| The mammalian gut epithelium forms a dynamic barrier between the body and the luminal gastrointestinal contents, including diet, microorganisms, digestive products and drugs. Maintenance of its integrity, which turns over every3-5days, is dependent on stringent regulations of pathways involving proliferation, migration, differentiation and apoptosis. Coordinated activation of these signaling molecules involved in the regulation these process is critical to maintain intestinal homeostasis and prevent the development of inflammatory bowel diseases (IBD). IBD, such as ulcerative colitis and Crohn’s disease, are associated with chronic relapsing and unresolved inflammation of the intestinal tract, and often result in excessive tissue injury, inadequate regeneration. One of the most serious complications of IBD is colorectal cancer, which has been shown the clearest link between inflammation and colon colorectal in patients with IBD. Although normal maintenance of the intestinal epithelium by many signaling passways has been well characterized, little is known about the molecular events that regulate restoration of intestinal tissue homeostasis in response to external stress or injury.Tumor necrosis factor-α induced protein-8(TNFAIP8or Tipe) was the first identified member of Tipe family. Many studies show that Tipe may prevent apoptosis in certain tumor cell lines and increase their oncogenic potential. Overexpression of Tipe gene in tumor cells correlates with enhanced proliferation, invasion and experimental metastasis. Tipe has been reported to interact with activated Gαi3to regulate cell death and transformation. Tipe gene polymorphism or over-expression is associated with susceptibility to Staphylococcus aureus infection, cancer development and psoriasis. However, the precise roles of Tipe in physiological condition and diseases are pooly understood. Given the potential roles of Tipe in regulating cell death and proliferation, in the current study, we utilize a well-characterized model of dextran sodium sulfate (DSS)-induced colitis to understand better the role of Tipe in regulating response to epithelial injury and mucosal inflammation. Our data indicate that Tipe plays a crucial role in maintaining colonic homeostasis during acute colitis. Objective1. To generate Tipe deficiency mice and investigate the phenotypes of TIPE knockout mice.2.To investigate the role of Tipe in the development of DSS-induced acute colitis and the potential mechanisms.Methods1. Generation and Genotyping of Tipe-/-Mice The C57BL/6(B6) embryonic stem (ES) cell line with a disrupted Tipe gene was obtained from the Texas A&M Institute for Genomic Medicine. The gene-trapping vector that disrupts Tipe gene was inserted into the only intron of the gene. The ES cells were injected into mouse blastocysts to generate chimeras. Six chimeras, one female and five males, were produced. The germline transmission was obtained from one of the male chimeras. Heterozygous mice for the disrupted Tipe locus were bred to produce homozygous Tipe-/-animals. Once identified, they were further tested by PCR to ensure that that did not express TIPE mRNA.2. Phenotyping of Tipe-/-MiceSix-to-eight-week mice were sacrificed and their immune organs were collected and weighted. Single cell suspensions were prepared by homogenizing, and total cell numbers from each organ were determined using the cell counter. Flow cytometry was performed after staining cells with anti-mouse CD4, CD8, B220, Gr-1, CD11B, CD11C, NK1.1, CD25, CD44, Foxp3, CD62L, and CD69for frequency analysis.3. DSS-induced colitisExperimental colitis was induced by adding DSS to the drinking water at a concentration of3%or4%(w/v) in8-12weeks old male mice. For survival studies, animals were fed with4%DSS water for5days and then allowed to recover by regular drinking water for additional9days. For all other experimental read-outs, colitis was induced by feeding mice3%DSS during5days, and then switched to regular drinking water until the end of the experiment on day7. Animals were checked every day and euthanized on day7or judged as moribund before day7and immediately euthanized.4. Evaluation of DSS-induced colitisMice were examined daily to determine their clinical Disease Activity Index (DAI), which was based on the degree of body weight loss, stool consistency, and fecal blood (ranging from0to12). Briefly, DAI was scored as follows:weight loss (no change=0;<5%=1;6-10%=2;11-20%=3;>20%=4); stool (normal=0; soft, well-formed=1; soft without pellets=2; diarrhea=4); blood (no blood=0; visible blood in rectum=1; grossing bleeding in rectum=2; visible blood on fur=4). For histological analysis, the distal colonic specimens were fixed in buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin (HE), and pathological scores, ranging from0to6(combining inflammatory cell infiltration score and tissue damage score), determined as follows. Inflammatory cell infiltration in the lamina propria (occasional inflammatory cells=0; increased inflammatory cells=1; confluence of inflammatory cells extending to the submucosa=2; transmural extension=3) and tissue damage (no mucosal damage=0; lymphoepithelial lesions=1; surface mucosal erosion=2; extensive mucosal damage and extension into deeper structures of the bowel wall=3).5. Generation of bone marrow chimeric miceBone marrow cells were harvested by flushing the femurs and tibias from donor WT and Tipe-/-mice that express either CD45.1or CD45.2. Recipient mice were sublethally irradiated twice, at a dose of4.5Gy. Following the second irradiation, bone marrow cells were transferred into WT and Tipe-/-mice,10million cells/mouse, intravenously via the tail vein. Four chimera groups were generated:WT→WT (WT cells expressing CD45.2into WT mice expressing CD45.1);Tipe-/-→WT (Tipe-/-cells expressing CD45.2into WT mice expressing CD45.1); WT→Tipe-/-(WT cells expressing CD45.1into Tipe-/-mice expressing CD45.2); Tipe-/-→Tipe-/-(Tipe-/-cells expressing CD45.2into Tipe-/-mice expressing CD45.2). Six weeks after the transplantation, the degree of bone marrow reconstitution was determined by staining peripheral blood eukocytes with PE-conjugated anti-CD45.1and PercpCy5.5-conjugated anti-CD45.2. 6. Real-time quantitative PCRTotal RNA was extracted from tissues or cells with TRIzol reagent. Two micrograms of total RNA was reverse transcribed. Real time quantitative PCR was carried out. The relative changes in gene expression were calculated using GAPDH as the loading control.7. ELISASera were collected and stored at-80℃before analysis. Colon was homogenized in CellLyticM buffer supplemented with complete protease inhibitors mixture. Protein concentration was measured by BCA assay. Cytokine content was presented as pg/mg of total colon protein. Quantitative ELISA was performed according to the manufacturer’s instructions.8. Histopathology and ImmunohistochemistryThe entire colon was excised to measure the length. The distal colons were washed, fixed in10%buffered formalin and embedded in paraffin. Tissue sections were stained with hematoxylin&eosin (H&E). For Ki-67and active caspase3staining, mice were treated with DSS and colons were collected, washed, and fixed in10%formalin. Colon sections were then stained with Ki-67and active caspase3antibodies.9. Isolation of Colon Epithelial CellsColons were dissected, washed with cold PBS, and cut into small pieces. Colon segments were incubated in PBS supplemented with EDTA/DTT for30min at37℃with gentle shaking. Cells in supernatants were filtered through a70μm cell strainer and washed twice. Enrichment for colonic epithelial cells was confirmed by staining cells for the epithelial cell-specific marker EpCam.70-80%of isolated cells stained positive for EpCam. If needed, the isolated colonic epithelial cells were cultured in epithelial cell culture medium (ECM) in vitro.10. Colon leukocytes isolationTo isolate lamina propria leukocytes, intestinal epithelial cells and intraepithelial lymphocytes were first stripped by shaking colonic sections in EDTA/DTT for30 minutes at37℃. The remaining colon tissue was cut into small pieces and incubated in DMEM containing penicilin/streptomycin, collagenase, Dnase and Dispase, for20minutes at37℃with gentle shaking. Supernatant was filtered with70μm strainers to obtain single cell suspensions.11. Apoptosis assayColon samples were processed and epithelial cells were obtained. The isolated colonic epithelial cells were cultured in epithelial cell culture medium overnight. Cells were treated with DSS for24hours and cell death was determined by7AAD and Annexin V staining.12. Bacterial cultureThe terminal3-cm segments of distal colon was washed, weighed, homogenized, and serially diluted. Different dilutions of the suspensions were plated on Brain Heart Infusion (BHI) agar and Blood agar plates, and incubated at37℃for24hours, to quantify the bacterial colonies.13. Assessment of intestinal permeabilityIntestinal barrier permeability in vivo was measured with a FITC-labeled dextran method. In brief, WT and TIPE2-/-mice were deprived of water and food overnight, and were fed with permeability tracer FITC-dextran at400mg/kg body weight by oral gavage. Blood was collected four hours later by retro-orbital bleeding. Fluorescence intensity of the serum was measured using a fluorescence spectrophotometer with an excitation wavelength of490nm and an emission wavelength of530nm. FITC-dextran concentrations were calculated using standard curves generated by a serial dilution of FITC-dextran.14. Blood cell countsBefore euthanizing the DSS-treated mice, blood was collected, and whole blood cell counts were determined by Drew Hemavet950FS.Results1. Generation and phenotyping of Tipe-deficient Mice Tipe-/-mice were generated using gene trap technology to target Tipe gene in B6ES cells. The gene-trapping vector is composed of a LTR (viral long terminal repeat), a splice acceptor site, a Neomycin-resistant gene, and a polyA tail that stops Tipe gene transcription. This disrupted Tipe gene allele results in a complete loss of the short isoform of Tipe mRNA and a truncated long isoform of Tipe mRNA that encodes the first N-terminal24amino acids. Homozygous offsprings were identified by PCR. Tipe gene mutation did not affect the gross growth and development of mice. Homozygous Tipe-/-mice were born with the expected Mendelian ratios with no detectable developmental abnormalities. The weight, structure, and cellular compositions of lymphoid organs are normal. Downregulation of Tipe was reported to protect thymocytes against glucocorticoid-mediated apoptosis. However, no significant differences between WT and Tipe-/-groups were detected in thymocyte death induced by dexamethasone.2. Tipe-/-mice are hyper-sensitive to DSS-induced ColitisTipe-/-mice did not appear to develop more spontaneous diseases than WT littermates. However, after drinking DSS-containing water, despite similar amount of water consumption, they developed more severe colitis than WT mice. They began to die on day six after the DSS treatment. By day ten, all of the Tipe-/-animals had died while40%of WT animals were still alive (p<0.01). The Tipe-/-mice suffered greater body weight loss (p<0.05) and increased overall disease manifestations (p<0.01). To further assess the severity of colitis, colon length was also measured. The DSS-fed Tipe-/-mice had significantly shorter and lighter colons (p<0.01). Consistent with these findings, histopathological examination of H&E-stained colons of knockout mice revealed more severe colitis characterized by crypt loss and infiltrating leukocytes than in the controls.3. Enhanced inflammatory responses in Tipe-/-Mice with colitisTo further identify the roles of Tipe in DSS-induced colitis, we examined the hematological and immunological aspects of the disease in age-and sex-matched Tipe-/-mice and WT controls. Significantly increased neutrophil counts were detected in Tipe-/-blood samples. At the same time, the spleens of Tipe-/-mice became smaller while more leukocytes were found in the colon of these mice. Enhanced levels of IL-17A and IL-6were also found in the sera of Tipe-/-mice. Similarly, enhanced levels of IL-6, IL-17A, and IL-1β proteins and/or mRNAs were detected in the Tipe-/-colons Increased CXCL2mRNA expression was also detected in Tipe-/-colons.4. Tipe-deficiency in non-hematopoietic cells exacerbates colitisAlthough enhanced inflammatory responses were detected in the DSS-challenged Tipe-/-mice, both hematopoietic and non-hematopoietic compartments could contribute to the exacerbated colitis phenotype observed above. To assess which compartment contributes directly to the exacerbation of colitis in Tipe-/-mice, we generated four groups of bone marrow chimeras. After recovering for eight weeks, mice were then subjected to five days of DSS treatment followed by two days of normal drinking water. After DSS treatment, the WT mice receiving wild type or Tipe-/-bone marrow cells demonstrated similar changes in body weight, disease index, and colon length indicating that hematopoietic cells are not major contributors to the exacerbation of colitis observed in Tipe-/-mice. By contrast, when WT bone marrow cells were injected into Tipe-/-or WT recipient mice, after DSS treatment, Tipe-/-recipients developed more severe colitis than the WT recipients indicating that the non-hematopoietic compartment plays a major role in the disease exacerbation.5. Increased bacterial invasion of the colonic tissue in Tipe-deficient miceThe disruption of mucosal barrier following gastrointestinal epithelial cell injury can lead to commensal bacterial infection and potent inflammatory responses against them, which is believed to be part of the pathogenic mechanisms of DSS-induced colitis. We next checked bacterial numbers in the colons of WT and Tipe-/-mice with colitis. We found significantly more bacteria in the colons of Tipe-/-mice than in WT mice, indicating enhanced dissemination of the commensal bacteria.6. Tipe-deficiency in Epithelial Cells Affects Their Survival and ProliferationThe increased mortality in Tipe-/-mice could be explained by increased cell death and/or decreased proliferation of colonic epithelial cells during colitis. To test whether Tipe-deficiency affects epithelial cell death and proliferation, epithelial cells were isolated from normal and DSS-fed WT and Tipe-deficient colons and the cell death was determined by7-AAD and Annexin V staining. Increased cell death was detected in the Tipe-/-group as compared to WT controls. PI3K-AKT pathway is important for regulating cell death and immunity. A significantly reduced level of activated AKT was detected in the Tipe-/-colon samples, indicating that the increased cell death might be due to the downregulation of the PI3K-AKT signaling. Ki67is an endogenous marker for cell proliferation. Significantly reduced Ki67staining was found in DSS-fed Tipe-/-colons compared to those of the WT, suggesting that Tipe protein is required for epithelial cell proliferation after DSS-induced colon damage. Increased active caspase3staining was also observed in the Tipe-/-colons, demonstrating augmented cell death. Taking together, increased death of Tipe-/-epithelial cells, combined with compromised proliferation, may be responsible for the increased mortality in Tipe-deficient mice treated with DSS.Conclusion1. Tipe deficiency does not affect T cell development in the thymus; Tipe deficiency does not affect dexamethasone-induced thymocytes apoptosis.2. Tipe-/-mice are hyper-sensitive to DSS-induced Colitis.3. Tipe-deficiency in non-hematopoietic cells exacerbates colitis.4. Increased apoptosis of Tipe-/-colonic epithelial cells, combined with compromised proliferation, may be responsible for crucial roles of Tipe in the development of DSS-induced colitis.Innovations and significances1. It is the first time to generate Tipe-/-mice and investigate the phenotypes of this mice2. It is the first time to report crucial roles of TNFAIP family proteins Tipe in the development of DSS-induced colitis, which is great helpful to investigate biological functions of Tipe protein under pathophysiological conditions.3. The current study preliminarily elucidates the mechanisms of Tipe action in the pathogenesis of colitis. Tipe may serve as a potential drug target for treating intestinal diseases. Inflammatory bowel diseases (IBD), comprising ulcerative colitis and Crohn’s diseases, constitute a major health problem in humans, and are associated with an increased risk of cancer of the colon and rectum. Ulcerative colitis exhibits a characteristic profile of chronic, relapsing and remitting inflammation involving the distal colon and rectum and is generally recognized as an immune-mediated disorder resulting from abnormal interaction between colonic microflora and mucosal immune cells. The production of inflammatory mediators resulting from excessive inflammatory and immune responses has been implicated in the pathogenesis of IBD in humans and experimental colitis. Although the precise etiology of ulcerative colitis is not well understood, several animal models of experimental colitis have been developed to help investigate the molecular and cellular mechanisms of the disease. Of these, the dextran sodium sulfate (DSS)-induced experimental colitis model is one of the best studied. DSS-induced colitis causes severe epithelial injury limited to the colonic mucosa, disruption of the epithelial barrier and a robust inflammatory response in the presence of both commensal microflora and myeloid cells. DSS-induced colitis is limited to the colonic mucosa characterised by weight loss, diarrhea, bloody faeces, and a histopathological picture of inflammation, mainly consisting of infiltrating macrophages and granulocytes, and colonic ulceration as seen in human IBD.TIPE2(tumor necrosis factor-a-induced protein-8like-2, also known as TNFAIP8L2) is a newly identified immune regulator of the TNFAIP8family and is preferentially expressed in inflamed organs and lymphoid tissues, such as thymus, spleen, lymph node, intestine and myeloid cells. TIPE2plays an essential role in the maintenance of immune homeostasis through negatively regulating innate and adaptive immune responses. Further studies demonstrated that abnormal expression of TIPE2has been found in patients with autoimmune disease, chronic inflammatory disease, infectious disease, metabolic disease and tumor development. In addition, our recent results show that TIPE2may control innate immunity to bacteria and dsRNA viruses by targeting the Rac GTPases. However, the role of TIPE2in IBD has not been reported. Based on this bacteria-dependent IBD model, we hypothesize that TIPE2plays an important role in the development of IBD by regulating immune responses to commensal microflora in the colon.ObjectiveTo investigate the role of TIPE2in the development of DSS-induced acute colitis and the potential mechanisms.MethodsRefer to the first partResults1. Expression of TIPE family members in the murine colon We first examined the colon phenotypes for TIPE2-/-mice. Unlike TIPE2-/-129mice that develop systemic inflammation starting from approximately8weeks of age, young TIPE2-/-mice of the B6background are relatively healthy. TIPE2-/-mice on the B6background did not develop spontaneous IBD and had a normal gastrointestinal tract. The mRNA expression of TIPE2was readily detected in the colon tissue homogenates from WT but not TIPE2-/-mice. Among the TIPE family members, both TIPE and TIPE1were abundant in the colon when compared with TIPE2expression. On the contrary, TIPE3was expressed at only minimal levels. In addition, there was no significant alteration in the mRNA expression of the other members in the colon of TIPE2-/-mice.2. TIPE2-/-mice are resistant to DSS-induced colitisTo determine the potential role of TIPE2in colonic inflammation, WT and TIPE2-/-mice were fed with4%DSS for5days, to induce acute colitis. TIPE2deficiency did not affect water consumption, but markedly reduced the severity of the colitis. The mortality rate was reduced from80%in the WT group to0%in the TIPE2-/-group (p<0.01). Consistent with the mortality data, TIPE2-/-mice lost considerably less weight than the WT controls commencing on day5. The body weight difference increased gradually until the end of the study (p<0.05). Consistent with these results, the clinical manifestation of the disease, reflected by DAI, was significantly less severe for TIPE2-/-mice compared with WT mice (p<0.01). After DSS treatment, colon weight of TIPE2-/-mice was23%more than that of WT mice (p<0.01), while colon length of TIPE2-/-mice was27%longer than that of WT mice (p<0.01). Histological examination was also performed to validate the clinical data. DSS treatment induced significant histopathological changes in the colons of WT mice characterized by massive inflammatory infiltrates and disruption of mucosal structures. However, TIPE2-/-mice displayed less severe injury compared with WT mice. The histopathological score of TIPE2-/-mice was significantly lower than that of WT mice (4.6±0.6in WT versus2.6±0.4in TIPE2-/-mice).3. Colonic inflammation is reduced in TIPE2-/-miceTo determine the effect of TIPE2deficiency on the production of proinflammatory cytokines at the site of DSS-induced inflammation, we examined the expression levels of several cytokines. We found that proinflammatory cytokines such as TNF-a, IL-6, and IL-12were markedly increased in WT mice after DSS treatment. However, TIPE2-/-mice produced significantly less of these cytokines relative to WT mice. On the other hand, TIPE2-/-mice produced comparable amounts of IL-1β, IL-4, and IFN-y as compared with WT mice. To characterize the inflammatory cells involved, leukocytes isolated from lamina propria of the colon were analyzed. In comparison with WT colon, the TIPE2+colon had markedly less neutrophils (8.89±0.55v.s.2.92±0.32,p<0.001), macrophages (12.63±0.63v.s.6.92±0.69,p<0.001), and dendritic cells (24.04±3.65v.s.11.97±0.27, p<0.05). Importantly, a significant proportion of the leukocytic infiltrate in both WT and TIPE2-/-mice consisted of Ly6G-CD11b+macrophages. By contrast, Ly6G+CD11b+neutrophils and CD11c+dendritic cells were responsible for only a small component of the inflammatory infiltration in both WT and TIPE2-/-mice.4. TIPE2deficiency in hematopoietic cells ameliorates colitisTo determined the cell populations that are critical for TIPE2-mediated effect, we generated four groups of bone marrow chimeras. Six weeks after bone marrow transplantation, we found that more than90%of the blood leukocytes were of donor origin, confirming successful engraftment. Following DSS treatment, KO→KO chimeric mice developed the least severe colitis. Compared with WT→WT chimeric mice, transplant of KO bone marrow to WT recipient mice (KO→WT) markedly reduced body weight loss, improved DAI, and preserved the colon length, as well as reduced histopathologcial score. These results indicate that TIPE2-deficient hematopoietic cells are responsible for the reduced colonic inflammation in the TIPE2knockout mice.5. Reduced local dissemination of commensal microflora and systemic inflammation in TIPE2-/-miceBecause we observed reduced inflammatory response in the DSS-induced colitis of TIPE2-/-mice, we asked whether TIPE2deficiency affected commensal microflora dissemination in mice. Significantly less bacteria were detected in the colon of TIPE2-/-mice relative to DSS-treated WT mice. We also found a significant decrease in plasma FITC fluorescence in TIPE2-/-mice after DSS-induced injury compared to WT mice, indicating less colon tissue disruption and less permeability. In agreement with the reduced bacterial dissemination in TIPE2-/-mice, the numbers of total peripheral blood leukocytes and monocytes were markedly reduced in TIPE2-/-mice relative to DSS-treated WT mice. Furthermore, serum concentrations of the proinflammatory cytokine IL-6was also lower in TIPE2-/-mice when compared to WT mice.6.TIPE2deficiency does not affect colonic bacterial compositionThe total DNA in stool was extracted and real-time PCR was conducted using specific16S rRNA primers for the following major groups:the Eubacterium rectale-Clostridium coccoides, Bacteroides sp. and Enterobacteriaceae. In addition, the total bacterial (eubacteria) numbers were also determined using standard curves constructed with reference bacteria specific for each group. We found no significant differences in the numbers of total bacteria or the three representative bacterial groups, between naive WT and TIPE2-/-mice of6-8weeks of age, indicating that bacterial composition was not affected by TIPE2deficiency.Conclusion1. Using the acute dextran sodium sulfate (DSS) colitis model, we found that TIPE2-/-mice were significantly protected from colitis.2. Adoptive transfer of TIPE2deficient bone marrow cells was capable of rescuing colonic injury phenotype in WT mice.3. The ameliorated colitis in TIPE2-/-mice was associated with reduced local dissemination of commensal bacteria and the systemic inflammatory response.Innovations and significances1. It is the first time to investigate the relationship between colitis and anti-inflammatory TIPE2protein, which is a natural extension of our study to know critical roles of TIPE2in different disease models.2. This study further confirms important roles of commensal bacteria in the pathogenesis of IBD. It may ameliorate colitis by effectively controlling the development of IBD through regulating local proliferation and dissemination of commensal bacteria in intestinal tract.3. The current study elucidates further the immunological inflammtory mechanisms in the development of IBD. The data significantly advance our understanding of the pathogenesis of colitis. TIPE2may serve as a potential drug target for treating intestinal diseases. |
PDF Full Text Request