Modulation of the Immunological Milieu in Acute Aneurysmal Subarachnoid Hemorrhage: The Potential Role of Monocytes Through CXCL10 Secretion.

Emerging evidence indicates that aneurysmal subarachnoid hemorrhage (aSAH) elicits a response from both innate and adaptive immune systems. An upregulation of CD8 + CD161 + cells has been observed in the cerebrospinal fluid (CSF) after aSAH, yet the precise role of these cells in the context of aSAH is unkown. CSF samples from patients with aSAH and non-aneurysmal SAH (naSAH) were analyzed. Single-cell RNA sequencing (scRNAseq) was performed on CD8 + CD161 + sorted samples from aSAH patients. Cell populations were identified using "clustering." Gene expression levels of ten previously described genes involved in inflammation were quantified from aSAH and naSAH samples using RT-qPCR. The study focused on the following genes: CCL5, CCL7, APOE, SPP1, CXCL8, CXCL10, HMOX1, LTB, MAL, and HLA-DRB1. Gene clustering analysis revealed that monocytes, NK cells, and T cells expressed CD8 + CD161 + in the CSF of patients with aSAH. In comparison to naSAH samples, aSAH samples exhibited higher mRNA levels of CXCL10 (median, IQR = 90, 16-149 vs. 0.5, 0-6.75, p = 0.02). A trend towards higher HMOX1 levels was also observed in aSAH (median, IQR = 12.6, 9-17.6 vs. 2.55, 1.68-5.7, p = 0.076). Specifically, CXCL10 and HMOX1 were expressed by the monocyte subpopulation. Monocytes, NK cells, and T cells can potentially express CD8 + CD161 + in patients with aSAH. Notably, monocytes show high levels of CXCL10. The elevated expression of CXCL10 in aSAH compared to naSAH indicates its potential significance as a target for future studies.

Keratinocyte FABP5-VCP complex mediates recruitment of neutrophils in psoriasis.

One of the hallmarks of intractable psoriasis is neutrophil infiltration in skin lesions. However, detailed molecular mechanisms of neutrophil chemotaxis and activation remain unclear. Here, we demonstrate a significant upregulation of epidermal fatty acid binding protein (E-FABP, FABP5) in the skin of human psoriasis and psoriatic mouse models. Genetic deletion of FABP5 in mice by global knockout and keratinocyte conditional (Krt6a-Cre) knockout, but not myeloid cell conditional (LysM-Cre) knockout, attenuates psoriatic symptoms. Immunophenotypic analysis shows that FABP5 deficiency specifically reduces skin recruitment of Ly6G+ neutrophils. Mechanistically, activated keratinocytes produce chemokines and cytokines that trigger neutrophil chemotaxis and activation in an FABP5-dependent manner. Proteomic analysis further identifies that FABP5 interacts with valosin-containing protein (VCP), a key player in NF-κB signaling activation. Silencing of FABP5, VCP, or both inhibits NF-κB/neutrophil chemotaxis signaling. Collectively, these data demonstrate dysregulated FABP5 as a molecular mechanism promoting NF-κB signaling and neutrophil infiltration in psoriasis pathogenesis.

Lateral Septal Circuits Govern Schizophrenia-Like Effects of Ketamine on Social Behavior.

Schizophrenia is marked by poor social functioning that can have a severe impact on quality of life and independence, but the underlying neural circuity is not well understood. Here we used a translational model of subanesthetic ketamine in mice to delineate neural pathways in the brain linked to social deficits in schizophrenia. Mice treated with chronic ketamine (30 mg/kg/day for 10 days) exhibit profound social and sensorimotor deficits as previously reported. Using three- dimensional c-Fos immunolabeling and volume imaging (iDISCO), we show that ketamine treatment resulted in hypoactivation of the lateral septum (LS) in response to social stimuli. Chemogenetic activation of the LS rescued social deficits after ketamine treatment, while chemogenetic inhibition of previously active populations in the LS (i.e. social engram neurons) recapitulated social deficits in ketamine-naïve mice. We then examined the translatome of LS social engram neurons and found that ketamine treatment dysregulated genes implicated in neuronal excitability and apoptosis, which may contribute to LS hypoactivation. We also identified 38 differentially expressed genes (DEGs) in common with human schizophrenia, including those involved in mitochondrial function, apoptosis, and neuroinflammatory pathways. Chemogenetic activation of LS social engram neurons induced downstream activity in the ventral part of the basolateral amygdala, subparafascicular nucleus of the thalamus, intercalated amygdalar nucleus, olfactory areas, and dentate gyrus, and it also reduces connectivity of the LS with the piriform cortex and caudate-putamen. In sum, schizophrenia-like social deficits may emerge via changes in the intrinsic excitability of a discrete subpopulation of LS neurons that serve as a central hub to coordinate social behavior via downstream projections to reward, fear extinction, motor and sensory processing regions of the brain.

Selective neuroimmune modulation by type I interferon drives neuropathology and neurologic dysfunction following traumatic brain injury.

Accumulating evidence suggests that type I interferon (IFN-I) signaling is a key contributor to immune cell-mediated neuropathology in neurodegenerative diseases. Recently, we demonstrated a robust upregulation of type I interferon-stimulated genes in microglia and astrocytes following experimental traumatic brain injury (TBI). The specific molecular and cellular mechanisms by which IFN-I signaling impacts the neuroimmune response and neuropathology following TBI remains unknown. Using the lateral fluid percussion injury model (FPI) in adult male mice, we demonstrated that IFN α/ß receptor (IFNAR) deficiency resulted in selective and sustained blockade of type I interferon-stimulated genes following TBI as well as decreased microgliosis and monocyte infiltration. Molecular alteration of reactive microglia also occurred with diminished expression of genes needed for MHC class I antigen processing and presentation following TBI. This was associated with decreased accumulation of cytotoxic T cells in the brain. The IFNAR-dependent modulation of the neuroimmune response was accompanied by protection from secondary neuronal death, white matter disruption, and neurobehavioral dysfunction. These data support further efforts to leverage the IFN-I pathway for novel, targeted therapy of TBI.

CDK4/6-MEK Inhibition in MPNSTs Causes Plasma Cell Infiltration, Sensitization to PD-L1 Blockade, and Tumor Regression.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are lethal, Ras-driven sarcomas that lack effective therapies. We investigated effects of targeting cyclin-dependent kinases 4 and 6 (CDK4/6), MEK, and/or programmed death-ligand 1 (PD-L1) in preclinical MPNST models. EXPERIMENTAL DESIGN: Patient-matched MPNSTs and precursor lesions were examined by FISH, RNA sequencing, IHC, and Connectivity-Map analyses. Antitumor activity of CDK4/6 and MEK inhibitors was measured in MPNST cell lines, patient-derived xenografts (PDX), and de novo mouse MPNSTs, with the latter used to determine anti-PD-L1 response. RESULTS: Patient tumor analyses identified CDK4/6 and MEK as actionable targets for MPNST therapy. Low-dose combinations of CDK4/6 and MEK inhibitors synergistically reactivated the retinoblastoma (RB1) tumor suppressor, induced cell death, and decreased clonogenic survival of MPNST cells. In immune-deficient mice, dual CDK4/6-MEK inhibition slowed tumor growth in 4 of 5 MPNST PDXs. In immunocompetent mice, combination therapy of de novo MPNSTs caused tumor regression, delayed resistant tumor outgrowth, and improved survival relative to monotherapies. Drug-sensitive tumors that regressed contained plasma cells and increased cytotoxic T cells, whereas drug-resistant tumors adopted an immunosuppressive microenvironment with elevated MHC II-low macrophages and increased tumor cell PD-L1 expression. Excitingly, CDK4/6-MEK inhibition sensitized MPNSTs to anti-PD-L1 immune checkpoint blockade (ICB) with some mice showing complete tumor regression. CONCLUSIONS: CDK4/6-MEK inhibition induces a novel plasma cell-associated immune response and extended antitumor activity in MPNSTs, which dramatically enhances anti-PD-L1 therapy. These preclinical findings provide strong rationale for clinical translation of CDK4/6-MEK-ICB targeted therapies in MPNST as they may yield sustained antitumor responses and improved patient outcomes.

Updating "Dataset of transcriptomic changes that occur in human preadipocytes over a 3-day course of exposure to 3,3',4,4',5-Pentachlorobiphenyl (PCB126)" with additional data on exposure to 2,2',5,5'-tetrachlorobiphenyl (PCB52) or its 4-hydroxy metabolite (4-OH-PCB52).

Polychlorinated biphenyls (PCBs) were used extensively in building materials, including those used in schools. PCBs accumulate in fat, and exposure to PCBs is associated with the development of cancer, neurodevelopmental disorders, cardiovascular disease, obesity, and diabetes. The non-dioxin-like PCB congener, PCB52 (2,2',5,5'-tetrachlorobiphenyl), is found at one of the highest levels of any congener in school air. PCB52 is oxidized in the liver to hydroxylated forms, mainly 4-OH-PCB52 (2,2',5,5'-tetrachlorobiphenyl-4-ol). In a previous study, we reported on RNAseq data generated from exposure of human preadipocytes to the dioxin-like PCB congener, PCB126. In this new dataset, we used identical techniques to examine alterations in gene transcript levels in human preadipocytes exposed to PCB52 or 4-OH-PCB52 over a time course. This updated set of data provides a comprehensive transcriptional profile of changes that occur in preadipocytes exposed to PCB52 or 4-OH-PCB52 over time and allows for comparison of these changes between the parent compound and its hydroxy metabolite. The datasets will allow others to explore how PCB52 and 4-OH-PCB52 impact biological pathways in preadipocytes. Further studies can be performed to determine how these changes might lead to disease.

Selective neuroimmune modulation by type I interferon drives neuropathology and neurologic dysfunction following traumatic brain injury.

Accumulating evidence suggests that type I interferon (IFN-I) signaling is a key contributor to immune cell-mediated neuropathology in neurodegenerative diseases. Recently, we demonstrated a robust upregulation of type I interferon-stimulated genes in microglia and astrocytes following experimental traumatic brain injury (TBI). The specific molecular and cellular mechanisms by which IFN-I signaling impacts the neuroimmune response and neuropathology following TBI remains unknown. Using the lateral fluid percussion injury model (FPI) in adult male mice, we demonstrated that IFN α/ß receptor (IFNAR) deficiency resulted in selective and sustained blockade of type I interferon-stimulated genes following TBI as well as decreased microgliosis and monocyte infiltration. Phenotypic alteration of reactive microglia also occurred with diminished expression of molecules needed for MHC class I antigen processing and presentation following TBI. This was associated with decreased accumulation of cytotoxic T cells in the brain. The IFNAR-dependent modulation of the neuroimmune response was accompanied by protection from secondary neuronal death, white matter disruption, and neurobehavioral dysfunction. These data support further efforts to leverage the IFN-I pathway for novel, targeted therapy of TBI.

The impact of sex and physical activity on the local immune response to muscle pain.

Induction of muscle pain triggers a local immune response to produce pain and this mechanism may be sex and activity level dependent. The purpose of this study was to measure the immune system response in the muscle following induction of pain in sedentary and physically active mice. Muscle pain was produced via an activity-induced pain model using acidic saline combined with fatiguing muscle contractions. Prior to induction of muscle pain, mice (C57/BL6) were sedentary or physically active (24hr access to running wheel) for 8 weeks. The ipsilateral gastrocnemius was harvested 24hr after induction of muscle pain for RNA sequencing or flow cytometry. RNA sequencing revealed activation of several immune pathways in both sexes after induction of muscle pain, and these pathways were attenuated in physically active females. Uniquely in females, the antigen processing and presentation pathway with MHC II signaling was activated after induction of muscle pain; activation of this pathway was blocked by physical activity. Blockade of MHC II attenuated development of muscle hyperalgesia exclusively in females. Induction of muscle pain increased the number of macrophages and T-cells in the muscle in both sexes, measured by flow cytometry. In both sexes, the phenotype of macrophages shifted toward a pro-inflammatory state after induction of muscle pain in sedentary mice (M1 + M1/2) but toward an anti-inflammatory state in physically active mice (M2 + M0). Thus, induction of muscle pain activates the immune system with sex-specific differences in the transcriptome while physical activity attenuates immune response in females and alters macrophage phenotype in both sexes.

Hydroxylation markedly alters how the polychlorinated biphenyl (PCB) congener, PCB52, affects gene expression in human preadipocytes.

Polychlorinated biphenyls (PCBs) accumulate in adipose tissue and are linked to obesity and diabetes. The congener, PCB52 (2,2',5,5'-tetrachorobiphenyl), is found at high levels in school air. Hydroxylation of PCB52 to 4-OH-PCB52 (4-hydroxy-2,2',5,5'-tetrachorobiphenyl) may increase its toxicity. To understand PCB52's role in causing adipose dysfunction, we exposed human preadipocytes to PCB52 or 4-OH-PCB52 across a time course and assessed transcript changes using RNAseq. 4-OH-PCB52 caused considerably more changes in the number of differentially expressed genes as compared to PCB52. Both PCB52 and 4-OH-PCB52 upregulated transcript levels of the sulfotransferase SULT1E1 at early time points, but cytochrome P450 genes were generally not affected. A set of genes known to be transcriptionally regulated by PPARα were consistently downregulated by PCB52 at all time points. In contrast, 4-OH-PCB52 affected a variety of pathways, including those involving cytokine responses, hormone responses, focal adhesion, Hippo, and Wnt signaling. Sets of genes known to be transcriptionally regulated by IL17A or parathyroid hormone (PTH) were found to be consistently downregulated by 4-OH-PCB52. Most of the genes affected by PCB52 and 4-OH-PCB52 were different and, of those that were the same, many were changed in an opposite direction. These studies provide insight into how PCB52 or its metabolites may cause adipose dysfunction to cause disease.

Dataset of transcriptomic changes that occur in human preadipocytes over a 3-day course of exposure to 3,3',4,4',5-Pentachlorobiphenyl (PCB126).

Exposure to polychlorinated biphenyls (PCBs) has been associated with the development of metabolic syndrome, a cluster of diseases that includes obesity, diabetes, liver steatosis, and cardiovascular problems. PCBs accumulate and fat and are known to act on adipocytes and their precursors, termed preadipocytes. The PCB congener, PCB126, has been shown to activate the aryl hydrocarbon receptor (AhR) as well as proinflammatory genes. Here, we used RNAseq to assess gene transcript changes that occur in PCB126-exposed human preadipocytes over a time course. RNA was collected from 4 replicates of PCB126-exposed and control-treated preadipocytes at 9 h, 24 h, and 72 h post-exposure. RNA was processed for RNAseq analysis using a NovaSeq 6000 with an obtained minimum of 25 million paired-end 50 bp reads per sample. Reads were aligned using the salmon aligner and transcript expression values were summarized to the gene level using tximport. Gene transcript level counts comparing treated- versus control-treated cells were used for differential expression analysis using DESeq2. Differential expression Excel tables (one for each time point) were generated displaying average differential expression (log2 fold change) of the 4 replicates of treated versus control samples with cutoffs of 0.3 log2 fold change (increase or decrease) and p-values of less than 0.05. FastQ, raw, and differential expression tables were uploaded to GEO. A heat map of genes that were changed in common across all time points was generated using GraphPrism. The data generated from this analysis provides a full transcriptional profile of changes that occur over time in preadipocytes that have been exposed to PCB126. The rich datasets can be mined by other researchers to understand how PCB126 and other dioxin-like compounds, including other PCB congeners such as PCB77 and PCB118, affect biological pathways in preadipocytes and other cell types to cause disease.

Transcriptome sequencing of 3,3',4,4',5-Pentachlorobiphenyl (PCB126)-treated human preadipocytes demonstrates progressive changes in pathways associated with inflammation and diabetes.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that accumulate in adipose tissue and have been associated with cardiometabolic disease. We have previously demonstrated that exposure of human preadipocytes to the dioxin-like PCB126 disrupts adipogenesis via the aryl hydrocarbon receptor (AhR). To further understand how PCB126 disrupts adipose tissue cells, we performed RNAseq analysis of PCB126-treated human preadipocytes over a 3-day time course. The most significant predicted upstream regulator affected by PCB126 exposure at the early time point of 9 h was the AhR. Progressive changes occurred in the number and magnitude of transcript levels of genes associated with inflammation, most closely fitting the pathways of cytokine-cytokine-receptor signaling and the AGE-RAGE diabetic complications pathway. Transcript levels of genes involved in the IL-17A, IL-1ß, MAP kinase, and NF-κB signaling pathways were increasingly dysregulated by PCB126 over time. Our results illustrate the progressive time-dependent nature of transcriptional changes caused by toxicants such as PCB126, point to important pathways affected by PCB126 exposure, and provide a rich dataset for further studies to address how PCB126 and other AhR agonists disrupt preadipocyte function. These findings have implications for understanding how dioxin-like PCBs and other dioxin-like compounds are involved in the development of obesity and diabetes.

T cells, particularly activated CD4+ cells, maintain anti-CD20-mediated NK cell viability and antibody dependent cellular cytotoxicity.

Anti-CD20 monoclonal antibody (mAb) therapy is a mainstay of therapy for B cell malignancies, however many patients fail to respond or eventually develop resistance. The current understanding of mechanisms responsible for this resistance is limited. When peripheral blood mononuclear cells of healthy donors were cultured with Raji cells for 7 days, rituximab (RTX) induced NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), enhanced NK cell viability and increased or maintained NK expression of CD56, CD16, CD57 and KIR. T cells, mainly CD4+, mediated these changes in a contact-dependent manner, with local T cell production of IL2 playing a central role. Similar findings were found when autologous B cells were used as target cells demonstrating the need for T cell help was not due to allogenic reaction. Results with other anti-CD20 and anti-EGFR antibodies were consistent. Small numbers of T cells activated by anti-CD3/CD28 beads or bispecific antibody enhanced RTX-mediated NK cell ADCC, viability and phenotypical changes. Pathway analysis of bulk NK cell mRNA sequencing after activation by RTX with and without T cells was consistent with T cells maintaining the viability of the activated NK cells. These findings suggest T cell help, mediated in large part by local production of IL2, contributes to NK cell ADCC and viability, and that activating T cells in the tumor microenvironment, such as through the use of anti-CD3 based bispecific antibodies, could enhance the efficacy of anti-CD20 and other mAb therapies where NK-mediated ADCC is a primary mechanism of action.

The Effects of Benoxacor on the Liver and Gut Microbiome of C57BL/6 Mice.

The toxicity of many "inert" ingredients of pesticide formulations, such as safeners, is poorly characterized, despite evidence that humans may be exposed to these chemicals. Analysis of ToxCast data for dichloroacetamide safeners with the ToxPi tool identified benoxacor as the safener with the highest potential for toxicity, especially liver toxicity. Benoxacor was subsequently administered to mice via oral gavage for 3 days at concentrations of 0, 0.5, 5, and 50 mg/kg bodyweight (b.w.). Bodyweight-adjusted liver and testes weights were significantly increased in the 50 mg/kg b.w. group. There were no overt pathologies in either the liver or the intestine. 16S rRNA analysis of the cecal microbiome revealed no effects of benoxacor on α- or ß-diversity; however, changes were observed in the abundance of certain bacteria. RNAseq analysis identified 163 hepatic genes affected by benoxacor exposure. Benoxacor exposure expressed a gene regulation profile similar to dichloroacetic acid and the fungicide sedaxane. Metabolomic analysis identified 9 serum and 15 liver metabolites that were affected by benoxacor exposure, changes that were not significant after correcting for multiple comparisons. The activity of antioxidant enzymes was not altered by benoxacor exposure. In vitro metabolism studies with liver microsomes and cytosol from male mice demonstrated that benoxacor is enantioselectively metabolized by cytochrome P450 enzymes, carboxylesterases, and glutathione S-transferases. These findings suggest that the minor toxic effects of benoxacor may be due to its rapid metabolism to toxic metabolites, such as dichloroacetic acid. This result challenges the assumption that inert ingredients of pesticide formulations are safe.

Traumatic brain injury results in unique microglial and astrocyte transcriptomes enriched for type I interferon response.

BACKGROUND: Traumatic brain injury (TBI) is a leading cause of death and disability that lacks neuroprotective therapies. Following a TBI, secondary injury response pathways are activated and contribute to ongoing neurodegeneration. Microglia and astrocytes are critical neuroimmune modulators with early and persistent reactivity following a TBI. Although histologic glial reactivity is well established, a precise understanding of microglia and astrocyte function following trauma remains unknown. METHODS: Adult male C57BL/6J mice underwent either fluid percussion or sham injury. RNA sequencing of concurrently isolated microglia and astrocytes was conducted 7 days post-injury to evaluate cell-type-specific transcriptional responses to TBI. Dual in situ hybridization and immunofluorescence were used to validate the TBI-induced gene expression changes in microglia and astrocytes and to identify spatial orientation of cells expressing these genes. Comparative analysis was performed between our glial transcriptomes and those from prior reports in mild TBI and other neurologic diseases to determine if severe TBI induces unique states of microglial and astrocyte activation. RESULTS: Our findings revealed sustained, lineage-specific transcriptional changes in both microglia and astrocytes, with microglia showing a greater transcriptional response than astrocytes at this subacute time point. Microglia and astrocytes showed overlapping enrichment for genes related to type I interferon signaling and MHC class I antigen presentation. The microglia and astrocyte transcriptional response to severe TBI was distinct from prior reports in mild TBI and other neurodegenerative and neuroinflammatory diseases. CONCLUSION: Concurrent lineage-specific analysis revealed novel TBI-specific transcriptional changes; these findings highlight the importance of cell-type-specific analysis of glial reactivity following TBI and may assist with the identification of novel, targeted therapies.

Direct and indirect immune effects of CMP-001, a virus-like particle containing a TLR9 agonist.

BACKGROUND: CMP-001, also known as vidutolimod, is a virus-like particle containing a TLR9 agonist that is showing promise in early clinical trials. Our group previously demonstrated that the immunostimulatory effects of CMP-001 are dependent on an anti-Qß antibody response which results in opsonization of CMP-001 and uptake by plasmacytoid dendritic cells (pDCs) that then produce interferon (IFN)-α. IFN-α then leads to an antitumor T-cell response that is responsible for the in vivo efficacy of CMP-001. Here, we explore mechanisms by which the initial effects of CMP-001 on pDCs activate other cells that can contribute to development of an antitumor T-cell response. METHODS: Uptake of CMP-001 by various peripheral blood mononuclear cell (PBMC) populations and response to anti-Qß-coated CMP-001 were evaluated by flow cytometry and single-cell RNA sequencing. Purified monocytes were treated with anti-Qß-coated CMP-001 or recombinant IFN-α to evaluate direct and secondary effects of anti-Qß-coated CMP-001 on monocytes. RESULTS: Monocytes had the highest per cell uptake of anti-Qß-coated CMP-001 with lower levels of uptake by pDCs and other cell types. Treatment of PBMCs with anti-Qß-coated CMP-001 induced upregulation of IFN-responsive genes including CXCL10, PDL1, and indoleamine-2,3-dioxygenase (IDO) expression by monocytes. Most of the impact of anti-Qß-coated CMP-001 on monocytes was indirect and mediated by IFN-α, but uptake of anti-Qß-coated CMP-001 altered the monocytic response to IFN-α and resulted in enhanced expression of PDL1, IDO, and CD80 and suppressed expression of CXCL10. These changes included an enhanced ability to induce autologous CD4 T-cell proliferation. CONCLUSIONS: Anti-Qß-coated CMP-001 induces IFN-α production by pDCs which has secondary effects on a variety of cells including monocytes. Uptake of anti-Qß-coated CMP-001 by monocytes alters their response to IFN-α, resulting in enhanced expression of PDL1, IDO and CD80 and suppressed expression of CXCL10. Despite aspects of an immunosuppressive phenotype, these monocytes demonstrated increased ability to augment autologous CD4 T-cell proliferation. These findings shed light on the complexity of the mechanism of action of anti-Qß-coated CMP-001 and provide insight into pathways that may be targeted to further enhance the efficacy of this novel approach to immunotherapy.

Differential gene expression analysis for multi-subject single-cell RNA-sequencing studies with aggregateBioVar.

MOTIVATION: Single-cell RNA-sequencing (scRNA-seq) provides more granular biological information than bulk RNA-sequencing; bulk RNA sequencing remains popular due to lower costs which allows processing more biological replicates and design more powerful studies. As scRNA-seq costs have decreased, collecting data from more than one biological replicate has become more feasible, but careful modeling of different layers of biological variation remains challenging for many users. Here, we propose a statistical model for scRNA-seq gene counts, describe a simple method for estimating model parameters and show that failing to account for additional biological variation in scRNA-seq studies can inflate false discovery rates (FDRs) of statistical tests. RESULTS: First, in a simulation study, we show that when the gene expression distribution of a population of cells varies between subjects, a naïve approach to differential expression analysis will inflate the FDR. We then compare multiple differential expression testing methods on scRNA-seq datasets from human samples and from animal models. These analyses suggest that a naïve approach to differential expression testing could lead to many false discoveries; in contrast, an approach based on pseudobulk counts has better FDR control. AVAILABILITY AND IMPLEMENTATION: A software package, aggregateBioVar, is freely available on Bioconductor (https://www.bioconductor.org/packages/release/bioc/html/aggregateBioVar.html) to accommodate compatibility with upstream and downstream methods in scRNA-seq data analysis pipelines. SUPPLEMENTARY INFORMATION: Raw gene-by-cell count matrices for pig scRNA-seq data are available as GEO accession GSE150211. Supplementary data are available at Bioinformatics online.

Characterization of Type I Interferon-Associated Chemokines and Cytokines in Lacrimal Glands of Nonobese Diabetic Mice.

Type I interferons (IFNs) are required for spontaneous lacrimal gland inflammation in the nonobese diabetic (NOD) mouse model of Sjögren's disease, but the consequences of type I IFN signaling are not well-defined. Here, we use RNA sequencing to define cytokine and chemokine genes upregulated in lacrimal glands of NOD mice in a type I IFN-dependent manner. Interleukin (IL)-21 was the highest differentially expressed cytokine gene, and Il21 knockout NOD mice were relatively protected from lacrimal gland inflammation. We defined a set of chemokines upregulated early in disease including Cxcl9 and Cxcl10, which share a receptor, CXCR3. CXCR3+ T cells were enriched in lacrimal glands with a dominant proportion of CXCR3+ regulatory T cells. Together these data define the early cytokine and chemokine signals associated with type I IFN-signaling in the development of lacrimal gland inflammation in NOD mice providing insight into the role of type I IFN in autoimmunity development.

TAZ-CAMTA1 and YAP-TFE3 alter the TAZ/YAP transcriptome by recruiting the ATAC histone acetyltransferase complex.

Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma that metastasizes early in its clinical course and lacks an effective medical therapy. The TAZ-CAMTA1 and YAP-TFE3 fusion proteins are chimeric transcription factors and initiating oncogenic drivers of EHE. A combined proteomic/genetic screen in human cell lines identified YEATS2 and ZZZ3, components of the Ada2a-containing histone acetyltransferase (ATAC) complex, as key interactors of both fusion proteins despite the dissimilarity of the C terminal fusion partners CAMTA1 and TFE3. Integrative next-generation sequencing approaches in human and murine cell lines showed that the fusion proteins drive a unique transcriptome by simultaneously hyperactivating a TEAD-based transcriptional program and modulating the chromatin environment via interaction with the ATAC complex. Interaction of the ATAC complex with both fusion proteins indicates that it is a key oncogenic driver and unifying enzymatic therapeutic target for this sarcoma. This study presents an approach to mechanistically dissect how chimeric transcription factors drive the formation of human cancers.

The proliferation of human cells is tightly regulated to ensure that enough cells are made to build and repair organs and tissues, while at the same time stopping cells from dividing uncontrollably and damaging the body. To get the right balance, cells rely on physical and chemical cues from their environment that trigger the biochemical signals that regulate two proteins called TAZ and YAP. These proteins control gene activity by regulating the rate at which genes are copied to produce proteins. If this process becomes dysregulated, cells can grow uncontrollably, causing cancer. In cancer cells, it is common to find TAZ and YAP fused to other proteins. In epithelioid hemangioendothelioma, a rare cancer that grows in the blood vessels, cancerous growth can be driven by a version of TAZ fused to the protein CAMTA1, or a version of YAP fused to the protein TFE3. While the role of TAZ and YAP in promoting gene activity is known, it is unclear how CAMTA1 and TFE3 contribute to cell growth becoming dysregulated. Merritt, Garcia et al. studied sarcoma cell lines to show that these two fusion proteins, TAZ-CAMTA1 and YAP-TFE3, change the pattern of gene activity seen in the cells compared to TAZ or YAP alone. An analysis of molecules that interact with the two fusion proteins identified a complex called ATAC as the cause of these changes. This complex adds chemical markers to DNA-packaging proteins, which control which genes are available for activation. The fusion proteins combine the ability of TAZ and YAP to control gene activity and the ability of CAMTA1 and TFE3 to make DNA more accessible, allowing the fusion proteins to drive uncontrolled cancerous growth. Similar TAZ and YAP fusion proteins have been found in other cancers, which can activate genes and potentially alter DNA packaging. Targeting drug development efforts at the proteins that complex with TAZ and YAP fusion proteins may lead to new therapies.

Toll-Like Receptor 7 Is Required for Lacrimal Gland Autoimmunity and Type 1 Diabetes Development in Male Nonobese Diabetic Mice.

Sjögren syndrome (SS) is an immunologically complex, chronic autoimmune disease targeting lacrimal and salivary glands. Nonobese diabetic (NOD) mice spontaneously develop inflammation of lacrimal and salivary glands with histopathological features similar to SS in humans including focal lymphocytic infiltrates in the affected glands. The innate immune signals driving lymphocytic infiltration of these glands are not well-defined. Here we evaluate the role of Toll-like receptor (TLR) 7 in the development of SS-like manifestations in NOD mice. We created a Tlr7 knockout NOD mouse strain and performed histological and gene expression studies to characterize the effects of TLR7 on autoimmunity development. TLR7 was required for male-specific lacrimal gland inflammation but not for female-specific salivary gland inflammation. Moreover, TLR7 was required for type 1 diabetes development in male but not female NOD mice. RNA sequencing demonstrated that TLR7 was associated with a type I interferon (IFN) response and a type I IFN-independent B cell response in the lacrimal glands. Together these studies identify a previously unappreciated pathogenic role for TLR7 in lacrimal gland autoimmunity and T1D development in male NOD mice adding to the growing body of evidence supporting sex differences in mechanisms of autoimmune disease in NOD mice.

Systemic Mesenchymal Stem Cell Treatment Mitigates Structural and Functional Retinal Ganglion Cell Degeneration in a Mouse Model of Multiple Sclerosis.

Purpose: The purpose of this study was to determine mesenchymal stem cell (MSC) therapy efficacy on rescuing the visual system in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) and to provide new mechanistic insights. Methods: EAE was induced in female C57BL6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG)35-55, complete Freund's adjuvant, and pertussis toxin. The findings were compared to sham-immunized mice. Half of the EAE mice received intraperitoneally delivered stem cells (EAE + MSC). Clinical progression was monitored according to a five-point EAE scoring scheme. Pattern electroretinogram (PERG) and retinal nerve fiber layer (RNFL) thickness were measured 32 days after induction. Retinas were harvested to determine retinal ganglion cell (RGC) density and prepared for RNA-sequencing. Results: EAE animals that received MSC treatment seven days after EAE induction showed significantly lower motor-sensory impairment, improvement in the PERG amplitude, and preserved RNFL. Analysis of RNA-sequencing data demonstrated statistically significant differences in gene expression in the retina of MSC-treated EAE mice. Differentially expressed genes were enriched for pathways involved in endoplasmic reticulum stress, endothelial cell differentiation, HIF-1 signaling, and cholesterol transport in the MSC-treated EAE group. Conclusions: Systemic MSC treatment positively affects RGC function and survival in EAE mice. Better cholesterol handling by increased expression of Abca1, the cholesterol efflux regulatory protein, paired with the resolution of HIF-1 signaling activation might explain the improvements seen in PERG of EAE animals after MSC treatment. Translational Relevance: Using MSC therapy in a mouse model of MS, we discovered previously unappreciated biochemical pathways associated with RGC neuroprotection, which have the potential to be pharmacologically targeted as a new treatment regimen.