Cross-species single-cell analysis uncovers the immunopathological mechanisms associated with IgA nephropathy progression
Chen et al. report single-cell transcriptomic characteristics of IgA nephropathy across species and identify key cell types and molecular pathways associated with disease progression. The cover art sho ws the recruitment of bone marrow–derived immune cells, especially macrophages, that infiltrate into kidney tissue through blood vessels and secrete immune factors to activate mesangial cells, thereby promoting IgA nephropathy immune injury. Image credit: Yiyao Deng.
-
Technical Advance
×
Abstract
Spinal 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 hypothesized that bone’s dielectric properties will enhance TTFields-mediated suppression of tumor growth in spinal 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, the bioluminescence imaging (BLI) signal decreased to 10%–80% of baseline, while control cultures displayed a 4.48- to 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 with 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 spinal metastases. This provides the groundwork for future studies investigating TTFields for patients with treatment-refractory spinal metastases.
Authors
Daniel Ledbetter, Romulo Augusto Andrade 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
-
Physician-Scientist Development
×
Abstract
Previous studies on attrition from MD-PhD programs have shown that students who self-identify as Black are more likely to withdraw before graduating than Hispanic students and students not from groups underrepresented in medicine (non-UIM). Here, we analyzed data collected for the National MD-PhD Program Outcomes Study, a national effort to track the careers of over 10,000 individuals who have graduated from MD-PhD programs over the past 60 years. On average, Black trainees took slightly longer to graduate, were less likely to choose careers in academia, and were more likely to enter nonacademic clinical practice; although, none of these differences were large. Black graduates were also more likely to choose careers in surgery or internal medicine, or entirely forego residency, and less likely to choose pediatrics, pathology, or neurology. Among those in academia, average research effort rates self-reported by Black, Hispanic, and non-UIM alumni were indistinguishable, as were rates of obtaining research grants and mentored training awards. However, the proportion of Black and Hispanic alumni who reported having NIH research grants was lower than that of non-UIM alumni, and the NIH career development to research project grant (K-to-R) conversion rate was lower for Black alumni. We propose that the reasons for these differences reflect experiences before, during, and after training and, therefore, conclude with action items that address each of these stages.
Authors
Myles H. Akabas, Lawrence F. Brass
-
Research Articles
×
Abstract
Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent antiinflammatory and antitumor activity in several preclinical models and has proven both safe and efficacious in clinical trials for advanced gastrointestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared with posttransplant cyclophosphamide (PTCy). Strikingly, we found Minnelide improved survival, weight loss, and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC–matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score, and colonic length. Notably, in addition to decreased donor T cell infiltration early after aHSCT, several regulatory cell populations, including Tregs, ILC2s, and myeloid-derived stem cells in the colon were increased, which together may account for Minnelide’s GVHD suppression after aHSCT. Importantly, Minnelide’s GVHD prevention was accompanied by preservation of graft-versus-tumor activity. As Minnelide possesses anti–acute myeloid leukemia (anti-AML) activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for patients with AML undergoing aHSCT.
Authors
Sabrina N. Copsel, Vanessa T. Garrido, Henry Barreras, Cameron S. Bader, Brent Pfeiffer, Beatriz Mateo-Victoriano, Dietlinde Wolf, Miguel Gallardo, Sophie Paczesny, Krishna V. Komanduri, Cara L. Benjamin, Alejandro V. Villarino, Ashok K. Saluja, Robert B. Levy
×Abstract
T cells are required for protective immunity against Mycobacterium tuberculosis. We recently described a cohort of Ugandan household contacts of tuberculosis cases who appear to “resist” M. tuberculosis infection (resisters; RSTRs) and showed that these individuals harbor IFN-γ–independent T cell responses to M. tuberculosis–specific peptide antigens. However, T cells also recognize nonprotein antigens via antigen-presenting systems that are independent of genetic background, known as donor-unrestricted T cells (DURTs). We used tetramer staining and flow cytometry to characterize the association between DURTs and “resistance” to M. tuberculosis infection. Peripheral blood frequencies of most DURT subsets were comparable between RSTRs and latently infected controls (LTBIs). However, we observed a 1.65-fold increase in frequency of MR1-restricted T (MR1T) cells among RSTRs in comparison with LTBIs. Single-cell RNA sequencing of 18,251 MR1T cells sorted from 8 donors revealed 5,150 clonotypes that expressed a common transcriptional program, the majority of which were private. Sequencing of the T cell receptor α/T cell receptor δ (TCRα/δ) repertoire revealed several DURT clonotypes were expanded among RSTRs, including 2 MR1T clonotypes that recognized mycobacteria-infected cells in a TCR-dependent manner. Overall, our data reveal unexpected donor-specific diversity in the TCR repertoire of human MR1T cells as well as associations between mycobacteria-reactive MR1T clonotypes and resistance to M. tuberculosis infection.
Authors
Deborah L. Cross, Erik D. Layton, Krystle K.Q. Yu, Malisa T. Smith, Melissa S. Aguilar, Shamin Li, Elise C. Wilcox, Aude G. Chapuis, Harriet Mayanja-Kizza, Catherine M. Stein, W. Henry Boom, Thomas R. Hawn, Philip Bradley, Evan W. Newell, Chetan Seshadri
×Abstract
The importance of the proper localization of most receptors at the cell surface is often underestimated, although this feature is essential for optimal receptor response. Endospanin 1 (Endo1) (also known as OBRGRP or LEPROT) is a protein generated from the same gene as the human leptin receptor and regulates the trafficking of proteins to the surface, including the leptin receptor. The systemic role of Endo1 on whole-body metabolism has not been studied so far. Here, we report that general Endo1-KO mice fed a high-fat diet develop metabolically healthy obesity with lipid repartitioning in organs and preferential accumulation of fat in adipose tissue, limited systematic inflammation, and better controlled glucose homeostasis. Mechanistically, Endo1 interacts with the lipid translocase CD36, thus regulating its surface abundance and lipid uptake in adipocytes. In humans, the level of Endo1 transcripts is increased in the adipose tissue of patients with obesity, but low levels rather correlate with a profile of metabolically healthy obesity. We suggest here that Endo1, most likely by controlling CD36 cell surface abundance and lipid uptake in adipocytes, dissociates obesity from diabetes and that its absence participates in metabolically healthy obesity.
Authors
Arturo Roca-Rivada, Marcio Do Cruzeiro, Raphaël G.P. Denis, Qiang Zhang, Christine Rouault, Yves Rouillé, Jean-Marie Launay, Céline Cruciani-Guglielmacci, Virginie Mattot, Karine Clément, Ralf Jockers, Julie Dam
×Abstract
Lung cancer is the leading cause of cancer-related deaths in the world, and non–small cell lung cancer (NSCLC) is the most common subset. We previously found that infiltration of tumor inflammatory monocytes (TIMs) into lung squamous carcinoma (LUSC) tumors is associated with increased metastases and poor survival. To further understand how TIMs promote metastases, we compared RNA-Seq profiles of TIMs from several LUSC metastatic models with inflammatory monocytes (IMs) of non–tumor-bearing controls. We identified Spon1 as upregulated in TIMs and found that Spon1 expression in LUSC tumors corresponded with poor survival and enrichment of collagen extracellular matrix signatures. We observed SPON1+ TIMs mediate their effects directly through LRP8 on NSCLC cells, which resulted in TGF-β1 activation and robust production of fibrillar collagens. Using several orthogonal approaches, we demonstrated that SPON1+ TIMs were sufficient to promote NSCLC metastases. Additionally, we found that Spon1 loss in the host, or Lrp8 loss in cancer cells, resulted in a significant decrease of both high-density collagen matrices and metastases. Finally, we confirmed the relevance of the SPON1/LRP8/TGF-β1 axis with collagen production and survival in patients with NSCLC. Taken together, our study describes how SPON1+ TIMs promote collagen remodeling and NSCLC metastases through an LRP8/TGF-β1 signaling axis.
Authors
Kristina M. Whately, Nisitha Sengottuvel, Lincy Edatt, Sonal Srivastava, Allison T. Woods, Yihsuan S. Tsai, Alessandro Porrello, Matthew P. Zimmerman, Aaron C. Chack, Stuart R. Jefferys, Gabriella Yacovone, Dae Joong Kim, Andrew C. Dudley, Antonio L. Amelio, Chad V. Pecot
×Abstract
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 has been largely unexplored. Here, we show that CyD was upregulated in normal wounds and venous ulcers, likely adaptive as CyD inhibition impaired reepithelialization, granulation tissue formation, and wound closure in both human and pig models. Overexpression of CyD increased keratinocyte migration and fibroblast proliferation, while 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-KO 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.
Authors
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
×Abstract
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 double-knockout (mdx:utrn–/–) mice in a 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 showed that reducing SLN expression improved mitochondrial structure and SR-mitochondria interactions in dystrophic cardiomyocytes. These findings indicate that SLN upregulation plays a substantial role in the pathogenesis of cardiomyopathy and that reducing SLN expression has clinical implications in the treatment of DMD cardiomyopathy.
Authors
Satvik Mareedu, Nadezhda Fefelova, Cristi L. Galindo, Goutham Prakash, Risa Mukai, Junichi Sadoshima, Lai-Hua Xie, Gopal J. Babu
×Abstract
Vaccination of malaria-naive volunteers with a high dose of Plasmodium falciparum sporozoites chemoattenuated by chloroquine (CQ) (PfSPZ-CVac [CQ]) has previously demonstrated full protection against controlled human malaria infection (CHMI). However, lower doses of PfSPZ-CVac [CQ] resulted in incomplete protection. This provides the opportunity to understand the immune mechanisms needed for better vaccine-induced protection by comparing individuals who were protected with those not protected. Using mass cytometry, we characterized immune cell composition and responses of malaria-naive European volunteers who received either lower doses of PfSPZ-CVac [CQ], resulting in 50% protection irrespective of the dose, or a placebo vaccination, with everyone becoming infected following CHMI. Clusters of CD4+ and γδ T cells associated with protection were identified, consistent with their known role in malaria immunity. Additionally, EMRA CD8+ T cells and CD56+CD8+ T cell clusters were associated with protection. In a cohort from a malaria-endemic area in Gabon, these CD8+ T cell clusters were also associated with parasitemia control in individuals with lifelong exposure to malaria. Upon stimulation with P. falciparum–infected erythrocytes, CD4+, γδ, and EMRA CD8+ T cells produced IFN-γ and/or TNF, indicating their ability to mediate responses that eliminate malaria parasites.
Authors
Yoanne D. Mouwenda, Simon P. Jochems, Vincent Van Unen, Madeleine Eunice Betouke Ongwe, Wouter A.A. de Steenhuijsen Piters, Koen A. Stam, Marguerite Massinga Loembe, Betty Kim Lee Sim, Meral Esen, Stephen L. Hoffman, Peter G. Kremsner, Rolf Fendel, Benjamin Mordmüller, Maria Yazdanbakhsh
×Abstract
Childhood-onset essential hypertension (COEH) is an uncommon form of hypertension that manifests in childhood or adolescence and, in the United States, disproportionately affects children of African ancestry. The etiology of COEH is unknown, but its childhood onset, low prevalence, high heritability, and skewed ancestral demography suggest the potential to identify rare genetic variation segregating in a Mendelian manner among affected individuals and thereby implicate genes important to disease pathogenesis. However, no COEH genes have been reported to date. Here, we identify recessive segregation of rare and putatively damaging missense variation in the spectrin domain of spectrin repeat containing nuclear envelope protein 1 (SYNE1), a cardiovascular candidate gene, in 3 of 16 families with early-onset COEH without an antecedent family history. By leveraging exome sequence data from an additional 48 COEH families, 1,700 in-house trios, and publicly available data sets, we demonstrate that compound heterozygous SYNE1 variation in these COEH individuals occurred more often than expected by chance and that this class of biallelic rare variation was significantly enriched among individuals of African genetic ancestry. Using in vitro shRNA knockdown of SYNE1, we show that reduced SYNE1 expression resulted in a substantial decrease in the elasticity of smooth muscle vascular cells that could be rescued by pharmacological inhibition of the downstream RhoA/Rho-associated protein kinase pathway. These results provide insights into the molecular genetics and underlying pathophysiology of COEH and suggest a role for precision therapeutics in the future.
Authors
Ian Copeland, Edmond Wonkam-Tingang, Monesha Gupta-Malhotra, S. Shahrukh Hashmi, Yixing Han, Aarti Jajoo, Nancy J. Hall, Paula P. Hernandez, Natasha Lie, Dan Liu, Jun Xu, Jill Rosenfeld, Aparna Haldipur, Zelene Desire, Zeynep H. Coban-Akdemir, Daryl A. Scott, Qing Li, Hsiao-Tuan Chao, Ana M. Zaske, James R. Lupski, Dianna M. Milewicz, Sanjay Shete, Jennifer E. Posey, Neil A. Hanchard
×Intimomedial tears of the aorta heal by smooth muscle cell–mediated fibrosis without atherosclerosis
Abstract
BACKGROUND Disease of the aorta varies from atherosclerosis to aneurysms, with complications including rupture, dissection, and poorly characterized limited tears. We studied limited tears without any mural hematoma, termed intimomedial tears, to gain insight into aortic vulnerability to excessive wall stresses. Our premise is that minimal injuries in aortas with sufficient medial resilience to prevent tear progression correspond to initial mechanisms leading to complete structural failure in aortas with significantly compromised medial resilience.METHODS Intimomedial tears were macroscopically identified in 9 of 108 ascending aortas after surgery and analyzed by histology and immunofluorescence confocal microscopy.RESULTS Nonhemorrhagic, nonatheromatous tears correlated with advanced aneurysmal disease and most lacked distinctive symptoms or radiological signs. Tears traversed the intima and part of the subjacent media, while the resultant defects were partially or completely filled with neointima characterized by differentiated smooth muscle cells, scattered leukocytes, dense fibrosis, and absent elastic laminae despite tropoelastin synthesis. Healed lesions contained organized fibrin at tear edges without evidence of plasma and erythrocyte extravasation or lipid accumulation.CONCLUSION These findings suggest a multiphasic model of aortic wall failure in which primary lesions of intimomedial tears either heal if the media is sufficiently resilient or progress as dissection or rupture by medial delamination and tear completion, respectively. Moreover, mural incorporation of thrombus and cellular responses to injury, two historically important concepts in atheroma pathogenesis, contribute to vessel wall repair with adequate conduit function, but even together are not sufficient to induce atherosclerosis.FUNDING NIH (R01-HL146723, R01-HL168473) and Yale Department of Surgery.
Authors
Abdulrahman H.M. Hassab, David J. Hur, Prashanth Vallabhajosyula, George Tellides, Roland Assi
×Abstract
IgA nephropathy (IgAN) represents the main cause of renal failure, while the precise pathogenetic mechanisms have not been fully determined. Herein, we conducted a cross-species single-cell survey on human IgAN and mouse and rat IgAN models to explore the pathogenic programs. Cross-species single-cell RNA sequencing (scRNA-Seq) revealed that the IgAN mesangial cells (MCs) expressed high levels of inflammatory signatures CXCL12, CCL2, CSF1, and IL-34 and specifically interacted with IgAN macrophages via the CXCL12/CXCR4, CSF1/IL-34/CSF1 receptor, and integrin subunit alpha X/integrin subunit alpha M/complement C3 (C3) axes. IgAN macrophages expressed high levels of CXCR4, PDGFB, triggering receptor expressed on myeloid cells 2, TNF, and C3, and the trajectory analysis suggested that these cells derived from the differentiation of infiltrating blood monocytes. Additionally, protein profiling of 21 progression and 28 nonprogression IgAN samples revealed that proteins CXCL12, C3, mannose receptor C-type 1, and CD163 were negatively correlated with estimated glomerular filtration rate (eGFR) value and poor prognosis (30% eGFR as composite end point). Last, a functional experiment revealed that specific blockade of the Cxcl12/Cxcr4 pathway substantially attenuated the glomerulus and tubule inflammatory injury, fibrosis, and renal function decline in the mouse IgAN model. This study provides insights into IgAN progression and may aid in the refinement of IgAN diagnosis and the optimization of treatment strategies.
Authors
Xizhao Chen, Tiantian Wang, Lei Chen, Yinghua Zhao, Yiyao Deng, Wanjun Shen, Lin Li, Zhong Yin, Chaoran Zhang, Guangyan Cai, Min Zhang, Xiangmei Chen
×Abstract
Diagnostic challenges continue to impede development of effective therapies for successful management of alcohol-associated hepatitis (AH), creating an unmet need to identify noninvasive biomarkers for AH. In murine models, complement contributes to ethanol-induced liver injury. Therefore, we hypothesized that complement proteins could be rational diagnostic/prognostic biomarkers in AH. Here, we performed a comparative analysis of data derived from human hepatic and serum proteome to identify and characterize complement protein signatures in severe AH (sAH). The quantity of multiple complement proteins was perturbed in liver and serum proteome of patients with sAH. Multiple complement proteins differentiated patients with sAH from those with alcohol cirrhosis (AC) or alcohol use disorder (AUD) and healthy controls (HCs). Serum collectin 11 and C1q binding protein were strongly associated with sAH and exhibited good discriminatory performance among patients with sAH, AC, or AUD and HCs. Furthermore, complement component receptor 1-like protein was negatively associated with pro-inflammatory cytokines. Additionally, lower serum MBL associated serine protease 1 and coagulation factor II independently predicted 90-day mortality. In summary, meta-analysis of proteomic profiles from liver and circulation revealed complement protein signatures of sAH, highlighting a complex perturbation of complement and identifying potential diagnostic and prognostic biomarkers for patients with sAH.
Authors
Moyinoluwa Taiwo, Emily Huang, Vai Pathak, Annette Bellar, Nicole Welch, Jaividhya Dasarathy, David Streem, Craig J. McClain, Mack C. Mitchell, Bruce A. Barton, Gyongyi Szabo, Srinivasan Dasarathy, Alcohol Hepatitis Network (AlcHepNet) Consortium, Esperance A. Schaefer, Jay Luther, Le Z. Day, Xinshou Ouyang, Arumugam Suyavaran, Wajahat Z. Mehal, Jon M. Jacobs, Russell P. Goodman, Daniel M. Rotroff, Laura E. Nagy
×Abstract
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+ and 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 below 200 cells/μL, 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 patients 35 years of age and older. Inflammatory biomarkers were higher in CH carriers compared with noncarriers, 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.
Authors
Joseph M. Rocco, Yifan Zhou, Nicholas S. Liu, Elizabeth Laidlaw, Frances Galindo, Megan V. Anderson, Adam Rupert, Silvia L. Lage, Ana M. Ortega-Villa, Shiqin Yu, Andrea Lisco, Maura Manion, George S. Vassiliou, Cynthia E. Dunbar, Irini Sereti
×Abstract
mRNA vaccines are likely to become widely used for the prevention of infectious diseases in the future. Nevertheless, a notable gap exists in mechanistic data, particularly concerning the potential effects of sequential mRNA immunization or preexisting immunity on the early innate immune response triggered by vaccination. In this study, healthy adults, with or without documented prior SARS-CoV-2 infection, were vaccinated with the BNT162b2/Comirnaty mRNA vaccine. Prior infection conferred significantly stronger induction of proinflammatory and type I IFN–related gene signatures, serum cytokines, and monocyte expansion after the prime vaccination. The response to the second vaccination further increased the magnitude of the early innate response in both study groups. The third vaccination did not further increase vaccine-induced inflammation. In vitro stimulation of PBMCs with TLR ligands showed no difference in cytokine responses between groups, or before or after prime vaccination, indicating absence of a trained immunity effect. We observed that levels of preexisting antigen-specific CD4 T cells, antibody, and memory B cells correlated with elements of the early innate response to the first vaccination. Our data thereby indicate that preexisting memory formed by infection may augment the innate immune activation induced by mRNA vaccines.
Authors
Fredrika Hellgren, Anja Rosdahl, Rodrigo Arcoverde Cerveira, Klara Lenart, Sebastian Ols, Yong-Dae Gwon, Seta Kurt, Anna Maria Delis, Gustav Joas, Magnus Evander, Johan Normark, Clas Ahlm, Mattias N.E. Forsell, Sara Cajander, Karin Loré
×Abstract
BACKGROUND. Upper-body obesity (UBO) results in insulin resistance with regards to free fatty acid (FFA) release; how this differs by fat depot and sex between adults with UBO and lean adults is unknown. We tested the hypothesis that insulin suppression of FFA release from the splanchnic bed, leg fat, and upper-body nonsplanchnic (UBNS) adipose tissue would be impaired in UBO. METHODS. Fourteen volunteers with UBO (7 men and 7 women) and 14 healthy volunteers with normal weight (7 men and 7 women) participated in studies that included femoral artery, femoral vein, and hepatic vein catheterization. We then measured leg and splanchnic plasma flow as well as FFA kinetics (using isotopic tracers) under overnight fasting as well as low- and high-dose insulin infusion using the insulin clamp technique. RESULTS. We found the expected insulin resistance in UBO; the most quantitatively important difference between adults with UBO and lean adults was greater FFA release from UBNS adipose tissue when plasma insulin concentrations were in the postprandial, physiological range. There were obesity, but not sex, differences in the regulation of splanchnic FFA release and sex differences in the regulation of leg FFA release. CONCLUSION. Reversing the defects in insulin-regulated UBNS adipose tissue FFA release would have the greatest effect on systemic FFA abnormalities in UBO. FUNDING. These studies were supported by the US Public Health Service (grants DK45343 and DK40484), the Novo Nordic Foundation (grant NNF18OC0031804 and NNF16OC0021406), and the Independent Research Fund Denmark (grant 8020-00420B).
Authors
Søren Nielsen, Michael D. Jensen
×Abstract
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, which 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.
Authors
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, Yun You, Brennon O’Callaghan, Huda Y. Zoghbi, Marija Cvetanovic, Vanessa C. Wheeler, James M. Ervasti, Michael D. Koob, Harry T. Orr
×Abstract
The viral kinetics of documented SARS-CoV-2 infections exhibit a high degree of interindividual variability. We identified 6 distinct viral shedding patterns, which differed according to peak viral load, duration, expansion rate, and clearance rate, by clustering data from 768 infections in the National Basketball Association cohort. Omicron variant infections in previously vaccinated individuals generally led to lower cumulative shedding levels of SARS-CoV-2 than other scenarios. We then developed a mechanistic mathematical model that recapitulated 1,510 observed viral trajectories, including viral rebound and cases of reinfection. Lower peak viral loads were explained by a more rapid and sustained transition of susceptible cells to a refractory state during infection as well as by an earlier and more potent late, cytolytic immune response. Our results suggest that viral elimination occurs more rapidly during Omicron infection, following vaccination, and following reinfection due to enhanced innate and acquired immune responses. Because viral load has been linked with COVID-19 severity and transmission risk, our model provides a framework for understanding the wide range of observed SARS-CoV-2 infection outcomes.
Authors
Katherine Owens, Shadisadat Esmaeili, Joshua T. Schiffer
×Abstract
Recent studies have uncovered that noncoding sequence variants may relate to Axenfeld-Rieger syndrome (ARS), a rare developmental anomaly with genetic heterogeneity. However, how these genomic regions are functionally and structurally associated with ARS is still unclear. In this study, we performed genome-wide linkage analysis and whole-genome sequencing in a Chinese family with ARS and identified a heterozygous deletion of about 570 kb (termed LOH-1) in the intergenic sequence between paired-like homeodomain transcription factor 2 (PITX2) and family with sequence similarity 241 member A. Knockout of LOH-1 homologous sequences caused ARS phenotypes in mice. RNA-Seq and real-time quantitative PCR revealed a significant reduction in Pitx2 gene expression in LOH-1–/– mice, while forkhead box C1 expression remained unchanged. ChIP-Seq and bioinformatics analysis identified a potential enhancer region (LOH-E1) within LOH-1. Deletion of LOH-E1 led to a substantial downregulation of the PITX2 gene. Mechanistically, we found a sequence (hg38 chr4:111,399,594–111,399,691) that is on LOH-E1 could regulate PITX2 by binding to RAD21, a critical component of the cohesin complex. Knockdown of RAD21 resulted in reduced PITX2 expression. Collectively, our findings indicate that a potential enhancer sequence that is within LOH-1 may regulate PITX2 expression remotely through cohesin-mediated loop domains, leading to ARS when absent.
Authors
Yizheng Jiang, Yu Peng, Qi Tian, Zhe Cheng, Bei Feng, Junping Hu, Lu Xia, Hui Guo, Kun Xia, Liang Zhou, Zhengmao Hu
×Abstract
Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer characterized by a poor outcome and an increasing incidence. A significant majority (>80%) of newly diagnosed cases are deemed unresectable, leaving chemotherapy as the sole viable option, though with only moderate success. This necessitates the identification of improved therapeutic options for PDA. We hypothesized that there are temporal variations in cancer-relevant processes within PDA tumors, offering insights into the optimal timing of drug administration — a concept termed chronotherapy. In this study, we explored the presence of the circadian transcriptome in PDA using patient-derived organoids and validated these findings by comparing PDA data from The Cancer Genome Atlas with noncancerous healthy pancreas data from GTEx. Several PDA-associated pathways (cell cycle, stress response, Rho GTPase signaling) and cancer driver hub genes (EGFR and JUN) exhibited a cancer-specific rhythmic pattern intricately linked to the circadian clock. Through the integration of multiple functional measurements for rhythmic cancer driver genes, we identified top chronotherapy targets and validated key findings in molecularly divergent pancreatic cancer cell lines. Testing the chemotherapeutic efficacy of clinically relevant drugs further revealed temporal variations that correlated with drug-target cycling. Collectively, our study unravels the PDA circadian transcriptome and highlights a potential approach for optimizing chrono-chemotherapeutic efficacy.
Authors
Deepak Sharma, Darbaz Adnan, Mostafa K. Abdel-Reheem, Ron C. Anafi, Daniel D. Leary, Faraz Bishehsari
×Abstract
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 participating in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children cohort. Twenty FLG LoF variants were identified, including 1 novel variant and 9 variants not previously associated with AD. FLG LoF variants were found in the cohort. Among these children, the presence of 1 or more FLG LoF variants was associated with moderate/severe AD compared with those with mild AD. Children with FLG LoF variants had a higher SCORing for Atopic Dermatitis (SCORAD) and higher likelihood of food allergy within the first 2.5 years of life. LoF variants were associated with higher transepidermal water loss (TEWL) in both lesional and nonlesional skin. Collectively, our study identifies established and potentially novel AD-associated FLG LoF variants and associates FLG LoF variants with higher TEWL in lesional and nonlesional skin.
Authors
Samuel J. Virolainen, Latha Satish, Jocelyn M. Biagini, Hassan Chaib, Wan Chi Chang, Phillip J. Dexheimer, Michael R. Dixon, Katelyn Dunn, David Fletcher, Carmy Forney, Marissa Granitto, Matthew S. Hestand, Makenna Hurd, Kenneth Kauffman, Lucinda Lawson, Lisa J. Martin, Loren D.M. Peña, Kieran J. Phelan, Molly Shook, Matthew T. Weirauch, Gurjit K. Khurana Hershey, Leah C. Kottyan
×Abstract
Atopic dermatitis (AD) is an inflammatory skin condition with a childhood prevalence of up to 25%. Microbial dysbiosis is characteristic of AD, with Staphylococcus aureus the most frequent pathogen associated with disease flares and increasingly implicated in disease pathogenesis. Therapeutics to mitigate the effects of S. aureus have had limited efficacy and S. aureus–associated temporal disease flares are synonymous with AD. An alternative approach is an anti–S. aureus vaccine, tailored to AD. Experimental vaccines have highlighted the importance of T cells in conferring protective anti–S. aureus responses; however, correlates of T cell immunity against S. aureus in AD have not been identified. We identify a systemic and cutaneous immunological signature associated with S. aureus skin infection (ADS.aureus) in a pediatric AD cohort, using a combined Bayesian multinomial analysis. ADS.aureus was most highly associated with elevated cutaneous chemokines IP10 and TARC, which preferentially direct Th1 and Th2 cells to skin. Systemic CD4+ and CD8+ T cells, except for Th2 cells, were suppressed in ADS.aureus, particularly circulating Th1, memory IL-10+ T cells, and skin-homing memory Th17 cells. Systemic γδ T cell expansion in ADS.aureus was also observed. This study suggests that augmentation of protective T cell subsets is a potential therapeutic strategy in the management of S. aureus in AD.
Authors
Julianne Clowry, Daniel J. Dempsey, Tracey J. Claxton, Aisling M. Towell, Mary B. Turley, Martin Sutton, Joan A. Geoghegan, Sanja Kezic, Ivone Jakasa, Arthur White, Alan D. Irvine, Rachel M. McLoughlin
×Abstract
Fibroblast growth factor 23 (FGF23) production has recently been shown to increase downstream of Gαq/11-PKC signaling in osteocytes. Inactivating mutations in the gene encoding Gα11 (GNA11) cause familial hypocalciuric hypercalcemia (FHH) due to impaired calcium-sensing receptor signaling. We explored the effect of Gα11 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 also showed evidence of systemic inflammation with elevated serum IL-1β levels. Furin gene expression was significantly increased in the Gna11–/– liver, suggesting enhanced FGF23 cleavage despite the observed rise in circulating 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, Gα11 ablation caused systemic inflammation and excess serum FGF23 in mice, suggesting that patients with FHH — at least those with GNA11 mutations — may be at risk for these complications.
Authors
Birol Ay, Sajin Marcus Cyr, Kaitlin Klovdahl, Wen Zhou, Christina M. Tognoni, Yorihiro Iwasaki, Eugene P Rhee, Alpaslan Dedeoglu, Petra Simic, Murat Bastepe
In-Press Preview - More
Abstract
Thermogenesis in beige/brown adipose tissues can be leveraged to combat metabolic disorders such as type 2 diabetes and obesity. The complement system plays pleiotropic roles in metabolic homeostasis and organismal energy balance with canonical effects on immune cells and non-canonical effects on non-immune cells. The adipsin/C3a/C3aR1 pathway stimulates insulin secretion and sustains pancreatic beta cell mass. However, its role in adipose thermogenesis has not been defined. Here, we show that male Adipsin/Cfd knockout mice exhibit increased energy expenditure and white adipose tissue (WAT) browning. In addition, male adipocyte-specific C3aR1 knockout mice exhibit enhanced WAT thermogenesis and increased respiration. In stark contrast, female adipocyte-specific C3aR1 knockout mice display decreased brown fat thermogenesis and are cold intolerant. Female mice express lower levels of Adipsin in thermogenic adipocytes and adipose tissues than males. C3aR1 is also lower in female subcutaneous adipose tissue than males. Collectively, these results reveal sexual dimorphism in the adipsin/C3a/C3aR1 axis in regulating adipose thermogenesis and defense against cold stress. Our findings establish a newly discovered role of the alternative complement pathway in adaptive thermogenesis and highlight sex-specific considerations in potential therapeutic targets for metabolic diseases.
Authors
Lunkun Ma, Ankit Gilani, Alfonso Rubio-Navarro, Eric Cortada, Ang Li, Shannon M. Reilly, Liling Tang, James C. Lo
Abstract
Inhibition of Bruton's tyrosine kinase (BTK) through covalent modifications of its active site (e.g., ibrutinib [IBT]) is a preferred treatment for multiple B cell malignancies. However, IBT-treated patients are more susceptible to invasive fungal infections, although the mechanism is poorly understood. Neutrophils are the primary line of defense against these infections; therefore, we examined the impact of IBT on primary human neutrophil effector activity against Aspergillus fumigatus. IBT significantly impaired the ability of neutrophils to kill A. fumigatus and potently inhibited reactive oxygen species (ROS) production, chemotaxis, and phagocytosis. Importantly, exogenous TNFα fully compensated for defects imposed by IBT and newer-generation BTK inhibitors and restored the ability of neutrophils to contain A. fumigatus hyphal growth. Blocking TNFα did not impact ROS production in healthy neutrophils but prevented exogenous TNFα from rescuing the phenotype of IBT-treated neutrophils. The restorative capacity of TNFα was independent of transcription. Moreover, the addition of TNFα immediately rescued ROS production in IBT-treated neutrophils indicating that TNFα worked through a BTK-independent signaling pathway. Finally, TNFα restored effector activity of primary neutrophils from patients on IBT therapy. Altogether, our data indicate that TNFα rescues the antifungal immunity block imposed by inhibition of BTK in primary human neutrophils.
Authors
Diego A. Vargas-Blanco, Olivia W. Hepworth, Kyle J. Basham, Patricia Simaku, Arianne J. Crossen, Kyle D. Timmer, Alex Hopke, Hannah Brown Harding, Steven R. Vandal, Kirstine N. Jensen, Daniel J. Floyd, Jennifer L. Reedy, Christopher Reardon, Michael K. Mansour, Rebecca A. Ward, Daniel Irimia, Jeremy S. Abramson, Jatin M. Vyas
Abstract
Astrocyte activation is a common feature of neurodegenerative diseases. However, the ways in which dying neurons influence the activity of astrocytes is poorly understood. Receptor interacting protein kinase-3 (RIPK3) signaling has recently been described as a key regulator of neuroinflammation, but whether this kinase mediates astrocytic responsiveness to neuronal death has not yet been studied. Here, we used the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease to show that activation of astrocytic RIPK3 drives dopaminergic cell death and axon damage. Transcriptomic profiling revealed that astrocytic RIPK3 promoted gene expression associated with neuroinflammation and movement disorders, and this coincided with significant engagement of damage associated molecular pattern (DAMP) signaling. In mechanistic experiments, we show that factors released from dying neurons signal through receptor for advanced glycation endproducts (RAGE) to induce astrocytic RIPK3 signaling, which conferred inflammatory and neurotoxic functional activity. These findings highlight a mechanism of neuron-glia crosstalk in which neuronal death perpetuates further neurodegeneration by engaging inflammatory astrocyte activation via RIPK3.
Authors
Nydia P. Chang, Evan M. DaPrano, Marissa Lindman, Irving Estevez, Tsui-Wen Chou, Wesley R. Evans, Marialaina Nissenbaum, Micheal McCourt, Diego Alzate, Colm Atkins, Alexander W. Kusnecov, Rafiq Huda, Brian P. Daniels
Abstract
Multiple myeloma is a largely incurable and life-threatening malignancy of antibody-secreting plasma cells. An effective and widely available animal model that recapitulates human myeloma and related plasma cell disorders is lacking. We show that busulfan-conditioned hIL-6 transgenic NSG mice (NSG+hIL6) reliably support the engraftment of malignant and pre-malignant human plasma cells including from patients diagnosed with monoclonal gammopathy of undetermined significance, pre- and post-relapse myeloma, plasma cell leukemia, and AL amyloidosis. Consistent with human disease, NSG+hIL6 mice engrafted with patient-derived myeloma cells, developed serum M spikes, and a majority developed anemia, hypercalcemia, and/or bone lesions. Single cell RNA sequencing showed non-malignant and malignant cell engraftment, the latter expressing a wide array of mRNAs associated with myeloma cell survival and proliferation. Myeloma engrafted mice given CAR T-cells targeting plasma cells or bortezomib experienced reduced tumor burden. Our results established NSG+hIL6 mice as an effective patient derived xenograft model for study and preclinical drug development of multiple myeloma and related plasma cell disorders.
Authors
Zainul S. Hasanali, Alfred L. Garfall, Lisa Burzenski, Leonard D. Shultz, Yan Tang, Siddhant Kadu, Neil C. Sheppard, Wei Liu, Derek Dopkin, Dan T. Vogl, Adam D. Cohen, Adam J. Waxman, Sandra P. Susanibar-Adaniya, Martin Carroll, Edward A. Stadtmauer, David Allman
Abstract
While sclerostin-neutralizing antibodies (Scl-Ab) transiently stimulate bone formation by activating Wnt signaling in osteoblast lineage cells, they exert sustained inhibition of bone resorption, suggesting an alternate signaling pathway by which Scl-Ab control osteoclast activity. Since sclerostin can activate platelet-derived growth factor receptors (PDGFRs) in osteoblast lineage cells in vitro and PDGFR signaling in these cells induces bone resorption through M-CSF secretion, we hypothesized that the prolonged anti-catabolic effect of Scl-Ab could result from PDGFR inhibition. We show here that inhibition of PDGFR signaling in osteoblast lineage cells is sufficient and necessary to mediate prolonged Scl-Ab effect on M-CSF secretion and osteoclast activity in mice. Indeed, sclerostin co-activates PDGFRs independently of Wnt/β-catenin signaling inhibition, by forming a ternary complex with LRP6 and PDGFRs in pre-osteoblasts. In turn, Scl-Ab prevents sclerostin-mediated co-activation of PDGFR signaling and consequent M-CSF up-regulation in pre-osteoblast cultures, thereby inhibiting osteoclast activity in pre-osteoblast/osteoclast co-culture assays. These results provide a new potential mechanism explaining the dissociation between anabolic and anti-resorptive effects of long-term Scl-Ab.
Authors
Cyril Thouverey, Pierre Apostolides, Julia Brun, Joseph Caverzasio, Serge Ferrari
