Study Of Milk Fat Globule Epidermal-Growth Factor8in The Pathogenesis Of Ulcerative Colitis

BackgroundUlcerative colitis (UC) is a chronic and relapsing inflammatory disorder involving the mucosal layer of the colon and rectum. The patients complain about abdominal pain, diarrhea, bloody stool, etc. Most patients have active and remission phases alternatively. However the underlying molecular basis of the disease has not been completely elucidated.Milk fat globule-epidermal growth factor8(MFG-E8), also named lactadherin, BA46or SED1, is a secreted glycoprotein present in several cell types, including macrophages, mammary epithelial cells, and epidermal keratinocytes. It participates in engulfing apoptotic cells. MFG-E8-null mice show severe autoimmune disease like human systemic lupus erythematosis because of accumulation of uncleared apoptotic cells. MFG-E8also participates in other cell events including the promotion of mammary gland branching and facilitation of sperm-egg binding.Several studies of animal models demonstrated that MFG-E8play an important role in intestinal homeostasis. In mice with sepsis, MFG-E8promoted enterocyte migration along the crypt-villus axis and accelerated intestinal mucosal healing. As well, MFG-E8expression was impaired in inflamed mouse colon during the acute phase of dextran sodium sulfate (DSS)-induced colitis, but pre-treatment with recombinant MFG-E8had a protective role against inflammation. Moreover, a recent study reported that recombinant MFG-E8administered during the recovery phase of DSS-induced colitis in mouse could attenuate inflammation and enhance epithelial repair, which suggests a therapeutic role for MFG-E8in DSS-induced colitis. But we lack reports of MFG-E8activity in humans with UC.Objectives1. To evaluate MFG-E8expression in normal human colon and inflamed colon in UC patients.2. To analyze the correlation of MFG-E8levels with colonic mucosal inflammation gradings and patients'disease activity indecies in UC patients.Methods1. Colonic biopsy samplingWe enrolled26patients with UC who underwent colonoscopy for disease surveillance. The diagnosis was based on well-established clinical, endoscopy and histopat-hological criteria in China. Control subjects were26volunteers matched by age and sex who underwent colonoscopy for physical examination, surveillance of polyp recurrence or functional abdominal pain. Our study was approved by the Clinical Ethical Committee of Qilu Hospital of Shandong University. All subjects had given their written informed consent before biopsy sampling.During colonoscopies, intestinal mucosal biopsies were obtained from the macroscopically inflamed area for UC patients or from normal areas for healthy controls in the sigmoid colon or rectum. For each subject, we obtained2adjacent biopsies, one for histology and anotherr for RNA or protein analysis. For histology, biopsies were immediately fixed in10%neutral buffered formaldehyde and those for RNA or protein analysis were snap-frozen in liquid nitrogen and stored at-80℃Biopsies from all of the subjects underwent routine histology, and all biopsies from healthy controls were confirmed as histologically normal.2. Inflammation grading and calculation of disease activity index Mucosal inflammation severity was classified as mild, moderate or severe by neutrophil infiltration according to Matt's grade. The Mayo disease activity index (0-12scale) calculated for each UC patient at the time of colonoscopy was based on frequency of bowel movements, rectal bleeding, endoscopy findings and the physician's overall assessment.3. RNA isolation, semi-quantitative RT-PCR and real-time quantitative PCRA total of25biopsy specimens (12from UC patients and13from controls) were used for RNA analysis. Total RNA was isolated by the Trizol reagent method. RNA was reverse-transcribed using MMLV-derived reverse transcriptase and oligodT primer. Semi-quantitative RT-PCR was performed with ExTaq DNA polymerase in a Mastercycler thermal cycler. For real-time PCR, cDNA was amplified with SYBR Green reagent in a Roche fluorescence thermo-cycler. Human beta-actin was used as an internal control. Targeted gene mRNA levels were normalized to those of beta-actin by the2-ΔΔCT method.4. Protein extraction and western blottingTotal protein was extracted from27biopsy samples (14from UC patients and13from controls) in RIPA buffer. Protein was quantified by the use of a BCA protein quantification kit. Protein was separated by SDS-PAGEs and transferred to PVDF membrane (0.22μm pore). The membrane was incubated with primary antibodies and HRP secondary antibodies sequentially. Densitometry of protein bands was quantified by the use of Quantity One4.6.2.5. Immunohistological analysisParaffin-embedded tissues were cut into3-μm-thick sections and underwent routine deparaffinizing and rehydrating. HE staining was used for routine histological analysis. For MFG-E8immunostaining, the Envision-two-step method was used.Results1. Demographic features of participantsNo statistical differences of age or sex ratio between26ulcerative patients and26control subjects were observed. Among26patients, there are3with ulcerative proclitis,14with left-sided colitis and7with ulcerative pancolitis.2. Colonic MFG-E8expression in ulcerative colitisSemi-quantitative RT-PCR proved the existence of MFG-E8mRNA in colonic biopsies from ulcerative colitis patients and controls. Real-time quantitative PCR revealed that colonic MFG-E8mRNA expression was significantly lowered in patients than controls (P<0.01). Western blotting results indicated that MFG-E8protein was dramatically decreased in ulcerative colitis patients than controls (P<0.01).3. Correlation analysis of colonic MFG-E8expression with mucosal inflammation and disease activity index in ulcerative colitisColonic MFG-E8expression in ulcerative colitis, was much lower in biopsies with moderate or severe inflammation than those with mild inflammation (P<0.01). Spearman correlation analysis revealed that MFG-E8expression was inversely correlated with disease activity index in ulcerative colitis (r=-0.7713, P<0.01).4. ImmunohistochemistryWe used immunohistochemistry to investigate the cellular origin of MFG-E8in intestinal tissues. The intestinal epithelium of healthy controls showed strong positive staining for MFG-E8. MFG-E8was present in both of the surface epithelium and crypts of normal intestinal tissues. However, the intestinal epithelium of UC patients showed less staining for MFG-E8.Conclusions1. MFG-E8is present in human colonic mucosa and the intestinal epithelial cell is its mam origin.2. Colonic MFG-E8mRNA and protein expression is decreased in ulcerative colitis patients compared with controls.3. Colonic MFG-E8expression in UC patients is inversely correlated with mucosa inflammation gradings and the Mayo disease activity indecies. BackgroundUlcerative colitis is characterized by elevated production of pro-inflammatory mediators, repeated intestinal injury and cell restitution and increased apoptosis of intestinal epithelial cells (IECs), which leads to impaired mucosal barrier function. However the underlying molecular basis has not been completely elucidated.IECs are a pivotal physiological barrier between the environment and the host. They play a key role in gut innate immunity and contribute to inflammatory progression in inflammatory bowel disease by secreting various chemokines and cytokines. IEC apoptosis has a major role in intestinal epithelial homeostasis and pathogenic mechanisms. Increased IEC apoptosis has been observed in acute inflammatory sites of patients with IBD, which led to impaired intestinal barrier function and contributed to disease development. The intestinal epithelium is exposed to various stimuli, and IEC injury is constant and inevitable. Especially in patients with IBD, repeated intestinal epithelial injury is typical. During intestinal epithelium injury, IECs rapidly depolarize and migrate to cover the denuded area, independent of proliferation. This process is called restitution, and rapid restitution is required to maintain the integrity of intestinal barrier.MFG-E8expression was impaired in inflamed mouse colons during the acute phase of dextran sodium sulfate (DSS)-induced colitis, but pre-treatment with recombinant MFG-E8had a protective role against inflammation. Our previous study has proved that MFG-E8was present in human intestinal epithelium and colonic MFG-E8was dramatically decreased in ulcerative colitis. Additionally, MFG-E8 expression in ulcerative colitis is inversely correlated with mucosal inflammation grading and disease activity index.Overall, we presumed that decreased MFG-E8expression in IECs may be related to increased apoptosis, abnormal inflammatory responsiveness and impaired wound healing in UC patients. Objectives1. To acquire MFG-E8knockdown intestinal epithelial cells (IECs) in vitro to represent IECs in ulcerative colitis.2. To detect apoptosis and apoptosis-related proteins expression in IECs after MFG-E8knockdown.3. To detect wound healing and intestinal restitution-related factors in MFG-E8knockdown IECs.4. To detect the inflammatory responses of IECs after MFG-E8knockdown.Methods1. Cell culture and lentivirus mediated MFG-E8knockdownHuman colorectal cancer-derived IEC lines Caco-2and HT-29were cultured in Dulbecco's modified Eagle's medium supplemented with10%(v/v) fetal calf serum. Lentiviral particles encoding short hairpin RNA (shRNA) sequences targeting human MFG-E8mRNA and control lentiviral particles encoding a scrambled shRNA sequence were from Santa Cruz Biotechnology. Lentiviruses all carried a puromycin-resistant gene. When cells were at50%to60%confluence, lentiviral vectors were added with10μg/ml polybrene. Puromycin was added to get stable-transduced clones. MFG-E8-knockdown levels were validated by real-time quantitative PCR and western blot analysis.2. Cellular RNA isolation and real-time quantitative PCRCellular RNA was isolated with Trizol reagent. The following were similar with procedures in Part Ⅰ.3. Cellular protein extraction and western blottingCells were lysised in RIPA buffer. Lysis was centrifuged by14000rpm at4℃for 10min. Supernatants were collected. Protein concentration was quantified with BCA protein assay kit. Total protein was separated by SDS-PAGEs and transferred to PVDF membrane later. The membrane was then incubated with different antibodies. An enhanced chemiluminiscent substrate was used to detect the protein bands.4. Apoptosis detectionApoptosis was detected by flowcytometry with an Annexin-V/FITC kit. Briefly, floating and adherent cells were washed with phosphate buffered saline. After cells were resuspended in binding buffer, FITC-conjugated annexin V and propidium iodide were added to incubate for10min in the dark. Annexin-V binding was determined in a BD FACSCalibur flow cytometer (Becton-Dickinson). Apoptotic nucleus changes were viewed under a fluorescence microscope after incubation with Hoechst33342staining solution.5. Wound healing assayHT-29cells transduced with LV-C or LV-M were seeded into6-well plates and allowed to reach confluence overnight, then wounds were made by scratching cell monolayers with a sterile pipette tip. Detached cells were rinsed off3times with PBS. Serum-deprived culture medium (DMED with0.1%FCS) was added for further culture. Cells were photographed0and24hr after wounding. Distances covered by migrated cells were quantified.Results1. MFG-E8in IEC lines and validation of MFG-E8knockdownMFG-E8mRNA and protein were present in the IEC lines HT-29and Caco-2. After transduction with LV-M, MFG-E8mRNA levels were decreased by93.2%and92.7%in HT-29and Caco-2cells. As well, MFG-E8protein levels were decreased by78%and73%in HT-29and Caco-2cells, respectively.2. MFG-E8has no effect on IEC inflammatory responsesThe basal IL-8mRNA expression levels in HT-29and Caco-2cells with MFG-E8-knockdown was similar to that with LV-C transduction. After stimulation with100ng/ml TNF-alpha or flagellin for12hr, IL-8mRNA levels were both upregulated in MFG-E8-knockdown cells and LV-C-transduced cells. And IL-8mRNA expression induced by TNF-alpha or flagellin was not altered in MFG-E8-knockdown IECs. To further investigate the role of MFG-E8in flagellin induced inflammatory response in IECs, we used recombinant human MFG-E8to treat Caco-2cells (Since Caco-2is more responsive to flagellin than HT-29) before flagellin addition. And pre-incubation with rhMFG-E8from lOng/ml to500ng/ml for12hr didn't alter IL-8mRNA levels induced by flagellin in Caco-2cells.3. Apoptosis induction in MFG-E8knockdown IECsAnnexin V positive apoptotic cell proportions were significantly higher in MFG-E8knockdown IECs. The pro-apoptotic protein BAX and cleaved caspase-3expression were upregulated after MFG-E8knockdown. The anti-apoptotic protein BCL-2level was downregulated after MFG-E8knockdown.4. MFG-E8knockdown impaired wound healing and TFF3expression in IECsMFG-E8knockdown greatly attenuated wound closure of scratched HT-29cell monolayers24hr after wounding. In addition, expression of TFF3, a pivotal factor in intestinal restitution, was decreased after MFG-E8knockdown.Conclusions1. MFG-E8knockdown can promote basal apoptosis in IEC lines accompanied by the induction of cleaved caspase-3and BAX and the suppression of BCL-2.2. MFG-E8knockdown can impair wound healing in IEC lines as well as the mRNA level of TFF3.3. MFG-E8has no effect on inflammatory responses in IECs induced by TNF-alpha or flagellin.