Aims Despite clinical evidence of liver involvement in patients with coeliac disease (CeD), there is a lack of a method to prove this association.
Methods Of 146 treatment-naive patients with CeD, 26 had liver dysfunction. Liver biopsies and corresponding small intestinal biopsies were obtained from these 26 patients. Multicolour immunohistochemical and immunofluorescence confocal microscopic studies were performed on paraffin-embedded tissue to detect the IgA/anti-TG2 deposits. Follow-up liver biopsies were taken after a gluten-free diet.
Results Twenty-six out of the 146 patients (17.8%) with suspected coeliac-associated liver disease on histological examination revealed irregular sinusoidal dilatation in 15 (57.6%), steatohepatitis in 4 (15.3%), non-specific chronic hepatitis in 3 (11.5%), autoimmune hepatitis in 2 (7.6%) biopsies, including cirrhosis in one of them, irregular perisinusoidal fibrosis and changes of non-cirrhotic portal fibrosis in one biopsy each (3.8%). IgA/anti-tTG deposits were observed in 22 (84.6%) liver biopsies by dual immunohistochemistry technique, and in 24 (92.3%) by confocal immunofluorescence technique and in all corresponding duodenal biopsies (100%). Overall, IgA/anti-tTG deposits showed 100% sensitivity, 77% specificity and 85% positive predictive value for establishing an association of extraintestinal pathology and CeD using archived tissues. Follow-up liver biopsies could be obtained in five patients; four of them showed not only resolution of the histological lesions but disappearance of IgA/anti-tTG co-localisation.
Conclusions Data of the present study adds to the body of evidence that liver lesions in patients with CeD are disease related and may have been caused by a similar pathogenic mechanism that causes intestinal changes.
- coeliac disease
- diagnostic techniques and procedures
- liver diseases
Data availability statement
Data are available on reasonable request.
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- coeliac disease
- diagnostic techniques and procedures
- liver diseases
In patients with coeliac disease (CeD) both intestinal and extraintestinal manifestations have been described.1 The hypersensitivity to gluten peptides in patients with CeD once thought to be limited to the small intestine, is now known to affect many other organs including skin, liver, kidney, bone and brain.2–5 Hence, CeD is now considered a disorder altogether and systemic manifestation can be seen in up to 60% adult and children with CeD.1 2 There are several levels of evidence that suggest an association between CeD and liver diseases. Some of these are asymptomatic increase in serum transaminases in up to 27% patients, reversible in 63%–90% patients with a gluten-free diet (GFD); reversibility of liver failure GFD and presence of coeliac specific serological tests and enteropathy in patients with cryptogenic liver diseases.6–10 Patients with CeD have been known to have coexistent autoimmune hepatitis (AIH), non-alcoholic fatty liver disease (NAFLD) and vascular liver diseases. While a diagnosis of CeD is based on a combination of clinical manifestations, positive serological test and presence of villous abnormalities on histology; the diagnosis of liver disease in them is based merely on their coexistence, exclusion of other causes of liver dysfunction and reversal of liver disease post-GFD.3 4 At present, there is no tissue-specific marker/staining/cellular repertoire to confirm the gluten-specificity of liver abnormalities in patients with CeD. Then, how to establish if the liver dysfunction is contributed by CeD? One possibility is to demonstrate the gluten-reactive immune cells in the liver, or demonstration of the cross-reactivity of the circulating IgA anti-TG2 antibody with hepatic tissue. IgA TG2 deposits have been reported in CeD, potential CeD, and seronegative CeD in the intestinal biopsies. In a recent meta-analysis of 23 studies, Gatti et al have reported IgA anti-TG2 deposits in symptomatic adults (100%) and children (73.2%–100%) with CeD; potential CeD (64.7%–100%) and in patients with dermatitis herpetiformis (63%–79%).11–15
Therefore, demonstration of IgA anti-TG2 Ab deposits by colocalising these antibodies in liver biopsies may be one of the evidence to suggest the correlation of liver pathology and CeD.7–9 We, therefore, systematically recruited treatment-naïve patients with CeD and screened them for evidence of liver dysfunction. Detailed morphological evaluations, followed-by IgA/anti-TG2 antibody colocalisation studies, were performed to establish the causality by dual-colour indirect immunofluorescence (IF) confocal microscopy and dual-colour immunohistochemistry (IHC) techniques. Since one of the valid requirements for establishing the association between liver dysfunctions and CeD is to demonstrate the reversibility of the liver damage by GFD; they were followed-up and liver biopsies were repeated from partially symptomatic patients and all tests were repeated.
Patients and methods
One hundred and forty-six consecutive treatment naïve patients with CeD presenting to Coeliac Disease Clinic between October 2015 to December 2017 were screened for evidence of liver dysfunction, based on the presence of one or more of the following criteria: (1) the presence of either or a combination of alanine aminotransferase or aspartate transaminases >1.5 times normal, (2) fibroscan showing liver stiffness measurement of >12 kPa on fibroscan, coarse liver echotexture or evidence of cirrhosis ultrasound of the abdomen and (3) hepatic vein block on colour Doppler ultrasound. In the absence of any established clinical criteria to define the coeliac-associated liver disease, the group of criteria was formulated in-house. All the patients having liver dysfunction underwent detailed etiological evaluation including alcoholism and drug intake, and screening for hepatitis B virus (hepatitis B surface antigen (HBsAg) and HBV-DNA), hepatitis C virus (anti-HCV Ab, HCV-RNA), autoimmune serology (antinuclear antibody, antismooth muscle antibody, antiliver-kidney microsomal antibody), evaluation for Wilson’s disease (serum ceruloplasmin), serum ferritin and lipid profile, to rule out any contributing factor for the liver dysfunction. All of them underwent ultrasound examination, fibroscan analysis and 26 patients (17.8%) fulfilling the above criteria underwent percutaneous (n=21) or transjugular (n=5) liver biopsies as applicable.
Control liver biopsies
As non-coeliac controls to compare the IgA/anti-TG2 deposits, archived liver biopsies from patients diagnosed with NAFLD (n=1), chronic hepatitis, not otherwise specified (n=1), chronic HBV infection (n=1), HBV-induced cirrhosis (n=1), and AIH (n=1) were included as disease controls.
Corresponding small intestinal mucosal biopsies
Corresponding small intestinal mucosal biopsies from the recruited patients with liver dysfunction (n 26) were included in this study to document the histological changes and compare the IgA/anti-TG2 deposits.
Control small intestinal biopsies from other known diseases
To compare the intestinal IgA/anti-TG2 deposits, duodenal biopsies (n-36) from already diagnoses patients with giardiasis (n=5), common variable immune deficiency (n=2), tropical sprue (n=16) and non-specific duodenitis (n=13) were also included.
Morphological changes in the liver biopsies
Liver biopsies showing changes of chronic hepatitis were graded and staged using the modified Ishak grading system.16 Steatosis, if found was graded and staged according to the non-alcoholic steatohepatitis)-clinical research network grading and staging system.17 In the case of AIH, the modified Ishak staging system was applied, as per the recommendation of the American Association of Studies of Liver Diseases.18 The presence of any other histopathological findings in the liver biopsies included was also looked for (figure 1).
Demonstration of IgA/anti-TG2 antibody deposits by colocalisation studies
Demonstration by dual colour-IHC technique
In short, 4 µm thick sections cut from the formalin-fixed paraffin-embedded (FFPE) blocks were subjected to dual-colour IHC by using peroxidase-labelled rabbit polyclonal anti-human anti-TG2 antibody (1:400 dilution, ab421; ABCAM; chromogen: DAB (Spring Bio, UK)) and alkaline phosphatase labelled monoclonal mouse antihuman IgA antibody (1:1000 dilution, clone M24A; MERCK; chromogen: Vector Blue (Vector Lab, Burlingame, USA)). The CeD-specific IgA/anti-TG2 dual deposits were then analysed and scored as follows: negative (0), mild (1), moderate (2) and strongly positive (3). Scores were calculated individually in different areas of the biopsies and then summed up, to arrive at a cumulative score. Areas analysed included hepatocytes, sinusoids and vascular endothelial cells. Cumulative scores were finally graded as Grade 0 (cumulative score of 0–3; negative or mild positive); grade 1 (cumulative score: 4–6; moderately positive); grade 2 (cumulative score: 6–9, strongly positive). While brown colour indicated anti-TG2 deposits and blue colour indicated IgA deposit; the areas of colocalisation, showed a unique bluish-brown colour. The portal structures, hepatocytes, sinusoidal lining cells, blood vessels and fibrous architecture were examined for antibody deposits and the whole liver core was examined microscopically. Similarly, IgA/anti-TG2 deposits in the corresponding duodenal and control biopsies were also examined (figures 2 and 3).
Demonstration by direct confocal dual IF technique
Dual IF study was performed on FFPE liver biopsies. Mucosal tissue transglutaminase (TG2) was analysed using rabbit polyclonal antibody against human TG2 (1:400 dilution, ab421; Abcam, Cambridge, USA) and was detected by goat anti-rabbit IgG (Heavy +Light chains) cross-adsorbed secondary antibody labelled with Alexa Fluor 594 (1:200 dilution, A-11012; Invitrogen). The same section was thereafter stained with mouse antihuman monoclonal IgA antibody, clone M24A, heavy chain, fluorescein isothiocyanate conjugated (1:1000 dilution, CBL 114F; MERCK, New York, USA). 4′, 6′-diamidino-2-phenylindole was used as a nuclear stain. The CeD-specific IgA/anti-TG2 dual deposits were then analysed by using a confocal microscope using the NIKON NIS AR software. The Pearson’s value (ranging from −1 to +1) as calculated by the software was recorded and graded as follows: 0 (none +mild) (Pearson’s index <0.3), 1+ (moderate) (Pearson’s index >0.3-<0.6), and 2+ (strong) (Pearson’s index >0.6–1). Biopsy sections with only 1+ and 2+ positivity were considered as having IgA/anti-TG2 dual deposits. Red IF indicated anti-TG2 deposits and green IF indicated IgA deposit, while IgA/anti-TG2 deposits showed colocalised yellow fluorescence. The portal structures, hepatocytes, sinusoidal lining cells, blood vessels and fibrous architecture were examined for antibody deposits and the whole liver core was examined microscopically. Similarly, IgA/anti-TG2 deposits in the corresponding duodenal and control biopsies were also examined (figures 4–6).
Follow up of patients on GFD
All patients were put on GFD under the supervision of a nutritionist with a special interest in counselling and management of CeD. These patients were then followed up by a dedicated clinician and their liver function tests were monitored. Their liver function tests were also monitored. After completion of 1 year of GFD, the patients were invited to undergo follow-up liver biopsy to assess the status of pathological changes and IgA/anti-TG2 antibody deposits. Only five of them agreed to undergo a repeat liver biopsy.
Research ethics and patient consent
Informed, written consent was taken from all the participants.
Data analysis was done using Stata V.14 StataCorp. 2015. (Stata Statistical software: Release14.) and presented in mean, SD and median (minimum, maximum), as appropriate. Categorical data were expressed as frequency and percentage. P<0.05 was considered as statistically significant.
Demographic, clinical, serological, and intestinal biopsy characteristics of the patients
Twenty-six out of 146 patients with treatment naïve CeD screened in this study showed clinical features of liver dysfunction (17.8%). Their characteristics have been summarised in table 1.
Provisional clinical diagnoses made in the study patient cohort (N 26)
Of 26 patients with CeD having liver abnormalities, nine had known causes including AIH in four, hepatic vein outflow tract obstruction, chronic hepatitis B, drug-induced liver injury, alcoholic liver disease and extrahepatic portal vein obstruction in one patients each. In other 17 patients, the cause of liver disease could not be established even after complete aetiological evaluation and they were labelled as having cryptogenic liver disease (table 2).
Histopathological changes in the liver biopsies from patients with suspected coeliac-associated liver disease
The histological changes in the liver biopsies of patients with coeliac-associated liver diseases noted were irregular sinusoidal dilatation in 15 (57.6%), steatohepatitis in 4 (15.3%), non-specific chronic hepatitis in 3 (11.5%), AIH in 2 (7.6%) biopsies, including changes of cirrhosis in one of them, irregular perisinusoidal fibrosis and changes of non-cirrhotic portal fibrosis in one liver biopsy each (3.8%) (table 2 and figure 1). In some cases, there were overlapping histological findings; hence, the total percentage does not sum up to 100%.
Histopathological changes in corresponding duodenal mucosal biopsies from patients with suspected coeliac-associated liver disease
The corresponding duodenal biopsies in all these 26 patients with suspected coeliac-associated liver disease had modified Marsh grade 3 changes, including 7 (26.9%) with modified Marsh 3a, 15 (57.6%) with modified Marsh 3b and 4 (15.3%) with modified Marsh 3c changes.
Site of IgA/anti-TG2 deposits in the liver biopsies
While anti-TG2 antibody deposition was observed in hepatocytes, Kupffer cells, focally in sinusoidal lining cells and in areas of fibrosis, and IgA deposit was noted mostly in the hepatic sinusoidal lining cells and focally in hepatocytes; the IgA/anti-TG2 dual antibody deposits were noted primarily in the space of Disse, sinusoidal lining cells as well as in vascular endothelial lining cells by both the techniques. In a subset of patients, the dual deposits were also noted in the hepatocyte cytoplasm (figures 2 and 5).
IgA/anti-TG2 dual deposits in the liver biopsies from patients with CeD and controls
Out of 26 suspected patients with coeliac-associated liver disease, IgA/anti-TG2 antibody deposits were observed in liver biopsies of 22 (84.6%) patients by dual IHC technique (figure 2) and in 24 (92.3%) by the dual confocal IF technique (figure 5). Strong (2+) positivity was observed in 8 (30.7%) and 7 (26.9%) liver biopsies by dual IHC and dual confocal IF techniques, respectively (table 2). Of the four liver biopsies where IgA/anti-TG2 antibody deposits were noted only with dual confocal IF technique, raised serum anti-TG Ab titers (fold-rise values 6.4, 10.7, 4 and 9.9, respectively) and irregular sinusoidal dilatation was identified in the liver biopsies on histological examination. The first three of these patients had mucosal modified Marsh 3a changes and the fourth patient had modified Marsh 3b changes on the duodenal mucosal biopsies.
IgA/anti-TG2 dual deposits in the liver biopsies from disease controls
Only one liver biopsy had mild (1+) IgA/anti-TG2 deposit with dual IHC technique, and two of the control liver biopsies showed mild (1+) IgA/anti-TG2 deposit with dual confocal IF technique (table 2, figure 5C).
IgA/anti-TG2 antibody dual deposits in corresponding intestinal biopsies from patients with suspected CeD associated liver disease and in non-coeliac enteropathy controls
All corresponding duodenal mucosal biopsies taken at baseline from 26 patients with the suspected coeliac-associated liver disease showed IgA/anti-TG2 antibody deposits by the dual IHC technique (100%) (figure 3). With dual confocal IF technique 25 of these liver biopsies (96.1%) showed IgA/anti-TG2 dual deposits (figures 4 and 6). Isolated green fluorescent anti-TG2 and red fluorescent IgA positivities were noted in the epithelial basement membranes, vascular lining cells and enterocyte cytoplasm. Anti-TG2 deposit besides was noted in the muscularis mucosae and the areas of mucosal fibrosis. IgA/anti-tTG dual antibody deposits were noted in the mucosal epithelium, sub-epithelial basement membrane, in mucosal vascular lining cells, and the muscularis mucosae (table 3).
IgA/anti-TG2 dual deposits in intestinal biopsies from disease controls
Duodenal mucosal biopsies from patients with non-coeliac enteropathies included showed IgA/anti-TG2 antibody deposits in only three patients (8.3%) by dual IHC technique, and in 4 (11.1%) by dual confocal IF technique (table 3).
Diagnostic efficacy of the IgA/anti-TG2 dual antibody deposits for coeliac-associated liver disease
The overall sensitivity, specificity, positive predictive value and negative predictive value of dual IHC and confocal dual IF techniques for detection of IgA/anti-TG2 dual antibody deposits were 100%, 76.9%, 85.7% and 100%, respectively, and 94.4%, 69.2%, 80.9% and 90%, respectively.
Overall agreement between the dual IHC and dual confocal if techniques for detection of IgA/anti-TG2 dual antibody deposits
The weighted kappa value for dual IHC and dual confocal IF based techniques on FFPE sections for detection of IgA/anti-TG2 deposit was 0.82, suggesting a strong agreement between these two techniques.
Clinical and histological findings in the suspected cohort of patients with coeliac associated liver disease on follow-up
Of the 26 patients with suspected coeliac-associated liver diseases, 23 patients were followed up in our clinic and rest three lost to follow-up. Normalisation of serum transaminases was observed in 17 (73.9%) of them after a median follow-up of 17.7 weeks (11–27 weeks). Of six patients with persistent elevation of serum transaminases level on follow-up, one had AIH which progressed to acute on chronic liver failure and the patient died despite the use of immunosuppressive and strict GFD. The other two of these six patients were non-compliant to GFD and one of these six patients continued to consume alcohol while on the last follow-up. Five of them agreed to undergo follow-up liver biopsies. Follow-up liver biopsies from patients with liver biopsies who showed normalisation of serum transaminases were ethically not justifiable. In the five symptomatic patients who underwent liver biopsies, there were histological improvements in comparison to the baseline changes (table 4). In patient number five irregular sinusoidal dilatation and mild steatosis were identified in follow-up liver biopsies (table 4).
Status of IgA/anti-TG2 dual antibody deposits in the follow-up liver biopsies
Out of the five symptomatic patients who underwent follow-up liver biopsies, IgA/anti-TG2 deposit (1+) was identified with both the techniques in patient number 3 and by dual IF confocal technique in patient number 5 additionally (figures 7 and 8). In patient number 3 in whom IgA/anti-TG2 dual deposit was noted in follow-up biopsy had histological changes of non-cirrhotic portal fibrosis with mild periportal fibrosis in the liver. The serum anti-tTG titre was eight times the normal during follow-up biopsies than 20 times the serology titre noted at baseline. As already stated, liver biopsy from patient five showed irregular sinusoidal dilatation and mild steatosis (table 4).
In the present study including 146 patients, 26 (17.8%) treatment naïve patients with CeD had either biochemical and/or structural liver abnormalities. Liver biopsies performed in these 26 patients with the suspected coeliac-associated liver disease showed a varying spectrum of histological changes and IgA/anti-TG2 deposits in the sinusoidal lining cells and hepatocyte cytoplasm focally in 24 (92.3%) liver biopsies by multicolour IHC and confocal multicolour IF techniques. The corresponding duodenal biopsies from all these 26 patients also showed colocalised IgA/anti-TG2 antibody deposits. The CeD unrelated diseased liver and intestinal controls showed only insignificant IgA/anti-TG2 deposits. Follow-up liver biopsies could be done in five symptomatic patients; four of them showed improvement of histological changes and the disappearance of IgA/anti-TG2 deposits.
There is a body of evidence that suggests that the liver is also a target organ in patients with CeD. Liver diseases in some patients with CeD may occur because of the ongoing systemic autoimmune inflammation and manifest as AIH, primary biliary cirrhosis or primary sclerosing cholangitis, but in many, the liver involvement is independent of the systemic autoimmune inflammation. At present, there is no available specific test that can establish a link between CeD and liver dysfunctions. Hence, the clinical diagnosis is based on clinical suspicion when the other aetiological factors are excluded and reversal of signs post-GFD.19–21 While the circulating IgA anti-TG2 Ab in the blood is the hallmark for screening, the implication of IgA/anti-TG2 deposits in extraintestinal tissues, especially in the archived liver tissue is lacking. An occasional report in this regard including a few patients has demonstrated these deposits in intestinal frozen tissue and reduced deposits post-GFD in patients with CeD, potential CeD, and seronegative CeD.22–26
Taking clues from these initial reports, we hypothesised that demonstration of IgA/anti- TG2 antibody deposits by IgA/anti-TG2 colocalisation studies in liver biopsies from patients with suspected coeliac-associated liver disease may establish a pathogenetic link between these two concurrent conditions, backed up with corresponding duodenal biopsies from patients, and control duodenal and liver biopsies. We systematically evaluated patients with CeD having concurrent liver dysfunctions and ruled out other aetiological association, documented the morphological changes, and demonstrated colocalised IgA/anti-TG2 antibody deposits by two parallelly done techniques on archival FFPE tissue and demonstrated resolution of the lesions in a subset of follow-up biopsies after GFD. The IgA/anti-TG2 antibody complex was mostly located in the vascular endothelial cells, including hepatic sinusoid lining cells and space of Disse, in addition to focal deposits in the hepatocytes and ductal epithelial cells. Deposits were also noted in the duodenal subepithelial basement membrane and enterocyte cytoplasm (figures 2–6). This observation shows that coeliac-associated liver changes and the intestinal pathology in CeD have a common pathogenetic link.
How the liver gets affected in patients with CeD, however, is not completely understood and needs further experimental studies. The earlier belief was that the extraintestinal manifestations in patients with CeD are consequences of intestinal malabsorption, due to increased intestinal permeability, deficiency of micronutrients and macronutrients, and general immune activation. While gliadin peptide activated T-cell mediated injury of the intestinal epithelial cells is well established, in this study we also have identified local intestinal and extra-intestinal deposit of circulating IgA/anti-TG2, which might lead to local inflammation, tissue injury and fibrosis. Like in the intestine, gliadin peptide-induced T-cell-mediated injury of the hepatocytes could also be possible like in the intestine, that needs to be further examined. If that is a reality, the local IgA/anti-TG2 deposits may act as the welcoming factor for the circulating activated T-cells. We agree with the fact that the interesting observations of this study give rise to more questions, rather than answers in this regard, and future researches could be targeted accordingly.26–28
The observations of the present study not only suggest an insight into the common pathogenesis of CeD and its extraintestinal manifestations in the liver; but also brings out the possibility of using IgA/anti-TG2 colocalisation test to demonstrate the pathogenetic link of CeD and its ever-expanding extraintestinal manifestations. We were only allowed ethically to perform follow-up liver biopsies from symptomatic patients. Out of the six symptomatic patients on follow-up five patients gave consent to undergo liver biopsies. The rest of the 17 patients on follow-up improved substantially with GFD and were asymptomatic during the follow-up.
To conclude, the data of the present study adds to the body of evidence that small intestinal pathology and liver lesions in patients with CeD are pathogenetically linked. IgA/anti-TG2 dual antibody deposits demonstrated by the IgA and anti-TG2 antibody colocalisation studies can be one of the possible mechanisms of liver dysfunction in patients with CeD. In the absence of an available laboratory test to establish a link between the CeD and extraintestinal manifestation, the IgA/anti-TG2 colocalisation test seems promising and can be performed on archival biopsies.
Take home messages
The diagnosis of coeliac-associated liver disease is based primarily on the coexistence of liver function abnormalities in patients with coeliac disease and its reversibility with a gluten-free diet.
IgA/anti-tissue transglutaminase (TG2) dual antibody deposits in the duodenal biopsies are characteristic of coeliac disease and they are present even in the absence of serum anti-tTG Ab.
IgA/anti-TG2 deposits were identified in liver biopsies from patients with coeliac-associated liver disease, supporting a pathogenetic link between these two conditions.
IgA/anti-TG2 dual antibody deposits can be used for exploring the association between coeliac disease and other extra-intestinal tissues such even using archival formalin-fixed paraffin-embedded tissues.
Data availability statement
Data are available on reasonable request.
Patient consent for publication
The study design was approved by the Institutional Ethics Committee (reference No. IEC-390/01.08.2016). The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution’s human research committee.
Handling editor Dhirendra Govender.
RD and AI contributed equally.
Correction notice The article has been corrected since it was published online first. Incorrect version of figure 4 was published which has been replaced with the correct one. Author names of Jayanth Kumar Palanichamy, Ashish Chauhan and Ashish Aggarwal have also been corrected.
Contributors RD and AI were involved in patient screening, recruitment, follow-up, patients’ management and performing all techniques applied in this article. PD, SDG, VA and GKM conceived and planned the study and supervised the whole process. JKP was responsible for supervising the confocal microscopy work. AS, WM, AC and AA were responsible for acquiring patient’s data and counseling them. They were also responsible for scheduling patients’ follow-up visits. VS was responsible for statistical analyses. PD is the overall guarantor of this article.
Funding The study was supported by the Intramural Research Grant of the All India Institute of Medical Sciences, New Delhi, India [A641, 2018].
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.