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Abstract
Airway neutrophilia occurs in approximately 50% of patients with asthma and is associated with particularly severe disease. Unfortunately, this form of asthma is usually refractory to corticosteroid treatment, and there is an unmet need for new therapies. Pulmonary neutrophilic inflammation is associated with Th17 cells, whose differentiation is controlled by the nuclear receptor, RORγt. Here, we tested whether VTP-938, a selective inverse agonist of this receptor, can reduce disease parameters in animal models of neutrophilic asthma. When administered prior to allergic sensitization through the airway, the RORγt inverse agonist blunted allergen-specific Th17 cell development in lung-draining lymph nodes and attenuated allergen-induced production of IL-17. VTP-938 also reduced pulmonary production of IL-17 and airway neutrophilia when given during the allergen challenge of the model. Finally, in an environmentally relevant model of allergic responses to house dust extracts, VTP-938 suppressed production of IL-17 and neutrophilic inflammation, and also markedly diminished airway hyperresponsiveness. Together, these findings suggest that orally available inverse agonists of RORγt might provide an effective therapy to treat glucocorticoid-resistant neutrophilic asthma.
Authors
Gregory S. Whitehead, Hong Soon Kang, Seddon Y. Thomas, Alexander Medvedev, Tadeusz P. Karcz, Gentaro Izumi, Keiko Nakano, Sergei S. Makarov, Hideki Nakano, Anton M. Jetten, Donald N. Cook
Abstract
Mutations in BSCL2 gene underlie human type 2 Berardinelli-Seip Congenital Lipodystrophy (BSCL2) disease. Global Bscl2−/− mice recapitulate human BSCL2 lipodystrophy and develop insulin resistance and hypertrophic cardiomyopathy. The pathological mechanisms underlying the development of lipodystrophy and cardiomyopathy in BSCL2 are controversial. Here we report that Bscl2−/− mice develop cardiac hypertrophy due to increased basal IGF1 receptor (IGF1R)-mediated PI3K/AKT signaling. Bscl2−/− hearts exhibited increased adipose triglyceride lipase (ATGL) protein stability and expression causing drastic reduction of glycerolipids. Excessive fatty acid oxidation was overt in Bscl2−/− hearts, partially attributing to the hyperacetylation of cardiac mitochondrial proteins. Intriguingly, pharmacological inhibition or genetic inactivation of ATGL could rescue adipocyte differentiation and lipodystrophy in Bscl2−/− cells and mice. Restoring a small portion of fat mass by ATGL partial deletion in Bscl2−/− mice not only reversed the systemic insulin resistance, but also ameliorated cardiac protein hyperacetylation, normalized cardiac substrate metabolism and improved contractile function. Collectively, our study uncovers novel pathways underlying lipodystrophy-induced cardiac hypertrophy and metabolic remodeling and pinpoints ATGL as a downstream target of BSCL2 in regulating the development of lipodystrophy and its associated cardiomyopathy.
Authors
Hongyi Zhou, Xinnuo Lei, Yun Yan, Todd Lydic, Jie Li, Neal L. Weintraub, Huabo Su, Weiqin Chen
Abstract
Progression of fibrosis and the development of cirrhosis are responsible for the liver related morbidity and mortality associated with chronic liver diseases. There is currently a great unmet need for effective anti-fibrotic strategies. Stem cells play a central role in wound healing responses to restore liver homeostasis following injury. Here we tested the hypothesis that extracellular vesicles (EVs) isolated from induced pluripotent stem cells (iPSC) modulate hepatic stellate cell (HSCs) activation and may have anti-fibrotic effects. Human iPSCs were generated by reprogramming primary skin fibroblasts. EVs were isolated by differential centrifugation, quantified by flow cytometry (FACS) and characterized by dynamic light scattering (DLS) and electron microscopy (TEM). Primary human HSCs were activated with TGFβ (10 ng/mL) and exposed to iPSC-EVs. Efficacy of iPSC-EVs was tested on HSC in vitro and in two murine models of liver injury (CCl4 and bile duct ligation). Characterization of iPSC-derived EVs by flow cytometry identified a large population of EVs released by iPSC, primarily with a diameter of 300 nm and that could be visualized by TEM as round, cup-shaped objects. Fluorescent tracing assays detected iPSC-EVs in HSC cytosol after a short incubation and EV uptake by HSCs resulted in both decrease of pro-fibrogenic markers αSMA, CollagenIα1, Fibronectin and TIMP-1 and HSC pro-fibrogenic responses such as chemotaxis and proliferation. Genomics analyses of iPSC-EV miRNA cargo revealed 22 highly expressed miRNAs, among which miR-92a-3p resulted the most abundant. Transcriptome analysis identified 60 genes down-modulated and 235 up-regulated in TGF-β-primed HSC in presence or absence of iPSC-EVs. Intravenous injection of iPSC-EVs in CCl4 and bile duct ligation-induced liver fibrosis resulted in anti-fibrotic effects at protein and gene levels. Results of this study identify iPSC-EVs as a novel anti-fibrotic approach that may reduce or reverse liver fibrosis in patients with chronic liver disease.
Authors
Davide Povero, Eva M. Pinatel, Aleksandra Leszczynska, Nidhi P. Goyal, Takahiro Nishio, Jihoon Kim, David Kneiber, Lucas de Araujo Horcel, Akiko Eguchi, Paulina M. Ordonez, Tatiana Kisseleva, Ariel E. Feldstein
Abstract
BACKGROUND. Black individuals have lower natriuretic peptide levels and greater risk of heart failure (HF) than white individuals. Higher N-terminal-pro-B-type natriuretic peptide (NT-proBNP) is associated with increased risk of incident HF, but little information is available in black individuals. We examined race-specific differences in 1) the association of NT-proBNP with incident HF and 2) the predictive ability of NT-proBNP for incident HF across body mass index (BMI) and estimated glomerular filtration rate (eGFR) categories. METHODS. In a prospective case-cohort study, baseline NT-proBNP was measured among 687 participants with incident HF and 2,923 (weighted 20,075) non-case randomly selected participants. Multivariable Cox proportional hazard modeling was used to assess the objectives of our study. Global Wald Chi-square score estimated from multivariable Cox models was used to assess predictive ability of NT-proBNP across BMI and eGFR categories. RESULTS. In the multivariable model, a doubling of NT-proBNP concentration was associated with greater risk of incident HF among white individuals [hazard ratio (HR): 1.73; 95% CI: 1.55–1.94] than black individuals (HR: 1.51; 95% CI: 1.34–1.70); Pinteraction by race = 0.024. Higher NT-proBNP was the strongest predictor of incident HF across all BMI and eGFR categories among white individuals. By contrast, among black individuals with obesity (BMI ≥ 30 kg/m2) or eGFR < 60 mL/min/1.73 m2, the predictive ability of NT-proBNP for incident HF was attenuated. CONCLUSIONS. The magnitude of the association of higher NT-proBNP with incident HF risk was greater among white individuals than black individuals. The diminished ability of NT-proBNP to predict the risk of HF in black population with obesity or impaired kidney function highlights the need of further investigations.
Authors
Nirav Patel, Mary Cushman, Orlando M. Gutierrez, George Howard, Monika M. Safford, Paul Muntner, Raegan W. Durant, Sumanth D. Prabhu, Garima Arora, Emily B. Levitan, Pankaj Arora
Abstract
Tuberculosis patients and mice infected with live Mycobacterium tuberculosis (Mtb) accumulate high numbers of myeloid-derived suppressor cells (MDSCs). Here, we hypothesized that also dead Mtb vaccines may induce MDSCs that could impair the efficacy of vaccination. We found that repeated injections of Mtb vaccines (heat-killed Mtb in Incomplete Freund's Adjuvant, like Montanide) but not single or control vaccines without Mtb strongly expanded CD11b+ myeloid cells in the spleen, that suppressed T cell proliferation and killing ex vivo. Dead Mtb vaccination induced the generation of CD11b+ Ly-6Chigh CD115+ iNOS/Nos2+ monocytic MDSCs (M-MDSCs) upon application of inflammatory or microbial activation signals. In vivo these M-MDSCs positioned strategically in the spleen by infiltrating the splenic bridging channels and white pulp areas. Notably, within 6 to 24 hours in a Nos2-dependent fashion they produced NO to rapidly kill conventional and plasmacytoid dendritic cells (cDCs, pDCs) while, surprisingly, sparing T cells in vivo. Thus, we demonstrate that Mtb vaccine induced M-MDSCs to not directly suppress T cell in vivo but, instead, M-MDSCs directly target DC subpopulations thereby indirectly suppressing effector T cell responses. Collectively, we demonstrate that Mtb booster vaccines induce M-MDSCs in the spleen that can be activated to kill DCs cautioning to thoroughly investigate MDSC formation in individuals after Mtb vaccination in clinical trials.
Authors
Eliana Ribechini, Ina Eckert, Andreas Beilhack, Nelita Du Plessis, Gerhard Walzl, Ulrike Schleicher, Uwe Ritter, Manfred B. Lutz
Abstract
Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the non-neuronal cholinergic system is not well understood. To address the immunomodulatory role of the non-neuronal cholinergic system in the heart we used a previously validated diphtheria toxin (DT)-induced cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or Pyridostigmine Bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIlowCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared to diluent treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with ACh, nicotine and muscarine. Viewed together, these findings reveal a previously unappreciated immunomodulatory role for the non-neuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.
Authors
Cibele Rocha-Resende, Carla Weinheimer, Geetika Bajpai, Luigi Adamo, Scot J. Matkovich, Joel Schilling, Philip M. Barger, Kory J. Lavine, Douglas L. Mann
Abstract
BACKGROUND. Sphingolipids (SPs) are ubiquitous, structurally diverse molecules that include ceramides, sphingomyelins, and sphingosines. They are involved in various pathologies including obesity and type 2 diabetes mellitus (T2DM). Therefore, it is likely that perturbations in plasma concentrations of SPs are associated with disease. Identifying these associations may reveal useful biomarkers or provide insight into disease processes. METHODS. We performed a lipidomics evaluation of molecularly-distinct SPs in the plasma of 2,302 ethnically-Chinese Singaporeans using electrospray ionization mass spectrometry coupled with liquid chromatography. SP profiles were compared to clinical and biochemical characteristics, and subjects were evaluated by follow-up visits for 11 years. RESULTS. We found that ceramides correlate positively but hexosylceramides correlate negatively with body mass index (BMI) and homeostatic model assessment of insulin resistance (HOMA-IR). Furthermore, SPs with a d16:1 sphingoid backbone correlate more positively with BMI and HOMA-IR, while d18:2 SPs correlate less positively, relative to canonical d18:1 SPs. We also found that higher concentrations of two distinct sphingomyelins were associated with a higher risk of T2DM (HR 1.45, 95% CI 1.18–1.78 for SM d16:1/C18:0; and HR 1.40, 95% CI 1.17–1.68 for SM d18:1/C18:0). CONCLUSION. We identified significant associations between SPs and obesity/T2DM characteristics, specifically, that of hexosylceramides, d16:1 SPs, and d18:2 SPs. This suggests that the balance of SP metabolism, rather than ceramide accumulation, is associated with the pathology of obesity. We further identified two specific SPs that may represent prognostic biomarkers for T2DM. FUNDING. Funding sources are listed in the Acknowledgements section
Authors
Wee Siong Chew, Federico Torta, Shanshan Ji, Hyungwon Choi, Husna Begum, Xueling Sim, Chin Meng Khoo, Eric Yin Hao Khoo, Wei-Yi Ong, Rob M. Van Dam, Markus R. Wenk, E. Shyong Tai, Deron R. Herr
Abstract
Excess dietary salt contributes to inflammation and hypertension via poorly understood mechanisms. Antigen presenting cells including dendritic cells (DCs) play a key role in regulating intestinal immune homeostasis in part by surveying the gut epithelial surface for pathogens. Previously, we found that highly reactive γ-ketoaldehydes or isolevuglandins (IsoLGs) accumulate in DCs and act as neoantigens, promoting an autoimmune-like state and hypertension. We hypothesized that excess dietary salt alters the gut microbiome leading to hypertension and this is associated with increased immunogenic IsoLG-adduct formation in myeloid antigen presenting cells. To test this hypothesis, we performed fecal microbiome analysis and measured blood pressure of healthy human volunteers with salt intake above or below the American Heart Association recommendations. We also performed 16S rRNA analysis on cecal samples of mice fed normal or high salt diets. In humans and mice, high salt intake was associated with changes in the gut microbiome reflecting an increase in Firmicutes, Proteobacteria and genus Prevotella bacteria. These alterations were associated with higher blood pressure in humans and predisposed mice to vascular inflammation and hypertension in response to a sub-pressor dose of angiotensin II. Mice fed a high salt diet exhibited increased intestinal inflammation including the mesenteric arterial arcade and aorta, with a marked increase in the B7 ligand CD86 and formation of IsoLG-protein adducts in CD11c+ myeloid cells. Adoptive transfer of fecal material from conventionally housed high salt-fed mice to germ-free mice predisposed them to increased intestinal inflammation and hypertension. These findings provide novel insight into the mechanisms underlying inflammation and hypertension associated with excess dietary salt and may lead to interventions targeting the microbiome to prevent and treat this important disease.
Authors
Jane F. Ferguson, Luul A. Aden, Natalia R. Barbaro, Justin P. Van Beusecum, Liang Xiao, Alan J. Simmons, Cassandra Warden, Lejla Pasic, Lauren E. Himmel, Mary K. Washington, Frank L. Revetta, Shilin Zhao, Shivani Kumaresan, Matthew B. Scholz, Zhengzheng Tang, Guanhua Chen, Muredach P. Reilly, Annet Kirabo
Abstract
Background: Little is known about the genomic differences between metastatic urothelial carcinoma (LTUC) and upper tract urothelial carcinoma (UTUC). We compare genomic features of primary and metastatic UTUC and LTUC tumors in a cohort of patients with end stage disease. Methods: We performed whole exome sequencing on matched primary and metastatic tumor samples (N=37) from 7 patients with metastatic UC collected via rapid autopsy. Inter- and intra-patient mutational burden, mutational signatures, predicted deleterious mutations, and somatic copy alterations (sCNV) were analyzed. Results: We investigated 3 patients with UTUC (3 primary samples, 13 metastases) and 4 patients with LTUC (4 primary samples, 17 metastases). We found that sSNV burden was higher in metastatic LTUC compared to UTUC. Moreover, the APOBEC mutational signature was pervasive in metastatic LTUC and less so in UTUC. Despite a lower overall sSNV burden, UTUC displayed greater inter- and intra-individual genomic distances at the copy number level between primary and metastatic tumors than LTUC. Our data also indicate that metastatic UTUC lesions can arise from small clonal populations present in the primary cancer. Importantly, putative druggable mutations were found across patients with the majority shared across all metastases within a patient. Conclusions: Metastatic UTUC demonstrated a lower overall mutational burden but greater structural variability compared to LTUC. Our findings suggest that metastatic UTUC displays a greater spectrum of copy number divergence from LTUC. Importantly, we identified druggable lesions shared across metastatic samples, which demonstrate a level of targetable homogeneity within individual patients.
Authors
Brian R. Winters, Navonil De Sarkar, Sonali Arora, Hamid Bolouri, Sujata Jana, Funda Vakar-Lopez, Heather H. Cheng, Michael T. Schweizer, Evan Y. Yu, Petros Grivas, John K. Lee, Lori Kollath, Sarah K. Holt, Lisa McFerrin, Gavin Ha, Peter S. Nelson, Robert B. Montgomery, Jonathan L. Wright, Hung-Ming Lam, Andrew C. Hsieh
Abstract
Sex-based differences influence incidence and outcome of infectious disease. Women have a significantly greater incidence of urinary tract infection (UTI) than men, yet, conversely, male UTI is more persistent with greater associated morbidity. Mechanisms underlying these sex-based differences are unknown, in part due to a lack of experimental models. We optimized a model to transurethrally infect male mice and directly compared UTI in both sexes. Although both sexes were initially equally colonized by uropathogenic E. coli, only male and testosterone-treated female mice remained chronically infected for up to 4 weeks. Female mice had more robust innate responses, including higher IL-17 expression, and increased γδ T cells and group 3 innate lymphoid cells in the bladder following infection. Accordingly, neutralizing IL-17 abolished resolution in female mice, identifying a cytokine pathway necessary for bacterial clearance. Our findings support the concept that sex-based responses to UTI contribute to impaired innate immunity in males and provide a rationale for non-antibiotic-based immune targeting to improve the response to UTI.
Authors
Anna Zychlinsky Scharff, Matthieu Rousseau, Livia Lacerda Mariano, Tracy Canton, Camila Rosat Consiglio, Matthew L. Albert, Magnus Fontes, Darragh Duffy, Molly A. Ingersoll
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