In this issue Andrea Remuzzi, Sara Conti, and colleagues used scanning electron microscopy and computational models to characterize kidney disease-related ultrastructural and hemodynamic changes in the glomerular filtration barrier. The results of this study indicate that structural alterations result in abnormal hydrodynamic forces that tend to detach podocytes and show that angiotensin II inhibition can reducing hydraulic resistance by preserving CD151-α3 integrin complex assembly. The cover image shows a podocyte and its interdigitating foot processes of a rat kidney specimen observed by scanning electron microscopy.
Ribosomopathies are congenital disorders caused by mutations in the genes encoding ribosomal and other functionally related proteins. They are characterized by anemia, other hematopoietic and developmental abnormalities, and p53 activation. Ribosome assembly requires coordinated expression of many ribosomal protein (RP) genes; however, the regulation of RP gene expression, especially in hematopoietic stem cells (HSCs), remains poorly understood. MYSM1 is a transcriptional regulator essential for HSC function and hematopoiesis. We established that HSC dysfunction in Mysm1 deficiency is driven by p53; however, the mechanisms of p53 activation remained unclear. Here, we describe the transcriptome of Mysm1-deficient mouse HSCs and identify MYSM1 genome-wide DNA binding sites. We establish a direct role for MYSM1 in RP gene expression and show a reduction in protein synthesis in Mysm1–/– HSCs. Loss of p53 in mice fully rescues Mysm1–/– anemia phenotype but not RP gene expression, indicating that RP gene dysregulation is a direct outcome of Mysm1 deficiency and an upstream mediator of Mysm1–/– phenotypes through p53 activation. We characterize a patient with a homozygous nonsense MYSM1 gene variant, and we demonstrate reduced protein synthesis and increased p53 levels in patient hematopoietic cells. Our work provides insights into the specialized mechanisms regulating RP gene expression in HSCs and establishes a common etiology of MYSM1 deficiency and ribosomopathy syndromes.
Jad I. Belle, HanChen Wang, Amanda Fiore, Jessica C. Petrov, Yun Hsiao Lin, Chu-Han Feng, Thi Tuyet Mai Nguyen, Jacky Tung, Philippe M. Campeau, Uta Behrends, Theresa Brunet, Gloria Sarah Leszinski, Philippe Gros, David Langlais, Anastasia Nijnik
BACKGROUND Currently recommended traditional spirometry outputs do not reflect the relative contributions of emphysema and airway disease to airflow obstruction. We hypothesized that machine-learning algorithms can be trained on spirometry data to identify these structural phenotypes.METHODS Participants enrolled in a large multicenter study (COPDGene) were included. The data points from expiratory flow-volume curves were trained using a deep-learning model to predict structural phenotypes of chronic obstructive pulmonary disease (COPD) on CT, and results were compared with traditional spirometry metrics and an optimized random forest classifier. Area under the receiver operating characteristic curve (AUC) and weighted F-score were used to measure the discriminative accuracy of a fully convolutional neural network, random forest, and traditional spirometry metrics to phenotype CT as normal, emphysema-predominant (>5% emphysema), airway-predominant (Pi10 > median), and mixed phenotypes. Similar comparisons were made for the detection of functional small airway disease phenotype (>20% on parametric response mapping).RESULTS Among 8980 individuals, the neural network was more accurate in discriminating predominant emphysema/airway phenotypes (AUC 0.80, 95%CI 0.79–0.81) compared with traditional measures of spirometry, FEV1/FVC (AUC 0.71, 95%CI 0.69–0.71), FEV1% predicted (AUC 0.70, 95%CI 0.68–0.71), and random forest classifier (AUC 0.78, 95%CI 0.77–0.79). The neural network was also more accurate in discriminating predominant emphysema/small airway phenotypes (AUC 0.91, 95%CI 0.90–0.92) compared with FEV1/FVC (AUC 0.80, 95%CI 0.78–0.82), FEV1% predicted (AUC 0.83, 95%CI 0.80–0.84), and with comparable accuracy with random forest classifier (AUC 0.90, 95%CI 0.88–0.91).CONCLUSIONS Structural phenotypes of COPD can be identified from spirometry using deep-learning and machine-learning approaches, demonstrating their potential to identify individuals for targeted therapies.TRIAL REGISTRATION ClinicalTrials.gov NCT00608764.FUNDING This study was supported by NIH grants K23 HL133438 and R21EB027891 and an American Thoracic Foundation 2018 Unrestricted Research Grant. The COPDGene study is supported by NIH grants NHLBI U01 HL089897 and U01 HL089856. The COPDGene study (NCT00608764) is also supported by the COPD Foundation through contributions made to an Industry Advisory Committee comprising AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Novartis, and Sunovion.
Sandeep Bodduluri, Arie Nakhmani, Joseph M. Reinhardt, Carla G. Wilson, Merry-Lynn McDonald, Ramaraju Rudraraju, Byron C. Jaeger, Nirav R. Bhakta, Peter J. Castaldi, Frank C. Sciurba, Chengcui Zhang, Purushotham V. Bangalore, Surya P. Bhatt
Musculoskeletal disorders represent the third greatest burden in terms of death and disability in the developed world. Osteoarthritis is the single greatest cause of chronic pain, has no cure, and affects 8.5 and 27 million people in the UK and US, respectively. Osteoarthritis is most prevalent in older people, but as it commonly occurs after joint injury, young people with such injuries are also susceptible. Painful joints are often treated with steroid or hyaluronic acid (HA) injections, but treatments to prevent subsequent joint degeneration remain elusive. In animals, joint injury increases glutamate release into the joint, acting on nerves to cause pain, and joint tissues to cause inflammation and degeneration. This study investigated synovial fluid glutamate concentrations and glutamate receptor (GluR) expression in injured human joints and compared the efficacy of GluR antagonists with current treatments in a mouse model of injury-induced osteoarthritis (ACL rupture). GluRs were expressed in the ligaments and meniscus after knee injury, and synovial fluid glutamate concentrations ranged from 19 to 129 μM. Intra-articular injection of NBQX (GluR antagonist) at the time of injury substantially reduced swelling and degeneration in the mouse ACL rupture model. HA had no effect, and Depo-Medrone reduced swelling for 1 day but increased degeneration by 50%. Intra-articular administration of NBQX modified both symptoms and disease to a greater extent than current treatments. There is an opportunity for repurposing related drugs, developed for CNS disorders and with proven safety in humans, to prevent injury-induced osteoarthritis. This could quickly reduce the substantial burden associated with osteoarthritis.
Cleo S. Bonnet, Sophie J. Gilbert, Emma J. Blain, Anwen S. Williams, Deborah J. Mason
Multiple organ failure in sepsis is a progressive failure of several interdependent organ systems. Liver dysfunction occurs early during sepsis and is directly associated with patient death; however, the underlying mechanism of liver dysfunction is unclear. Platelet transfusion benefits patients with sepsis, and inhibition of complement activation protects liver function in septic animals. Herein, we explored the potential link between platelets, complement activation, and liver dysfunction in sepsis. We found that deletion of platelet C-type lectin-like receptor 2 (CLEC-2) exacerbated liver dysfunction in early sepsis. Platelet CLEC-2–deficient mice exhibited higher complement activation, more severe complement attack in the liver, and lower plasma levels of complement inhibitors at early time points after E. coli infection. Circulating monocytes expressed the CLEC-2 ligand podoplanin in early sepsis, and podoplanin binding induced release of complement inhibitors from platelets. Injection of complement inhibitors released from platelets reduced complement attack and attenuated liver dysfunction in septic mice. These findings indicate a new function of platelets in the regulation of complement activation during sepsis.
Zhanli Xie, Bojing Shao, Christopher Hoover, Michael McDaniel, Jianhua Song, Miao Jiang, Zhenni Ma, Fei Yang, Jingjing Han, Xia Bai, Changgeng Ruan, Lijun Xia
Genetic or acquired defects of the lymphatic vasculature often result in disfiguring, disabling, and, occasionally, life-threatening clinical consequences. Advanced forms of lymphedema are readily diagnosed clinically, but more subtle presentations often require invasive imaging or other technologies for a conclusive diagnosis. On the other hand, lipedema, a chronic lymphatic microvascular disease with pathological accumulation of subcutaneous adipose tissue, is often misdiagnosed as obesity or lymphedema; currently there are no biomarkers or imaging criteria available for a conclusive diagnosis. Recent evidence suggests that otherwise-asymptomatic defective lymphatic vasculature likely contributes to an array of other pathologies, including obesity, inflammatory bowel disease, and neurological disorders. Accordingly, identification of biomarkers of lymphatic malfunction will provide a valuable resource for the diagnosis and clinical differentiation of lymphedema, lipedema, obesity, and other potential lymphatic pathologies. In this paper, we profiled and compared blood plasma exosomes isolated from mouse models and from human subjects with and without symptomatic lymphatic pathologies. We identified platelet factor 4 (PF4/CXCL4) as a biomarker that could be used to diagnose lymphatic vasculature dysfunction. Furthermore, we determined that PF4 levels in circulating blood plasma exosomes were also elevated in patients with lipedema, supporting current claims arguing that at least some of the underlying attributes of this disease are also the consequence of lymphatic defects.
Wanshu Ma, Hyea Jin Gil, Noelia Escobedo, Alberto Benito-Martín, Pilar Ximénez-Embún, Javier Muñoz, Héctor Peinado, Stanley G. Rockson, Guillermo Oliver
Glaucoma surgeries, such as trabeculectomy, are performed to lower intraocular pressure to reduce risk of vision loss. These surgeries create a new passage in the eye that reroutes the aqueous humor outflow to the subconjunctival space, where the fluid is presumably absorbed by the conjunctival lymphatics. Here, we characterized the development and function of the ocular lymphatics using transgenic lymphatic reporter mice and rats. We found that the limbal and conjunctival lymphatic networks are progressively formed from a primary lymphatic vessel that grows from the nasal-side medial canthus region at birth. This primary lymphatic vessel immediately branches out, invades the limbus and conjunctiva, and bidirectionally encircles the cornea. As a result, the distribution of the ocular lymphatics is significantly polarized toward the nasal side, and the limbal lymphatics are directly connected to the conjunctival lymphatics. New lymphatic sprouts are produced mainly from the nasal-side limbal lymphatics, posing the nasal side of the eye as more responsive to fluid drainage and inflammatory stimuli. Consistent with this polarized distribution of the ocular lymphatics, a higher drainage efficiency was observed in the nasal side than the temporal side of the eye when injected with a fluorescent tracer. In contrast, blood vessels are evenly distributed at the anterior surface of the eyes. Also, we found that these distinct vascular distribution patterns were conserved in human eyes. Together, our study demonstrated that the ocular surface lymphatics are more densely present in the nasal side and uncovered the potential clinical benefits in selecting the nasal side as a glaucoma surgery site to improve fluid drainage.
Yifan Wu, Young Jin Seong, Kin Li, Dongwon Choi, Eunkyung Park, George H. Daghlian, Eunson Jung, Khoa Bui, Luping Zhao, Shrimika Madhavan, Saren Daghlian, Patill Daghlian, Desmond Chin, Il-Taeg Cho, Alex K. Wong, Martin Heur, Sandy Zhang-Nunes, James C. Tan, Masatsugu Ema, Tina T. Wong, Alex S. Huang, Young-Kwon Hong
Wnt/β-catenin signaling is active in small subpopulations of Ewing sarcoma cells, and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1–dependent transcriptional activity. Importantly, these β-catenin–activated Ewing sarcoma cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell-autonomous mechanisms, Wnt/β-catenin–active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from β-catenin–active versus –nonactive tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of β-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that β-catenin–activated Ewing cells secreted factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of proangiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-β. Mechanistically, Wnt/β-catenin signaling antagonizes EWS-FLI1–dependent repression of TGF-β receptor type 2, thereby sensitizing tumor cells to TGF-β ligands. Together, these findings suggest that Wnt/β-catenin–active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.
Allegra G. Hawkins, Elisabeth A. Pedersen, Sydney Treichel, Kelsey Temprine, Colin Sperring, Jay A. Read, Brian Magnuson, Rashmi Chugh, Elizabeth R. Lawlor
Heterotopic ossification (HO) is defined as abnormal differentiation of local stromal cells of mesenchymal origin, resulting in pathologic cartilage and bone matrix deposition. Cyr61, CTGF, Nov (CCN) family members are matricellular proteins that have diverse regulatory functions on cell proliferation and differentiation, including the regulation of chondrogenesis. However, little is known regarding CCN family member expression or function in HO. Here, a combination of bulk and single-cell RNA sequencing defined the dynamic temporospatial pattern of CCN family member induction within a mouse model of trauma-induced HO. Among CCN family proteins, Wisp1 (also known as Ccn4) was most upregulated during the evolution of HO, and Wisp1 expression corresponded with chondrogenic gene profile. Immunohistochemistry confirmed WISP1 expression across traumatic and genetic HO mouse models as well as in human HO samples. Transgenic Wisp1LacZ/LacZ knockin animals showed an increase in endochondral ossification in HO after trauma. Finally, the transcriptome of Wisp1-null tenocytes revealed enrichment in signaling pathways, such as the STAT3 and PCP signaling pathways, that may explain increased HO in the context of Wisp1 deficiency. In sum, CCN family members, and in particular Wisp1, are spatiotemporally associated with and negatively regulate trauma-induced HO formation.
Ginny Ching-Yun Hsu, Simone Marini, Stefano Negri, Yiyun Wang, Jiajia Xu, Chase Pagani, Charles Hwang, David Stepien, Carolyn A. Meyers, Sarah Miller, Edward McCarthy, Karen M. Lyons, Benjamin Levi, Aaron W. James
Whole-sporozoite vaccines engender sterilizing immunity against malaria in animal models and importantly, in humans. Gene editing allows for the removal of specific parasite genes, enabling generation of genetically attenuated parasite (GAP) strains for vaccination. Using rodent malaria parasites, we have previously shown that late liver stage–arresting replication-competent (LARC) GAPs confer superior protection when compared with early liver stage–arresting replication-deficient GAPs and radiation-attenuated sporozoites. However, generating a LARC GAP in the human malaria parasite Plasmodium falciparum (P. falciparum) has been challenging. Here, we report the generation and characterization of a likely unprecedented P. falciparum LARC GAP generated by targeted gene deletion of the Mei2 gene: P. falciparum mei2–. Robust exoerythrocytic schizogony with extensive cell growth and DNA replication was observed for P. falciparum mei2– liver stages in human liver-chimeric mice. However, P. falciparum mei2– liver stages failed to complete development and did not form infectious exoerythrocytic merozoites, thereby preventing their transition to asexual blood stage infection. Therefore, P. falciparum mei2– is a replication-competent, attenuated human malaria parasite strain with potentially increased potency, useful for vaccination to protect against P. falciparum malaria infection.
Debashree Goswami, William Betz, Navin K. Locham, Chaitra Parthiban, Carolyn Brager, Carola Schäfer, Nelly Camargo, Thao Nguyen, Spencer Y. Kennedy, Sean C. Murphy, Ashley M. Vaughan, Stefan H.I. Kappe
Acute respiratory distress syndrome (ARDS) results from overwhelming pulmonary inflammation. Prior bulk RNA sequencing provided limited insights into ARDS pathogenesis. We used single cell RNA sequencing to probe ARDS at a higher resolution. PBMCs of patients with pneumonia and sepsis with early ARDS were compared with those of sepsis patients who did not develop ARDS. Monocyte clusters from ARDS patients revealed multiple distinguishing characteristics in comparison with monocytes from patients without ARDS, including downregulation of SOCS3 expression, accompanied by a proinflammatory signature with upregulation of multiple type I IFN–induced genes, especially in CD16+ cells. To generate an ARDS risk score, we identified upregulation of 29 genes in the monocytes of these patients, and 17 showed a similar profile in cells of patients in independent cohorts. Monocytes had increased expression of RAB11A, known to inhibit neutrophil efferocytosis; ATP2B1, a calcium pump that exports Ca2+ implicated in endothelial barrier disruption; and SPARC, associated with processing of procollagen to collagen. These data show that monocytes of ARDS patients upregulate expression of genes not just restricted to those associated with inflammation. Together, our findings identify molecules that are likely involved in ARDS pathogenesis that may inform biomarker and therapeutic development.
Yale Jiang, Brian R. Rosborough, Jie Chen, Sudipta Das, Georgios D. Kitsios, Bryan J. McVerry, Rama K. Mallampalli, Janet S. Lee, Anuradha Ray, Wei Chen, Prabir Ray
Increased microvascular leakage is a cardinal feature of many critical diseases. Regular exercise is associated with improved endothelial function and reduced risk of cardiovascular disease. Irisin, secreted during exercise, contributes to many health benefits of exercise. However, the effects of irisin on endothelial function and microvascular leakage remain unknown. In this study, we found that irisin remarkably strengthened endothelial junctions and barrier function via binding to integrin αVβ5 receptor in LPS-treated endothelial cells. The beneficial effect of irisin was associated with suppression of the Src–MLCK–β-catenin pathway, activation of the AMPK-Cdc42/Rac1 pathway, and improvement of mitochondrial function. In preclinical models of microvascular leakage, exogenous irisin improved pulmonary function, decreased lung edema and injury, suppressed inflammation, and increased survival. In ARDS patients, serum irisin levels were decreased and inversely correlated with disease severity and mortality. In conclusion, irisin enhances endothelial barrier function and mitigates microvascular leakage–related diseases.
Jianbin Bi, Jia Zhang, Yifan Ren, Zhaoqing Du, Yuanyuan Zhang, Chang Liu, Yawen Wang, Lin Zhang, Zhihong Shi, Zheng Wu, Yi Lv, Rongqian Wu
Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disorder caused by a polyglutamine expansion in the androgen receptor (AR). Using gene expression analysis and ChIP sequencing, we mapped transcriptional changes in genetically engineered patient stem cell–derived motor neurons. We found that transcriptional dysregulation in SBMA can occur through AR-mediated histone modification. We detected reduced histone acetylation, along with decreased expression of genes encoding compensatory metabolic proteins and reduced substrate availability for mitochondrial function. Furthermore, we found that pyruvate supplementation corrected this deficiency and improved mitochondrial function and SBMA motor neuron viability. We propose that epigenetic dysregulation of metabolic genes contributes to reduced mitochondrial ATP production. Our results show a molecular link between altered epigenetic regulation and mitochondrial metabolism that contributes to neurodegeneration.
Naemeh Pourshafie, Ester Masati, Eric Bunker, Alec R. Nickolls, Parisorn Thepmankorn, Kory Johnson, Xia Feng, Tyler Ekins, Christopher Grunseich, Kenneth H. Fischbeck
Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel tick-borne bunyavirus that recently emerged in East Asian countries. SFTS is characterized by high fever, thrombocytopenia, leukopenia, multiorgan failure, and hemorrhage with case fatality rates of 6.3% to 30%. Neither antivirals nor vaccines are available at present. We previously demonstrated that neutralizing antibodies specific for SFTSV glycoprotein (Gn) played a vital role in the survival of patients with SFTS. Nanobodies from camels present unique properties, such as thermostability, high affinity, and low immunogenicity. In the current study, mammalian expressed SFTSV Gn was used to immunize a camel, and functional nanobodies were isolated from the B cell nanobody library constructed from the immunized animal. Clone SNB02 was selected for in-depth analysis for its inhibition of SFTSV replication both in vitro and in vivo. We showed that SNB02 potently inhibited SFTSV infection and prevented thrombocytopenia in a humanized mouse model and is a potential candidate for therapeutics.
Xilin Wu, Yanlei Li, Bilian Huang, Xiaohua Ma, Linjing Zhu, Nan Zheng, Shijie Xu, Waqas Nawaz, Changping Xu, Zhiwei Wu
Chronic kidney disease is the main cause of mortality in patients with tuberous sclerosis complex (TSC) disease. The mechanisms underlying TSC cystic kidney disease remain unclear, with no available interventions to prevent cyst formation. Using targeted deletion of TSC1 in nephron progenitor cells, we showed that cysts in TSC1-null embryonic kidneys originate from injured proximal tubular cells with high mTOR complex 1 activity. Injection of rapamycin to pregnant mice inhibited the mTOR pathway and tubular cell proliferation in kidneys of TSC1-null offspring. Rapamycin also prevented renal cystogenesis and prolonged the life span of TSC newborns. Gene expression analysis of proximal tubule cells identified sets of genes and pathways that were modified secondary to TSC1 deletion and rescued by rapamycin administration during nephrogenesis. Inflammation with mononuclear infiltration was observed in the cystic areas of TSC1-null kidneys. Dexamethasone administration during pregnancy decreased cyst formation by not only inhibiting the inflammatory response, but also interfering with the mTORC1 pathway. These results reveal mechanisms of cystogenesis in TSC disease and suggest interventions before birth to ameliorate cystic disease in offspring.
Morris Nechama, Yaniv Makayes, Elad Resnick, Karen Meir, Oded Volovelsky
BACKGROUND HVTN 098, a randomized, double-blind, placebo-controlled trial, evaluated the safety, tolerability, and immunogenicity of PENNVAX-GP HIV DNA vaccine, administered with or without plasmid IL-12 (pIL-12), via intradermal (ID) or intramuscular (IM) electroporation (EP) in healthy, HIV-uninfected adults. The study tested whether PENNVAX-GP delivered via ID/EP at one-fifth the dose could elicit equivalent immune responses to delivery via IM/EP and whether inclusion of pIL-12 provided additional benefit.METHODS Participants received DNA encoding HIV-1 env/gag/pol in 3 groups: 1.6 mg ID (ID no IL-12 group, n = 20), 1.6 mg ID + 0.4 mg pIL-12 (ID + IL-12 group, n = 30), 8 mg IM + 1 mg pIL-12 (IM + IL-12 group, n = 30), or placebo (n = 9) via EP at 0, 1, 3, and 6 months. Results of cellular and humoral immunogenicity assessments are reported.RESULTS Following vaccination, the frequency of responders (response rate) to any HIV protein based on CD4+ T cells expressing IFN-γ or IL-2 was 96% for both the ID + IL-12 and IM + IL-12 groups; CD8+ T cell response rates were 64% and 44%, respectively. For ID delivery, the inclusion of pIL-12 increased CD4+ T cell response rate from 56% to 96%. The frequency of responders was similar (≥90%) for IgG binding antibody to gp140 consensus Env across all groups, but the magnitude was higher in the ID + IL-12 group compared with the IM + IL-12 group.CONCLUSION PENNVAX-GP DNA induced robust cellular and humoral immune responses, demonstrating that immunogenicity of DNA vaccines can be enhanced by EP route and inclusion of pIL-12. ID/EP was dose sparing, inducing equivalent, or in some aspects superior, immune responses compared with IM/EP.TRIAL REGISTRATION ClinicalTrials.gov NCT02431767.FUNDING This work was supported by National Institute of Allergy and Infectious Diseases (NIAID), U.S. Public Health Service grants, an HIV Vaccine Design and Development Team contract, Integrated Preclinical/Clinical AIDS Vaccine Development Program, and an NIH award.
Stephen C. De Rosa, Srilatha Edupuganti, Yunda Huang, Xue Han, Marnie Elizaga, Edith Swann, Laura Polakowski, Spyros A. Kalams, Michael C. Keefer, Janine Maenza, Yiwen Lu, Megan C. Wise, Jian Yan, Matthew P. Morrow, Amir S. Khan, Jean D. Boyer, Laurent Humeau, Scott White, Michael Pensiero, Niranjan Y. Sardesai, Mark L. Bagarazzi, David B. Weiner, Guido Ferrari, Georgia D. Tomaras, David C. Montefiori, Lawrence Corey, M. Juliana McElrath, HIV Vaccine Trials Network (HVTN) 098 Study Team
In diabetic dyslipidemia, cholesterol accumulates in the plasma membrane, decreasing fluidity and thereby suppressing the ability of cells to transduce ligand-activated signaling pathways. Liver X receptors (LXRs) make up the main cellular mechanism by which intracellular cholesterol is regulated and play important roles in inflammation and disease pathogenesis. N, N-dimethyl-3β-hydroxy-cholenamide (DMHCA), a selective LXR agonist, specifically activates the cholesterol efflux arm of the LXR pathway without stimulating triglyceride synthesis. In this study, we use a multisystem approach to understand the effects and molecular mechanisms of DMHCA treatment in type 2 diabetic (db/db) mice and human circulating angiogenic cells (CACs), which are hematopoietic progenitor cells with vascular reparative capacity. We found that DMHCA is sufficient to correct retinal and BM dysfunction in diabetes, thereby restoring retinal structure, function, and cholesterol homeostasis; rejuvenating membrane fluidity in CACs; hampering systemic inflammation; and correcting BM pathology. Using single-cell RNA sequencing on lineage–sca1+c-Kit+ (LSK) hematopoietic stem cells (HSCs) from untreated and DMHCA-treated diabetic mice, we provide potentially novel insights into hematopoiesis and reveal DMHCA’s mechanism of action in correcting diabetic HSCs by reducing myeloidosis and increasing CACs and erythrocyte progenitors. Taken together, these findings demonstrate the beneficial effects of DMHCA treatment on diabetes-induced retinal and BM pathology.
Cristiano P. Vieira, Seth D. Fortmann, Masroor Hossain, Ana Leda Longhini, Sandra S. Hammer, Bright Asare-Bediako, David K. Crossman, Micheli S. Sielski, Yvonne Adu-Agyeiwaah, Mariana Dupont, Jason L. Floyd, Sergio Li Calzi, Todd Lydic, Robert S. Welner, Gary J. Blanchard, Julia V. Busik, Maria B. Grant
The epithelial filtration slit is a crucial component of the glomerular capillary membrane, which is essential for maintaining glomerular filtration function. Though chronic kidney diseases are an immense clinical problem, the mechanisms through which structural alterations reduce glomerular water filtration have not yet been understood completely. To investigate the mechanisms underlying filtration function loss, we studied rats with spontaneously occurring progressive kidney disease, either treated with angiotensin II antagonist or untreated, combining high-resolution electron microscopy of the glomerular capillary wall with theoretical water filtration modeling. Under pathological conditions, epithelial filtration pores and the extension of the subpodocyte space were larger than in normal controls. Numerical analyses indicated that these ultrastructural changes increased hydraulic resistance of the glomerular capillary wall by extending coverage of the filtration barrier by the subpodocyte space, with the changes in hydrodynamic forces acting on podocytes likely being responsible for their detachment. Angiotensin II inhibition normalized the subpodocyte space’s hydraulic resistance, restored mechanical podocyte load, and preserved CD151–α3 integrin complex assembly, improving podocyte adherence and survival. Our results show that ultrastructural changes in podocytes are major determinants of the hydraulic resistance of the glomerular capillary wall and highlight the mechanism of podocyte loss in kidney disease progression, as well as the mechanisms underlying angiotensin II inhibition.
Andrea Remuzzi, Sara Conti, Bogdan Ene-Iordache, Susanna Tomasoni, Paola Rizzo, Ariela Benigni, Giuseppe Remuzzi
Despite advances in lipid-lowering therapies, people with diabetes continue to experience more limited cardiovascular benefits. In diabetes, hyperglycemia sustains inflammation and preempts vascular repair. We tested the hypothesis that the receptor for advanced glycation end-products (RAGE) contributes to these maladaptive processes. We report that transplantation of aortic arches from diabetic, Western diet–fed Ldlr—/— mice into diabetic Ager—/— (Ager, the gene encoding RAGE) versus WT diabetic recipient mice accelerated regression of atherosclerosis. RNA-sequencing experiments traced RAGE-dependent mechanisms principally to the recipient macrophages and linked RAGE to interferon signaling. Specifically, deletion of Ager in the regressing diabetic plaques downregulated interferon regulatory factor 7 (Irf7) in macrophages. Immunohistochemistry studies colocalized IRF7 and macrophages in both murine and human atherosclerotic plaques. In bone marrow–derived macrophages (BMDMs), RAGE ligands upregulated expression of Irf7, and in BMDMs immersed in a cholesterol-rich environment, knockdown of Irf7 triggered a switch from pro- to antiinflammatory gene expression and regulated a host of genes linked to cholesterol efflux and homeostasis. Collectively, this work adds a new dimension to the immunometabolic sphere of perturbations that impair regression of established diabetic atherosclerosis and suggests that targeting RAGE and IRF7 may facilitate vascular repair in diabetes.
Laura Senatus, Raquel López-Díez, Lander Egaña-Gorroño, Jianhua Liu, Jiyuan Hu, Gurdip Daffu, Qing Li, Karishma Rahman, Yuliya Vengrenyuk, Tessa J. Barrett, M. Zahidunnabi Dewan, Liang Guo, Daniela Fuller, Aloke V. Finn, Renu Virmani, Huilin Li, Richard A. Friedman, Edward A. Fisher, Ravichandran Ramasamy, Ann Marie Schmidt
Alopecia areata (AA) is a common autoimmune condition, presenting initially with loss of hair without other overt skin changes. The unremarkable appearance of the skin surface contrasts with the complex immune activity occurring at the hair follicle. AA pathogenesis is due to the loss of immune privilege of the hair follicle, leading to autoimmune attack. Although the literature has focused on CD8+ T cells, vital roles for CD4+ T cells and antigen-presenting cells have been suggested. Here, we use single-cell sequencing to reveal distinct expression profiles of immune cells in murine AA. We found clonal expansions of both CD4+ and CD8+ T cells, with shared clonotypes across varied transcriptional states. The murine AA data were used to generate highly predictive models of human AA disease. Finally, single-cell sequencing of T cells in human AA recapitulated the clonotypic findings and the gene expression of the predictive models.
Nicholas Borcherding, Sydney B. Crotts, Luana S. Ortolan, Nicholas Henderson, Nicholas L. Bormann, Ali Jabbari
BACKGROUND Dysregulation of l-arginine metabolism has been proposed to occur in patients with severe asthma. The effects of l-arginine supplementation on l-arginine metabolite profiles in these patients are unknown. We hypothesized that individuals with severe asthma with low fractional exhaled nitric oxide (FeNO) would have fewer exacerbations with the addition of l-arginine to their standard asthma medications compared with placebo and would demonstrate the greatest changes in metabolite profiles.METHODS Participants were enrolled in a single-center, crossover, double-blind l-arginine intervention trial at UCD. Subjects received placebo or l-arginine, dosed orally at 0.05 mg/kg (ideal body weight) twice daily. The primary end point was moderate asthma exacerbations. Longitudinal plasma metabolite levels were measured using mass spectrometry. A linear mixed-effect model with subject-specific intercepts was used for testing treatment effects.RESULTS A cohort of 50 subjects was included in the final analysis. l-Arginine did not significantly decrease asthma exacerbations in the overall cohort. Higher citrulline levels and a lower arginine availability index (AAI) were associated with higher FeNO (P = 0.005 and P = 2.51 × 10–9, respectively). Higher AAI was associated with lower exacerbation events. The eicosanoid prostaglandin H2 (PGH2) and Nα-acetyl-l-arginine were found to be good predictors for differentiating clinical responders and nonresponders.CONCLUSIONS There was no statistically significant decrease in asthma exacerbations in the overall cohort with l-arginine intervention. PGH2, Nα-acetyl-l-arginine, and the AAI could serve as predictive biomarkers in future clinical trials that intervene in the arginine metabolome.TRIAL REGISTRATION ClinicalTrials.gov NCT01841281.FUNDING This study was supported by NIH grants R01HL105573, DK097154, UL1 TR001861, and K08HL114882. Metabolomics analysis was supported in part by a grant from the University of California Tobacco-Related Disease Research Program program (TRDRP).
Shu-Yi Liao, Megan R. Showalter, Angela L. Linderholm, Lisa Franzi, Celeste Kivler, Yao Li, Michael R. Sa, Zachary A. Kons, Oliver Fiehn, Lihong Qi, Amir A. Zeki, Nicholas J. Kenyon
Acute gastrointestinal (GI) graft-versus-host disease (GVHD) is a primary determinant of mortality after allogeneic hematopoietic stem cell transplantation (alloSCT). The condition is mediated by alloreactive donor CD4+ T cells that differentiate into pathogenic subsets expressing IFN-γ, IL-17A, or GM-CSF and is regulated by subsets expressing IL-10 and/or Foxp3. Developmental relationships between Th cell states during priming in mesenteric lymph nodes (mLNs) and effector function in the GI tract remain undefined at genome scale. We applied scRNA-Seq and computational modeling to a mouse model of donor DC-mediated GVHD exacerbation, creating an atlas of putative CD4+ T cell differentiation pathways in vivo. Computational trajectory inference suggested emergence of pathogenic and regulatory states along a single developmental trajectory in mLNs. Importantly, we inferred an unexpected second trajectory, categorized by little proliferation or cytokine expression, reduced glycolysis, and high tcf7 expression. TCF1hi cells upregulated α4β7 before gut migration and failed to express cytokines. These cells exhibited recall potential and plasticity following secondary transplantation, including cytokine or Foxp3 expression, but reduced T cell factor 1 (TCF1). Thus, scRNA-Seq suggested divergence of alloreactive CD4+ T cells into quiescent and effector states during gut GVHD exacerbation by donor DC, reflecting putative heterogeneous priming in vivo. These findings, which are potentially the first at a single-cell level during GVHD over time, may assist in examination of T cell differentiation in patients undergoing alloSCT.
Jessica A. Engel, Hyun Jae Lee, Cameron G. Williams, Rachel Kuns, Stuart Olver, Lianne I.M. Lansink, Megan S.F. Soon, Stacey B. Andersen, Joseph E. Powell, Valentine Svensson, Sarah A. Teichmann, Geoffrey R. Hill, Antiopi Varelias, Motoko Koyama, Ashraful Haque
Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting tenocyte proliferation and matrix synthesis during embryonic tendon development. However, the role of scleraxis in the growth and adaptation of adult tendons is not known. We hypothesized that scleraxis is required for tendon growth in response to mechanical loading and that scleraxis promotes the specification of progenitor cells into tenocytes. We conditionally deleted scleraxis in adult mice using a tamoxifen-inducible Cre-recombinase expressed from the Rosa26 locus (ScxΔ) and then induced tendon growth in Scx+ and ScxΔ adult mice via plantaris tendon mechanical overload. Compared with the WT Scx+ group, ScxΔ mice demonstrated blunted tendon growth. Transcriptional and proteomic analyses revealed significant reductions in cell proliferation, protein synthesis, and extracellular matrix genes and proteins. Our results indicate that scleraxis is required for mechanically stimulated adult tendon growth by causing the commitment of CD146+ pericytes into the tenogenic lineage and by promoting the initial expansion of newly committed tenocytes and the production of extracellular matrix proteins.
Jonathan P. Gumucio, Martin M. Schonk, Yalda A. Kharaz, Eithne Comerford, Christopher L. Mendias
Prostaglandin E2 and its cognate EP1–4 receptors play important roles in blood pressure (BP) regulation. Herein, we show that endothelial cell–specific (EC-specific) EP4 gene–knockout mice (EC-EP4–/–) exhibited elevated, while EC-specific EP4-overexpression mice (EC-hEP4OE) displayed reduced, BP levels compared with the control mice under both basal and high-salt diet–fed conditions. The altered BP was completely abolished by treatment with l–NG-nitro-l-arginine methyl ester (l-NAME), a competitive inhibitor of endothelial nitric oxide synthase (eNOS). The mesenteric arteries of the EC-EP4–/– mice showed increased vasoconstrictive response to angiotensin II and reduced vasorelaxant response to acetylcholine, both of which were eliminated by l-NAME. Furthermore, EP4 activation significantly reduced BP levels in hypertensive rats. Mechanistically, EP4 deletion markedly decreased NO contents in blood vessels via reducing eNOS phosphorylation at Ser1177. EP4 enhanced NO production mainly through the AMPK pathway in cultured ECs. Collectively, our findings demonstrate that endothelial EP4 is essential for BP homeostasis.
Hu Xu, Bingying Fang, Shengnan Du, Sailun Wang, Qingwei Li, Xiao Jia, Chengzhen Bao, Lan Ye, Xue Sui, Lei Qian, Zhilin Luan, Guangrui Yang, Feng Zheng, Nanping Wang, Lihong Chen, Xiaoyan Zhang, Youfei Guan
BACKGROUND Fatal cases of COVID-19 are increasing globally. We retrospectively investigated the potential of immunologic parameters as early predictors of COVID-19.METHODS A total of 1018 patients with confirmed COVID-19 were enrolled in our 2-center retrospective study. Clinical feature, laboratory test, immunological test, radiological findings, and outcomes data were collected. Univariate and multivariable logistic regression analyses were performed to evaluate factors associated with in-hospital mortality. Receiver operator characteristic (ROC) curves and survival curves were plotted to evaluate their clinical utility.RESULTS The counts of all T lymphocyte subsets were markedly lower in nonsurvivors than in survivors, especially CD8+ T cells. Among all tested cytokines, IL-6 was elevated most significantly, with an upward trend of more than 10-fold. Using multivariate logistic regression analysis, IL-6 levels of more than 20 pg/mL and CD8+ T cell counts of less than 165 cells/μL were found to be associated with in-hospital mortality after adjusting for confounding factors. Groups with IL-6 levels of more than 20 pg/mL and CD8+ T cell counts of less than 165 cells/μL had a higher percentage of older and male patients as well as a higher proportion of patients with comorbidities, ventilation, intensive care unit admission, shock, and death. Furthermore, the receiver operating curve of the model combining IL-6 (>20 pg/mL) and CD8+ T cell counts (<165 cells/μL) displayed a more favorable discrimination than that of the CURB-65 score. The Hosmer-Lemeshow test showed a good fit of the model, with no statistical significance.CONCLUSION IL-6 (>20 pg/mL) and CD8+ T cell counts (<165 cells/μL) are 2 reliable prognostic indicators that accurately stratify patients into risk categories and predict COVID-19 mortality.Funding This work was supported by funding from the National Natural Science Foundation of China (no. 81772477 and 81201848).
Miao Luo, Jing Liu, Weiling Jiang, Shuang Yue, Huiguo Liu, Shuang Wei
Rheumatoid arthritis (RA) is characterized by synovial joint inflammation, cartilage damage, and dysregulation of the adaptive immune system. While neutrophil extracellular traps (NETs) have been proposed to play a role in the generation of modified autoantigens and in the activation of synovial fibroblasts, it remains unknown whether NETs are directly involved in cartilage damage. Here, we report a new mechanism by which NET-derived elastase disrupts cartilage matrix and induces release of membrane-bound peptidylarginine deiminase-2 by fibroblast-like synoviocytes (FLSs). Cartilage fragments are subsequently citrullinated, internalized by FLSs, and then presented to antigen-specific CD4+ T cells. Furthermore, immune complexes containing citrullinated cartilage components can activate macrophages to release proinflammatory cytokines. HLA-DRB1*04:01 transgenic mice immunized with NETs develop autoantibodies against citrullinated cartilage proteins and display enhanced cartilage damage. Inhibition of NET-derived elastase rescues NET-mediated cartilage damage. These results show that NETs and neutrophil elastase externalized in these structures play fundamental pathogenic roles in promoting cartilage damage and synovial inflammation. Strategies targeting neutrophil elastase and NETs could have a therapeutic role in RA and in other inflammatory diseases associated with inflammatory joint damage.
Carmelo Carmona-Rivera, Philip M. Carlucci, Rishi R. Goel, Eddie James, Stephen R. Brooks, Cliff Rims, Victoria Hoffmann, David A. Fox, Jane H. Buckner, Mariana J. Kaplan
BACKGROUND. Identifying immune correlates of COVID-19 disease severity is an urgent need for clinical management, vaccine evaluation, and drug development. Here, we present a temporal analysis of key immune mediators, cytokines, and chemokines in blood of hospitalized COVID-19 patients from serial sampling and follow-up over 4 weeks. METHODS. A total of 71 patients with laboratory-confirmed COVID-19 admitted to Beijing You’an Hospital in China with either mild (53 patients) or severe (18 patients) disease were enrolled with 18 healthy volunteers. We measured 34 immune mediators, cytokines, and chemokines in peripheral blood every 4–7 days over 1 month per patient using a bioplex multiplex immunoassay. RESULTS. We found that the chemokine RANTES (CCL5) was significantly elevated, from an early stage of the infection, in patients with mild but not severe disease. We also found that early production of inhibitory mediators including IL-10 and IL-1RA were significantly associated with disease severity, and a combination of CCL5, IL-1 receptor antagonist (IL-1RA), and IL-10 at week 1 may predict patient outcomes. The majority of cytokines that are known to be associated with the cytokine storm in virus infections such as IL-6 and IFN-γ were only significantly elevated in the late stage of severe COVID-19 illness. TNF-α and GM-CSF showed no significant differences between severe and mild cases. CONCLUSION. Together, our data suggest that early intervention to increase expression of CCL5 may prevent patients from developing severe illness. Our data also suggest that measurement of levels of CCL5, as well as IL-1RA and IL-10 in blood individually and in combination, might be useful prognostic biomarkers to guide treatment strategies.
Yan Zhao, Ling Qin, Ping Zhang, Kang Li, Lianchun Liang, Jianping Sun, Bin Xu, Yanchao Dai, Xuemei Li, Chi Zhang, Yanchun Peng, Yingmei Feng, Ang Li, Zhongjie Hu, Haiping Xiang, Graham Ogg, Ling-Pei Ho, Andrew McMichael, Ronghua Jin, Julian C. Knight, Tao Dong, Yonghong Zhang