JCI insight最新文献

Accumulation of extracellular matrix (ECM) proteins in trabecular meshwork (TM), which leads to increased outflow resistance of aqueous humor and consequently high intraocular pressure, is a major cause of primary open-angle glaucoma (POAG). According to our preliminary research, the RapGAP protein superfamily member, signal-induced proliferation-associated 1-like 1 protein (SIPA1L1), which is involved in tissue fibrosis, may have an impact on POAG by influencing ECM metabolism of TM. This study aims to confirm these findings and identify effects and cellular mechanisms of SIPA1L1 on ECM changes and phagocytosis in human TM (HTM) cells. Our results showed that the expression of SIPA1L1 in HTM cells was significantly increased by TGF-β2 treatment in label-free quantitative proteomics. The aqueous humor and TM cell concentration of SIPA1L1 in POAG patients was higher than that of control. In HTM cells, TGF-β2 increased expression of SIPA1L1 along with accumulation of ECM, RhoA, and p-cofilin 1. The effects of TGF-β2 were reduced by si-SIPA1L1. TGF-β2 decreased HTM cell phagocytosis by polymerizing cytoskeletal actin filaments, while si-SIPA1L1 increased phagocytosis by disassembling actin filaments. Simultaneously, overexpressing SIPA1L1 alone exhibited comparable effects to that of TGF-β2. Our studies demonstrate that SIPA1L1 not only promotes the production of ECM, but also inhibits its removal by reducing phagocytosis. Targeting SIPA1L1 degradation may become a significant therapy for POAG.

Sepsis-induced acute lung injury (ALI) is prevalent in patients with sepsis and has a high mortality rate. Peptidyl arginine deiminase 2 (PADI2) and PADI4 play crucial roles in mediating the host's immune response in sepsis, but their specific functions remain unclear. Our study shows that Padi2-/- Padi4-/- double KO (DKO) improved survival, reduced lung injury, and decreased bacterial load in Pseudomonas aeruginosa (PA) pneumonia-induced sepsis mice. Using single-cell RNA-Seq (scRNA-Seq), we found that the deletion of Padi2 and Padi4 reduced the Nlrp3+ proinflammatory macrophages and fostered Chil3+ myeloid cell differentiation into antiinflammatory macrophages. Additionally, we observed the regulatory role of the NLRP3/Ym1 axis upon DKO, confirmed by Chil3 knockdown and Nlrp3-KO experiments. Thus, eliminating Padi2 and Padi4 enhanced the polarization of Ym1+ M2 macrophages by suppressing NLRP3, aiding in inflammation resolution and lung tissue repair. This study unveils the PADIs/NLRP3/Ym1 pathway as a potential target in treatment of sepsis-induced ALI.

Daptomycin is a last-resort lipopeptide antibiotic that disrupts cell membrane (CM) and peptidoglycan homeostasis. Enterococcus faecalis has developed a sophisticated mechanism to avoid daptomycin killing by redistributing CM anionic phospholipids away from the septum. The CM changes are orchestrated by a 3-component regulatory system, designated LiaFSR, with a possible contribution of cardiolipin synthase (Cls). However, the mechanism by which LiaFSR controls the CM response and the role of Cls are unknown. Here, we show that cardiolipin synthase activity is essential for anionic phospholipid redistribution and daptomycin resistance since deletion of the 2 genes (cls1 and cls2) encoding Cls abolished CM remodeling. We identified LiaY, a transmembrane protein regulated by LiaFSR, and Cls1 as important mediators of CM remodeling required for redistribution of anionic phospholipid microdomains. Together, our insights provide a mechanistic framework on the enterococcal response to cell envelope antibiotics that could be exploited therapeutically.

Brain arteriovenous malformations (bAVMs) are anomalies forming vascular tangles connecting the arteries and veins, which cause hemorrhagic stroke in young adults. Current surgical approaches are highly invasive, and alternative therapeutic methods are warranted. Recent genetic studies identified KRAS mutations in endothelial cells of bAVMs; however, the underlying process leading to malformation in the postnatal stage remains unknown. Here we established a mouse model of bAVM developing during the early postnatal stage. Among 4 methods tested, mutant KRAS specifically introduced in brain endothelial cells by brain endothelial cell-directed adeno-associated virus (AAV) and endothelial cell-specific Cdh5-CreERT2 mice successfully induced bAVMs in the postnatal period. Mutant KRAS led to the development of multiple vascular tangles and hemorrhage in the brain with increased MAPK/ERK signaling and growth in endothelial cells. Three-dimensional analyses in cleared tissue revealed dilated vascular networks connecting arteries and veins, similar to human bAVMs. Single-cell RNA-Seq revealed dysregulated gene expressions in endothelial cells and multiple cell types involved in the pathological process. Finally, we employed CRISPR/CasRx to knock down mutant KRAS expression, which efficiently suppressed bAVM development. The present model reveals pathological processes that lead to postnatal bAVMs and demonstrates the efficacy of therapeutic strategies with CRISPR/CasRx.

Urinary concentration is an energy-dependent process that minimizes body water loss by increasing aquaporin 2 (AQP2) expression in collecting duct (CD) principal cells. To investigate the role of mitochondrial (mt) ATP production in renal water clearance, we disrupted mt electron transport in CD cells by targeting ubiquinone (Q) binding protein QPC (UQCRQ), a subunit of mt complex III essential for oxidative phosphorylation. QPC-deficient mice produced less concentrated urine than controls, both at baseline and after type 2 vasopressin receptor stimulation with desmopressin. Impaired urinary concentration in QPC-deficient mice was associated with reduced total AQP2 protein levels in CD tubules, while AQP2 phosphorylation and membrane trafficking remained unaffected. In cultured inner medullary CD cells treated with mt complex III inhibitor antimycin A, the reduction in AQP2 abundance was associated with activation of 5' adenosine monophosphate-activated protein kinase (AMPK) and was reversed by treatment with AMPK inhibitor SBI-0206965. In summary, our studies demonstrated that the physiological regulation of AQP2 abundance in principal CD cells was dependent on mt electron transport. Furthermore, our data suggested that oxidative phosphorylation in CD cells was dispensable for maintaining water homeostasis under baseline conditions, but necessary for maximal stimulation of AQP2 expression and urinary concentration.

Graves' disease (GD) is an autoimmune condition that can progress to Graves' ophthalmopathy (GO), leading to irreversible damage to orbital tissues and potential blindness. The pathogenic mechanism is not fully understood. In this study, we conducted single-cell multi-omics analyses on healthy individuals, patients with GD without GO, newly diagnosed patients with GO, and treated patients with GO. Our findings revealed gradual systemic inflammation during GO progression, marked by overactivation of cytotoxic effector T cell subsets, and expansion of specific T cell receptor clones. Importantly, we observed a decline in the immunosuppressive function of activated Treg (aTreg) accompanied by a cytotoxic phenotypic transition. In vitro experiments revealed that dysfunction and transition of GO-autoreactive Treg were regulated by the yin yang 1 (YY1) upon secondary stimulation of thyroid stimulating hormone receptor (TSHR) under inflammatory conditions. Furthermore, adoptive transfer experiments of the GO mouse model confirmed infiltration of these cytotoxic Treg into the orbital lesion tissues. Notably, these cells were found to upregulate inflammation and promote pathogenic fibrosis of orbital fibroblasts (OFs). Our results reveal the dynamic changes in immune landscape during GO progression and provide direct insights into the instability and phenotypic transition of Treg, offering potential targets for therapeutic intervention and prevention of autoimmune diseases.

Human periosteal skeletal stem cells (P-SSCs) are critical for cortical bone maintenance and repair. However, their in vivo identity, molecular characteristics, and specific markers remain unknown. Here, single-cell sequencing revealed human periosteum contains SSC clusters expressing known SSC markers, podoplanin (PDPN) and PDGFRA. Notably, human P-SSCs, but not bone marrow SSCs, selectively expressed identified markers low density lipoprotein receptor-related protein 1 (LRP1) and CD13. These LRP1+CD13+ human P-SSCs were perivascular cells with high osteochondrogenic but minimal adipogenic potential. Upon transplantation into bone injuries in mice, they preserved self-renewal capability in vivo. Single-cell analysis of mouse periosteum further supported the preferential expression of LRP1 and CD13 in Prx1+ P-SSCs. When Lrp1 was conditionally deleted in Prx1 lineage cells, it led to severe bone deformity, short stature, and periosteal defects. By contrast, local treatment with an LRP1 agonist at the injury sites induced early P-SSC proliferation and bone healing. Thus, human and mouse periosteum contains unique osteochondrogenic stem cell subsets, and these P-SSCs express specific markers, LRP1 and CD13, with a regulatory mechanism through LRP1 that enhances P-SSC function and bone repair.

Airway remodeling is a critical factor determining the pathogenesis and treatment sensitivity of severe asthma (SA) or uncontrolled asthma (UA). The activation of epithelial-mesenchymal trophic units (EMTUs) regulated by airway epithelial cells (AECs) has been proven to induce airway remodeling directly. However, the triggers for EMTU activation and the underlying mechanism of airway remodeling are not fully elucidated. Here, we screened the differentially expressed gene cathepsin C (CTSC; also known as dipeptidyl peptidase 1 [DPP-1]) in epithelia of patients with SA and UA using RNA-sequencing data and further verified the increased expression of CTSC in induced sputum of patients with asthma, which was positively correlated with severity and airway remodeling. Moreover, direct instillation of exogenous CTSC induced airway remodeling. Genetic inhibition of CTSC suppressed EMTU activation and airway remodeling in two asthma models with airway remodeling. Mechanistically, increased secretion of CTSC from AECs induced EMTU activation through the p38-mediated pathway, further inducing airway remodeling. Meanwhile, inhibition of CTSC also reduced the infiltration of inflammatory cells and the production of inflammatory factors in the lungs of asthmatic mice. Consequently, targeting CTSC with compound AZD7986 protected against airway inflammation, EMTU activation, and remodeling in the asthma model. Based on the dual effects of CTSC on airway inflammation and remodeling, CTSC is a potential biomarker and therapeutic target for SA or UA.

Human studies linking metabolism with organism-wide physiologic function have been challenged by confounding, adherence, and precisionHere, we united physiologic and molecular phenotypes of metabolism during controlled dietary intervention to understand integrated metabolic-physiologic responses to nutrition. In an inpatient study of individuals who underwent serial 24-hour metabolic chamber experiments (indirect calorimetry) and metabolite profiling, we mapped a human metabolome onto substrate oxidation rates and energy expenditure across up to 7 dietary conditions (energy balance, fasting, multiple 200% caloric excess overfeeding of varying fat, protein, and carbohydrate composition). Diets exhibiting greater fat oxidation (e.g., fasting, high-fat) were associated with changes in metabolites within pathways of mitochondrial β-oxidation, ketogenesis, adipose tissue fatty acid liberation, and/or multiple anapleurotic substrates for tricarboxylic acid cycle flux, with inverse associations for diets with greater carbohydrate availability. Changes in each of these metabolite classes were strongly related to 24-hour respiratory quotient (RQ) and substrate oxidation rates (e.g., acylcarnitines related to lower 24-hour RQ and higher 24-hour lipid oxidation), underscoring links between substrate availability, physiology, and metabolism in humans. Physiologic responses to diet determined by gold-standard human metabolic chambers are strongly coordinated with biologically consistent, interconnected metabolic pathways encoded in the metabolome.

Mutations in the CLCNKB gene (1p36), encoding the basolateral chloride channel ClC-Kb, cause type 3 Bartter syndrome. We identified a family with a mixed Bartter/Gitelman phenotype and early-onset kidney failure and by employing a candidate gene approach, identified what we believe is a novel homozygous mutation (CLCNKB c.499G>T [p.Gly167Cys]) in exon 6 of CLCNKB in the index patient. We then validated these results with Sanger and whole-exome sequencing. Compared with wild-type ClC-Kb, the Gly167Cys mutant conducted less current and exhibited impaired complex N-linked glycosylation in vitro. We demonstrated that loss of Gly-167, rather than gain of a mutant Cys, impairs complex glycosylation, but that surface expression remains intact. Moreover, Asn-364 was necessary for channel function and complex glycosylation. Morphologic evaluation of human kidney biopsies revealed typical basolateral localization of mutant Gly167Cys ClC-Kb in cortical distal tubular epithelia. However, we detected attenuated expression of distal sodium transport proteins, changes in abundance of distal tubule segments, and hypokalemia-associated intracellular condensates from the index patient compared with control nephrectomy specimens. The present data establish what we believe are novel regulatory mechanisms of ClC-Kb activity and demonstrate nephron remodeling in humans, caused by mutant ClC-Kb, with implications for renal electrolyte handling, blood pressure control, and kidney disease.