Research
Abstract
Post-acute sequelae of SARS-CoV-2 (PASC) occurs in subsets of individuals, including those with pre-existing lung disease. To investigate PASC pathogenesis and therapeutics in a chronic bronchitis mouse model (Scnn1b-Tg), Scnn1b-Tg and WT mice were inoculated with a mouse adapted SARS-CoV-2 virus (SARS-CoV-2MA10) and followed for 60 days. Viral titer, histology, immunohistochemistry (IHC), single-cell RNA sequencing, RNA in situ hybridization, and spatial transcriptomic profiling characterized disease pathologies. Scnn1b-Tg mice inoculated with SARS-CoV-2MA10 exhibited lower viral titers and less weight loss than WT mice. Airway epithelia of Scnn1b-Tg mice were less infected than epithelia of WT mice, reflecting increased airway mucus and enhanced epithelial antiviral activities in Scnn1b-Tg mice. However, Scnn1b-Tg mice subsequently exhibited heterogeneous airway and parenchymal disease with elevated Il33 expression characteristic of human eosinophilic pneumonia. Cohorts of infected mice were administered a monoclonal antibody targeting the IL-33 receptor (ST2) or enteral prednisone. Administration of an anti-ST2 monoclonal antibody mitigated development of eosinophilic pneumonia while enteral prednisone suppressed IL33 expression and disease. The eosinophilic pneumonia in Scnn1b-Tg mice after SARS-CoV-2MA10 infection mimics reports of eosinophilic pneumonia in humans post-SARS-CoV-2, suggesting targeting of IL-33 may be beneficial in treating post-viral eosinophilic pneumonia in humans.
Authors
Padraig E. Hawkins, Sarah R. Leist, Hong Dang, Minako Saito, Lisa C. Morton, Rodney C. Gilmore, Stephen A. Schworer, Ella F. Burns, Jason R. Rock, Robert S. Hagan, James J. Pestka, Alexandra Schäfer, Kenichi Okuda, Lauren K. Heine, Jack R. Harkema, Wanda K. O'Neal, Alessandra Livraghi-Butrico, Raymond J. Pickles, Ralph S. Baric, Richard C. Boucher
Abstract
Plasma membrane repair is critical for tissue integrity, especially for elongated contractile muscle cells. Genetically-mediated defects in plasma membrane resealing produce persistent leak, leading to a disordered extracellular matrix. Loss of the membrane repair protein dysferlin slows sarcolemmal resealing and promotes excess leak. Annexin A6 is also implicated in sarcolemmal repair, forming repair caps at the site of membrane disruption. On its own, deletion of the gene for annexin A6, Anxa6, had little effect on muscle health. In contrast, combined loss of dysferlin and annexin A6 (DysfA6) generated muscle fibers with profoundly defective membrane leak. Strikingly, Anxa6 deletion in the context of loss of dystrophin (mdxA6) did not exacerbate muscle defects. The persistent membrane leak in DysfA6 muscle resulted in marked macrophage infiltration with disordered macrophage polarization. Injured muscle fibers were targets of macrophage efferocytosis. Loss of Anxa6 was associated with increased expression of annexins A1 and A2, both of which were heavily deposited into the extracellular matrix. In vitro, macrophages exposed to annexins A1 and A2 increased Csf1 expression, consistent with a model where excess leak results in annexins A1 and A2 in the extracellular matrix, where this protein composition influences macrophage proliferation and efferocytosis.
Authors
GaHyun Lee, Alexander J. Fitt, Ashlee M. Long, Lauren A. Vaught, Dorothy DeBiasse, Alexander R. Keeble, Jason M. Kwon, Patrick G.T. Page, Marie-Therese Daher, Michele Hadhazy, Alexander B. Willis, David Ceja Galindo, Maxwell C. McCabe, Connor Lantz, Kirk C. Hansen, Rachelle H. Crosbie, Edward B. Thorp, Alexis R. Demonbreun, Elizabeth M. McNally
Abstract
Extracellular vesicles (EVs)-mediated inter-organ communication represents a promising frontier in transplant immunology; however, its role in cardiac allograft rejection remains poorly characterized. We performed proteomic profiling of plasma-derived EVs in a rat heterotopic heart transplantation model and identified a distinct liver-predominant protein signature during acute rejection, with Antithrombin III (ATIII) emerging as a top candidate. Functional validation revealed that pharmacological EV inhibition intensified systemic and intragraft inflammation, whereas adeno-associated virus (AAV)-mediated silencing of hepatic ATIII directly accelerated allograft rejection. Conversely, AAV-mediated hepatocyte-specific ATIII overexpression attenuated rejection pathology, reduced immune cell recruitment, and markedly prolonged median graft survival. This protective effect was achieved without evidence of coagulopathic complications, indicating an immunomodulatory mechanism beyond ATIII’s canonical anticoagulant function. Mechanistically, ATIII overexpression was associated with upregulation of heme oxygenase-1 (HO-1) in the liver and suppression of proinflammatory cytokine expression in the graft. These findings highlight hepatocyte-derived EVs as important mediators of a liver-heart signaling axis in transplant rejection, and further implicate the protein ATIII as a contributor to this axis. Our study reveals a therapeutically targetable liver-heart signaling axis in transplant rejection, whereby enhancing liver-derived ATIII or its downstream pathways (such as HO-1) could attenuate acute cardiac allograft rejection.
Authors
Shiyu Dai, Wei Zhou, Fangyu Chen, Huanyu Zhang, Zhenchun Ji, Xuejing Zong, Wanruo Zhang, Jie Hu, Shumin Jiang, Fei Wang, Zhenya Shen
Abstract
Vaccine development for tuberculosis is a global priority. Our studies using Collaborative Cross (CC) mice show that genetic diversity influences the efficacy of BCG, the most widely used TB vaccine. BCG vaccination of CC042 mice reduced their lung bacillary burden and increased their survival following low-dose aerosol Mycobacterium tuberculosis infection (MTBI), despite impaired T cell trafficking due to a defective Itgal gene. BCG vaccination conferred early bacillary control which appeared to be independent of B cell or T cell recall responses following MTBI. In contrast, long term survival of BCG-vaccinated CC042 mice after MTBI required T cells. Thus, CC042 mice reveal two phases of immunity induced by BCG: an early phase mediated by innate immunity or innate-like T cells and a later phase mediated by conventional memory CD4 and/or CD8 T cells. Although measurement of vaccine-induced protection 30 days after MTBI is a standard measure of vaccine efficacy in the TB model, this time point might be independent of memory T cells in CC042 mice. Our results suggest that vaccine-elicited innate/innate-like responses could have a larger role in protection than previously considered. The concordance between lung CFU, pathology, and survival make CC042 mice useful for mechanistic studies on vaccine-induced immunity.
Authors
Abiola F. Ogunsola, Rocky Lai, Kelly Cavallo, Anthony V. Tran, Gillian L. Beamer, Samuel M. Behar
Abstract
Undifferentiated pleomorphic sarcoma (UPS) is one of the most common adult soft tissue sarcomas (STS), yet therapeutic progress remains limited due to the absence of recurrent oncogenic driver mutations. To identify tumor suppressors contributing to UPS pathogenesis, we performed a customized in vivo CRISPR/Cas9 screen in mice. This approach identified BRCA1-associated protein 1 (BAP1) as a potent tumor suppressor in STS. Integrative analyses using RNA sequencing, multiplex immunohistochemistry, and flow cytometry revealed that Bap1-deficient sarcomas exhibited a markedly immunosuppressive tumor microenvironment. Consistent with these findings, BAP1 protein expression was reduced in human UPS, whereas polo-like kinase 1 (PLK1) expression was elevated. Functional studies demonstrated that PLK1 was required for the growth and survival of Bap1-deficient sarcomas. Pharmacologic inhibition of PLK1 with volasertib significantly suppressed tumor growth in both syngeneic and autochthonous mouse models. Moreover, combining PLK1 inhibition with anti-PD-1 therapy enhanced tumor control and improved survival compared with either treatment alone. Together, these results identify PLK1 as a potential therapeutic vulnerability in BAP1-deficient sarcomas and support further evaluation of combined PLK1 inhibition and immune checkpoint blockade as a treatment strategy for a subset of STS.
Authors
Jianguo Huang, Xingliang Liu, Warren Floyd, William Haugh, Zhaoyu Sun, Melissa J. Kasiewicz, Yaping Wu, Brian Piening, John T. Welle, Wesley K. Rosales, Venkatesh Rajamanickam, So Young Kim, Eric S. Xu, Lixia Luo, Yan Ma, Rutulkumar Patel, Ziqiang Zhang, Brady Bernard, William L. Redmond, Walter J. Urba, R. Bryan Bell, David G. Kirsch
Abstract
Given the central role of peroxisomes in lipid metabolism and redox homeostasis, we hypothesized that peroxisomal activity is critical for sustaining β cell function and identity. Pex5 deletion models were employed to investigate loss of peroxisomal function on glucose-stimulated insulin secretion (GSIS), oxidative stress, and β cell maturity markers. Peroxisome deficiency in male mice resulted in elevated GSIS. Glucose intolerance developed despite increased insulin secretion. Ion mobility mass spectrometry revealed oxidation of insulin proteins, and a truncated insulin 2-derived peptide, in islets from mice with a tissue-specific deficiency in peroxisomes. Peroxisome loss of function increased multiple markers of oxidative stress, including altered metabolite profiles, lipid peroxidation, and protein carbonylation. These findings reveal that increased secretion of oxidized insulin protein is insufficient to regulate whole-body glucose homeostasis. Peroxisome deficiency also reduced markers of β cell maturity. Based on these outcomes, we identified the peroxisome organelle as a key regulatory component of glucose homeostasis by protecting insulin from oxidative modification and degradation and by supporting maintenance of mature β cells.
Authors
J. Jason Collier, Caroline R. Cothern, Maggie P. Ducote, Thomas M. Martin, Melissa A. Linden, Robert C. Noland, David H. Burk, Samuel D. Dupuy, Michael D. Karlstad, Krisztian Stadler, Sarah S. Hirschbeck, Thanh D. Do, Anastasia Coldren, Marcela Brissova, Teayoun Kim, Kirk M. Habegger, Sujoy Ghosh, Zane A. Vickery, Qudus Sarumi, Shawn R. Campagna, Susan J. Burke
Abstract
The distal nephron segments play a critical role in maintaining electrolyte balance, yet the mechanisms that preserve epithelial identity and segmental organization within this region remain poorly defined. Yes-associated protein (YAP), a key effector of Hippo signaling, is essential for kidney development, but its function in distal nephron epithelia is unknown. Using a genetic gain-of-function approach to activate YAP selectively in distal nephron segments, we found that sustained YAP activity profoundly disrupts epithelial organization and nephron patterning. Lineage tracing revealed that both distal convoluted tubule and connecting tubule cells originate from Slc12a3-expressing cells, and YAP activation in these segments led to increased proliferation, displacement of lineage-labeled cells beyond expected segment boundaries, and loss of segment-specific gene expression. These changes were accompanied by defects in apicobasal polarity and junctional integrity, consistent with epithelial plasticity. Unexpectedly, YAP activation in distal nephron segments also suppressed proximal tubule gene expression, indicating non-cell-autonomous effects on nephron differentiation. Together, these findings identify YAP as a critical regulator of epithelial identity in the distal nephron segments and reveal a previously unrecognized role for Hippo signaling in coordinating intersegmental organization during kidney development.
Authors
Zeinab Dehghani-Ghobadi, Eunah Chung, Mohammed Sayed, Christopher Ahn, Hyojin Alex Choi, Annissa Aamoum, Benjamin R. Thomson, Yueh-Chiang Hu, Hee-Woong Lim, Joo-Seop Park
Abstract
It is necessary for naïve CD8 T cells to be actively maintained in a quiescent metabolic state in order to respond robustly to infection while avoiding inappropriate activation during homeostasis. With age this quiescent state is lost and the CD8 T cell response to infection decreases. The factors regulating metabolic quiescence of CD8 T cells and how this regulation is lost during aging are not completely understood. Herein, we identify the transcription factor AFF3 as a regulator of metabolic quiescence in naïve CD8 T cells. While naïve AFF3 deficient CD8 T cells are more metabolically active prior to infection, they have reduced accumulation in response to viral infection, and this is correlated with a poor capacity to engage glycolysis. During aging in both murine and human CD8 T cells, AFF3 expression is decreased. In mice, this is associated with a loss of metabolic quiescence and reduced capacity to accumulate following infection. Our data highlight the role of metabolic regulation in CD8 T cell quiescence and identifies a transcription factor that may be a target to reinvigorate CD8 T cell responses during aging.
Authors
Molly E. Lumnitzer, Stefanie F. Valbon, Stephanie A. Condotta, Allison E. Norlander, Sheng Liu, Jun Wan, Martin J. Richer
Abstract
Spinal cord injury (SCI) leads to severe neurological and functional impairments, yet reliable biomarkers for assessing injury severity and predicting recovery remain limited. Cerebrospinal fluid (CSF) is in direct contact with the central nervous system and provides a valuable source for detecting molecular changes after SCI. Although exosomal microRNAs and proteins are increasingly recognized as mediators of intercellular communication, the role of human CSF exosomes in SCI has not been systematically investigated. To identify exosome-based biomarkers and potential therapeutic targets, we analyzed CSF and serum exosomes from patients with acute SCI using RNA sequencing and proteomic profiling. Weighted Gene Co-expression Network Analysis (WGCNA) identified six gene modules significantly associated with injury severity and neurological recovery at three months. Proteomic analysis revealed a five-protein panel that distinguished complete from incomplete SCI and a four-protein panel that predicted neurological improvement. Additionally, fifteen CSF-specific and nine serum-specific exosomal miRNAs were identified independent of injury severity. Among ten tested miRNAs associated with neurological recovery, seven regulated astrocyte proliferation, and six promoted neurite extension and synapse formation. Overall, this study provides a comprehensive characterization of CSF exosomal miRNAs and proteins in human SCI and identifies molecular signatures associated with injury severity and recovery.
Authors
Dallas L. Sheinberg, Haichao Wei, Joseph S. Withrow, Farshad Homayouni Moghadam, Chia-Chen Lu, Jyotirmoy Rakshit, Jennifer Zaragoza, John R. Williams, Wen Li, Jacques J. Morcos, Jia Qian Wu
Abstract
Polymorphonuclear neutrophils (PMNs) serve as frontline defenders against injury and infection, eliminating pathogens and initiating mucosal tissue repair. However, excessive PMN transepithelial migration (TEpM) contributes to chronic mucosal inflammatory disorders, including inflammatory bowel disease. PMN pro-inflammatory and pro-repair functions are regulated by incompletely defined signaling cascades involving kinases and phosphatases. Here, we determined how the protein tyrosine phosphatase CD45/PTPRC regulates PMN trafficking and effector functions in the gut. Pharmacologic inhibition of CD45 significantly reduced PMN colonic TEpM in vitro and in vivo and decreased intestinal PMN trafficking was observed in transgenic mice with PMN-specific deletion of CD45 (MRP8-Cre;Cd45fl/fl). Beyond limiting TEpM, CD45 depletion impaired key antimicrobial functions, including degranulation and phagocytosis, indicating broader effects on PMN effector activity. Importantly, recovery from dextran sodium sulfate (DSS)–induced colitis and biopsy-induced colonic wounding was delayed in MRP8-Cre;Cd45fl/fl mice, linking altered PMN function to defective mucosal healing. Mechanistically, CD45 depletion reduced surface expression of the β2 integrin CD11b/CD18 and inactivated the Src family kinase member Lyn. Together, data highlight an important CD45–CD11b–Lyn signaling axis that regulates PMN trafficking and effector functions in the intestine and identify CD45 as a promising target for modulating PMN function to promote mucosal tissue repair.
Authors
Jael Miranda, Dylan J. Fink, Zachary S. Wilson, Roland Hilgarth, Asma Nusrat, Charles A. Parkos, Jennifer C. Brazil
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