| Abstract|| |
Background: It is hypothesized that the duodenal mucosal damage in patients with celiac disease (CeD) is caused by the mucosa-infiltrating lymphoid cells. This study aimed to analyze the immune effective and regulatory T (Treg) cells in duodenal biopsies from treatment-naive adult patients with CeD having different histological grades and controls. Patients and Methods: Dual-color immunohistochemical staining was done in a total of 234 duodenal biopsies, including 132 controls and 102 adult patients with CeD using CD20, CD3:CD4, CD3:CD8, CD4:FoxP3, CD8:FoxP3, and TCRαβ:TCRγδ antibodies. The density of these lymphoid cells in lamina propria and mucosal epithelium was compared between controls and CeD, with different modified Marsh grades. Results: Densities of CD4+ T cells in lamina propria and CD8+γδ intraepithelial lymphocytes (IELs) were significantly more in biopsies from patients with CeD, than in controls. An increasing linear pattern of IELs, CD3+ T cells, and CD20+ B cells was observed with increasing grades of villous abnormalities. Although CD8+ FoxP3+ Treg cells were significantly more in biopsies from patients with CeD, there was no significant difference in CD4+ FoxP3+ Treg cell infiltrate between both the groups. Conclusion: Our finding in this observational study generates interest to study the local intestinal mucosal immunity in CeD in detail. A study to prove the failure of CD4+ FoxP3+ Treg cell recruitment in CeD and its direct functional impact may yield valuable information regarding loss of mucosal tolerance.
Keywords: Duodenum, immunohistochemistry, intraepithelial lymphocytes, mucosa, tolerance
|How to cite this article:|
S. Gahlot GP, Das P, Baloda V, Singh A, Vishnubhatla S, Gupta SD, Makharia GK. Duodenal mucosal immune cells in treatment-naive adult patients with celiac disease having different histological grades and controls. Indian J Pathol Microbiol 2019;62:399-404
|How to cite this URL:|
S. Gahlot GP, Das P, Baloda V, Singh A, Vishnubhatla S, Gupta SD, Makharia GK. Duodenal mucosal immune cells in treatment-naive adult patients with celiac disease having different histological grades and controls. Indian J Pathol Microbiol [serial online] 2019 [cited 2019 Aug 17];62:399-404. Available from: http://www.ijpmonline.org/text.asp?2019/62/3/399/263498
| Introduction|| |
The current understanding of immune pathogenesis in celiac disease (CeD) is intriguing and represents a complex interplay between genetic factors (both HLA and non-HLA genomic loci) and environmental factors. Tightly regulated local mucosal immunity has been found to be essential for protection against luminal microflora and tolerance to dietary proteins., Intestinal mucosal dendritic cells (DCs) are crucial in initiating a tolerogenic state in response to enterocyte-driven factors such as retinoic acid, transforming growth factor-β (TGF-β), by upregulating the tolerance producing mucosal regulatory T (Treg) cells, a subset of CD4/CD8+ T cells with high density of CD25, and transcription factor forkhead box P3 (FoxP3) epitopes. FoxP3 epitopes can be used as a highly specific intracellular marker of these Treg cells. The main histologic feature of the CeD is increased intraepithelial lymphocytes (IELs) with or without villous atrophy of the duodenal mucosa. Inflammatory cell infiltrates are also noted in lamina propria in intestinal biopsies from patients with CeD, which leads to high cytokine level and suppresses the tolerogenic property of the DCs, affecting Treg-mediated mucosal tolerance.,,,,,, Brown et al. noticed an increasing linear pattern of mucosal B cells and T cells in patients with CeD in comparison to controls, along with increasing histological Corazza grades. In CeD, starting from a low number of Treg cells in circulating blood to their suppressed function have been hypothesized as the possible cause of loss of mucosal immunity,,,,, while other studies demonstrated a higher number of mucosal FoxP3+ Treg cells in intestinal biopsies of patients with CeD in comparison to controls.,,, In this study, we aimed to examine the density of immune effector and Treg cells in duodenal mucosal biopsies from adult patients with CeD having different modified Marsh grades and in controls.
| Patients and Methods|| |
It was a cross-sectional study based on a total of 234 duodenal biopsies, including 132 controls and 102 adult patients with CeD, and informed consent was obtained before taking biopsies. The study was approved by the Institute's Ethics Committee.
Patients with CeD included in this study were diagnosed as per the European Society of Pediatric Gastroenterology, Hepatology and Nutrition criteria. Serum antitissue transglutaminase (anti-tTG) titer and presence of villous abnormalities by histology were examined in all. Patients already on a gluten-free diet (GFD), patients having coexistent systemic diseases, human immunodeficiency virus infection, active, or past tubercular infection, and patients unwilling to participate in the study were excluded.
Of the 132 control biopsies, 50 biopsies were collected from subjects with functional dyspepsia, in whom endoscopic and laboratory investigations excluded any organic disease. The remaining 82 biopsies were collected from hepatitis B virus carriers (n = 82) undergoing esophagogastroduodenoscopy for screening.
Anti-tTG titer was determined using anti-tTG antibody (Ab) enzyme-linked immunosorbent assay (ELISA) kits. During the execution of this work, as anti-tTG ELISA kits from different manufacturers were used, for uniformity an anti-tTG Ab fold rise value was estimated as the tTG value in a patient ÷ normal cut-off value for that particular ELISA kit.
Processing of duodenal mucosal biopsies
Four to six mucosal biopsies were obtained from the postampulla second part of the duodenum from all recruits after taking informed consent. Hematoxylin–eosin-stained slides were cut from paraffin blocks, and oriented areas of the biopsy fragments were analyzed. The modified Marsh grading system of Oberhuber et al. was used for grading the severity of villous abnormalities.
Dual-color immunohistochemistry (IHC) with CD20, CD3:CD4, CD3:CD8, CD4:FoxP3, CD8:FoxP3, and TCRαβ:TCRγδ cells characterized the lymphocytes in lamina propria and the IELs. The primary antibodies used were standardized as per the following dilutions in our laboratory: CD20 (1:400; Spring Bioscience, CA, USA), CD3 (1:200; Spring Bioscience), CD4 (1:200; Spring Bioscience), CD8 (1:200; Spring Bioscience), FoxP3 (1:100; Spring Bioscience), T-cell receptor αβ (1:400; Santa Cruz Biotechnology, Dallas, TX, USA), and T-cell receptor γδ (1:100; Santa Cruz Biotechnology). Appropriate control sections were used for all the immunohistochemical markers. Two experienced pathologists, blinded to the clinical profile of the patients, evaluated all the duodenal biopsy and the immunohistochemical stains.
In short, the dual IHC procedure was as follows: antigen retrieval was done by boiling the tissue sections in 0.01 M citrate buffer at pH 6. After blocking endogenous peroxidase activity and nonspecific antigen binding, the tissue sections were incubated overnight at 4°C with primary rabbit antihuman FoxP3 monoclonal Ab clone SP97 (catalog no. M3974) and detected using an alkaline phosphatase–conjugated secondary Ab with fast Red Substrate Kit (Rabbit Uno VueTM ALP/Perma Red Detection System, catalog no. UR100AR; DBS, USA). Then, the sections were treated with peroxidase block solution, followed by incubation for 2 h at 4°C with secondary primary rabbit antihuman CD4 monoclonal Ab clone SP35 (catalog no. M3354), followed by detection with horseradish peroxidase–conjugated secondary Ab and 3,3'-diaminobenzidine substrate kit. Tissue sections were cleared, counterstained with hematoxylin stain, and mounted with distyrene plasticizer xylene.
Interpretation of IHC
Positivity of >10% immune infiltrative lymphocytes was taken as significant. Percentage of immune positivity was calculated by visual estimation of positive immune cells in comparison to the immune-negative immune cell population, individually by two experienced gastrointestinal pathologists. The final results were noted down after correlation and rereviewing the stained slides as felt necessary. Dual colocalization was identified by reddish-brown color, while nonlocalized antibodies were identified either as red or brown staining separately. In the case of dual IHC staining, as an example for CD4:CD3 T cells, the percentage of CD3+ CD4+ T cells (reddish-brown) out of all CD3-positive (brown) T cells was also calculated. In any case/control where the staining quality was not satisfactory, or there was background staining, it was excluded from final interpretation.
Statistical analysis was performed by STATA 11 software (STATA Corp LP, College Station, TX, USA). The differences in the inflammatory cell types in the duodenal biopsies of control and patients with CeD were assessed using Wilcoxon rank-sum test. Bonferroni test examined the distribution across different modified Marsh grades. The distribution pattern among the control and diseased duodenal biopsies was analyzed. P value of <0.05 was taken as statistically significant.
| Results|| |
In this study, duodenal mucosal biopsies from 234 subjects including 132 control subjects (mean age 32 ± 10.9 years, M:F ratio 3.8:1) and 102 patients with CeD (mean age 26.5 ± 13.5 years and M:F ratio 3:1) were included.
Status of villous abnormalities
In all, 129 of 132 (98%) controls included had no evidence of villous abnormalities (modified Marsh grade 0), while 3 of them had mild villous abnormalities, consistent with changes in modified Marsh grade 1; however, IELs were not increased.
Patients with CeD
Patients suspected of having CeD had the following grades of villous abnormalities as assessed by modified Marsh grading system – grade 0: 14 (13.7%), grade 1–3 (2.9%), grade 2–6 (5.8%), grade 3a–21 (20.5%), grade 3b–28 (27.4%), and grade 3c–30 (29.4%).
The relationship between anti-tTG Ab and grade of villous abnormalities
The anti-tTG fold change in patients with CeD was 8.5 ± 8, and in controls was 0.4 ± 0.6. The rise in serum anti-tTG level showed a linear correlation with histological severity of villous abnormalities, as was assessed by the modified Marsh grades (P < 0.001).
Immunophenotype of the IELs
In comparison to controls, mucosal biopsies from patients with CeD had significantly higher counts of CD3+ IELs than the controls [13.4 ± 8/100 epithelial cells (ECs) vs. 45.9 ± 19.9/100 ECs]. The CD3+ T-cell IELs also showed an increasing linear pattern with increasing modified Marsh grades [Table 1]. Among the IELs, TCRγδ cells were the predominant cell population than the TCRαβ cells [Table 2].
|Table 1: Mean number of IELs per 100 ECs (CD3+ cells) according to different grades of modified Marsh classification system in biopsies from patients with CeD (n=102)|
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|Table 2: Comparison of distribution of mucosal immune cells between controls and patients with celiac disease|
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Distribution of lamina propria–infiltrating lymphoid cells
Lymphoid cells in the lamina propria of the mucosa of patients with CeD
While patients with CeD had a higher number of CD4+ T-helper cells in the lamina propria in comparison to controls, no significant difference, however, was observed in the number of CD8+ cytotoxic T cells in the lamina propria between the duodenal biopsies from patients with CeD and controls [Table 2]. Among the T-cell population, the numbers of TCRγδ and TCRαβ cells were almost similar in patients with CeD in lamina propria [Table 2]. The number of CD20-positive B cells was also significantly higher in biopsies from patients with CeD, in comparison to controls (P < 0.001) [Table 2].
Interestingly, although the number of CD8+ FoxP3+ Treg cell population was significantly higher in patients with CeD in comparison to controls, the number of CD4+ FoxP3+ Treg cells was almost similar in biopsies from patients and controls [Table 2] and [Figure 1], [Figure 2]. When the density of lamina propria infiltrate was assessed according to the modified Marsh grades, CD3+ T cells showed an increasing linear pattern with increasing severity of villous abnormality. While there was also an increasing pattern of lamina propria CD20+ B cells and CD4+ T cells with increasing modified Marsh grades, certain linearity was not found. The remaining immune infiltrate in lamina propria did not show any significant difference in density between biopsies from patients and controls [Table 3].
|Figure 1: Boxplot diagram showing more number of CD4+ T cells in intestinal biopsies of celiac disease, than in controls (a). The number of TCR γδ cells and CD20-positive B cells were also more in celiac disease than in controls (b, TCR γδ cells; c, CD20+ B cells). Serum anti-tTG antibody titer showed an increasing linear pattern with higher modified Marsh grades (d)|
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|Figure 2: CD4+ CD3+ cells (arrows) in comparison to only brown-positive CD3 cells in controls (a × 40; b × 100) and CeD (c × 200; d × 100). In CeD, CD8+ CD3+ T cells (arrows) were the predominant among IELs (e × 200). While the TCR γδ cells (arrows) were sparse in controls, in CeD they were significantly increased, in comparison to brown-positive TCRαβ cells (f and g × 100; h × 200). FoxP3+ CD4+ Treg cells (arrows) in biopsies from controls (i × 100) and patients with celiac disease (j × 100). The FoxP3+ CD8+ Treg cells were significantly more in biopsies from CeD, than in controls (k and l × 100)|
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|Table 3: Comparison of modified Marsh grades with density of mucosa-infiltrating lymphoid cells in biopsies of patients with celiac disease and controls|
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| Discussion|| |
In this observational study, duodenal mucosal biopsies collected from a cohort of controls and patients with CeD showed an increasing linear pattern of CD3+ T-cell infiltrate with increasing modified Marsh grades, along with a similar increasing pattern of helper CD4+ T cells and CD20-positive B-cell infiltrate in lamina propria. Among the IELs, the TCRγδ+ T cells were predominant in patients with CeD. Mucosal CD8+ FoxP3+ Treg cells though were significantly more in intestinal biopsies from patients with CeD than in controls, the density of strongly immune suppressive CD4+ FoxP3+ Treg cells did not differ between intestinal biopsies from controls and patients with CeD, despite luminal antigen activation.
The immunological alterations going on in intestinal mucosa in patients with CeD are complex, and understanding regarding this is still evolving. The orchestrated release of IL-21 from mucosal infiltrating gluten-specific CD4+ T cells and the IL-15 from mucosal enterocytes recruits the CD8+ IELs in a patient with CeD.,,,, The recruited CD8+ T cells equipped with NK-type receptors on binding to their ligands in intestinal epithelium and extraintestinal tissue lead to EC death and cell lysis. In steady physiological condition, while the TCRγδ+ T cells comprise only 1%–5% of peripheral blood mononuclear cells, 4%–5% of intestinal IELs, and 17%–18% of lamina propria lymphocytes, in CeD both IEL and lamina propria TCRγδ+ T cells are increased, which upon receiving signals from IL-15 secrete soluble proteins in an effort to maintain the epithelial integrity. On the other hand, the TCRαβ+ T cells cause enterocyte damage by releasing IFNγ, granzyme, or perforins.,, In this study, we also identified significantly more TCRγδ+ IELs in the mucosal surface in patients than in controls.,
In steady state, the intestinal mucosal DCs act as the tolerogenic cells and are responsible for the induction of mucosal Treg cell population. However, in CeD, due to high level of mucosal IL-15, IL-21, and IFNα, the DCs fail to induce the tolerogenic Treg cells and promote a proinflammatory condition. On in vitro studies, it has been found that IL-15 imparts resistance of the mucosa-infiltrating lymphocytes to the suppressive function of the circulating Treg cells. In intestinal biopsies from patients with CeD in this cohort, although the numbers of CD8+ FoxP3+ Treg cells were significantly higher than in controls, the mucosa-infiltrating CD4+ FoxP3+ Treg cells were not upregulated. Either the failure of CD4+ Treg cell recruitment or loss of their suppressive function as discussed could explain the loss of mucosal tolerance to dietary antigens in CeD. While the majority of Treg cells in intestinal mucosa are CD4+ FoxP3+ Treg cells, the CD8+ FoxP3+ Treg cell population constitutes only around 2% of the FoxP3 population and have a feeble immunosuppressive activity. Depletion of the mucosal CD4+ FoxP3+ Treg cells can also produce type 1 diabetes or systemic lupus erythematosus in the experimental animal models., This altered tolerogenic mucosal environment in CeD and the relative paucity of CD4+ FoxP3+ Treg cells in the intestinal mucosa along with their loss of tolerogenic potential could justify the increased global risk of malignancy in CeD and the need to continue the GFD possibly lifelong to ameliorate the risk of recurrence of a hostile immune environment. Similar studies on immune signature expression may be helpful in prognostication and possible therapeutic intervention in CeD.
However, we did not correlate the number of mucosa-infiltrating immune cells and Treg cells with the circulating monocyte population. Also, the number of biopsies where the immunohistochemical stains could be interpreted varies markedly, as we discarded all stained biopsy slides where the IHC staining pattern was not interpretable, the tissue exhausted during processing, or where there was background staining.
To conclude, this preliminary observational study shows that alteration in local intestinal mucosal immune cell population between patients with CeD and control biopsies is exciting and in-depth analysis with their functional assessment may yield vital clue regarding immune pathogenesis of CeD and the exact cause of loss of mucosal tolerance.
Financial support and sponsorship
The study was supported by the Intramural Research grant of the AIIMS, New Delhi, India.
Conflicts of interest
There are no conflicts of interest.
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Department of Pathology, AIIMS, New Delhi
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]