J. Anthony Brandon

Multifactorial patterns of gene expression in colonic epithelial cells predict disease phenotypes in experimental colitis

Background: The pathogenesis of inflammatory bowel disease (IBD) is complex and the need to identify molecular biomarkers is critical. Epithelial cells play a central role in maintaining intestinal homeostasis. We previously identified five “signature” biomarkers in colonic epithelial cells (CEC) that are predictive of disease phenotype in Crohns disease. Here we investigate the ability of CEC biomarkers to define the mechanism and severity of intestinal inflammation. Methods: We analyzed the expression of RelA, A20, pIgR, tumor necrosis factor (TNF), and macrophage inflammatory protein (MIP)‐2 in CEC of mice with dextran sodium sulfate (DSS) acute colitis or T‐cell‐mediated chronic colitis. Factor analysis was used to combine the five biomarkers into two multifactorial principal components (PCs). PC scores for individual mice were correlated with disease severity. Results: For both colitis models, PC1 was strongly weighted toward RelA, A20, and pIgR, and PC2 was strongly weighted toward TNF and MIP‐2, while the contributions of other biomarkers varied depending on the etiology of inflammation. Disease severity was correlated with elevated PC2 scores in DSS colitis and reduced PC1 scores in T‐cell transfer colitis. Downregulation of pIgR was a common feature observed in both colitis models and was associated with altered cellular localization of pIgR and failure to transport IgA. Conclusions: A multifactorial analysis of epithelial gene expression may be more informative than examining single gene responses in IBD. These results provide insight into the homeostatic and proinflammatory functions of CEC in IBD pathogenesis and suggest that biomarker analysis could be useful for evaluating therapeutic options for IBD patients. (Inflamm Bowel Dis 2012;)

Murine Syngeneic Graft-Versus-Host Disease Is Responsive to Broad-Spectrum Antibiotic Therapy

Murine syngeneic graft-versus-host disease (SGVHD) initiates colon and liver inflammation following lethal irradiation, reconstitution with syngeneic bone marrow transplantation, and therapy with the immunosuppressive agent cyclosporine A. Previous studies have demonstrated that the inducible disease is mediated by CD4+ T cells with increased reactivity of peripheral and liver-associated lymphocytes against intestinal microbial Ags. In the current report, studies were performed to analyze the specificity of the CD4+ T cell response of T cells isolated from diseased animals and to determine the in vivo role of the microbiota to the development of SGVHD. Increased major histocompatibility Ag (MHC) class II-restricted responsiveness of SGVHD CD4+ T cells against microbial Ags isolated from the ceca of normal animals was observed. The enhanced proliferative response was observed in the CD62L− memory population of CD4+ T cells. To determine the role of the bacterial microbiota in the development of murine SGVHD, control and CsA-treated bone marrow transplantation animals were treated with broad-spectrum antibiotics (metronidazole, ciprofloxacin) after transplantation. Cyclosporine A-treated animals that were given antibiotic therapy failed to develop clinical symptoms and pathological lesions in the target tissues characteristic of SGVHD. Furthermore, the reduction in intestinal bacteria resulted in the elimination of the enhanced antimicrobial CD4+ T cell response and significantly reduced levels of the inflammatory cytokines, IFN-γ, IL-17, and TNF-α. The elimination of the disease-associated inflammatory immune responses and pathology by treatment with broad-spectrum antibiotics definitively links the role of the microbiota and microbial-specific immunity to the development of murine SGVHD.

Development of a TH17 immune response during the induction of murine syngeneic graft-versus-host disease

Syngeneic graft-versus-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow (BM) and treatment with a 21 day course of the immunosuppressive agent cyclosporine A (CsA). Clinical symptoms of SGVHD appear 2-3 weeks post-CsA with inflammation occurring in the colon and liver. Previously we have demonstrated that CD4(+) T cells and a T helper cell type 1 cytokine response (T(H)1) are involved in the development of SGVHD associated intestinal inflammation. Studies have recently discovered an additional T cell lineage that produces IL-17 and is termed T(H)17. It has been suggested that inflammatory bowel disease is a result of a T(H)17 response rather than a T(H)1 response. This study was designed to investigate T(H)17 involvement in SGVHD-associated colitis. Following induction of SGVHD, the levels of T(H)17 and T(H)1 cytokine mRNA and protein were measured in control and SGVHD animals. In vivo cytokine neutralization was performed to determine the role of the prototypic T(H)17 cytokine, IL-17, in the disease process. We found that during CsA-induced murine SGVHD there was an increase in both T(H)17 and T(H)1 mRNA and cytokines within the colons of diseased mice. The administration of an anti-mouse IL-17A mAb did not alter the course of disease. However, neutralization of IL-17A resulted in an increased production of IL-17F, a related family member, with an overlapping range of effector activities. These results demonstrate that in the pathophysiology of SGVHD, there is a redundancy in the T(H)17 effector molecules that mediate the development of SGVHD.

Association between chronic liver and colon inflammation during the development of murine syngeneic graft-versus-host disease

The murine model of cyclosporine A (CsA)-induced syngeneic graft-versus-host disease (SGVHD) is a bone marrow (BM) transplantation model that develops chronic colon inflammation identical to other murine models of CD4(+) T cell-mediated colitis. Interestingly, SGVHD animals develop chronic liver lesions that are similar to the early peribiliary inflammatory stages of clinical chronic liver disease, which is frequently associated with inflammatory bowel disease (IBD). Therefore, studies were initiated to investigate the chronic liver inflammation that develops in the SGVHD model. To induce SGVHD, mice were lethally irradiated, reconstituted with syngeneic BM, and treated with CsA. All of the SGVHD animals that developed colitis also develop chronic liver inflammation. Liver samples from control and SGVHD animals were monitored for tissue pathology, RNA for inflammatory mediators, and phenotypic analysis and in vitro reactivity of the inflammatory infiltrate. Diseased animals developed lesions of intrahepatic and extrahepatic bile ducts. Elevated levels of mRNA for molecules associated with chronic liver inflammation, including mucosal cellular adhesion molecule -1, the chemokines CCL25, CCL28, CCR9, and T(H)1- and T(H)17-associated cytokines were observed in livers of SGVHD mice. CD4(+) T cells were localized to the peribiliary region of the livers of diseased animals, and an enhanced proliferative response of liver-associated mononuclear cells against colonic bacterial antigens was observed. The murine model of SGVHD colitis may be a valuable tool to study the entero-hepatic linkage between chronic colon inflammation and inflammatory liver disease.

Accumulation of CD4 T cells in the colon of CsA-treated mice following myeloablative conditioning and bone marrow transplantation

Syngeneic graft vs. host disease (SGVHD) was first described as a graft vs. host disease-like syndrome that developed in rats following syngeneic bone marrow transplantation (BMT) and cyclosporin A (CsA) treatment. SGVHD can be induced by reconstitution of lethally irradiated mice with syngeneic bone marrow cells followed by 21 days of treatment with the immunosuppressive agent CsA. Clinical symptoms of the disease appear 2-3 wk following cessation of CsA therapy, and disease-associated inflammation occurs primarily in the colon and liver. CD4(+) T cells have been shown to play an important role in the inflammatory response observed in the gut of SGVHD mice. Time-course studies revealed a significant increase in migration of CD4(+) T cells into the colon during CsA therapy, as well as significantly elevated mRNA levels of TNF-α, proinflammatory chemokines, and cell adhesion molecules in colonic tissue of CsA-treated animals compared with BMT controls, as early as day 14 post-BMT. Homing studies revealed a greater migration of labeled CD4(+) T cells into the gut of CsA-treated mice at day 21 post-BMT than control animals via CsA-induced upregulation of mucosal addressin cell adhesion molecule. This study demonstrates that, during the 21 days of immunosuppressive therapy, functional mechanisms are in place that result in increased homing of CD4(+) T effector cells to colons of CsA-treated mice.

Induction of murine syngeneic graft-versus-host disease by cells of recipient origin

Background. Syngeneic graft-versus-host disease (SGVHD) develops after lethal irradiation, reconstitution with syngeneic bone marrow (BM), and treatment with a 21-day course of the immunosuppressant cyclosporine A (CsA). Clinical symptoms of SGVHD appear 2-3 weeks after CsA treatment, with inflammation in the colon and liver. It has been demonstrated that CD4+ T cells and a T helper cell type 1 cytokine response (Th1) are involved in the development of SGVHD associated intestinal inflammation. The immune response associated with SGVHD is thought to be the result of the reconstitution of the recipient immune system with the syngeneic donor BM. However, definitive studies have not addressed this issue experimentally. Methods. To determine the origin of the effector cells that participate in SGVHD, C3H/HeN recipient mice were lethally irradiated and transplanted with BM from normal immunocompetent mice or from immunodeficient, severe combined immune deficient, or Rag-2−/− animals. Results. CsA-treated animals, but not control animals, developed inflammation characteristic of SGVHD in the colon and liver regardless of the source of the donor marrow. Furthermore, immunologically, all CsA treated animals responded similarly with increased production of inflammatory cytokines and an increase in activated CD4+ T cells in the periphery and colon relative to controls. Conclusion. These results demonstrate that after lethal irradiation and in the absence of donor T cells, T cells of recipient origin can expand and mediate the induction of CsA-induced SGVHD.

Anti-IL-23p19 therapy inhibits the adoptive transfer of syngeneic graft-versus-host disease

Syngeneic graft-versus-host disease (SGVHD), a chronic inflammatory disease, develops following irradiation, syngeneic bone marrow transplantation (BMT) and treatment with the immunosuppressive agent cyclosporine A (CsA). We have shown that TH1 and TH17 cytokine responses are increased during the development of SGVHD. The current study was designed to further investigate the involvement of TH17 immunity in SGVHD-associated colitis. IL-23 is a TH17 cytokine responsible for maintaining the effector functions of TH17 cells. The administration of anti-mouse IL-23p19 was shown to significantly reduce the clinical symptoms of primary and secondary SGVHD-associated colitis resulting in a significant reduction in both TH1 and TH17 associated cytokine expression. These results demonstrate that the TH17-associated cytokine, IL-23, may prove to be a beneficial therapeutic target in the treatment of chronic colon inflammation.

A gut feeling about murine syngeneic GVHD

Murine syngeneic graft-versus-host disease (SGVHD) results in chronic colon and liver inflammation following syngeneic bone marrow transplantation (BMT) and treatment with the calcineurin inhibitor, cyclosporine A (CsA). SGVHD was initially thought to arise as a result of an autoreactive immune response, but more recently it has been shown that enhanced antimicrobial responses develop in SGVHD mice. Consequently, we performed studies to analyze the role of the microbiota in the development of murine SGVHD. Treatment with broad-spectrum antibiotics eliminated disease-associated inflammatory immune responses and pathology, linking the role of the microbiota and microbial-specific immunity to the development of murine SGVHD. In a broader context, these results bring into question the role that anti-microbial immune responses play in post-transplant immune pathologies that develop following allogeneic stem cell transplantation and use of calcineurin inhibitors.

Non-invasive detection of adeno-associated viral gene transfer using a genetically encoded CEST-MRI reporter gene in the murine heart

Research into gene therapy for heart failure has gained renewed interest as a result of improved safety and availability of adeno-associated viral vectors (AAV). While magnetic resonance imaging (MRI) is standard for functional assessment of gene therapy outcomes, quantitation of gene transfer/expression relies upon tissue biopsy, fluorescence or nuclear imaging. Imaging of gene expression through the use of genetically encoded chemical exchange saturation transfer (CEST)-MRI reporter genes could be combined with clinical cardiac MRI methods to comprehensively probe therapeutic gene expression and subsequent outcomes. The CEST-MRI reporter gene Lysine Rich Protein (LRP) was cloned into an AAV9 vector and either administered systemically via tail vein injection or directly injected into the left ventricular free wall of mice. Longitudinal in vivo CEST-MRI performed at days 15 and 45 after direct injection or at 1, 60 and 90 days after systemic injection revealed robust CEST contrast in myocardium that was later confirmed to express LRP by immunostaining. Ventricular structure and function were not impacted by expression of LRP in either study arm. The ability to quantify and link therapeutic gene expression to functional outcomes can provide rich data for further development of gene therapy for heart failure.

Role of Oxidative Stress in the Colonic Complications of Murine Syngeneic Graft-versus-host Disease

Syngeneic graft-versus-host disease (SGVHD) develops in mice following lethal irradiation, reconstitution with syngeneic bone marrow (BM) and treatment with a short course of the immunosuppressive agent cyclosporine A (CsA). The development of SGVHD is a complex process resulting from the cooperative interaction of multiple effector cell populations and inflammatory mediators contributing to the pathogenesis of this inducible disease. Using gene ex- pression analysis, flow cytometric analysis and immunohistochemistry, time course studies revealed increased reactive oxygen and nitrogen species in the tissues of CsA-treated animals compared to bone marrow transplantation (BMT) con- trols during the induction of SGVHD (d0-21 post-BMT). Studies were undertaken to determine the effect of CsA-induced oxidative stress on the induction of SGVHD. In vivo treatment with the superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin (MnTBAP), during (d0-21 post BMT), or after CsA therapy (>d21 post-BMT), caused a reduction in the development of clinical symptoms of SGVHD (weight loss, diarrhea). Interestingly, treatment with MnTBAP resulted in a significant reduction in the deposition of peroxynitrite in the colons of CsA-MnTBAP-treated versus control CsA-treated SGVHD animals. These studies suggest a role for oxidative stress in the development of mur- ine SGVHD.