Intestinal cell damage and systemic immune activation in individuals reporting sensitivity to wheat in the absence of coeliac disease
Uhde M, Ajamian M, Caio G et al (2016)
Gut. e-pub ahead of print. doi:10.1136/gutjnl-2016-311964
Some individuals report a range of symptoms in response to the ingestion of wheat and other gluten-containing cereals, yet lack the serological or histological markers of coeliac disease (CD). The terms non-coeliac gluten sensitivity (NCGS) and non-coeliac wheat sensitivity (NCWS) have been used interchangeably to define this group of individuals. The symptoms associated with this condition are similar to coeliac disease, most commonly involving bloating, abdominal pain, diarrhoea, fatigue, headaches and cognitive difficulties.
Gut. e-pub ahead of print. doi:10.1136/gutjnl-2016-311964
Some individuals report a range of symptoms in response to the ingestion of wheat and other gluten-containing cereals, yet lack the serological or histological markers of coeliac disease (CD). The terms non-coeliac gluten sensitivity (NCGS) and non-coeliac wheat sensitivity (NCWS) have been used interchangeably to define this group of individuals. The symptoms associated with this condition are similar to coeliac disease, most commonly involving bloating, abdominal pain, diarrhoea, fatigue, headaches and cognitive difficulties.
Despite much scientific interest in this condition, the aetiology and mechanism of the associated symptoms are largely unknown and diagnostic biomarkers are yet to be identified. However, some studies suggest that a sub-set of individuals demonstrate intercellular junction and immune abnormalities. The authors of this study hypothesised that enhanced antibody response to native gliadin antibodies in NCWS individuals may be a consequence of ongoing epithelial barrier defects. The human intestinal epithelial surface is in constant contact with a large number of micro-organisms, furthermore, compromised intestinal epithelial integrity has been linked to systemic immune activation as a result of microbial translocation from the lumen to the circulation. Therefore, this study sought to investigate:
Eighty individuals who met the recently agreed ‘Salerno Experts Criteria’ for the diagnosis of NCGS1 were included in this study. Subjects completed a modified version of the Gastrointestinal Symptom Rating Scale (GSRS) to evaluate the presence of symptoms commonly associated with NCGS/NCWS, both during consumption of a diet free from wheat, rye and barley and during inclusion of these foods. Serum samples from all participants whilst on an unrestricted diet were available. Samples for 20 of the participants were also available both before and after a 6-month period of a self-monitored wheat, rye and barley free diet. The study also included serum samples from 40 biopsy-proven active CD patients and 40 samples from healthy controls (both groups on a normal/ unrestricted diet at the time of serum collection).
Markers of CD and immune reactivity to gluten
Twenty six percent of subjects in the NCWS group expressed HLA DQ2/8 (a rate similar to the general population). Small intestinal biopsy showed a normal mucosa in 60% of NCWS subjects and Marsh grade 1 (increased epithelial lymphocytes) in the remaining 40%. All CD subjects were positive for HLA DQ2/8 and presented with Marsh grade 3 intestinal histological findings. Amongst the NCWS group, IgG, IgA and IgM antibodies for native gliadin were all significantly higher compared to the healthy control group.
Systemic innate immune activation in response to microbial products
Subjects with NCWS had significantly higher serum levels of lipopolysaccharide binding protein (LBP) and soluble cluster of differentiation 14 (sCD14) compared to healthy controls and CD patients. There was a highly significant correlation between these two findings suggesting that these molecules, both important components of the innate immune system, were concurrently expressed in response to the same stimulus in NCWS individuals. LBP is a soluble acute-phase protein that binds to the lipopolysaccharide (LPS) on the surface of bacteria cells to elicit immune responses by presenting the LPS to CD14 and Toll-like Receptor 4 (TLR4) cell recognition receptors on the surface of macrophages and neutrophils and other cells - stimulating continued immune activation. Soluble CD14 is found in the general circulation and binds LPS in the presence of LBP to further activate TLR4. Translocated circulating LPS can result in rapid secretion of LBP by GI and hepatic epithelial cells, as well as sCD14 by monocyctes and macrophages.
The NCWS group also demonstrated significantly elevated levels of IgM endotoxin-core antibodies (EndoCAb IgM), released in order to neutralise circulating bacterial endotoxin. Furthermore, the levels of IgG and IgM antibodies to flagellin were significantly elevated in a correlated fashion amongst the NCWS group, compared to the healthy controls. Flagellin is the principal component of bacterial flagellum (the tail-like appendages found on bacteria). These findings provide evidence of the translocation of microbial products across the epithelial barrier and systemic innate and adaptive immune activation in response to this stimulus amongst NCWS subjects on an unrestricted diet. Ultimately, the signalling pathway that proceeds the activation of TLRs results in the expression of various pro-inflammatory cytokines that may exert deleterious localised and/or systemic effects, a model consistent with the generally rapid onset of reported symptoms in NCWS.
Link between systemic immune activation and intestinal epithelial damage
In comparison with healthy controls, serum levels of fatty acid-binding protein 2 (FAB2), a marker of intestinal epithelial damage, useful for identifying acute intestinal injury, were significantly elevated amongst NCWS and CD subjects. The FAB2 concentrations in the NCWS group also correlated with the levels of LBP, sCD14, EndoCAb IgM and anti-flagellin IgM, whereas in the CD group, FABP2 concentrations correlated with levels of IgA tTG antibodies. These findings show that markers of intestinal epithelial damage correlated with those of acute systemic immune activation in response to translocated microbial products. Despite evidence of gut epithelial damage, and in contrast to CD, this and previous studies have failed to find evidence of villous atrophy or mucosal architecture abnormalities within small intestinal biopsies of subjects with NCWS. A possible explanation for this is that the epithelial changes associated with NCWS may be more subtle than those observed in CD, furthermore FAB2 is expressed primarily by epithelial cells in the jejunum rather than the duodenum, suggesting an alternative potential site of mucosal damage in NCWS.
Response to dietary restriction
Levels of serum markers of immune activation and epithelial damage, both before and after the removal of wheat, rye and barley, were measured in 20 of the subjects with NCWS. All subjects reported an improvement of symptoms at the end of 6 months, reflected by a significant reduction in symptom scores, accompanied by a reduction in IgG, IgA and IgM anti-gliadin antibodies. Furthermore, these subjects demonstrated a significant reduction in serum levels of LBP, sCD14, EndoCAb IgM, anti-flagellin IgG and IgM and FAB2 after completion of the restricted diet. However, a limitation of this portion of the study was the lack of a healthy control group in order to assess the potential impact of dietary restriction in unaffected individuals.
Conclusions/ implications for clinical practice
The results of this study demonstrate the presence of objective markers of systemic immune activation and correlating serum markers of gut epithelial cell damage in individuals who report sensitivity to wheat, rye and barley in the absence of CD. However, this study does not address the potential mechanism or molecular triggers within wheat, rye and barley that are responsible for the loss of epithelial barrier integrity and consequent microbial translocation. Further research should seek to identify such mechanisms and assess the potential use of identified immune markers for the diagnosis of NCWS and monitoring of response to specific treatment strategies.
1 Catassi C, Elli L, Bonaz B et al. Diagnosis of non-coeliac gluten sensitivity (NCGS): the Salerno experts’ criteria. Nutrients 2015; 7: 4966-77
link to original paper
- Whether systemic immune activation in response to translocated microbial products may be a feature of NCWS
- Whether this systemic immune activation is linked to a compromised intestinal epithelium
- Whether this systemic immune activation or damage to the epithelium is responsive to the elimination of wheat and other gluten-related cereals.
Eighty individuals who met the recently agreed ‘Salerno Experts Criteria’ for the diagnosis of NCGS1 were included in this study. Subjects completed a modified version of the Gastrointestinal Symptom Rating Scale (GSRS) to evaluate the presence of symptoms commonly associated with NCGS/NCWS, both during consumption of a diet free from wheat, rye and barley and during inclusion of these foods. Serum samples from all participants whilst on an unrestricted diet were available. Samples for 20 of the participants were also available both before and after a 6-month period of a self-monitored wheat, rye and barley free diet. The study also included serum samples from 40 biopsy-proven active CD patients and 40 samples from healthy controls (both groups on a normal/ unrestricted diet at the time of serum collection).
Markers of CD and immune reactivity to gluten
Twenty six percent of subjects in the NCWS group expressed HLA DQ2/8 (a rate similar to the general population). Small intestinal biopsy showed a normal mucosa in 60% of NCWS subjects and Marsh grade 1 (increased epithelial lymphocytes) in the remaining 40%. All CD subjects were positive for HLA DQ2/8 and presented with Marsh grade 3 intestinal histological findings. Amongst the NCWS group, IgG, IgA and IgM antibodies for native gliadin were all significantly higher compared to the healthy control group.
Systemic innate immune activation in response to microbial products
Subjects with NCWS had significantly higher serum levels of lipopolysaccharide binding protein (LBP) and soluble cluster of differentiation 14 (sCD14) compared to healthy controls and CD patients. There was a highly significant correlation between these two findings suggesting that these molecules, both important components of the innate immune system, were concurrently expressed in response to the same stimulus in NCWS individuals. LBP is a soluble acute-phase protein that binds to the lipopolysaccharide (LPS) on the surface of bacteria cells to elicit immune responses by presenting the LPS to CD14 and Toll-like Receptor 4 (TLR4) cell recognition receptors on the surface of macrophages and neutrophils and other cells - stimulating continued immune activation. Soluble CD14 is found in the general circulation and binds LPS in the presence of LBP to further activate TLR4. Translocated circulating LPS can result in rapid secretion of LBP by GI and hepatic epithelial cells, as well as sCD14 by monocyctes and macrophages.
The NCWS group also demonstrated significantly elevated levels of IgM endotoxin-core antibodies (EndoCAb IgM), released in order to neutralise circulating bacterial endotoxin. Furthermore, the levels of IgG and IgM antibodies to flagellin were significantly elevated in a correlated fashion amongst the NCWS group, compared to the healthy controls. Flagellin is the principal component of bacterial flagellum (the tail-like appendages found on bacteria). These findings provide evidence of the translocation of microbial products across the epithelial barrier and systemic innate and adaptive immune activation in response to this stimulus amongst NCWS subjects on an unrestricted diet. Ultimately, the signalling pathway that proceeds the activation of TLRs results in the expression of various pro-inflammatory cytokines that may exert deleterious localised and/or systemic effects, a model consistent with the generally rapid onset of reported symptoms in NCWS.
Link between systemic immune activation and intestinal epithelial damage
In comparison with healthy controls, serum levels of fatty acid-binding protein 2 (FAB2), a marker of intestinal epithelial damage, useful for identifying acute intestinal injury, were significantly elevated amongst NCWS and CD subjects. The FAB2 concentrations in the NCWS group also correlated with the levels of LBP, sCD14, EndoCAb IgM and anti-flagellin IgM, whereas in the CD group, FABP2 concentrations correlated with levels of IgA tTG antibodies. These findings show that markers of intestinal epithelial damage correlated with those of acute systemic immune activation in response to translocated microbial products. Despite evidence of gut epithelial damage, and in contrast to CD, this and previous studies have failed to find evidence of villous atrophy or mucosal architecture abnormalities within small intestinal biopsies of subjects with NCWS. A possible explanation for this is that the epithelial changes associated with NCWS may be more subtle than those observed in CD, furthermore FAB2 is expressed primarily by epithelial cells in the jejunum rather than the duodenum, suggesting an alternative potential site of mucosal damage in NCWS.
Response to dietary restriction
Levels of serum markers of immune activation and epithelial damage, both before and after the removal of wheat, rye and barley, were measured in 20 of the subjects with NCWS. All subjects reported an improvement of symptoms at the end of 6 months, reflected by a significant reduction in symptom scores, accompanied by a reduction in IgG, IgA and IgM anti-gliadin antibodies. Furthermore, these subjects demonstrated a significant reduction in serum levels of LBP, sCD14, EndoCAb IgM, anti-flagellin IgG and IgM and FAB2 after completion of the restricted diet. However, a limitation of this portion of the study was the lack of a healthy control group in order to assess the potential impact of dietary restriction in unaffected individuals.
Conclusions/ implications for clinical practice
The results of this study demonstrate the presence of objective markers of systemic immune activation and correlating serum markers of gut epithelial cell damage in individuals who report sensitivity to wheat, rye and barley in the absence of CD. However, this study does not address the potential mechanism or molecular triggers within wheat, rye and barley that are responsible for the loss of epithelial barrier integrity and consequent microbial translocation. Further research should seek to identify such mechanisms and assess the potential use of identified immune markers for the diagnosis of NCWS and monitoring of response to specific treatment strategies.
1 Catassi C, Elli L, Bonaz B et al. Diagnosis of non-coeliac gluten sensitivity (NCGS): the Salerno experts’ criteria. Nutrients 2015; 7: 4966-77
link to original paper
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