Research
Abstract
Type 2 (T2) immune cells dominate the airways of mild-moderate asthma (MMA) patients with a more complex Type 1 (T1)-T2 mixed immune response evident in treatment-refractory severe asthma (SA). We hypothesized that comparing the transcriptomes of the airway epithelium of SA and MMA patients would reveal molecular signatures associated with more severe disease in the context of a complex immune response. Using our novel interpretable machine learning tool, SLIDE, meaningful latent factors (context-specific gene co-expression networks) were revealed that distinguished SA from MMA. Unexpectedly, an aberrant high expression of normally host-protective, membrane-tethered and IFN-inducible mucins, MUC1 and MUC4, was identified in SA. Gene networks in the significant latent factors discriminating SA from MMA corresponded to enrichment of a keratinization program in SA airways. Keratinization was marked by increased expression of the stress keratin KRT16, signifying squamous metaplasia suggesting adaptive reprogramming of the airway epithelium in response to chronic stress. These mucins and KRT16 were inversely associated with lung function in two separate asthma cohorts. Imaging of endobronchial biopsies revealed significantly higher KRT16 protein expression in SA compared to MMA that strongly correlated with MUC1 protein expression. Our study identifies dysregulated host-protective and maladaptive repair responses in SA distinguishing from MMA.
Authors
Sagar L. Kale, Augusta M. Vincent, Mark A. Ross, Isha Mehta, Michael J. Calderon, Richard P. Ramonell, Himanshu Setya, Jessica C. McCreary-Partyka, Huijuan Yuan, Stephanie A. Christenson, Prescott G. Woodruff, Mario Castro, Kaharu Sumino, Nizar N. Jarjour, Loren C. Denlinger, Benjamin Gaston, Eugene R. Bleecker, Deborah A. Meyers, Wendy C. Moore, Elliot Israel, Bruce D. Levy, David Mauger, Serpil Erzurum, Anthony Newbrough, Taylor J. Nee, Prabir Ray, Claudette M. St. Croix, Sally E. Wenzel, Jishnu Das, Anuradha Ray, Marc C. Gauthier
Abstract
Polycystic kidney disease (PKD) arises from mutations in cilia-associated genes, such as PKD1 and PKD2, expressed in renal epithelial cells, leading to progressive kidney dysfunction and end-stage kidney disease (ESKD). PKD patients exhibit significant heterogeneity in disease progression, largely due to genetic and environmental modifiers. Like patients, mouse models of PKD also exhibit significant heterogeneity with regards to the gene mutated, age of disease onset, and rate of disease progression. To elucidate the cellular and molecular consequences of these variables, we constructed an integrated single-cell RNA sequencing atlas across mouse models of PKD, mapping changes in cell type composition, gene expression, and intercellular signaling networks across the whole atlas and within individual models. Across models, single cell RNA sequencing (scRNAseq) data revealed increased Spp1 (osteopontin) expression and signaling from PKD-enriched clusters. Global deletion of Spp1 in Pkd1RC/RC mice resulted in a modest reduction in cyst severity and improved kidney function. From these studies, we created a freely available, searchable website (https://bmblx.bmi.osumc.edu/scPKD/) that can be used to identify cross- and intra-model changes in gene expression, guiding researchers to new therapeutic targets for treating PKD.
Authors
Sarah J. Miller, Hua Zhong, Weidong Wu, Audrey M. Cordova, Morgan E. Yashchenko, Alex Yashchenko, Zhang Li, Daniyal J. Jafree, Chelsea N. Zimmerman, Christa I. DeVette, Vicki Do, Maya E. Hignite, Yohan Park, Fariha Nusrat, Bibi Maryam, Sizhao Lu, Xiaoyan Li, Jenny R. Gipson, Xiaogang Li, David A. Long, Mary C.M. Weiser-Evans, Bradley K. Yoder, Benjamin D. Cowley, Jr., Katharina Hopp, Jason R. Stubbs, Qin Ma, Anjun Ma, Kurt A. Zimmerman
Abstract
Locally produced nonpituitary growth hormone (npGH) promotes DNA damage accumulation and epithelial-mesenchymal transition (EMT) in aging human colon epithelium. GH receptor (GHR) and npGH are expressed in normal human prostate and benign prostatic hyperplasia (BPH) prevalence increases with age. We hypothesized that local prostate GH action may promote EMT and contribute to BPH pathogenesis. We show here that the number of patients expressing npGH increases >10-fold after age 60, concordant with increased γH2AX, a marker of DNA damage, and EMT activation. GH-treated human primary prostate epithelial cells, normal prostate cells, and primary cell cultures derived from resected BPH specimens exhibited enhanced DNA damage and activated EMT, with induced TWIST2 and suppressed E-cadherin, as well as increased Ki67, cell motility, and proliferation. In mice, prostate tissue adjacent to allografted GH-expressing fibroblasts showed increased γH2AX, TWIST2, and Ki67 compared to controls, along with morphological changes consistent with BPH. While GH and GH-induced IGF-1 both activated EMT, GH triggered DNA damage independent of IGF-1. These results elucidate a novel role for local npGH in aging prostate tissue, whereby npGH increases DNA damage and promotes EMT to enable a microenvironment favoring BPH development. Prostate GHR signaling may be an attractive therapeutic target for BPH.
Authors
Masaki Ryuzaki, Svetlana Zonis, Neil A. Bhowmick, Sandrine Billet, Saravana Kumar Kailasam Mani, Stephen J. Freedland, Hyung L. Kim, Vera Chesnokova, Shlomo Melmed
Abstract
Schistosomiasis is a common cause of pulmonary hypertension (PH) worldwide. It is known that adaptive immunity and specifically CD4 T cells are necessary for experimental disease pathogenesis. The lectin complement system is activated in those infected with schistosomiasis. We tested the hypothesis that lectin complement promotes Th2 CD4 T cell activation, leading to PH in a schistosomiasis exposure model. Wildtype and transgenic mice lacking mannose binding lectin (MBL), and bone marrow chimeras, were experimentally exposed to Schistosoma mansoni eggs. PH severity was assessed by hemodynamics and vascular remodeling, and CD4 T cell density and phenotype was assessed by flow cytometry. Wildtype recipients of MBL knockout bone marrow (BM) were protected from Schistosoma-induced PH. The protection from PH was associated with fewer Th2 CD4 T cells. In wildtype mice exposed to Schistosoma, CD4 T cells expression of MBL increased. MBL-deficient CD4 T cells had a suppressed Th2 phenotype when exposed to Schistosoma antigens. Mice with deficiency of C4, which functions downstream of MBL in the lectin complement pathway, were not protected from Schistosoma-PH. Mice lacking MBL were not protected from PH caused by hypoxia exposure. MBL in CD4 T cells promotes Schistosoma-induced PH.
Authors
Claudia Mickael, Dara C. Fonseca Balladares, Rahul Kumar, Michael H. Lee, Kevin Nolan, Linda Sanders, Katie J. Tuscan, Ramraj Prasad, Pilar Londono, Fernanda P. Oliveira, Kennedi B. Pyper, Ari B. Molofsky, Rubin M. Tuder, Kurt R. Stenmark, Brian B. Graham
Abstract
The hypothalamic changes that occur after the loss of ovarian estrogen remain poorly characterized. Here, we performed a comprehensive temporal characterization of the mouse hypothalamus following ovariectomy (OVX), combining physiological measurements with bulk RNA-sequencing of the posterior hypothalamus (PH) and preoptic area (POA) at short-term (14 days) and long-term (4 months) post-OVX. Serum LH levels rose progressively and then declined, while core temperature peaked early and subsequently normalized, recapitulating the endocrine and thermoregulatory dynamics of reproductive aging in humans. Transcriptomic analysis revealed time-dependent activation of inflammatory pathways, glial markers, and KNDy neuron-related gene networks, with the most pronounced changes emerging at 4 months post-OVX, particularly in the PH. Immunofluorescence confirmed increased NKB release, declining KNDy neuronal activity, and heightened astrocytic reactivity in the arcuate nucleus after prolonged estrogen withdrawal. To contextualize these findings, we analyzed publicly available human hypothalamic RNA-seq data across chronological age. Age-related transcriptomic patterns in women, including progressive inflammatory signaling, glial activation, and altered KNDy gene expression, showed significant correlation with the OVX mouse model, particularly at the pathway level. These findings establish a temporal framework for hypothalamic molecular changes after estrogen withdrawal, identify conserved neuroinflammatory signatures across species, and provide a preclinical platform for testing interventions targeting menopausal-associated hypothalamic dysfunction.
Authors
Jordana C.B. Bloom, Encarnación Torres, Sidney A. Pereira, Liliana Arvizu-Sanchez, Audrey N. Fontes, Hadine Joffe, David C. Page, Victor M. Navarro
Abstract
The precise mechanisms underlying the pathogenesis of idiopathic inflammatory myopathy (IIM) remain undefined. However, there has been increasing recognition that tissue-resident memory cells (TRMs) play an important role in the pathogenesis of systemic autoimmune disease. In IIM, TRM-associated transcriptional signatures have been reported, but on a very limited basis. By using multimodal single-cell RNA sequencing analysis in our established murine model of histidyl-tRNA synthetase (HRS)-induced myositis, we identified a prominent population of CD4+ TRMs in inflamed skeletal muscle. Muscle CD4+ TRMs exhibited high expression of genes encoding Cd69, Cxcr6, Runx3, and Prdm1, alongside low expression of Klf2, Ccr7, Sell, S1pr1, and Tcf7 — a profile that is generally consistent with previous reports of TRM gene signature and that we validate through comparison to transcriptomic profiles of human muscle tissue. Detailed pathway analysis in our model indicates that muscle CD4+ TRMs contribute to innate immune regulatory pathways enriched for TNF and IFN-γ signaling. Furthermore, analysis of TCR clonotype distribution and CDR3 sequence similarity revealed pronounced clonal expansion of CD4+ TRMs relative to other T-cell subsets — a pattern that remained stable from 2 to 6 weeks post-immunization. Collectively, these results suggest a potential role for CD4+ TRMs in the pathogenesis of autoimmune myositis.
Authors
Decheng Li, Daniel P. Reay, Iago Pinal-Fernandez, Maria Casal-Dominguez, Andrew L. Mammen, Sarah L. Gaffen, Timothy B. Oriss, Dana P. Ascherman
Abstract
Neonatal sepsis is a predominant cause of neonatal mortality and long-term morbidity which severely effects preterm and low birth weight newborns. Antibiotic resistance and long-term developmental issues associated with neonatal sepsis necessitates finding new and improved treatment options. Interleukin-27 (IL-27) has diverse influences on the immune response, is elevated during the neonatal period compared to adulthood, and continues to rise further during infection. Elevated levels of IL-27 early in life predispose the host to impaired control of the pathogen burden and increased mortality. This study explored the therapeutic potential of IL-27p28 antibody administration to improve treatment outcomes during murine neonatal sepsis. Sepsis was induced by subcutaneous inoculation of K1-encapsulated Escherichia coli and the neonatal pups were rescued with IL-27p28 monoclonal antibody. Pups that received prophylactic antibody prior to the infection demonstrated superior bacterial clearance and significant weight gain compared to controls during infection. The combination of subclinical dose of gentamicin and IL-27p28 antibody administered 2h post-infection, significantly improved bacterial clearance, glucose homeostasis, with reduced serum levels of IL-6 and TNF-α, vital organ damage and significantly improved the survival rate of infected pups compared to gentamicin alone. These findings suggest that IL-27p28 antagonization represents a promising therapeutic tool for treatment of neonatal sepsis.
Authors
Madhavi Annamanedi, Jessica M. Povroznik, Samantha Arevalo-Marcano, Cory M. Robinson
Abstract
Renin cells are essential for survival and serve as key regulators of blood pressure and fluid-electrolyte homeostasis. Their function and identity are dependent on signals from their local microenvironment afforded by neighboring cells and nerves. Whether and how renin cells contribute to the development and maintenance of this microenvironment remains unclear. Because renin cells are rare -0.01 % of kidney cells- conventional histological approaches cannot capture their interaction with nerve fibers and surrounding cells within the nephron and its vasculature. Using high-resolution 3D imaging, cell-specific multicolor reporter mice, single-cell RNA-Seq, and conditional gene deletions, we mapped how renin cells assemble within arterioles and communicate with axon fibers to organize the growth and orientation of the kidney arterioles during development and disease. This co-inductive process is mediated by Ngf produced by renin cell precursors and is necessary for renin cell survival and innervation. Interestingly, renin enzymatic insufficiency elevates Ngf and drives arteriolar hypertrophy with aberrant axon sprouting and hyperinnervation. These findings indicate that renin cells regulate kidney neurovascular development revealing them as active organizers of their local neuroregulatory microenvironment in health and disease.
Authors
Manako Yamaguchi, Georgina Gyarmati, Liam McLaughlin, Hiroki Yamaguchi, Jason P. Smith, Lucas Ferreira de Almeida, Daisuke Matsuoka, Alexandre G. Martini, Sara M. Wilmsen, Sijie Hao, Kazuki Tainaka, Silvia Medrano, Sanjay Jain, Janos Peti-Peterdi, Maria Luisa S. Sequeira-Lopez, R. Ariel Gomez
Abstract
Systemic sclerosis (SSc) is a rare autoimmune disease characterized by vasculopathy and fibrosis of the skin and internal organs. Individuals with SSc often suffer from chronic acid reflux and dysphagia due to loss of esophageal motility. However, the pathogenesis of esophageal dysmotility in SSc is poorly understood. To determine whether distinct changes in esophageal epithelial cells contribute to esophageal involvement in SSc, we investigated the stratified squamous esophageal epithelium from proximal and distal biopsies using single-cell RNA sequencing (n=306,372 cells) in individuals with SSc compared those with gastroesophageal reflux disease (GERD) and healthy controls. The proportion of epithelial cells in the apical, superficial compartment of the esophageal epithelium was reduced in SSc (9.4% vs 21.6% in HCs). Differential gene expression in SSc was primarily limited to the superficial compartment (3,572 genes vs. 232 in all other compartments, based on pseudobulk analysis), with significant upregulation of extracellular matrix and keratinization genes. These cellular and molecular changes in SSc were highly correlated with those seen in GERD, indicating they were secondary to reflux; however, their magnitudes were more pronounced in the proximal esophagus, suggesting that esophageal dysmotility leads to greater proximal acid exposure, which may contribute to aspiration. SSc-specific gene dysregulation implicated immunoregulatory pathways likely pertinent to pathogenic mechanisms. Ligand-receptor interaction analysis revealed enhanced pro-fibrotic signaling between fibroblasts and epithelial cells in SSc. Cell type localization and SSc-specific changes were confirmed by spatial molecular imaging. By offering a comprehensive view of transcriptional dysregulation at single-cell resolution in human esophageal epithelial cells in SSc compared to GERD and healthy tissue, this work clarifies the state of epithelial cells in SSc-induced esophageal dysfunction.
Authors
Matthew Dapas, Margarette H. Clevenger, Hadijat-Kubura M. Makinde, Tyler Therron, Dustin A. Carlson, Mary Carns, Kathleen Aren, Cenfu Wei, Kainat Mian, Lutfiyya N. Muhammad, Carrie L. Richardson, Parambir S Dulai, Monique Hinchcliff, John E. Pandolfino, Harris R. Perlman, Deborah R. Winter, Marie-Pier Tetreault
Microbiome-Derived Metabolites Shape CD4⁺ T-Cell Differentiation and Immune Aging in HIV-1 Infection
Abstract
The role of aromatic gut-derived bacterial metabolites (GDBMs) in shaping immune cell metabolism and function remains poorly explored. Using ex vivo metabolomic profiling of paired plasma and CD4⁺ T-cells from people living with HIV-1 (PLWH), we identified a network of aromatic GDBMs whose cell-associated abundance, rather than systemic levels, was linked to broad alterations in CD4⁺ T-cell metabolic and functional states. Among these, p-cresol sulfate (PCS) emerged as a mechanistic prototype. Ex vivo flow cytometry and single-cell RNA sequencing of CD4⁺ T-cells stratified by cell-associated PCS levels revealed dose-dependent enrichment of transcriptional programs associated with impaired differentiation, regulatory-like identity, and cellular senescence. In vitro transcriptomic and proteomic analyses of PCS-exposed CD4⁺ T cells demonstrated induction of cell-cycle arrest, mitochondrial dysfunction, and senescence-associated programs, including upregulation of p16 and p21. Integration of these immunometabolic findings with HIV-1 reservoir measurements revealed that CD4⁺ T-cell states defined by cell-associated GDBMs track with intact proviral DNA levels in vivo. These findings define a microbiome-derived axis that reshapes CD4⁺ T-cell metabolism and fate, promotes immune aging in PLWH, and may foster immunometabolic states linked to long-term HIV-1 reservoir persistence.
Authors
Amanda Cabral da Silva, Luke Flantzer, Jaclyn Weinberg, Shuya Kyu, Lisa P. Daley-Bauer, Anyce Godoy, Ana Carolina Santana, Aarthi Talla, Amber Rittgers, Sarah Welbourn, David E. Gordon, Jeffrey A. Tomalka, Vincent C. Marconi, Dean P. Jones, Souheil-Antoine Younes
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