Abstract
Aims
Atherosclerotic cardiovascular disease (ACVD) is a major cause of mortality and morbidity worldwide, and increased low-density lipoproteins (LDLs) play a critical role in development ...and progression of atherosclerosis. Here, we examined for the first time gut immunomodulatory effects of the microbiota-derived metabolite propionic acid (PA) on intestinal cholesterol metabolism.
Methods and results
Using both human and animal model studies, we demonstrate that treatment with PA reduces blood total and LDL cholesterol levels. In apolipoprotein E−/− (Apoe−/−) mice fed a high-fat diet (HFD), PA reduced intestinal cholesterol absorption and aortic atherosclerotic lesion area. Further, PA increased regulatory T-cell numbers and interleukin (IL)-10 levels in the intestinal microenvironment, which in turn suppressed the expression of Niemann-Pick C1-like 1 (Npc1l1), a major intestinal cholesterol transporter. Blockade of IL-10 receptor signalling attenuated the PA-related reduction in total and LDL cholesterol and augmented atherosclerotic lesion severity in the HFD-fed Apoe−/− mice. To translate these preclinical findings to humans, we conducted a randomized, double-blinded, placebo-controlled human study (clinical trial no. NCT03590496). Oral supplementation with 500 mg of PA twice daily over the course of 8 weeks significantly reduced LDL −15.9 mg/dL (−8.1%) vs. −1.6 mg/dL (−0.5%), P = 0.016, total −19.6 mg/dL (−7.3%) vs. −5.3 mg/dL (−1.7%), P = 0.014 and non-high-density lipoprotein cholesterol levels PA vs. placebo: −18.9 mg/dL (−9.1%) vs. −0.6 mg/dL (−0.5%), P = 0.002 in subjects with elevated baseline LDL cholesterol levels.
Conclusion
Our findings reveal a novel immune-mediated pathway linking the gut microbiota-derived metabolite PA with intestinal Npc1l1 expression and cholesterol homeostasis. The results highlight the gut immune system as a potential therapeutic target to control dyslipidaemia that may introduce a new avenue for prevention of ACVDs.
Graphical Abstract
Graphical Abstract
The figure illustrates the proposed model of the cholesterol-lowering and atheroprotective properties of propionate. A high-fat high-cholesterol diet causes a disbalance of immune cells in the small intestinal microenvironment, with reduced regulatory T cell frequencies and interleukin-10 concentrations. Altered regulatory T cell levels are rescued upon exogenous propionate supplementation, with increased local levels. This in turn modulates NPC1L1 expression and membrane density, with a subsequent reduction in cholesterol absorption, ultimately leading to reduced atherogenesis. The illustration was adopted from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Common Attribution 3.0 Generic License. HFD, high-fat diet; IL-10, interleukin-10; NPC1L1, Niemann-Pick C1-like 1; PA, propionic acid; Treg, regulatory T cell.
Immunomodulatory Foxp3+ regulatory T cells (Tregs) form a heterogeneous population consisting of subsets with different activation states, migratory properties and suppressive functions. Recently, ...expression of the IL-33 receptor ST2 was shown on Tregs in inflammatory settings. Here we report that ST2 expression identifies highly activated Tregs in mice even under homeostatic conditions. ST2+ Tregs preferentially accumulate at non-lymphoid sites, likely mediated by their high expression of several chemokine receptors facilitating tissue homing. ST2+ Tregs exhibit a Th2-biased character, expressing GATA-3 and producing the Th2 cytokines IL-5 and IL-13 -especially in response to IL-33. Yet, IL-33 is dispensable for the generation and maintenance of these cells in vivo. Furthermore, ST2+ Tregs are superior to ST2- Tregs in suppressing CD4+ T cell proliferation in vitro independent of IL-33. This higher suppressive capacity is partially mediated by enhanced production and activation of the anti-inflammatory cytokines IL-10 and TGFβ. Thus, ST2 expression identifies a highly activated, strongly suppressive Treg subset preferentially located in non-lymphoid tissues. Here ST2+ Tregs may be well positioned to immediately react to IL-33 alarm signals. Their specific properties may render ST2+ Tregs useful targets for immunomodulatory therapies.
Interactions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated ...responses of human CD4+ T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation.
We collected samples of peripheral blood mononuclear cells and intestinal tissues from healthy individuals (controls, n = 13−30) and patients with inflammatory bowel diseases (n = 119; 59 with ulcerative colitis and 60 with Crohn’s disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium, and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4+ T cells. We sequenced T-cell receptor Vβ genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4+ T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative polymerase chain reaction.
Circulating and gut-resident CD4+ T cells from controls responded to bacteria at frequencies of 40−4000 per million for each bacterial species tested. Microbiota-reactive CD4+ T cells were mainly of a memory phenotype, present in peripheral blood mononuclear cells and intestinal tissue, and had a diverse T-cell receptor Vβ repertoire. These cells were functionally heterogeneous, produced barrier-protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A, interferon gamma, and tumor necrosis factor. In patients with inflammatory bowel diseases, microbiota-reactive CD4+ T cells were reduced in the blood compared with intestine; T-cell responses that we detected had an increased frequency of interleukin 17A production compared with responses of T cells from blood or intestinal tissues of controls.
In an analysis of peripheral blood mononuclear cells and intestinal tissues from patients with inflammatory bowel diseases vs controls, we found that reactivity to intestinal bacteria is a normal property of the human CD4+ T-cell repertoire, and does not necessarily indicate disrupted interactions between immune cells and the commensal microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing barrier-protective cytokines and providing a large pool of T cells that react to pathogens.
Non-lymphoid tissues (NLTs) harbor a pool of adaptive immune cells with largely unexplored phenotype and development. We used single-cell RNA-seq to characterize 35,000 CD4+ regulatory (Treg) and ...memory (Tmem) T cells in mouse skin and colon, their respective draining lymph nodes (LNs) and spleen. In these tissues, we identified Treg cell subpopulations with distinct degrees of NLT phenotype. Subpopulation pseudotime ordering and gene kinetics were consistent in recruitment to skin and colon, yet the initial NLT-priming in LNs and the final stages of NLT functional adaptation reflected tissue-specific differences. Predicted kinetics were recapitulated using an in vivo melanoma-induction model, validating key regulators and receptors. Finally, we profiled human blood and NLT Treg and Tmem cells, and identified cross-mammalian conserved tissue signatures. In summary, we describe the relationship between Treg cell heterogeneity and recruitment to NLTs through the combined use of computational prediction and in vivo validation.
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•scRNA-seq reveals Treg cell tissue-specific signatures•The phenotype of Treg cells shows progressive adaptation to barrier tissues•Treg cells share transcriptional dynamics of adaptation to colon and skin•Modules of peripheral Treg cell identity are conserved between mouse and human
Treg cells are known to display tissue-specific heterogeneity. Miragaia and colleagues probe Treg cells in lymphoid and barrier tissues by single-cell transcriptomics. They show that Treg cells segregate into subpopulations along a continuum of tissue adaptation and present conserved expression programs between homeostasis and disease and mouse and human.
Significance The alarmin IL-33 amplifies immune responses of Th2 and CD8 ⁺ cytotoxic T cells against invading pathogens; however, little is known about a potential role of IL-33 in Th1 cell ...responses. This study demonstrates that activated Th1 effector cells transiently express the IL-33 receptor ST2 upon differentiation in vitro and in vivo during viral infection. ST2 expression on virus-specific Th1 cells depended on the Th1-associated transcription factors T-bet and STAT4. ST2-deficient virus-specific CD4 ⁺ T cells showed impaired expansion and Th1 effector function upon viral infection, indicating a direct impact of IL-33 on antiviral Th1 cell responses. These observations redefine the role of ST2 in Th cell activation, with implications for the design of adjuvants and therapies targeting the IL-33–ST2 pathway.
During infection, the release of damage-associated molecular patterns, so-called “alarmins,” orchestrates the immune response. The alarmin IL-33 plays a role in a wide range of pathologies. Upon release, IL-33 signals through its receptor ST2, which reportedly is expressed only on CD4 ⁺ T cells of the Th2 and regulatory subsets. Here we show that Th1 effector cells also express ST2 upon differentiation in vitro and in vivo during lymphocytic choriomeningitis virus (LCMV) infection. The expression of ST2 on Th1 cells was transient, in contrast to constitutive ST2 expression on Th2 cells, and marked highly activated effector cells. ST2 expression on virus-specific Th1 cells depended on the Th1-associated transcription factors T-bet and STAT4. ST2 deficiency resulted in a T-cell–intrinsic impairment of LCMV-specific Th1 effector responses in both mixed bone marrow-chimeric mice and adoptive cell transfer experiments. ST2-deficient virus-specific CD4 ⁺ T cells showed impaired expansion, Th1 effector differentiation, and antiviral cytokine production. Consequently, these cells mediated little virus-induced immunopathology. Thus, IL-33 acts as a critical and direct cofactor to drive antiviral Th1 effector cell activation, with implications for vaccination strategies and immunotherapeutic approaches.
Differentiated T helper (Th) cell lineages are thought to emerge from alternative cell fate decisions. However, recent studies indicated that differentiated Th cells can adopt mixed phenotypes during ...secondary immunological challenges. Here we show that natural primary immune responses against parasites generate bifunctional Th1 and Th2 hybrid cells that co-express the lineage-specifying transcription factors T-bet and GATA-3 and co-produce Th1 and Th2 cytokines. The integration of Th1-promoting interferon (IFN)-γ and interleukin (IL)-12 signals together with Th2-favoring IL-4 signals commits naive Th cells directly and homogeneously to the hybrid Th1/2 phenotype. Specifically, IFN-γ signals are essential for T-bet(+)GATA-3(+) cells to develop in vitro and in vivo by breaking the dominance of IL-4 over IL-12 signals. The hybrid Th1/2 phenotype is stably maintained in memory cells in vivo for months. It resists reprogramming into classic Th1 or Th2 cells by Th1- or Th2-promoting stimuli, which rather induce quantitative modulations of the combined Th1 and Th2 programs without abolishing either. The hybrid phenotype is associated with intermediate manifestations of both Th1 and Th2 cell properties. Consistently, hybrid Th1/2 cells support inflammatory type-1 and type-2 immune responses but cause less immunopathology than Th1 and Th2 cells, respectively. Thus, we propose the self-limitation of effector T cells based on the stable cell-intrinsic balance of two opposing differentiation programs as a novel concept of how the immune system can prevent excessive inflammation.
FOXP3(+) regulatory T cells (Treg cells) are abundant in the intestine, where they prevent dysregulated inflammatory responses to self and environmental stimuli. It is now appreciated that Treg cells ...acquire tissue-specific adaptations that facilitate their survival and function; however, key host factors controlling the Treg response in the intestine are poorly understood. The interleukin (IL)-1 family member IL-33 is constitutively expressed in epithelial cells at barrier sites, where it functions as an endogenous danger signal, or alarmin, in response to tissue damage. Recent studies in humans have described high levels of IL-33 in inflamed lesions of inflammatory bowel disease patients, suggesting a role for this cytokine in disease pathogenesis. In the intestine, both protective and pathological roles for IL-33 have been described in murine models of acute colitis, but its contribution to chronic inflammation remains ill defined. Here we show in mice that the IL-33 receptor ST2 is preferentially expressed on colonic Treg cells, where it promotes Treg function and adaptation to the inflammatory environment. IL-33 signalling in T cells stimulates Treg responses in several ways. First, it enhances transforming growth factor (TGF)-β1-mediated differentiation of Treg cells and, second, it provides a necessary signal for Treg-cell accumulation and maintenance in inflamed tissues. Strikingly, IL-23, a key pro-inflammatory cytokine in the pathogenesis of inflammatory bowel disease, restrained Treg responses through inhibition of IL-33 responsiveness. These results demonstrate a hitherto unrecognized link between an endogenous mediator of tissue damage and a major anti-inflammatory pathway, and suggest that the balance between IL-33 and IL-23 may be a key controller of intestinal immune responses.
The immune system in the intestine is highly adapted to resist invading pathogens while residing peacefully with the abundant and diverse commensal bacteria that colonize the gastrointestinal tract. ...In turn, bacterial signals shape immunity in the intestine, promoting intestinal homeostasis in part by inducing and expanding specialized regulatory T (Treg) cells that prevent aberrant inflammatory responses to self and environmental stimuli (1). On pages 989 and 993 of this issue, Ohnmacht et al. (2) and Sefik et al. (3), respectively, report the development and function of a subpopulation of Treg cells found primarily in the large intestine, and characterized by expression of the nuclear hormone receptor retinoic acid receptor-related orphan receptor gamma t (ROR gamma t). This is surprising because ROR gamma t classically promotes the differentiation of T helper 17 (TH17) cells, a population associated with tissue inflammation in many inflammatory diseases (4). Both studies show that microbiota-derived signals induce the expression of ROR gamma t in Treg cells that control intestinal inflammation (see the figure). These findings highlight the diversity of colonic Treg cells, their complex transcriptional programs, and their important role in the intestine.
Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are complex chronic inflammatory conditions of the gastrointestinal tract that are driven by perturbed ...cytokine pathways. Anti-tumor necrosis factor-α (TNF) antibodies are mainstay therapies for IBD. However, up to 40% of patients are nonresponsive to anti-TNF agents, which makes the identification of alternative therapeutic targets a priority. Here we show that, relative to healthy controls, inflamed intestinal tissues from patients with IBD express high amounts of the cytokine oncostatin M (OSM) and its receptor (OSMR), which correlate closely with histopathological disease severity. The OSMR is expressed in nonhematopoietic, nonepithelial intestinal stromal cells, which respond to OSM by producing various proinflammatory molecules, including interleukin (IL)-6, the leukocyte adhesion factor ICAM1, and chemokines that attract neutrophils, monocytes, and T cells. In an animal model of anti-TNF-resistant intestinal inflammation, genetic deletion or pharmacological blockade of OSM significantly attenuates colitis. Furthermore, according to an analysis of more than 200 patients with IBD, including two cohorts from phase 3 clinical trials of infliximab and golimumab, high pretreatment expression of OSM is strongly associated with failure of anti-TNF therapy. OSM is thus a potential biomarker and therapeutic target for IBD, and has particular relevance for anti-TNF-resistant patients.
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