Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA

Purpose: To investigate the role of thioredoxin (TRX), a novel regulator of extracellular transglutaminase 2 (TG2), in celiac patients IgA (CD IgA) mediated TG2 enzymatic activation. Methods: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs) under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA) were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. Results: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. Conclusions: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX

Endothelial extracellular transglutaminase 2 (TG2) activity analyses by monodansylcadaverine (MDC) incorporation.

<p>The relative extracellular TG2 activity (<b>A</b>) at different time points and with (<b>B</b>) increasing concentrations of class A immunoglobulins are shown. The dashed line indicates TG2 activity in untreated endothelial cells (HUVECs). Bars represent mean MDC incorporation as fold of control and error bars indicate standard error of the mean. P-value < 0.05 was considered significant ( _**p<0.001 and _*p<0.01). Data derived from three independent experiments, repeated in quadruplicate. HUVECs were treated with celiac disease patient (CD IgA) and non-celiac subject’s immunoglobulin-A (H IgA). Extracellular TG2 activity was inhibited by administering a non-permeable, site directed TG2 inhibitor, R281.</p

Celiac IgA CD IgA) mediated activation of extracellular transglutaminase 2 (TG2) by thioredoxin (TRX).

<p>(<b>A</b>) The relative surface expression of TG2 as well as (<b>B</b>) the relative TG2 activity on HUVECs treated with CD IgA autoantibodies was investigated by ELISA prior preincubation of endothelial cells with TRX inhibitor, PX12. The dashed line indicates TG2 expression and activity in untreated HUVECs. Bars represent mean values as fold of control and error bars indicate standard error of the mean. P-value < 0.05 was considered significant ( _*p<0.01 and _**p<0.001). (<b>C</b>) The secretion of TRX in endothelial cell (HUVECs) culture media was analysed by Western blotting in the presence of a specific TRX inhibitor, PX12 (1-methylpropyl 2-imidazolyl disulfide). Representative Western blots of HUVECs culture supernatants show amounts of TRX and ƴ-tubulin used as loading control for quality sample. Data derived from at least three independent experiments, repeated in quadruplicate are shown. Endothelial cells were treated with CD IgA and non-celiac subject’s immunoglobulin-A (H IgA).</p

Celiac IgA (CD IgA) promotes secretion of thioredoxin.

<p><b>(TRX) and decreases its endothelial surface expression</b>. (<b>A</b>) Representative Western blots from total cell lysates and supernatants show amounts of TRX and ƴ-tubulin used as loading control for protein extracts. (<b>B</b>) Relative surface expression of TRX was studied by ELISA. The dashed line indicates TRX surface expression in untreated HUVECs. Bars represent mean TRX expression as fold of control and error bars indicate standard error of the mean. P-value <0.05 was considered significant ( _*p<0.05 and _**p<0.001). Data derived from three independent experiments, repeated in quadruplicate are shown. Endothelial cells were treated with CD IgA and non-celiac subject’s immunoglobulin-A (H IgA). Extracellular TG2 activity was inhibited by administering a site directed, non-permeable inhibitor, R281.</p

Celiac patient immunoglobulins A (CD IgA) increase transglutaminase 2 (TG2) surface deposition on endothelial cells.

<p>(<b>A</b>) The relative surface expression of TG2 on non-permeabilized endothelial cells (HUVECs) was studied by ELISA. The dashed line indicates TG2 surface expression in untreated HUVECs. Bars represent mean TG2 expression as fold of control and error bars indicate standard error of the mean. P-value <0.05 was considered significant ( _*p<0.001). Data derived from three independent experiments, repeated in quadruplicate are shown. (<b>B</b>) Surface expression of TG2 on non-permeabilized HUVECs was also investigated by immunofluorescence (IF) with the monoclonal TG2 antibody, 4G3. Representative IF stainings are shown. Control non-celiac subject’s immunoglobulin-A (H IgA). Extracellular TG2 activity was inhibited by administering a site directed, non-permeable inhibitor, R281.</p

Celiac disease IgA (CD IgA) can activate fibronectin-bound transglutaminase 2 (FN-TG2) under reducing conditions.

<p>The effect of CD IgA on TG2 enzymatic activity was assessed by EZ-Link Pentylamine-Biotin (5-BP) incorporation on FN. In the absence of both the reducing agent, dithiothreitol (DTT) and Ca²⁺, TG2, alone or with any IgA, was enzymatically inactive. Maximal TG2 activity was obtained in the presence of DTT and Ca²⁺ even in the presence of class A immunoglobulins. On the contrary, in the presence of a reducing agent, DTT, but without exogenous Ca²⁺, CD IgA was able to induce the activation of TG2 in contrast to non-celiac IgA (H IgA). Bars represent mean 5-BP incorporation on FN as fold of TG2 in the absence of DTT and Ca²⁺. Error bars indicate standard error of the mean; P-value < 0.05 was considered significant ( _***p<0.001, _**p<0.01 and _*p<0.05). Data derived from three independent experiments, repeated in quadruplicate are shown.</p

Anti-microbial antibodies in celiac disease: Trick or treat?

AIM: To determine the prevalence of a new set of anti-glycan and anti-outer membrane protein (anti-OMP) antibodies in a Hungarian cohort of adult Celiac disease (CD) patients

Outcome measures in coeliac disease trials: the Tampere recommendations

A gluten-free diet is the only treatment option of coeliac disease, but recently an increasing number of trials have begun to explore alternative treatment strategies. We aimed to review the literature on coeliac disease therapeutic trials and issue recommendations for outcome measures

The role of gluten consumption at an early age in celiac disease development: a further analysis of the prospective PreventCD cohort study

Background: We previously found that the introduction of small quantities of gluten at 4-6 mo of age did not reduce the risk of celiac disease (CD) in a group of high-risk children. However, the consumption of high amounts of gluten early in life has been suggested to increase CD risk.Objective: The aim of this study was to evaluate this hypothesis by using data from the previous study of the PreventCD trial (www.preventcd.com).Design: Gluten intake was prospectively quantified by using specific food records between 11 and 36 mo of age in 715 children positive for the human leukocyte antigen (HLA)-DQ2 and/or HLA-DQ8 from 5 European countries. According to the PreventCD protocol, infants received 100 mg immunologically active gluten/d or placebo from 4 to 6 mo of age, with a stepwise and fixed gluten increase until age 10 mo and unrestricted intake thereafter. The primary outcome of the present study was the impact of the amount of gluten consumed from age 10 mo onward on CD development.Results: Mean daily gluten intakes from 10 mo onward were significantly different between countries for children at all ages (P 0.05). The variables country, sex, intervention group, and gluten consumption pattern did not show significant associations with CD development risk (HRs not significant). In addition, the interaction between HLA risk group and gluten consumption pattern showed no significant risk on CD development, except for the DQ2.2/DQ7 haplotype (HR: 5.81; 95% CI: 1.18, 28.74; P = 0.031).Conclusions: Gluten consumption patterns as well as the amount of gluten consumed at 11-36 mo of age do not influence CD development for most related HLA genotypes in children with a genetic risk. This study reports the gluten consumption pattern in children at risk of CD from different European countries. This trial was registered at www.controlled-trials.com as ISRCTN74582487