathologies avec de lourdes conséquences pour l’hôte. Les acteurs et les mécanismes à l’origine de ce déséquilibre immunitaire doivent être étudiés. Dans cette étude, nous montrons que l'infection pulmonaire par le gammaherpèsvirus murin 4 (MuHV-4), un γHV infectant la souris, entraîne le recrutement dans les voies aériennes de monocytes Ly6Chi qui contrôlent la réponse immunitaire de l'hôte. En effet, l'absence de ces monocytes provoque une immunopathologie sévère induite par le virus, associée à la libération systémique de médiateurs inflammatoires. D'un point de vue mécanistique, les monocytes suite à l’infection MuHV-4 recrutent des lymphocytes T CD4 dans les voies respiratoires et déclenchent des voies de signalisation immunosuppressives par l'intermédiaire de l'axe PD-L1/PD-1, freinant ainsi l'activation délétère des lymphocytes T CD4 cytotoxiques. Ces résultats mettent en évidence un nouveau rôle des monocytes Ly6Chi dans la modulation des fonctions des lymphocytes T CD4 et révèlent des voies clés qui pourraient être ciblées pour interférer avec la cascade immunopathologique néfaste induite au cours des infections virales respiratoires.Abstract: Gammaherpesviruses (γHVs) have co-evolved with their host, leading to a remarkably high infection prevalence and establishment of latency. The lifelong persistence of γHVs in hosts appears to broadly shape host immunity, and we show here that pulmonary infection with Murid herpesvirus 4 (MuHV-4), a mouse γHV, drives the recruitment of Ly6Chi monocytes (MOs) into the airway, thereby modulating the host immune response. The absence of Ly6Chi MOs is associated with severe virus-induced immunopathology and the systemic release of inflammatory mediators. Mechanistically, MuHV-4-imprinted MOs recruit CD4 T cells to the airways and trigger immunosuppressive signalling pathways through the PD-L1/PD-1 axis, thereby dampening the deleterious activation of cytotoxic CD4 T cells. These results uncover a role for Ly6Chi MOs in modulating CD4 T cell functions and reveal pathways that could be targeted therapeutically to reduce detrimental immunopathological responses associated with respiratory viral infections. One Sentence Summary: γHV-imprinted monocytes limit lethal infection by orchestrating the balance between regulatory and detrimental cytotoxic properties of CD4 T cells

Functional Phenotyping of Lung Mouse CD4+ T Cells Using Multiparametric Flow Cytometry Analysis.

peer reviewedGammaherpesviruses such as Epstein-Barr virus (EBV) are major modulators of the immune responses of their hosts. In the related study (PMID: 35857578), we investigated the role for Ly6Chi monocytes in shaping the function of effector CD4+ T cells in the context of a murine gammaherpesvirus infection (Murid gammaherpesvirus 4) as a model of human EBV. In order to unravel the polyfunctional properties of CD4+ T-cell subsets, we used multiparametric flow cytometry to perform intracellular staining on lung cells. As such, we have developed herein an intracellular staining workflow to identify on the same samples the cytotoxic and/or regulatory properties of CD4+ lymphocytes at the single-cell level. Briefly, following perfusion, collection, digestion, and filtration of the lung to obtain a single-cell suspension, lung cells were cultured for 4 h with protein transport inhibitors and specific stimulation media to accumulate cytokines of interest and/or cytotoxic granules. After multicolor surface labeling, fixation, and mild permeabilization, lung cells were stained for intracytoplasmic antigens and analyzed with a Fortessa 4-laser cytometer. This method of quantifying cytotoxic mediators as well as pro- or anti-inflammatory cytokines by flow cytometry has allowed us to decipher at high resolution the functional heterogeneity of lung CD4+ T cells recruited after a viral infection. Therefore, this analysis provided a better understanding of the importance of CD4+ T-cell regulation to prevent the development of virus-induced immunopathologies in the lung. Key features • High-resolution profiling of the functional properties of lung-infiltrating CD4+ T cells after viral infection using conventional multiparametric flow cytometry. • Detailed protocol for mouse lung dissection, preparation of single-cell suspension, and setup of multicolor surface/intracellular staining. • Summary of optimal ex vivo restimulation conditions for investigating the functional polarization and cytokine production of lung-infiltrating CD4+ T cells. • Comprehensive compilation of necessary biological and technical controls to ensure reliable data analysis and interpretation

Ly6Chi monocytes balance regulatory and cytotoxic CD4 T cell responses to control virus-induced immunopathology.

peer reviewedGammaherpesviruses (γHVs) have coevolved with their host, leading to a remarkably high infection prevalence and establishment of latency. The lifelong persistence of γHVs in hosts appears to broadly shape host immunity, and we show here that pulmonary infection with Murid herpesvirus 4 (MuHV-4), a mouse γHV, drives the recruitment of Ly6Chi monocytes (MOs) into the airway, thereby modulating the host immune response. The absence of Ly6Chi MOs is associated with severe virus-induced immunopathology and the systemic release of inflammatory mediators. Mechanistically, MuHV-4-imprinted MOs recruit CD4 T cells to the airways and trigger immunosuppressive signaling pathways through the PD-L1/PD-1 axis, thereby dampening the deleterious activation of cytotoxic CD4 T cells. These results uncover a role for Ly6Chi MOs in modulating CD4 T cell functions and reveal pathways that could be targeted therapeutically to reduce detrimental immunopathological responses associated with respiratory viral infections

Ly6Chi monocytes are key orchestrators of gammaherpesvirus lifecycle

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Role of monocytes in Murid gammaherpesvirus 4 lifecycle

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Ly6Chi monocytes are key immunoregulators of gammaherpesvirus infection

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