Cellular heterogeneity and dynamics of the human uterus in healthy premenopausal women.

The human uterus is a complex and dynamic organ whose lining grows, remodels, and regenerates every menstrual cycle or upon tissue damage. Here, we applied single-cell RNA sequencing to profile more the 50,000 uterine cells from both the endometrium and myometrium of five healthy premenopausal individuals, and jointly analyzed the data with a previously published dataset from 15 subjects. The resulting normal uterus cell atlas contains more than 167K cells, representing the lymphatic endothelium, blood endothelium, stromal, ciliated epithelium, unciliated epithelium, and immune cell populations. Focused analyses within each major cell type and comparisons with subtype labels from prior studies allowed us to document supporting evidence, resolve naming conflicts, and propose a consensus annotation system of 39 subtypes. We release their gene expression centroids, differentially expressed genes, and messenger Ribonucleic Acid (mRNA) patterns of literature-based markers as a shared community resource. We identify multiple potential progenitor cells: compartment-wide progenitors for each major cell type and potential cross-lineage multipotent stromal progenitors that may replenish the epithelial, stromal, and endothelial compartments. Furthermore, many cell types and subtypes exhibit shifts in cell number and transcriptomes across different phases of the menstrual cycle. Finally, comparisons between premenopausal, postpartum, and postmenopausal samples revealed substantial alterations in tissue composition, particularly in the proportions of stromal, endothelial, and immune cells. The cell taxonomy and molecular markers we report here are expected to inform studies of both basic biology of uterine function and its disorders.

Obesity Inhibits Alveolar Macrophage Responses to Pseudomonas aeruginosa Pneumonia via Upregulation of Prostaglandin E2 in Male, but Not Female, Mice.

Obesity is associated with increased morbidity and mortality during bacterial pneumonia. Cyclooxygenase-2 (COX-2) and PGE2 have been shown to be upregulated in patients who are obese. In this study, we investigated the role of obesity and PGE2 in bacterial pneumonia and how inhibition of PGE2 improves antibacterial functions of macrophages. C57BL/6J male and female mice were fed either a normal diet (ND) or high-fat diet (HFD) for 16 wk. After this time, animals were infected with Pseudomonas aeruginosa in the lung. In uninfected animals, alveolar macrophages were extracted for either RNA analysis or to be cultured ex vivo for functional analysis. HFD resulted in changes in immune cell numbers in both noninfected and infected animals. HFD animals had increased bacterial burden compared with ND animals; however, male HFD animals had higher bacterial burden compared with HFD females. Alveolar macrophages from HFD males had decreased ability to phagocytize and kill bacteria and were shown to have increased cyclooxygenase-2 and PGE2. Treating male, but not female, alveolar macrophages with PGE2 leads to increases in cAMP and decreased bacterial phagocytosis. Treatment with lumiracoxib-conjugated nanocarriers targeting alveolar macrophages improves bacterial phagocytosis and clearance in both ND and HFD male animals. Our study highlights that obesity leads to worse morbidity during bacterial pneumonia in male mice because of elevated PGE2. In addition, we uncover a sex difference in both obesity and infection, because females produce high basal PGE2 but because of a failure to signal via cAMP do not display impaired phagocytosis.

RSV enhances Staphylococcus aureus bacterial growth in the lung.

Patients coinfected with respiratory syncytial virus (RSV) and bacteria have longer hospital stays, higher risk of intensive care unit admission, and worse outcomes. We describe a model of RSV line 19F/methicillin-resistant Staphylococcus aureus (MRSA) USA300 coinfection that does not impair viral clearance, but prior RSV infection enhances USA300 MRSA bacterial growth in the lung. The increased bacterial burden post-RSV correlates with reduced accumulation of neutrophils and impaired bacterial killing by alveolar macrophages. Surprisingly, reduced neutrophil accumulation is likely not explained by reductions in phagocyte-recruiting chemokines or alterations in proinflammatory cytokine production compared with mice infected with S. aureus alone. Neutrophils from RSV-infected mice retain their ability to migrate toward chemokine signals, and neutrophils from the RSV-infected lung are better able to phagocytize and kill S. aureus ex vivo on a per cell basis. In contrast, while alveolar macrophages could ingest USA300 post-RSV, intracellular bacterial killing was impaired. The RSV/S. aureus coinfected lung promotes a state of overactivation in neutrophils, demonstrated by increased production of reactive oxygen species (ROS) that can drive formation of neutrophil extracellular traps (NETs), resulting in cell death. Mice with RSV/S. aureus coinfection had increased extracellular DNA and protein in bronchoalveolar lavage fluid and histological evidence confirmed NETosis in vivo. Taken together, these data highlight that prior RSV infection can prime the overactivation of neutrophils leading to cell death that impairs neutrophil accumulation in the lung. Additionally, alveolar macrophage killing of bacteria is impaired post-RSV. Together, these defects enhance USA300 MRSA bacterial growth in the lung post-RSV.

Myeloid- and Epithelial-derived Heparin-Binding Epidermal Growth Factor-like Growth Factor Promotes Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a poorly understood, progressive lethal lung disease with no known cure. In addition to alveolar epithelial cell (AEC) injury and excessive deposition of extracellular matrix proteins, chronic inflammation is a hallmark of IPF. Literature suggests that the persistent inflammation seen in IPF primarily consists of monocytes and macrophages. Recent work demonstrates that monocyte-derived alveolar macrophages (moAMs) drive lung fibrosis, but further characterization of critical moAM cell attributes is necessary. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an important epidermal growth factor receptor ligand that has essential roles in angiogenesis, wound healing, keratinocyte migration, and epithelial-mesenchymal transition. Our past work has shown HB-EGF is a primary marker of profibrotic M2 macrophages, and this study seeks to characterize myeloid-derived HB-EGF and its primary mechanism of action in bleomycin-induced lung fibrosis using Hbegff/f;Lyz2Cre+ mice. Here, we show that patients with IPF and mice with pulmonary fibrosis have increased expression of HB-EGF and that lung macrophages and transitional AECs of mice with pulmonary fibrosis and humans all express HB-EGF. We also show that Hbegff/f;Lyz2Cre+ mice are protected from bleomycin-induced fibrosis and that this protection is likely multifactorial, caused by decreased CCL2-dependent monocyte migration, decreased fibroblast migration, and decreased contribution of HB-EGF from AEC sources when HB-EGF is removed under the Lyz2Cre promoter.

The ADP-Heptose Biosynthesis Enzyme GmhB is a Conserved Gram-Negative Bacteremia Fitness Factor.

Klebsiella pneumoniae is a leading cause of Gram-negative bacteremia, which is a major source of morbidity and mortality worldwide. Gram-negative bacteremia requires three major steps: primary site infection, dissemination to the blood, and bloodstream survival. Because K. pneumoniae is a leading cause of health care-associated pneumonia, the lung is a common primary infection site leading to secondary bacteremia. K. pneumoniae factors essential for lung fitness have been characterized, but those required for subsequent bloodstream infection are unclear. To identify K. pneumoniae genes associated with dissemination and bloodstream survival, we combined previously and newly analyzed insertion site sequencing (InSeq) data from a murine model of bacteremic pneumonia. This analysis revealed the gene gmhB as important for either dissemination from the lung or bloodstream survival. In Escherichia coli, GmhB is a partially redundant enzyme in the synthesis of ADP-heptose for the lipopolysaccharide (LPS) core. To characterize its function in K. pneumoniae, an isogenic knockout strain (ΔgmhB) and complemented mutant were generated. During pneumonia, GmhB did not contribute to lung fitness and did not alter normal immune responses. However, GmhB enhanced bloodstream survival in a manner independent of serum susceptibility, specifically conveying resistance to spleen-mediated killing. In a tail-vein injection of murine bacteremia, GmhB was also required by K. pneumoniae, E. coli, and Citrobacter freundii for optimal fitness in the spleen and liver. Together, this study identifies GmhB as a conserved Gram-negative bacteremia fitness factor that acts through LPS-mediated mechanisms to enhance fitness in blood-filtering organs.

M2 macrophages have unique transcriptomes but conditioned media does not promote profibrotic responses in lung fibroblasts or alveolar epithelial cells in vitro.

Macrophages are critical regulators of pulmonary fibrosis. Their plasticity, proximity, and ability to cross talk with structural cells of the lung make them a key cell type of interest in the regulation of lung fibrosis. Macrophages can express a variety of phenotypes, which have been historically represented through an "M1-like" to "M2-like" delineation. In this classification, M1-like macrophages are proinflammatory and have increased phagocytic capacity compared with alternatively activated M2-like macrophages that are profibrotic and are associated with wound healing. Extensive evidence in the field in both patients and animal models aligns pulmonary fibrosis with M2 macrophages. In this study, we performed RNA sequencing (RNAseq) to fully characterize M1- vs. M2-skewed bone marrow-derived macrophages (BMDMs) and investigated the profibrotic abilities of M2 BMDM conditioned media (CM) to promote fibroblast migration and proliferation, alveolar epithelial cell (AEC) apoptosis, and mRNA expression of key fibrotic genes in both fibroblasts and AECs. Although M2 CM-treated fibroblasts had increased migration and M2 CM-treated fibroblasts and AECs had increased expression of profibrotic proteins over M1 CM-treated cells, all differences can be attributed to M2 polarization reagents IL-4 and IL-13 also present in the CM. Collectively, these data suggest that the profibrotic effects associated with M2 macrophage CM in vitro are attributable to effects of polarization cytokines rather than additional factors secreted in response to those polarizing cytokines.

Influenza-induced immune suppression to methicillin-resistant Staphylococcus aureus is mediated by TLR9.

Bacterial lung infections, particularly with methicillin-resistant Staphylococcus aureus (MRSA), increase mortality following influenza infection, but the mechanisms remain unclear. Here we show that expression of TLR9, a microbial DNA sensor, is increased in murine lung macrophages, dendritic cells, CD8+ T cells and epithelial cells post-influenza infection. TLR9-/- mice did not show differences in handling influenza nor MRSA infection alone. However, TLR9-/- mice have improved survival and bacterial clearance in the lung post-influenza and MRSA dual infection, with no difference in viral load during dual infection. We demonstrate that TLR9 is upregulated on macrophages even when they are not themselves infected, suggesting that TLR9 upregulation is related to soluble mediators. We rule out a role for elevations in interferon-γ (IFNγ) in mediating the beneficial MRSA clearance in TLR9-/- mice. While macrophages from WT and TLR9-/- mice show similar phagocytosis and bacterial killing to MRSA alone, following influenza infection, there is a marked upregulation of scavenger receptor A and MRSA phagocytosis as well as inducible nitric oxide synthase (Inos) and improved bacterial killing that is specific to TLR9-deficient cells. Bone marrow transplant chimera experiments and in vitro experiments using TLR9 antagonists suggest TLR9 expression on non-hematopoietic cells, rather than the macrophages themselves, is important for regulating myeloid cell function. Interestingly, improved bacterial clearance post-dual infection was restricted to MRSA, as there was no difference in the clearance of Streptococcus pneumoniae. Taken together these data show a surprising inhibitory role for TLR9 signaling in mediating clearance of MRSA that manifests following influenza infection.

Lung Microbiota Contribute to Pulmonary Inflammation and Disease Progression in Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) causes considerable global morbidity and mortality, and its mechanisms of disease progression are poorly understood. Recent observational studies have reported associations between lung dysbiosis, mortality, and altered host defense gene expression, supporting a role for lung microbiota in IPF. However, the causal significance of altered lung microbiota in disease progression is undetermined. Objectives: To examine the effect of microbiota on local alveolar inflammation and disease progression using both animal models and human subjects with IPF. Methods: For human studies, we characterized lung microbiota in BAL fluid from 68 patients with IPF. For animal modeling, we used a murine model of pulmonary fibrosis in conventional and germ-free mice. Lung bacteria were characterized using 16S rRNA gene sequencing with novel techniques optimized for low-biomass sample load. Microbiota were correlated with alveolar inflammation, measures of pulmonary fibrosis, and disease progression. Measurements and Main Results: Disruption of the lung microbiome predicts disease progression, correlates with local host inflammation, and participates in disease progression. In patients with IPF, lung bacterial burden predicts fibrosis progression, and microbiota diversity and composition correlate with increased alveolar profibrotic cytokines. In murine models of fibrosis, lung dysbiosis precedes peak lung injury and is persistent. In germ-free animals, the absence of a microbiome protects against mortality. Conclusions: Our results demonstrate that lung microbiota contribute to the progression of IPF. We provide biological plausibility for the hypothesis that lung dysbiosis promotes alveolar inflammation and aberrant repair. Manipulation of lung microbiota may represent a novel target for the treatment of IPF.

Loss of myeloid-specific protein phosphatase 2A enhances lung injury and fibrosis and results in IL-10-dependent sensitization of epithelial cell apoptosis.

Protein phosphatase 2A (PP2A), a ubiquitously expressed Ser/Thr phosphatase is an important regulator of cytokine signaling and cell function. We previously showed that myeloid-specific deletion of PP2A (LysMcrePP2A-/-) increased mortality in a murine peritoneal sepsis model. In the current study, we assessed the role of myeloid PP2A in regulation of lung injury induced by lipopolysaccharide (LPS) or bleomycin delivered intratracheally. LysMcrePP2A-/- mice experienced increased lung injury in response to both LPS and bleomycin. LysMcrePP2A-/- mice developed more exuberant fibrosis in response to bleomycin, elevated cytokine responses, and chronic myeloid inflammation. Bone marrow-derived macrophages (BMDMs) from LysMcrePP2A-/- mice showed exaggerated inflammatory cytokine release under conditions of both M1 and M2 activation. Notably, secretion of IL-10 was elevated under all stimulation conditions, including activation of BMDMs by multiple Toll-like receptor ligands. Supernatants collected from LPS-stimulated LysMcrePP2A-/- BMDMs induced epithelial cell apoptosis in vitro but this effect was mitigated when IL-10 was also depleted from the BMDMs by crossing LysMcrePP2A-/- mice with systemic IL-10-/- mice (LysMcrePP2A-/- × IL-10-/-) or when IL-10 was neutralized. Despite these findings, IL-10 did not directly induce epithelial cell apoptosis but sensitized epithelial cells to other mediators from the BMDMs. Taken together our results demonstrate that myeloid PP2A regulates production of multiple cytokines but that its effect is most pronounced on IL-10 production. Furthermore, IL-10 sensitizes epithelial cells to apoptosis in response to myeloid-derived mediators, which likely contributes to the pathogenesis of lung injury and fibrosis in this model.

IL-17 in the lung: the good, the bad, and the ugly.

The IL-17 family of cytokines has emerged over the last two decades as a pleiotropic group of molecules that function in a wide variety of both beneficial and detrimental (pathological) processes, mainly in mucosal barrier tissue. The beneficial effects of IL-17 expression are especially important in the lung, where exposure to foreign agents is abundant. IL-17A plays an important role in protection from both extracellular bacteria and fungi, as well as viruses that infect cells of the mucosal tracts. IL-17 coregulated cytokines, such as IL-22, are involved in maintaining epithelial cell homeostasis and participate in epithelial cell repair/regeneration following inflammatory insults. Thus, the IL-17/IL-22 axis is important in both responding to, and recovering from, pathogens. However, aberrant expression or overexpression of IL-17 cytokines contributes to a number of pathological outcomes, including asthma, pneumonitis, and generation or exacerbation of pulmonary fibrosis. This review covers the good, bad, and ugly aspects of IL-17 in the lung.

Loss of CCR2 signaling alters leukocyte recruitment and exacerbates γ-herpesvirus-induced pneumonitis and fibrosis following bone marrow transplantation.

CCR2-expressing leukocytes are required for the progression of fibrosis in models of induced lung injury as well as models of bone marrow transplant (BMT)-related idiopathic pneumonia syndrome. Infection with murid γ-herpesvirus-68 (γHV-68) results in severe pneumonitis and pulmonary fibrosis following syngeneic BMT; however, the roles that various proinflammatory leukocyte populations play in this process remain unclear. Deletion of CCR2 in both non-BMT and BMT mice increased early lytic viral replication and resulted in a reduction in the numbers of lung-infiltrating GR1+,F4/80+ and CXCR1+ cells, while maintaining robust neutrophil infiltration. Similarly, in γHV-68-infected CCR2(-/-) BMT mice, recruitment of monocytes and lymphocytes were reduced whereas neutrophil recruitment was increased compared with wild-type (WT) BMT mice. Interestingly, levels of profibrotic IL-17 were increased in infected CCR2 BMT mice compared with WT BMT. Furthermore, an increase in lung-associated collagen was detected even though there was an overall decrease in the number of profibrotic CCR2+ fibrocytes detected in the lungs of CCR2(-/-) BMT mice. These data indicate that, contrary to most models of fibrosis, deletion of CCR2 offers no protection from γ-herpesvirus-induced pneumonitis and fibrosis, and, indeed, CCR2+ cells play a suppressive role during the development of pulmonary fibrosis following γ-herpesvirus infection post-BMT by limiting IL-7 and collagen production.

Deletion of the human cytomegalovirus US17 gene increases the ratio of genomes per infectious unit and alters regulation of immune and endoplasmic reticulum stress response genes at early and late times after infection.

Human cytomegalovirus (HCMV) employs numerous strategies to combat, subvert, or co-opt host immunity. One evolutionary strategy for this involves capture of a host gene and then its successive duplication and divergence, forming a family of genes, many of which have immunomodulatory activities. The HCMV US12 family consists of 10 tandemly arranged sequence-related genes in the unique short (US) region of the HCMV genome (US12 to US21). Each gene encodes a protein possessing seven predicted transmembrane domains, patches of sequence similarity with cellular G-protein-coupled receptors, and the Bax inhibitor 1 family of antiapoptotic proteins. We show that one member, US17, plays an important role during virion maturation. Microarray analysis of cells infected with a recombinant HCMV isolate with a US17 deletion (the ΔUS17 mutant virus) revealed blunted host innate and interferon responses at early times after infection (12 h postinfection [hpi]), a pattern opposite that previously seen in the absence of the immunomodulatory tegument protein pp65 (pUL83). Although the ΔUS17 mutant virus produced numbers of infectious particles in fibroblasts equal to the numbers produced by the parental virus, it produced >3-fold more genome-containing noninfectious viral particles and delivered increased amounts of pp65 to newly infected cells. These results suggest that US17 has evolved to control virion composition, to elicit an appropriately balanced host immune response. At later time points (96 hpi), ΔUS17 mutant-infected cells displayed aberrant expression of several host endoplasmic reticulum stress response genes and chaperones, some of which are important for the final stages of virion assembly and egress. Our results suggest that US17 modulates host pathways to enable production of virions that elicit an appropriately balanced host immune response.

Identification of human cytomegalovirus genes important for biogenesis of the cytoplasmic virion assembly complex.

UNLABELLED: Human cytomegalovirus (HCMV) has many effects on cells, including remodeling the cytoplasm to form the cytoplasmic virion assembly complex (cVAC), the site of final virion assembly. Viral tegument, envelope, and some nonstructural proteins localize to the cVAC, and cytoskeletal filaments radiate from a microtubule organizing center in the cVAC. The endoplasmic reticulum (ER)-to-Golgi intermediate compartment, Golgi apparatus, and trans-Golgi network form a ring that outlines the cVAC. The center of the cVAC ring is occupied by numerous vesicles that share properties with recycling endosomes. In prior studies, we described the three-dimensional structure and the extensive remodeling of the cytoplasm and shifts in organelle identity that occur during development of the cVAC. The objective of this work was to identify HCMV proteins that regulate cVAC biogenesis. Because the cVAC does not form in the absence of viral DNA synthesis, we employed HCMV-infected cells transfected with synthetic small interfering RNAs (siRNAs) that targeted 26 candidate early-late and late protein-coding genes required for efficient virus replication. We identified three HCMV genes (UL48, UL94, and UL103) whose silencing had major effects on cVAC development, including failure to form the Golgi ring and dispersal of markers of early and recycling endosomes. To confirm and extend the siRNA results, we constructed recombinant viruses in which pUL48 and pUL103 are fused with a regulatable protein destabilization domain (dd-FKBP). In the presence of a stabilizing ligand (Shield-1), the cVAC appeared to develop normally. In its absence, cVAC development was abrogated, verifying roles for pUL48 and pUL103 in cVAC biogenesis. IMPORTANCE: Human cytomegalovirus (HCMV) is an important human pathogen that causes disease and disability in immunocompromised individuals and in children infected before birth. Few drugs are available for treatment of HCMV infections. HCMV remodels the interior of infected cells to build a factory for assembling new infectious particles (virions), the cytoplasmic virion assembly complex (cVAC). Here, we identified three HCMV genes (UL48, UL94, and UL103) as important contributors to cVAC development. In addition, we found that mutant viruses that express an unstable form of the UL103 protein have defects in cVAC development and production of infectious virions and produce small plaques and intracellular virions with aberrant appearances. Of these, only the reduced production of infectious virions is not eliminated by chemically stabilizing the protein. In addition to identifying new functions for these HCMV genes, this work is a necessary prelude to developing novel antivirals that would block cVAC development.

Cellular heterogeneity and dynamics of the human uterus in healthy premenopausal women.

The human uterus is a complex and dynamic organ whose lining grows, remodels, and regenerates in every menstrual cycle or upon tissue damage. Here we applied single-cell RNA sequencing to profile more the 50,000 uterine cells from both the endometrium and myometrium of 5 healthy premenopausal individuals, and jointly analyzed the data with a previously published dataset from 15 subjects. The resulting normal uterus cell atlas contains more than 167K cells representing the lymphatic endothelium, blood endothelium, stromal, ciliated epithelium, unciliated epithelium, and immune cell populations. Focused analyses within each major cell type and comparisons with subtype labels from prior studies allowed us to document supporting evidence, resolve naming conflicts, and to propose a consensus annotation system of 39 subtypes. We release their gene expression centroids, differentially expressed genes, and mRNA patterns of literature-based markers as a shared community resource. We find many subtypes show dynamic changes over different phases of the cycle and identify multiple potential progenitor cells: compartment-wide progenitors for each major cell type, transitional cells that are upstream of other subtypes, and potential cross-lineage multipotent stromal progenitors that may be capable of replenishing the epithelial, stromal, and endothelial compartments. When compared to the healthy premenopausal samples, a postpartum and a postmenopausal uterus sample revealed substantially altered tissue composition, involving the rise or fall of stromal, endothelial, and immune cells. The cell taxonomy and molecular markers we report here are expected to inform studies of both basic biology of uterine function and its disorders. SIGNIFICANCE: We present single-cell RNA sequencing data from seven individuals (five healthy pre-menopausal women, one post-menopausal woman, and one postpartum) and perform an integrated analysis of this data alongside 15 previously published scRNA-seq datasets. We identified 39 distinct cell subtypes across four major cell types in the uterus. By using RNA velocity analysis and centroid-centroid comparisons we identify multiple computationally predicted progenitor populations for each of the major cell compartments, as well as potential cross-compartment, multi-potent progenitors. While the function and interactions of these cell populations remain to be validated through future experiments, the markers and their "dual characteristics" that we describe will serve as a rich resource to the scientific community. Importantly, we address a significant challenge in the field: reconciling multiple uterine cell taxonomies being proposed. To achieve this, we focused on integrating historical and contemporary knowledge across multiple studies. By providing detailed evidence used for cell classification we lay the groundwork for establishing a stable, consensus cell atlas of the human uterus.

Dendritic Cell - Fibroblast Crosstalk via TLR9 and AHR Signaling Drives Lung Fibrogenesis.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring and loss of lung function. With limited treatment options, patients succumb to the disease within 2-5 years. The molecular pathogenesis of IPF regarding the immunologic changes that occur is poorly understood. We characterize a role for non-canonical aryl-hydrocarbon receptor signaling (ncAHR) in dendritic cells (DCs) that leads to production of IL-6 and IL-17, promoting fibrosis. TLR9 signaling in myofibroblasts is shown to regulate production of TDO2 which converts tryptophan into the endogenous AHR ligand kynurenine. Mice with augmented ncAHR signaling were created by crossing floxed AHR exon-2 deletion mice (AHR Δex2 ) with mice harboring a CD11c-Cre. Bleomycin was used to study fibrotic pathogenesis. Isolated CD11c+ cells and primary fibroblasts were treated ex-vivo with relevant TLR agonists and AHR modulating compounds to study how AHR signaling influenced inflammatory cytokine production. Human datasets were also interrogated. Inhibition of all AHR signaling rescued fibrosis, however, AHR Δex2 mice treated with bleomycin developed more fibrosis and DCs from these mice were hyperinflammatory and profibrotic upon adoptive transfer. Treatment of fibrotic fibroblasts with TLR9 agonist increased expression of TDO2. Study of human samples corroborate the relevance of these findings in IPF patients. We also, for the first time, identify that AHR exon-2 floxed mice retain capacity for ncAHR signaling.

Horizontal transmission of gut microbiota attenuates mortality in lung fibrosis.

Pulmonary fibrosis is a chronic and often fatal disease. The pathogenesis is characterized by aberrant repair of lung parenchyma, resulting in loss of physiological homeostasis, respiratory failure, and death. The immune response in pulmonary fibrosis is dysregulated. The gut microbiome is a key regulator of immunity. The role of the gut microbiome in regulating the pulmonary immunity in lung fibrosis is poorly understood. Here, we determine the impact of gut microbiota on pulmonary fibrosis in substrains of C57BL/6 mice derived from different vendors (C57BL/6J and C57BL/6NCrl). We used germ-free models, fecal microbiota transplantation, and cohousing to transmit gut microbiota. Metagenomic studies of feces established keystone species between substrains. Pulmonary fibrosis was microbiota dependent in C57BL/6 mice. Gut microbiota were distinct by ß diversity and α diversity. Mortality and lung fibrosis were attenuated in C57BL/6NCrl mice. Elevated CD4+IL-10+ T cells and lower IL-6 occurred in C57BL/6NCrl mice. Horizontal transmission of microbiota by cohousing attenuated mortality in C57BL/6J mice and promoted a transcriptionally altered pulmonary immunity. Temporal changes in lung and gut microbiota demonstrated that gut microbiota contributed largely to immunological phenotype. Key regulatory gut microbiota contributed to lung fibrosis, generating rationale for human studies.

Cellular heterogeneity of human fallopian tubes in normal and hydrosalpinx disease states identified using scRNA-seq.

Fallopian tube (FT) homeostasis requires dynamic regulation of heterogeneous cell populations and is disrupted in infertility and ovarian cancer. Here, we applied single-cell RNA-seq to profile 59,738 FT cells from four healthy, pre-menopausal subjects. The resulting cell atlas contains 12 major cell types representing epithelial, stromal, and immune compartments. Re-clustering of epithelial cells identified four ciliated and six non-ciliated secretory epithelial subtypes, two of which represent potential progenitor pools: one leading to mature secretory cells and the other contributing to either ciliated cells or one of the stromal cell types. To understand how FT cell numbers and states change in a disease state, we analyzed 17,798 cells from two hydrosalpinx samples and observed shifts in epithelial and stromal populations and cell-type-specific changes in extracellular matrix and TGF-ß signaling; this underscores fibrosis pathophysiology. This resource is expected to facilitate future studies aimed at expanding understanding of fallopian tube homeostasis in normal development and disease.

Stem cell transplantation uncovers TDO-AHR regulation of lung dendritic cells in herpesvirus-induced pathology.

The aryl-hydrocarbon receptor (AHR) is an intracellular sensor of aromatic hydrocarbons that sits at the top of various immunomodulatory pathways. Here, we present evidence that AHR plays a role in controlling IL-17 responses and the development of pulmonary fibrosis in response to respiratory pathogens following bone marrow transplant (BMT). Mice infected intranasally with gamma-herpesvirus 68 (γHV-68) following BMT displayed elevated levels of the AHR ligand, kynurenine (kyn), in comparison with control mice. Inhibition or genetic ablation of AHR signaling resulted in a significant decrease in IL-17 expression as well as a reduction in lung pathology. Lung CD103+ DCs expressed AHR following BMT, and treatment of induced CD103+ DCs with kyn resulted in altered cytokine production in response to γHV-68. Interestingly, mice deficient in the kyn-producing enzyme indolamine 2-3 dioxygenase showed no differences in cytokine responses to γHV-68 following BMT; however, isolated pulmonary fibroblasts infected with γHV-68 expressed the kyn-producing enzyme tryptophan dioxygenase (TDO2). Our data indicate that alterations in the production of AHR ligands in response to respiratory pathogens following BMT results in a pro-Th17 phenotype that drives lung pathology. We have further identified the TDO2/AHR axis as a potentially novel form of intercellular communication between fibroblasts and DCs that shapes immune responses to respiratory pathogens.

TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice.

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.