LIVER STEATOSIS AND FIBROSIS IN NON-CELIAC WHEAT SENSITIVITY PATIENTS: A PROSPECTIVE STUDY

Many people with symptoms similar to inflammatory bowel syndrome (IBS) or functional dyspepsia (FD) follow a wheat-free diet (WFD) because it is subjectively better tolerated, even if they do not suffer from celiac disease (CeD) or wheat allergy. This condition, originally named non-celiac gluten sensitivity, has been redefined as non-celiac wheat sensitivity (NCWS), because its clinical manifestations can be triggered by a spectrum of non-gluten wheat proteins, such as wheat amylase-trypsin inhibitors (ATIs), that cause a delayed type - non-IgE-mediated food allergy associated with an intestinal mucosa barrier (IB) defect.

Metabolic dysfunction-associated steatotic liver disease (MASLD) has emerged as the most common liver disease in countries with excess nutrient supply. In addition to the main etiopathogenetic factors, other factors must be implicated: nutrition, intestinal microbiota, intestinal permeability (IP) and the gut-liver axis might play a key role due to the translocation of nutrient-derived peptides and microbial products into the intestinal lamina propria. Here, the liver is prominently exposed to the inflammatory intestinal signals via the mesenteric-portal venous system, that significantly contributes to the onset of MASLD, metabolic disfunction-associated steatohepatitis (MASH) and liver fibrosis.

In patients with NCWS, intake of wheat and wheat ATIs (Amylase-Trypsin Inhibitors) increases IP, promotes intestinal dysbiosis, and activates the gastrointestinal and extra-intestinal immune response.

Hypothesizing an intestinal barrier impairment as a common pathophysiological substrate of both NCWS and MAFLD, in a retrospective cohort study (data not yet published), demographic, clinical, laboratory and histology data of NCWS patients at the time of diagnosis, were analyzed and compared to control subjects with IBS/FD and freshly diagnosed CeD. NCWS diagnosis was performed by a double-blind placebo-controlled wheat challenge. Steatosis was confirmed by ultrasound examination. Our retrospective data showed that the frequency of liver of steatosis was lower in NCWS than in IBS patients. In addition, it seems that pre-diagnosis avoidance of wheat in NCWS correlates with protection from steatosis. A subset of NCWS patients, recently exposed to wheat, with clinical features suggesting increased IP, seems to be predisposed to liver steatosis and fibrosis.

To validate the results of the retrospective study, the researchers planned the present prospective study, to analyze the prevalence of liver steatosis and fibrosis, evaluated by ultrasound examination, FibroScan analysis [LSM (Liver Stiffness Measurement) and CAP (Controlled Attenuation Parameter) values], FIB-4 (Fibrosis-4) index, and NFS [Non-alcoholic fatty liver disease (NAFLD) Fibrosis Score], in patients with NCWS at the time of diagnosis, comparing them with two control populations of newly diagnosed IBS/FD and CeD patients.

The inclusion/exclusion criteria used to select the study population have been previously validated in other retrospective studies. Additional exclusion criteria related to steatosis and other liver diseases and specifically required for this study were adopted.

Inclusion criteria

for Non-Celiac Wheat Sensitivity (NCWS) patients

• age >18 and <65 years;
• subjects with wheat-dependent symptoms, both gastrointestinal and extra-intestinal;
• negativity of IgA and IgG anti-deamidated gliadin peptide (DPG) antibodies, immunoglobulin (Ig)A and IgG anti-tissue transglutaminase (tTG) antibodies, and anti-endomysial antibodies (EMA);
• absence of duodenal villous atrophy, documented in all patients carrying the human leukocyte antigen (HLA) DQ2 and/or DQ8 haplotypes (therefore regardless of the negativity of celiac disease (CeD)-specific serum antibodies), evaluated when the patients had consumed a minimum of 100g of pasta and/or bread a day, for at least 45 days;
• absence of IgE-mediated wheat allergy (WA): negative skin prick-test and/or specific serum IgE assay for wheat, gluten and gliadin);
• resolution of symptoms on a strict standard elimination diet (i.e. extended oligoantigenic, excluding wheat, cow's milk, egg, tomato and chocolate and other foods self-reported by the patient as causing symptoms), followed for at least 4 weeks, and the recurrence of the same symptoms after double-blind placebo-controlled challenge (DBPCC) with wheat (for further details see below);
• complete medical records;
• duration of follow-up longer than 12 months after initial diagnosis, with at least 2 outpatient visits during the follow-up period.

Inclusion criteria

for Irritable Bowel Syndrome/Functional Dyspepsia (IBS/FD) and other functional gastrointestinal disorders unrelated to NCWS or other food allergies/intolerances patients

• age >18 and <65 years;
• subjects diagnosed with IBS/FD and other functional gastrointestinal disorders, according to the Rome IV classification, 1 who did not specifically report symptoms/signs, whether gastrointestinal or extra-intestinal, following ingestion of wheat or other foods and who did not respond to gluten-free diet (GFD).

Inclusion criteria

for Celiac Disease (CeD) patients

• age >18 and <65 years;
• subjects with gastrointestinal and extra-intestinal wheat-dependent symptoms that meet the diagnostic criteria of CeD 2: positivity of anti-tTG IgA and/or IgG antibodies and evidence of villous atrophy, according to the Marsh-Oberhuber classification, demonstrated by histology on duodenal biopsy;
• clinical response to the GFD: resolution of gastrointestinal and/or extra-intestinal symptoms.

Exclusion criteria

for all the patients enrolled in the study

• self-exclusion of wheat from the diet and refusal to reintroduce it for diagnostic purposes, before entering the study;
• drug abuse;
• treatment with steroids and/or non-steroidal anti-inflammatory drugs in the 2 weeks before duodenal biopsy;
• pregnancy or breastfeeding;
• diagnosis of chronic inflammatory bowel disease or other organic pathologies affecting the digestive system (e.g., wheat allergy, microscopic colitis, diverticulitis, segmental colitis associated with diverticulosis, etc.), neurological diseases, major psychiatric disorders, infectious diseases, immunological deficiencies, and impairments limiting physical activity;
• incomplete medical records;
• lack of clinical follow-up for at least 12 months after diagnosis with >2 outpatient visits during the follow-up period.

Additional exclusion criteria related to liver steatosis and other liver diseases

• absence of abdominal ultrasound (US) imaging performed before diagnosis (i.e. before starting the wheat-free/gluten-free diet in NCWS and CeD patients, and before any lifestyle modifications and/or drug/prebiotic/probiotic intake in IBS/FD patients);
• incomplete clinical records, lacking the data considered for the present study;
• chronic alcohol intake (>30 g/day for men and >20 g/day for women);
• chronic hepatotropic virus infections [hepatitis B virus (HBV) and hepatitis C virus (HCV)];
• autoimmune liver diseases;
• congenital metabolic liver diseases (e.g. alpha-1 antitrypsin deficiency, hemochromatosis, Wilson's disease, porphyria, other storage diseases, etc.);
• chronic long-term treatment with drugs associated with both macrovesicular (glucocorticoids, estrogens, tamoxifen, amiodarone, methotrexate, and 5-fluorouracil) and microvesicular (glucocorticoids, valproic acid, tetracycline, and zidovudine) steatosis.