Gut microbiota (GM) dysbiosis is associated with inflammatory bowel diseases and also with cardiometabolic, neurologic, and autoimmune diseases. GM composition has a direct effect on the immune system, and vice versa, and particularly on regulatory T cell (Treg) homeostasis. Low-dose interleukin-2 (IL-2LD) stimulates Tregs and is a promising treatment for autoimmune and inflammatory diseases. We aimed to evaluate the impacts of IL-2LD on GM, and correlatively on the immune system. We used 16S ribosomal RNA profiling and metagenomics to characterize GM of mice and humans treated or not with IL-2LD. We performed faecal microbiota transplantation (FMT) from IL-2LD-treated to naïve recipient mice and evaluated its effects in models of gut inflammation and diabetes. IL-2LD markedly affects GM composition in mice and humans. Transfer of an IL-2-tuned microbiota by FMT protected C57BL/6J mice from dextran sulphate sodium-induced colitis and prevented diabetes in NOD mice. Metagenomic analyses highlighted a role for several species impacted by IL-2LD and for microbial pathways involved in the biosynthesis of amino acids, short-chain fatty acids, and L-arginine. Our results demonstrate that IL-2LD induces changes in GM that are involved in the immunoregulatory effects of IL-2LD and suggest a cross-talk between Tregs and GM. These results provide novel insights for understanding the mode of action of Treg-directed therapies.
Nicolas Tchitchek, Otriv Nguekap Tchoumba, Gabriel Pires, Sarah Dandou, Julien Campagne, Guillaume Churlaud, Gwladys Fourcade, Thomas W. Hoffmann, Francesco Strozzi, Camille Gaal, Christophe Bonny, Emmanuelle Le Chatelier, Stanislav Dusko Erlich, Harry Sokol, David Klatzmann
Cardiovascular diseases, especially atherosclerosis and its complications, are a leading cause of death. Inhibition of the non-canonical IκB kinases TBK1 and IKKε with amlexanox restores insulin sensitivity and glucose homeostasis in diabetic mice and human subjects. Here we report that amlexanox improves diet-induced hypertriglyceridemia and hypercholesterolemia in Western diet (WD)-fed Ldlr-/- mice, and protects against atherogenesis. Amlexanox ameliorates dyslipidemia, inflammation and vascular dysfunction through synergistic actions that involve upregulation of bile acid synthesis to increase cholesterol excretion. Transcriptomic profiling demonstrates an elevated expression of key bile acid synthesis genes. Furthermore, we found that amlexanox attenuates monocytosis, eosinophilia and vascular dysfunction during WD-induced atherosclerosis. These findings demonstrate the potential of amlexanox as a new therapy for hypercholesterolemia and atherosclerosis.
Peng Zhao, Xiaoli Sun, Zhongji Liao, Hong Yu, Dan Li, Zeyang Shen, Christopher K. Glass, Joseph L. Witztum, Alan R. Saltiel
The sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions such as myopathies. Here, we investigated whether the nuclear receptor subfamily 1 group D member (NR1D1, also called REV-ERBα) regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that NR1D1 deficiency in mice impairs SERCA-dependent SR calcium uptake. NR1D1 acts on calcium homeostasis by repressing the SERCA inhibitor myoregulin through direct binding to its promoter. Restoration of myoregulin counteracts the effects of NR1D1 overexpression on SR calcium content. Interestingly, myoblasts from Duchenne myopathy patients display lower NR1D1 expression, whereas pharmacological NR1D1 activation ameliorates SR calcium homeostasis, and improves muscle structure and function in dystrophic mdx/Utr+/- mice. Our findings demonstrate that NR1D1 regulates muscle SR calcium homeostasis, pointing to its therapeutic interest for mitigating myopathy.
Alexis Boulinguiez, Christian Duhem, Alicia Mayeuf-Louchart, Benoit Pourcet, Yasmine Sebti, Kateryna Kondratska, Valérie Montel, Stéphane Delhaye, Quentin Thorel, Justine Beauchamp, Aurore Hebras, Marion Gimenez, Marie Couvelaere, Mathilde Zecchin, Lise Ferri, Natalia Prevarskaya, Anne Forand, Christel Gentil, Jessica Ohana, France Piétri-Rouxel, Bruno Bastide, Bart Staels, Helene Duez, Steve Lancel
The blood-brain barrier is formed by capillary endothelial cells expressing Cx37, Cx40 and Cx43, and is joined by closely apposed astrocytes expressing Cx43 and Cx30. We investigated whether connexin-targeting peptides could limit barrier leakage triggered by LPS-induced systemic inflammation in mice. Intraperitoneal LPS increased endothelial and astrocytic Cx43 expression, elevated TNFα, IL1β, IFNγ and IL6 in plasma and IL6 in the brain, and induced barrier leakage recorded over 24h. Barrier leakage was largely prevented by global Cx43 knockdown and Cx43/Cx30 double-knockout in astrocytes, slightly diminished by endothelial Cx43 knockout and not protected by global Cx30 knockout. Intravenous administration of Gap27 or Tat-Gap19 just before LPS also prevented barrier leakage, and intravenous BAPTA-AM to chelate intracellular calcium was equally effective. Patch-clamp experiments demonstrated LPS-induced Cx43 hemichannel opening in endothelial cells, which was suppressed by Gap27, Gap19 and BAPTA. LPS additionally triggered astrogliosis that was prevented by intravenous Tat-Gap19 or BAPTA-AM. Cortically applied Tat-Gap19 or BAPTA-AM to primarily target astrocytes, also strongly diminished barrier leakage. In vivo dye uptake and in vitro patch-clamp showed Cx43 hemichannel opening in astrocytes that was induced by IL6 in a calcium-dependent manner. We conclude that targeting endothelial and astrocytic connexins is a powerful approach to limit barrier failure and astrogliosis.
Marijke De Bock, Maarten A.J. De Smet, Stijn Verwaerde, Hanane Tahiri, Steffi Schumacher, Valérie Van Haver, Katja Witschas, Christian Steinhäuser, Nathalie Rouach, Roosmarijn E. Vandenbroucke, Luc Leybaert
Acquired mutations in the ligand-binding domain (LBD) of the gene encoding Estrogen Receptor alpha (ESR1) are a common mechanism of endocrine therapy resistance in metastatic ER-positive breast cancer patients. ESR1 Y537S mutation, in particular, is associated with development of resistance to most endocrine therapies used to treat breast cancer. Employing a high-throughput screen of nearly 1200 Federal Drug Administration (FDA)-approved drugs, we show that OTX015, a bromodomain and extraterminal domain (BET) inhibitor, is one of the top suppressors of ESR1 mutant cell growth. OTX015 was more efficacious than fulvestrant, a selective ER degrader, in inhibiting ESR1 mutant xenograft growth. When combined with abemaciclib, a CDK4/6 inhibitor, OTX015 induced more potent tumor regression than current standard-of-care treatment of abemaciclib+fulvestrant. OTX015 has preferential activity against Y537S mutant breast cancer cells and blocks their clonal selection in competition studies with wild-type cells. Thus, BET inhibition has the potential to both prevent and overcome ESR1 mutant-induced endocrine therapy resistance in breast cancer.
Sm N. Udden, Qian Wang, Sunil Kumar, Venkat S. Malladi, Shwu-Yuan Wu, Shuguang Wei, Bruce A. Posner, Sophie Geboers, Noelle S. Williams, Yu-Lun Liu, Jayesh K. Sharma, Ram S. Mani, Srinivas Malladi, Karla Parra, Mia Hofstad, Ganesh V. Raj, Jose M. Larios, Reshma Jagsi, Max S. Wicha, Ben Ho Park, Gaorav P. Gupta, Arul M. Chinnaiyan, Cheng-Ming Chiang, Prasanna G. Alluri
Increased red cell distribution width (RDW), which measures erythrocyte volume (MCV) variability (anisocytosis), has been linked to early mortality in many diseases and in older adults through unknown mechanisms. Hypoxic stress has been proposed as a potential mechanism. However, experimental models to investigate the link between increased RDW and reduced survival are lacking. Here, we show that lifelong hypobaric hypoxia (~10% O2) increases erythrocyte numbers, hemoglobin and RDW, while reducing longevity in male mice. Compound heterozygous knockout (chKO) mutations in succinate dehydrogenase (Sdh; mitochondrial complex II) genes Sdhb, Sdhc and Sdhd reduce Sdh subunit protein levels, RDW, and increase healthy lifespan compared to wild-type (WT) mice in chronic hypoxia. RDW-SD, a direct measure of MCV variability, and the standard deviation of MCV (1SD-RDW) show the most statistically significant reductions in Sdh hKO mice. Tissue metabolomic profiling of 147 common metabolites shows the largest increase in succinate with elevated succinate to fumarate and succinate to oxoglutarate (2-ketoglutarate) ratios in Sdh hKO mice. These results demonstrate that mitochondrial complex II level is an underlying determinant of both RDW and healthy lifespan in hypoxia, and suggest that therapeutic targeting of Sdh might reduce high RDW-associated clinical mortality in hypoxic diseases.
Bora E. Baysal, Abdulrahman A. Alahmari, Tori C. Rodrick, Debra Tabaczynski, Leslie Curtin, Mukund Seshadri, Drew R. Jones, Sandra Sexton
Regulatory CD4+Foxp3+ T cells (Treg) restrain inflammation and immunity. However, the mechanisms underlying Treg suppressor function in inflamed non-lymphoid tissues remain largely unexplored. Here, we restricted immune responses to non-lymphoid tissues and used intravital microscopy to visualize Treg suppression of rejection by effector T cells (Teff) within inflamed allogeneic islet transplants. Despite their elevated motility, Treg preferentially contact antigen-presenting cells (APCs) over Teff. Interestingly, Treg specifically target APCs that are extensively and simultaneously contacted by Teff. In turn, Treg decrease MHC-II expression on APCs and hinder Teff function. Lastly, we demonstrate that Treg suppressor function within inflamed allografts requires ecto-nucleotidase CD73 activity, which generates the anti-inflammatory adenosine. Consequently, CD73-/- Treg exhibit reduced contacts with APCs within inflamed allografts compared to wt Treg, but not in spleen. Overall, our findings demonstrate that Treg suppress immunity within inflamed grafts through CD73 activity and suggest that Treg-APC direct contacts are central to this process.
Hehua Dai, Andressa Pena, Lynne Bauer, Amanda Williams, Simon c. Watkins, Geoffrey Camirand
Pathogenic variants in the human F Box and Leucine Rich Repeat Protein 4 (FBXL4) gene result in an autosomal recessive, multi-systemic, mitochondrial disorder involving variable mitochondrial depletion and respiratory chain (RC) complex deficiencies with lactic acidemia. As no FDA-approved effective therapies exist, we sought to characterize translational C. elegans and zebrafish animal models, as well as human fibroblasts, to study FBXL4-/- disease mechanisms and identify preclinical therapeutic leads. Developmental delay, impaired fecundity and neurologic and/or muscular activity, mitochondrial dysfunction, and altered lactate metabolism were identified in fbxl-1(ok3741) C. elegans. Detailed studies of a pyruvate dehydrogenase complex activator, dichloroacetate (DCA) in fbxl-1(ok3741) C. elegans demonstrated its beneficial effects on fecundity, neuromotor activity, and mitochondrial function. Validation studies were performed in fbxl4sa12470 zebrafish larvae and in FBXL4-/- human fibroblasts, which showed DCA efficacy in preventing brain damage, impairment of neurologic and/or muscular function, mitochondrial biochemical dysfunction, and stress-induced morphologic and ultrastructural mitochondrial defects. These data demonstrate that fbxl-1 (ok3741) C. elegans and fbxl4sa12470 zebrafish provide robust translational models to study mechanisms and identify preclinical therapeutic candidates for FBXL4-/- disease. Further, DCA is a lead therapeutic candidate with therapeutic benefit on diverse aspects of survival, neurologic and/or muscular function, and mitochondrial physiology that warrants rigorous clinical trial study in human subjects with FBXL4-/- disease.
Manuela Lavorato, Eiko Nakamaru-Ogiso, Neal D. Mathew, Elizabeth Herman, Nina K. Shah, Suraiya Haroon, Rui Xiao, Christoph Seiler, Marni J. Falk
Accumulation of activated natural killer (NK) cells in tissues during Ebola virus infection contributes to Ebola virus disease (EVD) pathogenesis. Yet, immunization with Ebola virus-like particles (VLPs) comprising glycoprotein (GP) and matrix protein VP40 provides rapid, NK cell-mediated protection against Ebola challenge. We used Ebola VLPs as the viral surrogates to elucidate the molecular mechanism by which Ebola virus triggers heightened NK cell activity. Incubation of human peripheral blood mononuclear cells (PBMCs) with Ebola VLPs or VP40 protein led to increased expression of IFN-γ, TNF-α, granzyme B, and perforin by CD3-CD56+ NK cells, along with concomitant increase in degranulation and cytotoxic activity of these cells. Optimal activation required accessory cells like CD14+ myeloid and CD14- cells and triggered increased secretion of numerous inflammatory cytokines. VP40-induced IFN-γ and TNF-α secretion by NK cells was dependent on IL-12 and IL-18 and suppressed by IL-10. In contrast, their increased degranulation was dependent on IL-12 with little influence of IL-18 or IL-10. These results demonstrate that Ebola VP40 stimulates NK cell functions in an IL-12 and IL-18 dependent manner that involves CD14+ and CD14- accessory cells. These novel findings may help in designing improved intervention strategies required to control viral transmission during Ebola outbreaks.
Hung Le, Paul Spearman, Stephen N. Waggoner, Karnail Singh
Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a post-translational regulator of the low-density lipoprotein receptor (LDLR). Recent studies have proposed a role for PCSK9 in regulating immune responses. Using RNA-sequencing-based variant discovery, we identified a novel psoriasis susceptibility locus at 1p32.3, located within PCSK9 (rs662145 C>T). This finding was verified in independently acquired genomic and RNA-sequencing datasets. Single-cell RNA-sequencing (scRNA-seq) identified keratinocytes as the primary source of PCSK9 in human skin. PCSK9 expression, however, was not uniform across keratinocyte subpopulations. scRNA-seq and immunohistochemistry demonstrated an epidermal gradient of PCSK9, with expression being highest in basal and early spinous layer keratinocytes and lowest in granular layer keratinocytes. IL-36G expression followed the opposite pattern, with expression highest in granular layer keratinocytes. PCSK9 siRNA knockdown experiments confirmed this inverse relationship between PCSK9 and IL36G expression. Other immune genes were also linked to PCSK9 expression including, IL27RA, IL1RL1, ISG20, and STX3. In both cultured keratinocytes and nonlesional human skin, homozygosity for PCSK9 SNP rs662145 C>T was associated with lower PCSK9 expression and higher IL36G expression, when compared to heterozygous skin or cell lines. Together these results support PCSK9 as a novel psoriasis susceptibility locus and establish a putative link between PCSK9 and inflammatory cytokine expression.
Alexander Merleev, Antonio Ji-Xu, Atrin Toussi, Lam C. Tsoi, Stephanie T. Le, Guillaume Luxardi, Xianying Xing, Rachael Wasikowski, William Liakos, Marie-Charlotte Brüggen, James T. Elder, Iannis E. Adamopoulos, Yoshihiro Izumiya, Annie Riera-Leal, Qinyuan Li, Nikolay Yu Kuzminykh, Amanda Kirane, Alina I. Marusina, Johann E. Gudjonsson, Emanual Maverakis
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