Iurii Koboziev

Role of the Enteric Microbiota in Intestinal Homeostasis and Inflammation

The mammalian intestine encounters many more microorganisms than any other tissue in the body thus making it the largest and most complex component of the immune system. Indeed, there are greater than 100 trillion (10(14)) microbes within the healthy human intestine, and the total number of genes derived from this diverse microbiome exceeds that of the entire human genome by at least 100-fold. Our coexistence with the gut microbiota represents a dynamic and mutually beneficial relationship that is thought to be a major determinant of health and disease. Because of the potential for intestinal microorganisms to induce local and/or systemic inflammation, the intestinal immune system has developed a number of immune mechanisms to protect the host from pathogenic infections while limiting the inflammatory tissue injury that accompanies these immune responses. Failure to properly regulate intestinal mucosal immunity is thought to be responsible for the inflammatory tissue injury observed in the inflammatory bowel diseases (IBD; Crohn disease, ulcerative colitis). An accumulating body of experimental and clinical evidence strongly suggests that IBD results from a dysregulated immune response to components of the normal gut flora in genetically susceptible individuals. The objective of this review is to present our current understanding of the role that enteric microbiota play in intestinal homeostasis and pathogenesis of chronic intestinal inflammation.

Gut-associated lymphoid tissue, T cell trafficking, and chronic intestinal inflammation

The etiologies of the inflammatory bowel diseases (IBD; Crohns disease, ulcerative colitis) have not been fully elucidated. However, there is very good evidence implicating T cell and T cell trafficking to the gut and its associated lymphoid tissue as important components in disease pathogenesis. The objective of this review is to provide an overview of the mechanisms involved in naive and effector T cell trafficking to the gut‐associated lymphoid tissue (GALT; Peyers patches, isolated lymphoid follicles), mesenteric lymph nodes and intestine in response to commensal enteric antigens under physiological conditions as well as during the induction of chronic gut inflammation. In addition, recent data suggests that the GALT may not be required for enteric antigen‐driven intestinal inflammation in certain mouse models of IBD. These new data suggest a possible paradigm shift in our understanding of how and where naive T cells become activated to yield disease‐producing effector cells.

Pharmacological intervention studies using mouse models of the inflammatory bowel diseases: translating preclinical data into new drug therapies.

&NA; Most therapeutic agents used in clinical practice today were originally developed and tested in animal models so that drug toxicity and safety, dose‐responses, and efficacy could be determined. Retrospective analyses of preclinical intervention studies using animal models of different diseases demonstrate that only a small percentage of the interventions reporting promising effects translate to clinical efficacy. The failure to translate therapeutic efficacy from bench to bedside may be due, in part, to shortcomings in the design of the clinical studies; however, it is becoming clear that much of the problem resides within the preclinical studies. One potential strategy for improving our ability to identify new therapeutics that may have a reasonable chance of success in clinical trials is to identify the most immunologically‐relevant mouse models of IBD and pharmacologic strategies that most closely mimic the clinical situation. This review presents a critical evaluation of the different mouse models and pharmacological approaches that may be used in intervention studies as well as discuss emerging issues related to study design and data interpretation of preclinical studies. (Inflamm Bowel Dis 2011)

Use of Humanized Mice to Study the Pathogenesis of Autoimmune and Inflammatory Diseases

Abstract:Animal models of disease have been used extensively by the research community for the past several decades to better understand the pathogenesis of different diseases and assess the efficacy and toxicity of different therapeutic agents. Retrospective analyses of numerous preclinical intervention studies using mouse models of acute and chronic inflammatory diseases reveal a generalized failure to translate promising interventions or therapeutics into clinically effective treatments in patients. Although several possible reasons have been suggested to account for this generalized failure to translate therapeutic efficacy from the laboratory bench to the patients bedside, it is becoming increasingly apparent that the mouse immune system is substantially different from the human. Indeed, it is well known that >80 major differences exist between mouse and human immunology; all of which contribute to significant differences in immune system development, activation, and responses to challenges in innate and adaptive immunity. This inconvenient reality has prompted investigators to attempt to humanize the mouse immune system to address important human-specific questions that are impossible to study in patients. The successful long-term engraftment of human hematolymphoid cells in mice would provide investigators with a relatively inexpensive small animal model to study clinically relevant mechanisms and facilitate the evaluation of human-specific therapies in vivo. The discovery that targeted mutation of the IL-2 receptor common gamma chain in lymphopenic mice allows for the long-term engraftment of functional human immune cells has advanced greatly our ability to humanize the mouse immune system. The objective of this review is to present a brief overview of the recent advances that have been made in the development and use of humanized mice with special emphasis on autoimmune and chronic inflammatory diseases. In addition, we discuss the use of these unique mouse models to define the human-specific immunopathological mechanisms responsible for the induction and perpetuation of chronic gut inflammation.

Role of the Gut-associated and Secondary Lymphoid Tissue in the Induction of Chronic Colitis

Background: It is well known that enteric bacterial antigens drive the development of chronic colitis in a variety of different mouse models of the inflammatory bowel diseases (IBD). The objective of this study was to evaluate the role of gut‐associated lymphoid tissue (GALT; Peyers patches, isolated lymphoid follicles), mesenteric lymph nodes (MLNs) and spleen in the pathogenesis of chronic colitis in mice. Methods: Surgical as well as genetic approaches were used to generate lymphopenic mice devoid of one or more of these lymphoid tissues. For the first series of studies, we subjected recombinase activating gene‐1‐deficient mice (RAG−/−) to sham surgery (Sham), mesenteric lymphadenectomy (MLNx), splenectomy (Splx) or both (MLNx/Splx). In a second series of studies we intercrossed lymphotoxin&bgr;‐deficient (LT&bgr;−/−) mice with RAG−/− animals to generate LT&bgr;−/− x RAG−/− offspring that were anticipated to contain functional MLNs but be devoid of GALT and most peripheral lymph nodes. Flow purified naïve (CD4+CD45RBhigh) T‐cells were adoptively transferred into the different groups of RAG−/− recipients to induce chronic colitis. Results: We found that at 3‐5 wks following T‐cell transfer, all four of the surgically‐manipulated RAG−/− groups (Sham, MLNx, Splx and MLNx/Splx) developed chronic colitis that was similar in onset and severity. Flow cytometric analysis revealed no differences among the different groups with respect to surface expression of different gut‐homing markers nor were there any differences noted in IFN‐&ggr; and IL‐17 generation by mononuclear cells isolated among these surgically‐manipulated mice. Although we anticipated that LT&bgr;−/− x RAG−/− mice would contain functional MLNs but be devoid of GALT and peripheral lymph nodes (PLNs), we found that LT&bgr;−/− x RAG−/−mice were in fact devoid of MLNs as well as GALT and PLNs. Adoptive transfer of CD45RBhigh T‐cells into LT&bgr;−/− x RAG−/− mice or their littermate controls (LT&bgr;+/+ x RAG−/−) induced rapid and severe colitis in both groups. Conclusions: Taken together, our data demonstrate that: a) neither the GALT, MLNs nor PLNs are required for induction of chronic gut inflammation in this model of IBD and b) T‐and/or B‐cells may be required for the development of MLNs in LT&bgr;−/− mice. (Inflamm Bowel Dis 2011;)

Protective and pro-inflammatory roles of intestinal bacteria

The intestinal mucosal surface in all vertebrates is exposed to enormous numbers of microorganisms that include bacteria, archaea, fungi and viruses. Coexistence of the host with the gut microbiota represents an active and mutually beneficial relationship that helps to shape the mucosal and systemic immune systems of both mammals and teleosts (ray-finned fish). Due to the potential for enteric microorganisms to invade intestinal tissue and induce local and/or systemic inflammation, the mucosal immune system has developed a number of protective mechanisms that allow the host to mount an appropriate immune response to invading bacteria, while limiting bystander tissue injury associated with these immune responses. Failure to properly regulate mucosal immunity is thought to be responsible for the development of chronic intestinal inflammation. The objective of this review is to present our current understanding of the role that intestinal bacteria play in vertebrate health and disease. While our primary focus will be humans and mice, we also present the new and exciting comparative studies being performed in zebrafish to model host-microbe interactions.

Role of LFA-1 in the activation and trafficking of T cells: implications in the induction of chronic colitis.

Introduction: We have previously demonstrated that adoptive transfer of naïve CD4+ T cells devoid of lymphocyte function‐associated antigen‐1‐deficient (LFA‐1; CD11a/CD18) into recombination activating gene‐1 (RAG‐1) deficient (RAG−/−) mice fails to induce chronic colitis whereas transfer of wild type (WT) T‐cells induces unrelenting and chronic disease. Methods: The objectives of this study were to assess the role of lymphocyte function‐associated antigen‐1 (LFA‐1) in enteric antigen (EAg)‐induced activation of T cells in vitro and in vivo and to define the importance of this integrin in promoting trafficking of T cells to the mesenteric lymph nodes (MLNs) and colon. Results: We found that EAg‐pulsed dendritic cells (DCs) induced proliferation of LFA‐1‐deficient (CD11a−/−) CD4+ T cells that was very similar to that induced using WT T cells, suggesting that LFA‐1 is not required for activation/proliferation of T cells in vitro. Coculture of WT or CD11a−/− T cells with EAg‐pulsed DCs induced the generation of similar amounts of interferon‐gamma, interleukin (IL)‐4, and IL‐10, whereas IL‐17A production was reduced ≈2‐fold in cocultures with CD11a−/− T cells. Short‐term (20–22 hours) trafficking studies demonstrated that while both WT and CD11a−/− T cells migrated equally well into the spleen, liver, lungs, small intestine, cecum, and colon, trafficking of CD11a−/− T cells to the MLNs was reduced by 50% when compared to WT T cells. When the observation period was extended to 3–7 days posttransfer, we observed ≈2–3‐fold more WT T cells within the MLNs and colon than CD11a−/− T cells, whereas T‐cell proliferation (as measured by CFSE dilution) was comparable in both populations. Conclusions: Taken together, our data suggest that LFA‐1 is not required for EAg‐induced activation of CD4+ T cells in vitro or in vivo but is required for trafficking of T cells to the MLNs and homing of colitogenic effector cells to the colon where they initiate chronic gut inflammation. (Inflamm Bowel Dis 2012;)

Differential Susceptibility to T Cell-Induced Colitis in Mice: Role of the Intestinal Microbiota

One of the best characterized mouse models of the inflammatory bowel diseases (IBD; Crohns disease, ulcerative colitis) is the CD4+CD45RBhigh T cell transfer model of chronic colitis. Following our relocation to Texas Tech University Health Sciences Center (TTUHSC), we observed a dramatic reduction in the incidence of moderate-to-severe colitis from a 16-year historical average of 90% at Louisiana State University Health Sciences Center (LSUHSC) to <30% at TTUHSC. We hypothesized that differences in the commensal microbiota at the 2 institutions may account for the differences in susceptibility to T cell-induced colitis. Using bioinformatic analyses of 16S rRNA amplicon sequence data, we quantified and compared the major microbial populations in feces from healthy and colitic mice housed at the 2 institutions. We found that the bacterial composition differed greatly between mice housed at LSUHSC vs TTUHSC. We identified several genera strongly associated with, and signficantly overrepresented in high responding RAG-/- mice housed at LSUHSC. In addition, we found that colonization of healthy TTUHSC RAG-/- mice with feces obtained from healthy or colitic RAG-/- mice housed at LSUHSC transferred susceptibility to T cell-induced colitis such that the recipients developed chronic colitis with incidence and severity similar to mice generated at LSUHSC. Finally, we found that the treatment of mice with preexisting colitis with antibiotics remarkably attenuated disease. Taken together, our data demonstrate that specific microbial communities determine disease susceptibility and that manipulation of the intestinal microbiota alters the induction and/or perpetuation of chronic colitis.

S1772 LFA-1 is Required for T-Cell Homing to the Colon but Not for Enteric Antigen-Induced Activation of T-Cells During the Induction of Chronic Colitis

peripheral (PBMC) and lamina propria mononuclear cell (LPMC) activation, cell cycling, and apoptosis. To do so, T cells were isolated from control, Crohns disease, and ulcerative colitis specimens. After a 48-72h In Vitro stimulation in the presence of either 10 g/ml IFX, 50 g/ml IFX, 10 g/ml ADA, 50 g/ml ADA, or w/o any agents, the cells were fixed and FACS analysis was performed to determine activation (CD25), cell cycling (cyclin B1, PI), apoptosis/ necrosis (Annexin-V/PI), and expansion (CFDA). Immunoblotting was performed to determine key cell cycle regulators and NOTCH-1 expression. Transfection with siRNA was used to silence NOTCH-1mRNA levels. RESULTS: Both ADA and IFX significantly and comparably inhibited activation, cell cycle progression and expansion of anti-CD3-activated control, CD and UC PBMC and LPMC. Western blotting revealed that in all cell populations investigated, ADA and IFX robustly inhibit Rb phosphorylation, up-regulate the cell cycle inhibitor p21, but decrease p53 expression. As NOTCH-1 links cell cycling and apoptosis, both ADA and IFX increased NOTCH-1 activation. Silencing NOTCH-1 almost completely averted the TNF-a blocker-induced T cell cycle arrest. Interestingly, NOTCH-1 silencing also decreased the potency of ADA and IFX to induce T cell apoptosis. CONCLUSION: ADA and IXF both regulate T cell cycle promoter and inhibitor function. They potently restrict activation, cycling and expansion in normal, but also in IBD tissue, where unrestricted T cell expansion initiates and perpetuates the disease. By uncovering that NOTCH-1 mediates the inhibitory effects of ADA and IFX on T cell cycling, we provide not only a new mode of action, but also an underlying signalling pathway by which biologicals act in IBD.