Allergen recognition by specific effector Th2 cells enables IL-2-dependent activation of regulatory T-cell responses in humans.

Type 2 allergen-specific T cells are essential for the induction and maintenance of allergies to foods, and Tregs specific for these allergens are assumed to be involved in their resolution. However, it has not been convincingly demonstrated whether allergen-specific Treg responses are responsible for the generation of oral tolerance in humans. We observed that sustained food allergen exposure in the form of oral immunotherapy resulted in increased frequency of Tregs only in individuals with lasting clinical tolerance. We sought to identify regulatory components of the CD4+ T-cell response to food allergens by studying their functional activation over time in vitro and in vivo. Two subsets of Tregs expressing CD137 or CD25/OX40 were identified with a delayed kinetics of activation compared with clonally enriched pathogenic effector Th2 cells. Treg activation was dependent on IL-2 derived from effector T cells. In vivo exposure to peanut in the form of an oral food challenge of allergic subjects induced a delayed and persistent activation of Tregs after initiation of the allergen-specific Th2 response. The novel finding of our work is that a sustained wave of Treg activation is induced by the release of IL-2 from Th2 effector cells, with the implication that therapeutic administration of IL-2 could improve current OIT approaches.

Acetate supplementation rescues social deficits and alters transcriptional regulation in prefrontal cortex of Shank3 deficient mice.

BACKGROUND: The pathophysiology of autism spectrum disorder (ASD) involves genetic and environmental factors. Mounting evidence demonstrates a role for the gut microbiome in ASD, with signaling via short-chain fatty acids (SCFA) as one mechanism. Here, we utilize mice carrying deletion to exons 4-22 of Shank3 (Shank3KO) to model gene by microbiome interactions in ASD. We identify SCFA acetate as a mediator of gut-brain interactions and show acetate supplementation reverses social deficits concomitant with alterations to medial prefrontal cortex (mPFC) transcriptional regulation independent of microbiome status. METHODS: Shank3KO and wild-type (Wt) littermates were divided into control, Antibiotic (Abx), Acetate and Abx + Acetate groups upon weaning. After six weeks, animals underwent behavioral testing. Molecular analysis including 16S and metagenomic sequencing, metabolomic and transcriptional profiling were conducted. Additionally, targeted serum metabolomic data from Phelan McDermid Syndrome (PMS) patients (who are heterozygous for the Shank3 gene) were leveraged to assess levels of SCFA's relative to ASD clinical measures. RESULTS: Shank3KO mice were found to display social deficits, dysregulated gut microbiome and decreased cecal levels of acetate - effects exacerbated by Abx treatment. RNA-sequencing of mPFC showed unique gene expression signature induced by microbiome depletion in the Shank3KO mice. Oral treatment with acetate reverses social deficits and results in marked changes in gene expression enriched for synaptic signaling, pathways among others, even in Abx treated mice. Clinical data showed sex specific correlations between levels of acetate and hyperactivity scores. CONCLUSION: These results suggest a key role for the gut microbiome and the neuroactive metabolite acetate in regulating ASD-like behaviors.

Leveraging -omics for asthma endotyping.

Asthma is a highly heterogeneous disease, often manifesting with wheeze, dyspnea, chest tightness, and cough as prominent symptoms. The eliciting factors, natural history, underlying molecular biology, and clinical management of asthma vary highly among affected subjects. Because of this variation, many efforts have gone into subtyping asthma. Endotypes are subtypes of disease based on distinct pathophysiologic mechanisms. Endotypes can be clinically useful because they organize our mechanistic understanding of heterogeneous diseases and can direct treatment toward modalities that are likely to be the most effective. Asthma endotyping can be shaped by clinical features, laboratory parameters, and/or -omics approaches. We discuss the application of -omics approaches, including transcriptomics, epigenomics, microbiomics, metabolomics, and proteomics, to asthma endotyping. -Omics approaches have provided supporting evidence for many existing endotyping paradigms and also suggested novel ways to conceptualize asthma endotypes. Although endotypes based on single -omics approaches are relatively common, their integrated multi-omics application to asthma endotyping has been more limited thus far. We discuss paths forward to integrate multi-omics with clinical features and laboratory parameters to achieve the goal of precise asthma endotypes.

Pancreatic and Islet Remodeling in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Knockout Ferrets.

In cystic fibrosis (CF), there is early destruction of the exocrine pancreas, and this results in a unique form of diabetes that affects approximately half of adult CF individuals. An animal model of cystic fibrosis-related diabetes has been developed in the ferret, which progresses through phases of glycemic abnormalities because of islet remodeling during and after exocrine destruction. Herein, we quantified the pancreatic histopathological changes that occur during these phases. There was an increase in percentage ductal, fat, and islet area in CF ferrets over time compared with age-matched wild-type controls. We also quantified islet size, shape, islet cell composition, cell proliferation (Ki-67), and expression of remodeling markers (matrix metalloprotease-7, desmin, and α-smooth muscle actin). Pancreatic ducts were dilated with scattered proliferating cells and were surrounded by activated stellate cells, indicative of tissue remodeling. The timing of islet and duct proliferation, stellate cell activation, and matrix remodeling coincided with the previously published stages of glycemic crisis and inflammation. This mapping of remodeling events in the CF ferret pancreas provides insights into early changes that control glycemic intolerance and subsequent recovery during the evolution of CF pancreatic disease.

Depletion of Airway Submucosal Glands and TP63+KRT5+ Basal Cells in Obliterative Bronchiolitis.

RATIONALE: Obliterative bronchiolitis (OB) is a major cause of mortality after lung transplantation. Depletion of airway stem cells (SCs) may lead to fibrosis in OB. OBJECTIVES: Two major SC compartments in airways are submucosal glands (SMGs) and surface airway p63 (also known as TP63 [tumor protein 63])-positive/K5 (also known as KRT5 [keratin 5])-positive basal cells (BCs). We hypothesized that depletion of these SC compartments occurs in OB. METHODS: Ferret orthotopic left lung transplants were used as an experimental model of OB, and findings were corroborated in human lung allografts. Morphometric analysis was performed in ferret and human lungs to evaluate the abundance of SMGs and changes in the expression of phenotypic BC markers in control, lymphocytic bronchiolitis, and OB airways. The abundance and proliferative capacity of proximal and distal airway SCs was assessed using a clonogenic colony-forming efficiency assay. MEASUREMENTS AND MAIN RESULTS: Ferret allografts revealed significant loss of SMGs with development of OB. A progressive decline in p63+/K5+ and increase in K5+/K14+ and K14+ BC phenotypes correlated with the severity of allograft rejection in large and small ferret airways. The abundance and proliferative capacity of basal SCs in large allograft airways declined with severity of OB, and there was complete ablation of basal SCs in distal OB airways. Human allografts mirrored phenotypic BC changes observed in the ferret model. CONCLUSIONS: SMGs and basal SC compartments are depleted in large and/or small airways of lung allografts, and basal SC proliferative capacity declines with progression of disease and phenotypic changes. Global airway SC depletion may be a mechanism for pulmonary allograft failure.

Infection Is Not Required for Mucoinflammatory Lung Disease in CFTR-Knockout Ferrets.

RATIONALE: Classical interpretation of cystic fibrosis (CF) lung disease pathogenesis suggests that infection initiates disease progression, leading to an exuberant inflammatory response, excessive mucus, and ultimately bronchiectasis. Although symptomatic antibiotic treatment controls lung infections early in disease, lifelong bacterial residence typically ensues. Processes that control the establishment of persistent bacteria in the CF lung, and the contribution of noninfectious components to disease pathogenesis, are poorly understood. OBJECTIVES: To evaluate whether continuous antibiotic therapy protects the CF lung from disease using a ferret model that rapidly acquires lethal bacterial lung infections in the absence of antibiotics. METHODS: CFTR (cystic fibrosis transmembrane conductance regulator)-knockout ferrets were treated with three antibiotics from birth to several years of age and lung disease was followed by quantitative computed tomography, BAL, and histopathology. Lung disease was compared with CFTR-knockout ferrets treated symptomatically with antibiotics. MEASUREMENTS AND MAIN RESULTS: Bronchiectasis was quantified from computed tomography images. BAL was evaluated for cellular differential and features of inflammatory cellular activation, bacteria, fungi, and quantitative proteomics. Semiquantitative histopathology was compared across experimental groups. We demonstrate that lifelong antibiotics can protect the CF ferret lung from infections for several years. Surprisingly, CF animals still developed hallmarks of structural bronchiectasis, neutrophil-mediated inflammation, and mucus accumulation, despite the lack of infection. Quantitative proteomics of BAL from CF and non-CF pairs demonstrated a mucoinflammatory signature in the CF lung dominated by Muc5B and neutrophil chemoattractants and products. CONCLUSIONS: These findings implicate mucoinflammatory processes in the CF lung as pathogenic in the absence of clinically apparent bacterial and fungal infections.

Wnt Signaling Regulates Airway Epithelial Stem Cells in Adult Murine Submucosal Glands.

Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016;34:2758-2771.

Glandular Proteome Identifies Antiprotease Cystatin C as a Critical Modulator of Airway Hydration and Clearance.

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel lead to viscous secretions from submucosal glands that cannot be properly hydrated and cleared by beating cilia in cystic fibrosis (CF) airways. The mechanisms by which CFTR, and the predominant epithelial sodium channel (ENaC), control the hydration and clearance of glandular secretions remain unclear. We used a proteomics approach to characterize the proteins contained in CF and non-CF submucosal gland fluid droplets and found that differentially regulated proteases (cathepsin S and H) and their antiprotease (cystatin C) influenced the equilibration of fluid on the airway surface and tracheal mucociliary clearance (MCC). Contrary to prevailing models of airway hydration and clearance, cystatin C, or raising the airway surface liquid (ASL) pH, inhibited cathepsin-dependent ENaC-mediated fluid absorption and raised the height of ASL, and yet decreased MCC velocity. Importantly, coupling of both CFTR and ENaC activities were required for effective MCC and for effective ASL height equilibration after volume challenge. Cystatin C-inhibitable cathepsins controlled initial phases of ENaC-mediated fluid absorption, whereas CFTR activity was required to prevent ASL dehydration. Interestingly, CF airway epithelia absorbed fluid more slowly owing to reduced cysteine protease activity in the ASL but became abnormally dehydrated with time. Our findings demonstrate that, after volume challenge, pH-dependent protease-mediated coupling of CFTR and ENaC activities are required for rapid fluid equilibration at the airway surface and for effective MCC. These findings provide new insights into how glandular fluid secretions may be equilibrated at the airway surface and how this process may be impaired in CF.

Defective innate immunity and hyperinflammation in newborn cystic fibrosis transmembrane conductance regulator-knockout ferret lungs.

Mucociliary clearance (MCC) and submucosal glands are major components of airway innate immunity that have impaired function in cystic fibrosis (CF). Although both of these defense systems develop postnatally in the ferret, the lungs of newborn ferrets remain sterile in the presence of a functioning cystic fibrosis transmembrane conductance regulator gene. We evaluated several components of airway innate immunity and inflammation in the early CF ferret lung. At birth, the rates of MCC did not differ between CF and non-CF animals, but the height of the airway surface liquid was significantly reduced in CF newborn ferrets. CF ferrets had impaired MCC after 7 days of age, despite normal rates of ciliogenesis. Only non-CF ferrets eradicated Pseudomonas directly introduced into the lung after birth, whereas both genotypes could eradicate Staphylococcus. CF bronchoalveolar lavage fluid (BALF) had significantly lower antimicrobial activity selectively against Pseudomonas than non-CF BALF, which was insensitive to changes in pH and bicarbonate. Liquid chromatography-tandem mass spectrometry and cytokine analysis of BALF from sterile Caesarean-sectioned and nonsterile naturally born animals demonstrated CF-associated disturbances in IL-8, TNF-α, and IL-ß, and pathways that control immunity and inflammation, including the complement system, macrophage functions, mammalian target of rapamycin signaling, and eukaryotic initiation factor 2 signaling. Interestingly, during the birth transition, IL-8 was selectively induced in CF BALF, despite no genotypic difference in bacterial load shortly after birth. These results suggest that newborn CF ferrets have defects in both innate immunity and inflammatory signaling that may be important in the early onset and progression of lung disease in these animals.

Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets.

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 µg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.

Lung phenotype of juvenile and adult cystic fibrosis transmembrane conductance regulator-knockout ferrets.

Chronic bacterial lung infections in cystic fibrosis (CF) are caused by defects in the CF transmembrane conductance regulator chloride channel. Previously, we described that newborn CF transmembrane conductance regulator-knockout ferrets rapidly develop lung infections within the first week of life. Here, we report a more slowly progressing lung bacterial colonization phenotype observed in juvenile to adult CF ferrets reared on a layered antibiotic regimen. Even on antibiotics, CF ferrets were still very susceptible to bacterial lung infection. The severity of lung histopathology ranged from mild to severe, and variably included mucus obstruction of the airways and submucosal glands, air trapping, atelectasis, bronchopneumonia, and interstitial pneumonia. In all CF lungs, significant numbers of bacteria were detected and impaired tracheal mucociliary clearance was observed. Although Streptococcus, Staphylococcus, and Enterococcus were observed most frequently in the lungs of CF animals, each animal displayed a predominant bacterial species that accounted for over 50% of the culturable bacteria, with no one bacterial taxon predominating in all animals. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry fingerprinting was used to quantify lung bacteria in 10 CF animals and demonstrated Streptococcus, Staphylococcus, Enterococcus, or Escherichia as the most abundant genera. Interestingly, there was significant overlap in the types of bacteria observed in the lung and intestine of a given CF animal, including bacterial taxa unique to the lung and gut of each CF animal analyzed. These findings demonstrate that CF ferrets develop lung disease during the juvenile and adult stages that is similar to patients with CF, and suggest that enteric bacterial flora may seed the lung of CF ferrets.

Sox2 modulates Lef-1 expression during airway submucosal gland development.

Tracheobronchial submucosal glands (SMGs) are derived from one or more multipotent glandular stem cells that coalesce to form a placode in surface airway epithelium (SAE). Wnt/ß-catenin-dependent induction of lymphoid enhancer factor (Lef-1) gene expression during placode formation is an early event required for SMG morphogenesis. We discovered that Sox2 expression is repressed as Lef-1 is induced within airway SMG placodes. Deletion of Lef-1 did not activate Sox2 expression in SMG placodes, demonstrating that Lef-1 activation does not directly inhibit Sox2 expression. Repression of Sox2 protein in SMG placodes occurred posttranscriptionally, since the activity of its endogenous promoter remained unchanged in SMG placodes. Thus we hypothesized that Sox2 transcriptionally represses Lef-1 expression in the SAE and that suppression of Sox2 in SMG placodes activates Wnt/ß-catenin-dependent induction of Lef-1 during SMG morphogenesis. Consistent with this hypothesis, transcriptional reporter assays, ChIP analyses, and DNA-protein binding studies revealed a functional Sox2 DNA binding site in the Lef-1 promoter that is required for suppressing ß-catenin-dependent transcription. In polarized primary airway epithelium, Wnt induction enhanced Lef-1 expression while also inhibiting Sox2 expression. Conditional deletion of Sox2 also enhanced Lef-1 expression in polarized primary airway epithelium, but this induction was significantly augmented by Wnt stimulation. Our findings provide the first evidence that Sox2 acts as a repressor to directly modulate Wnt-responsive transcription of the Lef-1 gene promoter. These studies support a model whereby Wnt signals and Sox2 dynamically regulate the expression of Lef-1 in airway epithelia and potentially also during SMG development.

Anti-correlated feature selection prevents false discovery of subpopulations in scRNAseq.

While sub-clustering cell-populations has become popular in single cell-omics, negative controls for this process are lacking. Popular feature-selection/clustering algorithms fail the null-dataset problem, allowing erroneous subdivisions of homogenous clusters until nearly each cell is called its own cluster. Using real and synthetic datasets, we find that anti-correlated gene selection reduces or eliminates erroneous subdivisions, increases marker-gene selection efficacy, and efficiently scales to millions of cells.

Bioelectric characterization of epithelia from neonatal CFTR knockout ferrets.

Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (ISC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin-stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH-101)-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin-inducible ISC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport.

Redox-dependent Igfbp2 signaling controls Brca1 DNA damage response to govern neural stem cell fate.

Neural stem cell (NSC) maintenance and functions are regulated by reactive oxygen species (ROS). However, the mechanisms by which ROS control NSC behavior remain unclear. Here we report that ROS-dependent Igfbp2 signaling controls DNA repair pathways which balance NSC self-renewal and lineage commitment. Ncf1 or Igfbp2 deficiency constrains NSCs to a self-renewing state and prevents neurosphere formation. Ncf1-dependent oxidation of Igfbp2 promotes neurogenesis by NSCs in vitro and in vivo while repressing Brca1 DNA damage response genes and inducing DNA double-strand breaks (DDSBs). By contrast, Ncf1-/- and Igfbp2-/- NSCs favor the formation of oligodendrocytes in vitro and in vivo. Notably, transient repression of Brca1 DNA repair pathway genes induces DDSBs and is sufficient to rescue the ability of Ncf1-/- and Igfbp2-/- NSCs to lineage-commit to form neurospheres and neurons. NSC lineage commitment is dependent on the oxidizable cysteine-43 residue of Igfbp2. Our study highlights the role of DNA damage/repair in orchestrating NSC fate decisions downstream of redox-regulated Igfbp2.

Merged Affinity Network Association Clustering: Joint multi-omic/clinical clustering to identify disease endotypes.

Although clinical and laboratory data have long been used to guide medical practice, this information is rarely integrated with multi-omic data to identify endotypes. We present Merged Affinity Network Association Clustering (MANAclust), a coding-free, automated pipeline enabling integration of categorical and numeric data spanning clinical and multi-omic profiles for unsupervised clustering to identify disease subsets. Using simulations and real-world data from The Cancer Genome Atlas, we demonstrate that MANAclust's feature selection algorithms are accurate and outperform competitors. We also apply MANAclust to a clinically and multi-omically phenotyped asthma cohort. MANAclust identifies clinically and molecularly distinct clusters, including heterogeneous groups of "healthy controls" and viral and allergy-driven subsets of asthmatic subjects. We also find that subjects with similar clinical presentations have disparate molecular profiles, highlighting the need for additional testing to uncover asthma endotypes. This work facilitates data-driven personalized medicine through integration of clinical parameters with multi-omics. MANAclust is freely available at https://bitbucket.org/scottyler892/manaclust/src/master/.

Downregulation of exhausted cytotoxic T cells in gene expression networks of multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) presents with fever, inflammation and pathology of multiple organs in individuals under 21 years of age in the weeks following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although an autoimmune pathogenesis has been proposed, the genes, pathways and cell types causal to this new disease remain unknown. Here we perform RNA sequencing of blood from patients with MIS-C and controls to find disease-associated genes clustered in a co-expression module annotated to CD56dimCD57+ natural killer (NK) cells and exhausted CD8+ T cells. A similar transcriptome signature is replicated in an independent cohort of Kawasaki disease (KD), the related condition after which MIS-C was initially named. Probing a probabilistic causal network previously constructed from over 1,000 blood transcriptomes both validates the structure of this module and reveals nine key regulators, including TBX21, a central coordinator of exhausted CD8+ T cell differentiation. Together, this unbiased, transcriptome-wide survey implicates downregulation of NK cells and cytotoxic T cell exhaustion in the pathogenesis of MIS-C.

Cytotoxic lymphocytes are dysregulated in multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) presents with fever, inflammation and multiple organ involvement in individuals under 21 years following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To identify genes, pathways and cell types driving MIS-C, we sequenced the blood transcriptomes of MIS-C cases, pediatric cases of coronavirus disease 2019, and healthy controls. We define a MIS-C transcriptional signature partially shared with the transcriptional response to SARS-CoV-2 infection and with the signature of Kawasaki disease, a clinically similar condition. By projecting the MIS-C signature onto a co-expression network, we identified disease gene modules and found genes downregulated in MIS-C clustered in a module enriched for the transcriptional signatures of exhausted CD8 + T-cells and CD56 dim CD57 + NK cells. Bayesian network analyses revealed nine key regulators of this module, including TBX21 , a central coordinator of exhausted CD8 + T-cell differentiation. Together, these findings suggest dysregulated cytotoxic lymphocyte response to SARS-Cov-2 infection in MIS-C.

PyMINEr Finds Gene and Autocrine-Paracrine Networks from Human Islet scRNA-Seq.

Toolsets available for in-depth analysis of scRNA-seq datasets by biologists with little informatics experience is limited. Here, we describe an informatics tool (PyMINEr) that fully automates cell type identification, cell type-specific pathway analyses, graph theory-based analysis of gene regulation, and detection of autocrine-paracrine signaling networks in silico. We applied PyMINEr to interrogate human pancreatic islet scRNA-seq datasets and discovered several features of co-expression graphs, including concordance of scRNA-seq-graph structure with both protein-protein interactions and 3D genomic architecture, association of high-connectivity and low-expression genes with cell type enrichment, and potential for the graph structure to clarify potential etiologies of enigmatic disease-associated variants. We further created a consensus co-expression network and autocrine-paracrine signaling networks within and across islet cell types from seven datasets. PyMINEr correctly identified changes in BMP-WNT signaling associated with cystic fibrosis pancreatic acinar cell loss. This proof-of-principle study demonstrates that the PyMINEr framework will be a valuable resource for scRNA-seq analyses.