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Abstract
ADAMTS9 mutations cause the ciliopathies nephronophthisis and Joubert syndrome. Here we demonstrated that deletion of ADAMTS9 in the proximal nephron led to polycystic kidney development in mice. In males, Adamts9 deletion caused kidneys to become highly cystic while remaining small without undergoing enlargement. In contrast, female mice developed cystic kidneys at a slower rate. ADAMTS9 deletion disrupted ciliogenesis through the loss of cleavage of the ciliary transition zone (TZ) protein TMEM67, which led to loss of the MKS/B9 module – a key component of the ciliary gate. Functional analysis of all eight ciliopathy patient variants of ADAMTS9 identified to date showed TMEM67 C-terminus failed to localize to the TZ, thus disrupting a key regulatory mechanism in patient renal ciliogenesis. Modeling ADAMTS9-mediated TMEM67 cleavage utilizing TMEM67-cleavage deficient mice revealed loss of TZ formation, but not elevated canonical Wnt signaling as the underlying mechanism driving cystogenesis. Adamts9 deletion led to comparatively intense interstitial collagen deposition, which likely restricted kidney enlargement and resulted in the characteristically small kidney phenotype seen in nephronophthisis. By comparative analysis of four interconnected polycystic kidney models, in addition to Pkd1 and Pkd2 deleted kidneys, we identified differential collagen homeostasis as a principle factor determining cystic kidney size and type.
Authors
Sydney Fischer, Karyn L. Robert, Manu Ahmed, Griffin I. Kane, Matthew A. Kavanaugh, Wei Wang, Pamela V. Tran, Prabhani U. Atukorale, Sumeda Nandadasa
Abstract
Early antiretroviral therapy (ART) limits viral reservoir establishment in infants but also constrains viral-specific immunity required to clear reactivated cells. In the early ART context, we evaluated whether combining adeno-associated virus serotype 9 (AAV9)–vectored eCD4-IgG1 with pharmacologic latency reversal using the second mitochondria-derived activator of caspases (SMAC) mimetic AZD5582 could promote reservoir reduction and control in simian immunodeficiency virus (SIV)–infected infant macaques. AAV9 delivery achieved sustained eCD4-IgG1 expression and AZD5582 induced on-ART viremia, but the combination did not reduce intact SIV proviral DNA relative to controls. Partial post-intervention control of viremia during Analytical Treatment Interruption (ATI) occurred in 2/6 infants receiving AAV9-eCD4-IgG1 + AZD5582 with restricted reservoir expansion during recrudescence at week 13. Intact reservoir size during ATI correlated inversely with on-ART viremia during AZD5582 treatment. Controllers did not show increased eCD4-IgG1 expression nor increased on-ART viremia during AZD5582 treatment, but harbored less pre-ART intact SIV DNA in PBMCs than non-controllers. Controllers also exhibited higher frequencies and greater polyfunctionality of virus-specific CD8+ T-cells prior to ATI. These findings implicate immune control of reservoir size and post-ART viral dynamics in early-treated infants and suggest that AAV9-eCD4-IgG1 + AZD5582 increases the likelihood of post-ART viral control.
Authors
Jairo A. Fonseca, Alexis C. King, Kaleaha S. Davis, Daniel O'Hagan, Adrian Khoei, Lucas Alves Britto Da Costa, Camryn Cockerham, Mackenzie Cottrell, Stephanie Ehnert, Jennifer S. Wood, Michael D. Alpert, Matthew R. Gardner, Jeffrey D. Lifson, Maud Mavigner, Mauricio A. Martins, Ann Chahroudi
Abstract
Congenital and early-life Zika virus (ZIKV) infection can result in neurologic deficits. Precise mechanisms of injury, especially in the more subtle presentation of postnatal infection, are not fully elucidated. Here, we defined the effects of ZIKV on the developing brain using single cell transcriptomics, histopathology and design-based stereology, diffusion MRI, and neurobehavioral assessments in infant rhesus macaques. ZIKV upregulated interferon-stimulated genes in activated microglia and cell death pathways in neurons and downregulated metabolism and differentiation genes in mature oligodendrocytes. Abnormal micro-organization of the corpus collosum and limbic white matter tracts was seen on diffusion weighted imaging. A curated gene set associated with autism spectrum disorder risk was negatively enriched in inhibitory and excitatory neurons from ZIKV-infected infants, with increased emotional reactivity already evident two weeks following infection. From single cells to organism-level behaviors, these results define the pathways and processes disrupted by early-life ZIKV infection.
Authors
Venkata-Viswanadh Edara, Sienna Freeman, Maureen Sampson, Kathryn M. Moore, Rebecca Richardson, Nils Schoof, Divine Burgess, Michelle J. Lee, Gregory K. Tharp, Roza Vlasova, Shane Taylor, Ariana Hodjatzadeh, Yihana P. Melendez-Alejandro, Samia M. Abdallah, Esther W. Ndungu, Ava K. Mascarenhas, Chao-Hsiung Hsu, Tsang-Wei Tu, Martin Styner, Mehul S. Suthar, Mar M. Sanchez, Steven E. Bosinger, Mark W. Burke, Steven A. Sloan, Jessica Raper, Ann Chahroudi
Abstract
Ubiquitination is an important post-translational modification associated with essential cellular processes and implicated in regulation of immunity. Here we show that deletion of the E3 ubiquitin ligase Hectd3, germline or in hematopoietic compartment, including in CD11c+ cells, causes a more severe DSS-induced colitis and increased production of proinflammatory cytokines. Hectd3 mRNA levels were found reduced in colonic tissues of patients with ulcerative colitis (UC), which highlights a potential role for Hectd3 in regulating inflammatory responses in UC. We identified Myd88, a central adaptor in the TLR/IL1R signaling and inflammatory response, as a target for Hectd3 ubiquitination. We demonstrate that Hectd3 directly ubiquitinates Myd88 through K27-linked Poly-Ub chains in a nondegradative manner. Hectd3 KO GM-CSF-bone marrow derived cells treated with the TLR4 ligand lipopolysaccharide (LPS) produced more proinflammatory cytokines, show elevated phosphorylation of NF-κB and IRAK4, as well as elevated association of Myd88 with IRAK4, demonstrating that Hectd3 controls Myd88-IRAK4-NF-κB axis. Inhibition of Myd88 activity rescued colitis severity in the Hectd3 KO mice, including the elevated proinflammatory cytokine production. Thus, our results establish Myd88 as a new target for Hectd3 non-degradative polyubiquitination and restriction of immune colonic inflammation.
Authors
Shamima Islam, Shahnewaj Mannan, Ashley Zuniga, Upasana Parthasarathy, Valeriu B. Cismasiu, Leonardo Silvane, Sayan Chakraborty, Divya Priyanka Talada, Raghwendra Pratap Singh, Tomas Zelenka, Amreen Naveen, Sephra Chyanne Vickers, Michael G.M. Grant, Jonathan J. Cho, Theodore Drashansky, Alexander J. Kwiatkowski, Xintong Liu, Ross Tomaino, Olga A. Guryanova, Mariola J. Ferraro, Sang Yong Kim, Christian Jobin, Benjamin G. Keselowsky, Hongmin Li, Paulo C. Rodriguez, Martina Molgora, Amer A. Beg, Timothy I. Shaw, Zheng Ruan, Lixin Wan, Dorina Avram
Abstract
Right ventricular pressure overload (RVPO) is a critical pathophysiological feature of numerous pediatric cardiovascular diseases. Transverse tubules (T-tubules) form the foundation for efficient excitation-contraction coupling in mature cardiomyocytes. We hypothesized that RVPO impairs T-tubule maturation through the regulatory protein BIN1 (Bridging Integrator 1). In right ventricular samples from children with tetralogy of Fallot, characterized by RVPO, and in a neonatal rat RVPO model induced by pulmonary artery banding (PAB), T-tubule maturation was disrupted. RNA sequencing revealed significant downregulation of T-tubule–associated genes, with Bin1 among the most suppressed. Bin1 overexpression restored T-tubule maturation in PAB rats. Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) showed reduced chromatin accessibility at Bin1 loci; motif analysis identified Mef2d (myocyte enhancer factor 2D) as the top enriched transcription factor. Mef2d knockdown rescued Bin1 expression and T-tubule maturation, and mutation of the Mef2d binding sites within the Bin1 promoter abolished the inhibitory effect of Mef2d on Bin1 promoter activity. This study delineates a phenomenon and a mechanism of cardiomyocyte maturation under pathological stress. The findings not only advance our understanding of this most pivotal event in postnatal cardiac development but also unveil a potential therapeutic direction for pediatric cardiovascular diseases associated with RVPO.
Authors
Yuqing Hu, Yiting Xue, Xudong Chen, Linghui Kong, Debao Li, Zheng Wang, Sixie Zheng, Siqi She, Hao Li, Sijuan Sun, Hao Chen, Lijun Chen, Peisen Ruan, Kai Wang, Lincai Ye
Abstract
Sex-specific mechanisms in chronic obstructive pulmonary disease (COPD) remain poorly defined. Sphingolipids are bioactive mediators that regulate airway epithelial integrity, inflammation, and bronchial smooth muscle tone, and their dysregulation contributes to airway hyperresponsiveness. We tested whether sphingolipid metabolism is regulated by sex in COPD. Lung tissue and paired plasma from female and male never-smoking controls (NSC), ever-smoking controls (SC), and COPD subjects were analyzed for sphingolipid enzyme expression and metabolite levels. Bronchial rings from mice and human bronchial smooth muscle cells (HBSMCs) were exposed to estradiol or cigarette smoke extract (CSE) ± ceramidase or sphingosine kinase inhibitors. Expression of sphingolipid regulators ASAH1 and ORMDL3 was increased in COPD airways, significantly higher in women, and correlated with airway smooth muscle remodeling. Plasma from females with COPD showed increased sphingosine-1-phosphate (S1P), whereas males exhibited higher ceramide levels. Female mice displayed greater CSE-induced bronchial reactivity, attenuated by sphingosine kinase inhibition. In male-donor HBSMCs, estradiol increased ASAH1, ORMDL3, S1P, and smooth muscle protein expression and enhanced contractility. CSE further augmented molecular responses in estradiol-treated cells but reduced collagen-gel contraction, whereas ceramidase inhibition attenuated estradiol-dependent effects. These findings suggest that estrogen-dependent alterations in sphingolipid metabolism contribute to airway hyperreactivity and remodeling in females with COPD.
Authors
Elisabetta Granato, Xiaoyun Wang, Yun Zhang, Joselyn Rojas-Quintero, Ida Cerqua, Scott A. Ochsner, Jeff Thomas Kue, Luca Cecchetto, Maor Sauler, Farrah Kheradmand, Joshua Malo, Fiorentina Roviezzo, Irina Petrache, Francesca Polverino
Abstract
Type 2 innate lymphoid cells (ILC2) are a population of lineage-negative cells in the lung, gastrointestinal tract, and skin which have emerged as a significant component of type 2 allergic inflammation. The regulation of cyclooxygenase-2 (COX-2) metabolites is critical to the pathophysiology of many inflammatory disorders, including allergic asthma. While COX-2 regulates Th9 and Th17 cell differentiation and function during allergic lung inflammation, it remains unknown whether COX-2 also regulates ILC2 cell differentiation and function under similar conditions. To address this question, we examined lung ILC2 cells from COX-2+/+ and COX-2-/- mice after ovalbumin (OVA)- or Alternaria-induced allergic lung inflammation. ILC2 cells were significantly increased in COX-2-/- lungs compared with COX-2+/+ lungs after OVA exposure in vivo. The increase in ILC2 cells was accompanied by an increase in expression of the cytokines IL-5 and IL-13, and the transcription factor GATA3. Both genetic disruption and selective inhibition of COX-2 significantly increased ILC2 cell differentiation from isolated common lymphoid progenitor cells (CLP) in vitro. Furthermore, COX-2-derived PGE2 acting via EP2 receptors significantly reduced IL-33 and TSLP expression, and attenuated ILC2 cell differentiation in vitro and in vivo. Thus, during allergic lung inflammation, COX-2-derived PGE2 signals through the EP2 receptor to negatively regulate lung ILC2 cell differentiation and function.
Authors
Hong Li, Matthew L. Edin, Daniel Menendez, J. Alyce Bradbury, Joan P. Graves, Artiom Gruzdev, Gregory S. Whitehead, Maria I Sifre, Laura M. DeGraff, Darryl C. Zeldin
Abstract
Sterile tissue injury triggers a rapid neutrophil response that can be either pathogenic or protective, reflecting substantial functional heterogeneity of neutrophils; however, the neutrophil subsets underlying these divergent functions remain poorly defined. Here, using a well-established sterile corneal injury model, we delineate the time-dependent functional and transcriptional heterogeneity of neutrophils following sterile injury. Temporal neutrophil depletion revealed that neutrophils recruited at day 1, but not day 7, are essential for suppressing inflammation, promoting epithelial healing, and preserving nerve density. Single-cell RNA sequencing uncovered substantial transcriptional heterogeneity among circulating neutrophils and their rapid reprogramming within 24 hours after injury. Specifically, Il1r2 was highly enriched in these early injury-responsive neutrophils. Functional validation demonstrated that adoptive transfer of IL-1R2+ neutrophils markedly attenuated inflammation and accelerated epithelial and nerve repair, restoring tissue integrity, whereas IL-1R2- neutrophils exacerbated inflammatory responses. Together, these findings identify IL-1R2+ neutrophils as an early protective neutrophil subset expanded by sterile injury that restrains excessive inflammation and preserves tissue homeostasis, providing mechanistic insight into injury-induced neutrophil reprogramming and highlighting a potential therapeutic target for enhancing tissue repair.
Authors
Hyun Ju Lee, Jung Hwa Ko, Joo Youn Oh
Abstract
Both elevated plasma cell-free hemoglobin (CFH) and heparanase-driven endothelial glycocalyx shedding are contributors to microvascular dysfunction and organ injury in sepsis. However, the mechanisms governing heparanase activation, and the potential role of CFH in this process, are not understood. Utilizing patient samples, mice with cecal slurry-induced (CS) peritonitis and elevated CFH, and human lung microvascular endothelial cells (HLMVECs), we tested the hypothesis that CFH upregulates heparanase production to drive endothelial glycocalyx degradation. In human sepsis, elevated circulating CFH was associated with higher heparanase and heparan sulfate levels, which in turn correlated with adverse clinical outcomes. CS+CFH-treated mice had increased plasma heparanase, glycocalyx degradation, and pulmonary and systemic inflammation; endothelial heparanase deletion abrogated these effects. Additionally, in both pulmonary endothelial cells isolated from CS+CFH-treated mice and HLMVECs exposed to CFH and TNF, heparanase transcription and active enzyme production were increased. The deleterious effects of CFH were attenuated by acetaminophen, a hemoprotein reductant. In summary, we demonstrate that CFH oxidation stimulates endothelial heparanase expression and activation during sepsis, leading to endothelial glycocalyx degradation, which may disrupt the endothelial barrier and result in organ injury. Our findings highlight the CFH-heparanase axis as a potential therapeutic target for endothelial glycocalyx preservation in sepsis.
Authors
Avery M. Bogart, Anisa S. Haffizulla, Jason Lin, Nathan D. Putz, Han Noo Ri Lee, David M. Aslaner, Samantha K. Gonski, Nancy Wickersham, Kyle Riedmann, Jamie E. Meegan, Kaori Oshima, Ciara M. Shaver, Julie A. Bastarache, Eric P. Schmidt, Lorraine B. Ware
Abstract
TGF-β is a central driver of kidney fibrosis, a common pathological hallmark of chronic kidney disease (CKD). Initiation of TGF-β signaling requires not only its synthesis but also the conversion of latent TGF-β to its bioactive form. However, the mechanisms governing TGF-β activation in the kidney and their contribution to kidney fibrosis remain poorly understood. Glycoprotein A repetitions predominant (GARP) anchors latent TGF-β on the cell surface and facilitates its bioactive release. Here, we show that GARP-mediated TGF‐β activation promotes kidney fibrosis. GARP was upregulated in both human and mouse CKD kidneys, predominantly in fibroblasts, and was induced by TNF in an NF-kB-dependent fashion. In multiple mouse models of kidney fibrosis, either global or fibroblast-specific deletion of GARP significantly reduced fibrosis. Mechanistically, GARP enables sustained production of active TGF-β, thereby amplifying fibroblast stimulation. Deletion of GARP in kidney fibroblasts lowered active TGF-β levels and attenuated fibroblast activation, whereas GARP overexpression enhanced TGF-β signaling. Notably, tamoxifen-induced deletion of GARP after fibrosis onset attenuated kidney fibrosis. Together, our findings identify GARP-mediated release of active TGF‐β as a critical step in sustaining fibroblast activation during kidney fibrosis and highlight GARP as a promising therapeutic target for CKD.
Authors
Yintong Chen, Weiwei Xu, Jieli Yu, Pei Deng, Nianping Liu, Yinyin Li, Hui Zhou, Hong Zhou, Jianchuan Wang, Bo Zhao, Florian Winau, Fan Fan Hou, Yu Hu
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