The Regulatory Effects Of Low Dose5-fluorouracil On T Helper Cells

Background and AimsInflammatory bowel disease(IBD)refers to the non-specific inflammatory diseases with unclear etiology, including ulcerative colitis(UC) and Crohn’s disease(CD), categorized as autoimmune disease. In western countries, there has always been a high incidence of IBD, with an increasing number of reports of over120thousand cases. IBD has become a common digestive disease and the main pathogenic for chronic diarrhea. Most patients suffer the reoccurrence of IBD and the deferment of curability or even need surgical treatment. In this case, they are not only in great pain but also at the Risk of developing into cancer patients. Therefore, it is critical to treat the IBD. It has been known that the inflammation caused by abdominal Intestines mucous membrane immunoreaction plays an important role in IBD. Nowadays, most researchers believe that IBD is associated with environmental, genetic, inflectional and immunological factors. It involves multi-gene factors acting on immune system and target organ. Immunological factors play an essential role in IBD. It is our research focus to find a key factor among various factors and mediums involved in immune inflammation.It has been generally accepted that CD4+Helper T Cell plays a role in the development of IBD. CD4+Helper T Cell consists of at least four subsets:Thl cell subset, Th2cell subset, Th2cell subset and Treg.Until recently,IBD had been regarded as mediated by IFN-γ producing helper Thl and IL4/IL-13producing Th2cells.However,several studies have recently shown that the inflamed GI mucosa of patients with IBD have a massive infiltration of Th cells. Several studies have showed that Th17-associated cytokine were upregulated in IBD patients.Tissue biopsies from inflamed colons show levels of IL-17A, IL-17F, IL-21, IL-22that are elevated compared with those of healthy controls, and elevated levels of cytokines in the serum of patients with active disease.Currently, there are no medical cures for IBD. However, several different medications have proven to be effective in helping to control it. Medical therapy for IBD has three main goals:Inducing remission (periods of time that are symptom-free), Maintaining remission (preventing flare-ups of disease) and Improving the patient’s quality of life.To achieve these goals, therapy must suppress the chronic intestinal inflammation that causes the symptoms of IBD. Currently, there are three basic categories of medications used in the treatment of IBD:aminosalicylates, corticosteroids and immunomodulators. Biologic therapies offer a distinct advantage in IBD treatment. Their mechanism of action is targeted. Unlike corticosteroids, which tend to suppress the entire immune system and thereby produce major side effects, biologic agents act selectively. The newest class of drugs to be used in IBD include Adalim umab, Certoliz Mab pegol, Golim Mab, Infliximab, and Nataliz umab. Biologics are genetically engineered medications made from living organisms and their products, such as proteins, genes, and antibodies. Biologics interfere with the body’s inflammatory response in IBD by targeting specific molecular players in the process such as cytokines—specialized proteins that play a role in increasing or decreasing inflammation. Promising targets include t Mor necrosis factor (TNF)-alpha, interleukins, adhesion molecules, colony-stimulating factors, and others. But they can induce serious infections which lead patients to die.5-FU is widely used in the treatment of colorectal cancer. Antimetabolite drugs work by inhibiting essential biosynthetic processes, or by being incorporated into macromolecules, such as DNA and RNA, and inhibitingtheir normal function. The fluoropyrimidine5-fluorouracil (5-FU) does both.5-FU is an analogue of uracil with a fluorine atom at the C-5position in place of hydrogen.The mechanism of cytotoxicity of5-FU has been ascribed to the misincorporation of fluoronucleotides into RNA and DNA and to the inhibition of the nucleotide synthetic enzyme thymidylate synthase (TS). It rapidly enters the cell using the same facilitated transport mechanism as uracil.5-FU isconverted intracellularly to several active metabolites:fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP)—these active metabolites disrupt RNA synthesis and the action of TS. The rate-limiting enzyme in5-FU catabolism is dihydropyrimidine dehydrogenase (DPD), which converts5-FU to dihydrofluorouracil (DHFU).The most side effects of high-dose5-FU are bone marrow suppression and gastroenterology reaction when high-dose5-FU used as chemotherapy in colon cancer patients. As there are no specific metabolic pathways found in t Mor cells, and the difference between normal cells and t Mor cell growth fraction of anti-metabolic drugs theoretically able to kill t Mor cells without affecting normal cells. With5-FU in clinical increasingly widely used, researchers have gradually broadened the scope of drug treatment in many diseases in the field of application of the medium or low doses of5-FU have achieved good results.Max Schlaak et@1al found that Low-dose5-fluorouracil of4weeks in combination with salicylic cid shows significant effects in the treatment of actinic keratosis and no serious side effects occurred.Our group found that underwent preoperative5-FU chemotherapy in patients with colorectal cancer, the intestinal tissue of T-bet, RORyt were significantly lower than the level before chemotherapy, And T-bet and RORyt are the master transcription factors of Thl and Th47respectivly which may indicate that5-FU suppress the role of Thl and Thl7. However,5-FU is cytotoxic drugs and high-doses of5-FU have significant bone marrow and gastrointestinal reactions in the patients. We suppose that low dose of5-FU with selective inhibition of immune cells Thland Th17to provide an effective basis for new ideas and new ways to treat IBD.Research Contents1. collect tissue and serum specimen from colon cancer patients who received5-FU treatment before surgery, measure the expression of markers of Thl and Th17.2. Induce animal enteritis model, measure the expression of markers of Thl and Th17of the mice which received the low-dose5-FU treatment.3. Measure the expression of markers of Thl and Thl7of the T helper cells which received the low-dose5-FU treatment in vitro.4. Investigate the mechanism of the regulatory effects of low-dose5-FU on T helper cells.5. Evaluate the dose range of5-FU on T helper cells and colon cancer cells using modelling model.Subjects and methods1. Hospitalized patients with colon canecr who received5-FU chemotherapy before surgery in Nanfang Hospital from2011-2013were included. Clinical data, imagine, endoscopy and pathology records were recorded. Tissue specimens were obtained from normal mucosa or lesions when patients underwent endoscopy or quickly after surgery,then washedtwice with chilled phosphate buffered saline, immediately isolated and stored in liquid nitrogen and then at-80℃in tissue bank until further use. Venous blood was collected after an overnight fast. Routine blood tests were soon analyzed by Department of Laboratory, Nanfang Hospital. Serum was obtained after15minutes,1800×g centrifuged and the aliquots of the serum were stored at-80℃until analysis.2. For each tissue sample from patients, a histological examination withH-E staining was given. Paraffin biopsies and serum samples were studied by immunohistochemistry and ELISA to detect both IFN-y and IL-17A. RT-PCR was performed to detect the level of markers of Th1,Th17and Treg mRNA.3. A total of20male C57BL6mice (6-8weeks) were divided randomly into the PBS group, high-dose5-FU group, low-dose5-FU group, all mice were all given 3%DSS in distilled water for6days, and were sacrificed on day7. mice were separately given PBS,5-FU50mg/g (body weight) and5-FU10mg/g(body weight)from Day1through Intraperitoneal injection(I.P).Mice weight and stool characters were recorded and a disease activity index (DAI) was determined daily by an investigator blinded to the protocol.4. Make Adoptive transfer models.we performed adoptive transfer experiments using CD45RBhiCD25-cells from WT to induce colitis in RAG1KO (Rag1-/-) mice. After3weeks, mice were separately given PBS and5-FU10mg/g (body weight) every three days through Intraperitoneal injection(I.P).Mice weight and stool characters were recorded and a disease activity index (DAI) was determined daily by an investigator blinded to the protocol.5. Mice were sacrificed after anesthesia. Blood was collected from the hearts and the colons were removed through a midline incision. For each tissue sample, a histological examination withH-E staining was given. Spleen and Mesenteric lymph nodes of mice were re-stimulated with PMA/ionomycin for5h and stained for intracellular IL-17and IFN-y and analysed by flow cytometry.Reverse transcription PCR (RT-PCR) was employed to detect the level of markers of Thl and Thl7and enzyme-linked immunosorbent assay (ELISA) were used to measure the cytokines of Thl and Th17in each mouse serum.6. Naive CD4+T cells from wild-type were stimulated with plate-bound anti-CD3and anti-CD28antibodies and under Th1,Th17and Treg polarizing conditions.After3days of stimulation, The cells were re-stimulated with PMA/ionomycin for5h and stained for intracellular IL-17, IFN-y and FOXP3and analysed by flow cytometry. Total RNA was extracted and analysed by RT-PCR for mRNA expression of markers of Thl, Th17and Treg. And IL-17A and IFN-y protein secretion in culture supernatants was analysed by ELISA. Western Blot performed to evaluate the expression of transcription factors of Th1and Th17.7. Transfection and luciferase reporter assay.293T cells were transiently transfected with an IL-17promoter luciferase reporter plasmid together with RORyt in the presence of5-FU at deferent concentrations. For each transfection,2.0μg of plasmid was mixed with100μl DMEM (without ser M and antibiotics) and4.0μl Lipofectamine2000reagent. The mixture was incubated at room temperature for20min and added to12-well plates containing cells and complete medi M. The cells were incubated for30h and harvested using reporter lysis buffer (Promega) for determination of luciferase activity. Cells were cotransfected with a b-galactosidase reporter plasmid to normalize experiments for transfection efficiency.8. Chromatin immunoprecipitation assay. The ChIP procedure was performed using an assay kit following the manufacturer’s instruction (Upstate Biotechnology). Naive CD4+T cells from wild-type were stimulated with plate-bound anti-CD3and anti-CD28antibodies and under Thl and Th17polarizing conditions. After3days of stimulation, cells were then crosslinked by1%formaldehyde for10min at37℃. Nuclei were prepared and subjected to sonication to obtain DNA fragments. Chromatin fractions were precleared with protein A-agarose beads followed by immunoprecipitation overnight at4℃with3μg of anti-STAT3or control antibody. Crosslinking was reversed at65℃for4h, followed by proteinase K digestion. DNA was purified and subjected to qPCR. The input DNA was diluted200times before PCR amplification. The input and immunoprecipitated DNA were amplified by qPCR using primers (5’-CAGCCCTGGTCCTTAAACTG-3’and5’-TCACTTTCGTTGTGCCTTTG-3’) encompassing the CNS2region of the mouse IL-17promoter.9. For experiments measuring biomarkers, statistical analysis was performed with SPSS17.0software (Chicago, IL, USA). Descriptive statistics were calculated with means and standard deviation. When Data are normally distributed and the variances are equal, Independent samples T-test for comparing means between two groups and One-way ANOVA test for among three or more groups (with LSD method for post Hoc Multiple Comparisons) were used; when the variances are not equal, Satterthwaite’s approximate t test and Welch’s F test (with Dunnett’s T3method for post Hoc Multiple Comparisons) were used respectively; but when data aren’t normally distributed, Mann-Whitney U test and Kruskal-Wallis H test were used respectively. The en Meration data were calculated with sample n Mber and percentage and compared with the χ2test. Comparisons of Ranked data were determined by Mann-Whitney U Test (between two independent groups) or Kruskal-Wallis H Test (among multiple independent groups). Spearman’s correlation analysis was employed to define associations. A Pvalue less than0.05was considered significant. The extracted data for systematic review and meta-analysis in Part3were analyzed using RevMan (Version5.2, Cochrane Collaboration, Denmark) and Comprehensive Meta-Analysis software (Version2.2023, Englewood, NJ, USA). Binary outcomes were compared by odd ratio (OR) and continuous outcomes by weighted mean difference (WMD), and then presented in forest plots. The95%confidence interval (CI) was used and p value<0.05was ass Med to be showing a statistically significant difference. The statistical significance for heterogeneity was assessed by use of the Chibased Q statistic and the Istatistic for the extent of heterogeneity. Heterogeneity was considered significant if p<0.1and was more than50%. The fixed effect model was employed in pooling data where there was no evidence of heterogeneity and where there was evidence of heterogeneity, the random effects model was used instead. Sensitivity analyses were performed by excluding studies with unclear quality. Possible publication bias or other small study effects were evaluated using the Egger test.Results1. compared to control group, the expressions of Thl and Th17markers are significantly decreased in colon tissue of the patients who received5-FU chemotherapy before surgery.2. In two enteritis groups, significant symptoms (including weight loss, diarrhea, rectal bleeding) were observed. When the dose of5-FU at50mg/kg, the5-FU group began losing weight earlier and lost significantly more weight than PBS group. Parallel histological studies of colon sections from5-FU group mice revealed more severe inflammatory cell infiltrates and significantly higher pathological scores than PBS group. But when the dose of5-FU change to10mg/kg, the situation went to the opposite side. 3. In transfer experiments using CD4+CD45RBhi cells from WT mice to induce colitis in RAG1KO (Ragl-/-) mice. PBS group mice began losing weight earlier and lost more weight than mice of5-FU group. Parallel histological studies of colon sections from Ragl-/-mice of PBS group mice revealed more severe inflam-matory cell infiltrates and significantly higher pathological scores than those observed in sections from mice of5-FU group. In addition, PBS group mice had a significantly higher percentage of IL-17-producing cells than5-FU group by flow cytometry.4. Naive CD4+T cells from WT mice were differentiated under ThO, Thl, Th17and Treg condition and with the treatment of5-FU at the dose of0.5M for3days. TheTreatment of low-dose5-FU suppresses the transcription factor of Thl, Th17but not Treg by Western Blot. Accordingly, the transcription factor mRNA expression was decreased with the treatment of low-dose5-FU by Q-PCR. The cells of5-FU treatment had a significantly lower percentage of IL-17-producing cells than control by flow cytometry. And the cytokine secretion expression of Thl and Th17were decreased significantly performed by ELISA.5.293T cells were transfected with RORΓT, T-bet and STAT3with the dose of5-FU at1,5,10μM for48h. The RORΓT, T-bet and STAT3protein expression were decreased by the treatment of5-FU in a dose-dependent manner. Then293T cells were co-transfected with an IL-17promoter reporter and RORΓT plasmid with the dose of5-FU at1,5,10μM for48h. we confirmed that5-FU can suppress the expression of RORΓT in293T cells in a dose-dependent manner by luciferace reporter assay.6. Naive WT CD4+T cells were stimulated under Thl and Thl7polarizing conditions for60h and ChIP assay was performed as above. The precipitated chromatin DNA was analysed by PCR using primers covering the CNS2region of the IL-17promoter. Similarly, STAT3antibody specifically pulled down the CNS2region sequences of IL-17in Thl7cells. And STAT4antibody specifically pulled down the CNS2region sequences of in Th1cells.7. The dose of5-FU which can induce SW620cell ranges from10μM to20μM, while the dose of5-FU which can induce T helper cell ranges from1μM to2.5μM, which analyzed by modellingConclusionsHigh-dose5-FU chemotherapy can significantly decreased the expression of transcription factor and cytokine of Thl and Th17cells in colon cancer patients colon tissue, which may show that low dose5-FU suppress the expression of Thl and Th17cells in human. Low dose5-FU can significantly alleviate the progression of colitis both in chemical colitis model and in adoptive transfer colitis model. The mechanism of Low dose5-FU inhibiting colitis and protecting colon mucosa andselectively suppressing the expression of Thl and Th17cells in vivo and vitro may be through inhibiting the binding of stat4and T-bet promoter and stat3and IL-17and RORyt promoter seperately.