Loss-of-Function (LoF) variants in the filaggrin (FLG) gene are the strongest known genetic risk factor for atopic dermatitis (AD), but the impact of these variants on AD outcomes is poorly understood. We comprehensively identified genetic variants through targeted region sequencing of FLG in children (n = 438) participating in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children (MPAACH) cohort. Twenty FLG LoF variants were identified, including one novel variant and nine variants not previously associated with AD. FLG LoF variants were found in 13.6% of the cohort. Among these children, the presence of one or more FLG LoF variants was associated with moderate/severe AD (odds ratio (OR) = 2.00 (95% CI, 1.23–3.68) compared to those with mild AD. Children with FLG LoF variants had a higher SCORAD (SCORing for Atopic Dermatitis (SCORAD); P = 0.012) and higher likelihood of food allergy within the first 2.5 years of life (OR = 2.81, 1.50–5.26). LoF variants were associated with higher transepidermal Water Loss (TEWL) in both lesional (P = 0.018) and non-lesional skin (P = 0.015). Collectively, our study identifies established and novel AD-associated FLG LoF variants and associates FLG LoF with higher TEWL in lesional and non-lesional skin.
Samuel J. Virolainen, Latha Satish, Jocelyn M. Biagini, Hassan Chaib, Wan Chi Chang, Phillip J. Dexheimer, Michael R. Dixon, Katelyn A. Dunn, David Fletcher, Carmy Forney, Marissa Granitto, Matthew S. Hestand, Makenna Hurd, Kenneth Kaufman, Lucinda P. Lawson, Lisa J. Martin, Loren D.M. Peña, Kieran J. Phelan, Molly S. Shook, Matthew T. Weirauch, Gurjit K. Khurana Hershey, Leah C. Kottyan
Central for wound healing is the formation of granulation tissue, which largely consists of collagen and whose importance stretches past wound healing, including being implicated in both fibrosis and skin aging. Cyclophilin D (CyD) is a mitochondrial protein that regulates the permeability transition pore, known for its role in apoptosis and ischemia-reperfusion. To date, the role of CyD in human wound healing and collagen generation ihas been largely unexplored. Here, we show that CyD was upregulated in normal wounds and venous ulcers, likely adaptive as CyD inhibition impaired re-epithelialization, granulation tissue formation, and wound closure in both human and pig models. Overexpression of CyD increased keratinocyte migration and fibroblast proliferation, whilst its inhibition reduced migration. Independent of wound healing, CyD inhibition in fibroblasts reduced collagen secretion and caused endoplasmic reticulum collagen accumulation, while its overexpression increased collagen secretion. This was confirmed in a Ppif knockout mouse model, which showed a reduction in skin collagen. Overall, this study revealed previously unreported roles of CyD in skin, with implications for wound healing and beyond.
Ritu Bansal, Monica Torres, Matthew Hunt, Nuoqi Wang, Margarita Chatzopoulou, Mansi Manchanda, Evan P. Taddeo, Cynthia Shu, Orian S. Shirihai, Etty Bachar-Wikstrom, Jakob D. Wikstrom
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease associated with cardiomyopathy. DMD-cardiomyopathy is characterized by abnormal intracellular Ca2+ homeostasis and mitochondrial dysfunction. We used dystrophin and utrophin null (mdx:utrn–/–) mice in sarcolipin (SLN) heterozygous knockout (sln+/–) background to examine the effect of SLN reduction on mitochondrial function in the dystrophic myocardium. Germline reduction of SLN expression in mdx:utrn–/– mice improved cardiac sarco/endoplasmic reticulum (SR) Ca2+ cycling, reduced cardiac fibrosis, and improved cardiac function. At the cellular level, reducing SLN expression prevented mitochondrial Ca2+ overload, reduced mitochondrial membrane potential loss, and improved mitochondrial function. Transmission electron microscopy of myocardial tissues and proteomic analysis of mitochondria-associated membranes show that reducing SLN expression improved mitochondrial structure and SR-mitochondria interactions in dystrophic cardiac myocytes. These findings indicate that SLN upregulation plays a significant role in the pathogenesis of cardiomyopathy and that reducing SLN expression has clinical implications in the treatment of DMD-cardiomyopathy.
Satvik Mareedu, Nadezhda Fefelova, Cristi L. Galindo, Goutham Prakash, Risa Mukai, Junichi Sadoshima, Lai-Hua Xie, Gopal J. Babu
People with HIV (PWH) have a higher age-adjusted mortality due to chronic immune activation and age-related comorbidities. PWH also have higher rates of clonal hematopoiesis (CH) than age-matched non-HIV cohorts, however, risk factors influencing the development and expansion of CH in PWH remain incompletely explored. We investigated the relationship between CH, immune biomarkers, and HIV-associated risk factors (CD4, CD8 T-cells, nadir CD4 count, opportunistic infections [OIs], and immune reconstitution inflammatory syndrome [IRIS]) in a diverse cohort of 197-PWH with median age of 42-years, using a 56-gene panel. Seventy-nine percent had a CD4 nadir < 200, 58.9% had prior OIs, and 34.5% had a history of IRIS. The prevalence of CH was high (27.4%), even in younger individuals, and CD8 T-cells and nadir CD4 counts strongly associated with CH after controlling for age. A history of IRIS was associated with CH in a subgroup analysis of ≥ 35-years-old patients. Inflammatory biomarkers were higher in CH carriers compared to non-carriers supporting a dysregulated immune state. These findings suggest PWH with low nadir CD4 and/or inflammatory complications may be at high risk of CH regardless of age and represent a high-risk group that could benefit from risk reduction and potentially targeted immunomodulation.
Joseph M. Rocco, Yifan Zhou, Nicholas S. Liu, Elizabeth Laidlaw, Frances Galindo, Megan V. Anderson, Adam Rupert, Silvia Lucena Lage, Ana M. Ortega-Villa, Shiqin Yu, Andrea Lisco, Maura Manion, George S. Vassiliou, Cynthia E. Dunbar, Irini Sereti
Fibroblast Growth Factor 23 (FGF23) production has recently been shown to increase downstream of G⍺q/11-PKC signaling in osteocytes. Inactivating mutations in the gene encoding G⍺11 (GNA11) cause familial hypocalciuric hypercalcemia (FHH) due to impaired calcium-sensing receptor signaling. We explored the impact of G⍺11 deficiency on FGF23 production in mice with heterozygous (Gna11+/–) or homozygous (Gna11–/–) ablation of Gna11. Both Gna11+/– and Gna11–/– mice demonstrated hypercalcemia and mildly raised parathyroid hormone levels, consistent with FHH. Strikingly, these mice also displayed increased serum levels of total and intact FGF23 and hypophosphatemia. Gna11–/– mice showed augmented Fgf23 mRNA levels in the liver and heart, but not in bone or bone marrow, and evidence of systemic inflammation with elevated serum IL1β levels. Furin gene expression was significantly increased in the Gna11–/– liver, suggesting enhanced FGF23 cleavage despite the observed rise in intact FGF23 levels. Gna11–/– mice had normal renal function and reduced serum levels of glycerol-3-phosphate, excluding kidney injury as the primary cause of elevated intact FGF23 levels. Thus, G⍺11 ablation caused systemic inflammation and excess serum FGF23 in mice, suggesting that FHH patients, at least those with GNA11 mutations, may be at risk for these complications.
Birol Ay, Sajin Marcus Cyr, Kaitlin Klovdahl, Wen Zhou, Christina M. Tognoni, Yorihiro Iwasaki, Eugene P. Rhee, Alpaslan Dedeoglu, Petra Simic, Murat Bastepe
Skeletal muscle wasting results from numerous pathological conditions impacting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss of function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 results in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fibre integrity. We further implicate a disruption in atrogin-1 mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We reveal that BiP is not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorates pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicates atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy, and highlights atrogin-1’s essential role in maintaining muscle homeostasis.
Avnika A. Ruparelia, Margo Montandon, Jo Merriner, Cheng Huang, Siew Fen Lisa Wong, Carmen Sonntag, Justin P. Hardee, Gordon S. Lynch, Lee B. Miles, Ashley Siegel, Thomas E. Hall, Ralf B. Schittenhelm, Peter D. Currie
Novel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections. We applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modelling and network correlation analyses identified 118 differentially expressed proteins, significant through three complementary analytical pipelines. Candidate biomarkers were subsequently analysed in two validation cohorts of differing ethnicity using antibody-based proximity extension assays. TB-specific host biomarkers were confirmed. A six-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14 and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts. This biomarker panel exceeds the World Health Organisation Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.
Hannah F. Schiff, Naomi F. Walker, Cesar Ugarte-Gil, Marc Tebruegge, Antigoni Manousopoulou, Spiros D. Garbis, Salah Mansour, Pak Ho Wong, Gabrielle Rockett, Paolo Piazza, Mahesan Niranjan, Andres F. Vallejo, Christopher H. Woelk, Robert J. Wilkinson, Liku B. Tezera, Diana Garay-Baquero, Paul Elkington
Spine metastases can result in severe neurologic compromise and decreased overall survival. Despite treatment advances, local disease progression is frequent, highlighting the need for novel therapies. Tumor treating fields (TTFields) impair tumor cell replication and are influenced by properties of surrounding tissue. We hypothesize bone’s dielectric properties will enhance TTFields mediated suppression of tumor growth in spine metastasis models. Computational modeling of TTFields intensity was performed following surgical resection of a spinal metastasis and demonstrated enhanced TTFields intensity within the resected vertebral body. Additionally, luciferase-tagged human KRIB osteosarcoma and A549 lung adenocarcinoma cell lines were cultured in demineralized bone grafts and exposed to TTFields. Following TTFields exposure, BLI signal decreased 10-80% of baseline while control cultures displayed 4.48-9.36 fold increase in signal. Lastly, TTFields were applied in an orthotopic murine model of spinal metastasis. After 21 days of treatment, control mice demonstrated a 5-fold increase in BLI signal compared to TTFields treated mice. TTFields similarly prevented tumor invasion into the spinal canal and development of neurologic symptoms. Our data suggest that TTFields can be leveraged as a local therapy within minimally-conductive bone of spine metastases. This provides the groundwork for future studies investigating TTFields for patients with treatment-refractory spine metastases.
Daniel Ledbetter, Romulo de Almeida, Xizi Wu, Ariel Naveh, Chirag B. Patel, Queena Gonzalez, Thomas H. Beckham, Robert North, Laurence Rhines, Jing Li, Amol Ghia, David Aten, Claudio Tatsui, Christopher Alvarez-Breckenridge
HIPK2 is a multifunctional kinase that acts as a key pathogenic mediator of chronic kidney disease and fibrosis. It acts as a central effector of multiple signaling pathways implicated in kidney injury, such as TGF-β/Smad3-mediated extracellular matrix accumulation, NF-κB-mediated inflammation, and p53-mediated apoptosis. Thus, a better understanding of the specific HIPK2 regions necessary for distinct downstream pathway activation is critical for optimal drug development for CKD. Our study now shows that Caspase 6-mediated removal of the C-terminal region of HIPK2 (HIPK2-CT) leads to hyperactive p65 NF-κB transcriptional response in kidney cells. In contrast, the expression of cleaved HIPK2-CT fragment can restrain p65 NF-κB transcriptional activity by cytoplasmic sequestration NF-κB signaling component, p65 NF-κB, and attenuation of IκBα degradation. Therefore, we examined whether HIPK2-CT expression can be exploited to restrain renal inflammation in vivo. The induction of HIPK2-CT overexpression in kidney tubular cells attenuated p65 nuclear translocation, expression of inflammatory cytokines, and macrophage infiltration in the kidney of mice with unilateral ureteral obstruction and lipopolysaccharide-induced acute kidney injury. Collectively, our findings indicate that the C-terminal region of HIPK2 is involved in the regulation of nuclear NF-κB transcriptional activity and that HIPK2-CT or its analogs could be further exploited as potential anti-inflammatory agents to treat kidney disease.
Ye Feng, Zhengzhe Li, Heather Wang, Bi-Cheng Liu, Kyung Lee, John Cijiang He
Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, that showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks-of-age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks-of-age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.
Lisa Duvick, W. Michael Southern, Kellie A. Benzow, Zoe N. Burch, Hillary P. Handler, Jason S. Mitchell, Hannah Kuivinen, Udaya Gadiparthi, Praseuth Yang, Alyssa Soles, Carrie A. Sheeler, Orion Rainwater, Shannah Serres, Erin B. Lind, Tessa Nichols-Meade, Brennon O'Callaghan, Huda Y. Zoghbi, Marija Cvetanovic, Vanessa C. Wheeler, James M. Ervasti, Michael D. Koob, Harry T. Orr
Evaluating the response to immune checkpoint inhibitors (ICIs) remains an unmet challenge in triple-negative breast cancer (TNBC). The requirement of cholesterol for activation and function of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged the PET radiotracer, eFNP-59. eFNP-59 is an analog of cholesterol that our group validated as an imaging biomarker for cholesterol uptake in pre-clinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing ICI-responsive and non-responsive tumors directly, uptake of fluorescent cholesterol and eFNP-59 increased in T cells from ICI-responsive tumors. We discovered that accumulation of cholesterol by T cells increased in ICI-responding tumors that received anti-PD-1 checkpoint immunotherapy. In patients with TNBC, tumors containing cycling T cells had features of cholesterol uptake and trafficking within those populations. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells detects T cell activation and has potential to assess the success of ICI therapy.
Nicholas G. Ciavattone, Jenny Nan Guan, Alex Farfel, Jenelle Stauff, Timothy J. Desmond, Benjamin L. Viglianti, Peter J.H. Scott, Allen F. Brooks, Gary D. Luker
Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA) and the uPA receptor (uPAR), were dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression in a destabilization of the medial meniscus (DMM) mouse model, while genetic deficiency of plasmin activator inhibitor (PAI-1), or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans, induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, PDK1, AKT, and ERK signaling cascades, and activates matrix metalloproteinases (pro-MMPs) to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.
Qian Wang, Guoqiang Shao, Xiaoyi Zhao, Heidi H. Wong, Kate Chin, Mackenzie Zhao, Audrey Bai, Michelle S. Bloom, Zelda Z. Love, Constance R. Chu, Zhen Cheng, William H. Robinson
BACKGROUND. Survivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans. METHODS. We measured the levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from a cohort of 341 patients with respiratory failure and 13 healthy control patients, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We flow-cytometry sorted neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing. RESULTS. Microglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines. CONCLUSIONS. Prolonged lung inflammation results in sustained elevations in circulating cytokines patients with SARS-CoV-2 pneumonia compared to those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction. FUNDING. SCRIPT U19AI135964.
Rogan A. Grant, Taylor A. Poor, Lango Sichizya, Estefani Diaz, Joseph I. Bailey, Sahil Soni, Karolina J. Senkow, Xóchitl G. Pérez-Leonor, Hiam Abdala-Valencia, Ziyan Lu, Helen K. Donnelly, Lacy M. Simons, Egon A. Ozer, Robert M. Tighe, Jon W. Lomasney, Richard G. Wunderink, Benjamin D. Singer, Alexander V. Misharin, G.R. Scott Budinger
BACKGROUND COVID-19 convalescent plasma (CCP) viral specific antibody levels that translate into recipient post-transfusion antibody levels sufficient to prevent disease progression is not defined. METHODS This secondary analysis correlated donor and recipient antibody levels to hospitalization risk among unvaccinated, seronegative CCP recipients within the outpatient, double blind, randomized clinical trial that compared CCP to control plasma. The majority of COVID-19 CCP arm hospitalizations (15/17, 88%) occurred in this unvaccinated, seronegative subgroup. A functional cutoff to delineate recipient high versus low post-transfusion antibody levels was established by two methods: 1) analyzing virus neutralization-equivalent anti-Spike-receptor-binding-domain immunoglobulin G (anti-S-RBD IgG) responses in donors or 2) receiver operated curve (ROC) analysis. RESULTS SARS-CoV-2 anti-S-RBD IgG antibody was volume diluted 21.3 fold into post-transfusion seronegative recipients from matched donor units. Viral specific antibody delivered approximated 1.2 mg. The high antibody recipients transfused early (symptom onset within 5 days) had no hospitalizations. A CCP recipient analysis for antibody thresholds correlated to reduced hospitalizations found a statistical significant association between early transfusion and high antibodies versus all other CCP recipients (or control plasma) with antibody cutoffs established by both methods-donor-based virus neutralization cutoff in post-transfusion recipients: (0/85; 0% versus 15/276; 5.6%) p=0.03 or ROC based cutoff: (0/94; 0% versus 15/267; 5.4%) p=0.01. CONCLUSION In unvaccinated, seronegative CCP recipients, early transfusion of plasma units in the upper 30% of study donors antibody levels reduced outpatient hospitalizations. High antibody level plasma units, given early, should be reserved for therapeutic use. Trial registration: NCT04373460 FUNDING Defense Health Agency and others.
Han-Sol Park, Anna Yin, Caelan Barranta, John S. Lee, Christopher A. Caputo, Jaiprasath Sachithanandham, Maggie Li, Steve Yoon, Ioannis Sitaras, Anne Jedlicka, Yolanda Eby, Malathi Ram, Reinaldo E. Fernandez, Owen R. Baker, Aarthi G. Shenoy, Giselle S. Mosnaim, Yuriko Fukuta, Bela Patel, Sonya L. Heath, Adam C. Levine, Barry R. Meisenberg, Emily S. Spivak, Shweta Anjan, Moises A. Huaman, Janis E. Blair, Judith S. Currier, James H. Paxton, Jonathan M. Gerber, Joann R. Petrini, Patrick B. Broderick, William Rausch, Marie Elena Cordisco, Jean Hammel, Benjamin Greenblatt, Valerie C. Cluzet, Daniel Cruser, Kevin Oei, Matthew Abinante, Laura L. Hammitt, Catherine G. Sutcliffe, Donald N. Forthal, Martin S. Zand, Edward R. Cachay, Jay S. Raval, Seble G. Kassaye, Christi E. Marshall, Anusha Yarava, Karen Lane, Nichol A. McBee, Amy L. Gawad, Nicky Karlen, Atika Singh, Daniel E. Ford, Douglas A. Jabs, Lawrence J. Appel, David M. Shade, Bryan Lau, Stephan Ehrhardt, Sheriza N. Baksh, Janna R. Shapiro, Jiangda Ou, Yu Bin Na, Maria D. Knoll, Elysse Ornelas-Gatdula, Netzahualcóyotl Arroyo-Currás, Thomas J. Gniadek, Patrizio Caturegli, Jinke Wu, Nelson Ndahiro, Michael J. Betenbaugh, Alyssa Ziman, Daniel F. Hanley, Arturo Casadevall, Shmuel Shoham, Evan M. Bloch, Kelly A. Gebo, Aaron A.R. Tobian, Oliver Laeyendecker, Andrew Pekosz, Sabra L. Klein, David J. Sullivan
The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected >5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may impact cardiac contractility.
Colleen M. Kelly, Jody L. Martin, Michael J. Previs
Glioblastoma (GBM) remains an incurable disease, requiring more effective therapies. Through interrogation of publicly available CRISPR and RNAi library screens, we identified the alpha-ketoglutarate dehydrogenase (OGDH) gene, which encodes for an enzyme that is part of the tricarboxylic acid cycle (TCA cycle) as essential for GBM growth. Moreover, by combining a transcriptome and metabolite screening analyses we discovered that loss of function of OGDH by the clinically validated drug compound, CPI-613, was synthetically lethal with Bcl-xL inhibition (genetically and through the clinically validated BH3-mimetic, ABT263) in patient-derived xenograft as well neurosphere GBM cultures. CPI-613 mediated energy deprivation drove an integrated stress response with an up-regulation of the BH3-only domain protein, Noxa in an ATF4 dependent manner as demonstrated by genetic loss of function experiments. Consistently, silencing of Noxa attenuated cell death induced by CPI-613 in model systems of GBM. In patient-derived xenograft models of GBM in mice, the combination treatment of ABT263 and CPI-613 suppressed tumor growth and extended animal survival more potently than each compound on its own. Therefore, combined inhibition of Bcl-xL along with interference of the TCA-cycle might be a treatment strategy for GBM.
Trang T.T. Nguyen, Consuelo Torrini, Enyuan Shang, Chang Shu, Jeong-Yeon Mun, Qiuqiang Gao, Nelson Humala, Hasan O. Akman, Guoan Zhang, Mike-Andrew Westhoff, Georg Karpel-Massler, Jeffrey N. Bruce, Peter Canoll, Markus D. Siegelin
Both anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) originate from thyroid follicular epithelial cells, but ATC has a significantly worse prognosis and shows resistance to conventional therapies. However, clinical trials found that immunotherapy works better in ATC than late-stage PTC. Here, we utilized single-cell RNA sequencing (scRNA-seq) to generate a single-cell atlas of thyroid cancer. Differences in ATC and PTC tumor microenvironment (TME) components (including malignant cells, stromal cells, and immune cells) leading to the polarized prognoses were identified. Intriguingly, we found that CXCL13+ T lymphocytes were enriched in ATC samples and might promote the development of early tertiary lymphoid structure (TLS). Lastly, murine experiments and scRNA-seq analysis of a treated patient’s tumor demonstrated that Famitinib plus anti-PD-1 antibody could advance TLS in thyroid cancer. Conclusively, we displayed the cellular landscape of ATC and PTC, finding that CXCL13+ T cells and early TLS might make ATC more sensitive to immunotherapy.
Pei-Zhen Han, Wei-Dong Ye, Peng-Cheng Yu, Li-Cheng Tan, Xiao Shi, Xu-Feng Chen, Cong He, Jia-Qian Hu, Wen-Jun Wei, Zhong-Wu Lu, Ning Qu, Yu Wang, Qing-Hai Ji, Dong-Mei Ji, Yu-Long Wang
IL-17C is an epithelial cell-derived proinflammatory cytokine whose transcriptional regulation remains unclear. Analysis of the IL17C promoter region identified TCF4 as putative regulator and siRNA knockdown of TCF4 in human keratinocytes (KCs) increased IL17C. IL-17C stimulation of KCs (along with IL-17A and TNF-α) decreased TCF4 and increased NFKBIZ and ZC3H12A expression in an IL-17RA/RE-dependent manner thus creating a feedback loop. ZC3H12A (MCPIP1/Regnase-1), a transcriptional immune-response regulator also increased following TCF4 siRNA knockdown and siRNA knockdown of ZC3H12A decreased NFKBIZ, IL1B, IL36G, CCL20, and CXCL1, revealing a proinflammatory role for ZC3H12A. Examination of lesional skin from the KC-Tie2 inflammatory dermatitis mouse model identified decreases in TCF4 protein concomitant with increases in IL-17C and Zc3h12a, that reversed following the genetic elimination of Il17c, Il17ra, and Il17re and improvement in the skin phenotype. Conversely, interference with Tcf4 in KC-Tie2 mouse skin increased Il17c and exacerbated the inflammatory skin phenotype. Together these findings identify a role for TCF4 in the negative regulation of IL-17C, which alone and with TNF-α and IL-17A, feedback to decrease TCF4 in an IL-17RA/RE-dependent manner. This loop is further amplified by IL-17C-TCF4 autocrine regulation of ZC3H12A and IL-17C regulation of NFKBIZ to promote self-sustaining skin inflammation.
Yanyun Jiang, Dennis Gruszka, Chang Zeng, William R. Swindell, Christa Gaskill, Christian Sorensen, Whitney Brown, Roopesh Singh Gangwar, Lam C. Tsoi, Joshua Webster, Sigrun Laufey Sigurdardottir, Mrinal K. Sarkar, Ranjitha Uppala, Austin Kidder, Xianying Xing, Olesya Plazyo, Enze Xing, Allison C. Billi, Emanual Maverakis, J. Michelle Kahlenberg, Johann Gudjonsson, Nicole L. Ward
IKK2-NF𝜅B pathway mediated-inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2-NF𝜅B pathway in medial calcification remains to be elucidated. In this study, we found that CKD induces inflammatory pathways through the local activation of the IKK2-NF𝜅B pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2-NF𝜅B pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NF𝜅B by SMC-specific I𝜅B-α deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2-NF𝜅B pathway induced cell death of VSMCs by reducing anti-cell death gene expression, whereas activation of NF𝜅B reduced CKD-dependent vascular cell death. In addition, increased calcifying extracellular vesicles through the inhibition of the IKK2-NF𝜅B pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death in vitro and in vivo. This study reveals that activation of the IKK2-NF𝜅B pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles.
Shinobu Miyazaki-Anzai, Masashi Masuda, Audrey L. Keenan, Yuji Shiozaki, Jose G. Miranda, Makoto Miyazaki
Excessive lipolysis in white adipose tissues (WAT) leads to insulin resistance (IR) and ectopic fat accumulation in insulin-sensitive tissues. However, the impact of Gi-coupled receptors in restraining adipocyte lipolysis through inhibition of cAMP production remained poorly elucidated. Given that the Gi-coupled P2Y13 receptor (P2Y13-R) is a purinergic receptor expressed in WAT, we investigated its role in adipocyte lipolysis and its effect on IR and metabolic dysfunction-associated steatotic liver disease (MASLD). In human, mRNA expression of P2Y13-R in WAT was negatively correlated to adipocytes lipolysis. In mice, adipocytes lacking P2Y13-R displayed higher intracellular cAMP levels, indicating impaired Gi signaling. Consistently, the absence of P2Y13-R was linked to increased lipolysis in adipocytes and WAT explants via hormone-sensitive lipase activation. Metabolic studies indicate that mice lacking P2Y13-R show a greater susceptibility to diet-induced IR, systemic inflammation, and MASLD compared to their wild-type counterparts. Assays conducted on precision-cut liver slices exposed to WAT conditioned medium and on liver-specific P2Y13-R knockdown mice suggested that P2Y13-R activity in WAT protects from hepatic steatosis, independently of liver P2Y13-R expression. In conclusion, our findings support the idea that targeting adipose P2Y13-R activity may represent a pharmacological strategy to prevent obesity-associated disorders, including type 2 diabetes and MASLD.
Thibaut Duparc, Emilia Gore, Guillaume Combes, Diane Beuzelin, Julie Pires Da Silva, Vanessa Bouguetoch, Marie-Adeline Marquès, Ana Velazquez, Nathalie Viguerie, Geneviève Tavernier, Peter Arner, Mikael Rydén, Dominique Langin, Nabil Sioufi, Mohamad Nasser, Cendrine Cabou, Souad Najib, Laurent O. Martinez