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Xinyun Qiu, Mingming Zhang, Xiaotong Yang, Na Hong, Chenggong Yu, Faecalibacterium prausnitzii upregulates regulatory T cells and anti-inflammatory cytokines in treating TNBS-induced colitis, Journal of Crohn's and Colitis, Volume 7, Issue 11, December 2013, Pages e558–e568, https://doi.org/10.1016/j.crohns.2013.04.002
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Abstract
Background and aims:Faecalibacterium prausnitzii (F. prausnitzii) is a common anaerobic bacteria colonized in the human gut and inflammatory bowel disease (IBD) patients are usually lack of F. prausnitzii. The aims of this study were to evaluate the anti-inflammatory and immunomodulatory capacity of F. prausnitzii by comparing it with Bifidobacterium longum (B. longum) in both cellular and animal experiments.
Methods: Human peripheral blood mononuclear cells (PBMCs) and 2, 4, 6-trinitrobenzenesulphonic acid (TNBS)-induced colitis rat models were treated with F. prausnitzii, B. longum, F. prausnitzii supernatant or F. prausnitzii medium, respectively. Interleukin (IL)-10, TGF-β1 and IL-12p70 in human PBMCs culture supernatant and rat blood serum were detected. The frequency of CD25+Foxp3+Treg in human PBMCs, rat PBMCs and rat splenocytes were investigated. Besides, the T-bet, GATA-3, ROR-γt and Foxp3 mRNA in human PBMCs, histopathologic characteristics of the intestinal mucosal and weight loss in the rat models were examined.
Results:F. prausnitzii, B. longum and F. prausnitzii supernatant clearly facilitated the induction of IL-10 and TGF-β1, while induced relatively mild production of IL-12p70 in both cellular and animal models. The F. prausnitzii, B. longum and supernatant differed in their capacity to induce T-bet, GATA-3 and ROR-γt mRNA expression in human PBMCs (both bacterial strains inhibited the expression of ROR-γt while supernatant inhibited the T-bet and GATA-3). However, all of them induced the Foxp3 and Treg production and ameliorated the TNBS-induced colitis. In addition, F. prausnitzii supernatant exhibited the supreme anti-inflammatory capacity.
Conclusions:F. prausnitzii and its unidentified metabolites in the supernatant are promising candidates in treating IBD, and further research remains necessary to elucidate the safety, efficacy, optimum and mechanism of this bacterium in the clinical practice.
1 Introduction
The human intestinal tract harbors 10–100 trillion microorganisms,1 most of which colonize in the colon and shape a close relationship with the host in maintaining the human health.2 The microbes colonized in the gut are suggested to be divided into two parts3: the “protective” bacteria versus “harmful” bacteria. Both parties form a micro-ecological balance and play critical roles in shaping immune homeostasis in the gut.3 And the substantial changes in the composition of them could cause the chronic intestinal inflammation, such as the inflammatory bowel disease (IBD).2
F. prausnitzii, as an abundant anaerobic bacteria present in the human gut, was demonstrated to play an important role in maintaining the intestinal health and provide energy to the coloncytes.4,5 Accumulating evidences have revealed a diminished prevalence and abundance of F. prausnitzii in the fecal samples of patients with IBD, and what's more, the F. prausnitzii level was even much lower when the disease activity increased.5–8 Therefore, hypothesis that the bacteria might be closely related with the progress of IBD aroused a lot of interest of the researchers. Sokol et al.5 first demonstrated that both the bacteria and its supernatant (containing the metabolites secreted by the microbe) can attenuate the TNBS-induced intestinal colitis through their promotion of some anti-inflammatory cytokines and blockage the excretion of several key pro-inflammatory cytokines if they were prophylactically administered to the mice models. Interestingly, the supernatant showed a stronger anti-inflammatory capacity than the bacteria itself.5 Their findings broaden our visions on the pathogenesis of TNBS-induced intestinal colitis and afford a potential prevention strategy on the management of IBD. However, the therapeutic effects of this bacterium in treating the experimental colitis have not been verified, and available literatures on the immune-modulatory capacity of F. prausnitzii are still limited.
Nowadays, detecting cytokines in cellular or animal models which treated with a special bacterium was widely used to estimate the protective effects of the microbe. The ratio of anti-inflammatory cytokine (IL-10) to pro-inflammatory cytokine (IL-12) excreted by the immunocytes are frequently used to tell from an anti-inflammatory (high ratio) versus pro-inflammatory (low ratio) capacity exhibited by this bacterium.5 On the other hand, studies on the influences of bacteria on the differentiation of CD4+T cell clones (Th1, Th2, Th17 and Treg lymphocytes) and their relationships with the auto-immune diseases are other immediate areas of research focus. The Th1, Th2 and Th17 cells have been demonstrated to be three effector CD4+T lymphocytes which can facilitate the initiation and development of IBD.9,10 While the Treg, which are recognized as regulatory and suppressive cells, can restrain the progression of this disorder.11,12
In this study, we compared F. prausnitzii with a well-known probiotic-B. longum to investigate their effects on the cytokine production and the CD4+T cell differentiation and evaluate their protective ability when therapeutically used in TNBS-induced rat colitis.
2 Materials and methods
2.1 Bacterial strains and growth conditions
F. prausnitzii (ATCC 27766) was purchased from American type culture collection(ATCC), grown anaerobically at 37 °C in LYHBHI medium (Brain-heart infusion medium supplemented with 0.5% yeast extract (Difco) , 1 mg/ml cellobiose (Sigma), 5 mg/L hemin (Sigma-Aldrich) and 0.5 mg/ml cysteine (Sigma)). B. longum, which was kindly provided by Shanghai Sine Pharmaceutical Company (China), was cultivated in MRS medium supplemented with 0.05% L-cysteine-hydrochloride (Sigma). For stimulating PBMCs, bacteria were grown till the end of the exponential phase, washed three times and resuspended at 1 × 109 CFU/ml in phosphate buffered saline (PBS) containing 20% glycerol, and then stored at − 80 °C until used for experiments. For oral delivery to rats, bacteria were grown for 24 h, washed three times and resuspended at 1 × 109 CFU/ml in sterile PBS. F. prausnitzii supernatant was collected by centrifugation and separated from the bacteria through a 0.22 μm sterile filter (Millipore).
2.2 PBMC isolation
PBMCs were isolated from blood of healthy adult volunteers (median age: 39.3 years; range: 23–65 years) using Ficoll–Isopaue density gradient centrifugation (Ficoll–Paque, MP Biomedicals, Carlsbad, USA). Then, adjust the PBMCs to 2 × 106 cells/ml in RPMI 1640 supplemented with L-glutamine (2 mmol/L), penicillin (100 μg/ml) , streptomycin (100 μg/ml) and 10% fetal calf serum (FCS) (Gibco-BRL).
2.3 Induction of cytokine release
One milliliter of PBMCs (2 × 106 cells) were seeded in 24-well plates, F. prausnitzii (2 × 107 CFU), B. longum (2 × 107 CFU), F. prausnitzii supernatant(20 μl) or F. prausnitzii medium(20 μl) were incubated with the PBMCs at 37 °C in a atmosphere of air with 5% CO2 for 24 h. Culture supernatant were collected and stored at − 80 °C until cytokine analysis. Interleukin (IL)-10, IL-12p70 and transforming growth factor (TGF)-β1 were measured by enzyme-linked immunosorbent assay (ELISA) using commercially available ELISA kits (eBioscience, San Diego, CA).
2.4 Induction for flow cytometry and real-time quantitative PCR
One milliliter of PBMCs (1 × 106 cells) were cultured with F. prausnitzii (1 × 107 CFU), B. longum (1 × 107 CFU), F. prausnitzii supernatant (10 μl) and F. prausnitzii medium (10 μl) respectively for 168 h in 24-well plates which were pre-coated with 1.0 μg/ml anti-CD3 (OKT3, eBioscience) and 2.0 μg/ml anti-CD28 (CD28.2, eBioscience) overnight at 4 °C. In addition, recombinant human IL-2 (eBioscience) was added to the cultures of PBMCs at a concentration of 40 U/ml.
One part of the PBMCs was used for flow cytometry. Human regulatory T cell staining kit (eBioscience), which including FITC-labeled CD4 antibody, APC-labeled CD25 antibody and PE-labeled Foxp3 antibody, were used to detect the frequency of Treg (CD25+Foxp3+/CD4+ cells) in the PBMCs.
The rest part of PBMCs was used to measure the relative transcription factor mRNA of Th1, Th2, Th17 and Treg cells (T-bet, GATA-3, ROR-γt and Foxp3), respectively. RNA isolated from PBMCs was first subjected to reverse-transcription, and then specific Th1/Th2/Treg/Th17 cells real-time quantitative PCR array kits (TAKARO, Japan) were used to examine the mRNA levels of the transcription factor just mentioned.13 The oligonucleotide primer specific for T-bet, GATA-3, ROR-γt, Foxp3 and housekeeping gene β-actin were summarized in Table 1 .
2.5 Animals
Sixty adult male Sprague–Dawley (SD) rats (200–250 g) were obtained from the animal experimental center of Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School. These rodents were maintained under SPF conditions and fed rodent food and water according to the Animal Regulations of Jiangsu Province, China.
2.6 TNBS-induced colitis
Five days were provided to the rats that make them acclimatize to facility before the experimentation. Sixty rats were randomly assigned to 6 groups (n = 10). Five groups among them were used to build the colitis models by rectum injected with TNBS (Sigma Aldrich), which was dissolved in ethanol (50:50 vol/vol) at a dose of 80 mg/kg body weight. After 48 h of induced colitis, F. prausnitzii (109 CFU in 1 ml), B. longum (109 CFU in 1 ml), F. prausnitzii supernatant concentrated by five times (1 ml), F. prausnitzii medium concentrated by five times (1 ml) and PBS (1 ml) was orally administered to rats respectively in the next 7 days. The last group was used as no colitis control by intrarectally received PBS and ethanol (50:50 vol/vol) at a dose of 80 mg/kg body weight once in the first 48 h, and was orally delivered PBS (1 ml) once daily in the next 7 days. Rats were weighed before TNBS administration and sacrificed by cervical dislocation.
2.7 Cytokines detection in rat serum
Cytokine concentrations (TGF-β1, IL-10 and IL-12p70) in rat serum were quantified using ELISA kits (eBioscience) according to manufacturer recommendations.
2.8 FACS analysis of Treg in murine blood and spleen
Mononuclear cells were isolated from murine blood and spleens (by mechanical dissociation) using Ficoll–Isopaue density gradient centrifugation (Ficoll–Paque, Haoyang BioScience Corporation, Tianjin, China). Flow cytometry followed routine procedures by using 1 × 106 cells per sample. The cells were labeled with FITC anti-rat CD4 (eBioscience), APC anti-rat CD25 (eBioscience) and PE anti-rat Foxp3 (eBioscience). The stained cells were tested by flow cytometry (Becton Dickinson) and analyzed by using the Cell Quest software (BD Bioscience).
2.9 Inflammation scoring and body weight
Rats were weight before and 9 days after TNBS administration. When rats were sacrificed, colon tissues were removed, washed and opened, fixed in 10% neutral buffered formalin solution (Sigma Aldrich), embedded in paraffin, cut into tissue sections, and stained with hematoxylin/eosin (H&E). Inflammation grading was carried out by two independent blinded observers, using a previously published grading system14 (Table 2 ).
2.10 Statistical analysis
All data are expressed as mean ± SEM. Statistical analysis was performed with Statistical Package for Social Science version 16.0 software (SPSS Inc., Chicago, IL, USA). Non-parametric one-way analysis of variance and Mann–Whitney U-tests were used to compare the cytokine levels, the Treg number and relative mRNA expression level of T-bet, GATA-3, ROR-γt, Foxp3 and β-actin. P less than 0.05 were considered to be statistically significant.
3 Results
3.1 Bacterial strains and supernatant differ in their potential to induce cytokines release
The PBMCs stimulated in vitro by F. prausnitzii, F. prausnitzii supernatant and B. Longum showed distinct IL-10 and TGF-β1 release compared with the medium control (IL-10: P < 0.001, P < 0.001 and P = 0.006, respectively. TGF-β1: P = 0.019, 0.014 and 0.046, respectively). The IL-12p70 production induced by F. prausnitzii and B. Longum was increased significantly compared with the medium group (P < 0.001 and P = 0.046, respectively.). It is noteworthy that F. prausnitzii supernatant did not irritate the IL-12p70 excretion (P = 0.345). In addition, The IL-10/IL-12p70 ratio in F. prausnitzii, F. prausnitzii supernatant and B. Longum groups were significantly higher than that in the medium group (P < 0.001 in all treated group),. Interestingly, the F. prausnitzii supernatant elicit the highest IL-10/IL-12p70 ratio, revealing that F. prausnitzii supernatant exhibited the strongest anti-inflammatory capacity (Fig. 1 ).
In the in vivo experiments, we tested IL-10, IL-12p70 and TGF-β1 in the rat serum. F. prausnitzii supernatant can significantly dampened the IL-12p70 (P = 0.042) and up-regulated the IL-10 (P = 0.011) level when compared with the PBS-treated group. Besides, the TGF-β1 production by the F. prausnitzii, B. longum and F. prausnitzii supernatant were significantly promoted in comparison with the PBS-treated group (P = 0.002, 0.014 and < 0.001, respectively). F. prausnitzii and B. longum induced the IL-10 and prohibit the IL-12p70 excretion without statistical differences, which may due to the limited sample size. Of note, both the bacterium and supernatant displayed strikingly higher IL-10/IL-12p70 ratio compared with the PBS-treated group (F. prausnitzii: P = 0.013; B. longum: P = 0.004; F. prausnitzii supernatant: P < 0.001), and moreover, F. prausnitzii supernatant showed the highest IL-10/IL-12p70 ratio (Fig. 2 ).
3.2 Bacterial strains and supernatant differ in their potential to induce transcription factors mRNA expression
T-bet and GATA-3 mRNA expression were significantly suppressed by F. prausnitzii supernatant (P < 0.0001 and P = 0.001, respectively) when compared with the medium control, while was not obviously effected by the F. prausnitzii and B. longum. ROR-γt mRNA level was remarkably downregulated by F. prausnitzii and B. longum (P = 0.0003 and 0.008, respectively), but upregulated by F. prausnitzii supernatant without statistical difference. However, Foxp3 mRNA was notably overexpressed in F. prausnitzii, F. prausnitzii supernatant and B. longum treated PBMCs in comparison with the medium control (P = 0.002, < 0.0001 and = 0.006, respectively). Interestingly, the F. prausnitzii supernatant treated group showed the supreme Foxp3 mRNA level (Fig. 3 ).
3.3 Flow cytometric analysis
In the in vitro studies, CD4+CD25+Foxp3+ subpopulation in the PBMCs was consistently up-regulated by the F. prausnitzii, F. prausnitzii supernatant and B. longum compared with the medium control (P = 0.016, 0.003 and 0.021, respectively). However, F. prausnitzii supernatant induced the highest percentage of CD25+Foxp3+Treg in the CD4+ T cells (Fig. 4 ).
In the in vivo studies, the numbers of CD25+Foxp3+Treg in rat peripheral blood cells and splenocytes in the PBS treated group were statistically lower than that in the no colitis control (peripheral blood: P = 0.0079; spleen: P = 0.0009). After one week treatment of F. prausnitzii, F. prausnitzii supernatant or B. longum, the numbers of CD25+Foxp3+Treg in the peripheral blood cells and splenocytes were both increased compared with the PBS and the medium treated groups (Figs. 5 and 6 ) Remarkably, F. prausnitzii supernatant was also the strongest inducer in elevating the frequency of CD25+Foxp3+ Treg, mirrored it did in the in vitro experiment.
3.4 Body weight and colon morphological score
Histological assessment of colonic samples from the TNBS-induced colitis control group revealed severe transmural disruption of the normal architecture of the colon with marked degree of lymphocytes infiltration and high vascular density, giving a score of 3.5 ± 1.2 (P < 0.001 vs. no colitis control). Histological analysis of colonic specimens from rats treated with F. prausnitzii, F. prausnitzii supernatant and B. longum showed significant pronounced recovery in the intestinal architecture than that in the colitis control, with scores of 2.40 ± 0.97 (P = 0.025 vs colitis control), 2.10 ± 1.16 (P = 0.039 vs colitis control) and 2.25 ± 1.52 (P = 0.017 vs colitis control), respectively. While the F. prausnitzii medium did not show the difference with PBS treated group, giving a score of 3.13 ± 0.89 (P = 0.236 vs. PBS-treated group) (Figs. 7 and 8A ) F. prausnitzii supernatant and B. longum remarkably dampened the weight loss in the colitis models (P < 0.05 vs. PBS-treated group) (Fig. 8B).
4 Discussion
In this study, we have found that F. prausnitzii, as well as its supernatant (contains some unidentified metabolites), can attenuate the rat chemical colitis by regulating the inflammatory and tolerogenic T cell subsets differentiation and the relevant cytokines excretion. And we display for the first time, to our knowledge, that F. prausnitzii and its supernatant can facilitate the augmentation of Treg in peripheral blood cells and splenocytes.
In the past two decades, growing studies have shown that IBD patients are usually associated with the abnormality of cytokine secretion either as a cause or a consequence in their plasma or local inflammatory intestinal mucosa.15,16 While probiotics, such as Lactobacillus and Bifidobacterium, play a protective role against colitis by effectively inducing anti-inflammatory cytokines(e.g., IL-10,IL-4 and TGF-β,17–19 etc.) as well as suppressing the pro-inflammatory cytokines (e.g., IL-6, IL-12, IFN-γ and TNF-α18,20–23 etc.) production when treated to colitis animal models or humans. In our research, we compared F. prausnitzii with B. longum, and found that both bacteria played a coincident role in the cellular and animal models, giving a significantly higher IL-10/IL-12p70 ratio and attenuate the TNBS-induced inflammation when compared with the medium control. Intriguingly, F. prausnitzii supernatant displayed the supreme IL-10/IL-12p70 ratio in comparison with the other treating groups and revealed the strongest protective ability against the experimental colitis, which is accordant to the research previously reported in the mice models.5
It is widely accepted that IBD was driven by the Th1, Th2 or Th17 cells mediated immune responses,24 while suppressed by the Treg.25,26 IBD patients were proved to be increased in the Th1, Th2 or Th17 while decreased in the Treg cells in their peripheral blood.27–29 Thus, many investigators highlight the importance of reestablishment CD4+T cell homeostasis in controlling the development of IBD.29–31
Because T-bet, GATA-3, ROR-γt and Foxp3 are the lineage-defining factors in the CD4+T cells polarization25 , they are considered to be the surrogate markers to measure the Th1, Th2, Th17 and Treg skewings.13,32 We first studied how the F. prausnitzii, B. longum and F. prausnitzii supernatant worked, in the in vitro experiments, in affecting the polarization of CD4+T cells. The F. prausnitzii and B. longum tended to suppress the Th17-mediated reaction, for they can lead to a significant decrease in the production of ROR-γt (Th17), while had no obvious effects on the expression of T-bet (Th1) and GATA-3 (Th2). On the other hand, F. prausnitzii supernatant can remarkably reduce the expression of T-bet (Th1) and GATA-3 (Th2), whereas had no significant influence on the ROR-γt (Th17) expression. It is noteworthy that all of them steered the Foxp3 (Treg) production compared with the medium group, among which, F. prausnitzii supernatant group displayed the highest Foxp3 mRNA level.
Treg are important contributors to prevent colitis in the previous studies. Sakaguchi et al.33 have shown that transfer of naive CD4+ T cells into immune deficient mice which are lacking of Treg will cause colitis, while transfer of Treg into the colitis rodents can significantly attenuate the intestinal inflammation.34,35 Many probiotic strains manipulation have successfully reduced disease severity by augmenting Treg differentiation in both basic and clinical research.36–38 We successively tested the percentage of CD25+Foxp3+Treg (of CD4+ T cells) in cellular and animal models to investigate whether the bacterium or supernatant has the ability in augmenting the polarization of Treg. Interestingly, all of them consistently increased the Treg frequency in the in vitro and in vivo experiments and Treg induction was more pronounced by the F. prausnitzii supernatant. In our research, both the bacteria and supernatant displayed increased Treg frequency in the presence of higher IL-10 and TGF-β1, considering both cytokines were reported to be stimulative factors in the production of Treg,39 we hypothesize that IL-10 and TGF-β1provide a positive cytokine setting in the polarization of Treg.
The findings presented expand our current knowledge on the pathogenesis of TNBS-induced intestinal colitis, and reconfirm that F. prausnitzii, as an anti-inflammatory bacterium in treating TNBS-induced colitis, could be used in the management of IBD. In addition, the metabolites in F. prausnitzii supernatant also play an immunoregulatory role in our study, but there are still a large gap in our understanding of the real substance in the supernatant that effectively work. As microbiota were mostly colonized in the intestinal tract and shape a continuously dynamic effect with the mucosal immune cells,40 the efficacy that how the F. prausnitzii exhibited on intestinal mucosal and gut-associated lymphoid T cell differentiation need to be investigated in our ongoing studies. In conclusion, F. prausnitzii could be a potential new application of a candidate probiotic in treating IBD, and further research remains necessary to elucidate the safety, efficacy, optimum and mechanism of this bacterium in the clinical practice.
Funding
This study is supported by National Nature Science Foundation Grant of China (No. 81170359). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgment
We thank Shanghai Sine Pharmaceutical Company for providing the B. longum strain and the School of Life and Health Science, Nanjing University for affording the anaerobic culture incubator.
References
Gene | Specific sequence | Product length(bp) |
T-bet | F:5′-AAC ACA GGA GCG CAC TGG AT-3′ | 164 |
R:5′-TCT GGC TCT CCG TCG TTC A-3′ | ||
GATA-3 | F:5′-ACC GGC TTC GGA TGC AA-3′ | 210 |
R:5′-TGC TCT CCT GGC TGC AGA C-3′ | ||
ROR-γt | F:5′-GGC TCC CTG GAT GAA TAG AAT G -3′ | 190 |
F:5′- AGG CAG AGG CAG AAA ATG TAA AG-3′ | ||
Foxp3 | F:5′-GAG AAG GAG AAG CTG AGT GCC AT-3′ | 96 |
R:5′-AGC AGG AGC CCT TGT CGG AT-3′ | ||
β-actin | F:5′-GGC CAA CCG CGA GAA GAT-3′ | 134 |
R:5′-CGT CAC CGG AGT CCA TCA-3′ |
Gene | Specific sequence | Product length(bp) |
T-bet | F:5′-AAC ACA GGA GCG CAC TGG AT-3′ | 164 |
R:5′-TCT GGC TCT CCG TCG TTC A-3′ | ||
GATA-3 | F:5′-ACC GGC TTC GGA TGC AA-3′ | 210 |
R:5′-TGC TCT CCT GGC TGC AGA C-3′ | ||
ROR-γt | F:5′-GGC TCC CTG GAT GAA TAG AAT G -3′ | 190 |
F:5′- AGG CAG AGG CAG AAA ATG TAA AG-3′ | ||
Foxp3 | F:5′-GAG AAG GAG AAG CTG AGT GCC AT-3′ | 96 |
R:5′-AGC AGG AGC CCT TGT CGG AT-3′ | ||
β-actin | F:5′-GGC CAA CCG CGA GAA GAT-3′ | 134 |
R:5′-CGT CAC CGG AGT CCA TCA-3′ |
Gene | Specific sequence | Product length(bp) |
T-bet | F:5′-AAC ACA GGA GCG CAC TGG AT-3′ | 164 |
R:5′-TCT GGC TCT CCG TCG TTC A-3′ | ||
GATA-3 | F:5′-ACC GGC TTC GGA TGC AA-3′ | 210 |
R:5′-TGC TCT CCT GGC TGC AGA C-3′ | ||
ROR-γt | F:5′-GGC TCC CTG GAT GAA TAG AAT G -3′ | 190 |
F:5′- AGG CAG AGG CAG AAA ATG TAA AG-3′ | ||
Foxp3 | F:5′-GAG AAG GAG AAG CTG AGT GCC AT-3′ | 96 |
R:5′-AGC AGG AGC CCT TGT CGG AT-3′ | ||
β-actin | F:5′-GGC CAA CCG CGA GAA GAT-3′ | 134 |
R:5′-CGT CAC CGG AGT CCA TCA-3′ |
Gene | Specific sequence | Product length(bp) |
T-bet | F:5′-AAC ACA GGA GCG CAC TGG AT-3′ | 164 |
R:5′-TCT GGC TCT CCG TCG TTC A-3′ | ||
GATA-3 | F:5′-ACC GGC TTC GGA TGC AA-3′ | 210 |
R:5′-TGC TCT CCT GGC TGC AGA C-3′ | ||
ROR-γt | F:5′-GGC TCC CTG GAT GAA TAG AAT G -3′ | 190 |
F:5′- AGG CAG AGG CAG AAA ATG TAA AG-3′ | ||
Foxp3 | F:5′-GAG AAG GAG AAG CTG AGT GCC AT-3′ | 96 |
R:5′-AGC AGG AGC CCT TGT CGG AT-3′ | ||
β-actin | F:5′-GGC CAA CCG CGA GAA GAT-3′ | 134 |
R:5′-CGT CAC CGG AGT CCA TCA-3′ |
Score | Criteria |
0 | No inflammation |
1 | Low level of lymphocyte infiltration with infiltration seen in a < 10% high-power field (hpf),no structural changes observed |
2 | Moderate lymphocyte infiltration with infiltration seen in 10–25% hpf, crypt elongation, bowel wall thickening which does not extend beyond mucosal layer, no evidence of ulceration |
3 | High level of lymphocyte infiltration with infiltration seen in 25–50% hpf, high vascular density, thickening of bowel wall which extends beyond mucosal layer |
4 | Marked degree of lymphocyte infiltration with infiltration seen in > 50% hpf, high vascular density, crypt elongation with distortion, transmural bowel wall-thickening with ulceration |
Score | Criteria |
0 | No inflammation |
1 | Low level of lymphocyte infiltration with infiltration seen in a < 10% high-power field (hpf),no structural changes observed |
2 | Moderate lymphocyte infiltration with infiltration seen in 10–25% hpf, crypt elongation, bowel wall thickening which does not extend beyond mucosal layer, no evidence of ulceration |
3 | High level of lymphocyte infiltration with infiltration seen in 25–50% hpf, high vascular density, thickening of bowel wall which extends beyond mucosal layer |
4 | Marked degree of lymphocyte infiltration with infiltration seen in > 50% hpf, high vascular density, crypt elongation with distortion, transmural bowel wall-thickening with ulceration |
Score | Criteria |
0 | No inflammation |
1 | Low level of lymphocyte infiltration with infiltration seen in a < 10% high-power field (hpf),no structural changes observed |
2 | Moderate lymphocyte infiltration with infiltration seen in 10–25% hpf, crypt elongation, bowel wall thickening which does not extend beyond mucosal layer, no evidence of ulceration |
3 | High level of lymphocyte infiltration with infiltration seen in 25–50% hpf, high vascular density, thickening of bowel wall which extends beyond mucosal layer |
4 | Marked degree of lymphocyte infiltration with infiltration seen in > 50% hpf, high vascular density, crypt elongation with distortion, transmural bowel wall-thickening with ulceration |
Score | Criteria |
0 | No inflammation |
1 | Low level of lymphocyte infiltration with infiltration seen in a < 10% high-power field (hpf),no structural changes observed |
2 | Moderate lymphocyte infiltration with infiltration seen in 10–25% hpf, crypt elongation, bowel wall thickening which does not extend beyond mucosal layer, no evidence of ulceration |
3 | High level of lymphocyte infiltration with infiltration seen in 25–50% hpf, high vascular density, thickening of bowel wall which extends beyond mucosal layer |
4 | Marked degree of lymphocyte infiltration with infiltration seen in > 50% hpf, high vascular density, crypt elongation with distortion, transmural bowel wall-thickening with ulceration |
- anti-inflammatory agents
- cytokine
- inflammatory bowel disease
- colitis
- interleukin-10
- intestines
- animal model
- rna, messenger
- trinitrobenzenesulfonic acid
- bacteria
- mucous membrane
- rats
- antigens, cd25
- regulatory t-lymphocytes
- metabolites
- microbial colonization
- peripheral blood mononuclear cell
- gata3 gene
- foxp3 gene