Sylvia L.F. Pender

Chronic inflammation induces telomere dysfunction and accelerates ageing in mice

Chronic inflammation is associated with normal and pathological ageing. Here we show that chronic, progressive low-grade inflammation induced by knockout of the nfkb1 subunit of the transcription factor NF-κB induces premature ageing in mice. We also show that these mice have reduced regeneration in liver and gut. nfkb1−/− fibroblasts exhibit aggravated cell senescence because of an enhanced autocrine and paracrine feedback through NF-κB, COX-2 and ROS, which stabilizes DNA damage. Preferential accumulation of telomere-dysfunctional senescent cells in nfkb1−/− tissues is blocked by anti-inflammatory or antioxidant treatment of mice, and this rescues tissue regenerative potential. Frequencies of senescent cells in liver and intestinal crypts quantitatively predict mean and maximum lifespan in both short- and long-lived mice cohorts. These data indicate that systemic chronic inflammation can accelerate ageing via ROS-mediated exacerbation of telomere dysfunction and cell senescence in the absence of any other genetic or environmental factor.

Expression of Transforming Growth Factor-β1 by Pancreatic Stellate Cells and Its Implications for Matrix Secretion and Turnover in Chronic Pancreatitis

Pancreatic stellate cells mediate fibrosis in chronic pancreatitis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs)-1 and -2 are crucial modulators of fibrosis. Transforming growth factor-beta (TGF-beta) is a key regulator of extracellular matrix production and myofibroblast proliferation. We have examined MMP and TIMP synthesis by transformed cultured pancreatic stellate cells and their regulation by TGF-beta 1. By Northern analysis they expressed mRNAs for procollagen 1, TIMP-1, TIMP-2, and MMP-2. Expression of membrane type-1 MMP was confirmed by Western blotting. By immunohistochemistry these enzymes localized to fibrotic areas in human chronic pancreatitis. Active TGF-beta 1 constitutes 2 to 5% of total TGF-beta 1 secreted by pancreatic stellate cells; they express TGF-beta receptors I and II. Exogenous TGF-beta 1 (10 ng/ml) significantly increased procollagen-1 mRNA by 69% and collagen protein synthesis by 34%. Similarly TGF-beta 1 at 0.1, 1, and 10 ng/ml significantly reduced cellular proliferation rate by 37%, 44%, and 44%, respectively, whereas pan-TGF-beta-neutralizing antibody increased proliferation by 40%. TGF-beta1 (10 ng/ml) down-regulated MMP-9 by 54% and MMP-3 by 34% whereas TGF-beta 1-neutralizing antibody increased MMP-9 expression by 39%. Pancreatic stellate cells express both mediators of matrix remodeling and the regulatory cytokine TGF-beta 1 that, by autocrine inhibition of MMP-3 and MMP-9, may enhance fibrogenesis by reducing collagen degradation.

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.

Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

Background and Aims: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor β (TGFβ) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFβ signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn’s disease (CD). Methods: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFβ blocking antibody or TGFβ1. TGFβ transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. Results: TGFβ transcripts, phosphorylated Smad2–Smad3 (pSmad2–3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFβ transcripts, a greater pSmad2–3 response to TGFβ, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFβ blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. Conclusions: Changes in TGF-β signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

Role of interferon α in promoting T helper cell type 1 responses in the small intestine in coeliac disease

Coeliac disease (CD) is caused by a CD4 T helper cell type 1 (Th1) response in the small intestinal mucosa to dietary gluten. As the major Th1 inducing cytokine, interleukin 12, is undetectable in CD gut mucosa, the mechanism by which Th1 effector cells are generated remains unknown. Interferon (IFN) α, a cytokine capable of promoting IFN-γ synthesis, has been implicated in the development of Th1 mediated immune diseases. Here we report a case of CD-like enteropathy in a patient receiving IFN-α for chronic myeloid leukaemia. Morphological assessment of duodenal biopsies taken from the patient showed total villous atrophy, crypt cell hyperplasia, and a high number of CD3+ intraepithelial lymphocytes. Both antigliadin antibodies and antiendomysial antibodies were positive. RNA analysis revealed pronounced expression of IFN-γ. Withdrawal of gluten from the diet resulted in a patchy improvement in intestinal morphology, normalisation of laboratory parameters, and resolution of clinical symptoms. By western blot analysis, IFN-α protein was seen in the duodenal mucosa from untreated CD patients but not in controls. This was associated with marked expression of IFN-γ protein in CD mucosa. Collectively, these results suggest a role for IFN-α in promoting Th1 responses to gluten.

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.

Enhancing lamina propria Th1 cell responses with interleukin 12 produces severe tissue injury

BACKGROUND & AIMS Interleukin (IL)-12 is believed to modulate local T-cell response in human colitis. A direct functional relationship between IL-12 and tissue injury in human intestine has not been reported. The aim of this study was to examine changes that take place in explant cultures of human fetal gut after stimulation of T cells with anti-CD3 in the presence of exogenous IL-12/IL-18. METHODS T cells in explants of fetal gut were activated with anti-CD3 antibody and/or IL-12 or IL-18. Mucosal pathology was determined by immunohistochemistry. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to determine cytokine synthesis, and the production of matrix metalloproteinases was analyzed by RT-PCR and Western blotting. RESULTS Activation of T cells in explants with anti-CD3 antibody elicited very little interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and no tissue injury. Addition of graded doses of IL-12 with anti-CD3 resulted in a significant increase in both IFN-gamma and TNF-alpha. This change was associated with a massive increase in stromelysin-1 expression and severe tissue injury, which was inhibitable by a stromelysin-1 inhibitor. Costimulation of explants with anti-CD3 and IL-18 induced only IFN-gamma and no tissue injury. CONCLUSIONS IL-12 can convert a physiological T-cell signal into a strong signal with the downstream effect of elevating tissue stromelysin-1 concentration and mucosal degradation.

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.

Upregulation of matrix metalloproteinases in a model of T cell mediated tissue injury in the gut: analysis by gene array and in situ hybridisation

Background and aim: Matrix metalloproteinases (MMPs) have been implicated in tissue remodelling and ulceration in inflammatory bowel disease and coeliac disease. Studies to date have concluded that stromelysin 1 is functionally involved in mucosal degradation. However, there are many other MMPs whose function in the gut is currently unknown. This work had two aims: firstly, to use gene array technology to measure changes in MMP and tissue inhibitor of metalloproteinase (TIMP) expression in a model of T cell mediated injury in the gut, and secondly, to correlate data from gene arrays with that generated by in situ hybridisation. Methods: T cells in explants of human fetal gut were activated with pokeweed mitogen or anti-CD3 plus interleukin 12. Gene array analysis and in situ hybridisation were performed to investigate changes in MMP gene expression. Results: Both gene array analysis and in situ hybridisation indicated marked upregulation of stromelysin 2 and macrophage metalloelastase expression in the explants associated with mucosal destruction. The arrays also confirmed our previous observation that interstitial collagenase (MMP-1), stromelysin 1 (MMP-3), and gelatinase B (MMP-9) are upregulated but there was no change in MMP-2, -7, -8, -9, -11, -13, -14–17, or -19. Following T cell activation, transcripts for TIMPs were reduced. Conclusions: These results show that there is differential upregulation of MMPs during T cell responses in the gut and suggest that further studies on the role of stromelysin 2 and macrophage metalloelastase may show that they have a functional role. In addition, the increase in MMPs and reduction in TIMPs suggest that the protease/antiprotease balance in the mucosa may determine the extent of mucosal degradation.

Interferon-α drives T cell-mediated immunopathology in the intestine

The ability of interferon (IFN)‐α to induce autoimmunity and exacerbate Th1 diseases is well known. We have recently described enhanced expression of IFN‐α in the mucosa of patients with celiac disease (CD), a gluten‐sensitive Th1‐mediated enteropathy, characterized by villous atrophy and crypt cell hyperplasia. Previous studies from this laboratory have shown that T cell activation in explant cultures of human fetal gut can also result in villous atrophy and crypt cell hyperplasia. We have, therefore, examined changes that take place in explant cultures of human fetal gut after activation of T cells with anti‐CD3 and/or IFN‐α. We show that activation of T cells with anti‐CD3 alone elicits a small IFN‐γ and TNF‐α response with no tissue injury. Similarly, no changes are seen in explants cultured with IFN‐α alone. However, addition of IFN‐α with anti‐CD3 results in enhanced Th1 response and crypt cell hyperplasia. This is associated with enhanced phosphorylation of STAT1, STAT3, and Fyn, a Src homology tyrosine kinase, which interacts with both TCR and IFN‐α signal components. Together these data indicate that IFN‐α can facilitate activation of Th1‐reactive cells in the gut and drive immunopathology.