TNF-α and IFN-γ synergistically inhibit the repairing ability of mesenchymal stem cells on mice colitis and colon cancer.
ABSTRACT: BACKGROUND:Mesenchymal stem cells (MSCs) can be efficiently recruited to wound, inflammatory and tumor sites to repair and regenerate tissue. However, its role in colitis and colitis associated colon cancer is still controversial. This study was designed to evaluate the role and mechanisms of inflammatory cytokines-activated-MSCs in mice colitis and colon cancer. METHODS:We selected two well-characterized pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), to expand the inflammatory microenvironment of MSCs. The severity of colitis and colon cancer was evaluated by measuring colon length, Myeloperoxidase (MPO) activity, Hematoxylin-eosin staining, Western Blot, Immunohistochemistry and Immunofluorescence. These techniques were also performed to analyze the mechanisms of inflammatory cytokines-activated-MSCs in mice colitis and colon cancer. Real-time PCR and Enzyme-linked Immunosorbent Assay (ELISA) were used to measure the secretion of pro-inflammatory factors. RESULTS:We found that the incubation of MSCs with TNF-α and IFN-γ aggravates colitis, where high levels of pro-inflammatory factors, such as interleukin (IL)-17, IL-8, IL-12, IL-1β, transforming growth factor (TGF)-β, TNF-α and IFN-γ, were secreted. Furthermore, this phenomenon was associated with the activation of the nuclear factor-kappa-B (NF-κB)/Signal transducer and activator of transcription three (STAT3) pathway. In addition, our study demonstrated that TNF-α and IFN-γ pretreated MSCs synergistically exacerbated mice colon cancer, which was closely associated with angiogenesis. CONCLUSIONS:Taken together, these results indicate that TNF-α and IFN-γ pretreatment effectively inhibited the repair ability of MSCs and accelerated inflammation and tumor progression involving NF-κB/STAT3 pathway and angiogenesis-related factors.
Project description:BACKGROUND: Mice deficient in interleukin-2 (IL-2) develop inflammatory bowel disease resembling ulcerative colitis in humans. Recent studies provided evidence that alpha beta T cells, particularly CD4 T cells, rather than B cells, are involved in the pathogenesis of bowel inflammation of IL-2 deficient mice. AIM: To analyse the pattern of expression of cytokine mRNA in intestinal tissue of normal and IL-2 deficient mice. METHODS: Expression of beta-actin, IL-1 alpha, IL-1 beta, IL-6, IL-10, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma) and transforming growth factor beta 1 (TGF-beta 1) mRNA was analysed in colon and small intestinal tissue of both IL-2 deficient (IL-2-/-) mice and normal (wild type) litter mates (IL-2+/+) at different ages by using qualitative, as well as semiquantitative, competitive reverse transcription polymerase chain reaction (RT-PCR). Results were correlated with the phase of progression of the disease, as determined by histology. RESULTS: IL-2-/- mice had expressed low levels of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, and IFN-gamma mRNA in the colon by 1.5 weeks of age. In advance of the development of histologically and clinically detectable bowel inflammation, expression of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, IFN-gamma, and IL-10, but not TGF-beta 1, mRNA increased in the colon of IL-2 deficient mice. In contrast, IL-2+/+ mice expressed TGF-beta 1 mRNA in colon tissue at 13 and 23 weeks of age, but not IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, IL-10, or IFN-gamma mRNA. Levels of expression of cytokine mRNA in tissue from the small intestine were comparable in IL-2-/- and IL-2+/+ mice. CONCLUSIONS: Bowel inflammation in IL-2 deficient mice is preceded by an increase in IL-1 alpha, IL-1 beta, TNF-alpha, and IFN-gamma mRNA expression in colon tissue. Low levels of TGF-beta 1, but high levels of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, IFN-gamma, and IL-10 mRNA expression correlate with the manifestation of severe colitis, and suggest that T cells and macrophages are involved in bowel inflammation of IL-2 deficient mice.
Project description:Therapeutic mesenchymal stromal cells (MSCs) are attractive in part due to their immunomodulatory properties, achieved by their paracrine secretion of factors including prostaglandin E2 (PGE2). Despite promising pre-clinical data, demonstrating clinical efficacy has proven difficult. The current studies were designed to develop approaches to pre-induce desired functions from naïve MSCs and examine MSC donor variability, two factors contributing to this disconnect. MSCs from six human donors were pre-activated with interleukin 1 beta (IL-1?) at a concentration and duration identified as optimal or interferon gamma (IFN-?) as a comparator. Their secretion of PGE2 after pre-activation and secondary exposure to pro-inflammatory molecules was measured. Modulation of tumor necrosis factor alpha (TNF-?) secretion from M1 pro-inflammatory macrophages by co-cultured pre-activated MSCs was also measured. Our results indicated that pre-activation of MSCs with IL-1? resulted in upregulated PGE2 secretion post exposure. Pre-activation with IL-1? or IFN-? resulted in higher sensitivity to induction by secondary stimuli compared to no pre-activation. While IL-1? pre-activation led to enhanced MSC-mediated attenuation of macrophage TNF-? secretion, IFN-? pre-activation resulted in enhanced TNF-? secretion. Donor variability was noted in PGE2 secretion and upregulation and the level of improved or impaired macrophage modulation.
Project description:Mesenchymal stem cells derived from Wharton's jelly of the umbilical cord (UC-MSCs) have immunomodulatory properties. The aim of this study was to explore whether extracts of MSCs (MSC-Ex) could augment the low therapeutic efficacy of the whole cells in an Aspergillus fumigatus (Af)-induced atopic dermatitis (AD) model. LPS- or TNF-α/IFN-γ-stimulated keratinocytes (HaCaT cells) were treated with MSC-Ex, and the Af-induced AD model was established in BALB/c mice. In HaCaT cells, MSC-Ex treatment significantly reduced the inflammatory cytokine (IL-6, IL-1β, IL-4, IL-5 and TNF-α), iNOS and NF-κB levels, and upregulated the anti-inflammatory cytokines (IL-10 and TGF-β1). In the AD mice, the MSC-Ex group showed greatly reduced dermatitis, and lower clinical symptom scores and IgE levels. The histological dermatitis scores were also markedly lower in the MSC-Ex-treated animals compared with the MSC-treated group. Decreased levels of IFN-γ (Th1) and IL-17 (Th17), IL-4 and IL-13 (Th2) were detected in T cells and the skin tissue from the MSC-Ex treated AD mice. The therapeutic capacity of MSC-Ex was preserved after lyophilization and reconstitution. MSC-Ex treatment reproducibly suppresses dermatitis and inhibits the induction of inflammatory cytokines in the skin of AD mice. MSC-Ex is therefore a potential new treatment agent for AD.
Project description:Background and Objectives:The exosomes released by mesenchymal stromal cells (MSCs) in classical FBS-containing media have been demonstrated as an alternative, cell-free therapy in various diseases including inflammatory bowel disease (IBD). It has been found that the function of exosomes is affected by culture condition. We previously developed a serum-free, xeno-free and chemically defined medium, and umbilical cord-derived MSCs in this medium retained the immunosuppressive capability. Methods:In this study, we evaluated the immunosuppressive function of exosomes from MSCs (MSC-Exo) in defined medium and their therapeutic effect on treating colitis. Results and Conclusions:In vitro studies indicated that MSC-Exo reduced the concentration of pro-inflammatory cytokines IFN-?, TNF-? and IL-1?, and increased the secretion of anti-inflammatory cytokines TGF-?1 and IL-10, but no significant change of inhibitory effect on peripheral blood mononuclear cells proliferation was shown. In vivo experimental colitis showed that administration of MSC-Exo was able to significantly ameliorate the disease activity index score, weight loss, colon shortening, and the histological colitis score through up-regulation anti-inflammatory responses and down-regulation of inflammatory responses. Moreover, the use of MSC-Exo (200 µg) led to an improved therapeutic efficacy when compared with MSCs at a dose of 1×106 cells. Our findings indicate that the exosomes from MSCs in defined medium possess a certain degree of immunosuppressive effect in vitro and exhibit a therapeutic capability in a mouse model of DSS-induced colitis through suppressing inflammation mechanism.
Project description:We have previously reported human fetal cartilage progenitor cells (hFCPCs) as a novel source of therapeutic cells showing high proliferation and stem cell properties superior to those of adult mesenchymal stem cells (MSCs). In this study, we investigated the immunophenotype and immune-modulatory activities of hFCPCs. With institutional review board approval, hFCPCs were isolated from fetuses at 11-13 weeks of gestation. hFCPCs showed strong expression of HLA class I molecules but low or no expression of HLA class II and co-stimulatory molecules, which was not changed significantly after 4 days of IFN-γ treatment. In a mixed lymphocyte reaction (MLR), hFCPCs showed no allogeneic immune response to peripheral blood lymphocytes (PBLs) and suppressed concanavalin A (Con A)-mediated proliferation of PBLs in a dose-dependent manner. In addition, hFCPCs inhibited Con A-induced secretion of pro-inflammatory cytokines TNF-α and IFN-γ from PBLs but showed no significant decrease of secretion of IL-10, anti-inflammatory cytokine. Co-culture of hFCPCs with stimulated PBLs for 4 days resulted in a significant increase in CD4+CD25+FoxP3+ T regulatory cells (Tregs). hFCPCs expressed LIF, TGF-β1, TSG-6, and sHLA-G5 but did not express IDO and HGF. Stimulation of hFCPCs with TNF-α for 12 h showed slight induction in the expression of LIF, TSG-6, IDO, and HGF, whereas stimulation with IFN-γ did not affect expression of any of these factors. These results suggest that hFCPCs have low allogeneic immunogenicity and immune-modulatory activity in vitro, comparable to those of MSCs. However, compared with MSCs, hFCPCs were less responsive to TNF-α and IFN-γ, and the mechanisms underlying responses to these two cell types appeared distinct.
Project description:Mesenchymal stem cells (MSCs), which are modulated by cytokines present in the tumor microenvironment, play an important role in tumor progression. It is well documented that inflammation is an important part of the tumor microenvironment, so we investigated whether stimulation of MSCs by inflammatory cytokines would contribute to their ability to promote tumor growth. We first showed that MSCs could increase C26 colon cancer growth in mice. This growth-promoting effect was further accelerated when the MSCs were pre-stimulated by inflammatory factors IFN-? and TNF-?. At the same time, we demonstrated that MSCs pre-stimulated by both inflammatory factors could promote tumor angiogenesis in vivo to a greater degree than untreated MSCs or MSCs pre-stimulated by either IFN-? or TNF-? alone. A hen egg test-chorioallantoic membrane (HET-CAM) assay showed that treatment of MSC-conditioned medium can promote chorioallantoic membrane angiogenesis in vitro, especially treatment with conditioned medium of MSCs pretreated with IFN-? and TNF-? together. This mechanism of promoting angiogenesis appears to take place via an increase in the expression of vascular endothelial growth factor (VEGF), which itself takes place through an increase in signaling in the hypoxia-inducible factor 1? (HIF-1?)-dependent pathway. Inhibition of HIF-1? in MSCs by siRNA was found to effectively reduce the ability of MSC to affect the growth of colon cancer in vivo in the inflammatory microenviroment. These results indicate that MSCs stimulated by inflammatory cytokines such as IFN-? and TNF-? in the tumor microenvironment express higher levels of VEGF via the HIF-1? signaling pathway and that these MSCs then enhance tumor angiogenesis, finally leading to colon cancer growth in mice.
Project description:Oxidative stress is involved in the pathogenesis and progression of inflammatory bowel disease. Consumption of aronia berry inhibits T cell transfer colitis, but the antioxidant mechanisms pertinent to immune function are unclear. We hypothesized that aronia berry consumption could inhibit inflammation by modulating the antioxidant function of immunocytes and gastrointestinal tissues. Colitis was induced in recombinase activating gene-1 deficient (Rag1-/-) mice injected with syngeneic CD4+CD62L+ naïve T cells. Concurrent with transfer, mice consumed either 4.5% w/w aronia berry-supplemented or a control diet for five weeks. Aronia berry inhibited intestinal inflammation evidenced by lower colon weight/length ratios, 2-deoxy-2-[18F]fluoro-d-glucose (FDG) uptake, mRNA expressions of tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) in the colon. Aronia berry also suppressed systemic inflammation evidenced by lower FDG uptake in the spleen, liver, and lung. Colitis induced increased colon malondialdehyde (MDA), decreased colon glutathione peroxidase (GPx) activity, reduced glutathione (rGSH) level, and suppressed expression of antioxidant enzymes in the colon and mesenteric lymph node (MLN). Aronia berry upregulated expression of antioxidant enzymes, prevented colitis-associated depletion of rGSH, and maintained GPx activity. Moreover, aronia berry modulated mitochondria-specific antioxidant activity and decreased splenic mitochondrial H2O2 production in colitic mice. Thus, aronia berry consumption inhibits oxidative stress in the colon during T cell transfer colitis because of its multifaceted antioxidant function in both the cytosol and mitochondria of immunocytes.
Project description:MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis.By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1beta, TNF-alpha, and IFN-gamma was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-gamma, TNF-alpha, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis.Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.
Project description:BACKGROUND AND AIMS: Little is known about the intestinal epithelial expression and secretion of CXCL10 (IP-10), a chemokine involved in recruiting T cells and monocytes. We aimed to study CXCL10 gene expression and regulation by the pro-inflammatory cytokines interleukin (IL)-1beta, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in intestinal epithelial cell lines. MATERIALS AND METHODS: CXCL10 expression and secretion kinetics were assessed in Caco-2, HT-29 and DLD1 human colon epithelial cells, treated with IL-1beta, TNF-alpha, IFN-gamma alone or in combination with each other by real-time polymerase chain reaction (PCR), Northern blotting and enzyme-linked immunoabsorbent assay (ELISA). Transient transfections with TGL-IP10 (CXCL10 promoter) and TGL-IP10-kappaB2 mutant promoter and gelshifts and supershifts for nuclear factor (NF)-kappaB were also performed. RESULTS: Real-time PCRs and ELISA experiments revealed that IL-1beta was the strongest and earliest inducer of CXCL10 messenger ribonucleic acid (mRNA) expression and protein secretion in Caco-2 cell line, whereas INF-gamma had a delayed kinetics. There was a strong synergistic effect of either TNF-alpha or IL-1beta with IFN-gamma both on CXCL10 mRNA expression and protein secretion in all three cell lines. Real-time PCR and ELISA experiments using a specific NF-kappaB inhibitor and transfection experiments with a NF-kappaB-binding defective CXCL10 promoter construct revealed that the induction of CXCL10 by IL-1beta and its synergism with IFN-gamma is NF-kappaB dependent. CONCLUSION: These data demonstrate that in colonic epithelial cells, depending on the cellular context and utilizing the NF-kappaB pathway, IL-1beta alone and/or in synergism with IFN-gamma may play a major role in the induction of CXCL10.
Project description:We have investigated the effects of the pro-inflammatory cytokines interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on the iron metabolism of the human monocytic cell line U937. Cells were treated with each cytokine for up to 24 h, and then iron uptake from diferric transferrin was determined. The intracellular distribution of this iron, the expression of the transferrin receptor and levels of mRNA for the two ferritin subunits were also studied. IL-1 beta, TNF alpha and IFN gamma all decreased transferrin-iron uptake into cells, and all three cytokines had effects on the proportion of iron associated with ferritin. With TNF alpha there was a marked enhancement of the fraction incorporated into ferritin. Transferrin-receptor expression was diminished by TNF alpha and IL-1 beta, but not IFN gamma, suggesting different effector mechanisms. Both TNF alpha and IFN gamma increased the amount of cellular mRNA for ferritin H-chain, but not the L-chain; IL-1 beta affected mRNA for neither ferritin. These data demonstrate that cytokines, which can be present at high concentrations in inflammation, have the capacity to affect macrophage iron uptake, transferrin receptor expression, intracellular iron handling and the relative abundance of ferritin-subunit mRNA, and may therefore be important mediators in the observed perturbations of iron metabolism in inflammatory diseases.