Olga Turovskaya

Interleukin 10 acts on regulatory T cells to maintain expression of the transcription factor Foxp3 and suppressive function in mice with colitis

Regulatory T cells (Treg cells) that express the transcription factor Foxp3 suppress the activity of other cells. Here we show that interleukin 10 (IL-10) produced by CD11b+ myeloid cells in recombination-activating gene 1–deficient (Rag1−/−) recipient mice was needed to prevent the colitis induced by transferred CD4+CD45RBhi T cells. In Il10−/−Rag1−/− mice, Treg cells failed to maintain Foxp3 expression and regulatory activity. The loss of Foxp3 expression occurred only in recipients with colitis, which indicates that the requirement for IL-10 is manifested in the presence of inflammation. IL-10 receptor–deficient (Il10rb−/−) Treg cells also failed to maintain Foxp3 expression, which suggested that host IL-10 acted directly on the Treg cells. Our data indicate that IL-10 released from myeloid cells acts in a paracrine manner on Treg cells to maintain Foxp3 expression.

RAGE, carboxylated glycans and S100A8/A9 play essential roles in colitis-associated carcinogenesis.

Patients with inflammatory bowel diseases are at increased risk for colorectal cancer, but the molecular mechanisms linking inflammation and cancer are not well defined. We earlier showed that carboxylated N-glycans expressed on receptor for advanced glycation end products (RAGE) and other glycoproteins mediate colitis through activation of nuclear factor kappa B (NF-κB). Because NF-κB signaling plays a critical role in the molecular pathogenesis of colitis-associated cancer (CAC), we reasoned that carboxylated glycans, RAGE and its ligands might promote CAC. Carboxylated glycans are expressed on a subpopulation of RAGE on colon cancer cells and mediate S100A8/A9 binding to RAGE. Colon tumor cells express binding sites for S100A8/A9 and binding leads to activation of NF-κB and tumor cell proliferation. Binding, downstream signaling and tumor cell proliferation are blocked by mAbGB3.1, an anti-carboxylate glycan antibody, and by anti-RAGE. In human colon tumor tissues and in a mouse model of CAC, we found that myeloid progenitors expressing S100A8 and S100A9 infiltrate regions of dysplasia and adenoma. mAbGB3.1 administration markedly reduces chronic inflammation and tumorigenesis in the mouse model of CAC and RAGE-deficient mice are resistant to the onset of CAC. These findings show that RAGE, carboxylated glycans and S100A8/A9 play essential roles in tumor–stromal interactions, leading to inflammation-associated colon carcinogenesis.

NIK-dependent RelB Activation Defines a Unique Signaling Pathway for the Development of Vα14i NKT Cells

A defect in RelB, a member of the Rel/nuclear factor (NF)-κB family of transcription factors, affects antigen presenting cells and the formation of lymphoid organs, but its role in T lymphocyte differentiation is not well characterized. Here, we show that RelB deficiency in mice leads to a selective decrease of NKT cells. RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells. Like RelB-deficient mice, aly/aly mice with a mutation for the NF-κB–inducing kinase (NIK), have reduced NKT cell numbers. An analysis of NK1.1 and CD44 expression on NKT cells in the thymus of aly/aly mice reveals a late block in development. In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors. This link between NIK and RelB was further demonstrated in vivo by analyzing RelB+/− × aly/+ compound heterozygous mice. After stimulation with α-GalCer, an antigen recognized by NKT cells, these compound heterozygotes had reduced responses compared with either RelB+/− or aly/+ mice. These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.

A crucial role for HVEM and BTLA in preventing intestinal inflammation

The interaction between the tumor necrosis factor (TNF) family member LIGHT and the TNF family receptor herpes virus entry mediator (HVEM) co-stimulates T cells and promotes inflammation. However, HVEM also triggers inhibitory signals by acting as a ligand that binds to B and T lymphocyte attenuator (BTLA), an immunoglobulin super family member. The contribution of HVEM interacting with these two binding partners in inflammatory processes remains unknown. In this study, we investigated the role of HVEM in the development of colitis induced by the transfer of CD4+CD45RBhigh T cells into recombination activating gene (Rag)−/− mice. Although the absence of HVEM on the donor T cells led to a slight decrease in pathogenesis, surprisingly, the absence of HVEM in the Rag−/− recipients led to the opposite effect, a dramatic acceleration of intestinal inflammation. Furthermore, the critical role of HVEM in preventing colitis acceleration mainly involved HVEM expression by radioresistant cells in the Rag−/− recipients interacting with BTLA. Our experiments emphasize the antiinflammatory role of HVEM and the importance of HVEM expression by innate immune cells in preventing runaway inflammation in the intestine.

A Lymphotoxin-IFN-β Axis Essential for Lymphocyte Survival Revealed during Cytomegalovirus Infection

The importance of lymphotoxin (LT) βR (LTβR) as a regulator of lymphoid organogenesis is well established, but its role in host defense has yet to be fully defined. In this study, we report that mice deficient in LTβR signaling were highly susceptible to infection with murine CMV (MCMV) and early during infection exhibited a catastrophic loss of T and B lymphocytes, although the majority of lymphocytes were themselves not directly infected. Moreover, bone marrow chimeras revealed that lymphocyte survival required LTα expression by hemopoietic cells, independent of developmental defects in lymphoid tissue, whereas LTβR expression by both stromal and hemopoietic cells was needed to prevent apoptosis. The induction of IFN-β was also severely impaired in MCMV-infected LTα−/− mice, but immunotherapy with an agonist LTβR Ab restored IFN-β levels, prevented lymphocyte death, and enhanced the survival of these mice. IFN-αβR−/− mice were also found to exhibit profound lymphocyte death during MCMV infection, thus providing a potential mechanistic link between type 1 IFN induction and lymphocyte survival through a LTαβ-dependent pathway important for MCMV host defense.

Molecular imaging of murine intestinal inflammation with 2-deoxy-2-[18F]fluoro-D-glucose and positron emission tomography.

BACKGROUND & AIMS 2-Deoxy-2-[(18)F]fluoro-d-glucose (FDG) uptake by positron emission tomography (PET), a measure of glucose transporter activity, has been used to detect mucosal inflammation. However, there is limited understanding of the biologic basis of mucosal FDG uptake. METHODS A contrast-based computed tomographic isocontour method was developed to identify intestinal anatomic regions, and FDG uptake was integrated over these regions to achieve reproducible quantification during longitudinal assessment of individual mice. Intestinal FDG uptake was compared with histologic scores and with glucose transporter 1 levels in mucosal immune cells by flow cytometry. RESULTS Intestinal FDG uptake quantitatively correlated with disease activity in mild (C3H/HeJ.IL-10(-/-)) and severe (129.Galphai2(-/-), CD4(+) CD45RB(high), and Galphai2(-/-) CD3(+) transfer) murine colitis models at all time points examined (P < .05) and was sufficiently sensitive to detect preclinical inflammation. FDG uptake was correlated by flow cytometric detection of glucose transporter 1 levels in mucosal CD4(+) T lymphocyte but not other intestinal immune cell types. CD4(+) T-cell transfer in vivo confirmed that mucosal FDG uptake was associated with the activated but not quiescent state. When intestinal inflammation was increased by treatment with piroxicam and decreased with anti-TL1A treatment, FDG uptake was correspondingly altered. CONCLUSIONS This study clarifies the cellular basis of FDG signal in intestinal inflammation and introduces computed tomographic isocontour analysis of FDG-PET imaging for standardized quantitation of immune colitis.

Carboxylated Glycans Mediate Colitis through Activation of NF-κB

The role of carbohydrate modifications of glycoproteins in leukocyte trafficking is well established, but less is known concerning how glycans influence pathogenesis of inflammation. We previously identified a carboxylate modification of N-linked glycans that is recognized by S100A8, S100A9, and S100A12. The glycans are expressed on macrophages and dendritic cells of normal colonic lamina propria, and in inflammatory infiltrates in colon tissues from Crohn’s disease patients. We assessed the contribution of these glycans to the development of colitis induced by CD4+CD45RBhigh T cell transfer to Rag1−/− mice. Administration of an anti-carboxylate glycan Ab markedly reduced clinical and histological disease in preventive and early therapeutic protocols. Ab treatment reduced accumulation of CD4+ T cells in colon. This was accompanied by reduction in inflammatory cells, reduced expression of proinflammatory cytokines and of S100A8, S100A9, and receptor for advanced glycation end products. In vitro, the Ab inhibited expression of LPS-elicited cytokines and induced apoptosis of activated macrophages. It specifically blocked activation of NF-κB p65 in lamina propria cells of colitic mice and in activated macrophages. These results indicate that carboxylate-glycan-dependent pathways contribute to the early onset of colitis.

Elevated serum anti-I2 and anti-OmpW antibody levels in children with IBD

Background Bacteria are implicated as important factors in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to seek evidence of possible bacterial targets of the immune response related to IBD in children. Methods Seventy‐eight children referred to the Department of Paediatrics at Tampere University Hospital on suspicion of IBD were included in the study. Upper and lower gastrointestinal endoscopies with biopsies were performed on all children. Sera from 75 children were tested for antibodies to the Pseudomonas fluorescens‐associated sequence I2, a Bacteroides caccae TonB‐linked outer membrane protein, OmpW, anti‐Saccharomyces cerevisiae, and perinuclear anti‐neutrophil cytoplasmic antibodies. Results The IBD diagnosis was confirmed in 35 children (18 with Crohns disease [CD], 12 with ulcerative colitis [UC], and 5 with indeterminate colitis [IC]); 43 children were found to have no inflammation in the gut. Forty‐three percent (15 of 35) of those with IBD evinced positive seroreactivity to I2 and 46% (16 of 35) to OmpW. In CD, seroreactivity to I2 and OmpW was 50% (9 of 18) and 61% (11 of 18), respectively. Serum anti‐I2 and anti‐OmpW immunoglobulin A levels were significantly elevated in children with CD in comparison with the non‐IBD group (P = 0.007 and P = 0.001, respectively). A combination of OmpW, I2, and anti‐S cerevisiae tests identified 94% of CD patients, and a combination of OmpW, I2, and perinuclear anti‐neutrophil cytoplasmic antibodies detected 83% of UC cases. Conclusions Among children with IBD, strong serological responses to microbial antigens can be found, suggesting that P fluorescens and B caccae antigens have a potential role in the microbiology and immunology of the disease. Furthermore, serologic reactivity to the set of antigens studied here seems to be applicable in the initial differential diagnosis of children with CD and UC.

Villous B Cells of the Small Intestine Are Specialized for Invariant NK T Cell Dependence

B cells are important in mucosal microbial homeostasis through their well-known role in secretory IgA production and their emerging role in mucosal immunoregulation. Several specialized intraintestinal B cell compartments have been characterized, but the nature of conventional B cells in the lamina propria is poorly understood. In this study, we identify a B cell population predominantly composed of surface IgM+ IgD+ cells residing in villi of the small intestine and superficial lamina propria of the large intestine, but distinct from the intraepithelial compartment or organized intestinal lymphoid structures. Small intestinal (villous) B cells are diminished in genotypes that alter the strength of BCR signaling (Bruton tyrosine kinasexid, Gαi2−/−), and in mice lacking cognate BCR specificity. They are not dependent on enteric microbial sensing, because they are abundant in mice that are germfree or genetically deficient in TLR signaling. However, villous B cells are reduced in the absence of invariant NK T cells (Jα18−/− or CD1d−/− mice). These findings define a distinct population of conventional B cells in small intestinal villi, and suggest an immunologic link between CD1-restricted invariant NK T cells and this B cell population.

Integration of B cells and CD8 + T in the protective regulation of systemic epithelial inflammation

Mechanisms that control abnormal CD4(+) T cell-mediated tissue damage are a significant factor in averting and resolving chronic inflammatory epithelial diseases. B cells can promote such immunoregulation, and this is thought to involve interaction with MHC II- or CD1-restricted regulatory T cells. The purpose of this study is to genetically define the interacting cells targeted by protective B cells, and to elucidate their regulatory mechanisms in CD4(+) T cell inflammation. Transfer of G alpha i2-/- CD3(+) T cells into lymphopenic mice causes a dose-dependent multi-organ inflammatory disease including the skin, intestine, and lungs. Disease activity is associated with elevated levels of serum TNF-alpha and IFN-gamma, and an activated IL-17 producing CD4(+) T cell population. Mesenteric node B cells from wild type mice suppress disease activity, serum cytokine expression, and levels of CD4(+) T cells producing TNF-alpha IFN-gamma, and IL-17. The protective function of B cells requires genetic sufficiency of IL-10, MHC I and TAP1. Regulatory B cells induce the expansion and activation of CD8(+) T cells, which is correlated with disease protection. These results demonstrate that CD8(+) T cells can ameliorate lymphopenic systemic inflammatory disease, through peptide/MHC I-dependent B cell interaction.