Concordant B and T Cell Heterogeneity Inferred from the Multiomic Landscape of Peripheral Blood Mononuclear Cells in a Crohn's Disease Cohort.

BACKGROUND AND AIMS: Crohn's disease is characterized by inflammation in the gastrointestinal tract due to a combination of genetic, immune, and environmental factors. Transcriptomic and epigenomic profiling of intestinal tissue of Crohn's disease patients have revealed valuable insights into pathology, however have not been conducted jointly on less invasive peripheral blood mononuclear cells (PBMCs). Furthermore, the heterogeneous responses to treatments among individuals with Crohn's disease imply hidden diversity of pathological mechanisms. METHODS: We employed single nucleus multiomic analysis, integrating both snRNA-seq and snATAC-seq of PBMCs with a variety of open source bioinformatics applications. RESULTS: Our findings reveal a diverse range of transcriptional signatures among individuals, highlighting the heterogeneity in PBMC profiles. Nevertheless, striking concordance between three heterogeneous groups was observed across B cells and T cells. Differential gene regulatory mechanisms partially explain these profiles, notably including a signature involving TGFß signaling in two individuals with Crohn's disease. A mutation mapped to a transcription factor binding site within a differentially accessible peak associated with the expression of this pathway, with implications for a personalized approach to understanding disease pathology. CONCLUSIONS: This study highlights how multiomic analysis can reveal common regulatory mechanisms that underlie heterogeneity of PBMC profiles, one of which may be specific to inflammatory disease.

Landscape of pharmacogenetic variants associated with non-insulin antidiabetic drugs in the Indian population.

INTRODUCTION: Genetic variants contribute to differential responses to non-insulin antidiabetic drugs (NIADs), and consequently to variable plasma glucose control. Optimal control of plasma glucose is paramount to minimizing type 2 diabetes-related long-term complications. India's distinct genetic architecture and its exploding burden of type 2 diabetes warrants a population-specific survey of NIAD-associated pharmacogenetic (PGx) variants. The recent availability of large-scale whole genomes from the Indian population provides a unique opportunity to generate a population-specific map of NIAD-associated PGx variants. RESEARCH DESIGN AND METHODS: We mined 1029 Indian whole genomes for PGx variants, drug-drug interaction (DDI) and drug-drug-gene interactions (DDGI) associated with 44 NIADs. Population-wise allele frequencies were estimated and compared using Fisher's exact test. RESULTS: Overall, we found 76 known and 52 predicted deleterious common PGx variants associated with response to type 2 diabetes therapy among Indians. We report remarkable interethnic differences in the relative cumulative counts of decreased and increased response-associated alleles across NIAD classes. Indians and South Asians showed a significant excess of decreased metformin response-associated alleles compared with other global populations. Network analysis of shared PGx genes predicts high DDI risk during coadministration of NIADs with other metabolic disease drugs. We also predict an increased CYP2C19-mediated DDGI risk for CYP3A4/3A5-metabolized NIADs, saxagliptin, linagliptin and glyburide when coadministered with proton-pump inhibitors (PPIs). CONCLUSIONS: Indians and South Asians have a distinct PGx profile for antidiabetes drugs, marked by an excess of poor treatment response-associated alleles for various NIAD classes. This suggests the possibility of a population-specific reduced drug response in atleast some NIADs. In addition, our findings provide an actionable resource for accelerating future diabetes PGx studies in Indians and South Asians and reconsidering NIAD dosing guidelines to ensure maximum efficacy and safety in the population.

Genetically transitional disease: conceptual understanding and applicability to rheumatic disease.

In genomic medicine, the concept of genetically transitional disease (GTD) refers to cases in which gene mutation is necessary but not sufficient to cause disease. In this Perspective, we apply this novel concept to rheumatic diseases, which have been linked to hundreds of genetic variants via association studies. These variants are in the 'grey zone' between monogenic variants with large effect sizes and common susceptibility alleles with small effect sizes. Among genes associated with rare autoinflammatory diseases, many low-frequency and/or low-penetrance variants are known to increase susceptibility to systemic inflammation. In autoimmune diseases, hundreds of HLA and non-HLA genetic variants have been revealed to be modest- to moderate-risk alleles. These diseases can be reclassified as GTDs. The same concept could apply to many other human diseases. GTD could improve the reporting of genetic testing results, diagnostic yields, genetic counselling and selection of therapy, as well as facilitating research using a novel approach to human genetic diseases.

The role of admixture in the rare variant contribution to inflammatory bowel disease.

BACKGROUND: Identification of rare variants involved in complex, polygenic diseases like Crohn's disease (CD) has accelerated with the introduction of whole exome/genome sequencing association studies. Rare variants can be used in both diagnostic and therapeutic assessments; however, since they are likely to be restricted to specific ancestry groups, their contributions to risk assessment need to be evaluated outside the discovery population. Prior studies implied that the three known rare variants in NOD2 are absent in West African and Asian populations and only contribute in African Americans via admixture. METHODS: Whole genome sequencing (WGS) data from 3418 African American individuals, 1774 inflammatory bowel disease (IBD) cases, and 1644 controls were used to assess odds ratios and allele frequencies (AF), as well as haplotype-specific ancestral origins of European-derived CD variants discovered in a large exome-wide association study. Local and global ancestry was performed to assess the contribution of admixture to IBD contrasting European and African American cohorts. RESULTS: Twenty-five rare variants associated with CD in European discovery cohorts are typically five-fold lower frequency in African Americans. Correspondingly, where comparisons could be made, the rare variants were found to have a predicted four-fold reduced burden for IBD in African Americans, when compared to European individuals. Almost all of the rare CD European variants were found on European haplotypes in the African American cohort, implying that they contribute to disease risk in African Americans primarily due to recent admixture. In addition, proportion of European ancestry correlates the number of rare CD European variants each African American individual carry, as well as their polygenic risk of disease. Similar findings were observed for 23 mutations affecting 10 other common complex diseases for which the rare variants were discovered in European cohorts. CONCLUSIONS: European-derived Crohn's disease rare variants are even more rare in African Americans and contribute to disease risk mainly due to admixture, which needs to be accounted for when performing cross-ancestry genetic assessments.

Cell-based versus corticosteroid injections for knee pain in osteoarthritis: a randomized phase 3 trial.

Various types of cellular injection have become a popular and costly treatment option for patients with knee osteoarthritis despite a paucity of literature establishing relative efficacy to each other or corticosteroid injections. Here we aimed to identify the safety and efficacy of cell injections from autologous bone marrow aspirate concentrate, autologous adipose stromal vascular fraction and allogeneic human umbilical cord tissue-derived mesenchymal stromal cells, in comparison to corticosteroid injection (CSI). The study was a phase 2/3, four-arm parallel, multicenter, single-blind, randomized, controlled clinical trial with 480 patients with a diagnosis of knee osteoarthritis (Kellgren-Lawrence II-IV). Participants were randomized to the three different arms with a 3:1 distribution. Arm 1: autologous bone marrow aspirate concentrate (n = 120), CSI (n = 40); arm 2: umbilical cord tissue-derived mesenchymal stromal cells (n = 120), CSI (n = 40); arm 3: stromal vascular fraction (n = 120), CSI (n = 40). The co-primary endpoints were the visual analog scale pain score and Knee injury and Osteoarthritis Outcome Score pain score at 12 months versus baseline. Analyses of our primary endpoints, with 440 patients, revealed that at 1 year post injection, none of the three orthobiologic injections was superior to another, or to the CSI control. In addition, none of the four groups showed a significant change in magnetic resonance imaging osteoarthritis score compared to baseline. No procedure-related serious adverse events were reported during the study period. In summary, this study shows that at 1 year post injection, there was no superior orthobiologic as compared to CSI for knee osteoarthritis. ClinicalTrials.gov Identifier: NCT03818737.

Differential response of mesenchymal stromal cells (MSCs) to type 1 ex vivo cytokine priming: implications for MSC therapy.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are polymorphic, adherent cells with the capability to stimulate tissue regeneration and modulate immunity. MSCs have been broadly investigated for potential therapeutic applications, particularly immunomodulatory properties, wound healing and tissue regeneration. The exact physiologic role of MSCs, however, remains poorly understood, and this gap in knowledge significantly impedes the rational development of therapeutic cells. Here, we considered interferon γ (IFN-γ) and tumor necrosis factor alpha (TNF-α), two cytokines likely encountered physiologically and commonly used in cell manufacturing. For comparison, we studied interleukin-10 (IL-10) (anti-inflammatory) and interleukin-4 (IL-4) (type 2 cytokine). METHODS: We directly assessed the effects of these cytokines on bone marrow MSCs by comparing RNA Seq transcriptional profiles. Western blotting and flow cytometry were also used to evaluate effects of cytokine priming. RESULTS: The type 1 cytokines (IFN-γ and TNF-α) induced striking changes in gene expression and remarkably different profiles from one another. Importantly, priming MSCs with either of these cytokines did not increase variability among multiple donors beyond what is intrinsic to non-primed MSCs from different donors. IFN-γ-primed MSCs expressed IDO1 and chemokines that recruit activated T cells. In contrast, TNF-α-primed MSCs expressed genes in alternate pathways, namely PGE2 and matrix metalloproteinases synthesis, and chemokines that recruit neutrophils. IL-10 and IL-4 priming had little to no effect. CONCLUSIONS: Our data suggest that IFN-γ-primed MSCs may be a more efficacious immunosuppressive therapy aimed at diseases that target T cells (ie, graft-versus-host disease) compared with TNF-α-primed or non-primed MSCs, which may be better suited for therapies in other disease settings. These results contribute to our understanding of MSC bioactivity and suggest rational ex vivo cytokine priming approaches for MSC manufacturing and therapeutic applications.

Exploring the Landscape of the PP7 Virus-like Particle for Peptide Display.

Self-assembling virus-like particles (VLPs) can tolerate a wide degree of genetic and chemical manipulation to their capsid protein to display a foreign molecule polyvalently. We previously reported the successful incorporation of foreign peptide sequences in the junction loop and onto the C-terminus of PP7 dimer VLPs, as these regions are accessible for surface display on assembled capsids. Here, we report the implementation of a library-based approach to test the assembly tolerance of PP7 dimer capsid proteins to insertions or terminal extensions of randomized 15-mer peptide sequences. By performing two iterative rounds of assembly-based selection, we evaluated the degree of favorability of all 20 amino acids at each of the 15 randomized positions. Deep sequencing analysis revealed a distinct preference for the inclusion of hydrophilic peptides and negatively charged amino acids (Asp and Glu) and the exclusion of positively charged peptides and bulky and hydrophobic amino acid residues (Trp, Phe, Tyr, and Cys). Within the libraries tested here, we identified 4000 to 22,000 unique 15-mer peptide sequences that can successfully be displayed on the surface of the PP7 dimer capsid. Overall, the use of small initial libraries consisting of no more than a few million members yielded a significantly larger number of unique and assembly-competent VLP sequences than have been previously characterized for this class of nucleoprotein particle.

Understanding heterogeneity of human bone marrow plasma cell maturation and survival pathways by single-cell analyses.

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASCs) to long-lived plasma cells (LLPCs). We provide single-cell transcriptional resolution of 17,347 BM ASCs from five healthy adults. Fifteen clusters are identified ranging from newly minted ASCs (cluster 1) expressing MKI67 and high major histocompatibility complex (MHC) class II that progress to late clusters 5-8 through intermediate clusters 2-4. Additional ASC clusters include the following: immunoglobulin (Ig) M predominant (likely of extra-follicular origin), interferon responsive, and high mitochondrial activity. Late ASCs are distinguished by G2M checkpoints, mammalian target of rapamycin (mTOR) signaling, distinct metabolic pathways, CD38 expression, utilization of tumor necrosis factor (TNF)-receptor superfamily members, and two distinct maturation pathways involving TNF signaling through nuclear factor κB (NF-κB). This study provides a single-cell atlas and molecular roadmap of LLPC maturation trajectories essential in the BM microniche. Altogether, understanding BM ASC heterogeneity in health and disease enables development of new strategies to enhance protective ASCs and to deplete pathogenic ones.

Gene representation in scRNA-seq is correlated with common motifs at the 3' end of transcripts.

One important characteristic of single-cell RNA sequencing (scRNA-seq) data is its high sparsity, where the gene-cell count data matrix contains high proportion of zeros. The sparsity has motivated widespread discussions on dropouts and missing data, as well as imputation algorithms of scRNA-seq analysis. Here, we aim to investigate whether there exist genes that are more prone to be under-detected in scRNA-seq, and if yes, what commonalities those genes may share. From public data sources, we gathered paired bulk RNA-seq and scRNA-seq data from 53 human samples, which were generated in diverse biological contexts. We derived pseudo-bulk gene expression by averaging the scRNA-seq data across cells. Comparisons of the paired bulk and pseudo-bulk gene expression profiles revealed that there indeed exists a collection of genes that are frequently under-detected in scRNA-seq compared to bulk RNA-seq. This result was robust to randomization when unpaired bulk and pseudo-bulk gene expression profiles were compared. We performed motif search to the last 350 bp of the identified genes, and observed an enrichment of poly(T) motif. The poly(T) motif toward the tails of those genes may be able to form hairpin structures with the poly(A) tails of their mRNA transcripts, making it difficult for their mRNA transcripts to be captured during scRNA-seq library preparation, which is a mechanistic conjecture of why certain genes may be more prone to be under-detected in scRNA-seq.

Utilization of Targeted RNA-Seq for the Resolution of Variant Pathogenicity and Enhancement of Diagnostic Yield in Dysferlinopathy.

For inherited diseases, obtaining a definitive diagnosis is critical for proper disease management, family planning, and participation in clinical trials. This can be challenging for dysferlinopathy due to the significant clinical overlap between the 30+ subtypes of limb-girdle muscular dystrophy (LGMD) and the large number of variants of unknown significance (VUSs) that are identified in the dysferlin gene, DYSF. We performed targeted RNA-Seq using a custom gene-panel in 77 individuals with a clinical/genetic suspicion of dysferlinopathy and evaluated all 111 identified DYSF variants according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. This evaluation identified 11 novel DYSF variants and allowed for the classification of 87 DYSF variants as pathogenic/likely pathogenic, 8 likely benign, while 16 variants remained VUSs. By the end of the study, 60 of the 77 cases had a definitive diagnosis of dysferlinopathy, which was a 47% increase in diagnostic yield over the rate at study onset. This data shows the ability of RNA-Seq to assist in variant pathogenicity classification and diagnosis of dysferlinopathy and is, therefore, a type of analysis that should be considered when DNA-based genetic analysis is not sufficient to provide a definitive diagnosis.

Longitudinal DNA methylation profiling of the rectal mucosa identifies cell-specific signatures of disease status, severity and clinical outcomes in ulcerative colitis cell-specific DNA methylation signatures of UC.

BACKGROUND: In peripheral blood, DNA methylation (DNAm) patterns in inflammatory bowel disease patients reflect inflammatory status rather than disease status. Here, we examined DNAm in diseased rectal mucosa from ulcerative colitis (UC) patients, focusing on constituent cell types with the goal of identifying therapeutic targets for UC other than the immune system. We profiled DNAm of rectal mucosal biopsies of pediatric UC at diagnosis (n = 211) and non-IBD control (n = 85) patients and performed epigenome-wide association studies (EWAS) of specific cell types to understand DNAm changes in epithelial, immune and fibroblast cells across disease states, course, and clinical outcomes. We also examined longitudinal analysis on follow-up samples (n = 73), and comparisons were made among patients with clinical outcomes including those undergoing colectomy versus those who did not. Additionally, we included RNA-seq from the same subjects to assess the impact of CpG sites on the transcription of nearby genes during the disease course. RESULTS: At diagnosis, UC rectal mucosa exhibited a lower proportion of epithelial cells and fibroblasts, and higher proportion of immune cells, in conjunction with variation in the DNAm pattern. While treatment had significant effects on the methylation signature of immune cells, its effects on fibroblasts and epithelial cells were attenuated. Individuals who required colectomy exhibited cell composition and DNAm patterns at follow-up more similar to disease onset than patients who did not require colectomy. Combining these results with gene expression profiles, we identify CpG sites whose methylation patterns are most consistent with a contribution to poor disease outcomes and could thus be potential therapeutic targets. CONCLUSIONS: Cell-specific epigenetic changes in the rectal mucosa in UC are associated with disease severity and outcome. Current therapeutics may more effectively target the immune than the epithelial and fibroblast compartments.

Profiling the peripheral immune response to ex vivo TNF stimulation in untreated juvenile idiopathic arthritis using single cell RNA sequencing.

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) is an autoimmune disease with a heterogenous clinical presentation and unpredictable response to available therapies. This personalized transcriptomics study sought proof-of-concept for single-cell RNA sequencing to characterize patient-specific immune profiles. METHODS: Whole blood samples from six untreated children, newly diagnosed with JIA, and two healthy controls were cultured for 24 h with or without ex vivo TNF stimulation and subjected to scRNAseq to examine cellular populations and transcript expression in PBMCs. A novel analytical pipeline, scPool, was developed wherein cells are first pooled into pseudocells prior to expression analysis, facilitating variance partitioning of the effects of TNF stimulus, JIA disease status, and individual donor. RESULTS: Seventeen robust immune cell-types were identified, the abundance of which was significantly affected by TNF stimulus, which resulted in notable elevation of memory CD8 + T-cells and NK56 cells, but down-regulation of naïve B-cell proportions. Memory CD8 + and CD4 + T-cells were also both reduced in the JIA cases relative to two controls. Significant differential expression responses to TNF stimulus were also characterized, with monocytes showing more transcriptional shifts than T-lymphocyte subsets, while the B-cell response was more limited. We also show that donor variability exceeds the small degree of possible intrinsic differentiation between JIA and control profiles. An incidental finding of interest was association of HLA-DQA2 and HLA-DRB5 expression with JIA status. CONCLUSIONS: These results support the development of personalized immune-profiling combined with ex-vivo immune stimulation for evaluation of patient-specific modes of immune cell activity in autoimmune rheumatic disease.

Human Bone Marrow Plasma Cell Atlas: Maturation and Survival Pathways Unraveled by Single Cell Analyses.

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASC) to long-lived plasma cells (LLPC). We provide single cell transcriptional resolution of 17,347 BM ASC from 5 healthy adults. Fifteen clusters were identified ranging from newly minted ASC (cluster 1) expressing MKI67 and high MHC Class II that progressed to late clusters 5-8 through intermediate clusters 2-4. Additional clusters included early and late IgM-predominant ASC of likely extra-follicular origin; IFN-responsive; and high mitochondrial activity ASC. Late ASCs were distinguished by differences in G2M checkpoints, MTOR signaling, distinct metabolic pathways, CD38 expression, and utilization of TNF-receptor superfamily members. They mature through two distinct paths differentiated by the degree of TNF signaling through NFKB. This study provides the first single cell resolution atlas and molecular roadmap of LLPC maturation, thereby providing insight into differentiation trajectories and molecular regulation of these essential processes in the human BM microniche. This information enables investigation of the origin of protective and pathogenic antibodies in multiple diseases and development of new strategies targeted to the enhancement or depletion of the corresponding ASC. One Sentence Summary: The single cell transcriptomic atlas of human bone marrow plasma cell heterogeneity shows maturation of class-switched early and late subsets, specific IgM and Interferon-driven clusters, and unique heterogeneity of the late subsets which encompass the long-lived plasma cells.

Effect of all-but-one conditional analysis for eQTL isolation in peripheral blood.

Expression quantitative trait locus detection has become increasingly important for understanding how noncoding variants contribute to disease susceptibility and complex traits. The major challenges in expression quantitative trait locus fine-mapping and causal variant discovery relate to the impact of linkage disequilibrium on signals due to one or multiple functional variants that lie within a credible set. We perform expression quantitative trait locus fine-mapping using the all-but-one approach, conditioning each signal on all others detected in an interval, on the Consortium for the Architecture of Gene Expression cohorts of microarray-based peripheral blood gene expression in 2,138 European-ancestry human adults. We contrast these results with traditional forward stepwise conditional analysis and a Bayesian localization method. All-but-one conditioning significantly modifies effect-size estimates for 51% of 2,351 expression quantitative trait locus peaks, but only modestly affects credible set size and location. On the other hand, both conditioning approaches result in unexpectedly low overlap with Bayesian credible sets, with just 57% peak concordance and between 50% and 70% SNP sharing, leading us to caution against the assumption that any one localization method is superior to another. We also cross reference our results with ATAC-seq data, cell-type-specific expression quantitative trait locus, and activity-by-contact-enhancers, leading to the proposal of a 5-tier approach to further reduce credible set sizes and prioritize likely causal variants for all known inflammatory bowel disease risk loci active in immune cells.

Genetically transitional disease: a new concept in genomic medicine.

Traditional classification of genetic diseases as monogenic and polygenic has lagged far behind scientific progress. In this opinion article, we propose and define a new terminology, genetically transitional disease (GTD), referring to cases where a large-effect mutation is necessary, but not sufficient, to cause disease. This leads to a working disease nosology based on gradients of four types of genetic architecture: monogenic, polygenic, GTD, and mixed. We present four scenarios under which GTD may occur; namely, subsets of traditionally Mendelian disease, modifiable Tier 1 monogenic conditions, variable penetrance, and situations where a genetic mutational spectrum produces qualitatively divergent pathologies. The implications of the new nosology in precision medicine are discussed, in which therapeutic options may target the molecular cause or the disease phenotype.

Integrative polygenic analysis of the protective effects of fatty acid metabolism on disease as modified by obesity.

Dysregulation of fatty acid metabolites can play a crucial role in the progression of complex diseases, such as cardiovascular disease, digestive diseases, and metabolic diseases. Metabolites can have either protective or risk effects on a disease; however, the details of such associations remain contentious. In this study, we demonstrate an integrative PheWAS approach to establish high confidence, causally suggestive of metabolite-disease associations for three fatty acid metabolites, namely, omega-3 fatty acids, omega-6 fatty acids, and docosahexaenoic acid, for 1,254 disease endpoints. Metabolite-disease associations were established if there was a concordant direction of effect and significance for metabolite level and genetic risk score for the metabolite. There was enrichment for metabolite associations with diseases of the respiratory system for omega-3 fatty acids, diseases of the circulatory system and endocrine system for omega-6 fatty acids, and diseases of the digestive system for docosahexaenoic acid. Upon performing Mendelian randomization on a subset of the outcomes, we identified 3, 6, and 15 significant diseases associated with omega-3 fatty acids, omega-6 fatty acids, and docosahexaenoic acid, respectively. We then demonstrate a class of prevalence-risk relationships indicative of (de)canalization of disease under high and low fatty acid metabolite levels. Finally, we show that the interaction between the metabolites and obesity demonstrates that the degree of protection afforded by fatty acid metabolites is strongly modulated by underlying metabolic health. This study evaluated the disease architectures of three polyunsaturated fatty acids (PUFAs), which were validated by several PheWAS modes of support. Our results not only highlight specific diseases associated with each metabolite but also disease group enrichments. In addition, we demonstrate an integrative PheWAS methodology that can be applied to other components of the human metabolome or other traits of interest. The results of this study can be used as an atlas to cross-compare genetic with non-genetic disease associations for the three PUFAs investigated. The findings can be explored through our R shiny app at https://pufa.biosci.gatech.edu.

Mendelian Randomization Indicates a Causal Role for Omega-3 Fatty Acids in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal system. Omega-3 (ω3) fatty acids are polyunsaturated fatty acids (PUFAs) that are largely obtained from diet and have been speculated to decrease the inflammatory response that is involved in IBD; however, the causality of this association has not been established. A two-sample Mendelian randomization (MR) was used to assess genetic associations between 249 circulating metabolites measured in the UK Biobank as exposures and IBD as the outcome. The genome-wide association study summary level data for metabolite measurements and IBD were derived from large European ancestry cohorts. We observed ω3 fatty acids as a significant protective association with IBD, with multiple modes of MR evidence replicated in three IBD summary genetic datasets. The instrumental variables that were involved in the causal association of ω3 fatty acids with IBD highlighted an intronic SNP, rs174564, in FADS2, a protein engaged in the first step of alpha-linolenic acid desaturation leading to anti-inflammatory EPA and thence DHA production. A low ratio of ω3 to ω6 fatty acids was observed to be a causal risk factor, particularly for Crohn's disease. ω3 fatty acid supplementation may provide anti-inflammatory responses that are required to attenuate inflammation that is involved in IBD.

Angiotensin-converting Enzyme-2 (ACE2) Expression in Pediatric Liver Disease.

The membrane protein angiotensin-converting enzyme-2 (ACE2) has gained notoriety as the receptor for severe acute respiratory syndrome coronavirus 2. Prior evidence has shown ACE2 is expressed within the liver but its function has not been fully discerned. Here, we utilized novel methodology to assess ACE2 expression in pediatric immune-mediated liver disease to better understand its presence in liver diseases and its role during infections such as COVID-19. We stained liver tissue with ACE2-specific immunofluorescent antibodies, analyzed via confocal microscopy. Computational deep learning-based segmentation models identified nuclei and cells, allowing the quantification of mean cellular and cytosolic immunofluorescent. Spatial transcriptomics provided high-throughput gene expression analysis in tissue to determine cellular composition for ACE2 expression. ACE2 plasma expression was quantified via enzyme-linked immunosorbent assay. High ACE2 expression was seen at the apical surface of cholangiocytes, with lower expression within hepatocyte cytosol and nonparenchymal cells ( P <0.001). Children with liver disease had higher ACE2 hepatic expression than pediatric control tissue ( P <0.001). Adult control tissue had higher expression than pediatric control ( P <0.001). Plasma ACE2 was not found to be statistically different between samples. Spatial transcriptomics identified cell composition of ACE2-expressing spots containing antibody-secreting cells. Our results show ACE2 expression throughout the liver, with strongest localization to cholangiocyte membranes. Machine learning can be used to rapidly identify hepatic cellular components for histologic analysis. ACE2 expression in the liver may be increased in pediatric liver disease. Future work is needed to better understand the role of ACE2 in chronic disease and acute infections.

Dysregulated naive B cells and de novo autoreactivity in severe COVID-19.

Severe SARS-CoV-2 infection1 has been associated with highly inflammatory immune activation since the earliest days of the COVID-19 pandemic2-5. More recently, these responses have been associated with the emergence of self-reactive antibodies with pathologic potential6-10, although their origins and resolution have remained unclear11. Previously, we and others have identified extrafollicular B cell activation, a pathway associated with the formation of new autoreactive antibodies in chronic autoimmunity12,13, as a dominant feature of severe and critical COVID-19 (refs. 14-18). Here, using single-cell B cell repertoire analysis of patients with mild and severe disease, we identify the expansion of a naive-derived, low-mutation IgG1 population of antibody-secreting cells (ASCs) reflecting features of low selective pressure. These features correlate with progressive, broad, clinically relevant autoreactivity, particularly directed against nuclear antigens and carbamylated proteins, emerging 10-15 days after the onset of symptoms. Detailed analysis of the low-selection compartment shows a high frequency of clonotypes specific for both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against the glomerular basement membrane. We further identify the contraction of this pathway on recovery, re-establishment of tolerance standards and concomitant loss of acute-derived ASCs irrespective of antigen specificity. However, serological autoreactivity persists in a subset of patients with postacute sequelae, raising important questions as to the contribution of emerging autoreactivity to continuing symptomology on recovery. In summary, this study demonstrates the origins, breadth and resolution of autoreactivity in severe COVID-19, with implications for early intervention and the treatment of patients with post-COVID sequelae.