Functional proteomic profiling links deficient DNA clearance with increased mortality in individuals with severe COVID-19 pneumonia.

The factors that influence survival during severe infection are unclear. Extracellular chromatin drives pathology, but the mechanisms enabling its accumulation remain elusive. Here, we show that in murine sepsis models, splenocyte death interferes with chromatin clearance through the release of the DNase I inhibitor actin. Actin-mediated inhibition was compensated by upregulation of DNase I or the actin scavenger gelsolin. Splenocyte death and neutrophil extracellular trap (NET) clearance deficiencies were prevalent in individuals with severe COVID-19 pneumonia or microbial sepsis. Activity tracing by plasma proteomic profiling uncovered an association between low NET clearance and increased COVID-19 pathology and mortality. Low NET clearance activity with comparable proteome associations was prevalent in healthy donors with low-grade inflammation, implicating defective chromatin clearance in the development of cardiovascular disease and linking COVID-19 susceptibility to pre-existing conditions. Hence, the combination of aberrant chromatin release with defects in protective clearance mechanisms lead to poor survival outcomes.

Tumor-associated neutrophils suppress pro-tumoral IL-17+ γδ T cells through induction of oxidative stress.

Interleukin 17 (IL-17)-producing γδ T cells (γδ17 T cells) have been recently found to promote tumor growth and metastasis formation. How such γδ17 T-cell responses may be regulated in the tumor microenvironment remains, however, largely unknown. Here, we report that tumor-associated neutrophils can display an overt antitumor role by strongly suppressing γδ17 T cells. Tumor-associated neutrophils inhibited the proliferation of murine CD27- Vγ6+ γδ17 T cells via induction of oxidative stress, thereby preventing them from constituting the major source of pro-tumoral IL-17 in the tumor microenvironment. Mechanistically, we found that low expression of the antioxidant glutathione in CD27- γδ17 T cells renders them particularly susceptible to neutrophil-derived reactive oxygen species (ROS). Consistently, superoxide deficiency, or the administration of a glutathione precursor, rescued CD27- Vγ6+ γδ17 T-cell proliferation in vivo. Moreover, human Vδ1+ γδ T cells, which contain most γδ17 T cells found in cancer patients, also displayed low glutathione levels and were potently inhibited by ROS. This work thus identifies an unanticipated, immunosuppressive yet antitumoral, neutrophil/ROS/γδ17 T-cell axis in the tumor microenvironment.

De novo-induced self-antigen-specific Foxp3+ regulatory T cells impair the accumulation of inflammatory dendritic cells in draining lymph nodes.

Foxp3(+) regulatory T cell (Treg)-based immunotherapy holds promise for autoimmune diseases. However, this effort has been hampered by major caveats, including the low frequency of autoantigen-specific Foxp3(+) Tregs and lack of understanding of their molecular and cellular targets, in an unmanipulated wild-type (WT) immune repertoire. In this study, we demonstrate that infusion of myelin in WT mice results in the de novo induction of myelin-specific Foxp3(+) Tregs in WT mice and amelioration of experimental autoimmune encephalomyelitis. Myelin-specific Foxp3(+) Tregs exerted their effect both by diminishing Ag-bearing inflammatory dendritic cell (iDC) recruitment to lymph nodes and by impairing their function. Transcriptome analysis of ex vivo-isolated Treg-exposed iDCs showed significant enrichment of transcripts involved in functional properties of iDCs, including chemotaxis-related genes. To this end, CCR7 expression by iDCs was significantly downregulated in tolerant mice and this was tightly regulated by the presence of IL-10. Collectively, our data demonstrate a novel model for deciphering the Ag-specific Foxp3(+) Treg-mediated mechanisms of tolerance and delineate iDCs as a Foxp3(+) Treg cellular target in unmanipulated mice.

Cell-based modulation of autoimmune responses in multiple sclerosis and experimental autoimmmune encephalomyelitis: therapeutic implications.

Multiple sclerosis (MS) is a prototypic autoimmune inflammatory disorder of the central nervous system (CNS). MS pathogenesis is a complex phenomenon that is influenced by genetic and environmental factors that lead to the dysregulation of immune homeostasis and tolerance. It has been shown that pathogenic T lymphocyte subsets, such as T helper 1 (Th1) and Th17 cells, play a crucial role in the autoimmune cascade influencing disease initiation, progression and subsequent tissue damage during MS. On the other hand, several mechanisms have been described in both patients and animal models of MS with the potential to modulate myelin-specific autoimmune responses and to facilitate amelioration of disease pathology. To this end, regulatory T cells (Tregs) are considered to be a powerful cell subset not only in the maintenance of homeostasis but also in the re-establishment of tolerance. Along these lines, other cell subsets such as dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), γδ T cells and natural killer (NK) cells have been shown to regulate the autoimmune response in the CNS under certain circumstances. This review will attempt to summarize the relevant knowledge of the regulatory mechanisms exerted by immune cells in MS that could hold the promise for the design of novel therapeutic strategies.

In vivo ablation of plasmacytoid dendritic cells inhibits autoimmunity through expansion of myeloid-derived suppressor cells.

Autoimmunity ensues upon breakdown of tolerance mechanism and priming of self-reactive T cells. Plasmacytoid dendritic cells (pDCs) constitute a unique cell subset that participates in the activation of autoreactive T cells but also has been shown to be critically involved in the induction of self-tolerance. However, their functional importance during the priming phase of an organ-specific autoimmune response remains unclear. In this study, we demonstrate that absence of pDCs during myelin antigenic challenge resulted in amelioration of experimental autoimmune encephalomyelitis and reduced disease severity. This was accompanied by significantly decreased frequency of myelin-specific T cells in the draining lymph nodes and inhibition of Th1 and Th17 immune responses. Unexpectedly, in vivo ablation of pDCs increased myelopoiesis in the bone marrow and specifically induced the generation of CD11b(hi)Gr1(+) myeloid-derived suppressor cells (MDSCs). Furthermore, we demonstrate that pDC depletion enhanced the mobilization of MDSCs in the spleen, and that sorted MDSCs could potently suppress CD4(+) T cell responses in vitro. Importantly, pDC-depleted mice showed increased levels of MCP-1 in the draining lymph nodes, and in vivo administration of MCP-1 increased the frequency and absolute numbers of MDSCs in the periphery of treated mice. Together, our results reveal that absence of pDCs during the priming of an autoimmune response leads to increased mobilization of MDSCs in the periphery in an MCP-1-dependent manner and subsequent amelioration of autoimmunity.

Crucial role of granulocytic myeloid-derived suppressor cells in the regulation of central nervous system autoimmune disease.

There is a need in autoimmune diseases to uncover the mechanisms involved in the natural resolution of inflammation. In this article, we demonstrate that granulocytic myeloid-derived suppressor cells (G-MDSCs) abundantly accumulate within the peripheral lymphoid compartments and target organs of mice with experimental autoimmune encephalomyelitis prior to disease remission. In vivo transfer of G-MDSCs ameliorated experimental autoimmune encephalomyelitis, significantly decreased demyelination, and delayed disease onset through inhibition of encephalitogenic Th1 and Th17 immune responses. Exposure of G-MDSCs to the autoimmune milieu led to up-regulation of the programmed death 1 ligand that was required for the G-MDSC-mediated suppressive function both in vitro and in vivo. Importantly, myeloid-derived suppressor cells were enriched in the periphery of subjects with active multiple sclerosis and suppressed the activation and proliferation of autologous CD4(+) T cells ex vivo. Collectively, this study revealed a pivotal role for myeloid-derived suppressor cells in the regulation of multiple sclerosis, which could be exploited for therapeutic purposes.

Antidepressant and antipsychotic drug prescribing and diabetes outcomes: A systematic review of observational studies.

AIMS: Psychotropic medication may be associated with adverse effects, including among people with diabetes. We conducted a systematic review of observational studies investigating the association between antidepressant or antipsychotic drug prescribing and type 2 diabetes outcomes. METHODS: We systematically searched PubMed, EMBASE, and PsycINFO to 15th August 2022 to identify eligible studies. We used the Newcastle-Ottawa scale to assess study quality and performed a narrative synthesis. RESULTS: We included 18 studies, 14 reporting on antidepressants and four on antipsychotics. There were 11 cohort studies, one self-controlled before and after study, two case-control studies, and four cross-sectional studies, of variable quality with highly heterogeneous study populations, exposure definitions, and outcomes analysed. Antidepressant prescribing may be associated with increased risk of macrovascular disease, whilst evidence on antidepressant and antipsychotic prescribing and glycaemic control was mixed. Few studies reported microvascular outcomes and risk factors other than glycaemic control. CONCLUSIONS: Studies of antidepressant and antipsychotic drug prescribing in relation to diabetes outcomes are scarce, with shortcomings and mixed findings. Until further evidence is available, people with diabetes prescribed antidepressants and antipsychotics should receive monitoring and appropriate treatment of risk factors and screening for complications as recommended in general diabetes guidelines.

The negative costimulatory molecule PD-1 modulates the balance between immunity and tolerance via miR-21.

Disruption of the programmed death-1 (PD-1) pathway leads to breakdown of peripheral tolerance and initiation of autoimmunity. The molecular pathways that mediate this effect remain largely unknown. We report here that PD-1 knockout (PD-1(-/-) ) mice develop more severe and sustained Ag-induced arthritis (AIA) than WT animals, which is associated with increased T-cell proliferation and elevated levels of IFN-γ and IL-17 secretion. MicroRNA analysis of Ag-specific CD4(+) T cells revealed a significant upregulation of microRNA 21 (miR-21) in PD-1(-/-) T cells compared with WT controls. In addition, PD-1 inhibition, via siRNA, upregulated miR-21 expression and enhanced STAT5 binding in the miR-21 promoter area. Computational analysis confirmed that miR-21 targets directly the expression of programmed cell death 4 (PDCD4) and overexpression of miR-21 in cells harboring the 3'UTR of PDCD4 resulted in reduced transcription and PDCD4 protein expression. Importantly, in vitro delivery of antisense-miR-21 suppressed the Ag-specific proliferation and cytokine secretion by PD-1(-/-) T cells, whereas adoptive transfer of Ag-specific T cells, overexpressing miR-21, induced severe AIA. Collectively, our data demonstrate that breakdown of tolerance in PD-1(-/-) mice activates a signaling cascade mediated by STAT5, miR-21, and PDCD4 and establish their role in maintaining the balance between immune activation and tolerance.

Microbe capture by splenic macrophages triggers sepsis via T cell-death-dependent neutrophil lifespan shortening.

The mechanisms linking systemic infection to hyperinflammation and immune dysfunction in sepsis are poorly understood. Extracellular histones promote sepsis pathology, but their source and mechanism of action remain unclear. Here, we show that by controlling fungi and bacteria captured by splenic macrophages, neutrophil-derived myeloperoxidase attenuates sepsis by suppressing histone release. In systemic candidiasis, microbial capture via the phagocytic receptor SIGNR1 neutralizes myeloperoxidase by facilitating marginal zone infiltration and T cell death-dependent histone release. Histones and hyphae induce cytokines in adjacent CD169 macrophages including G-CSF that selectively depletes mature Ly6Ghigh neutrophils by shortening their lifespan in favour of immature Ly6Glow neutrophils with a defective oxidative burst. In sepsis patient plasma, these mediators shorten mature neutrophil lifespan and correlate with neutrophil mortality markers. Consequently, high G-CSF levels and neutrophil lifespan shortening activity are associated with sepsis patient mortality. Hence, by exploiting phagocytic receptors, pathogens degrade innate and adaptive immunity through the detrimental impact of downstream effectors on neutrophil lifespan.

Histones, DNA, and Citrullination Promote Neutrophil Extracellular Trap Inflammation by Regulating the Localization and Activation of TLR4.

Neutrophil extracellular traps (NETs) promote atherosclerosis by inducing proinflammatory cytokines, but the underlying mechanism remains unknown. NET DNA is immunogenic, but given the cytotoxicity of NET histones, it is unclear how it activates cells without killing them. Here, we show that histones, DNA, citrullination, and fragmentation synergize to drive inflammation below the histone cytotoxicity threshold. At low concentrations, nucleosomes induce cytokines, but high concentrations kill cells before cytokines are produced. The synergy between histones and DNA is critical for sub-lethal signaling and relies on distinct roles for histones and DNA. Histones bind and activate TLR4, whereas DNA recruits TLR4 to histone-containing endosomes. Citrullination is dispensable for NETosis but potentiates histone-mediated signaling. Consistently, chromatin blockade or PAD4 deficiency reduces atherosclerosis. Inflammation is also reduced in infected mice expressing GFP-tagged histones that block TLR4 binding. Thus, chromatin promotes inflammation in sterile disease and infection via synergistic mechanisms that use signals with distinct functions.

Type I IFN exacerbates disease in tuberculosis-susceptible mice by inducing neutrophil-mediated lung inflammation and NETosis.

Tuberculosis (TB) is a leading cause of mortality due to infectious disease, but the factors determining disease progression are unclear. Transcriptional signatures associated with type I IFN signalling and neutrophilic inflammation were shown to correlate with disease severity in mouse models of TB. Here we show that similar transcriptional signatures correlate with increased bacterial loads and exacerbate pathology during Mycobacterium tuberculosis infection upon GM-CSF blockade. Loss of GM-CSF signalling or genetic susceptibility to TB (C3HeB/FeJ mice) result in type I IFN-induced neutrophil extracellular trap (NET) formation that promotes bacterial growth and promotes disease severity. Consistently, NETs are present in necrotic lung lesions of TB patients responding poorly to antibiotic therapy, supporting the role of NETs in a late stage of TB pathogenesis. Our findings reveal an important cytokine-based innate immune effector network with a central role in determining the outcome of M. tuberculosis infection.

IL-23-producing IL-10Rα-deficient gut macrophages elicit an IL-22-driven proinflammatory epithelial cell response.

Cytokines maintain intestinal health, but precise intercellular communication networks remain poorly understood. Macrophages are immune sentinels of the intestinal tissue and are critical for gut homeostasis. Here, we show that in a murine inflammatory bowel disease (IBD) model based on macrophage-restricted interleukin-10 (IL-10) receptor deficiency (Cx3cr1Cre:Il10rafl/fl mice), proinflammatory mutant gut macrophages cause severe spontaneous colitis resembling the condition observed in children carrying IL-10R mutations. We establish macrophage-derived IL-23 as the driving factor of this pathology. Specifically, we report that Cx3cr1Cre:Il10rafl/fl:Il23afl/fl mice harboring macrophages deficient for both IL-10R and IL-23 are protected from colitis. By analyzing the epithelial response to proinflammatory macrophages, we provide evidence that T cells of colitic animals produce IL-22, which induces epithelial chemokine expression and detrimental neutrophil recruitment. Collectively, we define macrophage-specific contributions to the induction and pathogenesis of colitis, as manifested in mice harboring IL-10R deficiencies and human IBDs.

Transcriptional profiling unveils type I and II interferon networks in blood and tissues across diseases.

Understanding how immune challenges elicit different responses is critical for diagnosing and deciphering immune regulation. Using a modular strategy to interpret the complex transcriptional host response in mouse models of infection and inflammation, we show a breadth of immune responses in the lung. Lung immune signatures are dominated by either IFN-γ and IFN-inducible, IL-17-induced neutrophil- or allergy-associated gene expression. Type I IFN and IFN-γ-inducible, but not IL-17- or allergy-associated signatures, are preserved in the blood. While IL-17-associated genes identified in lung are detected in blood, the allergy signature is only detectable in blood CD4+ effector cells. Type I IFN-inducible genes are abrogated in the absence of IFN-γ signaling and decrease in the absence of IFNAR signaling, both independently contributing to the regulation of granulocyte responses and pathology during Toxoplasma gondii infection. Our framework provides an ideal tool for comparative analyses of transcriptional signatures contributing to protection or pathogenesis in disease.

Elimination of Granulocytic Myeloid-Derived Suppressor Cells in Lupus-Prone Mice Linked to Reactive Oxygen Species-Dependent Extracellular Trap Formation.

OBJECTIVE: Emerging evidence supports a crucial role of myeloid-derived suppressor cells (MDSCs) in the regulation of autoimmune diseases. However, their role in systemic lupus erythematosus (SLE) remains unknown. This study sought to address the role of MDSCs in the pathogenesis of SLE. METHODS: MDSCs from (NZB × NZW)F1 lupus-prone mice were assessed for phenotype by flow cytometry, and the function of MDSCs was analyzed by in vitro T cell proliferation assay and real-time quantitative polymerase chain reaction. Extracellular trap (ET) formation was evaluated by immunofluorescence and confocal microscopy. The production of reactive oxygen species (ROS) by Ly-6G+ cells was determined by fluorescence-activated cell sorting analysis. RESULTS: Expansion of MDSCs was impaired and the function of MDSCs was defective in the lymphoid organs of (NZB × NZW)F1 lupus-prone mice with established disease, in which involvement of predominantly the granulocytic MDSC (G-MDSC) cell subset was observed. More specifically, the results showed that increased elimination of G-MDSCs, driven by the inflammatory milieu of lupus, could be attributed to ET formation, and that cytokines, such as interferon-α (IFNα), IFNγ, and interleukin-6, play a role in this process. Induction of ET release by G-MDSCs was mediated by the production of ROS, since inhibition of ROS generation significantly reduced ET release. CONCLUSION: Collectively, the results of this study reveal that elimination of a crucial regulatory immune cell subset is a feature of the SLE microenvironment. These findings provide new insights into the pathogenetic mechanisms of the disease.

Inflammation. Neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis.

Secretion of the cytokine interleukin-1ß (IL-1ß) by macrophages, a major driver of pathogenesis in atherosclerosis, requires two steps: Priming signals promote transcription of immature IL-1ß, and then endogenous "danger" signals activate innate immune signaling complexes called inflammasomes to process IL-1ß for secretion. Although cholesterol crystals are known to act as danger signals in atherosclerosis, what primes IL-1ß transcription remains elusive. Using a murine model of atherosclerosis, we found that cholesterol crystals acted both as priming and danger signals for IL-1ß production. Cholesterol crystals triggered neutrophils to release neutrophil extracellular traps (NETs). NETs primed macrophages for cytokine release, activating T helper 17 (TH17) cells that amplify immune cell recruitment in atherosclerotic plaques. Therefore, danger signals may drive sterile inflammation, such as that seen in atherosclerosis, through their interactions with neutrophils.

Tpl2 kinase regulates FcγR signaling and immune thrombocytopenia in mice.

The MAPK3 Tpl2 controls innate and adaptive immunity by regulating TLR, TNF-α, and GPCR signaling in a variety of cell types. Its ablation gives rise to an anti-inflammatory phenotype characterized by resistance to LPS-induced endotoxin shock, DSS-induced colitis, and TNF-α-induced IBD. Here, we address the role of Tpl2 in autoimmunity. Our data show that the ablation and the pharmacological inhibition of Tpl2 protect mice from antiplatelet antibody-induced thrombocytopenia, a model of ITP. Thrombocytopenia in this model and in ITP is caused by phagocytosis of platelets opsonized with antiplatelet antibodies and depends on FcγR activation in splenic and hepatic myeloid cells. Further studies explained how Tpl2 inhibition protects from antibody-induced thrombocytopenia, by showing that Tpl2 is activated by FcγR signals in macrophages and that its activation by these signals is required for ERK activation, cytoplasmic Ca(2+) influx, the induction of cytokine and coreceptor gene expression, and phagocytosis.