Mutation of the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) was linked to Darier disease more than two decades ago; however, there remain no targeted therapies for this disorder causing recurrent skin blistering and infections. Since Atp2a2 knockout mice do not phenocopy its pathology, we established a human tissue model of Darier disease to elucidate its pathogenesis and identify potential therapies. Leveraging CRISPR/Cas9, we generated human keratinocytes lacking SERCA2, which replicated features of Darier disease, including weakened intercellular adhesion and defective differentiation in organotypic epidermis. To identify pathogenic drivers downstream of SERCA2 depletion, we performed RNA sequencing and proteomic analysis. SERCA2-deficient keratinocytes lacked desmosomal and cytoskeletal proteins required for epidermal integrity and exhibited excess MAP kinase signaling, which modulates keratinocyte adhesion and differentiation. Immunostaining patient biopsies substantiated these findings with lesions showing keratin deficiency, cadherin mis-localization, and ERK hyper-phosphorylation. Dampening ERK activity with MEK inhibitors rescued adhesive protein expression and restored keratinocyte sheet integrity despite SERCA2 depletion or chemical inhibition. In sum, coupling multi-omic analysis with human organotypic epidermis as a pre-clinical model, we found that SERCA2 haploinsufficiency disrupts critical adhesive components in keratinocytes via ERK signaling and identified MEK inhibition as a treatment strategy for Darier disease.
Shivam A. Zaver, Mrinal K. Sarkar, Shaun Egolf, Jonathan Zou, Afua Tiwaa, Brian C. Capell, Johann E. Gudjonsson, Cory L. Simpson
Acyl-CoA thioesterase 1 (ACOT1) catalyzes the hydrolysis of long-chain acyl-CoAs to free fatty acids and coenzyme A and is typically upregulated in obesity. Whether targeting ACOT1 in the setting of high-fat diet induced obesity would be metabolically beneficial is not known. Here we report that male and female ACOT1KO mice are partially protected from high-fat diet induced obesity, an effect associated with increased energy expenditure without alterations in physical activity or food intake. In males, ACOT1 deficiency increased mitochondrial uncoupling protein-2 (UCP2) protein abundance, while reducing 4-hydroxynonenal (4-HNE), a marker of oxidative stress, in white adipose tissue and liver of high-fat fed mice. Moreover, concurrent knockdown of UCP2 with ACOT1 in hepatocytes prevented increases in oxygen consumption observed with ACOT1 knockdown during high lipid loading, suggesting that UCP2-induced uncoupling may increase energy expenditure to attenuate weight gain. Together, these data indicate that targeting ACOT1 may be effective for obesity prevention during caloric excess by increasing energy expenditure.
Timothy D. Heden, Mallory P. Franklin, Christina Dailey, Mara T. Mashek, Chen Chen, Douglas G. Mashek
Metabolic mechanisms underlying the heterogeneity of major adverse cardiovascular events (MACE) risk in individuals with type 2 diabetes mellitus (T2D) remain unclear. We hypothesized that circulating metabolites reflecting mitochondrial dysfunction predict incident MACE in T2D. Targeted mass-spectrometry profiling of 60 metabolites was performed on baseline plasma from TECOS (discovery) and EXSCEL (validation) trial biomarker substudy cohorts. A principal components analysis metabolite factor comprised of medium-chain acylcarnitines was associated with MACE in TECOS and validated in EXSCEL, with higher levels associated with higher MACE risk. Meta-analysis showed that long-chain acylcarnitines and dicarboxylacylcarnitines were also associated with MACE. Metabolites remained associated with MACE in multivariate models and favorably changed with exenatide therapy. A third cohort (CATHGEN) with T2D assessed whether these metabolites improved discriminative capability multivariate for MACE; nine metabolites (medium- and long-chain acylcarnitines and one dicarboxylacylcarnitine) were associated with time-to-MACE in CATHGEN. Addition of these metabolites to clinical models minimally improved the discriminative capability for MACE but did significantly down reclassify risk. Thus, metabolites reporting on dysregulated mitochondrial fatty acid oxidation are higher in individuals with T2D who experience subsequent MACE. These biomarkers may improve CV risk prediction models, be therapy responsive, and highlight emerging risk mechanisms.
Jessica A. Regan, Robert J. Mentz, Maggie Nguyen, Jennifer B. Green, Lauren K. Truby, Olga Ilkayeva, Christopher Newgard, John B. Buse, Harald Sourij, C. David Sjöström, Naveed Sattar, Robert W. McGarrah, Yinggan Zheng, Darren K. McGuire, Eberhard Standl, Paul Armstrong, Eric Peterson, Adrian Hernandez, Rury R. Holman, Svati H. Shah
The growth of skeletal muscle relies on a delicate equilibrium between protein synthesis and degradation; however, how proteostasis is managed in the endoplasmic reticulum is largely unknown. Here, we report that the SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD) complex, the primary molecular machinery that degrades misfolded proteins in the ER is vital to maintain postnatal muscle growth and systemic energy balance. Myocyte-specific SEL1L deletion blunts the hypertrophic phase of muscle growth, resulting in a net zero gain of muscle mass during this developmental period and a 30% reduction in overall body growth. In addition, myocyte-specific SEL1L deletion triggered a systemic reprogramming of metabolism characterized by improved glucose sensitivity, enhanced beiging of adipocytes, and resistance to diet induced obesity. These effects were partially mediated by the upregulation of the myokine FGF21. These findings highlight the pivotal role of SEL1L-HRD1 ERAD activity in skeletal myocytes for postnatal muscle growth, and its physiological integration in maintaining whole-body energy balance.
Benedict Abdon, Yusheng Liang, Débora da Luz Scheffer, Mauricio Torres, Neha Shrestha, Rachel B. Reinert, You Lu, Brent Pederson, Amara Bugarin-Lapuz, Sander Kersten, Ling Qi
Cutaneous lupus erythematosus (CLE) is a disfiguring autoimmune skin disease characterized by an inflammatory infiltrate rich in T cells, which are strongly implicated in tissue damage. How these cells adapt to the skin environment and promote tissue inflammation and damage is not known. In lupus nephritis, we have previously identified an inflammatory gene program in kidney-infiltrating T cells that is dependent on hypoxia-inducible factor-1 (HIF-1), a transcription factor critical for the cellular and developmental response to hypoxia as well as inflammation-associated signals. In our current studies using a mouse model of lupus skin disease, we find that skin-infiltrating CD4+ and CD8+ T cells also express high levels of HIF-1. Skin-infiltrating T cells demonstrate a strong cytotoxic signature at the transcript and protein level, and HIF-1 inhibition abrogates skin and systemic disease in association with decreased T cell cytotoxic activity. We also demonstrate in human CLE tissue that the T cell rich inflammatory infiltrate exhibits increased amounts of HIF-1 and a cytotoxic signature. Granzyme B-expressing T cells are concentrated at sites of skin tissue damage in CLE, suggesting relevance of this pathway to human disease.
Alicia J. Little, Ping-Min Chen, Matthew D. Vesely, Rahanna N. Khan, Jacob Fiedler, James Garritano, Fahrisa M. Islam, Jennifer M. McNiff, Joseph E. Craft
The proportions and phenotypes of immune cell subsets in peripheral blood undergo continual and dramatic remodeling throughout the human lifespan, which complicates efforts to identify disease-associated immune signatures in type 1 diabetes (T1D). We conducted cross-sectional flow cytometric immune profiling on peripheral blood from 826 individuals [stage 3 T1D (n=240), their first-degree relatives (n=310), those with >2 islet autoantibodies (n=24), and autoantibody negative healthy controls (n=252)]. We constructed an immune-age predictive model in healthy participants and observed accelerated immune aging in the T1D cohort (p<0.001). We used generalized additive models for location, shape, and scale (GAMLSS) to obtain age-corrected data for 192 flow cytometry and complete blood count readouts, which can be visualized in our interactive portal (ImmScape; https://ufdiabetes.shinyapps.io/ImmScape/); 46 parameters were significantly associated with age only, 25 with T1D only, and 23 with both age and T1D. Phenotypes associated with accelerated immunological aging in T1D included increased CXCR3+ and PD-1+ frequencies in naïve and memory T cell subsets, despite reduced PD-1 expression levels (MFI) on memory T cells. Phenotypes associated with T1D after age-correction were predictive of T1D status (AUROC=82.3%). Our findings demonstrate advanced immune aging in T1D and highlight disease-associated phenotypes for biomarker monitoring and therapeutic interventions.
Melanie R. Shapiro, Xiaoru Dong, Daniel J. Perry, James M. McNichols, Puchong Thirawatananond, Amanda L. Posgai, Leeana D. Peters, Keshav Motwani, Richard S. Musca, Andrew Muir, Patrick Concannon, Laura M. Jacobsen, Clayton E. Mathews, Clive H. Wasserfall, Michael J. Haller, Desmond A. Schatz, Mark A. Atkinson, Maigan A. Brusko, Rhonda Bacher, Todd M. Brusko
The intricate interplay between maternal immune response to SARS-CoV-2 and the transfer of protective factors to the fetus remains unclear. By analyzing mother:neonate dyads from second and third trimester SARS-CoV-2 infections, our study shows that neutralizing antibodies (NAbs) are infrequently detected in cord blood. We uncovered that this is due to impaired IgG-NAbs placental transfer in symptomatic infection and to the predominance of maternal SARS-CoV-2 NAbs of the IgA and IgM isotypes, which are prevented from crossing the placenta. Crucially, the balance between maternal antiviral response and transplacental transfer of IgG-NAbs appears to hinge on IL-6 and IL-10 produced in response to SARS-CoV-2 infection. In addition, asymptomatic maternal infection was associated with expansion of anti-SARS-CoV-2 IgM and NK cell frequency. Our findings identify a protective role for IgA/IgM-NAbs in gestational SARS-CoV-2 infection and open the possibility that the maternal immune response to SARS-CoV-2 infection might benefit the neonate in two ways, first by skewing maternal immune response towards immediate viral clearance, and second by endowing the neonate with protective mechanisms to curtail horizontal viral transmission in the critical post-natal period, via the priming of IgA/IgM-NAbs to be transferred by the breast milk and via NK cell expansion in the neonate.
Juliana Gonçalves, Magda Melro, Marta Alenquer, Catarina Araújo, Júlia Castro-Neves, Daniela Amaral-Silva, Filipe Ferreira, José S. Ramalho, Nádia Charepe, Fátima Serrano, Carlos Pontinha, Maria Joao Amorim, Helena Soares
Nitric oxide (NO) is a critical signaling molecule that has been implicated in the pathogenesis of neurocognitive diseases. Both excessive and insufficient NO production have been linked to pathology. Previously, we have shown that argininosuccinate lyase deficiency (ASLD) is a novel model system to investigate cell-autonomous, nitric oxide synthase (NOS)-dependent NO deficiency. Humans with ASLD are at increased risk for developing hyperammonemia due to a block in ureagenesis. However, natural history studies have shown that individuals with ASLD have multi-systemic disease including neurocognitive deficits that can be independent of ammonia. Here, using ASLD as a model of NO deficiency, we investigated the effects of NO on brain endothelial cells in vitro and the blood-brain barrier (BBB) in vivo. Knockdown of ASL in human brain microvascular endothelial cells (HBMECs) led to decreased transendothelial electrical resistance (TEER), indicative of increased cell permeability. Mechanistically, treatment with an NO donor or inhibition of Claudin-1 improved barrier integrity in ASL-deficient HBMECs. Furthermore, in vivo assessment of a hypomorphic mouse model of ASLD showed increased BBB leakage, which was partially rescued by NO supplementation. Our results suggest that ASL-mediated NO synthesis is required for proper maintenance of brain microvascular endothelial cell functions as well as BBB integrity.
Jordan Kho, Urszula Polak, Ming-Ming Jiang, John D. Odom, Jill V. Hunter, Saima M. Ali, Lindsay C. Burrage, Sandesh C.S. Nagamani, Robia G. Pautler, Hannah P. Thompson, Akihiko Urayama, Zixue Jin, Brendan Lee
Eosinophilic esophagitis (EoE) is a chronic gastrointestinal disorder characterized by food antigen–driven eosinophilic inflammation and hyperproliferation of esophageal mucosa. By utilizing a large-scale, proteomic screen of esophageal biopsies, we aimed to uncover molecular drivers of the disease. Proteomic analysis by liquid chromatography–tandem mass spectrometry identified 402 differentially expressed proteins (DEPs) that correlated with the EoE transcriptome. Immune cell–related proteins were among the most highly upregulated DEPs in EoE compared to controls, whereas proteins linked to epithelial differentiation were primarily downregulated. Notably, in the inflamed esophageal tissue, all six subunits of the minichromosome maintenance (MCM) complex, a DNA helicase essential for genomic DNA replication, were significantly upregulated at the gene and protein levels. Furthermore, treating esophageal epithelial cells with a known inhibitor of the MCM complex (ciprofloxacin) blocked esophageal epithelial proliferation. In a murine model of EoE driven by overexpression of IL-13, ciprofloxacin treatment decreased basal zone thickness and reduced dilated intercellular spaces by blocking the transition of the epithelial cell through the S-phase of the cell cycle. Collectively, a broad-spectrum proteomic screen has identified the involvement of the MCM complex in EoE and has highlighted MCM inhibitors as potential therapeutic agents for the disease.
Mark Rochman, Yrina Rochman, Julie M. Caldwell, Lydia Mack, John A Besse, Nathan P Manes, Sung Hwan Yoon, Tetsuo Shoda, Aleksandra Nita-Lazar, Marc Rothenberg
X-linked hypophosphatemia (XLH) is characterized by high serum fibroblast growth factor 23 (FGF23) levels, resulting in impaired 1,25 dihydroxyvitamin D (1,25D) production. Adults with XLH develop a painful mineralization of the tendon-bone attachment site (enthesis), called enthesopathy. Treatment of mice with XLH (Hyp) with 1,25D or an anti-FGF23 antibody, both of which increase 1,25D signaling, prevents enthesopathy. Therefore, we undertook studies to determine a role for impaired 1,25D action in enthesopathy development. Entheses from mice lacking vitamin D 1-alpha-hydroxylase (Cyp27b1) (C-/-) had a similar enthesopathy to Hyp mice, while deletion of Fgf23 in Hyp mice prevented enthesopathy and deletion of both Cyp27b1 and Fgf23 in mice resulted in enthesopathy, demonstrating that the impaired 1,25D action due to high FGF23 levels underlied XLH enthesopathy development. Like Hyp mice, enthesopathy in C-/- mice was observed by P14 and was prevented, but not reversed, with 1,25D therapy. Deletion of the vitamin D receptor in scleraxis-expressing cells resulted in enthesopathy, indicating 1,25D acted directly on enthesis cells to regulate enthesopathy development. These results showed that 1,25D signaling was necessary for normal post-natal enthesis maturation and played a role in XLH enthesopathy development. Optimizing 1,25D replacement in pediatric XLH patients is necessary to prevent enthesopathy.
Rakshya Rana, Jiana T. Baker, Melissa Sorsby, Supriya Jagga, Shreya Venkat, Shaza Almardini, Eva S. Liu
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