Mona Bajaj-Elliott

Imbalance of stromelysin-1 and TIMP-1 in the mucosal lesions of children with inflammatory bowel disease

BACKGROUND Degradation of the extracellular matrix and ulceration of the mucosa are major features of inflammatory bowel disease (IBD). One of the most important enzymes in degrading the matrix and produced in excess by cytokine activated stromal cells, is stromelysin-1. The activity of stromelysin-1 is controlled by tissue inhibitor of metalloproteinase (TIMP-1), its natural inhibitor. In model systems excess stromelysin-1 produces mucosal degradation. METHODS Quantitative competitive RT-PCR was used to analyse stromelysin-1 and TIMP-1 transcripts; western blotting was used to measure the amount of stromelysin-1 and TIMP-1 protein in biopsy samples from children with IBD. RESULTS In biopsies from patients with active Crohns disease (n=24), ulcerative colitis (n=23), and controls (n=16), TIMP-1 transcripts and protein were abundant and unchanged. Stromelysin-1 transcripts and protein were markedly elevated in mucosal biopsies obtained from inflamed sites of patients with active IBD but were not elevated in adjacent endoscopically normal mucosa (n=10). Elevated levels of stromelysin-1 transcripts in active Crohns disease (n=5) returned to normal levels following treatment with enteral nutrition. CONCLUSIONS Stromelysin-1 is markedly overexpressed at inflamed sites in patients with IBD whereas TIMP-1 remains unaltered. Excess stromelysin-1 is likely to be responsible for loss of mucosal integrity in IBD.

Impaired Resistance and Enhanced Pathology During Infection with a Noninvasive, Attaching-Effacing Enteric Bacterial Pathogen, Citrobacter rodentium, in Mice Lacking IL-12 or IFN-γ

Mice infected with Citrobacter rodentium represent an excellent model in which to examine immune defenses against an attaching-effacing enteric bacterial pathogen. Colonic tissue from mice infected with C. rodentium harbors increased transcripts for IL-12 and IFN-γ and displays mucosal pathology compared with uninfected controls. In this study, the role of IL-12 and IFN-γ in host defense and mucosal injury during C. rodentium infection was examined using gene knockout mice. IL-12p40−/− and IFN-γ−/− mice were significantly more susceptible to mucosal and gut-derived systemic C. rodentium infection. In particular, a proportion of IL-12p40−/− mice died during infection. Analysis of the gut mucosa of IL-12p40−/− mice revealed an influx of CD4+ T cells and a local IFN-γ response. Infected IL-12p40−/− and IFN-γ−/− mice also mounted anti-Citrobacter serum and gut-associated IgA responses and strongly expressed inducible NO synthase (iNOS) in mucosal tissue, despite diminished serum nitrite/nitrate levels. However, iNOS does not detectably contribute to host defense against C. rodentium, as iNOS−/− mice were not more susceptible to infection. However, C57BL/6 mice infected with C. rodentium up-regulated expression of the mouse β-defensin (mBD)-1 and mBD-3 in colonic tissue. In contrast, expression of mBD-3, but not mBD-1, was significantly attenuated during infection of IL-12- and IFN-γ-deficient mice, suggesting mBD-3 may contribute to host defense. These studies are among the first to examine mechanisms of host resistance to an attaching-effacing pathogen and show an important role for IL-12 and IFN-γ in limiting bacterial infection of the colonic epithelium.

Interleukin 18 and associated markers of T helper cell type 1 activity in coeliac disease

Background: Coeliac disease (CD) is caused by a T helper cell type 1 (Th1) response in the small intestinal mucosa to dietary gluten. Paradoxically, interleukin (IL)-12, the major Th1 inducing factor, is undetectable in the mucosa of active CD. IL-18 is a recently described cytokine capable of promoting T cell interferon (IFN)-γ production and facilitating Th1 cell polarisation. Aim: To examine expression of IL-18 and IL-18-associated Th1 proteins in CD. Methods: IL-18 and IFN-γ RNA transcripts were determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). IL-18 and caspase-1 protein expression were assessed by western blotting. Caspase-1 activity was determined using a commercially available assay. RNA transcripts for the IL-18 receptor subunits, IL-1 receptor related protein (IL-1 Rrp) and accessory protein-like subunit (AcPL), and IL-18 induced Th1 specific T box transcription factor (T-bet) were measured by RT-PCR and Southern blotting. Results: IL-18 RNA transcripts were found in all mucosal samples analysed, with no difference between CD patients and controls. By western blot analysis, a large protein of approximately 24 kDa, corresponding to the immature IL-18, was detected in all mucosal samples from CD patients and controls. In contrast, mature IL-18 was only seen in CD patients. Immunoreactivity corresponding to both immature and mature caspase-1 was present in both CD and control samples. Tissue homogenates from CD patients and controls expressed similar levels of caspase-1 activity. IL-1Rrp and AcPL were seen in all samples but were expressed at greater levels in the mucosa of CD patients. T-bet was also upregulated in CD. Conclusions: Active IL-18 is expressed in CD as well as other markers of Th1 polarisation.

T cells orchestrate intestinal mucosal shape and integrity

T helper 1-type immune reactions in the gut cause important human diseases, such as Crohns disease and coeliac disease. Here, Tom MacDonald, Mona Elliott and Sylvia Pender suggest that the tissue injury in these diseases is mediated not by the immune cells, but by cytokine-induced changes in keratinocyte growth factor and matrix metalloproteinase production by resident gut myofibroblasts.

Human Peyer’s Patch T Cells Are Sensitized to Dietary Antigen and Display a Th Cell Type 1 Cytokine Profile

Animal studies have demonstrated that feeding Ags induces regulatory (Th2, Th3) cells in Peyer’s patches (PP), which migrate to the periphery and produce immunomodulatory cytokines such as IL-4, IL-10, or TGF-β. In this work we have attempted to extend this paradigm to man by analyzing the response of human PP T cells to in vitro challenge with the common dietary Ag β-lactoglobulin (βlg) of cow’s milk. PP T cells stimulated with βlg showed enhanced proliferation compared with blood T cells from the same patient. Increased expression of CD25 and the Th1-associated chemokine receptor CCR5 was also seen on CD4+ and CD8+ PP T cells, but not blood T cells, stimulated with βlg. By enzyme-linked immunospot assay and RT-PCR, the PP T cell recall response to βlg and casein was dominated by IFN-γ, with negligible IL-4, IL-5, IL-10, or TGF-β. To help explain the PP T cell response to βlg, we examined IL-12 expression. Both IL-12p40 and -p35 transcripts were abundantly expressed in PP, but not in adjacent normal ileal mucosa. Immunoreactive IL-12p40-containing cells were present below the PP dome epithelium. Furthermore, in culture, PP, but not paired PBMC, spontaneously released IL-12p70. These results suggest that the human response to oral Ags in the gut may be different from that in rodents.

Interactions between Stromal Cell-derived Keratinocyte Growth Factor and Epithelial Transforming Growth Factor in Immune-mediated Crypt Cell Hyperplasia

Immune reactions in the gut are associated with increased epithelial cell proliferation. Here we have studied the role of keratinocyte growth factor (KGF; FGF7) and transforming growth factor-alpha (TGF-alpha) in the epithelial cell hyperplasia seen in explants of fetal human small intestine after activation of lamina propria T cells with the superantigen Staphylococcus aureus enterotoxin B (SEB). After the addition of SEB to the explants there is a 10-fold increase in KGF mRNA by 72 h of culture. KGF transcripts were abundant in the lamina propria using in situ hybridization and the culture supernatants contained elevated amounts of KGF protein. SEB had no direct effect on KGF mRNA and protein production by cultured lamina propria mesenchymal cells, but both were upregulated by TNF-alpha. Accompanying the increase in KGF there was also an increase in TGF-alpha precursor proteins in the culture supernatants and the phosphorylated form of the EGFR receptor was also detected in the tissue. Increased TGF-alpha precursor proteins were also detected in the supernatants of control explants stimulated with KGF alone. The direct addition of KGF and TGF-alpha enhanced epithelial cell proliferation and antibodies against KGF and TGF-alpha partially inhibited SEB-induced crypt hyperplasia. These results suggest molecular cross-talk between the KGF/KGFR and the TGF-alpha/EGFR in immune-mediated crypt cell hyperplasia.

Increase in Campylobacter jejuni Invasion of Intestinal Epithelial Cells under Low-Oxygen Coculture Conditions That Reflect the In Vivo Environment

ABSTRACT Campylobacter jejuni infection often results in bloody, inflammatory diarrhea, indicating bacterial disruption and invasion of the intestinal epithelium. While C. jejuni infection can be reproduced in vitro using intestinal epithelial cell (IEC) lines, low numbers of bacteria invading IECs do not reflect these clinical symptoms. Performing in vitro assays under atmospheric oxygen conditions neither is optimal for microaerophilic C. jejuni nor reflects the low-oxygen environment of the intestinal lumen. A vertical diffusion chamber (VDC) model system creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured IECs, producing an in vitro system that closely mimics in vivo conditions in the human intestine. Ninefold increases in interacting and 80-fold increases in intracellular C. jejuni 11168H wild-type strain bacteria were observed after 24-h coculture with Caco-2 IECs in VDCs under microaerobic conditions at the apical surface, compared to results under aerobic conditions. Increased bacterial interaction was matched by an enhanced and directional host innate immune response, particularly an increased basolateral secretion of the proinflammatory chemokine interleukin-8 (IL-8). Analysis of the invasive ability of a nonmotile C. jejuni 11168H rpoN mutant in the VDC model system indicates that motility is an important factor in the early stages of bacterial invasion. The first report of the use of a VDC model system for studying the interactions of an invasive bacterial pathogen with IECs demonstrates the importance of performing such experiments under conditions that represent the in vivo situation and will allow novel insights into C. jejuni pathogenic mechanisms.

Brief Report: Innate lymphoid cells and T-cells contribute to the IL-17A signature detected in the synovial fluid of patients with Juvenile Idiopathic Arthritis

Evidence suggests that aberrant function of innate lymphoid cells (ILCs), whose functional and transcriptional profiles overlap with those of Th cell subsets, contributes to immune‐mediated pathologies. To date, analysis of juvenile idiopathic arthritis (JIA) immune pathology has concentrated on the contribution of CD4+ T cells; we have previously identified an expansion of Th17 cells within the synovial fluid (SF) of JIA patients. We undertook this study to extend this analysis to further investigate the role of ILCs and other interleukin‐17 (IL‐17)–producing T cell subsets in JIA.