Increased levels of GM-CSF and CXCL10 and low CD8+ memory stem T Cell count are markers of immunosenescence and severe COVID-19 in older people.

BACKGROUND: Ageing leads to altered immune responses, resulting in higher susceptibility to certain infections in the elderly. Immune ageing is a heterogeneous process also associated with inflammaging, a low-grade chronic inflammation. Altered cytotoxic T cell responses and cytokine storm have previously been described in severe COVID-19 cases, however the parameters responsible for such immune response failures are not well known. The aim of our study was to characterize CD8+ T cells and cytokines associated with ageing, in a cohort of patients aged over 70 years stratified by COVID-19 severity. RESULTS: One hundred and four patients were included in the study. We found that, in older people, COVID-19 severity was associated with (i) higher level of GM-CSF, CXCL10 (IP-10), VEGF, IL-1ß, CCL2 (MCP-1) and the neutrophil to lymphocyte ratio (NLR), (ii) increased terminally differentiated CD8+T cells, and (ii) decreased early precursors CD8+ T stem cell-like memory cells (TSCM) and CD27+CD28+. The cytokines mentioned above were found at higher concentrations in the COVID-19+ older cohort compared to a younger cohort in which they were not associated with disease severity. CONCLUSIONS: Our results highlight the particular importance of the myeloid lineage in COVID-19 severity among older people. As GM-CSF and CXCL10 were not associated with COVID-19 severity in younger patients, they may represent disease severity specific markers of ageing and should be considered in older people care.

Loss of hematopoietic progenitors heterogeneity is an adverse prognostic factor in lower-risk myelodysplastic neoplasms.

Myelodysplastic neoplasms (MDS) are characterized by clonal evolution starting from the compartment of hematopoietic stem and progenitors cells (HSPCs), leading in some cases to leukemic transformation. We hypothesized that deciphering the diversity of the HSPCs compartment may allow for the early detection of an emergent sub-clone that drives disease progression. Deep analysis of HSPCs repartition by multiparametric flow cytometry revealed a strong disorder of the hematopoietic branching system in most patients at diagnosis with different phenotypic signatures closely related to specific MDS features. In two independent cohorts of 131 and 584 MDS, the HSPCs heterogeneity quantified through entropy calculation was decreased in 47% and 46% of cases, reflecting a more advanced state of the disease with deeper cytopenias, higher IPSS-R risk and accumulation of somatic mutations. We demonstrated that patients with lower-risk MDS and low CD34 + CD38+HSPCs entropy had an adverse outcome and that this parameter is as an independent predictive biomarker for progression free survival, leukemia free survival and overall survival. Analysis of HSPCs repartition at diagnosis represents therefore a very powerful tool to identify lower-risk MDS patients with a worse outcome and valuable for clinical decision-making, which could be fully integrated in the MDS diagnostic workflow.

JAK2V617F mutation drives vascular resident macrophages toward a pathogenic phenotype and promotes dissecting aortic aneurysm.

JAK2V617F mutation is associated with an increased risk for athero-thrombotic cardiovascular disease, but its role in aortic disease development and complications remains unknown. In a cohort of patients with myeloproliferative neoplasm, JAK2V617F mutation was identified as an independent risk factor for dilation of both the ascending and descending thoracic aorta. Using single-cell RNA-seq, complementary genetically-modified mouse models, as well as pharmacological approaches, we found that JAK2V617F mutation was associated with a pathogenic pro-inflammatory phenotype of perivascular tissue-resident macrophages, which promoted deleterious aortic wall remodeling at early stages, and dissecting aneurysm through the recruitment of circulating monocytes at later stages. Finally, genetic manipulation of tissue-resident macrophages, or treatment with a Jak2 inhibitor, ruxolitinib, mitigated aortic wall inflammation and reduced aortic dilation and rupture. Overall, JAK2V617F mutation drives vascular resident macrophages toward a pathogenic phenotype and promotes dissecting aortic aneurysm.

Hematopoietic differentiation is characterized by a transient peak of entropy at a single-cell level.

BACKGROUND: Mature blood cells arise from hematopoietic stem cells in the bone marrow by a process of differentiation along one of several different lineage trajectories. This is often represented as a series of discrete steps of increasing progenitor cell commitment to a given lineage, but as for differentiation in general, whether the process is instructive or stochastic remains controversial. Here, we examine this question by analyzing single-cell transcriptomic data from human bone marrow cells, assessing cell-to-cell variability along the trajectories of hematopoietic differentiation into four different types of mature blood cells. The instructive model predicts that cells will be following the same sequence of instructions and that there will be minimal variability of gene expression between them throughout the process, while the stochastic model predicts a role for cell-to-cell variability when lineage commitments are being made. RESULTS: Applying Shannon entropy to measure cell-to-cell variability among human hematopoietic bone marrow cells at the same stage of differentiation, we observed a transient peak of gene expression variability occurring at characteristic points in all hematopoietic differentiation pathways. Strikingly, the genes whose cell-to-cell variation of expression fluctuated the most over the course of a given differentiation trajectory are pathway-specific genes, whereas genes which showed the greatest variation of mean expression are common to all pathways. Finally, we showed that the level of cell-to-cell variation is increased in the most immature compartment of hematopoiesis in myelodysplastic syndromes. CONCLUSIONS: These data suggest that human hematopoietic differentiation could be better conceptualized as a dynamical stochastic process with a transient stage of cellular indetermination, and strongly support the stochastic view of differentiation. They also highlight the need to consider the role of stochastic gene expression in complex physiological processes and pathologies such as cancers, paving the way for possible noise-based therapies through epigenetic regulation.

SLIT2/ROBO signaling in tumor-associated microglia and macrophages drives glioblastoma immunosuppression and vascular dysmorphia.

SLIT2 is a secreted polypeptide that guides migration of cells expressing Roundabout 1 and 2 (ROBO1 and ROBO2) receptors. Herein, we investigated SLIT2/ROBO signaling effects in gliomas. In patients with glioblastoma (GBM), SLIT2 expression increased with malignant progression and correlated with poor survival and immunosuppression. Knockdown of SLIT2 in mouse glioma cells and patient-derived GBM xenografts reduced tumor growth and rendered tumors sensitive to immunotherapy. Tumor cell SLIT2 knockdown inhibited macrophage invasion and promoted a cytotoxic gene expression profile, which improved tumor vessel function and enhanced efficacy of chemotherapy and immunotherapy. Mechanistically, SLIT2 promoted microglia/macrophage chemotaxis and tumor-supportive polarization via ROBO1- and ROBO2-mediated PI3K-γ activation. Macrophage Robo1 and Robo2 deletion and systemic SLIT2 trap delivery mimicked SLIT2 knockdown effects on tumor growth and the tumor microenvironment (TME), revealing SLIT2 signaling through macrophage ROBOs as a potentially novel regulator of the GBM microenvironment and immunotherapeutic target for brain tumors.

Endothelial Cell Indoleamine 2, 3-Dioxygenase 1 Alters Cardiac Function After Myocardial Infarction Through Kynurenine.

BACKGROUND: Ischemic cardiovascular diseases, particularly acute myocardial infarction (MI), is one of the leading causes of mortality worldwide. Indoleamine 2, 3-dioxygenase 1 (IDO) catalyzes 1 rate-limiting step of L-tryptophan metabolism, and emerges as an important regulator of many pathological conditions. We hypothesized that IDO could play a key role to locally regulate cardiac homeostasis after MI. METHODS: Cardiac repair was analyzed in mice harboring specific endothelial or smooth muscle cells or cardiomyocyte or myeloid cell deficiency of IDO and challenged with acute myocardial infarction. RESULTS: We show that kynurenine generation through IDO is markedly induced after MI in mice. Total genetic deletion or pharmacological inhibition of IDO limits cardiac injury and cardiac dysfunction after MI. Distinct loss of function of IDO in smooth muscle cells, inflammatory cells, or cardiomyocytes does not affect cardiac function and remodeling in infarcted mice. In sharp contrast, mice harboring endothelial cell-specific deletion of IDO show an improvement of cardiac function as well as cardiomyocyte contractility and reduction in adverse ventricular remodeling. In vivo kynurenine supplementation in IDO-deficient mice abrogates the protective effects of IDO deletion. Kynurenine precipitates cardiomyocyte apoptosis through reactive oxygen species production in an aryl hydrocarbon receptor-dependent mechanism. CONCLUSIONS: These data suggest that IDO could constitute a new therapeutic target during acute MI.

Multidimensional Proteomic Approach of Endothelial Progenitors Demonstrate Expression of KDR Restricted to CD19 Cells.

Endothelial progenitor cells (EPCs) are involved in vasculogenesis and cardiovascular diseases. However, the phenotype of circulating EPCs remains elusive but they are more often described as CD34+KDR+. The aim of the study was to extensively characterize circulating potential vasculogenic stem cell candidates in two populations of patients with cardiovascular disease by powerful multidimensional single cell complementary cytometric approaches (mass, imaging and flow). We identified cellular candidates in one patient before and after bioprosthetic total artificial heart implantation and results were confirmed in healthy peripheral and cord blood by mass cytometry. We also quantified cellular candidates in 10 patients with different COVID-19 severity. Both C-TAH implantation and COVID-19 at critical stage induce a redistribution of circulating CD34+ and CD19+ sub-populations in peripheral blood. After C-TAH implantation, circulating CD34+ progenitor cells expressed c-Kit stem marker while specific subsets CD34+CD133-/+CD45-/dimc-Kit+KDR- were mobilized. KDR was only expressed by CD19+ B-lymphocytes and CD14+ monocytes subpopulations in circulation. We confirmed by mass cytometry this KDR expression on CD19+ in healthy peripheral and cord blood, also with a VE-cadherin expression, confirming absence of endothelial lineage marker on CD34+ subtypes. In COVID-19, a significant mobilization of CD34+c-Kit+KDR- cells was observed between moderate and critical COVID-19 patients regardless CD133 or CD45 expression. In order to better evaluate EPC phenotype, we performed imaging flow cytometry measurements of immature CD34+KDR+ cells in cord blood and showed that, after elimination of non-circular events, those cells were all CD19+. During COVID-19, a significant mobilization of CD19+KDR+ per million of CD45+ cells was observed between moderate and critical COVID-19 patients regardless of CD34 expression. CD34+c-Kit+ cells are mobilized in both cardiovascular disease described here. KDR cells in peripheral blood are CD19 positive cells and are not classic vasculogenic stem and/or progenitor cells. A better evaluation of c-Kit and KDR expressing cells will lead to the redefinition of circulating endothelial progenitors.Graphical abstract Central illustration figure. Multidimensional proteomic approach of endothelial progenitors demonstrate expression of KDR restricted to CD19 cells. Endothelial progenitor cells (EPCs) are involved in cardiovascular diseases, however their phenotype remains elusive. We elucidated here EPCs phenotype by a deep characterization by multidimensional single cell complementary cytometric approaches after Bioprosthetic total artificial heart implantation and during COVID-19. We showed a redistribution of circulating CD34+ and CD19+ sub-populations in both situations. None of the immature cell population expresses KDR. Mobilized CD34+ expressed c-Kit. Imaging flow cytometry demonstrated that CD34+KDR+ cells, after elimination of non-circular events, are all CD19+. Our results suggest a new definition of circulating EPCs and emphasize involvement of CD19 cells in cardiovascular disease.

ORAI2 modulates store-operated calcium entry and T cell-mediated immunity.

Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels is critical for lymphocyte function and immune responses. CRAC channels are hexamers of ORAI proteins that form the channel pore, but the contributions of individual ORAI homologues to CRAC channel function are not well understood. Here we show that deletion of Orai1 reduces, whereas deletion of Orai2 increases, SOCE in mouse T cells. These distinct effects are due to the ability of ORAI2 to form heteromeric channels with ORAI1 and to attenuate CRAC channel function. The combined deletion of Orai1 and Orai2 abolishes SOCE and strongly impairs T cell function. In vivo, Orai1/Orai2 double-deficient mice have impaired T cell-dependent antiviral immune responses, and are protected from T cell-mediated autoimmunity and alloimmunity in models of colitis and graft-versus-host disease. Our study demonstrates that ORAI1 and ORAI2 form heteromeric CRAC channels, in which ORAI2 fine-tunes the magnitude of SOCE to modulate immune responses.

Regulation of Foxp3+ inducible regulatory T cell stability by SOCS2.

Suppressor of cytokine signaling (SOCS) proteins are key regulators of CD4(+) T cell differentiation, and in particular, we have recently shown that SOCS2 inhibits the development of Th2 cells and allergic immune responses. Interestingly, transcriptome analyses have identified SOCS2 as being preferentially expressed in both natural regulatory T cells (Tregs) and inducible Tregs (iTregs); however, the role of SOCS2 in Foxp3(+) Treg function or development has not been fully elucidated. In this study, we show that despite having no effect on natural Treg development or function, SOCS2 is highly expressed in iTregs and required for the stable expression of Foxp3 in iTregs in vitro and in vivo. Indeed, SOCS2-deficient CD4(+) T cells upregulated Foxp3 following in vitro TGF-ß stimulation, but failed to maintain stable expression of Foxp3. Moreover, in vivo generation of iTregs following OVA feeding was impaired in the absence of SOCS2 and could be rescued in the presence of IL-4 neutralizing Ab. Following IL-4 stimulation, SOCS2-deficient Foxp3(+) iTregs secreted elevated IFN-γ and IL-13 levels and displayed enhanced STAT6 phosphorylation. Therefore, we propose that SOCS2 regulates iTreg stability by downregulating IL-4 signaling. Moreover, SOCS2 is essential to maintain the anti-inflammatory phenotype of iTregs by preventing the secretion of proinflammatory cytokines. Collectively, these results suggest that SOCS2 may prevent IL-4-induced Foxp3(+) iTreg instability. Foxp3(+) iTregs are key regulators of immune responses at mucosal surfaces; therefore, this dual role of SOCS2 in both Th2 and Foxp3(+) iTregs reinforces SOCS2 as a potential therapeutic target for Th2-biased diseases.

Intestinal antimicrobial peptides during homeostasis, infection, and disease.

Antimicrobial peptides (AMPs), including defensins and cathelicidins, constitute an arsenal of innate regulators of paramount importance in the gut. The intestinal epithelium is exposed to myriad of enteric pathogens and these endogenous peptides are essential to fend off microbes and protect against infections. It is becoming increasingly evident that AMPs shape the composition of the commensal microbiota and help maintain intestinal homeostasis. They contribute to innate immunity, hence playing important functions in health and disease. AMP expression is tightly controlled by the engagement of pattern recognition receptors (PRRs) and their impairment is linked to abnormal host responses to infection and inflammatory bowel diseases (IBD). In this review, we provide an overview of the mucosal immune barriers and the intricate crosstalk between the host and the microbiota during homeostasis. We focus on the AMPs and pay particular attention to how PRRs promote their secretion in the intestine. Furthermore, we discuss their production and main functions in three different scenarios, at steady state, throughout infection with enteric pathogens and IBD.

Regulation of CD4+ T-cell polarization by suppressor of cytokine signalling proteins.

Suppressors of cytokine signalling (SOCS) proteins are induced in responses to many stimuli and by binding to cytokine receptors and associated janus kinase (JAK) proteins, directly regulate the activation of the signal transducers and activators of transcription (STATs). STAT proteins regulate the expression of many genes required for the differentiation of various CD4(+) T helper cell lineages, and there is now accumulating evidence that SOCS also play essential roles in the regulation and maintenance of CD4(+) T-cell polarization. As it is now clear that CD4(+) T cells are more plastic than initially thought, it is of particular importance to understand the molecular mechanisms regulating CD4(+) T-cell differentiation. Here we review the current understanding of how STATs and SOCS act in concert to influence the polarization of CD4(+) T cells and highlight the relevance of this in disease.

SOCS2 regulates T helper type 2 differentiation and the generation of type 2 allergic responses.

The incidence of allergy and asthma in developed countries is on the increase and this trend looks likely to continue. CD4(+) T helper 2 (Th2) cells are major drivers of these diseases and their commitment is controlled by cytokines such as interleukin 4, which are in turn regulated by the suppressor of cytokine signaling (SOCS) proteins. We report that SOCS2(-/-) CD4(+) T cells show markedly enhanced Th2 differentiation. SOCS2(-/-) mice, as well as RAG-1(-/-) mice transferred with SOCS2(-/-) CD4(+) T cells, exhibit elevated type 2 responses after helminth antigen challenge. Moreover, in in vivo models of atopic dermatitis and allergen-induced airway inflammation, SOCS2(-/-) mice show significantly elevated IgE, eosinophilia, type 2 responses, and inflammatory pathology relative to wild-type mice. Finally, after T cell activation, markedly enhanced STAT6 and STAT5 phosphorylation is observed in SOCS2(-/-) T cells, whereas STAT3 phosphorylation is blunted. Thus, we provide the first evidence that SOCS2 plays an important role in regulating Th2 cell expansion and development of the type 2 allergic responses.