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Skeletal muscle regeneration in adults is predominantly driven by satellite cells. Loss of satellite cell pool and function leads to skeletal muscle wasting in many conditions and disease states. Here, we demonstrate that the levels of fibroblast growth factor-inducible 14 (Fn14) were increased in satellite cells after muscle injury. Conditional ablation of Fn14 in Pax7-expressing satellite cells drastically reduced their expansion and skeletal muscle regeneration following injury. Fn14 was required for satellite cell self-renewal and proliferation as well as to prevent precocious differentiation. Targeted deletion of Fn14 inhibited Notch signaling but led to the spurious activation of STAT3 signaling in regenerating skeletal muscle and in cultured muscle progenitor cells. Silencing of STAT3 improved proliferation and inhibited premature differentiation of Fn14-deficient satellite cells. Furthermore, conditional ablation of Fn14 in satellite cells exacerbated myopathy in the mdx mouse model of Duchenne muscular dystrophy (DMD) whereas its overexpression improved the engraftment of exogenous muscle progenitor cells into the dystrophic muscle of mdx mice. Altogether, our study highlights the crucial role of Fn14 in the regulation of satellite cell fate and function and suggests that Fn14 can be a potential molecular target to improve muscle regeneration in muscular disorders.

Somatic activating mutations in KRAS can cause complex lymphatic anomalies (CLAs). However, the specific processes that drive KRAS-mediated CLAs have yet to be fully elucidated. Here, we used single-cell RNA sequencing to construct an atlas of normal and KrasG12D-malformed lymphatic vessels. We identified six subtypes of lymphatic endothelial cells (LECs) in the lungs of adult wild-type mice (Ptx3, capillary, collecting, valve, mixed, and proliferating). To determine when the LEC subtypes were specified during development, we integrated our data with data from four stages of development. We found that proliferating and Ptx3 LECs were prevalent during early lymphatic development and that collecting and valve LECs emerged later in development. Additionally, we discovered that the proportion of Ptx3 LECs decreased as the lymphatic network matured but remained high in KrasG12D mice. We also observed that the proportion of collecting and valve LECs was lower in KrasG12D mice than in wild-type mice. Last, we found that immature lymphatic vessels in young mice were more sensitive to the pathologic effects of KrasG12D than mature lymphatic vessels in older mice. Together, our results expand the current model for the development of the lymphatic system and suggest that KRAS mutations impair the maturation of lymphatic vessels.

Osteoarthritis (OA) shows various clinical manifestations depending on the status of its joint components. We aimed to identify the synovial cell subsets responsible for OA pathophysiology by comprehensive analyses of human synovium samples in single-cell resolution. Two distinct OA synovial tissue groups were classified by gene expression profiles in RNA-Seq: inflammatory and fibrotic. The inflammatory group exhibited high expression of inflammatory cytokines, histologically inflammatory infiltrate, and a more severe pain score. The fibrotic group showed higher expression of fibroblast growth factor (FGFs) and bone morphogenetic proteins (BMPs), showed histologically perivascular fibrosis, and showed a lower pain score. In single-cell RNA-Seq (scRNA-Seq) of synovial cells, MERTKloCD206lo macrophages and CD34hi fibroblasts were associated with the inflammatory and fibrotic groups, respectively. Among the 3 fibroblast subsets, CD34loTHY1lo and CD34loTHY1hi fibroblasts were influenced by synovial immune cells, whereas CD34hi fibroblasts were influenced by mural and endothelial cells. Particularly, in CD34hi fibroblast subsets, CD34hiCD70hi fibroblasts promoted proliferation of Tregs, potentially suppressing synovitis and protecting articular cartilage. Elucidation of the mechanisms underlying the regulation of these synovial cell subsets may lead to novel strategies for OA therapeutics.

Autoimmune uveitis (AU) is a sight-threatening ocular autoimmune disorder that often manifests as retinal vasculitis. Increased neutrophil infiltration around retinal vessels has been reported during the progression of AU, while how they function is not fully recognized. Neutrophil extracellular traps (NETs), produced by activated neutrophils, have been suggested to be detrimental in autoimmune diseases. Here, we found that NETs were elevated in patients with active AU, and this was verified in an experimental AU (EAU) mouse model. Depletion of neutrophils or degradation of NETs with deoxyribonuclease-I (DNase I) could decrease CD4+ effector T cell (Teff) infiltration in retina and spleen to alleviate EAU. Moreover, we found that the expression of adhesion molecules, selectin, and antigen-presenting molecules was elevated in EAU retina and in retinal microvascular endothelial cells (RMECs) cocultured with NETs. The stimulated RMECs further facilitated CD4+ T cell adhesion, activation, and differentiation into Teffs. Mechanistically, NETs trigger RMEC activation by hastening cell senescence through the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. Slowing down senescence or inhibiting the cGAS/STING pathway in RMECs reduces the activation and differentiation of CD4+ T cells. These results suggest a deleterious role of NETs in AU. Targeting NETs would offer an effective therapeutic method.

Diabetes mellitus can cause impaired and delayed wound healing, leading to lower extremity amputations; however, the mechanisms underlying the regulation of vascular endothelial growth factor-dependent (VEGF-dependent) angiogenesis remain unclear. In our study, the molecular underpinnings of endothelial dysfunction in diabetes are investigated, focusing on the roles of disabled-2 (Dab2) and Forkhead box M1 (FOXM1) in VEGF receptor 2 (VEGFR2) signaling and endothelial cell function. Bulk RNA-sequencing analysis identified significant downregulation of Dab2 in high-glucose-treated primary mouse skin endothelial cells. In diabetic mice with endothelial deficiency of Dab2, in vivo and in vitro angiogenesis and wound healing were reduced when compared with wild-type diabetic mice. Restoration of Dab2 expression by injected mRNA-containing, LyP-1-conjugated lipid nanoparticles rescued impaired angiogenesis and wound healing in diabetic mice. Furthermore, FOXM1 was downregulated in skin endothelial cells under high-glucose conditions as determined by RNA-sequencing analysis. FOXM1 was found to bind to the Dab2 promoter, regulating its expression and influencing VEGFR2 signaling. The FOXM1 inhibitor FDI-6 reduced Dab2 expression and phosphorylation of VEGFR2. Our study provides evidence of the crucial roles of Dab2 and FOXM1 in diabetic endothelial dysfunction and establishes targeted delivery as a promising treatment for diabetic vascular complications.

CD8+ T cells are critical for immune protection against severe COVID-19 during acute infection with SARS-CoV-2. However, the induction of antiviral CD8+ T cell responses varies substantially among infected people, and a better understanding of the mechanisms that underlie such immune heterogeneity is required for pandemic preparedness and risk stratification. In this study, we analyzed SARS-CoV-2-specific CD4+ and CD8+ T cell responses in relation to age, clinical status, and inflammation among patients infected primarily during the initial wave of the pandemic in France or Japan. We found that age-related contraction of the naive lymphocyte pool and systemic inflammation were associated with suboptimal SARS-CoV-2-specific CD4+ and, even more evidently, CD8+ T cell immunity in patients with acute COVID-19. No such differences were observed for humoral immune responses targeting the spike protein of SARS-CoV-2. We also found that the proinflammatory cytokine IL-18, concentrations of which were significantly elevated among patients with severe disease, suppressed the de novo induction and memory recall of antigen-specific CD8+ T cells, including those directed against SARS-CoV-2. These results potentially explain the vulnerability of older adults to infections that elicit a profound inflammatory response, exemplified by acute COVID-19.

Abdominal aortic aneurysms (AAA) are a life-threatening cardiovascular disease for which there is a lack of effective therapy preventing aortic rupture. During AAA formation, pathological vascular remodeling is driven by vascular smooth muscle cell (VSMC) dysfunction and apoptosis, for which the mechanisms regulating loss of VSMCs within the aortic wall remain poorly defined. Using single-cell RNA-Seq of human AAA tissues, we identified increased activation of the endoplasmic reticulum stress response pathway, PERK/eIF2α/ATF4, in aortic VSMCs resulting in upregulation of an apoptotic cellular response. Mechanistically, we reported that aberrant TNF-α activity within the aortic wall induces VSMC ATF4 activation through the PERK endoplasmic reticulum stress response, resulting in progressive apoptosis. In vivo targeted inhibition of the PERK pathway, with VSMC-specific genetic depletion (Eif2ak3fl/fl Myh11-CreERT2) or pharmacological inhibition in the elastase and angiotensin II-induced AAA model preserved VSMC function, decreased elastin fragmentation, attenuated VSMC apoptosis, and markedly reduced AAA expansion. Together, our findings suggest that cell-specific pharmacologic therapy targeting the PERK/eIF2α/ATF4 pathway in VSMCs may be an effective intervention to prevent AAA expansion.

T cells targeting a KRAS mutation can induce durable tumor regression in some patients with metastatic epithelial cancer. It is unknown whether T cells targeting mutant KRAS that are capable of killing tumor cells can be identified from peripheral blood of patients with pancreatic cancer. We developed an in vitro stimulation approach and identified HLA-A*11:01-restricted KRAS G12V-reactive CD8+ T cells and HLA-DRB1*15:01-restricted KRAS G12V-reactive CD4+ T cells from peripheral blood of 2 out of 6 HLA-A*11:01-positive patients with pancreatic cancer whose tumors expressed KRAS G12V. The HLA-A*11:01-restricted KRAS G12V-reactive T cell receptor (TCR) was isolated and validated to specifically recognize the KRAS G12V8-16 neoepitope. While T cells engineered to express this TCR specifically recognized all 5 tested human HLA-A*11:01+ and KRAS G12V+ pancreatic cancer organoids, the recognition was often modest, and tumor cell killing was observed in only 2 out of 5 organoids. IFN-γ priming of the organoids enhanced the recognition and killing by the TCR-engineered T cells. The TCR-engineered T cells could significantly slow the growth of an established organoid-derived xenograft in immunodeficient mice. Our data suggest that this TCR has potential for use in TCR-gene therapy, but additional strategies that enhance tumor recognition by the TCR-engineered T cells likely will be required to increase clinical activity.

Pediatric high-grade gliomas (pHGGs) are the most aggressive brain tumors in children, necessitating innovative therapies to improve outcomes. Unlike adult gliomas, recent research reveals that childhood gliomas have distinct biological features, requiring specific treatment strategies. Here, we focused on deciphering unique genetic dependencies specific to childhood gliomas. Using a pooled CRISPR/Cas9 knockout screening approach on 65 pediatric and 10 adult high-grade glioma (HGG) cell lines, myeloid cell leukemia 1 (MCL1) emerged as a key antiapoptotic gene essential in pediatric but not adult gliomas. We demonstrated that MCL1 is targetable using current small molecule inhibitors, and its inhibition leads to potent anticancer activity across pediatric HGG cell lines irrespective of genotype. Employing predictive modeling approaches on a large set of childhood cancer cell lines with multiomics data features, we identified a potentially previously unreported cluster of CpG sites in the antiapoptotic BCL-xL/BCL2L1 gene, which predicted MCL1 inhibitor response. We extended these data across multiple pediatric tumor types, showing that BCL2L1 methylation is a broad predictor of MCL1 dependency in vitro and in vivo. Overall, our multidimensional, integrated genomic approach identified MCL1 as a promising therapeutic target in several BCL2L1-methylated pediatric cancers, offering a translational strategy to identify patients most likely to benefit from MCL1 inhibitor therapy.

Urinary obstruction causes injury to the renal medulla, impairing the ability to concentrate urine, and increasing the risk of progressive kidney disease. However, the regenerative capacity of the renal medulla after reversal of obstruction is poorly understood. To investigate this, we developed a mouse model of reversible urinary obstruction. Despite robust regeneration and complete histological recovery of the renal medulla, these mice exhibited a permanent defect in urinary concentrating capacity. However, there were lasting changes in the composition, organization, and transcriptional profiles of epithelial, endothelial, and interstitial cells. Persistent inflammatory responses were also seen in patients with renal stone disease, but there were also adaptive responses to the increasingly hypoxic environment of the renal medulla that only occurred after reversal of obstruction. These findings indicate that while partial repair occurs after reversal of urinary obstruction, there are lasting structural and functional changes across all major cellular compartments of the renal medulla. These changes reflect shared and distinct responses to different renal medullary injuries in humans and mice.