oxazolone and Colitis

NOD2 has emerged as a critical player in the induction of both Th1 and Th2 responses for potentiation and polarisation of antigen-dependent immunity. Loss-of-function mutations in the NOD2-encoding gene and deregulation of its downstream signalling pathway have been linked to Crohn's disease. Although it is well documented that NOD2 is capable of sensing bacterial muramyl dipeptide, it remains counter-intuitive to link development of overt intestinal inflammation to a loss of bacterial-induced inflammatory response. We hypothesised that a T helper bias could also contribute to an autoimmune-like colitis different from inflammation that is fully fledged by Th1 type cells.. An oedematous bowel wall with a mixed Th1/Th2 response was induced in mice by intrarectal instillation of the haptenating agent oxazolone. Survival and clinical scoring were evaluated. At several time points after instillation, colonic damage was assessed by macroscopic and microscopic observations. To evaluate the involvement of NOD2 in immunochemical phenomena, quantitative polymerase chain reaction [PCR] and flow cytometry analysis were performed. Bone marrow chimera experimentation allowed us to evaluate the role of haematopoietic/non-hematopoietic NOD2-expressing cells.. Herein, we identified a key regulatory circuit whereby NOD2-mediated sensing of a muramyl dipeptide [MDP] by radio-resistant cells improves colitis with a mixed Th1/Th2 response that is induced by oxazolone. Genetic ablation of either Nod2 or Ripk2 precipitated oxazolone colitis that is predominantly linked to a lack of interferon-gamma. Bone marrow chimera experiments revealed that inactivation of Nod2 signalling in non-haematopoietic cells is causing a biased M1-M2 polarisation of macrophages and a decreased frequency of splenic regulatory T cells that correlates with an impaired activation of CD4 + T cells within mesenteric lymph nodes. Mechanistically, mice were protected from oxazolone-induced colitis upon administration of MDP in an interleukin-1- and interleukin-23-dependent manner.. These findings indicate that Nod2 signalling may prevent pathological conversion of T helper cells for maintenance of tissue homeostasis.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Colitis; Inflammation; Mice; Nod2 Signaling Adaptor Protein; Oxazolone; Signal Transduction

Clinical challenges in inflammatory bowel diseases require microscopic in vivo evaluation of inflammation. Here, label-free imaging holds great potential, and recently, our group demonstrated the advantage of using in vivo multiphoton endomicroscopy for longitudinal animal studies. This article extends our previous work by in-depth analysis of label-free tissue features in common colitis models quantified by the multiphoton colitis score (MCS).. Fresh mucosal tissues were evaluated from acute and chronic dextran sulfate sodium (DSS), TNBS, oxazolone, and transfer colitis. Label-free imaging was performed by using second harmonic generation and natural autofluorescence. Morphological changes in mucosal crypts, collagen fibers, and cellularity in the stroma were analyzed and graded.. Our approach discriminated between healthy (mean MCS = 2.5) and inflamed tissue (mean MCS > 5) in all models, and the MCS was validated by hematoxylin and eosin scoring of the same samples (85.2% agreement). Moreover, specific characteristics of each phenotype were identified. While TNBS, oxazolone, and transfer colitis showed high cellularity in stroma, epithelial damage seemed specific for chronic, acute DSS and transfer colitis. Crypt deformations were mostly observed in acute DSS.. Quantification of label-free imaging is promising for in vivo endoscopy. In the future, this could be valuable for monitoring of inflammatory pathways in murine models, which is highly relevant for the development of new inflammatory bowel disease therapeutics.

Topics: Animals; Colitis; Dextran Sulfate; Disease Models, Animal; Inflammation; Inflammatory Bowel Diseases; Mice; Oxazolone

The functions of basophils have not been elucidated until recently because of their rarity. However, with recent developments in basophil-specific antibodies and basophil-deficient animals, the roles of basophils in various diseases related to chronic inflammation have been clarified. In this study, we aimed to investigate the roles of basophils in human ulcerative colitis (UC) and oxazolone (OXA) colitis using genetically engineered Mcpt8. Immunohistochemical staining of human colon specimens was performed to examine the involvement of basophils in the pathogenesis of UC. We examined the correlation between the number of infiltrating basophils and the UC endoscopic index of severity (UCEIS), Mayo score, and Matts score. We also examined the correlation between eosinophil count and basophil infiltration. In murine experiments, we examined whether basophil infiltration was involved in OXA-induced colitis and whether basophil depletion improved inflammation in Mcpt8. Colonic basophil infiltration was significantly increased in patients with UC. There were significant correlations between UCEIS, Mayo score, Matts score, and the number of infiltrating basophils. In murine OXA-induced colitis, a significant increase in basophil infiltration was observed. When basophils were depleted by diphtheria toxin in Mcpt8. Basophil infiltration correlated with endoscopic, clinical, and pathological scores in human UC independently of eosinophil infiltration, and depletion of basophils ameliorated mucosal inflammation in murine OXA-induced colitis, collectively suggesting that basophils exert a proinflammatory role in chronic intestinal inflammation such as UC.

Topics: Animals; Basophils; Colitis; Colitis, Ulcerative; Humans; Inflammation; Intestines; Mice; Oxazolone

Topics: Anti-Inflammatory Agents; Atorvastatin; Colitis; Gene Expression; Humans; Interleukin-10; Interleukin-6; Mesalamine; Oxazolone

Crohn's disease (CD) and ulcerative colitis (UC) actually had different pathological mechanisms, as the former was mainly induced by Th1 and Th17 response and the latter by Th2 response. Our previous study found that oxazolone-induced Th2-mediated colitis could not be attenuated by vitamin D supplementation. This study investigated the influence of intestinal vitamin D receptor (VDR) knockout on oxazolone-induced colitis and explored the possible immunological mechanism. Intestinal VDR knockout mice had milder oxazolone-induced colitis than wildtype controls, as demonstrated by less body weight decrease and faster recovery, more intact local structure, reduced cell apoptosis, and better preserved barrier function. Th2-mediated inflammation was significantly inhibited by VDR deficiency. Meanwhile, the percentage of invariant natural killer T (iNKT) cells did not increase as much in intestinal VDR knockout mice as in wild-type controls, nor did the iNKT cells develop normally as in the controls. Intestinal VDR knockout protected against oxazolone-induced colitis in mice by blocking Th2 cell response and reducing the function of intestinal iNKT cells. Vitamin D status had no influence on the severity of colitis. This study may explain the diverse outcomes after vitamin D supplementation in literature and add some clue to the targeted therapy of IBD.

Topics: Adjuvants, Immunologic; Animals; Colitis; Disease Models, Animal; Female; Humans; Male; Mice; Mice, Knockout; Oxazolone; Survival Analysis; Vitamin D

Children born by cesarean section (CS) have an increased risk of developing inflammatory bowel disease (IBD), possibly due to skewed microbial colonization during birth and consequently impaired bacterial stimulation of the developing immune system. The aim of this study was to investigate the association between CS and experimental colitis in a murine model of IBD. It was hypothesized that CS aggravates colonic inflammation due to a change in gut microbiota (GM) composition. C57BL/6 mice, delivered by CS or vaginal delivery (VD), were intra-rectally challenged with oxazolone at 8 weeks of age and monitored for colitis symptoms. The results showed that CS delivered mice experienced an increased body weight loss and colon weight, together with higher colonic concentrations of TNF-α and MPO compared with VD mice. Increased infiltration of inflammatory cells was present in CS delivered mice, as well as a downregulation in expression of the gut integrity genes occludin and tight junction protein 1 indicative of an impaired barrier function. The GM from CS delivered mice without colitis partly contributed to the increase in colitis symptoms when inoculated into germ-free recipient mice. In conclusion, CS increased sensitivity to oxazolone induced colitis in mice.

Topics: Animals; Cesarean Section; Colitis; Colon; Disease Models, Animal; Fecal Microbiota Transplantation; Female; Gastrointestinal Microbiome; Inflammation Mediators; Intestinal Mucosa; Mice, Inbred C57BL; Oxazolone; Peroxidase; Pregnancy; Severity of Illness Index; Tumor Necrosis Factor-alpha

Chemokine systems modulate inflammatory and immune responses in inflammatory bowel disease (IBD). The colons of IBD patients show increased levels of fractalkine (FKN) and high numbers of FKN receptor-positive (CX3CR1+) cells; however, the FKN-CX3CR1 axis's role in intestinal inflammation, especially in intravascular leukocyte behaviors, still remains unclear. Here, we show that interruption of the FKN-CX3CR1 axis by anti-FKN monoclonal antibody (mAb) ameliorates murine colitis through regulation of intravascular monocyte behaviors in murine colitis models. FKN expression was detectable in vascular endothelium and CX3CR1+ macrophages accumulated in the mucosal lamina propria and submucosa of the inflamed colons. CD115+ monocytes tethered to the venous endothelium and expressed pro-inflammatory mediators. The anti-FKN mAb improved colitis symptoms, markedly reduced pro-inflammatory factors in the colon, maintained blood vessel integrity and reduced tethered monocytes in the inflamed veins. Intravital imaging revealed that CD115+Gr-1low/- monocytes crawled on the apical surfaces of venous endothelium, and anti-FKN mAb rapidly dislodged the crawling monocytes and inhibited their patrolling behavior. These findings suggest that the FKN-CX3CR1 axis triggers the patrolling behavior of crawling monocytes on the venous endothelium of inflamed colons, and accelerates the subsequent leukocyte activation and infiltration by locally producing inflammatory cytokines and chemokines. The mAb also ameliorated symptoms in another IBD model, T-cell-transferred colitis. Blocking the FKN-CX3CR1 axis with an anti-FKN mAb considerably inhibits the colitis-triggered inflammatory cascades, which may be an alternative strategy to treat IBD.

Topics: Administration, Rectal; Animals; Antibodies, Monoclonal; Chemokine CX3CL1; Colitis; CX3C Chemokine Receptor 1; Female; Humans; Male; Mice; Mice, Inbred BALB C; Monocytes; Oxazoles

Topics: Animals; Apoptosis; Artemisinins; Colitis; Disease Models, Animal; Enzyme Induction; Heme Oxygenase-1; Inflammatory Bowel Diseases; Lymphocyte Activation; Lymphocyte Subsets; Mice; Oxazolone; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Trinitrobenzenesulfonic Acid

Adenosine represents a powerful modulating factor, which has been shown to orchestrate the scope, duration, and remission of the inflammatory response through the activation of four specific receptors, classified as A

Topics: Adenosine A2 Receptor Agonists; Adjuvants, Immunologic; Animals; Anti-Inflammatory Agents, Non-Steroidal; Colitis; Colon; Disease Models, Animal; Furans; Male; Oxazolone; Rats; Rats, Sprague-Dawley

Although previous studies have suggested that appendix seems to be involved in the colitis, the role of this in the pathogenesis remains unclear. In this study, we assessed the importance of appendiceal lymphoid follicles, specifically the cecal patches (CP) in mice, using an experimental colitis model. Treatment with oxazolone resulted in ulcerations particularly at CP with follicular expansion as well as colitis. The colitis was attenuated by either appendectomy or the absence of mature B cells. We therefore established an intravital imaging system accompanied by the fluorescence resonance energy transfer technology to analyze the dynamic immune response of CP B cells. Our observation revealed frequent Ca

Topics: Animals; Appendix; B-Lymphocytes; Calcium Signaling; Cecum; Colitis; Colon; Disease Models, Animal; Humans; Intravital Microscopy; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Oxazolone; Tertiary Lymphoid Structures

Ulcerative colitis (UC) patients exhibit elevated histamine, but how histamine exacerbates disease is unclear as targeting histamine 1 receptor (H1R) or H2R is clinically ineffective. We hypothesized that histamine functioned instead through the other colon-expressed histamine receptor, H4R. In humans, UC patient biopsies exhibited increased H4R RNA and protein expression over control tissue, and immunohistochemistry showed that H4R was in proximity to immunopathogenic myeloperoxidase-positive neutrophils. To characterize this association further, we employed both the oxazolone (Ox)- and dextran sulfate sodium (DSS)-induced experimental colitis mouse models and also found upregulated H4R expression. Mast cell (MC)-derived histamine and H4R drove experimental colitis, as H4R

Topics: Adolescent; Adult; Aged; Animals; Cells, Cultured; Colitis; Colitis, Ulcerative; Colon; Dextran Sulfate; Disease Models, Animal; Female; Histamine; Histidine Decarboxylase; Humans; Intestinal Mucosa; Male; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Neutrophil Infiltration; Oxazolone; Receptors, Histamine H4; Young Adult

Inflammatory priming of immune cells in early life may optimize the response to a subsequent inflammatory challenge later in life. To prime the immune cells in the gut in vivo through a short inflammatory insult, we administered a low dose of dextran sulfate sodium (DSS) to 5-weeks-old BALB/c mice in the drinking water. We hypothesized that DSS-primed mice would show decreased inflammation and difference in immunological profiling, when subjected to presensitizing and oxazolone-induced colitis by rectal instillation at 9 weeks compared to non-DSS-primed control mice. In fact, this low-dose DSS priming apparently decreased the acute inflammation, as colitis scores along with IFNγ, IL-1ß, and IL-4 were significantly decreased with the same tendency for IL-5, TNFα, and IL-2 on day 3 post-induction compared to control mice. On day 7, both DSS-primed and control mice had significantly higher numbers of FoxP3

Topics: Adjuvants, Immunologic; Animals; Colitis; Colon; Dextran Sulfate; Inflammation; Inflammatory Bowel Diseases; Lymphocytes; Mice; Oxazolone; T-Lymphocytes, Regulatory

Oxazolone-induced colitis has been frequently used in literature as a model of IBD, but insights into the underlying immune response and pathological features are surprisingly still very limited. Vagus nerve stimulation (VNS) has proven to be effective in innate and Th1/Th17 predominant inflammatory models, including pre-clinical models of colitis, however to what extent VNS is also effective in ameliorating Th2-driven colitis remains to be studied. In the present study, we therefore further characterized the immune response in oxazolone-induced colitis and investigated the potential therapeutic effect of VNS.. Colitis was induced in Balb/c mice by cutaneous sensitization with 3% oxazolone followed by intracolonic administration of 1% oxazolone 7 days later. To evaluate the effect of VNS on the development of Th2-driven colitis, VNS and sham-treated mice were challenged with 1% oxazolone.. Intracolonic oxazolone administration resulted in a severe destruction of the colonic mucosa and a rapid drop in body temperature leading to a 65% mortality rate at day 5. Severe infiltration of neutrophils and monocytes was detected 6h after oxazolone administration which was associated with a Th2-type inflammatory response. VNS significantly improved survival rate which correlated with decreased levels of HMGB1 and reduced colonic (il6 and cxcl1 mRNA) and serum cytokine levels (IL-6, TNFα and CXCL1) compared to sham treated mice.. Oxazolone-induced colitis rather represents a model of sepsis and, at best, may resemble a severe type of ulcerative colitis, associated with early and severe mucosal damage and a high mortality rate. VNS reduces colonic inflammation and improves survival in this model, supporting the anti-inflammatory properties of VNS, even in an aggressive model as oxazolone-induced colitis.

Topics: Animals; Colitis; Cytokines; Disease Models, Animal; Female; Hypothermia; Inflammation; Intestinal Mucosa; Mice, Inbred BALB C; Natural Killer T-Cells; Oxazolone; Survival Analysis; Vagus Nerve Stimulation

Activated invariant natural killer T (iNKT) cells rapidly produce large amounts of cytokines, but how cytokine mRNAs are induced, stabilized and mobilized following iNKT activation is still unclear. Here we show that an endoplasmic reticulum stress sensor, inositol-requiring enzyme 1α (IRE1α), links key cellular processes required for iNKT cell effector functions in specific iNKT subsets, in which TCR-dependent activation of IRE1α is associated with downstream activation of p38 MAPK and the stabilization of preformed cytokine mRNAs. Importantly, genetic deletion of IRE1α in iNKT cells reduces cytokine production and protects mice from oxazolone colitis. We therefore propose that an IRE1α-dependent signaling cascade couples constitutive cytokine mRNA expression to the rapid induction of cytokine secretion and effector functions in activated iNKT cells.

Topics: Animals; Cells, Cultured; Colitis; Cytokines; Endoplasmic Reticulum Stress; Endoribonucleases; Gene Deletion; Lymphocyte Activation; Mice; Mice, Knockout; Natural Killer T-Cells; Oxazolone; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; RNA, Messenger; Signal Transduction; Unfolded Protein Response

Indigo naturalis (IND) is an herbal medicine that has been used as an anti-inflammatory agent to treat diseases including dermatitis and inflammatory bowel disease in China. However, the mechanism by which IND exerts its immunomodulatory effect is not well understood.. A murine model of dermatitis and inflammatory bowel disease, both induced by oxazolone (OXA), was treated with IND. The severity of dermatitis was evaluated based on ear thickness measurements and histological scoring. The severity of colitis was evaluated by measuring body weight, histological scoring, and endoscopic scoring. The expression of inflammatory cytokines in ear and colon tissue was evaluated using real-time PCR. 16S rRNA DNA sequencing of feces from OXA-induced colitis mice was performed before and after IND treatment. The effects of IND on OXA-induced colitis were also evaluated after depleting the gut flora with antibiotics to test whether alteration of the gut flora by IND influenced the course of intestinal inflammation in this model.. IND treatment ameliorated OXA dermatitis with a reduction in IL-4 and eosinophil recruitment. However, OXA colitis was significantly aggravated in spite of a reduction in intestinal IL-13, a pivotal cytokine in the induction of the colitis. It was found that IND dramatically altered the gut flora and IND no longer exacerbated colitis when colitis was induced after gut flora depletion.. Our data suggest that IND could modify the inflammatory immune response in multiple ways, either directly (i.e., modification of the allergic immune cell activity) or indirectly (i.e., alteration of commensal compositions).

Topics: Adjuvants, Immunologic; Animals; Colitis; Colon; Dermatitis, Allergic Contact; DNA, Bacterial; Feces; Gastrointestinal Microbiome; Indigo Carmine; Interleukin-13; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Oxazolone; Phytotherapy; Skin

Inflammatory bowel diseases (IBDs) result in diarrhea and abdominal pain with further potential complications such as tissue fibrosis and stenosis. Animal models help in understanding the immunopathogenesis of IBDs and in the design of novel therapeutic concepts. Here we present an updated version of a protocol we published in 2007 for key models of acute and chronic forms of colitis induced by 2,4,6-trinitro-benzene sulfonic acid (TNBS), oxazolone and dextran sulfate sodium (DSS). This protocol update describes an adaptation of the existing protocol that modifies the technique. This protocol has been used to generate improved mouse models that better reflect the nature of IBDs in humans. In TNBS and oxazolone colitis models, topical administration of hapten reagents results in T-cell-mediated immunity against haptenized proteins and luminal antigens. By contrast, to generate DSS colitis models, mice orally receive DSS, causing death of epithelial cells, compromising barrier function and causing subsequent inflammation. The analysis of the acute colitis models can be performed within 1-2 weeks, whereas that of the chronic models may take 2-4 months. The strengths of the acute models are that they are based on the analysis of short-lasting barrier alterations, innate immune effects and flares. The advantages of the chronic models are that they may offer better insight into adaptive immunity and complications such as neoplasia and tissue fibrosis. The protocol requires basic skills in laboratory animal research.

Topics: Animals; Chronic Disease; Colitis; Dextran Sulfate; Disease Models, Animal; Mice; Oxazolone; Trinitrobenzenesulfonic Acid

GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice.. We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n = 61) or ulcerative colitis (UC, n = 74) or from patients without inflammatory bowel diseases (n = 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated and analyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by in vivo imaging (endoscopy) of mice.. Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA, compared with mice given a control DNAzyme. Mice given hgd40 did not develop colitis after administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid; lamina propria cells from these mice expressed lower levels of IL6, IL9, and IL13 than cells from mice given the control DNAzyme. Mini-endoscopic images revealed that hgd40 and anti-TNF reduced colon inflammation over 3 days; hgd40 reduced colitis in TNFR double-knockout mice.. Levels of GATA3 are increased in patients with UC and correlate with production of inflammatory cytokines in mice and humans. A DNAzyme that prevents expression of GATA3 reduces colitis in mice, independently of TNF, and reduces levels of cytokines in the colon. This DNAzyme might be developed for treatment of patients with UC.

Topics: Administration, Rectal; Adolescent; Adult; Aged; Animals; Case-Control Studies; Child; Colitis; Colitis, Ulcerative; Colon; Crohn Disease; Cytokines; Disease Models, Animal; DNA, Catalytic; Female; GATA3 Transcription Factor; Humans; Intestinal Mucosa; Male; Mice; Mice, Knockout; Middle Aged; Oxazolone; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; RNA, Messenger; T-Lymphocytes; Trinitrobenzenesulfonic Acid; Young Adult

Inflammatory bowel disease results from chronic dysregulation of the mucosal immune system and aberrant activation of both the innate and adaptive immune responses. IL-19 is a member of the IL-10 family, and IL-10 plays an important role in inflammatory bowel disease. We have previously shown that IL-19 knockout mice are more susceptible to innate-mediated colitis. Next, we ask whether IL-19 contributes to T cells-mediated colitis. Here, we investigated the role of IL-19 in a mouse model of Th2 cell-mediated colitis. Inflammatory responses in IL-19-deficient mice were assessed using a Th2-mediated colitis induced by oxazolone. The colitis was evaluated by analyzing the body weight loss and histology of the colon. Lymph node cells were cultured in vitro to determine cytokine production. IL-19 knockout mice exacerbated oxazolone-induced colitis by stimulating the transport of inflammatory cells into the colon, and by increasing IgE production and the number of circulating eosinophil. The exacerbation of oxazolone-induced colonic inflammation following IL-19 knockout mice was accompanied by an increased production of IL-4 and IL-9, but no changes in the expression of IL-5 and IL-13 in lymph node cells. IL-19 plays an anti-inflammatory role in the Th2-mediated colitis model, suggesting that IL-19 may represent a potential therapeutic target for reducing colonic inflammation.

Topics: Animals; Cells, Cultured; Colitis; Colon; Disease Models, Animal; Genetic Predisposition to Disease; Inflammation Mediators; Interleukin-10; Interleukins; Lymph Nodes; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; Phenotype; Protective Factors; Th2 Cells; Time Factors

P2X7 receptor activation contributes to inflammation development in different pathologies. We previously reported that the P2X7 receptor is over-expressed in the gut mucosa of patients with inflammatory bowel disease, and that P2X7 inhibition protects against chemically induced colitis. Here, we investigated in detail the role of the P2X7 receptor in inflammatory bowel disease development, by treating P2X7 knockout (KO) and WT mice with two different (and established) colitis inductors. P2X7 KO mice were protected against gut inflammation induced by 2,4,6-trinitrobenzenesulfonic acid or oxazolone, with no weight loss or gut histological alterations after treatment. P2X7 receptor knockout induced regulatory T cell accumulation in the colon, as evaluated by qRT-PCR for FoxP3 expression and immunostaining for CD90/CD45RB

Topics: Animals; Colitis; Female; Immunity, Mucosal; Inflammation; Inflammatory Bowel Diseases; Intestinal Mucosa; Mice; Mice, Knockout; Oxazolone; Receptors, Purinergic P2X7; T-Lymphocytes; Trinitrobenzenesulfonic Acid

Mast cells in the gut play an important role in the innate and adaptive immune responses that are relevant to human inflammatory bowel disease. However, the contribution of mast cells to the development of inflammatory bowel disease is not well understood. This study aimed to determine the role of mast cells in oxazolone-induced colitis and to explore whether the mast cell membrane stabiliser tranilast could ameliorate colonic inflammation.. Wild-type rats and mast cell-deficient rats were sensitised and challenged with oxazolone, then treated with tranilast after challenge. Controls were treated with saline.. Mast cell-deficient rats presented a weak response to oxazolone, while wild-type rats showed severe ulcerative colitis after stimulation with oxazolone. The mast cell-deficient rats model had a significantly lower disease activity index score than wild-type rats model (1.8±1.64 vs. 8.3±0.58 respectively; P<0.01). Tranilast could reduce the secretion of cytokines, immunoglobulins and myeloperoxidase activity in tranilast treatment groups compared with the model group. The number of mast cells in the wild-type model was higher than in the other groups. There was no significant change in mast cell-deficient rats.. Mast cells play an important role in oxazolone-induced colitis. The mast cell membrane stabiliser tranilast can ameliorate oxazolone-induced colitis via a mast cell-dependent pathway.

Topics: Adjuvants, Immunologic; Animals; Colitis; Colon; Cytokines; Disease Models, Animal; Histamine H1 Antagonists; Inflammatory Bowel Diseases; Interleukin-13; Interleukin-33; Interleukin-6; Mast Cells; ortho-Aminobenzoates; Oxazolone; Rats

Elucidate the mechanism of action of the small molecule inhibitor of protein binding to glycosaminoglycans, RX-111 and assay its anti-inflammatory activity in animal models of inflammatory disease.. The glycosaminoglycan, heparin, was used in the mechanism of action study of RX-111. Human T lymphocytes and umbilical vein endothelial cells were used to assay the in vitro activity of RX-111. Mouse and rat models of disease were used to assay the anti-inflammatory activity of RX-111 in vivo.. Circular dichroism and UV/Vis absorption spectroscopy were used to study the binding of RX-111 to the glycosaminoglycan, heparin. T lymphocyte rolling on endothelial cells under shear flow was used to assay RX-111 activity in vitro. Delayed-type hypersensitivity (DTH) and tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in mice and experimental autoimmune encephalomyelitis (EAE) in rats were used to assay anti-inflammatory activity of RX-111 in vivo.. RX-111 was shown to bind directly to heparin. It inhibited leukocyte rolling on endothelial cells under shear flow and reduced inflammation in the mouse model of DTH. RX-111 was efficacious in the mouse model of inflammatory bowel disease, TNBS-induced colitis and the rat model of multiple sclerosis, EAE.. RX-111 exercises its broad spectrum anti-inflammatory activity by a singular mechanism of action, inhibition of protein binding to the cell surface GAG, heparan sulfate. RX-111 and related thieno[2,3-c]pyridine derivatives are potential therapeutics for the treatment of inflammatory and autoimmune diseases.

Topics: Animals; Anti-Inflammatory Agents; Cell Line, Tumor; Colitis; Encephalomyelitis, Autoimmune, Experimental; Heparitin Sulfate; Human Umbilical Vein Endothelial Cells; Humans; Hypersensitivity, Delayed; Leukocyte Rolling; Male; Mice, Inbred BALB C; Myelin Basic Protein; Oxazolone; Pyridines; Rats; Rats, Inbred Lew; T-Lymphocytes; Thiophenes; Treatment Outcome; Trinitrobenzenesulfonic Acid

Nerol is a natural monoterpene with antinociceptive and anti-inflammatory properties. Its possible beneficial effects in ulcerative colitis and its corresponding mechanism of action have not been determined to date. The aim of this study was to investigate whether nerol prevents the appearance of pathological markers and hyperalgesia in oxazolone-induced colitis, and protects against gastric damage produced by ethanol. The experimental design included groups of oxazolone-treated mice receiving nerol at 10-300 mg/kg, p.o., or a reference drug (sulfasalazine, 100 mg/kg, p.o.) compared to sham and untreated groups. Gastric damage was evaluated in the absolute ethanol-induced ulcer model in rats. Variables measured in animals with oxazolone-induced colitis included weight loss, stool consistency and macroscopic colon damage; mechanical nociception was determined by the use of von Frey filaments, whereas levels of inflammatory cytokines were assessed by enzyme-linked immunosorbent assay. Nerol (30-300 mg/kg, p.o.) prevented or significantly decreased the pathological alterations observed in the oxazolone- induced colitis model. It also showed antinociceptive effects and reduced the increased levels of inflammatory cytokines (IL-13 and TNF-α). Gastric damage was also prevented starting at 10 mg/kg, p.o. In conclusion, our results provide evidence for a beneficial effect of nerol after colitis induction involving tissue protection, antinociception and modulation of the immunological system, suggesting the therapeutic potential of this monoterpene as a novel alternative in controlling ulcerative colitis.

Topics: Analgesics; Animals; Biomarkers; Colitis; Colon; Cytokines; Cytoprotection; Gene Expression Regulation; Male; Mice; Mice, Inbred BALB C; Oxazolone; Rats

IL-1β and IL-17A are two cytokines with strong proinflammatory activities and are now known to be involved in a number of chronic inflammatory disorders. High-mobility group box 1 (HMGB1) is a nuclear protein regulating the expression of these proinflammatory cytokines. The NLRP3 inflammasome promotes the maturation of the IL-1β and its activation has been shown as a critical mechanism in the pathogenesis of inflammatory bowel disease (IBD). However, underlying mechanisms to modulate their production in IBD are still unclear. The aim of this study was to investigate the expression levels of mRNA for the NLRP3 inflammasome, HMGB1 and proinflammatory cytokines, IL-1β, IL-17A in the inflamed colon of rats with experimental oxazolone-induced colitis. Experiments were carried out on male wistar rats. IL-1β, IL-17A, HMGB1 and NLRP3 inflammasome mRNA expression were analyzed by real-time reverse transcriptase-polymerase chain reaction. Our results indicated that the expression levels of IL-1β, IL-17A, NLRP3 and HMGB1 were elevated in the inflamed colon of rats with oxazolone-induced colitis.

Topics: Animals; Colitis; Colon; Cytokines; Disease Models, Animal; HMGB1 Protein; Inflammation Mediators; Interleukin-17; Interleukin-1beta; Male; NLR Family, Pyrin Domain-Containing 3 Protein; Oxazolone; Rats, Wistar; RNA, Messenger; Up-Regulation

Carrageenan is a traditional ingredient that has been widely used in the food industry. In the present study, we propose a hypothesis that carrageenan is a conditional inflammatory agent. When the intestinal tract is in an "unhealthy" state such as that during bacterial infection or acute inflammation, carrageenan can synergistically enhance the inflammatory response.. BALB/C mice received κ-carrageenan via intragastric administration prior to the induction of oxazolone colitis. Weight changes, survival rate, histologic change, secretion of inflammatory cytokines, ratio of regulatory T cells (Tregs) in peripheral blood, and expression of genes and proteins involved in inflammation and cell proliferation in the colonic mucosa were examined.. Intragastric administration of κ-carrageenan to BALB/c mice prior to the induction of oxazolone colitis resulted in an aggravation of body weight loss, a decrease in the survival ratio, aggravation of colonic inflammation, and decrease in the ratio of CD4 + CD25+/CD4+. The secretion of interleukin-4 (IL-4), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) also significantly increased after κ-carrageenan administration. κ-Carrageenan, together with oxazolone, suppressed the expression of forkhead box p3 (FOXp3) and increased the expression of Toll-like receptor 4 (TLR4), Nuclear factor-κB (NF-κB), and proliferating cell nuclear antigen in the colonic mucosa. These results were confirmed by qRT-PCR and western blot analyses at the molecular and protein levels, respectively.. κ-Carrageenan aggravated oxazolone-induced intestinal inflammation in BALB/c mice. This effect is associated with an activation of the TLR4-NF-κB pathway, a decreased ratio of Tregs, and the induction of Th2-dependent immune responses.

Topics: Adjuvants, Immunologic; Animals; Blotting, Western; Carrageenan; Cell Proliferation; Colitis; Colon; Cytokines; Drug Synergism; Forkhead Transcription Factors; Inflammation; Interleukin-10; Interleukin-4; Interleukin-6; Intestinal Mucosa; Mice; Mice, Inbred BALB C; NF-kappa B; Oxazolone; Proliferating Cell Nuclear Antigen; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Inflammatory bowel diseases (IBDs) are chronic inflammatory gastrointestinal disorders caused by a dysregulated mucosal immune response and epithelial barrier disruption. Conventional treatment of IBD is currently limited to overcoming patient symptoms and is often associated with severe adverse effects from the drugs used. Modified Pulsatilla decoction has been used previously to treat ulcerative colitis (UC) in clinical practice in China, however, the underlying mechanism in the treatment of UC remains to be elucidated. In the present study, the efficiency and mechanisms of modified Pulsatilla decoction in the treatment of oxazolone‑induced colitis were investigated. Assessment of clinical colitis and histological examination found that the administration of modified Pulsatilla decoction attenuated the severity of oxazolone‑induced colitis in mice. Measurement of cytokine concentration, western blotting and reverse transcription‑quantitative polymerase chain reaction demonstrated modified Pulsatilla decoction treatment significantly reduced the secretion of pro‑inflammatory cytokines and restored alterations in tight junction proteins in the colon tissues. In addition, modified Pulsatilla decoction suppressed the activation of the nuclear factor‑κB signaling pathway. Thus, the findings of the present study demonstrated that modified Pulsatilla decoction offers an effective therapeutic approach for the treatment of IBD and revealed the underlying mechanisms of action offered by modified Pulsatilla decoction.

Topics: Animals; Colitis; Cytokines; Disease Models, Animal; Drugs, Chinese Herbal; Inflammation Mediators; Inflammatory Bowel Diseases; Intestinal Mucosa; Male; Mice; NF-kappa B; Oxazolone; Plant Extracts; Pulsatilla; Signal Transduction; Tight Junction Proteins

The inflammasomes induce maturation of pro-interleukin-1β (IL-1β) and pro-IL-18. We investigated roles of the NLRP3 inflammasome in the pathogenesis of ulcerative colitis (UC). After induction of oxazolone-induced colitis, a mouse UC model, colonic tissues were assayed for inflammatory mediators. Histological studies were performed on inflamed colonic tissue from mice and UC patients. Histological severity of murine colitis peaked on day 1, accompanied by an increase in the expression of Th2 cytokines including IL-4 and IL-13. Oxazolone treatment stimulated maturation of pro-caspase-1 and pro-IL-1β, while it reduced IL-18 expression. Either exogenous IL-1β or IL-18 ameliorated the colitis with or without reduction in Th2 cytokine expression, respectively. Induction of colitis decreased MUC2 expression, which was reversed by administration of IL-18, but not IL-1β. Compared to wild-type mice, NLRP3

Topics: Adult; Aged; Aged, 80 and over; Animals; Caspase 1; Colitis; Colitis, Ulcerative; Cytokines; Disease Models, Animal; Female; Gene Expression Regulation; Gene Knockout Techniques; Humans; Inflammasomes; Macrophages; Male; Mice; Middle Aged; NLR Family, Pyrin Domain-Containing 3 Protein; Oxazolone; Severity of Illness Index; Young Adult

Pulverized konjac glucomannan (PKGM) is a natural biologically active compound extracted from konjac, a Japanese traditional food. In the present study, we investigated the role of PKGM in intestinal immunity in a mouse model of oxazolone (OXA)-induced colitis.. C57BL/6(B6) mice were fed PKGM or control food from 2 weeks before the induction of OXA colitis. Body weight change, colon length, and histological change in the colon were examined. The mononuclear cells were purified from colon and stimulated with PMA/ionomycin. The levels of TNF-α, interferon (IFN)-γ, interleukin (IL)-4, and IL-13 from the supernatant were measured by ELISA.. Oral administration of PKGM prevented the body weight loss and shortening of colon length associated with OXA-induced colitis. Histological analysis revealed that the colonic inflammation was improved by the administration of PKGM. The levels of IL-4 and IL-13, the critical inflammatory cytokines in OXA colitis, derived from mononuclear cells from the lamina propria of the colon were significantly suppressed by PKGM administration. PKGM-fed mice showed a significantly lower IL-4/IFN-γ ratio in the colonic lamina propria compared with that in control-fed mice. Fluorescence-activated cell sorting analysis revealed that natural killer (NK) 1.1(+) T cells in the liver were significantly decreased in PKGM-fed mice. Finally, the preventive role of PKGM in OXA-induced colitis was not observed in invariant natural killer T cell-deficient mice.. PKGM ameliorated OXA-induced colitis in mice. This effect is associated with a decreased population of NK1.1(+) T cells and induction of Th1-polarized immune responses.

Topics: Adjuvants, Immunologic; Animals; Colitis; Colon; Diet; Female; Food Handling; Interferon-gamma; Interleukin-13; Interleukin-4; Japan; Mannans; Mice; Mice, Inbred C57BL; Mice, Knockout; Natural Killer T-Cells; Oxazolone; Tumor Necrosis Factor-alpha; Weight Loss

Helminth parasites provoke multicellular immune responses in their hosts that can suppress concomitant disease. The gut lumen-dwelling tapeworm Hymenolepis diminuta, unlike other parasites assessed as helminth therapy, causes no host tissue damage while potently suppressing murine colitis. With the goal of harnessing the immunomodulatory capacity of infection with H. diminuta, we assessed the putative generation of anti-colitic regulatory B cells following H. diminuta infection. Splenic CD19(+) B cells isolated from mice infected 7 [HdBc(7(d))] and 14 d (but not 3 d) previously with H. diminuta and transferred to naive mice significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-, oxazolone-, and dextran-sodium sulfate-induced colitis. Mechanistic studies with the DNBS model, revealed the anti-colitic HdBc(7(d)) was within the follicular B cell population and its phenotype was not dependent on IL-4 or IL-10. The HdBc(7(d)) were not characterized by increased expression of CD1d, CD5, CD23, or IL-10 production, but did spontaneously, and upon LPS plus anti-CD40 stimulation, produce more TGF-β than CD19(+) B cells from controls. DNBS-induced colitis in RAG1(-/-) mice was inhibited by administration of HdBc(7(d)), indicating a lack of a requirement for T and B cells in the recipient; however, depletion of macrophages in recipient mice abrogated the anti-colitic effect of HdBc(7(d)). Thus, in response to H. diminuta, a putatively unique splenic CD19(+) B cell with a functional immunoregulatory program is generated that promotes the suppression of colitis dominated by TH1, TH2, or TH1-plus-TH2 events, and may do so via the synthesis of TGF-β and the generation of, or cooperation with, a regulatory macrophage.

Topics: Animals; Antigens, CD19; Antigens, CD1d; B-Lymphocytes; Benzenesulfonates; CD40 Antigens; CD5 Antigens; Colitis; Dextran Sulfate; Homeodomain Proteins; Hymenolepiasis; Hymenolepis diminuta; Immunomodulation; Immunotherapy; Interleukin-10; Interleukin-4; Lipopolysaccharides; Macrophages; Male; Mast Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; Receptors, IgE; Th1 Cells; Th2 Cells; Transforming Growth Factor beta

IL-33, a member of the IL-1 cytokine family that signals through ST2, is upregulated in ulcerative colitis (UC); however, the role of IL-33 in colitis remains unclear. IL-33 augments type 2 immune responses, which have been implicated in UC pathogenesis. We sought to determine the role of IL-33 signaling in oxazolone (OXA) colitis, a type 2 cytokine-mediated murine model of UC.. Colon mucosal IL-33 expression was compared between pediatric and adult UC and non-IBD patients using immunohistochemistry and real-time PCR. OXA colitis was induced in WT, IL-33, and ST2 mice, and histopathology, cytokine levels, and goblet cells were assessed. Transepithelial resistance was measured across IL-33-treated T84 cell monolayers.. Colon mucosal IL-33 was increased in pediatric patients with active UC and in OXA colitis. IL-33 and ST2 OXA mice exhibited increased disease severity compared with WT OXA mice. OXA induced a mixed mucosal cytokine response, but few differences were observed between OXA WT and IL-33 or ST2 mice. Goblet cells were significantly decreased in IL-33 and ST2 OXA compared with WT OXA mice. IL-33 augmented transepithelial resistance in T84 cells, and this effect was blocked by the ERK1/2 inhibitor PD98,059.. OXA colitis is exacerbated in IL-33 and ST2 mice. Increased mucosal IL-33 in human UC and murine colitis may be a homeostatic response to limit inflammation, potentially through effects on epithelial barrier function. Further investigation of IL-33 protective mechanisms would inform the development of novel therapeutic approaches.

Topics: Adult; Animals; Child; Colitis; Colitis, Ulcerative; Colon; Cytokines; Goblet Cells; Humans; Immunohistochemistry; Interleukin-1 Receptor-Like 1 Protein; Interleukin-33; Intestinal Mucosa; Intestines; Mice; Oxazolone; Real-Time Polymerase Chain Reaction; Receptors, Interleukin; Signal Transduction; Up-Regulation

We studied the expression of AhR and ATG16L1 protein in experimental oxazolone-induced colitis in rats and anti-inflammatory action of recombinant antagonist of IL-1 receptors (ARIL-1) and simvastatin. The immunopositive cells were determined using an indirect immunofluorescence technique with using a monoclonal rat antibody. It has been established that development of colitis was accompanied by an increase of total number of ATG16L1-lymphocytes (by 30%, P < 0.05) in lymphoid structures of the colon. However the amount of AhR(+)-lymphocytes has not changed. At the same time has increased the concentration of ATG16L1 protein (by 4-11%, P < 0.05) in immunopositive cells. Administration of simvastatin and ARIL-1 during the development of experimental pathology was accompanied by decrease of total number of AhR(+) (by 24-38%, P < 0.05) and ATG16L1(+)-lymphocytes (by 43% - 2 fold, P < 0.05) in the colon.

Topics: Animals; Anti-Inflammatory Agents; Antibodies, Monoclonal; Autophagy-Related Proteins; Carrier Proteins; Colitis; Colon; Gene Expression Regulation; Immunophenotyping; Lymphocyte Count; Lymphocytes; Male; Oxazolone; Peptides; Rats; Rats, Wistar; Receptors, Aryl Hydrocarbon; Receptors, Interleukin-1; Recombinant Proteins; Simvastatin

Coevolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Here, we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host's endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood, and hosts are protected against experimental iNKT cell-mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids-exemplified by an isolated peak (MW = 717.6) called GSL-Bf717-reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive.

Topics: Animals; Animals, Newborn; Bacteroides fragilis; Cell Proliferation; Colitis; Colon; Glycosphingolipids; Mice; Mice, Inbred C57BL; Natural Killer T-Cells; Oxazolone

Tetramethylpyrazine (TMP) is an effective Chinese plant-derived medicine for colitis in the clinic, but the underlying molecular mechanisms of its use remain poorly understood. The purpose of this study was to investigate the mechanisms involved in its therapeutic action.. A colitis mouse model was induced by oxazolone enema. TMP was administered at 80 mg/kg/day and sulphasalazine (SASP) was used as positive control and administered at 100 mg/kg/day for the treatment of colitis. On the fourth day after enema, mice were sacrificed. The inflammatory response was assessed by the disease activity index and histology. Colon mucosa was isolated and biochemically analyzed. In addition, in vitro studies were performed to evaluate the activity of TMP in Caco-2 cells.. Our results showed that TMP improved the colonic inflammatory status as evidenced by histological findings, as well as SASP. These effects were associated with a decrease in nucleus translocation of NF-κB. Paired with this inhibitive activity, there was a decrease in downstream signaling, such as C-MYC, iNOS and COX-2. In vitro assays revealed that TMP inhibited NF-κB translocation and its downstream production of inflammatory factors, such as TNF-α, IL-6 and IL-8, and that ROS production that was induced by LPS in Caco-2 cells.. TMP improved the colitis induced by oxazoline, and its activity was associated with inhibition of NF-κB translocation, and subsequent inhibition of pro-inflammatory factor production and oxidative stress.

Topics: Animals; Caco-2 Cells; Colitis; Cytokines; Disease Models, Animal; Humans; Inflammation Mediators; Intestinal Mucosa; Male; Mice; NF-kappa B; Oxazolone; Oxidative Stress; Phytotherapy; Pyrazines; Signal Transduction

The mechanisms by which mucosal homeostasis is maintained are of central importance to inflammatory bowel disease. Critical to these processes is the intestinal epithelial cell (IEC), which regulates immune responses at the interface between the commensal microbiota and the host. CD1d presents self and microbial lipid antigens to natural killer T (NKT) cells, which are involved in the pathogenesis of colitis in animal models and human inflammatory bowel disease. As CD1d crosslinking on model IECs results in the production of the important regulatory cytokine interleukin (IL)-10 (ref. 9), decreased epithelial CD1d expression--as observed in inflammatory bowel disease--may contribute substantially to intestinal inflammation. Here we show in mice that whereas bone-marrow-derived CD1d signals contribute to NKT-cell-mediated intestinal inflammation, engagement of epithelial CD1d elicits protective effects through the activation of STAT3 and STAT3-dependent transcription of IL-10, heat shock protein 110 (HSP110; also known as HSP105), and CD1d itself. All of these epithelial elements are critically involved in controlling CD1d-mediated intestinal inflammation. This is demonstrated by severe NKT-cell-mediated colitis upon IEC-specific deletion of IL-10, CD1d, and its critical regulator microsomal triglyceride transfer protein (MTP), as well as deletion of HSP110 in the radioresistant compartment. Our studies thus uncover a novel pathway of IEC-dependent regulation of mucosal homeostasis and highlight a critical role of IL-10 in the intestinal epithelium, with broad implications for diseases such as inflammatory bowel disease.

Topics: Animals; Antigens, CD1d; Carrier Proteins; Colitis; Disease Models, Animal; Epithelial Cells; Female; HSP110 Heat-Shock Proteins; Humans; Immunity, Mucosal; Inflammation; Inflammatory Bowel Diseases; Interleukin-10; Intestinal Mucosa; Male; Mice; Natural Killer T-Cells; Oxazolone; STAT3 Transcription Factor

Natural killer T (NKT) cells share phenotypic and functional properties with both conventional natural killer cells and T cells. These cells might have an important role in the pathogenesis of ulcerative colitis (UC). The interaction of chemokine ligand 25 (CCL25) with chemokine receptor 9 (CCR9) is involved in gut-specific migration of leukocytes and induces regulatory T cells (Tregs) to migrate to the intestine in chronic ileitis.. In UC patients, NKT receptor CD161, CCL25, and CCR9 expression levels were evaluated by qRT-PCR. A murine model of oxazolone-induced colitis was induced in BALB/c mice. The mRNA levels of NK1.1, CCL25 and CCR9, and pro-inflammatory cytokines in mice were evaluated. The CCR9 expression on Type I or invariant NKT (iNKT) cells, and the iNKT cells chemotaxis are observed according to flow cytometry. NKT receptor CD161, CCL25 and CCR9 expression levels were significantly increased in UC patients. And, the mRNA expression levels of NK1.1, CCL25 and CCR9 were increased in oxazolone-induced colitis in mice. The production of pro-inflammatory cytokines was significantly increased, especially interleukin 4 (IL-4), IL-10 and IL-13. We observed significantly increased CCR9 expression on iNKT cells. Furthermore, we found an increased iNKT population and enhanced chemotaxis during oxazolone-induced colitis.. Our study suggests that CCL25/CCR9 interactions may promote the induction and function of iNKT cells during oxazolone-induced colitis. These findings may have important implications for UC treatment and suggest a role for CCR9 inhibitors.

Topics: Adult; Aged; Animals; Blotting, Western; Cells, Cultured; Chemokines, CC; Colitis; Colitis, Ulcerative; Female; Flow Cytometry; Humans; Male; Mice; Mice, Inbred BALB C; Middle Aged; Natural Killer T-Cells; Oxazolone; Real-Time Polymerase Chain Reaction; Receptors, CCR; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Young Adult

Induction of colitis in mice by administration of oxazolone is mediated by T-helper (Th) 2 cells and has features of human ulcerative colitis. We investigated whether activation of interleukin (IL)-4Rα on T and B cells determines their effector functions and mediates oxazolone-induced colitis.. We studied induction of colitis with oxazolone in wild-type mice and those with CD4(+) T cells that did not express IL-4Rα (Lck(cre)IL-4Rα(-/lox)). We also generated mice with B cells that did not express IL-4Rα (mb1(cre)IL-4Rα(-/lox)) and studied induction of colitis.. Lck(cre)IL-4Rα(-/lox) mice did not develop colitis in response to oxazolone, and their levels of IL-4, IL-13, and immunoglobulin (Ig) E were reduced. Adoptive transfer of naïve, wild-type CD4(+) Th cells depleted of natural killer T cells to Lck(cre)IL-4Rα(-/lox) mice restored their susceptibility to colitis. In contrast, Lck(cre)IL-4Rα(-/lox) mice maintained their protection against colitis when IL-13-deficient CD4(+) T cells were transferred. These findings indicate that development of colitis involves not only natural killer T-cell functions, but also requires IL-13 production by CD4(+) T helper cells. Mb1(cre)IL-4Rα(-/lox) mice, which cannot produce IgE, were also protected against oxazolone-induced colitis. Blocking IgE binding significantly reduced mast cell numbers in colons and protected wild-type BALB/c mice from the onset of colitis.. IL-4 appears to induce CD4(+) Th2 cells to produce IL-13 and B cells to produce IgE, which together mediate oxazolone-induced colitis in mice.

Topics: Adoptive Transfer; Animals; Antibodies, Neutralizing; B-Lymphocytes; Cells, Cultured; Colitis; Colon; Disease Models, Animal; Disease Susceptibility; Immunoglobulin E; Interleukin-13; Interleukin-4; Male; Mast Cells; Mice; Mice, Inbred BALB C; Mice, Knockout; Natural Killer T-Cells; Oxazolone; Receptors, Cell Surface; Signal Transduction; Th2 Cells; Time Factors

Extrinsic sensory neurons play a crucial role in aberrant immune responses in colitis. The activation of peptidergic sensory nerve fibres is accompanied by a release of the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP). SP levels increase whilst CGRP levels decrease in colon specimens from patients with inflammatory bowel disease; thus suggesting the pro- and anti-inflammatory roles, respectively, of these neuropeptides.. Oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one) colitis was induced in wild-type (WT), SP and CGRP knockout ((-/-)) mice. CGRP(-/-) mice were treated with the neurokinin 1-receptor antagonist CP-96345 (CP). The permeability of the mouse colon was evaluated by Evans Blue uptake. Cytokines produced by colonic lamina propria mononuclear cells were measured by ELISA.. Colons of WT, CGRP(-/-) and SP(-/-) mice showed similar tissue architecture and permeability. SP(-/-) mice were protected against oxazolone colitis, whereas CGRP(-/-) showed increased susceptibility to colitis compared to WT mice. SP(-/-) and CP-treated CGRP(-/-) mice showed no significant body weight loss during the period of sickness in contrast to untreated CGRP(-/-) and WT mice. Decreased production of IL-4, IL-5, and IL-13 by colonic lamina propria mononuclear cells of the protected SP(-/-) mice confirms the crucial role of these cytokines in oxazolone colitis.. We demonstrate that the neuropeptides CGRP and SP exert opposing effects in oxazolone colitis and provide further evidence for a prominent neuroimmune association in the gut.

Topics: Animals; Calcitonin Gene-Related Peptide; Colitis; Colon; Cytokines; Enzyme-Linked Immunosorbent Assay; Inflammatory Bowel Diseases; Mice; Mice, Knockout; Oxazolone; Substance P

In inflammatory bowel disease (IBD) the disruption of the intestinal barrier function and the strong presence of immune-related cells like macrophages in inflamed tissue allow the selective accumulation of particulate carrier systems at the site of action. We developed clodronate loaded nanoparticles (ClNP) based on a cationic polymethacrylate (Eudragit RL) using a modified solvent displacement method. Particle diameter of ClNP was around 120nm and dissolution experiments showed that ionic interactions with either the dissolution medium or mucin have to take place to enable complete drug release. In murine experimental colitis in-vivo, myeloperoxidase activity decreased significantly in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-colitis and oxazolone (OXA)-colitis models after treatment with ClNP while free clodronate did not show a mitigating effect. Similarly, alkaline phosphatase could be lowered significantly from 12.5±1.9 to 6.8±2.2ng/mg tissue in TNBS-colitis and from 16.6±6.2 to 11.8±2.7ng/mg tissue in OXA-colitis. In cultured RAW 264.7 cells, only ClNP but not clodronate alone led to a decrease in tumor necrosis factor-alpha and interleukin-6 secretion of the activated macrophages. The therapeutic benefit of ClNP was confirmed in-vivo although it is limited compared to data with other drugs. Cell culture experiments indicated that intracellular delivery of clodronate was necessary to obtain an anti-inflammatory effect.

Topics: Animals; Anti-Inflammatory Agents; Caco-2 Cells; Cell Line; Cell Survival; Clodronic Acid; Colitis; Disease Models, Animal; Humans; Mice; Nanoparticles; Oxazolone; Polymers; Trinitrobenzenesulfonic Acid

A recent paper suggests that reduced exposure to germs results in the expansion of a cell type called natural killer T cells, which predisposes to colitis and asthma. Such a scenario could explain the Hygiene Hypothesis, which has been a puzzle for decades.

Topics: Animals; Asthma; Cell Proliferation; Colitis; Environmental Exposure; Humans; Hygiene Hypothesis; Hypersensitivity; Interleukins; Metagenome; Mice; Natural Killer T-Cells; Oxazolone; Specific Pathogen-Free Organisms

Dysregulation of immune responses in inflammatory bowel diseases (IBD) results in intestinal inflammation and vascular injury while exacerbating systemic disease. CD39 is an ectonucleotidase, expressed by T regulatory cells and dendritic cells, that hydrolyzes extracellular nucleotides to modify those cellular immune responses implicated in IBD. Genetic polymorphisms of CD39 have been linked to Crohn's disease while gene deletion in mice exacerbates dextran sodium sulphate-induced colitis.. The aim of this study was to test how global deletion of CD39 in mice impacts other models of experimental colitis.. Colitis was induced in CD39-null and -wt mice, using trinitrobenzene sulfonic acid (TNBS, 125 mg/kg) administered intrarectally. Oxazolone colitis (1.5% oxazolone in 50% alcohol) was induced in comparable groups. Morphology, clinical and molecular parameters, and FACS analyses of lamina propria mononuclear cells (LPMC) were examined in CD39-null mice. CD39 expression was analyzed in human IBD biopsies.. Paradoxically, TNBS colitis in CD39-null mice was characterized by improved survival, favorable clinical scores, and decreased MPO activity, when compared to wt mice (P < 0.05). LPMC from TNBS colitis contained significantly increased amounts of T-cells (CD3(+) and CD4(+)) and TNF-α mRNA expression were increased over those in CD39 null mice (P < 0.05). In contrast, oxazolone treated CD39-null and wt mice had comparable outcomes. In both ulcerative colitis and Crohn's disease, CD39 is present at high levels in intestinal tissue biopsies.. TNBS colitis was attenuated in CD39-null mice whereas oxazolone-induced colitis was not impacted. Impaired adaptive cellular immune reactivity in the CD39-null environment appears protective in hapten-mediated Th1-type colitis. CD39 is expressed at high levels in clinical IBD tissues.

Topics: Animals; Antigens, CD; Apyrase; Colitis; Colon; Cytokines; Humans; Leukocytes, Mononuclear; Mice; Mice, Knockout; Oxazolone; Specific Pathogen-Free Organisms; Trinitrobenzenesulfonic Acid

Cannabinoid receptor 2 (CB2) activation is suggested to trigger the peroxisome proliferator-activated receptor-γ (PPARγ) pathway, and agonists of both receptors improve colitis. Recently, the plant metabolite (E)-β-caryophyllene (BCP) was shown to bind to and activate CB2. In this study, we examined the anti-inflammatory effect of BCP in dextran sulfate sodium (DSS)-induced colitis and analyzed whether this effect was mediated by CB2 and PPARγ. Oral treatment with BCP reduced disease activity, colonic macro- and microscopic damage, myeloperoxidase and N-acetylglucosaminidase activities, and levels and mRNA expression of colonic tumor necrosis factor-α, IL-1β, interferon-γ, and keratinocyte-derived chemokine. BCP treatment also inhibited the activation of extracellular signal-regulated kinase 1/2, nuclear factor κB, IκB-kinase α/β, cAMP response element binding and the expression of caspase-3 and Ki-67. Moreover, BCP enhanced IL-4 levels and forkhead box P3 mRNA expression in the mouse colon and reduced cytokine levels (tumor necrosis factor-α, keratinocyte-derived chemokine, and macrophage-inflammatory protein-2) in a culture of macrophages stimulated with lipopolysaccharide. The use of the CB2 antagonist AM630 or the PPARγ antagonist GW9662 significantly reversed the protective effect of BCP. Confirming our results, AM630 reversed the beneficial effect of BCP on pro-inflammatory cytokine expression in IEC-6 cells. These results demonstrate that the anti-inflammatory effect of BCP involves CB2 and the PPARγ pathway and suggest BCP as a possible therapy for the treatment of inflammatory bowel disease.

Topics: Animals; Caspase 3; Caspase Inhibitors; Claudin-4; Colitis; Colon; Cyclic AMP Response Element-Binding Protein; Cytokines; Dextran Sulfate; Extracellular Signal-Regulated MAP Kinases; I-kappa B Kinase; Inflammation Mediators; Ki-67 Antigen; Lipopolysaccharides; Macrophage Activation; Macrophages; Membrane Proteins; Mice; NF-kappa B; Oxazolone; Polycyclic Sesquiterpenes; PPAR gamma; Receptor, Cannabinoid, CB2; Sesquiterpenes; Signal Transduction

Patients with prolonged ulcerative colitis (UC) frequently develop colorectal adenocarcinoma for reasons that are not fully clear. To analyze inflammation-associated colonic tumorigenesis, we developed a chronic form of oxazolone-induced colitis in mice that, similar to UC, was distinguished by the presence of IL-13-producing NKT cells. In this model, the induction of tumors using azoxymethane was accompanied by the coappearance of F4/80+CD11b(high)Gr1(low) M2 macrophages, cells that undergo polarization by IL-13 and are absent in tumors that lack high level IL-13 production. Importantly, this subset of macrophages was a source of tumor-promoting factors, including IL-6. Similar to dextran sodium sulfate-induced colitis, F4/80+CD11b(high)Gr1(intermediate) macrophages were present in the mouse model of chronic oxazolone-induced colitis and may influence tumor development through production of TGF-β1, a cytokine that inhibits tumor immunosurveillance. Finally, while robust chronic oxazolone-induced colitis developed in myeloid differentiation primary response gene 88-deficient (Myd88-/-) mice, these mice did not support tumor development. The inhibition of tumor development in Myd88-/- mice correlated with cessation of IL-6 and TGF-β1 production by M2 and F4/80+CD11b(high)Gr1(intermediate) macrophages, respectively, and was reversed by exogenous IL-6. These data show that an UC-like inflammation may facilitate tumor development by providing a milieu favoring development of MyD88-dependent tumor-supporting macrophages.

Topics: Animals; Antigens, Ly; CD11b Antigen; CD8-Positive T-Lymphocytes; Colitis; Colitis, Ulcerative; Inflammation; Interleukin-13; Interleukin-6; Macrophages; Mice; Mice, Inbred BALB C; Mice, Transgenic; Myeloid Differentiation Factor 88; Oxazolone; Signal Transduction; Transforming Growth Factor beta1

Probiotic bacteria can potentially have beneficial effects on the clinical course of several intestinal disorders, but our understanding of probiotic action is limited. We have identified a probiotic bacteria-derived soluble protein, p40, from Lactobacillus rhamnosus GG (LGG), which prevents cytokine-induced apoptosis in intestinal epithelial cells. In the current study, we analyzed the mechanisms by which p40 regulates cellular responses in intestinal epithelial cells and p40's effects on experimental colitis using mouse models. We show that the recombinant p40 protein activated EGFR, leading to Akt activation. Activation of EGFR by p40 was required for inhibition of cytokine-induced apoptosis in intestinal epithelial cells in vitro and ex vivo. Furthermore, we developed a pectin/zein hydrogel bead system to specifically deliver p40 to the mouse colon, which activated EGFR in colon epithelial cells. Administration of p40-containing beads reduced intestinal epithelial apoptosis and disruption of barrier function in the colon epithelium in an EGFR-dependent manner, thereby preventing and treating DSS-induced intestinal injury and acute colitis. Furthermore, p40 activation of EGFR was required for ameliorating colon epithelial cell apoptosis and chronic inflammation in oxazolone-induced colitis. These data define what we believe to be a previously unrecognized mechanism of probiotic-derived soluble proteins in protecting the intestine from injury and inflammation.

Topics: Administration, Rectal; Animals; Apoptosis; Bacterial Proteins; Colitis; Dextran Sulfate; Drug Evaluation, Preclinical; Enzyme Activation; Epithelial Cells; ErbB Receptors; Hydrogels; Intestinal Mucosa; Lacticaseibacillus rhamnosus; Mice; Mice, Inbred C57BL; Microspheres; Oxazolone; Permeability; Probiotics; Proto-Oncogene Proteins c-akt; Quinazolines; Recombinant Proteins; Signal Transduction; Tyrphostins

Mycobacterium bovis Bacillus Calmette-Guérin (BCG), killed by extended freeze-drying (EFD), induces secretion of interleukin-10 and reduces lung inflammation in a mouse model of asthma. We investigated the effects of EFD BCG in mouse models of inflammatory bowel disease.. EFD BCG was administered subcutaneously to mice with colitis induced by dextran sodium sulfate (DSS), oxazolone, or adoptive transfer of CD4(+)CD45RB(high)Foxp3(-) T cells from C57Bl/6 Foxp3GFP mice to RAG2(-/-) mice.. EFD BCG, administered either before induction of DSS and oxazolone colitis or after development of acute or chronic DSS-induced colitis, reduced symptom scores, loss of body weight, and inflammation. Although transfer of CD4(+)CD45RB(high)Foxp3(-) cells induced colitis in RAG2(-/-) mice, administration of EFD BCG at the time of the transfer converted Foxp3(-) T cells to Foxp3(+) T cells and the mice did not develop colitis. EFD BCG protected mice from colitis via a mechanism that required expansion of T regulatory cells and production of interleukin-10 and transforming growth factor β. EFD BCG activated the retinoid X receptor (RXR)-α-peroxisome proliferator-activated receptor (PPAR)-γ heterodimer, blocked translocation of nuclear factor κB to the nucleus, and reduced colonic inflammation; it did not increase the number of colon tumors that formed in mice with chronic DSS-induced colitis.. EFD BCG controls severe colitis in mice by expanding T regulatory cell populations and PPAR-γ and might be developed to treat patients with inflammatory bowel disease.

Topics: Animals; BCG Vaccine; Colitis; Colon; Dextran Sulfate; Forkhead Transcription Factors; Freeze Drying; Interleukin-1; Interleukin-10; Interleukin-6; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mycobacterium bovis; NF-kappa B; Oxazolone; Peroxidase; PPAR gamma; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Weight Loss

To investigate the therapeutic effects of Bawei Xilei San (BWXLS), a compound traditional Chinese herbal medicine, on mice with oxazolone-induced colitis and to explore the mechanisms.. Thirty-two BALB/c mice were randomly divided into 4 groups (8 for each): normal control group, untreated group, hydrocortisone group and BWXLS group. Except for the mice in the normal control group, all mice were intrarectally administered with 3.0% oxazolone to induce colitis. Then the mice in the normal control group and untreated group were administered with 0.9% carboxymethyl cellulose sodium solution. Mice in the BWXLS group were intrarectally administered with 0.2 mg/g BWXLS and hydrocortisone group with 0.02 mg/g respectively for 5 days. The body weight and stool consistency and occult or gross blood were recorded to calculate the disease activity index (DAI). The mice were sacrificed at the 6th day. The macroscopic and histological changes of the colon were evaluated. The expressions of Toll-like receptor 4 (TLR4), nuclear factor-kappaB (NF-kappaB), and epithelial tight junction protein occludin were assessed by immunohistochemical method. The level of tumor necrosis factor-alpha (TNF-alpha) in colonic mucosa was evaluated by enzyme-linked immunosorbent assay.. The DAI, and macroscopic and histological changes in the BWXLS group were improved as compared with those in the untreated group (P<0.05) but were similar to those in the hydrocortisone group. The expression of occludin was significantly increased (P<0.05) while the expressions of TLR4, NF-kappaB and TNF-alpha were significantly decreased in the BWXLS group as compared with the untreated group, and were similar to those in the hydrocortisone group (P>0.05).. Up-regulating the expression of occludin and down-regulating the expressions of TLR4 and NF-kappaB, and hence inhibiting TNF-alpha expression and improving the mucosa barrier function may be part of the mechanisms of BWXLS in treating oxazolone-induced colitis in mice.

Topics: Animals; Colitis; Drugs, Chinese Herbal; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; NF-kappa B; Occludin; Oxazolone; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

We examined the change of protein tyrosine kinases (PTKs) expression levels in colonic epithelial cells isolated from mice in which colitis was induced by oxazolone administration, using the monoclonal antibody YK34, which cross-reacts with a wide variety of PTKs. We identified focal adhesion kinase (FAK) and found the expression level increased due to the induction of colitis. Furthermore, we found that there was a positive correlation between FAK expression and the severity of colitis. Also, FAK expression localized in the colonic epithelium but not in the lamina propria, implying FAK functions in epithelial cells during colitis formation and/or wound repairing.

Topics: Animals; Colitis; Colon; Disease Models, Animal; Focal Adhesion Kinase 1; Intestinal Mucosa; Male; Mice; Mice, Inbred BALB C; Oxazolone

Substantial data show that infection with helminth parasites ameliorates colitis; however, oxazolone-induced colitis is exaggerated in mice infected with the tapeworm, Hymenolepis diminuta. We tested the hypothesis that the IL-5 response to helminth infection enhances the severity of oxazolone-induced colitis. Mice were infected with H. diminuta and 8 days later were treated with oxazolone ± anti-IL-5 antibodies. Colitis was assessed 72 hours postoxazolone treatment by disease activity scores, myeloperoxidase activity, and histopathology. Other mice received injections of a replication-deficient adenovirus that carried the IL-5 (Ad.IL-5) gene or a control adenovirus (Ad.delete) ± oxazolone. The effect of H. diminuta+oxazolone in CCL11/CCL22 (eotaxin-1 and 2) knockout (KO) mice was determined. Helminth infection and Ad.IL-5 treatment increased IL-5 and eosinophil numbers. In vivo neutralization of IL-5 significantly reduced the severity of colitis in H. diminuta+oxazolone-treated mice, and H. diminuta did not exaggerate oxazolone-induced colitis in CCL11/CCL22 KO mice. Mice receiving Ad.IL-5 only had no colitis, while oxazolone-induced colitis was more severe in animals cotreated with Ad.IL-5 (Ad.delete + oxazolone was not significantly different from oxazolone only). Thus, while there is much to be gleaned about antiinflammatory mechanisms from rodent-helminth model systems, these data illustrate the caveat that infection with helminth parasites as a therapy could be contraindicated in patients with eosinophilia or elevated IL-5 unless coupled to appropriate measures to block IL-5 and/or eosinophil activity.

Topics: Animals; Antibodies; Chemokine CCL11; Chemokine CCL22; Colitis; Disease Progression; Eosinophils; Helminths; Hymenolepiasis; Hymenolepis diminuta; Immunotherapy, Adoptive; Interleukin-5; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Oxazolone; Receptors, Cell Surface

Previous studies have demonstrated that the Chinese medicine paeoniflorin, derived from the Ranunculaceae plant peony, peony, purple peony root, was able to have anti-inflammatory, anti-ulcer, anti-hypersusceptibility and anti-oxidation activity. In order to elucidate the pesticide effect and the mechanisms by which paeoniflorin exerts its effect of anti-inflammation and immunoregulation on oxazolone-induced colitic mice, disease activity index (DAI) and histological grading of colitis (HGC) were evaluated in animal model. Moreover, the expressions of HBD-2, IL-6 and IL-10 of mice with experimental colitis were observed with immunohistochemistry and RT-PCR in this study. Results showed that DAI and HGC of oxazolone control group was significantly higher than that of normal control group, and that paeoniflorin groups and 5-ASA group, compared with oxazolone control group, could alleviate the symptoms and histological damages of colitic mice (P < 0.05, P < 0.01). The expression of HBD-2 and IL-6 cytokine on the colon of colitic mice was higher than that of normal control, paeoniflorin and 5-ASA groups (P < 0.05, P < 0.01), but the expression of IL-10 is lower than that of normal control, paeoniflorin and 5-ASA groups (P < 0.05, P < 0.01). The positive correlations were demonstrated between the expression of (HBD-2 and IL-6) and DAI (Pearson r = 0.728, Pearson r = 0.758, P < 0.01, respectively), (HBD-2 and IL-6) and HGC (Pearson r = 0.819, Pearson r = 0.825, P < 0.01, respectively), whereas, the negative correlations were demonstrated between the expression of IL-10 and DAI (Pearson r = -0.789, P < 0.01), IL-10 and HGC (Pearson r = -0.725, P < 0.01). It can be concluded that to some extent paeoniflorin effectively alleviate the symptoms of oxazolone-induced colitis through regulating the expression of HBD-2, IL-6 and IL-10.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Benzoates; beta-Defensins; Bridged-Ring Compounds; Colitis; Colon; Female; Glucosides; Interleukin-10; Interleukin-6; Intestinal Mucosa; Mesalamine; Mice; Mice, Inbred BALB C; Monoterpenes; Oxazolone; Paeonia; Random Allocation; RNA, Messenger

Inflammatory bowel diseases are relatively common diseases of the gastrointestinal tract. The relative therapeutic efficacy of glucocorticoids used in inflammatory bowel diseases resides in part in their capability to inhibit activity of nuclear factor kappaB (NF-kappaB), a transcription factor central to the inflammatory process, and the consequent production of T-helper 1 (Th1)-type cytokines. Previous studies indicate that increased expression in transgenic mice of glucocorticoid-induced leucine zipper (GILZ), a gene rapidly induced by glucocorticoids, inhibits NF-kappaB and Th1 activity.. We performed experiments with the aim to test the susceptibility of GILZ transgenic (GILZ-TG) mice to dinitrobenzene sulfonic acid-induced colitis.. Consistent with a decreased Th1 response, GILZ-TG mice were less susceptible to colitis induction as compared with wild-type littermates, while they were more susceptible to Th2-mediated colitis. The inhibition was comparable to that obtained with dexamethasone treatment. Moreover, diminished intestinal tissue damage, associated with inhibition of NF-kappaB nuclear translocation, interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 production in CD4+ T lymphocytes of the lamina propria, was evident in GILZ-TG as compared with wild-type mice. In addition, inhibition of colitis development was also evident when GILZ fusion protein was delivered in vivo in dinitrobenzene sulfonic acid-treated WT animals as well as in interleukin-10 knockout mice.. Together these results demonstrate that GILZ mimics the effects of glucocorticoids, suggesting a contribution of this protein to the anti-inflammatory activity of glucocorticoids in Th1-induced colitis.

Topics: Animals; Colitis; Colon; Dinitrofluorobenzene; Disease Models, Animal; Genetic Predisposition to Disease; Glucocorticoids; Intercellular Adhesion Molecule-1; Interferon-gamma; Interleukin-1; Leucine Zippers; Mice; Mice, Knockout; Mice, Transgenic; NF-kappa B; Oxazolone; Th1 Cells; Th2 Cells; Transcription Factors; Tumor Necrosis Factor-alpha

The purpose of the study was to evaluate digestion of pectin/Kollicoat SR30D free films for colonic delivery in vitro and in vivo.. Free films containing different ratios of pectin to Kollicoat SR30D were prepared by casting/solvent evaporation method. An in-vitro comparison of swelling, degradation and permeability of the free films was carried out in simulated colon fluids containing caecal contents from normal rats with colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) or oxazolone. A comparative in-vivo evaluation of degradation was also conducted in normal and colitis-induced model rats.. The pectin within the mixed films was susceptible to rat colonic bacterial enzymes. The extent of digestion correlated with the amount of pectin present within the film. In vitro, the swelling index, drug permeability and extent of film digestion in simulated colon fluids with caecal contents obtained from normal rats were higher than from TNBS- or oxazolone-induced model rats, whereas in-vivo degradation was similar in the three groups of rats. The pectin/Kollicoat SR30D free films were completely degraded in the colitis-induced rats.. Pectic/Kollicoat SR30D films may be useful as coatings to target delivery of drugs to the colon.

Topics: Animals; Cecum; Colitis; Colon; Disease Models, Animal; Drug Delivery Systems; Fluorouracil; Glass; Oxazolone; Pectins; Permeability; Polyvinyls; Rats; Steam; Temperature; Trinitrobenzenesulfonic Acid

Ulcerative colitis (UC) is associated with intestinal and extra intestinal clinical manifestations. The profound organic changes in UC indicate that the colonic mechanical and mechanosensory functions are affected. The aim was to study acute morphological and biomechanical properties of the distal colon in oxazolone-induced UC in BALB/C mice. Six normal male BALB/C mice and 10 oxazolone-induced UC mice were studied. UC was induced by epicutaneous and intrarectal administration of oxazolone. The mechanical test was done as a distension experiment where the colon was distended up to 20 cmH2O. The pressure, outer diameter and length were recorded simultaneously. Circumferential and longitudinal stresses and strains were computed. The intestinal specimens were processed for histology. The mucosa was infiltrated with acute and chronic inflammatory cells. Mucosal bleeding, irregular ulcers crypt abscess, and destruction of the epithelial border were observed. Although, the mucosa in ulcers was much thinner than in the normal controls, the mucosa and submucosa around the ulcer were thicker than in the normal controls (P<0.05). Oxazolone-induced colitis increased the circumferences and wall cross-sectional area (P<0.01), the opening angle and residual strain at the serosa increased (P<0.01). Furthermore, the circumferential and longitudinal stiffness increased in the UC wall and was most pronounced in longitudinal direction. The opening angle and residual strain was linearly correlated to the wall thickness, area and inflammation degree. In conclusion, morphological and biomechanical changes of the colon occurred during the development of UC. The increased stiffness may contribute to the abnormal function in patients with UC.

Topics: Animals; Biomechanical Phenomena; Colitis; Male; Mice; Mice, Inbred BALB C; Oxazolone

To evaluate the therapeutic effects of a probiotic supplement (Clostridium butyricum, CGMCC0313) in a chemically-induced rat model of experimental colitis.. An experimental ulcerative colitis model was established by rectal injection of oxazolone into the colon of 40 Wistar rats randomly divided into four groups. The positive control group was sacrificed 3 d after colitis onset. The remaining groups were fed daily with either 2 mL of C. butyricum (2.3 x 10(11) CFU/L), 2 mL of mesalamine (100 g/L), or 1 mL of sodium butyrate (50 mmol/L) for 21 d. The animals' body weight, behavior, and bowel movements were recorded weekly. After sacrifice, visual and microscopic observations of pathological changes of colon tissue were made, body weight and wet colon mass index were measured and recorded, and serum levels of interleukin-23 (IL-23) and TNF-alpha were measured using ELISA. Expression of calcitonin gene-related peptide in colon tissue was measured by RT-PCR. Finally, changes in rat intestinal microflora status were measured in all groups.. We found that treatment with C. butyricum lowered the serum levels of both IL-23 and tumor necrosis factor-alpha (TNF-alpha) with similar or even better efficiency than that of mesalamine or sodium butyrate. The rat intestinal flora appeared to recover more quickly in the group treated with C. butyricum than in the mesalamine and sodium butyrate groups. Finally, we found that the expression level of calcitonin gene related peptide was elevated in colon tissue in the sodium butyrate treated group but not in the C. butyricum or mesalamine treated groups, indicating a sensitization of colon following sodium butyrate treatment.. In our experimental colitis model, treatment with C. butyricum CGMCC0313, a probiotic supplement, is at least as efficient as treatment with mesalamine.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Butyrates; Calcitonin Gene-Related Peptide; Clostridium butyricum; Colitis; Colon; Disease Models, Animal; Feces; Interleukin-23; Mesalamine; Organ Size; Oxazolone; Random Allocation; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha

Crohn's disease (CD) and ulcerative colitis (UC), both of which are referred to as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the gastrointestinal tract that have characteristic clinical, pathological, endoscopic, and radiologic features. Knowledge about the pathogenesis of IBD has dramatically increased in recent years based in part on the use of experimental models of IBD. Although none of these models exactly mimics the human disorder, they have proven to be useful for studying many important aspects of these conditions. Detailed in this unit is a description of the most commonly used chemically induced mouse models of IBD. These include trinitrobenzene sulfonic acid (TNBS), oxazolone, and acute or chronic dextran sodium sulfate (DSS) colitis models.

Topics: Animal Husbandry; Animals; Colitis; Dextran Sulfate; Disease Models, Animal; Mice; Oxazolone; Trinitrobenzenesulfonic Acid

The proinflammatory cytokine IL-6 seems to have an important role in the intestinal inflammation that characterizes inflammatory bowel diseases (IBDs) such as Crohn disease and ulcerative colitis. However, little is known about the molecular mechanisms regulating IL-6 production in IBD. Here, we assessed the role of the transcriptional regulator IFN regulatory factor-4 (IRF4) in this process. Patients with either Crohn disease or ulcerative colitis exhibited increased IRF4 expression in lamina propria CD3+ T cells as compared with control patients. Consistent with IRF4 having a regulatory function in T cells, in a mouse model of IBD whereby colitis is induced in RAG-deficient mice by transplantation with CD4+CD45RB(hi) T cells, adoptive transfer of wild-type but not IRF4-deficient T cells resulted in severe colitis. Furthermore, IRF4-deficient mice were protected from T cell-dependent chronic intestinal inflammation in trinitrobenzene sulfonic acid- and oxazolone-induced colitis. In addition, IRF4-deficient mice with induced colitis had reduced mucosal IL-6 production, and IRF4 was required for IL-6 production by mucosal CD90+ T cells, which it protected from apoptosis. Finally, the protective effect of IRF4 deficiency could be abrogated by systemic administration of either recombinant IL-6 or a combination of soluble IL-6 receptor (sIL-6R) plus IL-6 (hyper-IL-6). Taken together, our data identify IRF4 as a key regulator of mucosal IL-6 production in T cell-dependent experimental colitis and suggest that IRF4 might provide a therapeutic target for IBDs.

Topics: Adoptive Transfer; Adult; Animals; Apoptosis; CD4-Positive T-Lymphocytes; Colitis; Cytokines; DNA-Binding Proteins; Female; Gene Expression Regulation; Humans; Inflammatory Bowel Diseases; Interferon Regulatory Factors; Interleukin-6; Intestinal Mucosa; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Oxazolone; Receptors, Interleukin-6; Recombinant Fusion Proteins; T-Lymphocytes; Trinitrobenzenesulfonic Acid

To determine whether simvastatin is able to inhibit inflammation in trinitrobenzene sulfonic acid (TNBS)-induced or oxazalone (OXA)-induced colitis.. In the prophylactic protocol, simvastatin dose-dependently suppressed the decrease in body weight and inflammatory grade of TNBS-treated mice. In contrast, in the therapeutic protocol, no significant difference in body weight reduction was observed between simvastatin-treated and control mice. IFN-gamma release from LP cells was significantly suppressed in mice receiving high-dose simvastatin in the prophylactic protocol. In contrast to TNBS colitis, even high-dose prophylactic simvastatin had no suppressive effects on either weight reduction or the inflammatory grade in OXA colitis.. Our results indicate that simvastatin negatively regulates inflammation in TNBS-induced colitis, but not in OXA-induced colitis. In TNBS-induced colitis, simvastatin suppressed the Th1-polarized immune response. Our findings suggest that simvastatin has potential effects as a therapeutic agent in human inflammatory bowel disease, particularly Crohn's disease.

Topics: Animals; Colitis; Cytokines; Enzyme-Linked Immunosorbent Assay; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Male; Mice; Oxazolone; Simvastatin; Statistics, Nonparametric; Trinitrobenzenesulfonic Acid

A CD30-ligand (CD30L) is a 40-kilodalton, type II membrane-associated glycoprotein belonging to the tumor necrosis factor family. Serum levels of soluble CD30 increased in inflammatory bowel diseases (IBD), suggesting that CD30L/CD30 signaling is involved in the pathogenesis of IBD. In this study, we investigated the role of CD30L in oxazolone (OXA)- and trinitrobenzene sulfonic acid (TNBS)-induced colitis in CD30L knockout (KO) mice.. Colitis was induced by OXA or TNBS in CD30LKO mice with BALB/c or C57BL/6 background, respectively, and diverse clinical signs of the disease were evaluated. Cytokine production from lamina propria T cells of the colon was assessed by enzyme-linked immunosorbent assay. Anti-interleukin (IL)-4 monoclonal antibody (mAb) or agonistic anti-CD30 mAb was inoculated in mice with colitis induced by OXA or TNBS.. CD30LKO mice were susceptible to OXA-induced colitis but resistant to TNBS-induced acute colitis. The levels of T helper cell 2 type cytokines such as IL-4 and IL-13 in the LP T cells were significantly higher, but the levels of interferon gamma were lower in OXA- or TNBS-treated CD30LKO mice than in wild-type mice. In vivo administration of agonistic anti-CD30 mAb ameliorated OXA-induced colitis but aggravated TNBS-induced colitis in CD30LKO mice.. These results suggest that CD30L/CD30 signaling is involved in development of both OXA- and TNBS-induced colitis. Modulation of CD30L/CD30 signaling by mAb could be a novel biologic therapy for IBD.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; CD30 Ligand; Colitis; Cytokines; Disease Models, Animal; Genetic Predisposition to Disease; Inflammatory Bowel Diseases; Interleukin-4; Intestinal Mucosa; Ki-1 Antigen; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; Signal Transduction; Trinitrobenzenesulfonic Acid

Treatment with ex vivo expanded regulatory T cells (Tregs) is regarded as a promising therapeutic approach in inflammatory bowel disease but is hampered by impaired Treg accumulation and function at inflammatory sites. We aim to study whether antigen-specific redirected Tregs can overcome these limitations.. We developed transgenic mice whose T cells, including Tregs, express chimeric receptor (CR) made of antibody variable region as recognition unit and T-cell stimulatory and costimulatory domains to activate specifically in response to the predetermined model antigen 2,4,6-trinitrophenol (TNP).. TNP-specific CR-bearing Tregs were potently and specifically activated by exogenous TNP and suppressed effector T cells in the absence of costimulatory B7-CD28 interaction. TNP-specific transgenic (Tg) mice were resistant to 2,4,6-trinitrobenzene sulphonic acid (TNBS) colitis but not to other hapten-mediated colitis. Adoptive transfer of CR-bearing Tregs to wild-type mice with TNBS colitis was associated with significant amelioration of colitis and improved survival. Although TNP-specific CR-bearing Tregs did not suppress oxazolone colitis, they cured it after addition of traces of TNBS to oxazolone-inflamed colons, demonstrating a "bystander" effect. In vivo imaging of adoptively transferred CR-bearing Tregs demonstrated that they preferentially migrate to TNBS-induced colonic mucosal lesions within hours of induction of colitis.. Tregs can be redirected with specificity distinct from that of pathogenic lymphocytes, accumulate at colonic inflammatory lesions, and suppress effector T cells in a specific, nonmajor histocompatibility complex-restricted, and noncostimulatory-dependent manner, resulting in significant amelioration of colitis. Hopefully, this approach will lead to a novel therapy for inflammatory bowel disease, as well as other inflammatory diseases.

Topics: Animals; Bystander Effect; CD28 Antigens; Cell Line; Cell Movement; Colitis; Colon; Disease Models, Animal; Forkhead Transcription Factors; Histocompatibility Antigens; Immunity, Mucosal; Immunoglobulin Variable Region; Immunotherapy, Adoptive; Intestinal Mucosa; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Transgenic; Oxazolone; Picrates; Receptors, Antigen, T-Cell; Receptors, IgG; Recombinant Fusion Proteins; T-Lymphocytes, Regulatory; Time Factors; Trinitrobenzenesulfonic Acid

The role of protease-activated receptor-2 (PAR(2)) during intestinal inflammation is still unclear due to the fact that PAR(2)-activating peptide has both pro- and anti-inflammatory properties. The aim of this study was to investigate the effects of PAR(2) deficiency (using PAR(2)-deficient mice, PAR(2)(-/-)) in models of colitis, in order to elucidate the role of endogenous PAR(2) in the process of inflammation in the gut.. Colonic inflammation in wild-type and PAR(2)(-/-) mice was induced by dextran sodium sulfate, trinitrobenzene sulfonic acid (TNBS), a T helper-1 predominant model, or oxazolone, a T helper-2 predominant model. Leukocyte recruitment, assessed by intravital microscopy, and inflammatory parameters (myeloperoxidase (MPO), macroscopic and microscopic damage) were assessed during the development of colitis. Lastly, the protein levels of cyclooxygenases (COXs) and adhesion molecules (ICAM-1, VCAM-1, alpha-M, alpha-4) were assessed by using western blot analysis.. In all three models of colitis, MPO activity, macroscopic damage score and bowel thickness were significantly lower in PAR(2)(-/-) mice. Changes in vessel leukocyte recruitment parameters (rolling and adhesion) were also significantly reduced in PAR(2)(-/-) mice compared to wild-type mice after the induction of colitis. The protein expression of ICAM-1, VCAM-1 and alpha-4 was significantly attenuated, whereas the expression of COX-1 was significantly increased in PAR(2)(-/-) mice challenged with TNBS-induced colitis.. The role of endogenous PAR(2) in the gut is pro-inflammatory and independent of the T helper-1 or -2 cytokine profile. Endogenous PAR(2) activation controls leukocyte recruitment in the colon and thus appears as a new potential therapeutic target for the treatment of inflammatory bowel disease.

Topics: Animals; Cell Adhesion Molecules; Colitis; Dextran Sulfate; Disease Models, Animal; Mice; Mice, Inbred C57BL; Oxazolone; Peroxidase; Prostaglandin-Endoperoxide Synthases; Receptor, PAR-2; Trinitrobenzenesulfonic Acid

Topics: Animals; Autoimmune Diseases; Bystander Effect; Cell Movement; Colitis; Colon; Disease Models, Animal; Forkhead Transcription Factors; Immunity, Mucosal; Immunotherapy, Adoptive; Intestinal Mucosa; Lymphocyte Activation; Mice; Mice, Transgenic; Oxazolone; Picrates; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; T-Lymphocytes, Regulatory; Time Factors; Trinitrobenzenesulfonic Acid

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of several inflammatory diseases. Its expression is increased in inflamed mucosa of Crohn's disease patients and anti-TNF treatment improves mucosal inflammation. Besides neutralization, induction of apoptosis of monocytes/macrophages and T cells is thought to be an important mechanism of action of the anti-TNF monoclonal antibody therapy. The aim was to investigate the pathogenic role of TNF in hapten-induced colitis models and to study the relationship between apoptosis induction and disease remission.. In 2 murine colitis models (trinitrobenzene sulphonic acid, TNBS, and oxazolone colitis), mice were injected daily with anti-TNF monoclonal antibody (mAb). Macrophages were collected from lamina propria of TNBS colitis mice. 7AAD and anti-active-caspase-3 staining were used to study DNA degradation and intracellular caspase activation. A pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), was given to a subgroup of the colitis mice.. Treatment with anti-TNF effectively reduced intestinal mucosal inflammation in TNBS colitis but not in oxazolone colitis. Effectiveness was evidenced by a more rapid recovery of body weight and reduced cell infiltration, and downregulation of proinflammatory cytokines interferon-gamma (IFN-gamma), TNF, and IL-18 at the mRNA level. Apoptosis was induced in lamina propria macrophages after treatment with anti-TNF, and it was abrogated through short-term pretreatment with Z-VAD-FMK.. Anti-TNF downregulates proinflammatory cytokines and decreases cell infiltration in the bowel after TNBS application. The remission-inducing effect of anti-TNF may partly rely on apoptosis induction.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies, Monoclonal; Apoptosis; Caspase Inhibitors; Colitis; Down-Regulation; Inflammatory Bowel Diseases; Infliximab; Interferon-gamma; Interleukin-18; Male; Mice; Mice, Inbred C57BL; Oxazolone; Trinitrobenzenesulfonic Acid; Tumor Necrosis Factor-alpha

Animal models of intestinal inflammation are indispensable for our understanding of the pathogenesis of Crohn disease and ulcerative colitis, the two major forms of inflammatory bowel disease in humans. Here, we provide protocols for establishing murine 2,4,6-trinitro benzene sulfonic acid (TNBS)-, oxazolone- and both acute and chronic dextran sodium sulfate (DSS) colitis, the most widely used chemically induced models of intestinal inflammation. In the former two models, colitis is induced by intrarectal administration of the covalently reactive reagents TNBS/oxazolone, which are believed to induce a T-cell-mediated response against hapten-modified autologous proteins/luminal antigens. In the DSS model, mice are subjected several days to drinking water supplemented with DSS, which seems to be directly toxic to colonic epithelial cells of the basal crypts. The procedures for the hapten models of colitis and acute DSS colitis can be accomplished in about 2 weeks but the protocol for chronic DSS colitis takes about 2 months.

Topics: Animals; Colitis; Dextran Sulfate; Disease Models, Animal; Epithelial Cells; Mice; Oxazolone; Trinitrobenzenesulfonic Acid

There is convincing evidence from animal and human studies that infection with parasitic helminths can alleviate the histopathology and symptoms of colitis. Here the ability of the rat tapeworm Hymenolepis diminuta to affect the course of oxazolone-induced colitis (a TH2 model) was assessed.. Mice were infected with H diminuta and 8 days later they received oxazolone (3 mg in 50% EtOH, intrarectal). On autopsy (3 or 7 days postoxazolone), disease severity was assessed by macroscopic clinical scores, histologic damage scores, myeloperoxidase and eosinophil peroxidase activity, and cytokine synthesis.. As gauged by all markers of gut function, infection with H diminuta caused a significant exacerbation of oxazolone-induced colitis. Indeed, while mice receiving oxazolone only began to recover approximately 3-4 days posttreatment, the cotreated group continued to deteriorate. Helminth infection, independent of oxazolone administration, enhanced IL-4, IL-5, IL-10, and IL-13 production from in vitro stimulated immune cells and evoked increases in colonic eosinophil peroxidase of cotreated mice. Finally, while knockout of natural killer (NK) and NK-T cells by administration of a neutralizing NK1.1 antibody reduced the inflammation in oxazolone and oxazolone + H diminuta-treated animals, mice in the latter group still displayed significant colitis.. We have shown that H diminuta infection is beneficial in other models of colitis. The current data is presented as a caveat to the position that parasitic helminths in general can be considered as a therapy for heterogeneous inflammatory disorders without careful analysis of the immunologic basis of the condition.

Topics: Adjuvants, Immunologic; Animals; Antigens, Ly; Antigens, Surface; Biomarkers; Colitis; Colon; Disease Models, Animal; Disease Progression; Enzyme-Linked Immunosorbent Assay; Eosinophil Peroxidase; Follow-Up Studies; Hymenolepiasis; Hymenolepis diminuta; Interleukin-10; Interleukin-13; Interleukin-4; Interleukin-5; Killer Cells, Natural; Lectins, C-Type; Male; Mice; Mice, Inbred BALB C; NK Cell Lectin-Like Receptor Subfamily B; Oxazolone; Peroxidase; Survival Rate

Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious and autoimmune diseases. In studies of the mechanisms underlying such induction, we found that IL-13 induces transforming growth factor (TGF)-beta(1) in macrophages through a two-stage process involving, first, the induction of a receptor formerly considered to function only as a decoy receptor, IL-13Ralpha(2). Such induction requires IL-13 (or IL-4) and tumor necrosis factor (TNF)-alpha. Second, it involves IL-13 signaling through IL-13Ralpha(2) to activate an AP-1 variant containing c-jun and Fra-2, which then activates the TGFB1 promoter. In vivo, we found that prevention of IL-13Ralpha(2) expression reduced production of TGF-beta(1) in oxazolone-induced colitis and that prevention of IL-13Ralpha(2) expression, Il13ra2 gene silencing or blockade of IL-13Ralpha(2) signaling led to marked downregulation of TGF-beta(1) production and collagen deposition in bleomycin-induced lung fibrosis. These data suggest that IL-13Ralpha(2) signaling during prolonged inflammation is an important therapeutic target for the prevention of TGF-beta(1)-mediated fibrosis.

Topics: Animals; Bleomycin; Blotting, Western; Cell Lineage; Colitis; Collagen; Cytokines; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Etanercept; Fibrosis; Flow Cytometry; Gene Silencing; Genetic Vectors; Humans; Immunoglobulin G; Inflammation; Interleukin-13; Interleukin-13 Receptor alpha1 Subunit; Luciferases; Lung; Macrophages; Mice; Mice, Inbred C57BL; Monocytes; NF-kappa B; Oxazolone; Promoter Regions, Genetic; Receptors, Interleukin; Receptors, Interleukin-13; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Time Factors; Transcription Factor AP-1; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Up-Regulation

The MHC class II transactivator (CIITA) is the master transcriptional regulator of genes involved in MHC class II restricted antigen presentation. Previously we suggested another role of CIITA in Th1/Th2 balance by demonstrating that forced expression of CIITA in murine T cells repressed Th1 immunity both in vitro and in vivo. However, the results were contradictory to the report that CIITA functioned to suppress the production of Th2 cytokine by CD4+ T cells in CIITA deficient mice. In this study, we investigated the influence of constitutive expression of CIITA in T cells on Th2 immune response in vivo using murine experimental colitis model. In the dextran sodium sulfate-induced acute colitis, a disease involving innate immunity, CIITA transgenic mice and wild type control mice showed similar progression of the disease. However, the development of oxazolone-induced colitis, a colitis mediated by predominantly Th2 immune response, was aggravated in CIITA-transgenic mice. And, CD4+ T cells from the mesenteric lymph node of CIITA-transgenic mice treated with oxazolone exhibited a high level of IL-4 secretion. Together, these data demonstrate that constitutive expression of CIITA in T cells skews immune response to Th2, resulting in aggravation of Th2-mediated colitis in vivo.

Topics: Animals; Colitis; Interleukin-4; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nuclear Proteins; Oxazolone; T-Lymphocytes; Th2 Cells; Trans-Activators

In previous studies, we have shown that the oral administration of colonic proteins that have been haptenated (i.e., haptenated colonic proteins [HCPs]) with trinitrophenol (TNP) can protect mice from the subsequent induction of trinitrobenzene sulfonic acid colitis. Inasmuch as this protection was mediated by regulatory cells that express the antigen-non-specific suppressor factors transforming growth factor-beta and interleukin-10, we reasoned that TNP-HCP feeding would also "cross-protect" mice from colitis induced by a different hapten, oxazolone. Indeed, we found that feeding TNP-HCP protected mice from the development of oxazolone-colitis, albeit to a lesser extent than it protected mice from trinitrobenzene sulfonic acid colitis. In addition, we showed that protection was associated with the appearance of mononuclear cells producing regulatory cytokines. These data strongly imply that the cells induced by feeding 1 type of haptenated protein are capable of cross-reacting with antigens present in colitis produced by a second type of haptenated protein. The cross-protection demonstrated in this study holds promise for the treatment of humans with inflammatory bowel disease because it shows that an appropriate fed antigen can induce regulatory cells that have the potential to suppress an inflammation induced by the unknown antigens causing this disease.

Topics: Adjuvants, Immunologic; Administration, Oral; Animal Feed; Animals; Antigens; Colitis; Colon; Disease Models, Animal; Haptens; Interleukin-10; Male; Mice; Monocytes; Oxazolone; Picrates; Proteins; Transforming Growth Factor beta; Trinitrobenzenesulfonic Acid

This paper studies the disturbances in ionic secretion in the colon of rats with different models of acute and chronic colitis measured as changes in short-circuit current. The aim was to verify whether the reported inhibition of basal and stimulated secretion in the trinitrobenzene sulfonic acid and mytomicin C models are applicable to experimental colitis as such. All models showed remarkable similarity in ion transport as determined in Ussing chambers, with downregulated basal as well as carbachol evoked secretion. The EC(50) of carbachol was unchanged in all cases. Iodoacetamide and oxazolone colitis models were notable exceptions in that the dose response curves for carbachol were unaltered compared to controls. The reason is unclear but seems to be unrelated to either interferon gamma or interleukin 4 levels or to the severity of the inflammatory response.

Topics: Acetic Acid; Acute Disease; Alkylating Agents; Animals; Animals, Genetically Modified; Chloramines; Chronic Disease; Colitis; Dextran Sulfate; Disease Models, Animal; Epithelial Cells; Female; HLA-B27 Antigen; Indicators and Reagents; Iodoacetamide; Ions; Oxazolone; Patch-Clamp Techniques; Rats; Trinitrobenzenesulfonic Acid

Nanoparticles (NP) are proposed for targeted drug delivery to the inflammation site in severe cases of inflammatory bowel disease where state-of-the-art delivery devices fail. FK506 (tacrolimus) entrapped into NP was administered either orally or rectally to male Wistar rats suffering from a preexisting experimental colitis. Clinical activity score, colon/body weight index, and myeloperoxidase activity were determined to assess the inflammation. Tissue penetration experiments elucidated the processes involved in the proposed new therapeutic approach. The therapeutic effects of FK506 solutions as well as FK506-NP by oral route were minor. The myeloperoxidase activity and colon/body weight ratio decreased significantly (P < 0.05) only after the rectal administration of FK506-NP, whereas treatment by free drug was not different from colitis control in both 2,4,6-trinitrobenzenesulfonic acid and oxazolone colitis model. NP allows an enhanced and selective drug penetration into the inflammation site as opposed to surrounding healthy tissue (healthy: FK506, 109 +/- 18 nmol/cm2; FK506-NP, 51 +/- 13 nmol/cm2; colitis: FK506, 79 +/- 28 nmol/cm2; FK506-NP, 105 +/- 24 nmol/cm2), presumably by protecting the encapsulated drug against influences from efflux systems and mucosal metabolism. The relative drug penetration into the inflamed tissue is about 3-fold higher compared with healthy tissue when using NP as drug carriers. The use of drug-loaded NP offers several advantages compared with standard therapeutic strategies such as a higher selectivity in adhesion to and enhanced drug penetration into the inflamed tissue.

Topics: Animals; Colitis; Creatinine; Drug Delivery Systems; Male; Nanostructures; Oxazolone; Rats; Rats, Wistar; Tacrolimus; Trinitrobenzenesulfonic Acid

Human semicarbazide-sensitive amine oxidase (SSAO) or vascular adhesion protein-1 (VAP-1) is a copper-containing amine oxidase (AOC3, EC 1.4.3.6) that has both enzymatic and adhesive function. SSAO catalyzes the oxidative deamination of primary amines, resulting in the formation of the corresponding aldehyde and release of hydrogen peroxide and ammonia. Membrane-bound SSAO is an inflammation-inducible endothelial cell adhesion molecule that mediates the interaction between leukocytes and activated endothelial cells in inflamed vessels. Both the direct adhesive and enzymatic functions seem to be involved in the adhesion cascade. LJP 1207 [N'-(2-phenyl-allyl)-hydrazine hydrochloride] is a potent (human SSAO IC(50) = 17 nM), selective, and orally available SSAO inhibitor that blocks both the enzymatic and adhesion functions of SSAO/VAP-1. In a mouse model of ulcerative colitis, LJP 1207 significantly reduces mortality, loss of body weight, and colonic cytokine levels. Quantitative histopathological assessment of colitis activity in this model showed a highly significant suppression of inflammation, injury, and ulceration scores in the animals treated with the SSAO/VAP-1 inhibitor. LJP 1207 also reduced serum levels of tumor necrosis factor-alpha and interleukin 6 in lipopolysaccharide (LPS)-challenged mice and prolonged survival post-LPS-induced endotoxemia. Therapeutic and prophylactic administration of LJP 1207 in the rat carrageenan footpad model also markedly inhibited swelling and inflammation. Overall, the data suggest that small molecule SSAO/VAP-1 inhibitors may provide clinical benefit in the treatment of acute and chronic inflammatory diseases.

Topics: Amine Oxidase (Copper-Containing); Animals; Anti-Inflammatory Agents; Carrageenan; Cell Adhesion; Cloning, Molecular; Colitis; Cyclooxygenase 2; Cytokines; Edema; Endothelial Cells; Endotoxemia; Female; Hydrazines; Inflammation; Male; Mice; Mice, Inbred Strains; Monoamine Oxidase Inhibitors; Oxazolone; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction

Activation of colonic proteinase activated receptor-1 (PAR1) provokes colonic inflammation and increases mucosal permeability in mice. The mechanism of inflammation is not neurogenic like in the paw of rats but depends on PAR1-mediated activation monocytic cells. PAR1 activation in the colon increases the release of lymphocyte T helper-1 (TH1) cytokines. Moreover, PAR1 expression is increased in biopsies from patients with inflammatory bowel disease, and its activation during TH1-mediated colitis in mice increases all of the hallmarks of inflammation.. This study aimed to characterize the effects of PAR1 activation in oxazolone-mediated colitis, involving a TH2 cytokine profile.. Intracolonic administration of oxazolone increased myeloperoxidase activity, damage score, and interleukin (IL)-4, IL-10, tumor necrosis factor alpha, and IL-1beta mRNA expression but lowered interferon-gamma mRNA expression, indicating colonic inflammation of a TH2 profile. The concurrent intracolonic administration of a PAR1 agonist in oxazolone-treated mice inhibited colitis, resulting in a reduction of myeloperoxidase activity, damage score, and inflammatory cytokine mRNA expression. Using PAR1-deficient mice, we confirmed that the anti-inflammatory effects of PAR1 agonists were mediated by PAR1. Moreover, in PAR1-deficient mice or in mice treated with a PAR1 antagonist, oxazolone-induced colitis was exacerbated, showing an endogenous modulatory role for PAR1 in this TH2 cytokine profile of colitis.. Thus, as opposed to a previously shown proinflammatory role for PAR1 in a TH1 cytokine-mediated colitis, our new data show anti-inflammatory role for PAR1 activation in the setting of TH2 cytokine colitis model.

Topics: Animals; Colitis; Cytokines; Inflammation; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Oxazolone; Receptor, PAR-1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Th1 Cells

Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a cell surface molecule that has been proposed to negatively regulate T cell function. We have shown that CEACAM1 is associated with specific regulation of T helper cell (Th)1 pathways, T-bet-mediated Th1 cytokine signaling, and Th1-mediated immunopathology in vivo. Mice treated with anti-mouse CEACAM1-specific monoclonal antibody (mAb) CC1 during the effector phase exhibited a reduced severity of trinitrobenzene sulfonic acid colitis in association with decreased interferon (IFN)-gamma production. Although oxazolone colitis has been reported as Th2 mediated, mice treated with the CC1 mAb or a CEACAM1-Fc chimeric protein exhibited a reduced severity of colitis in association with a significant reduction of IFN-gamma and T-bet activation, whereas signal transducer and activator of antigen 4 activation was unaffected. Both interleukin-4 and IFN-gamma gene-deficient mice exhibited less severe colitis induction by oxazolone. Direct ligation of T cells in vitro with the murine hepatitis virus spike protein, a natural ligand for the N-domain of CEACAM1, inhibited the differentiation of naive cells into Th1 but not Th2 cells and activation of Th1 but not Th2 cytokine production. These results indicate that CEACAM1 isoforms are a novel class of activation-induced cell surface molecules on T cells that function in the specific regulation of Th1-mediated inflammation such as that associated with inflammatory bowel disease.

Topics: Animals; Antibodies, Monoclonal; Carcinoembryonic Antigen; Colitis; Disease Models, Animal; Female; Immunoglobulin Fc Fragments; Inflammation; Interferon-gamma; Interleukin-1; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; Recombinant Fusion Proteins; T-Lymphocytes, Helper-Inducer; Th1 Cells

Since glycolipid biosynthesis is potentially involved in immunological and inflammatory responses, we tested the effect of a novel inhibitor of intracellular glycolipid biosynthesis N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM) in two hapten-induced colitis models: trinitrobenzene sulphonic acid (TNBS)- and oxazolone (4-ethoxymethylene-2phenyl-2oxazoline-5-one; Oxa)-induced colitis. AMP-DNM was given either by intraperitoneal injection or orally via the diet. Mice treated with AMP-DNM had less severe colitis and a more rapid weight recovery, less edema and less wall thickness. Cellular infiltration, goblet cell loss and myeloperoxidase (MPO) activity were reduced in colons of AMP-DNM-treated animals. Intralesional IFN-gamma and IL-18 production were lower in mice of the AMP-DNM-treated groups. Furthermore, AMP-DNM treatment reduced the serum anti-TNBS and anti-Oxa antibody levels. Our findings show that the glycolipid biosynthesis inhibitor AMP-DNM has a strong anti-inflammatory and immune suppressive activity on both TNBS- and Oxa-induced colitis. The data also provide evidence that glycolipid biosynthesis is involved in the inflammatory cascade in these inflammatory bowel disease (IBD) models.

Topics: 1-Deoxynojirimycin; Adamantane; Animals; Anti-Inflammatory Agents; Antibodies; Body Weight; Colitis; Colon; Disease Models, Animal; Enzyme Inhibitors; Glycolipids; Haptens; Immunoglobulin G; Immunosuppressive Agents; Interferon-gamma; Interleukin-18; Male; Mice; Mice, Inbred C57BL; Oxazolone; Peroxidase; Trinitrobenzenesulfonic Acid

A number of experimental models of colitis have been proposed. However, few studies have presented T helper-2 (Th-2) type colitis models that substitute for human ulcerative colitis (UC). In recent years, the murine oxazolone (OXA)-induced colitis model came to be accepted as a Th-2 type model, but it has yet to be used in any pharmacological study. In the present study, we modified the OXA-induced colitis model in BALB/C mice to evaluate the efficacy of treatments for UC. Colitis was induced by intrarectal administration of OXA solution (7.5 mg/mL in 40% ethanol) in a BALB/C strain that is known to favor Th-2 immune responses. A lower mortality rate was obtained in the BALB/C strain than was found in the original method. Histological examination showed that there were morphological similarities to human UC. Increased mRNA expression of interleukin-13, a Th-2 cytokine, was observed in mesenteric lymph nodes. Intrarectal administration of 5-aminosalicylic acid or sodium prednisolone phosphate resulted in a significant improvement in the colitis. These results suggest that the OXA-induced colitis model in the BALB/C strain provides a new way to evaluate the efficacy of therapeutic agents for UC.

Topics: Animals; Colitis; Colitis, Ulcerative; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Male; Mesalamine; Mice; Mice, Inbred BALB C; Oxazolone; Prednisolone

Oxazolone colitis (OC) is an experimental colitis that has a histologic resemblance to human ulcerative colitis. Here we show that IL-13 production is a significant pathologic factor in OC since its neutralization by IL-13Ralpha2-Fc administration prevents colitis. We further show that OC is mediated by NK-T cells since it can be induced neither in mice depleted of NK-T cells nor in mice that cannot present antigen to NK-T cells and mice lacking an NK-T cell-associated TCR. Finally, we show that NK-T cells are the source of the IL-13, since they produce IL-13 upon stimulation by alpha-galactosylceramide, an NK-T cell-specific antigen. These data thus describe a cellular mechanism underlying an experimental colitis that may explain the pathogenesis of ulcerative colitis.

Topics: Adjuvants, Immunologic; Animals; Colitis; Colitis, Ulcerative; Disease Models, Animal; Humans; Interleukin-13; Killer Cells, Natural; Male; Mice; Mice, Inbred C57BL; Oxazolone; Th2 Cells

Trinitrobenzene sulfonic acid (TNBS)-induced colitis is an IL-12-driven, Th1 T cell-mediated colitis that resembles human Crohn's disease. In the present study, we showed initially that the oral administration of recombinant subunit B of cholera toxin (rCT-B) at the time of TNBS-induced colitis by intrarectal TNBS instillation inhibits the development of colitis or, at later time when TNBS-induced colitis is well established, brings about resolution of the colitis. Dose-response studies showed that a majority of mice (68%) treated with rCT-B at a dose of 100 microg (times four daily doses) exhibited complete inhibition of the development of colitis, whereas a minority (30%) treated with rCT-B at a dose of 10 microg (times four daily doses) exhibited complete inhibition; in both cases, however, the remaining mice exhibited some reduction in the severity of inflammation. In further studies, we showed that rCT-B administration is accompanied by prevention/reversal of increased IFN-gamma secretion (the hallmark of a Th1 response) without at the same time causing an increase in IL-4 secretion. This decreased IFN-gamma secretion was not associated with the up-regulation of the secretion of counterregulatory cytokines (IL-10 or TGF-beta), but was associated with a marked inhibition of IL-12 secretion, i.e., the secretion of the cytokine driving the Th1 response. Finally, we showed that rCT-B administration results in increased apoptosis of lamina propria cells, an effect previously shown to be indicative of IL-12 deprivation. From these studies, rCT-B emerges as a powerful inhibitor of Th1 T cell-driven inflammation that can conceivably be applied to the treatment of Crohn's disease.

Topics: Administration, Oral; Administration, Rectal; Animals; Apoptosis; Cells, Cultured; Cholera Toxin; Colitis; Colon; Disease Progression; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-4; Intestinal Mucosa; Leukocytes, Mononuclear; Male; Mice; Mice, Inbred Strains; Oxazolone; Recombinant Proteins; Transforming Growth Factor beta; Trinitrobenzenesulfonic Acid

Oxazolone-induced colitis in the rat is an immune-driven model of human colitis. The aim of the present study was to measure the changes in the absorptive and exudative permeabilites, oedema formation, and local blood flow in this model during the development of inflammation. We also assessed the effects of acute (<1 h), topical glucocorticosteroid (GCS) treatment on these factors.. Colitis was induced by local instillation of oxazolone in previously sensitized animals. Calculating the 40-min plasma-equivalent extravascular volume quantitated the plasma exudation rate. This was determined by using labelled albumin as marker for total tissue content of plasma and Evans blue content as marker for the intravascular volume. Absorptive permeability was simultaneously measured as uptake of rectally administered (51Cr)-labelled ethylenediaminetetraacetic acid (EDTA). In separate experiments regional blood flows were measured by means of the labelled microsphere method.. At both 3 and 24 h after challenge marked enhancements of both exudative and absorptive permeabilities were found. At 24 h there was also an increase in local blood flow. GCS treatment abolished all of the hyperaemia and the main part of the exudative response but had no significant effect on the absorptive permeability.. In this model immunologic mechanisms induce permeability and blood flow changes similar to those in the human disease. It seems suitable for the study of GCS and other anti-inflammatory or immune-modulating drugs.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents; Budesonide; Colitis; Colon; Disease Models, Animal; Edetic Acid; Endothelium; Epithelium; Female; Glucocorticoids; Humans; Intestine, Small; Oxazolone; Permeability; Rats

The intention of the present study was to develop a new hapten-based inflammatory bowel disease model in the rat, useful for pharmacologic screening of new substances with anti-inflammatory properties and immunomodulating capacities. It was considered important to avoid the use of an irritating barrier breaker, such as ethanol.. Dark Agouti rats were skin-sensitized with oxazolone and further challenged intra-rectally with oxazolone dissolved in carmellose sodium (Orabase)/peanut oil. The effects of treatment with budesonide, prednisolone, cyclosporin A, and 5-aminosalicylic acid (5-ASA) were studied.. The intra-rectal challenge with oxazolone in sensitized rats induced an inflammation with an increased colon wet weight, pronounced myeloperoxidase (MPO) activity, and hyperemia/ulcerations in the epithelial lining. Improvement was achieved by treatment with budesonide, prednisolone, and cyclosporin A but not with 5-ASA.. The model fulfills the criteria for a fast, reproducible animal model for human colon inflammation, suitable for pharmacologic screening and studies of an immune-driven colon inflammation.

Topics: Animals; Budesonide; Colitis; Colon; Cyclosporine; Disease Models, Animal; Female; Intestinal Mucosa; Mesalamine; Organ Size; Oxazolone; Peroxidase; Rats; Time Factors

In this study we describe oxazolone colitis, a new form of experimental colitis. This model is induced in SJL/J mice by the rectal instillation of the haptenating agent, oxazolone, and is characterized by a rapidly developing colitis confined to the distal half of the colon; it consists of a mixed neutrophil/lymphocyte infiltration limited to the superficial layer of the mucosa which is associated with ulceration. Oxazolone colitis is a T helper cell type 2 (Th2)-mediated process since stimulated T cells from lesional tissue produce markedly increased amounts of interleukin (IL)-4 and IL-5; in addition, anti-IL-4 administration leads to a striking amelioration of disease, whereas anti-IL-12 administration either has no effect or exacerbates disease. Finally, this proinflammatory Th2 cytokine response is counterbalanced by a massive transforming growth factor-beta (TGF-beta) response which limits both the extent and duration of disease: lesional (distal) T cells manifest a 20-30-fold increase in TGF-beta production, whereas nonlesional (proximal) T cells manifest an even greater 40-50-fold increase. In addition, anti-TGF-beta administration leads to more severe inflammation which now involves the entire colon. The histologic features and distribution of oxazolone colitis have characteristics that resemble ulcerative colitis (UC) and thus sharply distinguish this model from most other models, which usually resemble Crohn's disease. This feature of oxazolone colitis as well as its cytokine profile have important implications to the pathogenesis and treatment of UC.

Topics: Administration, Rectal; Animals; Antibodies; Colitis; Colitis, Ulcerative; Colon; Cytokines; Disease Models, Animal; Histocytochemistry; Humans; Inflammation; Interleukin-4; Interleukins; Mice; Mice, Inbred Strains; Oxazolone; Th2 Cells

Experimental colitis was induced in the rat, by ethanol-oxazolone injections into the distal colon, resulting in diarrhea together with edema, ulcers, and cell infiltration in the exposed colon. Colitic rats showed an elevated urinary recovery of the permeability marker [51Cr]EDTA after intragastric feeding, 19 +/- 10%, compared to 2.9 +/- 0.7% for control rats (P < 0.001). An increased retention of [51Cr]EDTA in the intestines and a decreased discharge in feces suggested an increased intestinal transit time in colitic rats. The in vitro permeability to [51Cr]EDTA and ovalbumin was not elevated in the severely inflamed distal colon, but was in the proximal, unaffected colon to ovalbumin (P < 0.05) and in the distal small intestine, to both [51Cr]EDTA (P < 0.01) and ovalbumin (P < 0.05), indicating that an inflammation in one part of the intestine could have permeability effects in other remote parts. In conclusion, the increased [51Cr]EDTA absorption in vivo during colitis was probably due to both an increased permeability and an increased intestinal transit time.

Topics: Animals; Chromium Radioisotopes; Colitis; Colon; Edetic Acid; Ethanol; Female; In Vitro Techniques; Intestinal Absorption; Ovalbumin; Oxazolone; Rats; Rats, Sprague-Dawley