The prevalence of obesity and type 2 diabetes is growing at an alarming rate, including among pregnant women. Low-calorie sweeteners (LCS) have increasingly been used as an alternative to sugar to deliver a sweet taste without the excessive caloric load. However, there is little evidence regarding their biological effects, particularly during development. Here, we used a mouse model of maternal LCS consumption to explore the impact of perinatal LCS exposure on the development of neural systems involved in metabolic regulations. We report that adult male, but not female, offspring from both aspartame- and rebaudioside A-exposed dams displayed increased adiposity and developed glucose intolerance. Moreover, maternal LCS consumption reorganized hypothalamic melanocortin circuits and disrupted parasympathetic innervation of pancreatic islets in male offspring. We then identified phenylacetylglycine (PAG) as a unique metabolite that is upregulated in the milk of LCS-fed dams and the serum of their pups. Furthermore, maternal PAG treatment recapitulates some of the key metabolic and neurodevelopmental abnormalities associated with maternal LCS consumption. Together, our data indicate that maternal LCS consumption has enduring consequences on the offspring's metabolism and neural development and that these effects are likely to be mediated through the gut microbial co-metabolite PAG.
Soyoung Park, Amine M. Belfoul, Marialetizia Rastelli, Alice Jang, Magali Monnoye, Hosung Bae, Anna Kamitakahara, Patrick Giavalisco, Shan Sun, Pierre-Yves Barelle, Jasmine Plows, Cholsoon Jang, Anthony Fodor, Michael I. Goran, Sebastien G. Bouret
Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1, an inwardly rectifying potassium channel that regulates membrane hyperpolarization, contributes to vasodilation in resistance arteries. First, we show phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data has implied PS may compete with PIP2 binding on Kir2.1. We found 83.33% of Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate PS blocks PIP2 activation of Kir2.1, and addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggests PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and demonstrates the intracellular lipid localization within endothelium is an important determinant of vascular function.
Claire A. Ruddiman, Richard G. Peckham, Melissa A. Luse, Yen-Lin Chen, Maniselvan Kuppusamy, Bruce A. Corliss, Jordan Hall, Chien-Jung Lin, Shayn M. Peirce, Swapnil K. Sonkusare, Robert P. Mecham, Jessica E. Wagenseil, Brant E. Isakson
Synovial Fibroblasts (SFs) are key pathogenic drivers in Rheumatoid arthritis (RA). Their in vivo activation by TNF is sufficient to orchestrate full arthritic pathogenesis in animal models and TNF blockade proved efficacious for a high percentage of RA patients albeit co-inducing rare but serious side effects. Aiming to find new potent therapeutics, we applied the L1000CDS2 search engine, in order to repurpose drugs that could reverse the pathogenic expression signature of arthritogenic human TNF transgenic (hTNFtg) SFs. We identified a neuroleptic drug, namely Amisulpride, which reduced SFs’ inflammatory potential while decreasing the clinical score of hTNFtg polyarthritis. Notably, we found that Amisulpride function is neither through its known targets Dopamine receptors 2 and 3 and Serotonin Receptor 7, nor through TNF-TNFRI binding inhibition. Through a click chemistry approach, novel potential targets of Amisulpride were identified, which were further validated to repress hTNFtg SFs’ inflammatory potential ex vivo (Ascc3 and Sec62), while phosphoproteomics analysis revealed that treatment altered important fibroblast activation pathways, such as adhesion. Thus, Amisulpride could prove beneficial to patients suffering from RA and the often-accompanying comorbid dysthymia, reducing SF pathogenicity along with its anti-depressive activity, serving further as a “lead” compound for the development of novel therapeutics against fibroblast activation.
Dimitra Papadopoulou, Fani Roumelioti, Christos Tzaferis, Panagiotis Chouvardas, Anna-Kathrine Pedersen, Filippos Charalampous, Eleni Christodoulou-Vafeiadou, Lydia Ntari, Niki Karagianni, Maria C. Denis, Jesper V. Olsen, Alexis Ν. Matralis, George Kollias
Although the expression of Mex3 RNA binding family member B (MEX3B) is upregulated in human nasal epithelial cells (HENCs) predominately in the eosinophilic chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) subtype, its functions as an RNA binding protein in airway epithelial cells remain unknown. Here, we revealed the role of MEX3B based on different subtypes of CRS, and demonstrated that MEX3B decreased TGF-β receptor III (TGFBR3) mRNA level by binding to its 3’ UTR and reducing its stability in HNECs. TGF-βR3 was found to be a TGF-β2 specific coreceptor in HNECs. Knocking down or overexpressing MEX3B promoted or inhibited TGF-β2-induced phosphorylation of Smad2 in HNECs, respectively. TGF-βR3 and p-Smad2 levels were downregulated in CRSwNP compared with controls and CRS without nasal polyps (CRSsNP), with a more prominent downregulation in the eosinophilic CRSwNP. TGF-β2 promoted collagen production in HNECs. Collagen abundance decreased and edema scores increased in CRSwNP compared to control, again more prominently in the eosinophilic type. Collagen expression in eosinophilic CRSwNP was negatively correlated with MEX3B but positively correlated with TGF-βR3. These results suggest that MEX3B inhibits tissue fibrosis in eosinophilic CRSwNP by downregulating epithelial cell TGFBR3 expression; consequently, MEX3B might be a valuable therapeutic target against eosinophilic CRSwNP.
Jin-Xin Liu, Chen Ao-Nan, Qihong Yu, Ke-Tai Shi, Yi-Bo Liu, Cui-Lian Guo, Zhe-Zheng Wang, Yin Yao, Li Pan, Xiang Lu, Kai Xu, Heng Wang, Ming Zeng, Chaohong Liu, Robert P. Schleimer, Ning Wu, Bo Liao, Zheng Liu
Vascular smooth muscle-derived Sca1+ adventitial progenitor (AdvSca1-SM) cells are tissue resident, multipotent stem cells that contribute to progression of vascular remodeling and fibrosis. Upon acute vascular injury, AdvSca1-SM cells differentiate into myofibroblasts and are embedded in perivascular collagen and extracellular matrix. While the phenotypic properties of AdvSca1-SM-derived myofibroblasts have been defined, the underlying epigenetic regulators driving the AdvSca1-SM-to-myofibroblast transition are unclear. We show that the chromatin remodeler, Smarca4/Brg1, facilitates AdvSca1-SM myofibroblast differentiation. Brg1 mRNA and protein was upregulated in AdvSca1-SM cells after acute vascular injury and pharmacological inhibition of Brg1 by the small molecule PFI-3 attenuated perivascular fibrosis and adventitial expansion. TGF-β1 stimulation of AdvSca1-SM cells in vitro reduced expression of stemness genes while inducing expression of myofibroblast genes that was associated with enhanced contractility; PFI blocked TGF-β1-induced phenotypic transition. Similarly, genetic knockdown of Brg1 in vivo reduced adventitial remodeling and fibrosis and reversed AdvSca1-SM-to-myofibroblast transition in vitro. Mechanistically, TGF-β1 promoted redistribution of Brg1 from distal intergenic sites of stemness genes and recruitment to promoter regions of myofibroblast-related genes, which was blocked by PFI-3. These data shed insight into epigenetic regulation of resident vascular progenitor cell differentiation and support that manipulating the AdvSca1-SM phenotype will provide important anti-fibrotic clinical benefit.
Austin J. Jolly, Sizhao Lu, Allison M. Dubner, Keith A. Strand, Marie F. Mutryn, Aaron Pilotti-Riley, Etienne P. Danis, Raphael A. Nemenoff, Karen S. Moulton, Mark W. Majesky, Mary C.M. Weiser-Evans
Invariant Natural Killer T (iNKT) cells act at the interface between lipid metabolism and immunity, due to their restriction to lipid antigens presented on CD1d by antigen presenting cells (APC). How foreign lipid antigens are delivered to APC remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), Isoglobotrihexosylceramide (iGb3) and OCH, a sphingosine-truncated analogue of αGalCer, with very-low-density (VLDL) and/or low-density (LDL) lipoproteins in vitro and in vivo. We demonstrate LDL receptor (LDLR)-mediated uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a novel mechanism of lipid antigen delivery to APCs, and provides further insight in the immunological capacities of circulating lipoproteins.
Suzanne E. Engelen, Francesca A. Ververs, Angela Markovska, B. Christoffer Lagerholm, Jordan M. Kraaijenhof, Laura I.E. Yousif, Yasemin-Xiomara Zurke, Can M.C. Gulersonmez, Sander Kooijman, Michael Goddard, Robert J. van Eijkeren, Peter J. Jervis, Gurdyal S. Besra, Saskia Haitjema, Folkert W. Asselbergs, Eric Kalkhoven, Hidde L. Ploegh, Marianne Boes, Vincenzo Cerundolo, G. Kees Hovingh, Mariolina Salio, Edwin C.A. Stigter, Patrick C.N. Rensen, Claudia Monaco, Henk S. Schipper
Loss of function mutations in the DNA methyltransferase 3A (DNMT3A) are seen in a large number of AML patients with normal cytogenetics and are frequently associated with poor prognosis. DNMT3A mutations are an early pre-leukemic event, which when combined with other genetic lesions result in full blown leukemia. Here, we show that loss of Dnmt3a in HSC/Ps results in myeloproliferation, which is associated with hyperactivation of the PI3Kinase pathway. PI3Kα/β or the PI3Kα/δ inhibitor treatment partially corrects myeloproliferation, although the partial rescue is more efficient in response to the PI3Kα/β inhibitor treatment. In vivo RNA-seq analysis on drug treated Dnmt3a–/– HSC/Ps showed a reduction in the expression of genes associated with chemokines, inflammation, cell attachment and extracellular matrix compared to controls. Remarkably, drug treated leukemic mice showed a reversal in the enhanced fetal liver HSC like gene signature observed in vehicle treated Dnmt3a–/– LSK cells as well as a reduction in the expression of genes involved in regulating actin cytoskeleton-based functions including the RHO/RAC GTPases. In a human PDX model bearing DNMT3A mutant AML, PI3Kα/β inhibitor treatment prolonged their survival and rescued the leukemic burden. Our results identify a new target for treating DNMT3A mutation driven myeloid malignancies.
Lakshmi Reddy Palam, Baskar Ramdas, Katelyn M. Pickerell, Santhosh Kumar Pasupuleti, Rahul Kanumuri, Annamaria Cesarano, Megan Szymanski, Bryce M. Selman, Utpal P. Davé, George Sandusky, Fabiana Perna, Sophie Paczesny, Reuben Kapur
Several pre-clinical studies have demonstrated that certain cytotoxic drugs enhance metastasis, but the importance of host responses triggered by chemotherapy in regulating cancer metastasis has not been fully explored. Here, we showed that multi-dose Gemcitabine (GEM) treatment promoted breast cancer lung metastasis in a transgenic spontaneous breast cancer model. GEM treatment significantly increased accumulation of CCR2+ macrophages and monocytes in the lungs of tumor-bearing as well as tumor-free mice. These changes were largely caused by chemotherapy induced reactive myelopoiesis that is biased toward monocyte development. Mechanistically, enhanced production of mitochondrial ROS was observed in GEM treated BM LSK cells and monocytes. Treatment with the mitochondrial targeted antioxidant abrogated GEM induced hyper-differentiation of BM progenitors. In addition, GEM treatment induced up-regulation of host cell derived CCL2, and knockout CCR2 signaling abrogated the pro-metastatic host response induced by chemotherapy. Furthermore, chemotherapy treatment resulted in the upregulation of coagulation factor X (FX) in lung interstitial macrophages. Targeting activated FX (FXa) using FXa inhibitor or F10 gene knockdown reduced pro-metastatic effect of chemotherapy. Together, these studies suggest a novel mechanism for chemotherapy induced metastasis via the host response induced accumulation of monocytes/macrophages and interplay between coagulation and inflammation in the lungs.
Caijun Wu, Qian Zhong, Rejeena Shrestha, Jingzhi Wang, Xiaoling Hu, Hong Li, Eric C. Rouchka, Jun Yan, Chuanlin Ding
BACKGROUND. Due to their immunoregulatory and tissue regenerative features, mesenchymal stromal cells (MSCs) are a promising novel tool for the management of ulcerative proctitis (UP). Here we report on a phase IIa clinical study to evaluate the impact of local MSC therapy in UP. METHODS. Thirteen refractory UP patients, with endoscopic Mayo score (EMS) 2 or 3, were included. Seven patients received 20-40 x 106 allogeneic MSCs (cohort 1), while six patients received 40-80 x 106 MSCs (cohort 2). Adverse events (AEs) were assessed at baseline and week 2, 6, 12, and 24. Clinical, endoscopic, and biochemical parameters were assessed at baseline, week 2 and 6. Furthermore, we evaluated the engraftment of MSCs, presence of donor-specific human leukocyte antigen (HLA) antibodies (DSAs), and we determined the impact of MSC therapy on the local immune compartment. RESULTS. No serious AEs were observed. The clinical Mayo score was significantly improved at week 2 and 6, and the EMS was significantly improved at week 6, compared to baseline. At week 6, donor MSCs were still detectable in rectum biopsies of 4/9 patients and DSAs against both HLA-class I and -class II were found. Mass cytometry showed a reduction of activated CD8+ T cells and CD16+ monocytes and an enrichment in mononuclear phagocytes and natural killer cells in biopsies after local MSC therapy. CONCLUSION. Local administration of allogeneic MSCs is safe, tolerable, and feasible for treatment of refractory UP and shows encouraging signs of clinical efficacy and modulation of local immune responses. This sets the stage for larger clinical trials. TRIAL REGISTRATION. clinicaltrialsregister.eu, EudraCT: 2017-003524-75, Dutch Trial register: NTR7205. FUNDING. ECCO grant 2020.
Laura F. Ouboter, Marieke C. Barnhoorn, Hein W. Verspaget, Leonie Plug, Emma S. Pool, Karoly Szuhai, Lukas J.A.C. Hawinkels, Melissa van Pel, Jaap Jan Zwaginga, Dave Roelen, Frits Koning, M. Fernanda Pascutti, Andrea van der Meulen - de Jong
BACKGROUND. Longitudinal investigations of murine acute kidney injury (AKI) suggest that injury and inflammation may persist long after the initial insult. However, the evolution of these processes and their prognostic values are unknown in patients with AKI. METHODS. In a prospective cohort of 656 participants hospitalized with AKI, we measured seven urine and two plasma biomarkers of kidney injury, inflammation, and tubular health at multiple timepoints from the diagnosis to 12 months after AKI. We used linear mixed-effect models to estimate biomarker changes over time, and used Cox proportional hazard regressions to determine their associations with a composite outcome of CKD incidence and progression. We compared the gene expression kinetics of biomarkers in murine models of repair and atrophy after ischemic reperfusion injury (IRI). RESULTS. After 4.3 years, 106 and 52 participants developed incident CKD and CKD progression, respectively. Each standard deviation increases in the change of urine KIM-1, MCP-1 and plasma TNFR1 from baseline to 12 months was associated with 2-3-fold increased risk for CKD, while the increase in urine UMOD was associated with 40% reduced risk for CKD. The trajectories of these biological processes were associated with progression to kidney atrophy in mice after IRI. CONCLUSION. Sustained tissue injury and inflammation, and slower restoration of tubular health are associated with higher risk of kidney disease progression. Further investigation into these ongoing biological processes may help understand and prevent the AKI-to-CKD transition. FUNDING. NIH and NIDDK (grants U01DK082223, U01DK082185, U01DK082192, U01DK082183, R01DK098233, R01DK101507, R01DK114014, K23DK100468, R03DK111881, K01DK120783, and R01DK093771).
Yumeng Wen, Leyuan Xu, Isabel A. Melchinger, Heather Thiessen-Philbrook, Dennis G. Moledina, Steven G. Coca, Chi-yuan Hsu, Alan S. Go, Kathleen D. Liu, Edward D. Siew, T. Alp Ikizler, Vernon M. Chinchilli, James S. Kaufman, Paul L. Kimmel, Jonathan Himmelfarb, Lloyd G. Cantley, Chirag R. Parikh
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