Research
Abstract
Obstructive nephropathy is a significant and preventable contributor to chronic kidney disease, yet no disease-modifying anti-fibrotic agents are currently available.We hypothesized that interferon regulatory factor 5 (IRF5) functions as a macrophage transcriptional regulator that directly transactivates matrix metalloproteinase 9 (MMP9) to initiate early extracellular matrix (ECM) remodeling. Analysis of 30 human obstructive nephropathy biopsy specimens demonstrated that IRF5+CD68+ macrophage density increased progressively with fibrosis severity and correlated significantly with α-smooth muscle actin (α-SMA) positive areas. In the murine unilateral ureteral obstruction (UUO) model, both global and myeloid-specific Irf5 deletion significantly attenuated collagen deposition, immune cell infiltration, and fibrotic gene expression compared with wild-type controls. Cleavage under targets and tagmentation (CUT&Tag) analysis demonstrated that IRF5 directly binds the Mmp9 enhancer region and increases chromatin accessibility. Consequently, myeloid-specific Irf5 knockout significantly reduced Mmp9 mRNA and MMP9 protein levels. Pharmacological inhibition using the IRF5 inhibitor N5-1 mitigated established fibrosis, down-regulated α-SMA and MMP9 expression, and reduced CD68+ macrophage infiltration. These findings identify the IRF5-MMP9 axis as a therapeutically targetable pathway driving macrophage-mediated ECM expansion and provide pre-clinical evidence supporting IRF5 inhibition as a potential treatment strategy for patients with obstructive nephropathy.
Authors
Jia Wei, Gengyu Du, Zixia Li, Min Yang, Ting Chen, Zihao Xu, Zhen Yuan, Yidan Zheng, Xiang Yan
Abstract
Metastatic prostate cancer is a clinically and molecularly heterogeneous disease. Under the selective pressure of androgen receptor (AR)–directed therapies, resistant phenotypes frequently emerge, posing significant diagnostic and therapeutic challenges. Neuroendocrine prostate cancer (NEPC) is a clinically important phenotype characterized by lineage plasticity, neuroendocrine features, visceral metastases and poor prognosis. Accurately diagnosing NEPC remains difficult due to its histologic and molecular complexity but has high clinical relevance. In this study, we developed a deep learning model that leverages interpretable cellular features to improve feature extraction from H&E-stained tissue sections (NEURAL-PC). By incorporating a multiple instance learning (MIL) framework, NEURAL-PC enables robust NEPC classification solely from H&E tumor images, achieving an area under the receiver operating characteristic curve (AUROC) of 0.921 in independent external validation. In addition to its diagnostic utility, NEURAL-PC provides prognostic information that enables further subclassification of advanced prostate cancer across diverse datasets supporting its strong prognostic value and generalizability. Broadly, our work highlights a hybrid approach that integrates features across different domains, offering a promising strategy for developing reliable deep learning tools in pathology. Built on this framework, NEURAL-PC represents an extensively validated diagnostic and prognostic model for advanced prostate cancer.
Authors
Zhijun Chen, Erolcan Sayar, Daniela Guevara, Helen Richards, Haoyue Zhang, Radhika A. Patel, Agnes C. Gawne, Lucas J. Liu, Ilsa Coleman, Ruth Dumpit, Colm Morrissey, Michael T. Schweizer, Ruben Raychaudhuri, Laura S. Graham, Evan Y. Yu, Heather H. Cheng, Chien-Kuang C. Ding, Yuzhuo Wang, Peter Choyke, Baris Turkbey, Chantal Chanel-Vos, Christina Fedorov, John R. Otilano III, Troy Kane, Jyothi Manohar, Michael Sigouros, Jones T. Nauseef, Ana Molina, David Nanus, Scott T. Tagawa, Juan Miguel Mosquera, Himisha P. Beltran, Ruth Etzioni, Peter S. Nelson, Rama Soundararajan, Ana M. Aparicio, Cora N. Sternberg, Michael C. Haffner, Stephanie A. Harmon
Abstract
Natural killer (NK) cells undergo stepwise differentiation from multipotent progenitors within secondary lymphoid tissues. Despite the central importance of the tissue microenvironment in their development, little is known about cell-cell interactions that regulate human NK cell trafficking and maturation. Here, we identify the chemokine receptor CXCR4 and its ligand CXCL12 as regulators of stromal-NK cell interactions required for NK cell maturation. We demonstrate that CXCR4 is expressed throughout human NK cell development in peripheral blood and tonsil, and CXCL12 is enriched in stromal niches containing developing NK cells. Pharmacologic blockade or genetic disruption of CXCR4 resulted in diminished adhesion to integrin ligands, and high-resolution imaging demonstrated crosstalk between CXCR4 and integrins, providing a mechanistic basis for chemokine-dependent modulation of adhesion. Further, CXCR4 blockade resulted in altered contact-dependent motility on stromal cells and integrin ligands, with decreased stable stromal engagement and increased cell speed. Consistent with a requirement for these interactions, treatment with the CXCR4 antagonist plerixafor (AMD3100) impaired NK cell generation from CD34+ precursors. Analysis of NK cells from WHIM syndrome patients with CXCR4 gain-of-function mutations treated with plerixafor revealed similar defects in migration and adhesion, supporting the in-vivo relevance of CXCR4-dependent regulation of NK cell adhesion and motility.
Authors
Shira E. Eisman, Francesca E. Grossberg, Batya S. Koenigsberg, David H. McDermott, Frédérique van den Haak, Luis A. Pedroza, Everardo Hegewisch-Solloa, Philip M. Murphy, Emily M. Mace
Abstract
Autoimmune Addison’s disease (AD) is a rare but life-threatening disorder caused by immune-mediated destruction of the adrenal cortex, and progress in therapy has been limited by insufficient mechanistic insight. Here, we establish a model of Experimental Autoimmune Adrenalitis (EAA) that recapitulates key features of AD and reveals sex-dependent differences in disease manifestation within the model. Immunization with peptides derived from the adrenal self-antigen CYP11A1 induces corticosterone insufficiency. We show that autoimmune adrenalitis is driven by IFNG produced by self-reactive CD4+ T cells, promoting granulomatous inflammation in the adrenal cortex. Together, these findings identify IFNG as a central effector of autoimmune adrenalitis and suggest that targeting the IFNG pathway may represent a potential therapeutic strategy for AD.
Authors
Arina Andreyeva, Juraj Michalik, Veronika Niederlova, Veronika Cimermanova, Ales Drobek, Radislav Sedlacek, Jan Prochazka, Juraj Labaj, Olha Fedosieieva, Waldemar Kanczkowski, Peter Draber, André Sulen, Ondrej Stepanek, Aleš Neuwirth
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
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