ORIGINAL ARTICLE Year : 2022  |  Volume : 11  |  Issue : 4  |  Page : 384-388

Comparative evaluation of interleukin-10, transforming growth factor-β, and interleukin-17 in gastrointestinal tuberculosis and crohn's disease

Anjali Gupta1, Kusum Sharma2, Vishal Sharma3, Jagdeep Singh1, Ritambhra Nada4, Biman Saikia1, Ranjana W Minz1, Shashi Anand1, Mahendra Kumar1
1 Department of Immunopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
4 Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Date of Submission19-Aug-2022Date of Decision27-Sep-2022Date of Acceptance15-Oct-2022Date of Web Publication10-Dec-2022

Correspondence Address:
Mahendra Kumar
Department of Immunopathology, Research Block-A, Room No-29, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ijmy.ijmy_131_22

Background: Gastrointestinal tuberculosis (GITB) and Crohn's disease (CD) are close mimickers and difficult to discriminate. Recent work has focused on the immunological differences between GITB and CD based on cytokines related to T-regulatory cells and Th17 cells. In the present cross-sectional study, suspected cases of GITB or CD underwent extensive clinical, radiological, endoscopic, histological, and microbiological assessment. The diagnosis was based on standard criteria and response to antitubercular therapy endoscopically. Methods: Interleukin (IL)-10, transforming growth factor-β (TGF-β), and IL-17 were measured and compared between GITB and CD along with other parameters. Fisher's exact test and Mann–Whitney U test were used as per the data type. Results: Of the 27 patients, 11 had CD, 9 had GITB, and 7 had other conditions. Chronic diarrhea, involvement of left and long segments of the colon, and aphthous ulcers were significantly more frequent in CD; however, transverse ulcers were in GITB. IL-10 was reduced in both GITB (median-interquartile range [IQR] 9.54 [3.65–24.04]) and CD (median-IQR 13.28 [6.91–22.50]) compared to control (median-IQR 26.72 [10.34–35.43]). TGF-β showed little variation, but IL-17 was below the detection limit in most cases. None of these cytokines were significantly different between CD and GITB. The sensitivity and specificity of multiplex Mycobacterium tuberculosis-polymerase chain reaction were 44.44% and 100%, respectively. Conclusion: Serum cytokine profiling (IL-10, IL-17, and TGF-β) could not significantly differentiate GITB and CD. Moreover, extensive molecular, transcriptomic, chemokines, and cytokine analyses may shed light on these aspects.

Keywords: Crohn's disease, cytokines, gastrointestinal tuberculosis, granuloma, interleukin-10, interleukin-17, transforming growth factor-β

How to cite this article:
Gupta A, Sharma K, Sharma V, Singh J, Nada R, Saikia B, Minz RW, Anand S, Kumar M. Comparative evaluation of interleukin-10, transforming growth factor-β, and interleukin-17 in gastrointestinal tuberculosis and crohn's disease. Int J Mycobacteriol 2022;11:384-8
How to cite this URL:
Gupta A, Sharma K, Sharma V, Singh J, Nada R, Saikia B, Minz RW, Anand S, Kumar M. Comparative evaluation of interleukin-10, transforming growth factor-β, and interleukin-17 in gastrointestinal tuberculosis and crohn's disease. Int J Mycobacteriol [serial online] 2022 [cited 2022 Dec 12];11:384-8. Available from: https://www.ijmyco.org/text.asp?2022/11/4/384/363151   Introduction 

Gastrointestinal tuberculosis (GITB) and Crohn's disease (CD) share many clinical, radiological, endoscopic, and histological characteristics, making differentiation between these conditions difficult.[1] The rising incidence of inflammatory bowel disease, including CD, in the developing world, makes it imperative to have effective tools to differentiate these two conditions.[2]

Various multiparametric models have been attempted, including demographic, radiological, endoscopic, and pathological characteristics. However, their utility is limited by complexity, lack of specificity, and sometimes sensitivity, lack of validation in different populations, and infrequent use in the clinics. Currently, the response to antitubercular therapy (ATT) by assessment of mucosal healing 2 months after initiation is used as a preferred approach to distinguish GITB from CD.[3] However, this leads to a time lag in determining the diagnosis, resulting in a significant delay in diagnosis and initiation of CD treatment. Misdiagnosis of GITB patients as CD can expose patients to the harmful effects of immunosuppressive medications.

Understanding of the immunological basis of tuberculosis and CD has expanded in recent decades. Peripheral blood of active pulmonary tuberculosis (PTB) has increased T-regulatory (Treg) cells and related immunosuppressive cytokines (interleukin-[IL]-10 and transforming growth factor-β [TGF-β]).[4] Nevertheless, findings related to Th17 cell and their cytokines IL-17 are inconsistent.[5],[6],[7] In CD patients, Treg cells decreased in peripheral blood along with a concomitant increase in tissue, suggesting pooling of Treg at the diseased site.[8] On the other hand, the Th17 population and IL-17 were increased in peripheral blood in active disease with variable alteration in tissue.[9],[10] Hence, cytokines profiling may provide immunological tools to differentiate GITB from CD.

  Methods 

Ethics and study design

We enrolled patients suspected of having either GITB or CD based on clinical, radiologic, or endoscopic findings. The institutional ethics committee approved the study, and informed consent was obtained from each participant, including healthy volunteers recruited to compare serological results. Exclusion criteria covered patients under 14 years, acute or chronic infection, HIV, pregnant/lactating women, and patients unwilling to consent. Samples were collected as per the Helsinki Declaration of 1975 (revised 2000). The results of all investigations led to the classification of patients into three major groups: patients with CD, patients with GITB, and patients with other diagnoses. Cases where confusion regarding GITB or CD was present and received standard ATT for 2 months, after which they were reassessed with a colonoscopy to document the ulcers' healing (or lack of). The present study collected demographic, clinical, radiological, histopathological, and microbiological details and measured cytokines assay.

Case definitions

Crohn's disease

The patients were diagnosed with CD based on the European Crohn's and Colitis Organization guidelines, combining clinical, endoscopic, and histological features.[11]

Gastrointestinal tuberculosis

An integrated evaluation of clinical, endoscopic, histological (caseating granulomas, confluent, and dense granulomas), and microbiological (multiplex Mycobacterium tuberculosis (MTB)-polymerase chain reaction (PCR) and culture) features helped diagnose GITB. The presence of caseating granulomas or demonstration of acid-fast bacilli (AFB) on biopsy/smear or evidence of AFB through culture confirmed the GITB.

Indeterminate cases

Indeterminate cases (suspicious for GITB but not confirmed) received an ATT trial response. Endoscopic healing of ulcers on a repeat colonoscopy at ≥2 months after initiation of treatment suggested GITB; however, lack of improvement along with the clinical and/or endoscopic response to CD-specific therapy suggested underlying CD.[3]

Histopathological evaluation: six to eight endoscopic biopsies from the involved area were taken. We sent a few biopsy pieces for histopathological examinations (hematoxylin, eosin, and AFB stain) and two for microbiological studies (multiplex MTB-PCR and culture/smear).

Cytokines assay

Serum was separated from peripheral blood (3 ml) and stored at −80°C until further testing for cytokines analysis. Quantitative assay of IL-10 (E-EL-H6154, Elabscience, USA), TGF-β (E-EL-0162, Elabscience, USA), and IL-17A (BMS2017, INVITROGEN, Vienna, Austria; Detection limit – 1.6–100 pg/mL) was performed through enzyme-linked immunosorbent assay as per the protocol provided with kits, and standard curved obtained was appropriate.

Microbiological assay for Mycobacterium tuberculosis

Smear and culture examination

Following standard procedures, we examined tissue samples using Ziehl–Neelsen smears and cultured them on liquid BACTEC (MGIT 960).

Multiplex Mycobacterium tuberculosis-PCR

Specific pair of primers was designed to amplify Protein B and IS6110 to identify MTB in the MTB complex. The band size for Protein B and IS6110 was about 419 bp and 123 bp, respectively. The sequences of primers used for: Protein B: Pb1:5'-TCCGCTGCCAGTCGTCTTCC-3', Pb2:5'-GTCCTCGCGAGT CTAGGCCA-3'and for IS6110: ISI: 5'-CCTGCGAGCGTAGGCGT-3' IS2: 5'-CTCGTCCAGCGCCGCTTCGG 3'.

Statistical analysis

The statistical analyses were conducted using IBM SPSS (Armonk, NY: IBM Corp: Released 2017) Statistics for Windows, version 25.0. A mean standard deviation was used for continuous data, and frequency and percentage were used for categorical data. Inferential statistics such as Fisher's exact test and Mann–Whitney U test were applied to compare the two groups; P < 0.05 was considered statistically significant.

  Results 

This prospective study recruited 27 clinically and/or radiologically (endoscopic) suspected cases of GITB and CD. The clinical, radiologic/endoscopic, histopathological, and microbiological evaluation of the cases classified them as CD (n = 11), GITB (n = 9), indeterminate (n = 01), and other diseases (n = 6; included adenocarcinoma, adenoma, solitary rectal ulcer syndrome, eosinophilic gastroenteritis, and two normal). [Table 1] compares various parameters between GITB and CD. Chronic diarrhea (eight [72%] vs. one [11%]), involvement of left (Six [54%] vs. 0) and long segments (five [45.5%] vs. 0) of colon, aphthous ulcers (Nine [81.8%] vs. 0%), and transverse ulcers (One [9.1%] vs. Six [66.7%]) were significantly different between CD and ITB. The frequency of granulomas was not too different (two in GITB [22%] vs. one in CD [9%]), but their nature was slightly different. Granulomas in GITB were large, compact, and submucosal in a location with lymphoid cuffing. However, granulomas in CD were small, vague with relatively fewer epithelioid cells, and mainly located in the lamina propria and muscular layer. Out of 15 clinically suspected cases of GITB, three were positive on multiplex PCR and one on both smear and multiplex PCR (for MTB). Six out of the remaining 11 microbiologically negative cases were nonresponsive to the ATT trial and grouped into CD, and the remaining five grouped into GITB as responded to ATT. None of the microbiological parameters were statistically significant between CD and GITB, but multiplex PCR had a sensitivity of 44.44% and specificity of 100% for GITB in the present study.

Table 1: Comparative evaluation of various parameters between gastrointestinal tuberculosis and Crohn's disease

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Figure 1 highlights the comparative evaluation of cytokines. Serum IL-10 was reduced in both GITB (median-interquartile range [IQR] 9.54 [3.65–24.04] pg/mL) and CD (median-IQR 13.28 [6.91–22.50] pg/mL) compared to control (median-IQR 26.72 [10.34–35.43] pg/mL) but reduction was more in GITB. TGF-β was found to be lower in CD patients (median-IQR 31.84 [23.04–40.93] ng/mL) and higher in GITB group (median-IQR 37.04 [29.64–44.74] ng/mL) as compared to control group (median-IQR 35.70 [31.31–39.12] ng/mL). Neither disease versus control groups nor the two disease groups showed significant associations. In most cases of GITB and CD, IL-17 was not detected (except one from each group: 2.59 pg/mL and 31.14 pg/mL). Seven of 15 controls had IL-17 within the normal range with a mean of 2.65 ± 3.49 pg/mL.

Figure 1: Serum levels of IL-10, TGF-β, and IL-17 in control, CD, and GITB P > 0.05 for any two groups. IL-10: Interleukin-10, IL: Interleukin-7, TGF-β: Transforming growth factor-β, CD: Crohn's disease, GITB: Gastrointestinal tuberculosis

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  Discussion 

The GITB and CD are closely related, and despite many efforts to differentiate the two, there is significant heterogeneity in prior research.[1],[12] Even some of those are validated in the present study, like chronic diarrhea, and involvement of the left and long segment of the colon was significantly more frequent in CD; however, transverse ulcers are significantly more common in GITB.[1] Identification of MTB through AFB stain, culture, and multiplex PCR has low sensitivity ranging from 19% to 70%.[1],[13],[14] None of the cases was positive on culture, but multiplex PCR was positive in four patients with sensitivity and specificity of 44.44% and 100% in the present study. The present study also supports the importance of the ATT trial by favoring the diagnosis of GITB in 60% of dilemma cases.[3],[15]

With the advancement in knowledge of immunology, researchers are trying hard to differentiate these two based on their immunopathogenesis. Immunologically GITB and PTB are similar and predominantly involve the Th1/Th17 pathway like the CD. In PTB, Th17 mainly plays a protective role through IL-17, which recruits neutrophils to promote granuloma formation and increases the expression of chemokines, CXCL9, CXCL10, and CXCL11, which will compel interferon-gamma-producing cells to the site of infection.[16] However, persistent overexpression of IL-17 may result in immunopathological damage to host tissue. The number of Th17 cells are higher in plural fluid compared to peripheral blood suggesting pooling at the infection site early in pathogenesis.[17] Even their number was found to be higher than the control. In chronic TB, functional laxity of Th17 cells with a decrease in IL-17A production has been documented, which might be due to exhaustion of T-cells mainly due to overexposure to MTB antigens and hyperexpression of programmed death 1 (PD-1).[7] Moreover, decreased PD-1 expression and increased IL-17A production after ATT support this hypothesis.[18]

In the initial stage of MTB infection, Treg has a negative impact by inhibiting the activation of T-cells, leading to a delay in the adaptive immune response.[19] However, in the chronic phase, it reduces excessive inflammation by inhibiting both effector T-cells and antigen-presenting cells.[19] It is not well understood whether Treg amplification is the host's counterbalance mechanism to excessive inflammation or due to the direct impact of MTB. However, few pieces of evidence support the direct role of MTB.[20] Many reports documented decreased Treg in peripheral blood of latent TB infection; however, it increased in PTB, supporting pooling of Treg at the site of the disease (lungs) in the early phase and subsequent reappearance in the periphery later.[4] Even the number of Treg inversely correlates with response to therapy, and few authors suggested it as a marker for the treatment response.[21] Treg-associated cytokines (IL-10 and TGF-β) are raised in PTB compared to healthy control, but others could not find any difference.[4],[22] It is controversial regarding response to therapy and levels of these cytokines; some reported reduced levels, whereas others failed to demonstrate any difference.[23],[24] PTB and GITB are immunologically similar, and the study also reported higher Treg in GITB.[25] In the present study, TGF-β was raised in GITB; however, IL-10 was much lower than in the control. Possibly, lower IL-10 may be due to the treatment response he might have received before enrolling with us.

Classically, the CD is considered a Th1-dominant immune-mediated disease, but the recently altered balance of Th17 and Treg is deemed to be a significant contributor to IBD's immunopathogenesis.[26] The proportion of IL-17-producing CD4+ T-cells and their mRNA expression is significantly higher in active UC and CD than in inactive and healthy control.[9],[10] Even increased fecal and serum IL-17A further support the role of IL-17 in CD pathogenesis.[9],[27] Other studies, including ours, could not find any difference.[28] Treg and T-regulatory1 (Tr1) cells produce suppressive cytokines (IL-10 and TGF-β) to maintain homeostasis.[26] Increased forkhead box P3 (FOXP3) transcript and Tregs (CD4+ CD25 hi Foxp3+ T cells) in inflamed mucosa compared to noninflamed mucosa along with the reduced frequency of CD4+ Foxp3+ T-cells in peripheral blood strengthen the role of Tregs in counteracting pro-inflammatory environment at disease site in CD patient.[8] Nevertheless, contrary reports are also available, which may be due to different surface markers used to characterize Treg cells and the plasticity of both Th17 and Treg cells.[29],[30] Contrary to the previous report, serum IL-10 and TGF-β were reduced compared to control in the present study [Figure 1].[31] The major limitation of our study was the small sample size, mainly attributed to the coronavirus disease (COVID-19)-related lockdown. Simultaneous immunological evaluation at the injury site would have provided better insight into this respect.

To summarize, the primary diagnostic strategy for GITB and CD remains an integrated approach that combines clinical, radiological, histopathological, and microbiological investigations. The present study highlights the role of multiplex PCR and ATT trial in diagnosing GITB. The present study could not demonstrate a significant difference between GITB and CD concerning IL-10, TGF-β, and IL-17. An immunological tool might be developed to differentiate GITB from CD by more holistic research involving systemic and lesional site assay of cytokines/chemokines and immune cells, including molecular profiling.

Ethical statement

Institute Ethic Committee of PGIMER, Chandigarh, India NK/6798/MD/541 dated June 10, 2021.

Authors' contributions

All authors fulfill authorship criteria as per the International Committee of Medical Journal Editors (ICMJE). All authors equally contributed to this article with the conception and design of the study, literature review and analysis, drafting and critical revision and editing, and final approval of the final version.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 

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  [Figure 1]
 
 
  [Table 1]