GM-CSF distinctly impacts human monocytes and macrophages via ERK1/2-dependent pathways.

Human monocytes and macrophages are two major myeloid cell subsets with similar and distinct functions in tissue homeostasis and immune responses. GM-CSF plays a fundamental role in myeloid cell differentiation and activation. Hence, we compared the effects of GM-CSF on the expression of several immune mediators by human monocytes and monocyte-derived macrophages obtained from healthy donors. We report that GM-CSF similarly elevated the expression of CD80 and ICAM-1 and reduced HLA-DR levels on both myeloid cell subsets. However, GM-CSF increased the percentage of macrophages expressing surface IL-15 but reduced the proportion of monocytes carrying surface IL-15. Moreover, GM-CSF significantly increased the secretion of IL-4, IL-6, TNF, CXCL10, and IL-27 by macrophages while reducing the secretion of IL-4 and CXCL10 by monocytes. We show that GM-CSF triggered ERK1/2, STAT3, STAT5, and SAPK/JNK pathways in both myeloid subsets. Using a pharmacological inhibitor (U0126) preventing ERK phosphorylation, we demonstrated that this pathway was involved in both the GM-CSF-induced increase and decrease of the percentage of IL-15+ macrophages and monocytes, respectively. Moreover, ERK1/2 contributed to GM-CSF-triggered secretion of IL-4, IL-6, TNF, IL-27 and CXCL10 by macrophages. However, the ERK1/2 pathway exhibited different roles in monocytes and macrophages for the GM-CSF-mediated impact on surface makers (CD80, HLA-DR, and ICAM-1). Our data demonstrate that GM-CSF stimulation induces differential responses by human monocytes and monocyte-derived macrophages and that some but not all of these effects are ERK-dependent.

Identification of SARS-CoV-2-specific immune alterations in acutely ill patients.

Dysregulated immune profiles have been described in symptomatic patients infected with SARS-CoV-2. Whether the reported immune alterations are specific to SARS-CoV-2 infection or also triggered by other acute illnesses remains unclear. We performed flow cytometry analysis on fresh peripheral blood from a consecutive cohort of (a) patients hospitalized with acute SARS-CoV-2 infection, (b) patients of comparable age and sex hospitalized for another acute disease (SARS-CoV-2 negative), and (c) healthy controls. Using both data-driven and hypothesis-driven analyses, we found several dysregulations in immune cell subsets (e.g., decreased proportion of T cells) that were similarly associated with acute SARS-CoV-2 infection and non-COVID-19-related acute illnesses. In contrast, we identified specific differences in myeloid and lymphocyte subsets that were associated with SARS-CoV-2 status (e.g., elevated proportion of ICAM-1+ mature/activated neutrophils, ALCAM+ monocytes, and CD38+CD8+ T cells). A subset of SARS-CoV-2-specific immune alterations correlated with disease severity, disease outcome at 30 days, and mortality. Our data provide an understanding of the immune dysregulation specifically associated with SARS-CoV-2 infection among acute care hospitalized patients. Our study lays the foundation for the development of specific biomarkers to stratify SARS-CoV-2-positive patients at risk of unfavorable outcomes and to uncover candidate molecules to investigate from a therapeutic perspective.

NKG2D and Its Ligand MULT1 Contribute to Disease Progression in a Mouse Model of Multiple Sclerosis.

NKG2D is an activating receptor expressed on the surface of immune cells including subsets of T lymphocytes. NKG2D binds multiple ligands (NKG2DL) whose expression are differentially triggered in a cell type and stress specific manner. The NKG2D-NKG2DL interaction has been involved in autoimmune disorders but its role in animal models of multiple sclerosis (MS) remains incompletely resolved. Here we show that NKG2D and its ligand MULT1 contribute to the pathobiology of experimental autoimmune encephalomyelitis (EAE). MULT1 protein levels are increased in the central nervous system (CNS) at EAE disease peak; soluble MULT1 is elevated in the cerebrospinal fluid of both active and passive EAE. We establish that such soluble MULT1 enhances effector functions (e.g., IFNγ production) of activated CD8 T lymphocytes from wild type but not from NKG2D-deficient (Klrk1-/-) mice in vitro. The adoptive transfer of activated T lymphocytes from wild type donors induced a significantly reduced EAE disease in Klrk1-/- compared to wild type (Klrk1+/+) recipients. Characterization of T lymphocytes infiltrating the CNS of recipient mice shows that donor (CD45.1) rather than endogenous (CD45.2) CD4 T cells are the main producers of key cytokines (IFNγ, GM-CSF). In contrast, infiltrating CD8 T lymphocytes include mainly endogenous (CD45.2) cells exhibiting effector properties (NKG2D, granzyme B and IFNγ). Our data support the notion that endogenous CD8 T cells contribute to passive EAE pathobiology in a NKG2D-dependent manner. Collectively, our results point to the deleterious role of NKG2D and its MULT1 in the pathobiology of a MS mouse model.

Peripheral human CD4+CD8+ T lymphocytes exhibit a memory phenotype and enhanced responses to IL-2, IL-7 and IL-15.

CD4+CD8+ T lymphocytes account for 1-2% of circulating human T lymphocytes, but their frequency is augmented in several diseases. The phenotypic and functional properties of these T lymphocytes are still ill-defined. We performed an ex vivo characterization of CD4+CD8+ T lymphocytes from the blood of healthy individuals. We observed that CD4+CD8+ T lymphocytes exhibit several characteristics associated with memory T lymphocytes including the expression of chemokine receptors (e.g. CCR7, CXCR3, CCR6) and activation markers (e.g. CD57, CD95). Moreover, we showed that a greater proportion of CD4+CD8+ T lymphocytes have an enhanced capacity to produce cytokines (IFNγ, TNFα, IL-2, IL-4, IL-17A) and lytic enzymes (perforin, granzyme B) compared to CD4+ and/or CD8+ T lymphocytes. Finally, we assessed the impact of three key cytokines in T cell biology on these cells. We observed that IL-2, IL-7 and IL-15 triggered STAT5 phosphorylation in a greater proportion of CD4+CD8+ T lymphocytes compared to CD4 and CD8 counterparts. We demonstrate that CD4+CD8+ T lymphocytes from healthy donors exhibit a phenotypic profile associated with memory T lymphocytes, an increased capacity to produce cytokines and lytic enzymes, and a higher proportion of cells responding to key cytokines implicated in T cell survival, homeostasis and activation.