A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification.

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

The aconitate decarboxylase 1/itaconate pathway modulates immune dysregulation and associates with cardiovascular disease markers in SLE.

Objective: The Krebs cycle enzyme Aconitate Decarboxylase 1 (ACOD1) mediates itaconate synthesis in myeloid cells.. Previously, we reported that administration of 4-octyl itaconate abrogated lupus phenotype in mice. Here, we explore the role of the endogenous ACOD1/itaconate pathway in the development of murine lupus as well as their relevance in premature cardiovascular damage in SLE. Methods: We characterized Acod1 protein expression in bone marrow-derived macrophages and human monocyte-derived macrophages, following a TLR7 agonist (imiquimod, IMQ). Wild type and Acod1-/- mice were exposed to topical IMQ for 5 weeks to induce an SLE phenotype and immune dysregulation was quantified. Itaconate serum levels were quantified in SLE patients and associated to cardiometabolic parameters and disease activity. Results: ACOD1 was induced in mouse bone marrow-derived macrophages (BMDM) and human monocyte-derived macrophages following in vitro TLR7 stimulation. This induction was partially dependent on type I Interferon receptor signaling and specific intracellular pathways. In the IMQ-induced mouse model of lupus, ACOD1 knockout (Acod1-/-) displayed disruptions of the splenic architecture, increased serum anti-dsDNA and proinflammatory cytokine levels, enhanced kidney immune complex deposition and proteinuria, when compared to the IMQ-treated WT mice. Consistent with these results, Acod1-/- BMDM exposed to IMQ showed higher proinflammatory features in vitro. Itaconate levels were decreased in SLE serum compared to healthy control sera, in association with specific perturbed cardiometabolic parameters and subclinical vascular disease. Conclusion: These findings suggest that the ACOD1/itaconate pathway plays important immunomodulatory and vasculoprotective roles in SLE, supporting the potential therapeutic role of itaconate analogs in autoimmune diseases.

Cholesterol Interference in the Assessment of Vitamin D Status: A Canadian Health Measures Survey Biobank Project.

BACKGROUND: Matrix effects are a known problem with immunoassays measuring serum 25-hydroxyvitamin D [25(OH)D]. OBJECTIVES: To determine if the inverse association between serum 25(OH)D and serum cholesterol concentrations is a function of assay method: Diasorin Liaison 25(OH) Vitamin D Total Assay (Liaison Total Assay), an immunoassay, compared with liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: Canadian Health Measures Survey data and biobank serum (White males aged 20-79 y, n = 392) were evaluated for bias in serum 25(OH)D using Bland-Altman plots. Differences in serum 25(OH)D (Liaison Total Assay - LC-MS/MS) were compared among non-HDL-cholesterol <4.2 (n = 295) compared with ≥4.2 (n = 97) mmol/L and total cholesterol groups <5.2 (n = 256) compared with ≥5.2 (n = 136) mmol/L, and associations tested between 25(OH)D and non-HDL-cholesterol or total cholesterol concentrations, using regression. RESULTS: Serum 25(OH)D measured using Liaison Total Assay ranged from 10.7 to 137.0 nmol/L and 14.4 to 137.9 nmol/L by LC-MS/MS. Liaison Total Assay - LC-MS/MS showed a negative bias of 5.5 (95% limits of agreement -23.8, 12.7) nmol/L. Differences in 25(OH)D were -4.0 ± 9.0 (±SD) nmol/L if non-HDL-cholesterol was <4.2 mmol/L and -10.2 ± 8.7 nmol/L if ≥4.2 mmol/L (P < 0.0001). Differences in 25(OH)D, if total cholesterol was <5.2 mmol/L, were -3.4 ± 8.6 nmol/L and -9.6 ± 9.3 nmol/L if ≥5.2 mmol/L (P < 0.0001). Serum non-HDL-cholesterol (beta -3.17, P = 0.0014) and total cholesterol (beta -2.77, P = 0.0046) were inversely associated with Liaison Total Assay 25(OH)D (adjusted for age, fasting, and body mass index), but not with LC-MS/MS measured 25(OH)D. Interference by these lipoproteins was not eliminated by standardization of the Liaison Total Assay. Similar associations were observed with triglycerides as for the lipoproteins. CONCLUSIONS: Total cholesterol inversely associates with 25(OH)D, which is likely due to elevated non-HDL-cholesterol lipoprotein or triglyceride interference with the Liaison Total Assay. This is important as elevated cholesterol is common, and an underestimation of vitamin D status could be an unnecessary cause for concern.

Regional impacts of warming on biodiversity and biomass in high latitude stream ecosystems across the Northern Hemisphere.

Warming can have profound impacts on ecological communities. However, explorations of how differences in biogeography and productivity might reshape the effect of warming have been limited to theoretical or proxy-based approaches: for instance, studies of latitudinal temperature gradients are often conflated with other drivers (e.g., species richness). Here, we overcome these limitations by using local geothermal temperature gradients across multiple high-latitude stream ecosystems. Each suite of streams (6-11 warmed by 1-15°C above ambient) is set within one of five regions (37 streams total); because the heating comes from the bedrock and is not confounded by changes in chemistry, we can isolate the effect of temperature. We found a negative overall relationship between diatom and invertebrate species richness and temperature, but the strength of the relationship varied regionally, declining more strongly in regions with low terrestrial productivity. Total invertebrate biomass increased with temperature in all regions. The latter pattern combined with the former suggests that the increased biomass of tolerant species might compensate for the loss of sensitive species. Our results show that the impact of warming can be dependent on regional conditions, demonstrating that local variation should be included in future climate projections rather than simply assuming universal relationships.

Risk of bias in cross-sectional studies: Protocol for a scoping review of concepts and tools.

Cross-sectional studies are commonly used to study human health and disease, but are especially susceptible to bias. This scoping review aims to identify and describe available tools to assess the risk of bias (RoB) in cross-sectional studies and to compile the key bias concepts relevant to cross-sectional studies into an item bank. Using the JBI scoping review methodology, the strategy to locate relevant RoB concepts and tools is a combination of database searches, prospective review of PROSPERO registry records; and consultation with knowledge users and content experts. English language records will be included if they describe tools, checklists, or instruments which describe or permit assessment of RoB for cross-sectional studies. Systematic reviews will be included if they consider eligible RoB tools or use RoB tools for RoB of cross-sectional studies. All records will be independently screened, selected, and extracted by one researcher and checked by a second. An analytic framework will be used to structure the extraction of data. Results for the scoping review are pending. Results from this scoping review will be used to inform future selection of RoB tools and to consider whether development of a new RoB tool for cross-sectional studies is needed.

Identification of Fibroinflammatory and Fibrotic Transcriptomic Subsets of Human Cutaneous Sclerotic Chronic Graft-Versus-Host Disease.

Cutaneous sclerotic chronic graft-versus-host disease (cGVHD) is a common and highly morbid complication of allogeneic hematopoietic stem cell transplantation. Our goals were to identify signals active in the skin of patients with sclerotic cGVHD in an effort to better understand how to treat this manifestation and to explore the heterogeneity of the disease. We identified genes that are significantly upregulated in the skin of patients with sclerotic cGVHD (n = 17) compared with those in the skin of patients who underwent allogeneic hematopoietic stem cell transplantation without cutaneous cGVHD (n = 9) by bulk RNA sequencing. Sclerotic cGVHD was most associated with T helper 1, phagocytic, and fibrotic pathways. In addition, different transcriptomic groups of affected patients were discovered: those with fibrotic and inflammatory/T helper 1 gene expression (the fibroinflammatory group) and those with predominantly fibrotic/TGFß-associated expression (the fibrotic group). Further study will help elucidate whether these gene expression findings can be used to tailor treatment decisions. Multiple proteins encoded by highly induced genes in the skin (SFRP4, SERPINE2, COMP) were also highly induced in the plasma of patients with sclerotic cGVHD (n = 16) compared with those in plasma of control patients who underwent allogeneic hematopoietic stem cell transplantation without sclerotic cGVHD (n = 17), suggesting these TGFß and Wnt pathway mediators as candidate blood biomarkers of the disease.

Microbial ligand-independent regulation of lymphopoiesis by NOD1.

Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. Here we demonstrate that NOD1, a cytosolic innate sensor of bacterial peptidoglycan, also functions in murine hematopoietic cells as a major regulator of both the generation and differentiation of lymphoid progenitors as well as peripheral T lymphocyte homeostasis. We further show that NOD1 mediates these functions by facilitating STAT5 signaling downstream of hematopoietic cytokines. In steady-state, loss of NOD1 resulted in a modest but significant decrease in numbers of mature T, B and natural killer cells. During systemic protozoan infection this defect was markedly enhanced, leading to host mortality. Lack of functional NOD1 also impaired T cell-dependent anti-tumor immunity while preventing colitis. These findings reveal that, in addition to its classical role as a bacterial ligand receptor, NOD1 plays an important function in regulating adaptive immunity through interaction with a major host cytokine signaling pathway.

RichPathR: a gene set enrichment analysis and visualization tool.

Gene set enrichment analysis (GSEA) is an important step for disease and drug discovery. Genomic, transcriptomics, proteomics and epigenetic analysis of tissue or cells generates gene lists that need to be further investigated in the known biological context. The advent of high-throughput technologies generates the vast number of gene lists that are up or down regulated together. One way of getting meaningful insights of the relationship of these genes is utilizing existing knowledge bases linking them with biological functions or phenotypes. Multiple public databases with annotated gene sets are available for GSEA, and enrichR is the most popular web application still requiring custom tools for large-scale mining. richPathR package is a collection of R functions that helps researchers carry out exploratory analysis and visualization of gene set enrichment using EnrichR.

Divergent roles for STAT4 in shaping differentiation of cytotoxic ILC1 and NK cells during gut inflammation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) require signal transducer and activator of transcription 4 (STAT4) to elicit rapid effector responses and protect against pathogens. By combining genetic and transcriptomic approaches, we uncovered divergent roles for STAT4 in regulating effector differentiation of these functionally related cell types. Stat4 deletion in Ncr1-expressing cells led to impaired NK cell terminal differentiation as well as to an unexpected increased generation of cytotoxic ILC1 during intestinal inflammation. Mechanistically, Stat4-deficient ILC1 exhibited upregulation of gene modules regulated by STAT5 in vivo and an aberrant effector differentiation upon in vitro stimulation with IL-2, used as a prototypical STAT5 activator. Moreover, STAT4 expression in NCR+ innate lymphocytes restrained gut inflammation in the dextran sulfate sodium-induced colitis model limiting pathogenic production of IL-13 from adaptive CD4+ T cells in the large intestine. Collectively, our data shed light on shared and distinctive mechanisms of STAT4-regulated transcriptional control in NK cells and ILC1 required for intestinal inflammatory responses.

CD271 activation prevents low to high-risk progression of cutaneous squamous cell carcinoma and improves therapy outcomes.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, showing a rapid increasing incidence worldwide. Although most cSCC can be cured by surgery, a sizeable number of cases are diagnosed at advanced stages, with local invasion and distant metastatic lesions. In the skin, neurotrophins (NTs) and their receptors (CD271 and Trk) form a complex network regulating epidermal homeostasis. Recently, several works suggested a significant implication of NT receptors in cancer. However, CD271 functions in epithelial tumors are controversial and its precise role in cSCC is still to be defined. METHODS: Spheroids from cSCC patients with low-risk (In situ or Well-Differentiated cSCC) or high-risk tumors (Moderately/Poorly Differentiated cSCC), were established to explore histological features, proliferation, invasion abilities, and molecular pathways modulated in response to CD271 overexpression or activation in vitro. The effect of CD271 activities on the response to therapeutics was also investigated. The impact on the metastatic process and inflammation was explored in vivo and in vitro, by using zebrafish xenograft and 2D/3D models. RESULTS: Our data proved that CD271 is upregulated in Well-Differentiated tumors as compared to the more aggressive Moderately/Poorly Differentiated cSCC, both in vivo and in vitro. We demonstrated that CD271 activities reduce proliferation and malignancy marker expression in patient-derived cSCC spheroids at each tumor grade, by increasing neoplastic cell differentiation. CD271 overexpression significantly increases cSCC spheroid mass density, while it reduces their weight and diameter, and promotes a major fold-enrichment in differentiation and keratinization genes. Moreover, both CD271 overexpression and activation decrease cSCC cell invasiveness in vitro. A significant inhibition of the metastatic process by CD271 was observed in a newly established zebrafish cSCC model. We found that the recruitment of leucocytes by CD271-overexpressing cells directly correlates with tumor killing and this finding was further highlighted by monocyte infiltration in a THP-1-SCC13 3D model. Finally, CD271 activity synergizes with Trk receptor inhibition, by reducing spheroid viability, and significantly improves the outcome of photodynamic therapy (PTD) or chemotherapy in spheroids and zebrafish. CONCLUSION: Our study provides evidence that CD271 could prevent the switch between low to high-risk cSCC tumors. Because CD271 contributes to maintaining active differentiative paths and favors the response to therapies, it might be a promising target for future pharmaceutical development.

Single-cell multiomic analysis reveals the involvement of Type I interferon-responsive CD8+ T cells in amyloid beta-associated memory loss.

Alzheimer's disease (AD) is the leading cause of dementia worldwide, but there are limited therapeutic options and no current cure. While the involvement of microglia in AD has been highly appreciated, the role of other innate and adaptive immune cells remains largely unknown, partly due to their scarcity and heterogeneity. This study aimed to study non-microglial immune cells in wild type and AD-transgenic mouse brains across different ages. Our results uncovered the presence of a unique CD8+ T cell population that were selectively increased in aging AD mouse brains, here referred to as "disease-associated T cells (DATs)". These DATs were found to express an elevated tissue-resident memory and Type I interferon-responsive gene signature. Further analysis of aged AD mouse brains showed that these CD8+ T cells were not present in peripheral or meningeal tissues. Preventing CD8+ T cell development in AD-transgenic mice via genetic deletion of beta-2 microglobulin ( B2m ) led to a reduction of amyloid-ß plaque formation in aged mice, and improved memory in AD-transgenic mice as early as four months of age. The integration of transcriptomic and epigenomic profiles at the single-cell level revealed potential transcription factors that reshape the regulomes of CD8+ T cells. These findings highlight a critical role for DATs in the progression of AD and provide a new avenue for treatment.

Risk of relapse of ANCA-associated vasculitis among patients homozygous for the proteinase 3 gene Val119Ile polymorphism.

BACKGROUND: The frequency of proteinase 3 gene (PRTN3) polymorphisms in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is not fully characterised. We hypothesise that the presence of a PRTN3 gene polymorphism (single nucleotide polymorphism (SNP) rs351111) is relevant for clinical outcomes. METHODS: DNA variant calling for SNP rs351111 (chr.19:844020, c.355G>A) in PRTN3 gene assessed the allelic frequency in patients with PR3-AAV included in the Rituximab in ANCA-Associated Vasculitis trial. This was followed by RNA-seq variant calling to characterise the mRNA expression. We compared clinical outcomes between patients homozygous for PRTN3-Ile119 or PRTN3-Val119. RESULTS: Whole blood samples for DNA calling were available in 188 patients. 75 patients with PR3-AAV had the allelic variant: 62 heterozygous PRTN3-Val119Ile and 13 homozygous for PRTN3-Ile119. RNA-seq was available for 89 patients and mRNA corresponding to the allelic variant was found in 32 patients with PR3-AAV: 25 heterozygous PRTN3-Val119Ile and 7 homozygous for PRTN3-Ile119. The agreement between the DNA calling results and mRNA expression of the 86 patients analysed by both methods was 100%. We compared the clinical outcomes of 64 patients with PR3-AAV: 51 homozygous for PRTN3-Val119 and 13 homozygous for PRTN3-Ile119. The frequency of severe flares at 18 months in homozygous PRTN3-Ile119 was significantly higher when compared with homozygous PRTN3-Val119 (46.2% vs 19.6%, p=0.048). Multivariate analysis identified homozygous PR3-Ile119 as main predictor of severe relapse (HR 4.67, 95% CI 1.16 to 18.86, p=0.030). CONCLUSION: In patients with PR3-AAV, homozygosity for PRTN3-Val119Ile polymorphism appears associated with higher frequency of severe relapse. Further studies are necessary to better understand the association of this observation with the risk of severe relapse.

Constitutively active Lyn kinase causes a cutaneous small vessel vasculitis and liver fibrosis syndrome.

Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN, p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of ß2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

Spleen tyrosine kinase inhibition restores myeloid homeostasis in COVID-19.

Spleen tyrosine kinase (SYK) is a previously unidentified therapeutic target that inhibits neutrophil and macrophage activation in coronavirus disease 2019 (COVID-19). Fostamatinib, a SYK inhibitor, was studied in a phase 2 placebo-controlled randomized clinical trial and was associated with improvements in many secondary end points related to efficacy. Here, we used a multiomic approach to evaluate cellular and soluble immune mediator responses of patients enrolled in this trial. We demonstrated that SYK inhibition was associated with reduced neutrophil activation, increased circulation of mature neutrophils (CD10+CD33-), and decreased circulation of low-density granulocytes and polymorphonuclear myeloid-derived suppressor cells (HLA-DR-CD33+CD11b-). SYK inhibition was also associated with normalization of transcriptional activity in circulating monocytes relative to healthy controls, an increase in frequency of circulating nonclassical and HLA-DRhi classical monocyte populations, and restoration of interferon responses. Together, these data suggest that SYK inhibition may mitigate proinflammatory myeloid cellular and soluble mediator responses thought to contribute to immunopathogenesis of severe COVID-19.

Chromatin Landscape Governing Murine Epidermal Differentiation.

Chromatin landscape and regulatory networks are determinants in lineage specification and differentiation. To define the temporospatial differentiation axis in murine epidermal cells in vivo, we generated datasets profiling expression dynamics (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin using sequencing), architecture (Hi-C), and histone modifications (chromatin immunoprecipitation followed by sequencing) in the epidermis. We show that many differentially regulated genes are suppressed during the differentiation process, with superenhancers controlling differentiation-specific epigenomic changes. Our data shows the relevance of the Dlx/Klf/Grhl combinatorial regulatory network in maintaining correct temporospatial gene expression during epidermal differentiation. We determined differential open compartments, topologically associating domain score, and looping in the basal cell and suprabasal cell epidermal fractions, with the evolutionarily conserved epidermal differentiation complex region showing distinct suprabasal cell-specific topologically associating domain and loop formation that coincided with superenhancer sites. Overall, our study provides a global genome-wide resource of chromatin dynamics that define unrecognized regulatory networks and the epigenetic control of Dlx3-bound superenhancer elements during epidermal differentiation.

Molecular mechanisms regulating wound repair: Evidence for paracrine signaling from corneal epithelial cells to fibroblasts and immune cells following transient epithelial cell treatment with Mitomycin C.

In this paper, we use RNAseq to identify senescence and phagocytosis as key factors to understanding how mitomyin C (MMC) stimulates regenerative wound repair. We use conditioned media (CM) from untreated (CMC) and MMC treated (CMM) human and mouse corneal epithelial cells to show that corneal epithelial cells indirectly exposed to MMC secrete elevated levels of immunomodulatory proteins including IL-1α and TGFß1 compared to cells exposed to CMC. These factors increase epithelial and macrophage phagocytosis and promote ECM turnover. IL-1α supplementation can increase phagocytosis in control epithelial cells and attenuate TGFß1 induced αSMA expression by corneal fibroblasts. Yet, we show that epithelial cell CM contains factors besides IL-1α that regulate phagocytosis and αSMA expression by fibroblasts. Exposure to CMM also impacts the activation of bone marrow derived dendritic cells and their ability to present antigen. These in vitro studies show how a brief exposure to MMC induces corneal epithelial cells to release proteins and other factors that function in a paracrine way to enhance debris removal and enlist resident epithelial and immune cells as well as stromal fibroblasts to support regenerative and not fibrotic wound healing.

A central role for STAT5 in the transcriptional programing of T helper cell metabolism.

Activated lymphocytes adapt their metabolism to meet the energetic and biosynthetic demands imposed by rapid growth and proliferation. Common gamma chain (cγ) family cytokines are central to these processes, but the role of downstream signal transducer and activator of transcription 5 (STAT5) signaling, which is engaged by all cγ members, is poorly understood. Using genome-, transcriptome-, and metabolome-wide analyses, we demonstrate that STAT5 is a master regulator of energy and amino acid metabolism in CD4+ T helper cells. Mechanistically, STAT5 localizes to an array of enhancers and promoters for genes encoding essential enzymes and transporters, where it facilitates p300 recruitment and epigenetic remodeling. We also find that STAT5 licenses the activity of two other key metabolic regulators, the mTOR signaling pathway and the MYC transcription factor. Building on the latter, we present evidence for transcriptome-wide cooperation between STAT5 and MYC in both normal and transformed T cells. Together, our data provide a molecular framework for transcriptional programing of T cell metabolism downstream of cγ cytokines and highlight the JAK-STAT pathway in mediating cellular growth and proliferation.

Perspective: Human Milk Composition and Related Data for National Health and Nutrition Monitoring and Related Research.

National health and nutrition monitoring is an important federal effort in the United States and Canada, and the basis for many of their nutrition and health policies. Understanding of child exposures through human milk (HM) remains out of reach due to lack of current and representative data on HM's composition and intake volume. This article provides an overview of the current national health and nutrition monitoring activities for HM-fed children, HM composition (HMC) and volume data used for exposure assessment, categories of potential measures in HM, and associated variability factors. In this Perspective, we advocate for a framework for collection and reporting of HMC data for national health and nutrition monitoring and programmatic needs, including a shared vision for a publicly available Human Milk Composition Data Repository (HMCD-R) to include essential metadata associated with HMC. HMCD-R can provide a central, integrated platform for researchers and public health officials for compiling, evaluating, and sharing HMC data. The compiled compositional and metadata in HMCD-R would provide pertinent measures of central tendency and variability and allow use of modeling techniques to approximate compositional profiles for subgroups, providing more accurate exposure assessments for purposes of monitoring and surveillance. HMC and related metadata could facilitate understanding the complexity and variability of HM composition, provide crucial data for assessment of infant and maternal nutritional needs, and inform public health policies, food and nutrition programs, and clinical practice guidelines.

A Bayesian method for identifying associations between response variables and bacterial community composition.

Determining associations between intestinal bacteria and continuously measured physiological outcomes is important for understanding the bacteria-host relationship but is not straightforward since abundance data (compositional data) are not normally distributed. To address this issue, we developed a fully Bayesian linear regression model (BRACoD; Bayesian Regression Analysis of Compositional Data) with physiological measurements (continuous data) as a function of a matrix of relative bacterial abundances. Bacteria can be classified as operational taxonomic units or by taxonomy (genus, family, etc.). Bacteria associated with the physiological measurement were identified using a Bayesian variable selection method: Stochastic Search Variable Selection. The output is a list of inclusion probabilities ([Formula: see text]) and coefficients that indicate the strength of the association ([Formula: see text]) for each bacterial taxa. Tests with simulated communities showed that adopting a cut point value of [Formula: see text] ≥ 0.3 for identifying included bacteria optimized the true positive rate (TPR) while maintaining a false positive rate (FPR) of ≤ 5%. At this point, the chances of identifying non-contributing bacteria were low and all well-established contributors were included. Comparison with other methods showed that BRACoD (at [Formula: see text] ≥ 0.3) had higher precision and a higher TPR than a commonly used center log transformed LASSO procedure (clr-LASSO) as well as higher TPR than an off-the-shelf Spike and Slab method after center log transformation (clr-SS). BRACoD was also less likely to include non-contributing bacteria that merely correlate with contributing bacteria. Analysis of a rat microbiome experiment identified 47 operational taxonomic units that contributed to fecal butyrate levels. Of these, 31 were positively and 16 negatively associated with butyrate. Consistent with their known role in butyrate metabolism, most of these fell within the Lachnospiraceae and Ruminococcaceae. We conclude that BRACoD provides a more precise and accurate method for determining bacteria associated with a continuous physiological outcome compared to clr-LASSO. It is more sensitive than a generalized clr-SS algorithm, although it has a higher FPR. Its ability to distinguish genuine contributors from correlated bacteria makes it better suited to discriminating bacteria that directly contribute to an outcome. The algorithm corrects for the distortions arising from compositional data making it appropriate for analysis of microbiome data.

Single-Cell Analysis Reveals the Range of Transcriptional States of Circulating Human Neutrophils.

Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently, neutrophils were considered homogeneous and transcriptionally inactive cells, but both concepts are being challenged. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However, the use of scRNA-seq to characterize neutrophils has proven technically difficult, explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline, rather than to the existing scRNA-seq chemistries, can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells, which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells, through a transitional phenotype (Nh1), into one of two end points defined by either relative transcriptional inactivity (Nh2) or high expression of type I IFN-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level, we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity, with potential implications for the characterization of neutrophils in health and disease.