Transglutaminase 2 expression is enhanced synergistically by interferon-γ and tumour necrosis factor-α in human small intestine

Transglutaminase 2 (TG2) is expressed ubiquitously, has multiple physiological functions and has also been associated with inflammatory diseases, neurodegenerative disorders, autoimmunity and cancer. In particular, TG2 is expressed in small intestine mucosa where it is up-regulated in active coeliac disease (CD). The aim of this work was to investigate the induction of TG2 expression by proinflammatory cytokines [interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and IL-15] and the signalling pathways involved, in human epithelial and monocytic cells and in intestinal tissue from controls and untreated CD patients. Here we report that IFN-γ was the most potent inducer of TG2 expression in the small intestinal mucosa and in four [Caco-2, HT-29, Calu-6 and human acute monocytic leukaemia cell line (THP-1)] of five cell lines tested. The combination of TNF-α and IFN-γ produced a strong synergistic effect. The use of selective inhibitors of signalling pathways revealed that induction of TG2 by IFN-γ was mediated by phosphoinositide 3-kinase (PI3K), while c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were required for TNF-α activation. Quantitative polymerase chain reaction (PCR), flow cytometry and Western blot analysis showed that TG2 expression was blocked completely when stimulation by either TNF-α or IFN-γ was performed in the presence of nuclear factor (NF)-κB inhibitors (sulphasalazine and BAY-117082). TG2 was up-regulated substantially by TNF-α and IFN-γ in intestinal mucosa in untreated CD compared with controls. This study shows that IFN-γ, a dominant cytokine in intestinal mucosa in active CD, is the most potent inducer of TG2, and synergism with TNF-α may contribute to exacerbate the pathogenic mechanism of CD. Selective inhibition of signalling pathways may be of therapeutic benefit. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.Fil: Bayardo, Mariela Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Punzi, F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Bondar, Constanza María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Chopita, Nestor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Chirdo, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; Argentin

High frequencies of telomeric associations, chromosome aberrations, and sister chromatid exchanges in ulcerative colitis

Chromosome instability provides a predisposing background to malignancy, contributing to the crucial genetic changes in multistep carcinogenesis. The aim of this work was to analyze chromosome instability in patients with ulcerative colitis (UC) to achieve a better understanding of the increased risk for colorectal cancer. METHODS: Peripheral blood lymphocyte cultures from 20 untreated UC patients and 24 controls were used to study chromosome instability by assessing telomeric associations (TAS), chromosome aberrations (CA), and sister chromatid exchanges (SCE). RESULTS: Mean frequencies of TAS and CA were significantly increased in UC patients compared to controls (p < 0.001). Chromosomes 10, 11, 21, 16, and 19 were the most frequently involved in TAS. A total of 104 CA clustered in 66 breakpoints could be exactly localized. Seven nonrandom bands significantly affected in UC patients were found (p < 0.004), showing a significant correlation with the location of cancer breakpoints (p < 0.003), particularly with colorectal carcinoma rearrangements. SCE analysis showed higher levels in patients compared to controls (p < 0.006), but no differences were observed in cell cycle kinetics. CONCLUSIONS: Our results demonstrate the presence of an unstable genome in UC patients that could be related to the cancer development observed in this disease.Fil: Cottliar, Alejandra. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Fundia, Ariela Freya. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Boerr, Luis. Hospital de Gastroenterología “Bonorino Udaondo,; ArgentinaFil: Sambuelli, Alicia. Hospital de Gastroenterología “Bonorino Udaondo,; ArgentinaFil: Negreira, Silvia. Hospital de Gastroenterología “Bonorino Udaondo,; ArgentinaFil: Gil, Anibal. Hospital de Gastroenterología “Bonorino Udaondo,; ArgentinaFil: Gomez, Juan Carlos. Provincia de Buenos Aires. Hospital Interzonal General de Agudos Gral. San Martin; ArgentinaFil: Chopita, Nestor. Provincia de Buenos Aires. Hospital Interzonal General de Agudos Gral. San Martin; ArgentinaFil: Bernedo, Alberto. Provincia de Buenos Aires. Hospital Interzonal General de Agudos Gral. San Martin; ArgentinaFil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentin

Broad MICA/B expression in the small bowel mucosa: a link between cellular stress and celiac disease

The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress, neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we identified MICA/B+ T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B+ B cells, plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which the stress response plays an active role.Fil: Allegretti, Yessica Lorena. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biologicas. Laboratorio de Investigaciones del Sistema Inmune; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bondar, Constanza María. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biologicas. Laboratorio de Investigaciones del Sistema Inmune; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Guzmán, Luciana. Provincia de Buenos Aires. Ministerio de Salud. Hospital de Niños "Sor María Ludovica" de la Plata; ArgentinaFil: Cueto Rua, Eduardo. Provincia de Buenos Aires. Ministerio de Salud. Hospital de Niños "Sor María Ludovica" de la Plata; ArgentinaFil: Chopita, Nestor. Provincia de Buenos Aires. Hospital Interzonal General de Agudos Gral. San Martin; ArgentinaFil: Fuertes, Mercedes Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Zwirner, Norberto Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; ArgentinaFil: Chirdo, Fernando Gabriel. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biologicas. Laboratorio de Investigaciones del Sistema Inmune; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy

Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs’ expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa

Induction of CXCL10 by IL-15 and poly I:C in the small intestine.

<p>Biopsies from celiac patients and non-CD controls were incubated for 3 h in the presence of IL-15 (<b>a</b>) or Poly I:C (<b>b</b>). A second biopsy from each patient was cultured with medium (NS). In non-CD controls, both IL-15 and poly I:C induced CXCL10 mRNA expression (p = 0.0100 and p = 0.0058, respectively; paired t-test). No significant changes were observed in the untreated CD group.</p

Role of CXCR3/CXCL10 Axis in Immune Cell Recruitment into the Small Intestine in Celiac Disease

<div><p>Lymphocytic infiltration in the <i>lamina propria</i> (LP), which is primarily composed of CD4⁺ Th1 cells and plasma cells, and increased numbers of intraepithelial lymphocytes (IELs), is a characteristic finding in active celiac disease (CD). Signals for this selective cell recruitment have not been fully established. CXCR3 and its ligands, particularly CXCL10, have been suggested to be one of the most relevant pathways in the attraction of cells into inflamed tissues. In addition, CXCR3 is characteristically expressed by Th1 cells. The aim of this work was to investigate the participation of the chemokine CXCL10/CXCR3 axis in CD pathogenesis. A higher concentration of CXCL10 was found in the serum of untreated CD patients. The mRNA levels of CXCL10 and CXCL11 but not CXCL9 were significantly higher in duodenal biopsies from untreated CD patients compared with non-CD controls or treated patients. The results demonstrate that CXCL10 is abundantly produced in untreated CD and reduced in treated patients, and the expression of CXCL10 was found to be correlated with the IFNγ levels in the tissue. Plasma cells and enterocytes were identified as CXCL10-producing cells. Moreover, the CXCL10 expression in intestinal tissues was upregulated by poly I:C and IL-15. IELs, LP T lymphocytes, and plasma cells, which infiltrate the intestinal mucosa in untreated CD, express CXCR3. The CXCR3/CXCL10 signalling axis is overactivated in the small intestinal mucosa in untreated patients, and this finding explains the specific recruitment of the major cell populations that infiltrate the epithelium and the LP in CD.</p></div

Infiltration of CXCR3⁺ cells in the <i>lamina propria</i> of small intestine mucosa.

<p><b>a.</b> Confocal immunofluorescence for CXCR3 was performed in sections of duodenal biopsies from controls (i), untreated celiac patients (ii), and treated celiac patients (iii). CXCR3 is shown in green, and nuclei are shown in red. Untreated celiac patients showed a higher number of positive cells infiltrating the <i>LP</i>. (Magnification, 630×). <b>b.</b> The number of CXCR3⁺ cells in the LP was higher in the duodenal mucosa of untreated celiac patients (n = 9) compared with control individuals (n = 6) and treated patients (n = 5) (unpaired t-test; p = 0.0089 and p = 0.0055, respectively). The positive cells in LP regions from sections of duodenal biopsies were counted using immunofluorescence microscopy. <b>c.</b> Representative flow cytometric analysis from the LP compartment of a duodenal sample of an untreated CD patient showing plasma cells (CD138⁺) that express CXCR3. <b>d.</b> Representative flow cytometric analysis from the LP compartment of a duodenal sample of an untreated CD patient showing LP lymphocytes (CD3⁺ or CD4⁺) that express CXCR3.</p

CXCL9, CXCL10, and CXCL11 mRNA levels in the duodenum.

<p><b>a.</b> The mRNA expression levels of CXCR3 ligands were determined by real-time PCR. Duodenal biopsies from celiac individuals at the time of diagnosis (n = 26), celiac individuals on a GFD (n = 6), and non-CD controls (n = 25) were included. The untreated celiac patients expressed significantly higher levels of CXCL10 and CXCL11 than the treated patients (p = 0.0436 and p = 0.0160, respectively) and controls (p = 0.0002 and p<0.0001, respectively). There was no difference in the CXCL9 mRNA levels between the groups. The results are shown as relative units in reference with the levels of the housekeeping gene β-actin. An unpaired t-test was used to assess the significance of the differences. <b>b.</b> The correlation between the mRNA levels of CXCL10 and the mRNA levels of CXCL11 in duodenal samples from CD patients (black circles) and non-CD controls (white circles) was analysed. The CXCL10 and CXCL11 expression levels were correlated significantly in untreated CD patients (r = 0.7463, p<0.0001) and in non-CD controls (r = 0.6690, p = 0.0003).</p

Cellular sources of CXCL10 in the small intestinal <i>lamina propria</i>.

<p>The cellular sources of CXCL10 in the duodenum were identified using immunofluorescence confocal microscopy. CD3⁺ cells were found to be negative for CXCL10 in both CD patients (i) and control subjects (ii). Numerous CD138⁺ cells that express CXCL10 were found in the celiac mucosa (iii). Plasma cells expressing CXCL10 were not found in the duodenum from non-CD controls (iv). HAM56⁺ cells did not produce CXCL10 in the celiac (v) and in the non-CD control intestinal mucosa (vi). CD3, CD138, and HAM56 are shown in green, CXCL10 is shown in red, and nuclei are shown in blue. (Magnification, 1071×).</p