Blood-based untargeted metabolomics in relapsing-remitting multiple sclerosis revealed the testable therapeutic target.

Metabolic aberrations impact the pathogenesis of multiple sclerosis (MS) and possibly can provide clues for new treatment strategies. Using untargeted metabolomics, we measured serum metabolites from 35 patients with relapsing-remitting multiple sclerosis (RRMS) and 14 healthy age-matched controls. Of 632 known metabolites detected, 60 were significantly altered in RRMS. Bioinformatics analysis identified an altered metabotype in patients with RRMS, represented by four changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these four pathways is the glycolysis pathway. Real-time bioenergetic analysis of the patient-derived peripheral blood mononuclear cells showed enhanced glycolysis, supporting the altered metabolic state of immune cells. Experimental autoimmune encephalomyelitis mice treated with the glycolytic inhibitor 2-deoxy-D-glucose ameliorated the disease progression and inhibited the disease pathology significantly by promoting the antiinflammatory phenotype of monocytes/macrophage in the central nervous system. Our study provided a proof of principle for how a blood-based metabolomic approach using patient samples could lead to the identification of a therapeutic target for developing potential therapy.

Hematopoietic Stem Cell Requirement for Macrophage Regeneration Is Tissue Specific.

Tissue-resident macrophages (TRMΦ) are important immune sentinels responsible for maintaining tissue and immune homeostasis within their specific niche. Recently, the origins of TRMΦ have undergone intense scrutiny, in which now most TRMΦ are thought to originate early during embryonic development independent of hematopoietic stem cells (HSCs). We previously characterized two distinct subsets of mouse peritoneal cavity macrophages (MΦ) (large and small peritoneal MΦ) whose origins and relationship to both fetal and adult long-term (LT) HSCs have not been fully investigated. In this study, we employ highly purified LT-HSC transplantation and in vivo lineage tracing to show a dual ontogeny for large and small peritoneal MΦ, in which the initial wave of peritoneal MΦ is seeded from yolk sac-derived precursors, which later require LT-HSCs for regeneration. In contrast, transplanted fetal and adult LT-HSCs are not able to regenerate brain-resident microglia. Thus, we demonstrate that LT-HSCs retain the potential to develop into TRMΦ, but their requirement is tissue specific in the peritoneum and brain.

Blood basophil activation is a reliable biomarker of allergic bronchopulmonary aspergillosis in cystic fibrosis.

The diagnosis of cystic fibrosis (CF) patients with allergic bronchopulmonary aspergillosis (ABPA) is clinically challenging, due to the absence of an objective biological test. Since blood basophils play a major role in allergic responses, we hypothesised that changes in their surface activation pattern discriminate between CF patients with and without ABPA.We conducted a prospective longitudinal study (Stanford cohort) comparing basophil activation test CD203c levels by flow cytometry before and after activation with Aspergillus fumigatus allergen extract or recombinant Asp f1 in 20 CF patients with ABPA (CF-ABPA) and in two comparison groups: CF patients with A. fumigatus colonisation (AC) but without ABPA (CF-AC; n=13) and CF patients without either AC or ABPA (CF; n=12). Patients were tested every 6 months and when ill with pulmonary exacerbation. We also conducted cross-sectional validation in a separate patient set (Dublin cohort).Basophil CD203c surface expression reliably discriminated CF-ABPA from CF-AC and CF over time. Ex vivo stimulation with A. fumigatus extract or recombinant Asp f1 produced similar results within the Stanford (p<0.0001) and the Dublin cohorts. CF-ABPA patients were likelier to have elevated specific IgE to A. fumigatus and were less frequently co-infected with Staphylococcus aureus.Basophil CD203c upregulation is a suitable diagnostic and stable monitoring biomarker of ABPA in CF.

Blood-based targeted metabolipidomics reveals altered omega fatty acid-derived lipid mediators in relapsing-remitting multiple sclerosis patients.

Unresolved and uncontrolled inflammation is considered a hallmark of pathogenesis in chronic inflammatory diseases like multiple sclerosis (MS), suggesting a defective resolution process. Inflammatory resolution is an active process partially mediated by endogenous metabolites of dietary polyunsaturated fatty acids (PUFA), collectively termed specialized pro-resolving lipid mediators (SPMs). Altered levels of resolution mediators have been reported in several inflammatory diseases and may partly explain impaired inflammatory resolution. Performing LC-MS/MS-based targeted lipid mediator profiling, we observed distinct changes in fatty acid metabolites in serum from 30 relapsing-remitting MS (RRMS) patients relative to 30 matched healthy subjects (HS). Robust linear regression revealed 12 altered lipid mediators after adjusting for confounders (p <0.05). Of these, 15d-PGJ2, PGE3, and LTB5 were increased in MS while PGF2a, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 12-HEPE, 14-HDoHE, and DHEA were decreased in MS compared to HS. In addition, 12,13-DiHOME and 12,13-DiHODE were positively correlated with expanded disability status scale values (EDSS). Using Partial Least Squares, we identified several lipid mediators with high VIP scores (VIP > 1: 32% - 52%) of which POEA, PGE3, DHEA, LTB5, and 12-HETE were top predictors for distinguishing between RRMS and HS (AUC =0.75) based on the XGBoost Classifier algorithm. Collectively, these findings suggest an imbalance between inflammation and resolution. Altogether, lipid mediators appear to have potential as diagnostic and prognostic biomarkers for RRMS.

Metabolomics as a promising tool for improving understanding of multiple sclerosis: A review of recent advances.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system that usually affects young adults. The development of MS is closely related to the changes in the metabolome. Metabolomics studies have been performed using biofluids or tissue samples from rodent models and human patients to reveal metabolic alterations associated with MS progression. This review aims to provide an overview of the applications of metabolomics that for the investigations of the perturbed metabolic pathways in MS and to reveal the potential of metabolomics in personalizing treatments. In conclusion, informative variations of metabolites can be potential biomarkers in advancing our understanding of MS pathogenesis for MS diagnosis, predicting the progression of the disease, and estimating drug effects. Metabolomics will be a promising technique for improving clinical care in MS.

CytofIn enables integrated analysis of public mass cytometry datasets using generalized anchors.

The increasing use of mass cytometry for analyzing clinical samples offers the possibility to perform comparative analyses across public datasets. However, challenges in batch normalization and data integration limit the comparison of datasets not intended to be analyzed together. Here, we present a data integration strategy, CytofIn, using generalized anchors to integrate mass cytometry datasets from the public domain. We show that low-variance controls, such as healthy samples and stable channels, are inherently homogeneous, robust against stimulation, and can serve as generalized anchors for batch correction. Single-cell quantification comparing mass cytometry data from 989 leukemia files pre- and post normalization with CytofIn demonstrates effective batch correction while recapitulating the gold-standard bead normalization. CytofIn integration of public cancer datasets enabled the comparison of immune features across histologies and treatments. We demonstrate the ability to integrate public datasets without necessitating identical control samples or bead standards for fast and robust analysis using CytofIn.

Development of immunosuppressive myeloid cells to induce tolerance in solid organ and hematopoietic cell transplant recipients.

Replacement of failed organs followed by safe withdrawal of immunosuppressive drugs has long been the goal of organ transplantation. We studied changes in the balance of T cells and myeloid cells in the blood of HLA-matched and -mismatched patients given living donor kidney transplants followed by total lymphoid irradiation, anti-thymocyte globulin conditioning, and donor hematopoietic cell transplant to induce mixed chimerism and immune tolerance. The clinical trials were based on a conditioning regimen used to establish mixed chimerism and tolerance in mice. In preclinical murine studies, there was a profound depletion of T cells and an increase in immunosuppressive polymorphonuclear (pmn) myeloid-derived suppressor cells (MDSCs) in the spleen and blood following transplant. Selective depletion of pmn MDSCs in mice abrogated mixed chimerism and tolerance. In our clinical trials, patients given an analogous tolerance conditioning regimen developed similar changes, including profound depletion of T cells and a marked increase in MDSCs in blood posttransplant. Posttransplant pmn MDSCs transiently increased expression of lectin-type oxidized LDL receptor-1, a marker of immunosuppression, and production of the T-cell inhibitor arginase-1. These posttransplant pmn MDSCs suppressed the activation, proliferation, and inflammatory cytokine secretion of autologous T-cell receptor microbead-stimulated pretransplant T cells when cocultured in vitro. In conclusion, we elucidated changes in receptors and function of immunosuppressive myeloid cells in patients enrolled in the tolerance protocol that were nearly identical to those of MDSCs required for tolerance in mice. These trials were registered at www.clinicaltrials.gov as #NCT00319657 and #NCT01165762.

Mixed chimerism and acceptance of kidney transplants after immunosuppressive drug withdrawal.

Preclinical studies have shown that persistent mixed chimerism is linked to acceptance of organ allografts without immunosuppressive (IS) drugs. Mixed chimerism refers to continued mixing of donor and recipient hematopoietic cells in recipient tissues after transplantation of donor cells. To determine whether persistent mixed chimerism and tolerance can be established in patients undergoing living donor kidney transplantation, we infused allograft recipients with donor T cells and hematopoietic progenitors after posttransplant lymphoid irradiation. In 24 of 29 fully human leukocyte antigen (HLA)-matched patients who had persistent mixed chimerism for at least 6 months, complete IS drug withdrawal was achieved without subsequent evidence of rejection for at least 2 years. In 10 of 22 HLA haplotype-matched patients with persistent mixed chimerism for at least 12 months, reduction of IS drugs to tacrolimus monotherapy was achieved. Withdrawal of tacrolimus during the second year resulted in loss of detectable chimerism and subsequent rejection episodes, unless tacrolimus therapy was reinstituted. Posttransplant immune reconstitution of naïve B cells and B cell precursors was more rapid than the reconstitution of naïve T cells and thymic T cell precursors. Robust chimerism was observed only among naïve T and B cells but not among memory T cells. No evidence of rejection was observed in all surveillance graft biopsies obtained from mixed chimeric patients withdrawn from IS drugs, and none developed graft-versus-host disease. In conclusion, persistent mixed chimerism established in fully HLA- or haplotype-matched patients allowed for complete or partial IS drug withdrawal without rejection.

Accelerated, but not conventional, radiotherapy of murine B-cell lymphoma induces potent T cell-mediated remissions.

Conventional local tumor irradiation (LTI), delivered in small daily doses over several weeks, is used clinically as a palliative, rather than curative, treatment for chemotherapy-resistant diffuse large B-cell lymphoma (DLBCL) for patients who are ineligible for hematopoietic cell transplantation. Our goal was to test the hypothesis that accelerated, but not conventional, LTI would be more curative by inducing T cell-mediated durable remissions. We irradiated subcutaneous A20 and BL3750 lymphoma tumors in mice with a clinically relevant total radiation dose of 30 Gy LTI, delivered in 10 doses of 3 Gy over 4 days (accelerated irradiation) or as 10 doses of 3 Gy over 12 days (conventional irradiation). Compared with conventional LTI, accelerated LTI resulted in more complete and durable tumor remissions. The majority of these mice were resistant to rechallenge with lymphoma cells, demonstrating the induction of memory antitumor immunity. The increased efficacy of accelerated LTI correlated with higher levels of tumor cell necrosis vs apoptosis and expression of "immunogenic cell death" markers, including calreticulin, heat shock protein 70 (Hsp70), and Hsp90. Accelerated LTI-induced remissions were not seen in immunodeficient Rag-2 -/- mice, CD8+ T-cell-depleted mice, or Batf-3 -/- mice lacking CD8α+ and CD103+ dendritic cells. Accelerated, but not conventional, LTI in immunocompetent hosts induced marked increases in tumor-infiltrating CD4+ and CD8+ T cells and MHCII+CD103+CD11c+ dendritic cells and corresponding reductions in exhausted PD-1+Eomes+CD8+ T cells and CD4+CD25+FOXP3+ regulatory T cells. These findings raise the possibility that accelerated LTI can provide effective immune control of human DLBCL.

Fetal Hematopoietic Stem Cell Transplantation Fails to Fully Regenerate the B-Lymphocyte Compartment.

B cells are key components of cellular and humoral immunity and, like all lymphocytes, are thought to originate and renew from hematopoietic stem cells (HSCs). However, our recent single-HSC transfer studies demonstrate that adult bone marrow HSCs do not regenerate B-1a, a subset of tissue B cells required for protection against pneumonia, influenza, and other infections. Since B-1a are regenerated by transfers of fetal liver, the question arises as to whether B-1a derive from fetal, but not adult, HSCs. Here we show that, similar to adult HSCs, fetal HSCs selectively fail to regenerate B-1a. We also show that, in humanized mice, human fetal liver regenerates tissue B cells that are phenotypically similar to murine B-1a, raising the question of whether human HSC transplantation, the mainstay of such models, is sufficient to regenerate human B-1a. Thus, our studies overtly challenge the current paradigm that HSCs give rise to all components of the immune system.

Earth Mover's Distance (EMD): A True Metric for Comparing Biomarker Expression Levels in Cell Populations.

Changes in the frequencies of cell subsets that (co)express characteristic biomarkers, or levels of the biomarkers on the subsets, are widely used as indices of drug response, disease prognosis, stem cell reconstitution, etc. However, although the currently available computational "gating" tools accurately reveal subset frequencies and marker expression levels, they fail to enable statistically reliable judgements as to whether these frequencies and expression levels differ significantly between/among subject groups. Here we introduce flow cytometry data analysis pipeline which includes the Earth Mover's Distance (EMD) metric as solution to this problem. Well known as an informative quantitative measure of differences between distributions, we present three exemplary studies showing that EMD 1) reveals clinically-relevant shifts in two markers on blood basophils responding to an offending allergen; 2) shows that ablative tumor radiation induces significant changes in the murine colon cancer tumor microenvironment; and, 3) ranks immunological differences in mouse peritoneal cavity cells harvested from three genetically distinct mouse strains.

AutoGate: automating analysis of flow cytometry data.

Nowadays, one can hardly imagine biology and medicine without flow cytometry to measure CD4 T cell counts in HIV, follow bone marrow transplant patients, characterize leukemias, etc. Similarly, without flow cytometry, there would be a bleak future for stem cell deployment, HIV drug development and full characterization of the cells and cell interactions in the immune system. But while flow instruments have improved markedly, the development of automated tools for processing and analyzing flow data has lagged sorely behind. To address this deficit, we have developed automated flow analysis software technology, provisionally named AutoComp and AutoGate. AutoComp acquires sample and reagent labels from users or flow data files, and uses this information to complete the flow data compensation task. AutoGate replaces the manual subsetting capabilities provided by current analysis packages with newly defined statistical algorithms that automatically and accurately detect, display and delineate subsets in well-labeled and well-recognized formats (histograms, contour and dot plots). Users guide analyses by successively specifying axes (flow parameters) for data subset displays and selecting statistically defined subsets to be used for the next analysis round. Ultimately, this process generates analysis "trees" that can be applied to automatically guide analyses for similar samples. The first AutoComp/AutoGate version is currently in the hands of a small group of users at Stanford, Emory and NIH. When this "early adopter" phase is complete, the authors expect to distribute the software free of charge to .edu, .org and .gov users.