Research
Abstract
Fibroblast-like synoviocytes (FLSs) are crucial in driving synovial inflammation and joint damage in rheumatoid arthritis (RA). This study explored the functions and underlying mechanisms of GALNT1-mediated O-glycosylation, which is markedly upregulated in RA FLSs, in synovial aggression and subsequent experimental joint damage. Targeted suppression of GALNT1 effectively curtailed migration and invasion in RA FLSs and mitigated arthritis severity in a rat collagen-induced arthritis (CIA) model. Mechanistically, NEK9 was identified as a pivotal substrate and downstream effector of GALNT1, affecting the aggressive phenotype of RA FLSs. In vitro experiments further demonstrated that O-glycosylation of NEK9, mediated by GALNT1, promotes the pathogenic phenotype of RA FLSs by promoting cytoskeleton reorganization and restraining excessive endoplasmic reticulum (ER) stress activation. Our study provides mechanistic insights into the activation of RA FLSs and identifies GALNT1 as a potential therapeutic target for RA.
Authors
Yaoyao Zou, Haobo Lin, Jianling Su, Jieying Wang, Qin Zeng, Tianxiao Feng, Yunxia Lei, Jianda Ma, Hudan Pan, Hanshi Xu, Lie Dai, Yang Li
Abstract
Aortic dissection (AD) is a catastrophic vascular emergency with high mortality, and current pharmacologic interventions to prevent its progression are limited. Vascular smooth muscle cells (VSMCs) undergo a pathological phenotypic switch from a contractile to a synthetic state during AD, compromising aortic wall integrity; however, the underlying metabolic mechanisms remain poorly understood. In this study, we performed integrative transcriptomic analyses and identified glutaminase 1 (GLS1) as a key regulator of VSMC phenotypic switching in AD. GLS1 expression was significantly downregulated in VSMCs from both human AD aortic tissues and mouse models. Functionally, GLS1 deficiency promoted PDGF-BB–induced VSMC dedifferentiation in vitro. Smooth muscle cells specific Gls1 knockout (Gls1SMKO) mice exhibited aggravated AD upon BAPN treatment, whereas VSMCs specific GLS1 overexpression improved the contractile phenotype and reduced AD incidence. Mechanistically, GLS1 downregulation impaired glutamate metabolism, leading to reduced levels of glutathione and α-ketoglutarate. This metabolic disruption promoted reactive oxygen species accumulation and mitochondrial dysfunction, ultimately triggering VSMC phenotypic switching. Furthermore, we found that GLS1 transcription was repressed by retinoic acid receptor-α (RARα). Pharmacologic inhibition of RARα with AR7 restored GLS1 expression, ameliorated VSMC phenotypic switching, and conferred protection against AD. These findings reveal a critical role of GLS1-mediated glutamate metabolism in VSMC phenotypic switching and suggest a promising therapeutic strategy for AD.
Authors
Wei Xie, Chen Ning, Chen Lu, Dongjin Wang, Shuang Zhao, Tianyu Song, Hailong Cao
Abstract
Inflammatory bowel disease (IBD) is frequently accompanied by intestinal fibrosis, with non-response (NR) to long-term anti-tumor necrosis factor α (anti-TNFα) therapy occurring in approximately 23-46% of patients. Integrated analysis of single-cell and bulk RNA sequencing datasets revealed an expansion of IL11⁺ fibroblasts in inflamed intestine and their significant enrichment in non-responders. We further identified IL11⁺ fibroblasts as a central communication hub that engaged in extensive crosstalk with monocytes and may contribute to inflammatory amplification and fibrotic remodeling. Additionally, we employed machine learning approaches including least absolute shrinkage and selection operator (LASSO), support vector machines (SVM), and random forest (RF) to derive an IL11⁺ fibroblast-related gene signature effectively predicting NR to anti-TNFα in validation and test cohorts. IHC further confirmed the overexpression of IL-11 in non-responders. The signature genes we found are not only associated with immune and inflammatory responses but also with fibrosis, indicating a robust association between fibrosis and anti-TNFα treatment failure. In summary, this study highlights the important role of IL11⁺ fibroblasts in orchestrating both inflammation and fibrosis and provides an applicable model for predicting NR to anti-TNFα in IBD, thereby laying the foundation for precision medicine and targeted therapeutic strategies.
Authors
Wangyue Li, Wei Huang, Jiaxin Wang, Yiwen Tu, Qidi Yang, Yao Zhou, Zile Zhang, Haiming Zhuang, Yubei Gu, Duowu Zou, Yao Zhang
Abstract
Inhibiting the mammalian target of rapamycin (mTOR) during acute viral infection generates highly functional memory CD8 T cells. We investigated the effects of inhibiting mTOR by using rapamycin during the effector and contraction phases of the immune response to a DNA prime and Modified Vaccinia Ankara (MVA) boost SIV vaccination in rhesus macaques. Rapamycin administered either during MVA boosts alone (DMR) or during both primes and boosts (DRMR) reduced the contraction of effector CD8 T cells, resulting in higher frequencies of SIV-specific memory CD8 T cells with enhanced quality as indicated by expression of Bcl-2 and CD127. Additionally, rapamycin reduced the frequency of proliferating CCR5+ CD4 T cells in the blood following the MVA boost. Post SIV251 infection, rapamycin-treated macaques demonstrated marked expansion of SIV-specific CD8 T cells (reaching up to 50% in blood and 25% in gut). The heightened expansion of SIV-specific CD8 T cells in the DMR group was associated with markedly lower (2-logs compared to unvaccinated and 1-log compared to DM) peak viral load in the gut and set-point viremia, along with improved survival post infection. Thus, inhibiting the mTOR pathway during MVA boosts of a DNA/MVA vaccine enhances vaccine efficacy by improving memory CD4 and CD8 T cell function.
Authors
Shanmugalakshmi Sadagopal, Kasey Stokdyk, Suefen Kwa, Rahul Basu, Sailaja Gangadhara, Rafi Ahmed, Smita S. Iyer, Koichi Araki, Rama Rao Amara
Abstract
Donnai-Barrow Syndrome (DBS) arises from loss-of-function (LoF) variants in the endocytic receptor LRP2/megalin and is characterized by low molecular weight (LMW) proteinuria and developmental abnormalities. Urinary proteomics of nine DBS patients revealed that the urinary proteome of a DBS patient with the missense variant LRP2 p.C1400R was indistinguishable from that of patients with splice site, nonsense, or frameshift mutations. A CRISPR mouse model of the variant was generated to determine the mechanism of LoF and proteinuria. The mutant LRP2 was expressed and observed to dimerize and localize to the proximal tubule apical membrane. However, both fluid-phase and receptor-mediated endocytosis were impaired in the context of a general perturbation of endocytic flux. Immunofluorescence revealed aberrant endocytic recycling with mislocalized RAB11+ and TFR1+ compartments and enlarged lysosomes. Structural modeling showed the LRP2 assembly likely tolerates the cysteine to arginine substitution at the cell surface, but at endosomal pH the variant introduced steric clashes that may disrupt intramolecular interfaces and disturb receptor recycling. These findings point to the importance of LRP2 recycling for global endocytic flux and offer a blueprint for leveraging patient-specific alleles to dissect proximal tubule function.
Authors
Andrew Beenken, Tian H. Shen, Aryan Ghotra, Hediye Erdjument-Bromage, Jeong Lee, Jared S. Kushner, Rachel E. Sturley, Atlas Khan, Jeffrey R. Arace, Leora Kronenberg, Lucy D. Shen, Gabriel H. Rahmani, Patricia K. Donahoe, Thomas A. Neubert, Frances A. High, Ora A. Weisz, Jonathan Barasch
Abstract
Chronic hyperglycemia induces microvascular complications in patients with type 2 diabetes (T2D), particularly diabetic retinopathy, nephropathy, and neuropathy. We revisited to examine such vascular damage in the pancreas in 3D. Using thick pancreatic tissue slices, we analyzed volumetric intraislet and peri-islet exocrine capillary density (vICD and vECD), as well as interface capillary counts along the islet periphery to quantify vascular integration between the islets and surrounding acinar cells. Contrary to the previous reports, vICD was not homogeneous, but highly heterogeneous across the five species studied (human, monkey, pig, ferret and mouse), especially in smaller islets (15%–80%). vICD became less variable with increasing islet size converging at approximately 20%. With this foundation of islet vascularization, pancreatic tissues from non-diabetic (ND) and T2D subjects consisting of eight age- and sex-matched pairs (age range of 35-65 years with various duration: 0-15 years) were examined. Strikingly, no significant differences in microvasculature were found, where mean vICD (~30%) and mean vECD (~15%) were nearly equivalent between the groups. Capillary integration with respect to islet size was comparable. It suggests that integrated pancreatic blood flow with robust crosstalk between the endocrine and exocrine pancreas may facilitate microvascular preservation in T2D via local distribution of insulin.
Authors
Alex M. Tollefson, Frank R. Marsico, Manami Hara
Abstract
Primary sclerosing cholangitis (PSC) is a chronic, idiopathic cholestatic liver disease characterized by inflammation and fibrosis of the bile ducts, yet the cellular crosstalk driving periductal fibrosis remains poorly defined. This study applied a multi-omics approach integrating spatial transcriptomics, RNA-seq, and proteomics to characterize fibrotic periductal regions and their cell–cell communications. Macrophages (MP) subsets, including monocyte-drived-(Mo)MP and lipid-associated-macrophage (LAM)-like, co-localized with cholangiocytes, lymphocytes, and hepatic stellate cells (HSC1). Cell niche analysis identified periductal regions with elevated fibrotic signals, where cell–cell communication analysis revealed potential MP–HSC interactions involving 17 fibrotic driver genes in MP, including ITGB2, GRN, and CCL21, and 6 fibrotic effector genes in HSC. In validation analyses, bulk RNA-seq data showed higher driver and effector gene expression in PSC with established fibrosis compared to early-stage PSC or healthy control (HC). Plasma proteins encoded by MP driver genes were elevated in PSC and in patients with elevated (≥3.29 kPa) liver stiffness on MR elastography. Furthermore, immunofluorescence and SHG imaging showed enrichment of CD68+/CD18+(ITGB2) macrophages in fibrotic regions of PSC liver biopsies. These findings revealed enrichment of MoMP and LAM-like macrophages in fibrotic regions and suggest that they likely contribute to fibrotic activation of nearby HSCs in PSC.
Authors
Yunguan Wang, David Adeleke, Xiangfei Xie, Zi F. Yang, Xiangya Wang, Giulia Loi, Annika Yang vom Hofe, Manavi Singh, Astha Malik, Ramesh Kudira, Cyd Castro-Rojas, Liva Pfuhler, Mosab Alquraish, Pamela Sylvestre, Jonathan R. Dillman, Andrew T. Trout, Emily R. Miraldi, Alexander G. Miethke
Abstract
High dietary salt intake elevates blood pressure and drives multi-organ damage. However, the molecular programs underlying progressive organ injury remain poorly defined. Here, we present a longitudinal multi-organ transcriptomic atlas of salt-induced hypertensive injury. We profiled kidney cortex, kidney medulla, heart, and liver across four stages spanning early hypertension to advanced pathology in Dahl salt-sensitive rats. We identified dynamic and tissue-specific molecular trajectories, including a shared early proliferative response that converges on proinflammatory and fibrotic remodeling. Notably, we uncovered compartment-specific renal responses, showing that the cortex and medulla, despite their proximity, follow distinct molecular trajectories during disease progression. We further identified 79 stage- and tissue-specific transcription factors that drive gene expression dynamics in salt-induced hypertensive injury. Integration with human genome-wide association studies revealed conserved pathways in endocrine signaling, ion transport, lipid metabolism, and detoxification, establishing cross-species relevance and highlighting mechanistic targets of clinical importance. Compound–transcriptome analysis revealed stage- and organ-specific therapeutic opportunities, prioritizing kinase and epigenetic modulators as candidates to rebalance maladaptive gene programs. Overall, this study provides a resource for understanding molecular mechanisms from early salt-induced hypertension to tissue-specific injury and underscores the need for precision interventions.
Authors
Ratnakar Tiwari, Olha Kravtsova, Lashodya V. Dissanayake, Melissa Lowe, Biyang Xu, Vladislav Levchenko, Steven Didik, Ruslan Bohovyk, Daria V. Ilatovskaya, Oleg Palygin, Alexander Staruschenko
Abstract
Extracellular matrix (ECM) disorder was considered as the result of fibrosis, but it is recently recognized that fibrotic ECM initiates a self-reinforcing circuit and contributes to development of fibrosis. Versican, an ECM component, participates in cell-ECM interaction and ECM regeneration. In pleura, versican is primarily derived from pleural mesothelial cells (PMCs). However, the role and mechanism of versican in pleural fibrosis remained unknown. In this study, versican and versican-mediated pleural viscoelasticity was found elevated in both human and murine pleural fibrotic tissues. Versican knockdown by shRNA prevented increases of viscoelasticity as well as pleural fibrosis. High level of versican and viscoelasticity promoted mesothelial to mesenchymal transition (MesoMT) in PMCs. Mechanistically, increased viscoelasticity induced pleural fibrosis through CD44/USP10/Smad4 mechanotransduction pathway. In conclusion, these results revealed that excessive versican in fibrotic pleural ECM enhanced ECM viscoelasticity, and consequently promoted progression of pleural fibrosis.
Authors
Zi-Heng Jia, Xin-Liang He, Xiao-Lin Cui, Qian Li, Pei-Pei Cheng, Li-Qin Zhao, Shu-Yi Ye, Shi-He Hu, Chen-Yue Lian, He-De Zhang, Li-Mei Liang, Lin-Jie Song, Fan Yu, Liang Xiong, Fei Xiang, Xiaorong Wang, Meng Wang, Xiyong Dai, Hong Ye, Wan-Li Ma
Abstract
Lipotoxicity associated with metabolic dysfunction-associated steatotic liver disease (MASLD) causes dysregulated fatty acid (FA) and glucose metabolism, inducing cellular energy imbalance, oxidative stress (OS), and hepatocellular injury. Interleukin (IL)-10 is altered in MASLD, including increased IL-10 transcripts in peripheral immune cells; however, its role in hepatic responses to lipotoxic stress remains unclear. We evaluated whether IL-10 treatment attenuates lipotoxic injury and MASLD-related phenotypes in vivo and in vitro to reveal MASLD treatment strategies. As MASLD models, in vivo high-fat diet mice and in vitro normal human hepatocytes under palmitic acid exposure, with confirmatory experiments in HepG2 cells, were used and treated with IL-10. We assessed FA and glucose metabolism, OS, and apoptosis with histological changes and mechanisms related to hepatocellular viability/metabolic activity and stress-responsive survival signaling in vitro. IL-10 modulated FA synthesis and β-oxidation, reducing lipid accumulation, and altered glucose metabolic pathways, consistent with improved glucose handling under lipotoxic stress. Furthermore, IL-10 reduced OS and cell death markers while enhancing antioxidant responses, consistent with hepatocellular protection. These data suggest that IL-10 attenuates lipotoxic injury by modulating hepatic response pathways, thereby improving MASLD-related phenotypes, and supports the potential of IL-10 as a therapeutic target for MASLD.
Authors
Akira Kado, Kazuya Okushin, Takeya Tsutsumi, Toshiyuki Kishida, Kazuhiko Ikeuchi, Hiroshi Yotsuyanagi, Kyoji Moriya, Kazuhiko Koike, Mitsuhiro Fujishiro
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