More fuel to the re: Some patients with non-celiac gluten sensitivity exhibit adaptive immunological responses in duodenal mucosa

In contrast to the well characterized Celiac Disease (CD), the clinical scenarios encompassed in non-celiac gluten sensitivity (NCGS) might be related to different antigens that trigger distinct immune-inammatory reactions. Although an increased number of intestinal intraepithelial lymphocytes is observed at the inception of both diseases, subsequent immunopathogenic pathways seems to be different. Aims To compare the immunological prole in the duodenal mucosa of patients with CD, self-reported gluten intolerant subjects and gluten tolerant patients with functional dyspepsia (GT-FD).


Introduction
Celiac disease (CD) and non-celiac gluten sensitivity (NCGS) are gluten related disorders (GRD) that share clinical characteristics but have marked serological and histological differences. [1][2][3] While autoantibodies and duodenal villus atrophy (VA) must be present in order to diagnose CD, they have to be absent in order to establish a presumptive diagnosis of NCGS.
Due to the lack of speci c biomarkers the diagnosis of NCGS is largely based upon clinical characteristics with symptoms improving during a gluten-free diet and symptomatic relapse while consuming a gluten-containing diet, along with normal duodenal biopsies and negative CD serology. In these cases, wheat allergy should be also discarded . 4,5 CD is a well-characterized disease with speci c histological and serological features and convincing immunopathology mechanisms triggered by the ingestion of gluten and related proteins. In genetically predisposed individuals, gluten ingestion results in mucosal in ammation and VA mediated by a series of processes orchestrated by CD4+ T helper 1 (Th1) and Th17 cells. [6][7][8][9][10] In contrast, NCGS is a poorly characterized disorder in which the role of gluten as the main antigen and the pathophysiologic mechanisms involved in tissue damage and symptoms development are debatable.
Actual evidence suggests a paramount role of the innate immune response, although some authors have found that the adaptive immune response may be involved. 6,[11][12][13][14][15] The main objective of this work is to compare the immune response in duodenal mucosa of celiac patients with that of self-described gluten intolerant subjects. Although an increased number of intestinal intraepithelial lymphocytes is observed at the inception of these entities, subsequent immunopathogenic pathways seems to be different. Therefore, we compared the immunologic pro le of the duodenal mucosa of subjects with CD, NCGS and a control group of gluten tolerant subjects with functional dyspepsia (GT-FD) focusing in the pro-in ammatory cytokine-expressing monocyte and monocyte-derived dendritic cells involved in innate immune activation, cytokine-driven polarization and maintenance of Th1 polarization and Th17/Th22, and anti-in ammatory/pro brogenic cytokines.

Patients and controls
In this blind and cross-sectional study, we included 15 consecutive untreated patients with active CD and 9 individuals with NCGS who attended to the Department of Gastroenterology outpatient clinic at the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, a tertiary care medical facility in Mexico City.
CD was diagnosed when patients met the following criteria: 1) Compatible clinical data: chronic diarrhea, weight loss, bloating, abdominal disconfort, fatigue or, nutrient de ciencies, 2) positive serum autoantibodies: anti-endomysium (EmA IgA), anti-transglutaminase (anti-tTg IgA) and anti-deamidated gliadin peptide antibodies (AGA-DGP IgA and IgG) and, 3) VA according to Marsh-Oberhuber criteria. 16 Diagnosis of NCGS was considered when patients presented with: 1) intestinal and extra intestinal symptoms associated with the ingestion of gluten-containing food, 2) a clear clinical response while they followed a gluten-free diet (GFD), 3) relapse of symptoms with the ingestion of gluten-containing foods, 4) negative CD (EmA IgA, anti-tTG IgA, AGA-DGP) and wheat allergy (IgE serological test) serological markers and, 5) normal duodenal biopsies.
All patients were evaluated by a quali ed nutritionist with expertise in CD. Symptoms severity (abdominal discomfort or pain, bloating, diarrhea, and constipation) was assessed using a visual analog scale (VAS: 0-10) at baseline, while on an unrestricted diet, 6 weeks after they followed a GFD and after concluding a 6 week challenge based on a diet adjusted to contain 10 grams of gluten par day. Diet compliance was evaluated at biweekly out-patient visits during the gluten-challenge. All serological tests (EmA IgA, anti-tTG IgA, AGA-DGP) were performed at baseline visit and at conclusion of the 6 weeks gluten challenge. The presence of headache, tingling or numbness on feet or hands, fatigue, musculoskeletal pain, brain fog, rash and oral ulcers were considered extra-intestinal symptoms and were speci cally evaluated.
We considered a good clinical response to the GFD when there was a decrease in the intensity of symptoms of at least 50% compared to the basal VAS. We did not perform a double-blind gluten/placebocontrolled trial challenge in any case.
We excluded patients with other gastrointestinal diseases, history of gastrointestinal surgery, active or previous infectious diseases, clotting disorders, renal insu ciency, pregnancy or breast feeding, active use of antimicrobial, probiotics, immunosuppressive drugs, non-steroidal anti-in ammatory drugs or corticosteroids.
As a control group we included 10 subjects that ful lled ROME III criteria for functional dyspepsia (FD) with negative CD serology and who had undergone an upper endoscopy with normal duodenal histology (GT-FD).

Biopsy Sampling
During upper endoscopy four tissue samples from the second portion of the duodenum were obtained, two of them were placed immediately in ice-chilled Hank buffer solution (HBSS) /5% fetal bovine serum (SFB, GIBCO). The others were xed in 10% formaldehyde and subsequently embedded in para n wax and cut into 4 μm thick sections.

Intraepithelial lymphocytes (IELs) Isolation from Duodenal Tissue
Duodenal mucosa samples (epithelium and lamina propria) were cut with a scalpel blade and incubated in phosphate buffer 1x (PBS) / ethylenediamine tetra acetic acid (EDTA) 2mM at 34ºC for 30 min while being agitated. After that, samples were treated with Collagenase IV (Sigma) at 60 U/ml for 1 h at 34°C while being agitated. The cell suspension was then passed through a 40μm cell strainer (Cell Strainer BD Falcon), washed with 2 ml of PBS, and centrifuged at 800 g for 10 min at 25°C. The resulting pellet was homogenized in 1 mL of PBS and incubated with 1 μL of Brefeldin A (BD Golgi Plug) for 1 h at 37°C with 5% CO2. Live-dead assay and cellular count was realized on cellular samples (>90%) on Neubauer chamber (trypan blue).

Immunohistochemistry
Tissues placed on positively charged slides were incubated with mouse monoclonal anti-human IL-1β, IL-6, IL-8, IL-10, IL-15, IL-22, IL-23, IFN-γ, TNF-α, and with rabbit polyclonal anti-human IL-2, IL-12p40, IL-17A, IL-21, or TGF-β1 antibody (Abcam, Cambridge, MA, USA) or anti-human IL-4 antibody (Bio Legend Inc., San Diego, CA, USA) at 10 µg/mL during 30 min. Binding was detected with Universal Dako labelled streptavidin biotin reagent+peroxidase for primary antibodies from rabbit, mouse and goat (Dako, Glostrup, Denmark). Spleen and ganglion samples were used as a positive control. Negative controls were carried out with normal human serum (1:100) and with the IHC universal negative control reagent (Enzo Life Sciences, Inc., Farmingdale, NY, USA), while phosphate buffer saline-egg albumin (SIGMA-Aldrich) was use in the reactive blank. Controls excluded nonspeci c staining or endogenous enzymatic activities. We examined three different sections of each biopsy. Cytokine-expressing cells were reported as the percentage of positive cells in three elds (X320) taken from the epithelium and lamina propria.
Results are expressed as the median, mean and 5 th /95 th percentiles.
We collected a sample of venous blood to isolate PBMCs by gradient centrifugation on Ficoll-Paque (Merck-Millipore). The bottom was resuspended in 1 mL of PBS 1x /Brefeldin A (BD GolgiPlug) and incubated at 37ºC in 5% CO 2 during 1h. Live-dead assay (trypan blue) and cellular count was realized on cellular samples (>90%). Flow Cytometry. For an auto uorescence control, we ran an unstained and permeabilized cell sample. An AbC anti-mouse bead kit (Invitrogen, UK) was used to adjust instrument settings, to set uorescence compensation, and to check instrument sensitivity. Fluorescence minus one (FMO) controls were stained in parallel. Samples were analyzed with an Attune Acoustic Focusing Cytometer Blue/Red (Life Technologies). We recorded more than 10,000 events for each sample, and they were analyzed with Attune® Cytometric Software v2.1.
Ethical Considerations. This work was performed according to the principles expressed in the Declaration of Helsinki. Our Institutional research and ethics committees approved the protocol (GAS-1298-14/15-1; August 11, 2014). Each patient signed a written informed consent.

Statistical analysis
Data were analyzed using GraphPad Prism for Windows (version 6.01 GraphPad software Inc. USA) and Kruskall Wallis non-parametric test. Immunohistochemical data are expressed as the median, mean and 5 th /95 th percentiles. We performed one-way ANOVA on ranks by Holm-Sidak method and Dunn's test for all pairwise multiple comparison procedures. A p value <0.05 was considered statistically signi cant.

Results
Demographic and clinical characteristics are summarized in Table 1 and 2. Diarrhea, abdominal pain and bloating were the most frequent symptoms in both, CD and NCGS patients. All NCGS patients' symptoms improved during the GFD but while on the gluten challenge, they presented with bloating and abdominal pain. One of them complained from mouth itching. Nonetheless all completed the challenge.
In 2 patients, the levels of AGA-DGP IgA (NCGS) and AGA-DGP IgG (control group) were above the upper limit of normal (ULN). None of them showed other alterations neither in serology nor in histopathology.

Percentage of mucosal subpopulations in duodenal tissue
The percentage of CD4+ mLs was higher in CD patients compared to the control group (P=0.003, Figure  1B). No differences were found between CD patients and NCGS nor between NCGS and the control group (GT-FD).
CD patients had a signi cantly higher CD4/FoxP3 percentage in duodenum compared to the control group. (P=0.039, Figure 1D). The NCGS CD4/FoxP3 percentage was similar to both, CD and control group.
T-bet and ROR-γt were higher in CD patients versus control group (P=0.036, Figure 1F and P=0.03, Figure  1J, respectively). No differences were observed between CD patients and NCGS. Neither there was any difference when comparing NCGS to the control group. Moreover, the percentage of IFN-γ− and IL-17A−expressing CD4 mucosal T cells in the CD group, although higher than NCGS, was not statistically signi cant amongst the three groups ( Figure 1H and 1L).

Pro-in ammatory cytokines in duodenal tissue
The percentage of IL-1β− and TNF-α−expressing cells in tissue of CD and NCGS patients was signi cantly higher compared to the control group. It is noteworthy, that tissue of NCGS patients had statistically signi cant lower levels of IL-1β− and TNF-α−expressing cells compared with CD patients (Figure 2A,B).
The number of IL-6− and IL-8−producing cells was signi cantly higher in CD patients compared with control group and NCGS patients. No differences in the number of IL-6+ or IL-8+ cells were found in the NCGS group compared to the control group ( Figure 2C,D).

Cytokines involved in the differentiation and maintenance of Th1 in duodenal tissue
The percentage of IL-2+ and IFN-γ+ cells of CD patients was higher compared to NCGS and control group. Anti-in ammatory/Pro brogenic cytokine expression in duodenal No differences were observed in IL-4 cell percentage when compared CD patients or NCGS versus control group ( Figure 5A). TGF-β1− and IL-10−expressing cells from CD or NCGS patients were higher versus control group ( Figure  6B,C). However, no statistically signi cant difference was found between CD and NCGS patients.

Discussion
CD is a well-characterized disease with speci c histological and serological features and established immuno-pathological mechanisms triggered by the ingestion of gluten and related proteins, affecting genetically predisposed individuals. 7,10 In contrast, NCGS is a disorder seeking its own identity. It is a condition that encompasses different clinical scenarios including subjects with irritable bowel syndrome (IBS) and patients with food intolerances. NCGS abdominal and extra-intestinal troubles trigger by foods containing gluten and symptoms typically improve with a GFD. 17,18 The ambiguity about its existence is based on the absence of speci c biological markers and histological characteristics. Its diagnosis has been based on information from complex and heterogeneous double-blind placebo-controlled challenges that used different vehicles and doses of gluten. 19,20 In an attempt to standardize NCGS diagnosis, a group of experts met in Salerno and based on the results of a double-blind placebo-controlled challenge using 8 grams of gluten administered over 2 periods of one week separated by one-week wash-up term proposed an expert´s diagnostic criteria. 21 Since the Salerno criteria is di cult to ful ll in the clinical setting, once CD and wheat allergy have been reasonably ruled out, the presumptive diagnosis of NCGS can be considered and is based merely on the clinical responses to a diet with and without gluten. 22 Despite these diagnostic caveats, we are con dent that our self-considered gluten intolerant patients and, here classi ed as NCGS, have wheat intolerance considering that all of them improved while on a GFD and relapsed when they followed a gluten-containing diet challenge. CD and wheat allergy were reasonable excluded through serological test and histological features. Since we did not perform a double-blind gluten/placebo-controlled challenge in any case, the role of other antigens besides the gluten present in wheat cannot be excluded.
While on an unrestricted diet, ow cytometry and immunohistochemical assessment showed that CD4+ cell subpopulations were quite different among groups.
As expected, in ow cytometry, CD patients exhibited a wealth range of innate and adaptive immune responses when compared to the control group. The percentage of in ammatory and regulatory cells CD4+ T, CD4+/Foxp3+, CD4+/T-bet+, CD4+/ROR-γt+ were higher in CD than in the control group and NCGS patients. Importantly, the percentage of in ammatory and regulatory CD4+ cells was higher in the NCGS patients compared to the control group but lower than that observed in CD subjects. Although this nding did not reach statistically signi cant difference, it coincides with prior reports, suggesting that an in ammatory process is present in these self-reported gluten intolerant subjects. 18 Immunohistochemical analysis showed the most noticeable changes. The percentage of proin ammatory cytokine-expressing cells in the duodenal mucosa was higher in patients than in controls. The in ammatory response was conspicuously higher in CD subjects. Pro-in ammatory cytokineexpressing cells were also evident in NCGS patients, except for IL-6-and IL-8-expressing cells.
Present evidence suggests that innate immune response plays a central key role in the pathophysiology of NCGS. Different authors have reported an increased expression of toll-like receptor 2 (TLR-2) and 4 (TLR-4), claudin 4 (CLD-4), IL-8 and TNF-α in subjects with self-reported gluten intolerance. 12,23,24 According to this concept, IL-1-and TNF-α-expressing cells were higher in NCGS patients compared to the control group. All these ndings support the widely demonstrated participation of innate immunity in both, CD and NCGS. 10,18 Th cell polarization from naïve precursor is a tightly controlled process, where IL-12 and IL-15 play a central role as factors involved in the differentiation of Th1 response. 25,26 The secreted IL-2 by activated antigen-speci c CD4+ and CD8´T cells is consumed at the same and distant sites by cells that express the IL-2Rα (e.g. effector T cells, NK cells and Treg). IL-2 acts via STAT5 and in uences the differentiation of Th1, Th2 and Th17 cell subsets, also it is important to maintain the transcriptional program for the Treg function. 27 In addition, IL-2 probably stimulates the differentiation of other cell groups while IL-12 does it for Th1 and, IL-22 and IL-23 for Th17. In contrast, IL-15 not only promotes the increase of IEL´s in CD but also supports Th1 and Th17 responses. 25,[28][29][30] Meeting these concepts, we observed that the percentage of cytokine expressing-cells that induce and maintain Th1 and Th17 polarization in the mucosa of CD patients was higher compared to the other groups. Interestingly, IL-2-and IL-12-expressing cells were also increased in NCGS group compared to control group, suggesting some participation of adaptive immunity components in these self-reported wheat intolerant subjects.
Recent evidence supports the participation of adaptive immune mechanisms in NCGS. Mucosal based studies have found high levels of IFN-γ in duodenum and rectum of NCGS patients challenged with gluten. (31,32) Moreover, it has been shown that these patients are able to produce speci c antibodies to native gliadins and, at least 50% of them compared to 30% of the general population, express HLA-DQ2/DQ8, haplotypes the risk for CD. (33) It should be noted that we found haplotypes of risk for CD in 6 of 8 patients with NCGS while all CD subjects had HLA DQ2/DQ8 genes.
TGB-β1-expressing cells were notably higher in both, CD and NCGS patients. It is very likely that this nding is an attempt to control the in ammatory process elicited by peptides from prolamines in the case of CD or other antigens recognize in NCGS patients (34). Interestingly IL-10-expressing cells were not increased neither in CD nor in NCGS. In fact, they were lower in NCGS than in the other groups.
Our ndings in CD patients are quite similar to those widely reported in the literature but some differences can be noted in our NCGS patients. At the present time the term NCGS represent an umbrella covering a wide range of unspeci c non-celiac clinical scenarios. Our patients with NCGS were diagnosed on clinical bases excluding wheat allergy and CD, however, we did not perform a double-blind gluten-placebo challenge in any case and the participation of other antigens, besides gluten, could not be excluded. It could be argued that in fact they could represent different forms of CD, perhaps seronegative CD, mainly because the majority present with HLA DQ2/DQ8 genotype, however all of them had a normal duodenal mucosa while on a gluten-containing diet. (35) Due to the mentioned NCGS diagnostic aws, these patients represent a true challenge in clinical practice. In the absence of a double-blind gluten/placebo-controlled challenge suggested by the Salerno expert consensus, it seems reasonable to consider them as patients with wheat intolerance as it has already been suggested (17,18). At the present time it seems clear that other wheat components like fermentable short-chain carbohydrates (FODMAP) or amylase-trypsin inhibitors (ATI) play a role in eliciting symptoms and in ammatory response mainly mediated by innate immunity (15,34). In this work we found that our patients with NCGS exhibited components of both innate and adaptive immunity. We believe that these ndings provide one more piece to the complicated puzzle of wheat sensitivity disorders.

Conclusions
Some self-reported gluten intolerant subjects have components of both, innate and adaptive immunity response in the duodenal mucosa. The clinical characteristics and HLA genotype of our patients resemble to that described in subjects with IBS-D sensible to wheat and it probable represent a distinct  Availability of data and materials The datasets during or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests
None to report.

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors. All authors read and approved the nal version of the manuscript to be published for the original study on which the present article is based *Give information separately for exposed and unexposed groups.