Faecal microbial transfer and complex carbohydrates mediate protection against COPD.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear. DESIGN: Using an in vivo mouse model of cigarette smoke (CS)-induced COPD and faecal microbial transfer (FMT), we characterised the faecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in 16 patients with COPD using a randomised, double-blind, placebo-controlled pilot study of inulin supplementation. RESULTS: FMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation, and transfer of CS-associated microbiota after antibiotic-induced microbiome depletion was sufficient to increase lung inflammation while suppressing colonic immunity in the absence of CS exposure. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in CS-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes. CONCLUSION: The gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically.

A unique human cord blood CD8+CD45RA+CD27+CD161+ T-cell subset identified by flow cytometric data analysis using Seurat.

Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.

Diet, commensal microbiota-derived extracellular vesicles, and host immunity.

The gut microbiota has co-evolved with its host, and commensal bacteria can influence both the host's immune development and function. Recently, a role has emerged for bacterial extracellular vesicles (BEVs) as potent immune modulators. BEVs are nanosized membrane vesicles produced by all bacteria, possessing the membrane characteristics of the originating bacterium and carrying an internal cargo that may include nucleic acid, proteins, lipids, and metabolites. Thus, BEVs possess multiple avenues for regulating immune processes, and have been implicated in allergic, autoimmune, and metabolic diseases. BEVs are biodistributed locally in the gut, and also systemically, and thus have the potential to affect both the local and systemic immune responses. The production of gut microbiota-derived BEVs is regulated by host factors such as diet and antibiotic usage. Specifically, all aspects of nutrition, including macronutrients (protein, carbohydrates, and fat), micronutrients (vitamins and minerals), and food additives (the antimicrobial sodium benzoate), can regulate BEV production. This review summarizes current knowledge of the powerful links between nutrition, antibiotics, gut microbiota-derived BEV, and their effects on immunity and disease development. It highlights the potential of targeting or utilizing gut microbiota-derived BEV as a therapeutic intervention.

GPR109A expressed on medullary thymic epithelial cells affects thymic Treg development.

Regulatory T cells (Treg) maintain immune homeostasis due to their anti-inflammatory functions. They can be generated either centrally in the thymus or in peripheral organs. Metabolites such as short-chain fatty acids produced by intestinal microbiota can induce peripheral Treg differentiation, by activating G-protein-coupled-receptors like GPR109A. In this study, we identified a novel role for GPR109A in thymic Treg development. We found that Gpr109a-/- mice had increased Treg under basal conditions in multiple organs compared with WT mice. GPR109A was not expressed on T cells but on medullary thymic epithelial cells (mTECs), as revealed by single-cell RNA sequencing in both mice and humans and confirmed by flow cytometry in mice. mTECs isolated from Gpr109a-/- mice had higher expression of autoimmune regulator (AIRE), the key regulator of Treg development, while the subset of mTECs that did not express Gpr109a in the WT displayed increased Aire expression and also enhanced signaling related to mTEC functionality. Increased thymic Treg in Gpr109a-/- mice was associated with protection from experimental autoimmune encephalomyelitis, with ameliorated clinical signs and reduced inflammation. This work identifies a novel role for GPR109A and possibly the gut microbiota, on thymic Treg development via its regulation of mTECs.

NK cell profiling in West Nile virus encephalitis reveals potential metabolic basis for functional inhibition.

Natural killer (NK) cells are cytotoxic lymphocytes important for viral defense. West Nile virus (WNV) infection of the central nervous system (CNS) causes marked recruitment of bone marrow (BM)-derived monocytes, T cells and NK cells, resulting in severe neuroinflammation and brain damage. Despite substantial numbers of NK cells in the CNS, their function and phenotype remain largely unexplored. Here, we demonstrate that NK cells mature from the BM to the brain, upregulate inhibitory receptors and show reduced cytokine production and degranulation, likely due to the increased expression of the inhibitory NK cell molecule, MHC-I. Intriguingly, this correlated with a reduction in metabolism associated with cytotoxicity in brain-infiltrating NK cells. Importantly, the degranulation and killing capability were restored in NK cells isolated from WNV-infected tissue, suggesting that WNV-induced NK cell inhibition occurs in the CNS. Overall, this work identifies a potential link between MHC-I inhibition of NK cells and metabolic reduction of their cytotoxicity during infection.

Photochemical Tissue Bonding of Amnion Allograft Membranes for Peripheral Nerve Repair: A Biomechanical Analysis.

BACKGROUND: Photochemical tissue bonding (PTB) is a technique for peripheral nerve repair in which a collagenous membrane is bonded around approximated nerve ends. Studies using PTB with cryopreserved human amnion have shown promising results in a rat sciatic nerve transection model including a more rapid and complete return of function, larger axon size, and thicker myelination than suture repair. Commercial collagen membranes, such as dehydrated amnion allograft, are readily available, offer ease of storage, and have no risk of disease transmission or tissue rejection. However, the biomechanical properties of these membranes using PTB are currently unknown in comparison to PTB of cryopreserved human amnion and suture neurorrhaphy. METHODS: Rat sciatic nerves (n = 10 per group) were transected and repaired using either suture neurorrhaphy or PTB with one of the following membranes: cryopreserved human amnion, monolayer human amnion allograft (crosslinked and noncrosslinked), trilayer human amnion/chorion allograft (crosslinked and noncrosslinked), or swine submucosa. Repaired nerves were subjected to mechanical testing. RESULTS: During ultimate stress testing, the repair groups that withstood the greatest strain increases were suture neurorrhaphy (69 ± 14%), PTB with crosslinked trilayer amnion (52 ± 10%), and PTB with cryopreserved human amnion (46 ± 20%), although the differences between these groups were not statistically significant. Neurorrhaphy repairs had a maximum load (0.98 ± 0.30 N) significantly greater than all other repair groups except for noncrosslinked trilayer amnion (0.51 ± 0.27 N). During fatigue testing, all samples repaired with suture, or PTBs with either crosslinked or noncrosslinked trilayer amnion were able to withstand strain increases of at least 50%. CONCLUSION: PTB repairs with commercial noncrosslinked amnion allograft membranes can withstand physiological strain and have comparable performance to repairs with human amnion, which has demonstrated efficacy in vivo. These results indicate the need for further testing of these membranes using in vivo animal model repairs.

Integrating transcriptomic datasets across neurological disease identifies unique myeloid subpopulations driving disease-specific signatures.

Microglia and bone marrow-derived monocytes are key elements of central nervous system (CNS) inflammation, both capable of enhancing and dampening immune-mediated pathology. However, the study-specific focus on individual cell types, disease models or experimental approaches has limited our ability to infer common and disease-specific responses. This meta-analysis integrates bulk and single-cell transcriptomic datasets of microglia and monocytes from disease models of autoimmunity, neurodegeneration, sterile injury, and infection to build a comprehensive resource connecting myeloid responses across CNS disease. We demonstrate that the bulk microglial and monocyte program is highly contingent on the disease environment, challenging the notion of a universal microglial disease signature. Integration of six single-cell RNA-sequencing datasets revealed that these disease-specific signatures are likely driven by differing proportions of unique myeloid subpopulations that were individually expanded in different disease settings. These subsets were functionally-defined as neurodegeneration-associated, inflammatory, interferon-responsive, phagocytic, antigen-presenting, and lipopolysaccharide-responsive cellular states, revealing a core set of myeloid responses at the single-cell level that are conserved across CNS pathology. Showcasing the predictive and practical value of this resource, we performed differential expression analysis on microglia and monocytes across disease and identified Cd81 as a new neuroinflammatory-stable gene that accurately identified microglia and distinguished them from monocyte-derived cells across all experimental models at both the bulk and single-cell level. Together, this resource dissects the influence of disease environment on shared immune response programmes to build a unified perspective of myeloid behavior across CNS pathology.

Early microglial response, myelin deterioration and lethality in mice deficient for very long chain ceramide synthesis in oligodendrocytes.

The sphingolipids galactosylceramide (GalCer), sulfatide (ST) and sphingomyelin (SM) are essential for myelin stability and function. GalCer and ST are synthesized mostly from C22-C24 ceramides, generated by Ceramide Synthase 2 (CerS2). To clarify the requirement for C22-C24 sphingolipid synthesis in myelin biosynthesis and stability, we generated mice lacking CerS2 specifically in myelinating cells (CerS2ΔO/ΔO ). At 6 weeks of age, normal-appearing myelin had formed in CerS2ΔO/ΔO mice, however there was a reduction in myelin thickness and the percentage of myelinated axons. Pronounced loss of C22-C24 sphingolipids in myelin of CerS2ΔO/ΔO mice was compensated by greatly increased levels of C18 sphingolipids. A distinct microglial population expressing high levels of activation and phagocytic markers such as CD64, CD11c, MHC class II, and CD68 was apparent at 6 weeks of age in CerS2ΔO/ΔO mice, and had increased by 10 weeks. Increased staining for denatured myelin basic protein was also apparent in 6-week-old CerS2ΔO/ΔO mice. By 16 weeks, CerS2ΔO/ΔO mice showed pronounced myelin atrophy, motor deficits, and axon beading, a hallmark of axon stress. 90% of CerS2ΔO/ΔO mice died between 16 and 26 weeks of age. This study highlights the importance of sphingolipid acyl chain length for the structural integrity of myelin, demonstrating how a modest reduction in lipid chain length causes exposure of a denatured myelin protein epitope and expansion of phagocytic microglia, followed by axon pathology, myelin degeneration, and motor deficits. Understanding the molecular trigger for microglial activation should aid the development of therapeutics for demyelinating and neurodegenerative diseases.

BioCaster in 2021: automatic disease outbreaks detection from global news media.

SUMMARY: BioCaster was launched in 2008 to provide an ontology-based text mining system for early disease detection from open news sources. Following a 6-year break, we have re-launched the system in 2021. Our goal is to systematically upgrade the methodology using state-of-the-art neural network language models, whilst retaining the original benefits that the system provided in terms of logical reasoning and automated early detection of infectious disease outbreaks. Here, we present recent extensions such as neural machine translation in 10 languages, neural classification of disease outbreak reports and a new cloud-based visualization dashboard. Furthermore, we discuss our vision for further improvements, including combining risk assessment with event semantics and assessing the risk of outbreaks with multi-granularity. We hope that these efforts will benefit the global public health community. AVAILABILITY AND IMPLEMENTATION: BioCaster web-portal is freely accessible at http://biocaster.org.

TrackSOM: Mapping immune response dynamics through clustering of time-course cytometry data.

Mapping the dynamics of immune cell populations over time or disease-course is key to understanding immunopathogenesis and devising putative interventions. We present TrackSOM, a novel method for delineating cellular populations and tracking their development over a time- or disease-course cytometry datasets. We demonstrate TrackSOM-enabled elucidation of the immune response to West Nile Virus infection in mice, uncovering heterogeneous subpopulations of immune cells and relating their functional evolution to disease severity. TrackSOM is easy to use, encompasses few parameters, is quick to execute, and enables an integrative and dynamic overview of the immune system kinetics that underlie disease progression and/or resolution.

Photosealing of dural defects using a biocompatible patch.

PURPOSE: Photosealing of many biological tissues can be achieved using a biocompatible material in combination with a dye that is activated by visible light to chemically bond over the tissue defect via protein cross-linking reactions. The aim of this study was to test the efficacy of photosealing using a commercially available biomembrane (AmnioExcel Plus) to securely close dural defects in comparison to another sutureless method (fibrin glue) in terms of repair strength. METHODS: Two-millimeter diameter holes were created in dura harvested from New Zealand white rabbits and repaired ex vivo using one of two methods: (1) in n = 10 samples, photosealing was used to bond a 6-mm-diameter AmnioExcel Plus patch over the dural defect, and (2) in n = 10 samples, fibrin glue was used to attach the same patch over the dural defect. Repaired dura samples were then subjected to burst pressure testing. Histological analysis was also performed of photosealed dura. RESULTS: The mean burst pressures of rabbit dura repaired with photosealing and fibrin glue were 302 ± 149 mmHg and 26 ± 24 mmHg, respectively. The increased repair strength using photosealing was statistically significant and considerably higher than the normal intracranial pressure of ~ 20 mmHg. Histology demonstrated a tight union at the interface between the dura surface and patch with no disruption of the dura structure. CONCLUSION: The results of this study suggest that photosealing performs better than fibrin glue for the fixation of a patch for ex vivo repair of small dural defects. Photosealing is worthy of testing in pre-clinical models for the repair of dural defects.

Evaluation of Novel Urinary Biomarkers in Beagle Dogs With Amphotericin B-Induced Kidney Injury.

Next-generation urinary protein biomarkers have been qualified to enable monitoring for drug-induced kidney injury in toxicology studies conducted in rats. However, there is limited literature on the utility of these biomarkers in dogs. To add to the existing body of knowledge on the utility of the next-generation drug-induced kidney injury (DIKI) biomarkers, we evaluated the value of these biomarkers for the early detection of DIKI in Beagle dogs using a differentiated nephrotoxicant, Amphotericin B (AmpB). In dogs with AmpB-induced kidney injury, we monitored the response of urinary albumin, total protein, clusterin, kidney injury molecule 1, neutrophil gelatinase-associated lipocalin and N-acetyl-beta-D-glucosaminidase. We also measured blood urea nitrogen, serum creatinine and cystatin C. The results showed that urinary clusterin (up to ∼ 112x) was much more sensitive to AmpB-induced kidney injury relative to other biomarkers. Moreover, other than urinary clusterin and to a much lesser extent urinary albumin and total protein, none of the other biomarkers analyzed in this study were more sensitive than blood urea nitrogen and serum creatinine. The AmpB related tubular alterations were characterized by minimal to mild, multifocal necrosis, degeneration, regeneration, dilatation and mineralization. The mild nature of these histopathologic findings further attested to the sensitivity of urinary clusterin to AmpB-induced kidney injury in dogs. These results will help drug developers make informed decisions when selecting urinary biomarkers for monitoring DIKI in dogs for toxicology studies.

Targeting inflammatory monocytes by immune-modifying nanoparticles prevents acute kidney allograft rejection.

Inflammatory monocytes are a major component of the cellular infiltrate in acutely rejecting human kidney allografts. Since immune-modifying nanoparticles (IMPs) bind to circulating inflammatory monocytes via the specific scavenger receptor MARCO, causing diversion to the spleen and subsequent apoptosis, we investigated the therapeutic potential of negatively charged, 500-nm diameter polystyrene IMPs to prevent kidney allograft rejection. Kidney transplants were performed from BALB/c (H2d) to C57BL/6 (H2b) mice in two groups: controls (allo) and allo mice infused with IMPs. Groups were studied for 14 (acute rejection) or 100 (chronic rejection) days. Allo mice receiving IMPs exhibited superior survival and markedly less acute rejection, with better kidney function, less tubulitis, and diminished inflammatory cell density, cytokine and cytotoxic molecule expression in the allograft and lower titers of donor-specific IgG2c antibody in serum at day 14, as compared to allo mice. Cells isolated from kidneys from allo mice receiving IMPs showed reduced Ly6Chi monocytes, CD11b+ cells and NKT+ cells compared to allo mice. IMPs predominantly bound CD11b+ cells in the bloodstream and CD11b+ and CD11c-B220+ marginal zone B cells in the spleen. In the spleen, IMPs were found predominantly in red pulp, colocalized with MARCO and expression of cleaved caspase-3. At day 100, allo mice receiving IMPs exhibited reduced macrophage M1 responses but were not protected from chronic rejection. IMPs afforded significant protection from acute rejection, inhibiting both innate and adaptive alloimmunity. Thus, our current experimental findings, coupled with our earlier demonstration of IMP-induced protection in kidney ischemia-reperfusion injury, identify IMPs as a potential induction agent in kidney transplantation.

Using single-cell cytometry to illustrate integrated multi-perspective evaluation of clustering algorithms using Pareto fronts.

MOTIVATION: Many 'automated gating' algorithms now exist to cluster cytometry and single cell sequencing data into discrete populations. Comparative algorithm evaluations on benchmark datasets rely either on a single performance metric, or a few metrics considered independently of one another. However, single metrics emphasise different aspects of clustering performance and do not rank clustering solutions in the same order. This underlies the lack of consensus between comparative studies regarding optimal clustering algorithms and undermines the translatability of results onto other non-benchmark datasets. RESULTS: We propose the Pareto fronts framework as an integrative evaluation protocol, wherein individual metrics are instead leveraged as complementary perspectives. Judged superior are algorithms that provide the best trade-off between the multiple metrics considered simultaneously. This yields a more comprehensive and complete view of clustering performance. Moreover, by broadly and systematically sampling algorithm parameter values using the Latin Hypercube sampling method, our evaluation protocol minimises (un)fortunate parameter value selections as confounding factors. Furthermore, it reveals how meticulously each algorithm must be tuned in order to obtain good results, vital knowledge for users with novel data. We exemplify the protocol by conducting a comparative study between three clustering algorithms (ChronoClust, FlowSOM and Phenograph) using four common performance metrics applied across four cytometry benchmark datasets. To our knowledge, this is the first time Pareto fronts have been used to evaluate the performance of clustering algorithms in any application domain. AVAILABILITY: Implementation of our Pareto front methodology and all scripts to reproduce this article are available at https://github.com/ghar1821/ParetoBench.

Contribution of STAT1 to innate and adaptive immunity during type I interferon-mediated lethal virus infection.

Signal transducers and activators of transcription (STAT) 1 is critical for cellular responses to type I interferons (IFN-Is), with the capacity to determine the outcome of viral infection. We previously showed that while wildtype (WT) mice develop mild disease and survive infection with lymphocytic choriomeningitis virus (LCMV), LCMV infection of STAT1-deficient mice results in a lethal wasting disease that is dependent on IFN-I and CD4+ cells. IFN-Is are considered to act as a bridge between innate and adaptive immunity. Here, we determined the relative contribution of STAT1 on innate and adaptive immunity during LCMV infection. We show that STAT1 deficiency results in a biphasic disease following LCMV infection. The initial, innate immunity-driven phase of disease was characterized by rapid weight loss, thrombocytopenia, systemic cytokine and chemokine responses and leukocyte infiltration of infected organs. In the absence of an adaptive immune response, this first phase of disease largely resolved resulting in survival of the infected host. However, in the presence of adaptive immunity, the disease progressed into a second phase with continued cytokine and chemokine production, persistent leukocyte extravasation into infected tissues and ultimately, host death. Overall, our findings demonstrate the key contribution of STAT1 in modulating innate and adaptive immunity during type I interferon-mediated lethal virus infection.

Integration, exploration, and analysis of high-dimensional single-cell cytometry data using Spectre.

As the size and complexity of high-dimensional (HD) cytometry data continue to expand, comprehensive, scalable, and methodical computational analysis approaches are essential. Yet, contemporary clustering and dimensionality reduction tools alone are insufficient to analyze or reproduce analyses across large numbers of samples, batches, or experiments. Moreover, approaches that allow for the integration of data across batches or experiments are not well incorporated into computational toolkits to allow for streamlined workflows. Here we present Spectre, an R package that enables comprehensive end-to-end integration and analysis of HD cytometry data from different batches or experiments. Spectre streamlines the analytical stages of raw data pre-processing, batch alignment, data integration, clustering, dimensionality reduction, visualization, and population labelling, as well as quantitative and statistical analysis. Critically, the fundamental data structures used within Spectre, along with the implementation of machine learning classifiers, allow for the scalable analysis of very large HD datasets, generated by flow cytometry, mass cytometry, or spectral cytometry. Using open and flexible data structures, Spectre can also be used to analyze data generated by single-cell RNA sequencing or HD imaging technologies, such as Imaging Mass Cytometry. The simple, clear, and modular design of analysis workflows allow these tools to be used by bioinformaticians and laboratory scientists alike. Spectre is available as an R package or Docker container. R code is available on Github (https://github.com/immunedynamics/spectre).

Microglia and monocytes in inflammatory CNS disease: integrating phenotype and function.

In neurological diseases, the actions of microglia, the resident myeloid cells of the CNS parenchyma, may diverge from, or intersect with, those of recruited monocytes to drive immune-mediated pathology. However, defining the precise roles of each cell type has historically been impeded by the lack of discriminating markers and experimental systems capable of accurately identifying them. Our ability to distinguish microglia from monocytes in neuroinflammation has advanced with single-cell technologies, new markers and drugs that identify and deplete them, respectively. Nevertheless, the focus of individual studies on particular cell types, diseases or experimental approaches has limited our ability to connect phenotype and function more widely and across diverse CNS pathologies. Here, we critically review, tabulate and integrate the disease-specific functions and immune profiles of microglia and monocytes to provide a comprehensive atlas of myeloid responses in viral encephalitis, demyelination, neurodegeneration and ischemic injury. In emphasizing the differential roles of microglia and monocytes in the severe neuroinflammatory disease of viral encephalitis, we connect inflammatory pathways common to equally incapacitating diseases with less severe inflammation. We examine these findings in the context of human studies and highlight the benefits and inherent limitations of animal models that may impede or facilitate clinical translation. This enables us to highlight common and contrasting, non-redundant and often opposing roles of microglia and monocytes in disease that could be targeted therapeutically.

Gliadin Nanoparticles Induce Immune Tolerance to Gliadin in Mouse Models of Celiac Disease.

BACKGROUND & AIMS: Celiac disease could be treated, and potentially cured, by restoring T-cell tolerance to gliadin. We investigated the safety and efficacy of negatively charged 500-nm poly(lactide-co-glycolide) nanoparticles encapsulating gliadin protein (TIMP-GLIA) in 3 mouse models of celiac disease. Uptake of these nanoparticles by antigen-presenting cells was shown to induce immune tolerance in other animal models of autoimmune disease. METHODS: We performed studies with C57BL/6; RAG1-/- (C57BL/6); and HLA-DQ8, huCD4 transgenic Ab0 NOD mice. Mice were given 1 or 2 tail-vein injections of TIMP-GLIA or control nanoparticles. Some mice were given intradermal injections of gliadin in complete Freund's adjuvant (immunization) or of soluble gliadin or ovalbumin (ear challenge). RAG-/- mice were given intraperitoneal injections of CD4+CD62L-CD44hi T cells from gliadin-immunized C57BL/6 mice and were fed with an AIN-76A-based diet containing wheat gluten (oral challenge) or without gluten. Spleen or lymph node cells were analyzed in proliferation and cytokine secretion assays or by flow cytometry, RNA sequencing, or real-time quantitative polymerase chain reaction. Serum samples were analyzed by gliadin antibody enzyme-linked immunosorbent assay, and intestinal tissues were analyzed by histology. Human peripheral blood mononuclear cells, or immature dendritic cells derived from human peripheral blood mononuclear cells, were cultured in medium containing TIMP-GLIA, anti-CD3 antibody, or lipopolysaccharide (controls) and analyzed in proliferation and cytokine secretion assays or by flow cytometry. Whole blood or plasma from healthy volunteers was incubated with TIMP-GLIA, and hemolysis, platelet activation and aggregation, and complement activation or coagulation were analyzed. RESULTS: TIMP-GLIA did not increase markers of maturation on cultured human dendritic cells or induce activation of T cells from patients with active or treated celiac disease. In the delayed-type hypersensitivity (model 1), the HLA-DQ8 transgenic (model 2), and the gliadin memory T-cell enteropathy (model 3) models of celiac disease, intravenous injections of TIMP-GLIA significantly decreased gliadin-specific T-cell proliferation (in models 1 and 2), inflammatory cytokine secretion (in models 1, 2, and 3), circulating gliadin-specific IgG/IgG2c (in models 1 and 2), ear swelling (in model 1), gluten-dependent enteropathy (in model 3), and body weight loss (in model 3). In model 1, the effects were shown to be dose dependent. Splenocytes from HLA-DQ8 transgenic mice given TIMP-GLIA nanoparticles, but not control nanoparticles, had increased levels of FOXP3 and gene expression signatures associated with tolerance induction. CONCLUSIONS: In mice with gliadin sensitivity, injection of TIMP-GLIA nanoparticles induced unresponsiveness to gliadin and reduced markers of inflammation and enteropathy. This strategy might be developed for the treatment of celiac disease.

High-parameter cytometry unmasks microglial cell spatio-temporal response kinetics in severe neuroinflammatory disease.

BACKGROUND: Differentiating infiltrating myeloid cells from resident microglia in neuroinflammatory disease is challenging, because bone marrow-derived inflammatory monocytes infiltrating the inflamed brain adopt a 'microglia-like' phenotype. This precludes the accurate identification of either cell type without genetic manipulation, which is important to understand their temporal contribution to disease and inform effective intervention in its pathogenesis. During West Nile virus (WNV) encephalitis, widespread neuronal infection drives substantial CNS infiltration of inflammatory monocytes, causing severe immunopathology and/or death, but the role of microglia in this remains unclear. METHODS: Using high-parameter cytometry and dimensionality-reduction, we devised a simple, novel gating strategy to identify microglia and infiltrating myeloid cells during WNV-infection. Validating our strategy, we (1) blocked the entry of infiltrating myeloid populations from peripheral blood using monoclonal blocking antibodies, (2) adoptively transferred BM-derived monocytes and tracked their phenotypic changes after infiltration and (3) labelled peripheral leukocytes that infiltrate into the brain with an intravenous dye. We demonstrated that myeloid immigrants populated only the identified macrophage gates, while PLX5622 depletion reduced all 4 subsets defined by the microglial gates. RESULTS: Using this gating approach, we identified four consistent microglia subsets in the homeostatic and WNV-infected brain. These were P2RY12hi CD86-, P2RY12hi CD86+ and P2RY12lo CD86- P2RY12lo CD86+. During infection, 2 further populations were identified as 'inflammatory' and 'microglia-like' macrophages, recruited from the bone marrow. Detailed kinetic analysis showed significant increases in the proportions of both P2RY12lo microglia subsets in all anatomical areas, largely at the expense of the P2RY12hi CD86- subset, with the latter undergoing compensatory proliferation, suggesting replenishment of, and differentiation from this subset in response to infection. Microglia altered their morphology early in infection, with all cells adopting temporal and regional disease-specific phenotypes. Late in disease, microglia produced IL-12, downregulated CX3CR1, F4/80 and TMEM119 and underwent apoptosis. Infiltrating macrophages expressed both TMEM119 and P2RY12 de novo, with the microglia-like subset notably exhibiting the highest proportional myeloid population death. CONCLUSIONS: Our approach enables detailed kinetic analysis of resident vs infiltrating myeloid cells in a wide range of neuroinflammatory models without non-physiological manipulation. This will more clearly inform potential therapeutic approaches that specifically modulate these cells.

Declining Life Expectancy in the United States: Missing the Trees for the Forest.

In recent years, life expectancy in the United States has stagnated, followed by three consecutive years of decline. The decline is small in absolute terms but is unprecedented and has generated considerable research interest and theorizing about potential causes. Recent trends show that the decline has affected nearly all race/ethnic and gender groups, and the proximate causes of the decline are increases in opioid overdose deaths, suicide, homicide, and Alzheimer's disease. A slowdown in the long-term decline in mortality from cardiovascular diseases has also prevented life expectancy from improving further. Although a popular explanation for the decline is the cumulative decline in living standards across generations, recent trends suggest that distinct mechanisms for specific causes of death are more plausible explanations. Interventions to stem the increase in overdose deaths, reduce access to mechanisms that contribute to violent deaths, and decrease cardiovascular risk over the life course are urgently needed to improve mortality in the United States.