Transcriptional modulation of pattern recognition receptors in chronic colitis in mice is accompanied with Th1 and Th17 response

Pattern recognition receptors (PRRs) may contribute to inflammatory bowel diseases (IBD) development due to their microbial-sensing ability and the unique microenvironment in the inflamed gut. In this study, the PRR mRNA expression profile together with T cell-associated factors in the colon was examined using a chronic colitis mice model. 8–12 week old C57BL/6 mice were exposed to multiple dextran sodium sulfate (DSS) treatments interspersed with a rest period to mimic the course of chronic colitis. The clinical features and histological data were collected. The mRNA expressions of colonic PRRs, T cell-associated components were measured. Finally, the colons were scored for Foxp3+ cells. During chronic colitis, the histological data, but not the clinical manifestations demonstrated characteristic inflammatory symptoms in the distal colon. In contrast to acute colitis, the expression of all Toll-like receptors (Tlrs), except Tlr5 and Tlr9, was unaffected after repeated DSS treatments. The expression of Nod1 was decreased, while Nod2 increased. After third DSS treatment, only the expressions of Tlr3 and Tlr4 were significantly enhanced. Unlike other PRRs, decreased Tlr5 and increased Tlr9 mRNA expression persisted during the chronic colitis period. As the colitis progress, only the mRNA expression of Ifnγ and Il17 staid increased during chronic colitis, while the acute colitis-associated increase of Il23, and Il10 and Il12 was abolished. Finally, increased histological score of Foxp3+ cell in colon was found during the chronic colitis period. This study provides an expression pattern of PRRs during chronic colitis that is accompanied by a Th1- and Th17 cell-mediated immune response.     

CCL25/CCR9 interactions regulate large intestinal inflammation in a murine model of acute colitis

Background & Aims

CCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine.

Methods

Acute inflammation and recovery in wild-type (WT) and CCR9^−/− mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level.

Results

CCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9^−/− mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9^−/− colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9^−/− animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNFα, IFNγ, IL2, IL-6 and IL-17A while down modulating IL-10 production.

Conclusions

Our results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation.     

Exacerbated intestinal inflammation in P2Y6 deficient mice is associated with Th17 activation

Extracellular nucleotides are released as constitutive danger signals by various cell types and activate nucleotide (P2) receptors such as P2Y₆ receptor. P2Y₆ activation on monocytes induces the secretion of the chemokine CXCL8 which may propagate intestinal inflammation. Also, P2Y₆ expression is increased in infiltrating T cells of Crohn's disease patients. As inflammatory bowel disease (IBD) is associated with immune cell recruitment, we hypothesised that P2Y₆ would participate to the establishment of inflammation in this disease. To address this, we used P2Y₆ deficient (P2ry6^‐−/−) mice in the dextran sodium sulfate (DSS) murine model of IBD. In disagreement with our hypothesis, P2Y₆ deficient mice were more susceptible to inflammation induced by DSS than WT mice. DSS treated-P2ry6^−/− mice showed increased histological damage and increased neutrophil and macrophage infiltration that correlated with increased mRNA levels of the chemokines KC and MCP-1. DSS treated-P2ry6^−/− mice exhibited also higher levels of Th17/Th1 lymphocytes in their colon which correlated with increased levels of IFN-γ and IL-17A in the sera as well as increased mRNA levels of IFN-γ, IL-17A, IL-6, IL-23 and IL-1β in P2ry6^−/− colons. This inflammation was also accompanied by a decreased cell proliferation and goblet cell number. Importantly, injection of anti-IL-17 intraperitoneally partially protected P2ry6^−/− mice from DSS-induced colitis. Taken together, in the absence of P2Y₆, an exacerbated intestinal inflammation to DSS was observed which correlated with increased recruitment of Th17/Th1 lymphocytes. These data suggest a protective role of P2Y₆ expressed on leukocytes in intestinal inflammation.     

Anthocyanin-containing purple potatoes ameliorate DSS-induced colitis in mice

Ulcerative colitis (UC), a major form of inflammatory bowel disease (IBD), is on the rise worldwide. Approximately three million people suffer from IBD in the United States alone, but the current therapeutic options (e.g., corticosteroids) come with adverse side effects including reduced ability to fight infections. Thus, there is a critical need for developing effective, safe and evidence-based food products with anti-inflammatory activity. This study evaluated the antiinflammatory potential of purple-fleshed potato using a dextran sodium sulfate (DSS) murine model of colitis. Mice were randomly assigned to control (AIN-93G diet), P15 (15% purple-fleshed potato diet) and P25 (25% purple-fleshed potato diet) groups. Colitis was induced by 2% DSS administration in drinking water for six days. The results indicated that purple-fleshed potato supplementation suppressed the DSS-induced reduction in body weight and colon length as well as the increase in spleen and liver weights. P15 and P25 diets suppressed the elevation in the intestinal permeability, colonic MPO activity, mRNA expression and protein levels of pro-inflammatory interleukins IL-6 and IL-17, the relative abundance of specific pathogenic bacteria such as Enterobacteriaceae, Escherichia coli (E. coli) and pks⁺ E. coli, and the increased flagellin levels induced by DSS treatment. P25 alone suppressed the elevated systemic MPO levels in DSS-exposed mice, and elevated the relative abundance of Akkermansia muciniphila (A. muciniphila) as well as attenuated colonic mRNA expression level of IL-17 and the protein levels of IL-6 and IL-1β. Therefore, the purple-fleshed potato has the potential to aid in the amelioration of UC symptoms.     

Faecalibacterium prausnitzii Inhibits Interleukin-17 to Ameliorate Colorectal Colitis in Rats

Background and Aims

It has been shown that Faecalibacterium prausnitzii (F. prausnitzii), one of the dominant intestinal bacterial flora, may protect colonic mucosa against the development of inflammation and subsequent inflammatory bowel disease (IBD), with the underlying mechanisms being unclear.

Methods

The impacts of F. prausnitzii and its metabolites on IL-23/Th17/IL-17 pathway markers were determined in human monocytes and a rat model of colitis induced by 2,4,6-trinitrobenzene sulfonic acid. F. prausnitzii and its culture medium (containing complete metabolites) were used to treat the rats in vivo, as well as rat splenocytes and human monocytes in vitro. Inflammatory cytokines were measured in colon tissue, plasma and cell culture medium.

Results

The culture supernatant of F. prausnitzii increased plasma anti-Th17 cytokines (IL-10 and IL-12)and suppressed IL-17 levels in both plasma and colonic mucosa, with ameliorated colonic colitis lesions. This inhibition of IL-17 release has also been observed in both rat splenocytes and human venous monocytes in vitro. The culture supernatant of F. prausnitzii also suppressed Th17 cell differentiation induced by cytokines (TGF-ß and IL-6) and bone marrow-derived dendritic cells (BMDCs) in vitro. The metabolites of F. prausnitzii in the culture supernatant exert a stronger anti-inflammatory effect than the bacterium itself. F. prausnitzii protected the colon mucosa against the development of IBD by its metabolites, suggesting a promising potential for the use of F. prausnitzii and its metabolic products in the treatment of IBD.     

Bioactive constituents of Mosla chinensis-cv. Jiangxiangru ameliorate inflammation through MAPK signaling pathways and modify intestinal microbiota in DSS-induced colitis mice

BackgroundMosla chinensis Maxim. cv. Jiangxiangru (JXR), a traditional Chinese medicine, commonly used for the therapy of cold, fever, diarrhea, digestive disorders, and other diseases. Inflammatory bowel disease (IBD) is a chronic disorder of the human gastrointestinal tract. Research about the effect of JXR on IBD and the active ingredient composition of JXR remains deficiency.PurposeThis study aims to determine the phytochemical composition and the anti-inflammatory property of JXR, as well as the possible anti-inflammatory mechanisms.MethodsThe bioactive profile of JXR extracts was determined by UPLC-LTQ-Orbitrap-MS. A DSS induced colitis mouse model was applied to explore the anti-inflammatory activity of JXR. The body weight, colon length and histopathological status of colon tissue were evaluated. The content of inflammatory mediators (nitric oxide (NO), prostaglandin E₂ (PGE₂)) and cytokines (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β)), corresponding mRNA and protein expression levels were analyzed. Oxidation pressure and gut microbial composition were also explored.ResultsTotally 63 constitutes were identified from JXR, among them, phenolic acids and flavonoids comprised a large part, and rosmarinic acid (RA) was the main compound. The results of DSS-induced colitis mice model indicated that JXR effectively ameliorated inflammation, restore the redox balance in the gut. JXR treatment significantly reduced the production of reactive oxygen species (ROS), increased the activity of antioxidative enzyme, suppressed the secretion of inflammatory mediators (NO, PGE₂) and cytokines (TNF-α, IL-6, IL-1β). JXR also restrained the activation of mitogen-activated protein kinases (MAPKs) signaling pathway. Furthermore, JXR could restore the microbial diversity by suppressing Bacteroidaceae, increasing Bifidobacteriales and Melainabacteria in DSS colitis mouse model.ConclusionsThis study demonstrated that JXR composed with various bioactive compounds, effectively ameliorated colitis, restored the redox balance and regulated gut microbiota. Results from the present study provide an insight of therapeutic potential of JXR in IBD based on its anti-inflammatory and antioxidant properties, also provide a scientific basis for using JXR as a functional ingredient to promote colon health.     

Cyclosporine A Impairs Nucleotide Binding Oligomerization Domain (Nod1)-Mediated Innate Antibacterial Renal Defenses in Mice and Human Transplant Recipients

Acute pyelonephritis (APN), which is mainly caused by uropathogenic Escherichia coli (UPEC), is the most common bacterial complication in renal transplant recipients receiving immunosuppressive treatment. However, it remains unclear how immunosuppressive drugs, such as the calcineurin inhibitor cyclosporine A (CsA), decrease renal resistance to UPEC. Here, we investigated the effects of CsA in host defense against UPEC in an experimental model of APN. We show that CsA-treated mice exhibit impaired production of the chemoattractant chemokines CXCL2 and CXCL1, decreased intrarenal recruitment of neutrophils, and greater susceptibility to UPEC than vehicle-treated mice. Strikingly, renal expression of Toll-like receptor 4 (Tlr4) and nucleotide-binding oligomerization domain 1 (Nod1), neutrophil migration capacity, and phagocytic killing of E. coli were significantly reduced in CsA-treated mice. CsA inhibited lipopolysaccharide (LPS)-induced, Tlr4-mediated production of CXCL2 by epithelial collecting duct cells. In addition, CsA markedly inhibited Nod1 expression in neutrophils, macrophages, and renal dendritic cells. CsA, acting through inhibition of the nuclear factor of activated T-cells (NFATs), also markedly downregulated Nod1 in neutrophils and macrophages. Silencing the NFATc1 isoform mRNA, similar to CsA, downregulated Nod1 expression in macrophages, and administration of the 11R-VIVIT peptide inhibitor of NFATs to mice also reduced neutrophil bacterial phagocytosis and renal resistance to UPEC. Conversely, synthetic Nod1 stimulating agonists given to CsA-treated mice significantly increased renal resistance to UPEC. Renal transplant recipients receiving CsA exhibited similar decrease in NOD1 expression and neutrophil phagocytosis of E. coli. The findings suggest that such mechanism of NFATc1-dependent inhibition of Nod1-mediated innate immune response together with the decrease in Tlr4-mediated production of chemoattractant chemokines caused by CsA may contribute to sensitizing kidney grafts to APN.     

过表达白细胞介素10的间充质干细胞对炎症性肠病的治疗作用

炎症性肠病(inflammatory bowel disease,IBD)是一种以T细胞浸润至结肠为特征的难治性炎性自身免疫疾病。间充质干细胞 (mesenchymal stem cells,MSCs) 具有免疫抑制能力,在IBD的治疗中具有一定的潜力。但是由于MSCs在体内的免疫调节能力不稳定,所以其治疗效果会受到影响。本研究构建了过表达白细胞介素10(interleukin 10,IL-10)的工程化MSCs,并对其在IBD小鼠模型中的治疗潜力进行评估。MSCs经编码IL-10的慢病毒(lentivirus,LV)转染后,其表型和细胞增殖率均不发生变化。免疫细胞和 MSCs体外共培养的结果表明,与未修饰的MSCs相比,同过表达IL-10的MSCs共培养的免疫细胞中辅助T细胞1(T helper 1 cells,Th1)和辅助T细胞17(T helper 17 cells,Th17)数量显著性降低(P<0.05),同过表达IL-10的MSCs共培养的巨噬细胞细胞培养上清液,TNF-α含量显著性降低(P<0.0001)。右旋糖酐硫酸钠(dextran sodium sulfate,DSS)诱导IBD小鼠模型中,尾静脉注射过表达IL-10的MSCs与注射未修饰MSCs相比,过表达IL-10的MSCs具有更好的治疗效果,结肠长度、疾病活动指数(disease activity index,DAI)和结肠炎性细胞因子表达共同证明这一差异。实验结果均具有统计学差异(P<0.05)。总体而言,经LV转染过表达IL-10的MSCs可能是IBD的一种有希望的替代治疗选择。     

Enhanced Inflammatory Potential of CD4+ T-Cells That Lack Proteasome Immunosubunit Expression,in a T-Cell Transfer-Based Colitis Model

Proteasomes play a fundamental role in intracellular protein degradation and therewith regulate a variety of cellular processes. Exposure of cells to (pro)inflammatory cytokines upregulates the expression of three inducible catalytic proteasome subunits, the immunosubunits, which incorporate into newly assembled proteasome complexes and alter the catalytic activity of the cellular proteasome population. Single gene-deficient mice lacking one of the three immunosubunits are resistant to dextran sulfate sodium (DSS)-induced colitis development and, likewise, inhibition of one single immunosubunit protects mice against the development of DSS-induced colitis. The observed diminished disease susceptibility has been attributed to altered cytokine production and CD4⁺ T-cell differentiation in the absence of immunosubunits. To further test whether the catalytic activity conferred by immunosubunits plays an essential role in CD4⁺ T-cell function and to distinguish between the role of immunosubunits in effector T-cells versus inflamed tissue, we used a T-cell transfer-induced colitis model. Naïve wt or immunosubunit-deficient CD4⁺ T-cells were adoptively transferred into RAG1^−/− and immunosubunit-deficient RAG1^−/− mice and colitis development was determined six weeks later. While immunosubunit expression in recipient mice had no effect on colitis development, transferred immunosubunit-deficient T- cells were more potent in inducing colitis and produced more proinflammatory IL17 than wt T-cells. Taken together, our data show that modifications in proteasome-mediated proteolysis in T-cells, conferred by lack of immunosubunit incorporation, do not attenuate but enhance CD4⁺ T-cell-induced inflammation.     

Overexpression of GATA-3 in T Cells Accelerates Dextran Sulfate Sodium-Induced Colitis

Ulcerative colitis (UC) is an inflammatory bowel disease, and its pathogenesis includes genetic, environmental, and immunological factors, such as T helper cells and their secreted cytokines. T helper cells are classified as Th1, Th2, and Th17 cells. However, it is unclear which T helper cells are important in UC. Dextran sulfate sodium (DSS)-induced colitis is a commonly used model of UC. In this study, we induced DSS colitis in Th1 dominant (T-bet transgenic (Tg)) mice, Th2 dominant (GATA-3 Tg) mice, and Th17 dominant (RORγt Tg) mice to elucidate the roles of T helper cell in DSS colitis. The results showed that GATA-3 Tg mice developed the most severe DSS colitis compared with the other groups. GATA-3 Tg mice showed a significant decreased in weight from day 1 to day 7, and an increased high score for the disease activity index compared with the other groups. Furthermore, GATA-3 Tg mice developed many ulcers in the colon, and many neutrophils and macrophages were detected on day 4 after DSS treatment. Measurement of GATA-3-induced cytokines demonstrated that IL-13 was highly expressed in the colon from DSS-induced GATA-3 Tg mice. In conclusion, GATA-3 overexpression in T-cells and IL-13 might play important roles in the development of DSS colitis.     

Trichinella spiralis Infection Suppressed Gut Inflammation with CD4+CD25+Foxp3+ T Cell Recruitment

In order to know the effect of pre-existing Trichinella spiralis infection on experimentally induced intestinal inflammation and immune responses, we induced colitis in T. spiralis-infected mice and observed the severity of colitis and the levels of Th1, Th2, and regulatory cytokines and recruitment of CD4⁺CD25⁺Foxp3⁺ T (regulatory T; T_reg) cells. Female C57BL/6 mice were infected with 250 muscle larvae; after 4 weeks, induction of experimental colitis was performed using 3% dextran sulfate sodium (DSS). During the induction period, we observed severity of colitis, including weight loss and status of stool, and evaluated the disease activity index (DAI). A significantly low DAI and degree of weight loss were observed in infected mice, compared with uninfected mice. In addition, colon length in infected mice was not contracted, compared with uninfected mice. We also observed a significant increase in production of pro-inflammatory cytokines, IL-6 and IFN-γ, in spleen lymphocytes treated with DSS; however, such an increase was not observed in infected mice treated with DSS. Of particular interest, production of regulatory cytokines, IL-10 and transforming growth factor (TGF)-β, in spleen lymphocytes showed a significant increase in mice infected with T. spiralis. A similar result was observed in mesenteric lymph nodes (MLN). Subsets of the population of T_reg cells in MLN and spleen showed significant increases in mice infected with T. spiralis. In conclusion, T. spiralis infection can inhibit the DSS-induced colitis in mice by enhancing the regulatory cytokine and T_reg cells recruitment.     

Dietary tryptophan alleviates dextran sodium sulfate-induced colitis through aryl hydrocarbon receptor in mice

Ulcerative colitis is the typical progression of chronic inflammatory bowel disease. Amino acids, particularly tryptophan, have been reported to exert a protective effect against colitis induced by dextran sodium sulfate (DSS), but the precise underlying mechanisms remain incompletely clarified. Tryptophan metabolites are recognized to function as endogenous ligands for aryl hydrocarbon receptor (Ahr), which is a critical regulator of inflammation and immunity. Thus, we conducted this study to investigate whether dietary tryptophan supplementation protects against DSS-induced colitis by acting through Ahr. Female wild-type (WT) and Ahr-deficient (knockout; KO) mice (10–12 weeks old) were divided into four groups and fed either a control or 0.5% tryptophan diet. The tryptophan diet ameliorated DSS-induced colitis symptoms and severity in WT mice but not in KO mice, and the diet reduced the mRNA expression of Il-6, Tnfα, Il-1β and the chemokines Ccl2, Cxcl1 and Cxcl2 in the WT groups. Furthermore, Il-22 and Stat3 mRNA expression in the colon was elevated in WT mice fed with the tryptophan diet, which mainly protected epithelial layer integrity, and Ahr also modulated immune homeostasis by regulating Foxp3 and Il-17 mRNA expression. These data suggest that tryptophan-containing diet might ameliorate DSS-induced acute colitis and regulate epithelial homeostasis through Ahr. Thus, tryptophan could serve as a promising preventive agent in the treatment of ulcerative colitis.     

Frequency and functional activity of Th17, Tc17 and other T-cell subsets in Systemic Lupus Erythematosus

To compare frequency and functional activity of peripheral blood (PB) Th(c)17, Th(c)1 and Treg cells and the amount of type 2 cytokines mRNA we recruited SLE patients in active (n = 15) and inactive disease (n = 19) and healthy age- and gender-matched controls (n = 15). The study of Th(c)17, Th(c)1 and Treg cells was done by flow cytometry and cytokine mRNA by real-time PCR. Compared to NC, SLE patients present an increased proportion of Th(c)17 cells, but with lower amounts of IL-17 per cell and also a decreased frequency of Treg, but with increased production of TGF-β and FoxP3 mRNA. Ιn active compared to inactive SLE, there is a marked decreased in frequency of Th(c)1 cells, an increased production of type 2 cytokines mRNA and a distinct functional profile of Th(c)17 cells. Our findings suggest a functional disequilibrium of T-cell subsets in SLE which may contribute to the inflammatory process and disease pathogenesis.