IRF4 regulates IL-17A promoter activity and controls ROR?t-dependent Th17 colitis in vivo

The transcription factor IRF4 is involved in several T-cell-dependent chronic inflammatory diseases. To elucidate the mechanisms for pathological cytokine production in colitis, we addressed the role of the IRF transcription factors in human inflammatory bowel disease (IBD) and experimental colitis.

The transcription factor IFN regulatory factor-4 controls experimental colitis in mice via T cell-derived IL-6

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.

Bone healing around titanium implants in two rat colitis models

OBJECTIVE Crohn's disease is a chronic inflammatory process that has recently been associated with a higher risk of early implant failure. Herein we provide information on the impact of colitis on peri-implant bone formation using preclinical models of chemically induced colitis. METHODS Colitis was induced by intrarectal instillation of 2,4,6-trinitro-benzene-sulfonic-acid (TNBS). Colitis was also induced by feeding rats dextran-sodium-sulfate (DSS) in drinking water. One week after disease induction, titanium miniscrews were inserted into the tibia. Four weeks after implantation, peri-implant bone volume per tissue volume (BV/TV) and bone-to-implant contacts (BIC) were determined by histomorphometric analysis. RESULTS Cortical histomorphometric parameters were similar in the control (n = 10), DSS (n = 10) and TNBS (n = 8) groups. Cortical BV/TV was 92.2 ± 3.7%, 92.0 ± 3.0% and 92.6 ± 2.7%. Cortical BIC was 81.3 ± 8.8%, 83.2 ± 8.4% and 84.0 ± 7.0%, respectively. No significant differences were observed when comparing the medullary BV/TV and BIC (19.5 ± 6.4%, 16.2 ± 5.6% and 15.4 ± 9.0%) and (48.8 ± 12.9%, 49.2 ± 6.2 and 41.9 ± 11.7%), respectively. Successful induction of colitis was confirmed by loss of body weight and colon morphology. CONCLUSIONS The results suggest bone regeneration around implants is not impaired in chemically induced colitis models. Considering that Crohn's disease can affect any part of the gastrointestinal tract including the mouth, our model only partially reflects the clinical situation.

Transcriptional analysis of left-sided colitis, pancolitis, and ulcerative colitis-associated dysplasia.

BACKGROUND It is unknown why patients with extensive ulcerative colitis (UC) have a higher risk of colorectal cancer compared with patients with left-sided UC. This study characterizes the inflammatory processes in left-sided UC, pancolitis, and UC-associated dysplasia at the transcriptional level to identify potential biomarkers and transcripts of importance for the carcinogenic behavior of chronic inflammation. METHODS The Affymetrix GeneChip Human Genome U133 Plus 2.0 was applied on colonic biopsies from UC patients with left-sided UC, pancolitis, dysplasia, and controls. Reverse transcription polymerase chain reaction and immunohistochemistry were performed for validating selected transcripts in the initial cohort and in 2 independent cohorts of patients with UC. Microarray data were analyzed by principal component analysis, and reverse transcription polymerase chain reaction and immunohistochemistry data by the Wilcoxon's rank-sum test. RESULTS The principal component analysis results revealed separate clusters for left-sided UC, pancolitis, dysplasia, and controls. Close clustering of dysplastic and pancolitic samples indicated similarities in gene expression. Indeed, 101 and 656 parallel upregulated and downregulated transcripts, respectively, were identified in specimens from dysplasia and pancolitis. Validation of selected transcripts hereof identified insulin receptor alpha (INSRA) and MAP kinase interacting serine/threonine kinase 2 (MKNK2) with an enhanced expression in dysplasia compared with left-sided UC and controls, whereas laminin γ2 (LAMC2) was found with a lower expression in dysplasia compared with the remaining 3 groups. CONCLUSIONS This study demonstrates pancolitis and left-sided UC as distinct inflammatory processes at the transcriptional level, and identifies INSRA, MKNK2, and LAMC2 as potential critical transcripts in the inflammation-driven preneoplastic process of UC.

CD4 T cells are required for both development and maintenance of disease in a new mouse model of reversible colitis

Current therapies to treat inflammatory bowel diseases have limited efficacy, significant side effects, and often wane over time. Little is known about the cellular and molecular mechanisms operative in the process of mucosal healing from colitis. To study such events, we developed a new model of reversible colitis in which adoptive transfer of CD4(+)CD45RB(hi) T cells into Helicobacter typhlonius-colonized lymphopenic mice resulted in a rapid onset of colonic inflammation that was reversible through depletion of colitogenic T cells. Remission was associated with an improved clinical and histopathological score, reduced immune cell infiltration to the intestinal mucosa, altered intestinal gene expression profiles, regeneration of the colonic mucus layer, and the restoration of epithelial barrier integrity. Notably, colitogenic T cells were not only critical for induction of colitis but also for maintenance of disease. Depletion of colitogenic T cells resulted in a rapid drop in tumor necrosis factor α (TNFα) levels associated with reduced infiltration of inflammatory immune cells to sites of inflammation. Although neutralization of TNFα prevented the onset of colitis, anti-TNFα treatment of mice with established disease failed to resolve colonic inflammation. Collectively, this new model of reversible colitis provides an important research tool to study the dynamics of mucosal healing in chronic intestinal remitting-relapsing disorders.Mucosal Immunology advance online publication 16 September 2015; doi:10.1038/mi.2015.93.

The Role of IL-25 in Colitis-Associated Colon Cancer and Pulmonary Inflammation

Thesis (Ph.D.)--University of Washington, 2016-06

Characterization of tumour-infiltrating lymphocytes and apoptosis in colitis-associated neoplasia: comparison with sporadic colorectal cancer

The development of colorectal cancer is a major complication for patients with chronic idiopathic colitis. Colitis-associated tumours tend to occur at a younger age and be more aggressive than sporadic colorectal cancers. While we have previously associated the presence of tumour-infiltrating lymphocytes (TILs) and increased apoptosis in sporadic colorectal cancer with high-level microsatellite instability and improved prognosis, little is known of the relationship between these variables in colitis-associated colorectal cancer. The aim of this study was to correlate TILs and tumour cell apoptosis in colitis-associated neoplasms stratified according to microsatellite instability. Twenty tumour and 11 dysplastic samples resected from 21 patients with long-standing colitis were analysed for microsatellite instability at 10 microsatellite markers. TIL distribution (CD3, CD8) and function (granzyme B) were quantified by immunohistochemistry. Neoplastic cell apoptosis was assessed using the M30 CytoDEATH antibody. These findings were compared with 40 microsatellite stable (MSS) sporadic colorectal cancers previously evaluated for TILs and neoplastic apoptosis. Low-level microsatellite instability was found in 1/20 colitis-associated tumours. All other colitis-associated lesions were designated MSS. CD3(+) and CD8(+) TIL counts were significantly higher in colitis-associated lesions compared with NISS sporadic colorectal cancer (p < 0.0001, p = 0.001 respectively). Despite their higher TIL density, colitis-associated tumours were more likely to present late (Dukes' stage C or D) (P = 0.02). Functionally, colitis-associated TILs demonstrated significantly less granzyme B expression compared to sporadic cancers (p = 0.002). The level of tumour cell apoptosis was similar between the two groups (sporadic, 1.53%; colitis cancers, 1.45%). In conclusion, NISS colitis-associated tumours have a higher prevalence of CD3(+)/CD8(+) TILs but no associated increase in tumour cell killing by apoptosis. Unlike cytotoxic T cells in sporadic colorectal cancer, TILs do not appear to enhance the prognosis of colitis-associated colorectal cancer. This may be related to an impairment of granzyme B expression within these lesions. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Comparison of prophylactic and therapeutic use of short-chain fatty acid enemas in diversion colitis: a study in Wistar rats

OBJECTIVES: To study the effect of short-chain fatty-acids on atrophy and inflammation of excluded colonic segments before and after the development of diversion colitis. INTRODUCTION: Diversion colitis is a chronic inflammatory process affecting the dysfunctional colon, possibly evolving with mucous and blood discharge. The most favored hypotheses to explain its development is short-chain fatty-acid deficiency in the colon lumen. METHODS: Wistar rats were submitted to colostomy with distal colon exclusion. Two control groups (A1 and B1) received rectally administered physiological saline, whereas two experimental groups (A2 and B2) received rectally administered short-chain fatty-acids. The A groups were prophylactically treated (5th to 40th days postoperatively), whereas the B groups were therapeutically treated (after post-operative day 40). The mucosal thickness of the excluded colon was measured histologically. The inflammatory reaction of the mucosal lamina propria and the lymphoid tissue response were quantified through established scores. RESULTS: There was a significant thickness recovery of the colonic mucosa in group B2 animals (p = 0.0001), which also exhibited a significant reduction in the number of eosinophilic polymorphonuclear cells in the lamina propria (p = 0.0126) and in the intestinal lumen (p = 0.0256). Group A2 showed no mucosal thickness recovery and significant increases in the numbers of lymphocytes (p = 0.0006) and eosinophilic polymorphonuclear cells in the lamina propria of the mucosa (p = 0.0022). CONCLUSION: Therapeutic use of short-chain fatty-acids significantly reduced eosinophilic polymorphonuclear cell numbers in the intestinal wall and in the colonic lumen; it also reversed the atrophy of the colonic mucosa. Prophylactic use did not impede the development of mucosal atrophy

Comparison of prophylactic and therapeutic use of short-chain fatty acid enemas in diversion colitis: a study in Wistar rats

OBJECTIVES: To study the effect of short-chain fatty-acids on atrophy and inflammation of excluded colonic segments before and after the development of diversion colitis. INTRODUCTION: Diversion colitis is a chronic inflammatory process affecting the dysfunctional colon, possibly evolving with mucous and blood discharge. The most favored hypotheses to explain its development is short-chain fatty-acid deficiency in the colon lumen. METHODS: Wistar rats were submitted to colostomy with distal colon exclusion. Two control groups (A1 and B1) received rectally administered physiological saline, whereas two experimental groups (A2 and B2) received rectally administered short-chain fatty-acids. The A groups were prophylactically treated (5th to 40th days postoperatively), whereas the B groups were therapeutically treated (after post-operative day 40). The mucosal thickness of the excluded colon was measured histologically. The inflammatory reaction of the mucosal lamina propria and the lymphoid tissue response were quantified through established scores. RESULTS: There was a significant thickness recovery of the colonic mucosa in group B2 animals (p = 0.0001), which also exhibited a significant reduction in the number of eosinophilic polymorphonuclear cells in the lamina propria (p = 0.0126) and in the intestinal lumen (p = 0.0256). Group A2 showed no mucosal thickness recovery and significant increases in the numbers of lymphocytes (p = 0.0006) and eosinophilic polymorphonuclear cells in the lamina propria of the mucosa (p = 0.0022). CONCLUSION: Therapeutic use of short-chain fatty-acids significantly reduced eosinophilic polymorphonuclear cell numbers in the intestinal wall and in the colonic lumen; it also reversed the atrophy of the colonic mucosa. Prophylactic use did not impede the development of mucosal atrophy

Development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to T cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/-recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts. This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

Quantitative electroencephalographic comodulation : an investigation of patterns in chronic fatigue syndrome

Introduction. This is a pilot study of quantitative electro-encephalographic (QEEG) comodulation analysis, which is used to assist in identifying regional brain differences in those people suffering from chronic fatigue syndrome (CFS) compared to a normative database. The QEEG comodulation analysis examines spatial-temporal cross-correlation of spectral estimates in the resting dominant frequency band. A pattern shown by Sterman and Kaiser (2001) and referred to as the anterior posterior dissociation (APD) discloses a significant reduction in shared functional modulation between frontal and centro-parietal areas of the cortex. This research attempts to examine whether this pattern is evident in CFS. Method. Eleven adult participants, diagnosed by a physician as having CFS, were involved in QEEG data collection. Nineteen-channel cap recordings were made in five conditions: eyes-closed baseline, eyes-open, reading task one, math computations task two, and a second eyes-closed baseline. Results. Four of the 11 participants showed an anterior posterior dissociation pattern for the eyes-closed resting dominant frequency. However, seven of the 11 participants did not show this pattern. Examination of the mean 8-12 Hz amplitudes across three cortical regions (frontal, central and parietal) indicated a trend of higher overall alpha levels in the parietal region in CFS patients who showed the APD pattern compared to those who did not have this pattern. All patients showing the pattern were free of medication, while 71% of those absent of the pattern were using antidepressant medications. Conclusions. Although the sample is small, it is suggested that this method of evaluating the disorder holds promise. The fact that this pattern was not consistently represented in the CFS sample could be explained by the possibility of subtypes of CFS, or perhaps co-morbid conditions. Further, the use of antidepressant medications may mask the pattern by altering the temporal characteristics of the EEG. The results of this pilot study indicate that further research is warranted to verify that the pattern holds across the wider population of CFS sufferers.

Exploring non-cancer pain conditions in a community sample : critiquing a current conceptual model of the acute to chronic pain transition and examining predictors of chronicity

This program of research examines the experience of chronic pain in a community sample. While, it is clear that like patient samples, chronic pain in non-patient samples is also associated with psychological distress and physical disability, the experience of pain across the total spectrum of pain conditions (including acute and episodic pain conditions) and during the early course of chronic pain is less clear. Information about these aspects of the pain experience is important because effective early intervention for chronic pain relies on identification of people who are likely to progress to chronicity post-injury. A conceptual model of the transition from acute to chronic pain was proposed by Gatchel (1991a). In brief, Gatchel’s model describes three stages that individuals who have a serious pain experience move through, each with worsening psychological dysfunction and physical disability. The aims of this program of research were to describe the experience of pain in a community sample in order to obtain pain-specific data on the problem of pain in Queensland, and to explore the usefulness of Gatchel’s Model in a non-clinical sample. Additionally, five risk factors and six protective factors were proposed as possible extensions to Gatchel’s Model. To address these aims, a prospective longitudinal mixed-method research design was used. Quantitative data was collected in Phase 1 via a comprehensive postal questionnaire. Phase 2 consisted of a follow-up questionnaire 3 months post-baseline. Phase 3 consisted of semi-structured interviews with a subset of the original sample 12 months post follow-up, which used qualitative data to provide a further in-depth examination of the experience and process of chronic pain from respondents’ point of view. The results indicate chronic pain is associated with high levels of anxiety and depressive symptoms. However, the levels of disability reported by this Queensland sample were generally lower than those reported by clinical samples and consistent with disability data reported in a New South Wales population-based study. With regard to the second aim of this program of research, while some elements of the pain experience of this sample were consistent with that described by Gatchel’s Model, overall the model was not a good fit with the experience of this non-clinical sample. The findings indicate that passive coping strategies (minimising activity), catastrophising, self efficacy, optimism, social support, active strategies (use of distraction) and the belief that emotions affect pain may be important to consider in understanding the processes that underlie the transition to and continuation of chronic pain.

A three species model to simulate application of hyperbaric oxygen therapy to chronic wounds

Chronic wounds are a significant socioeconomic problem for governments worldwide. Approximately 15% of people who suffer from diabetes will experience a lower-limb ulcer at some stage of their lives, and 24% of these wounds will ultimately result in amputation of the lower limb. Hyperbaric Oxygen Therapy (HBOT) has been shown to aid the healing of chronic wounds; however, the causal reasons for the improved healing remain unclear and hence current HBOT protocols remain empirical. Here we develop a three-species mathematical model of wound healing that is used to simulate the application of hyperbaric oxygen therapy in the treatment of wounds. Based on our modelling, we predict that intermittent HBOT will assist chronic wound healing while normobaric oxygen is ineffective in treating such wounds. Furthermore, treatment should continue until healing is complete, and HBOT will not stimulate healing under all circumstances, leading us to conclude that finding the right protocol for an individual patient is crucial if HBOT is to be effective. We provide constraints that depend on the model parameters for the range of HBOT protocols that will stimulate healing. More specifically, we predict that patients with a poor arterial supply of oxygen, high consumption of oxygen by the wound tissue, chronically hypoxic wounds, and/or a dysfunctional endothelial cell response to oxygen are at risk of nonresponsiveness to HBOT. The work of this paper can, in some way, highlight which patients are most likely to respond well to HBOT (for example, those with a good arterial supply), and thus has the potential to assist in improving both the success rate and hence the costeffectiveness of this therapy.

Collaborative recovery: An integrative model for working with individuals that experience chronic or recurring mental illness.

Objectives: Recovery is an emerging movement in mental health. Evidence for recovery-based approaches is not well developed and approaches to implement recovery-oriented services are not well articulated. The collaborative recovery model (CRM) is presented as a model that assists clinicians to use evidence-based skills with consumers, in a manner consistent with the recovery movement. A current 5 year multisite Australian study to evaluate the effectiveness of CRM is briefly described. Conclusion: The collaborative recovery model puts into practice several aspects of policy regarding recovery-oriented services, using evidence-based practices to assist individuals who have chronic or recurring mental disorders (CRMD). It is argued that this model provides an integrative framework combining (i) evidence-based practice; (ii) manageable and modularized competencies relevant to case management and psychosocial rehabilitation contexts; and (iii) recognition of the subjective experiences of consumers.