
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39266694
72736
10.1038/s41598-024-72736-x
Article
Interleukin-21 and Interleukin-23 levels in familial Mediterranean Fever before and after treatment: the role of cytokines in disease pathogenesis
Hizal Mutlu drmutluhizal@hotmail.com

1
Tufan Abdurrahman 2
Mercan Ridvan 2
Pasaoglu Ozge Tugce 3
Pasaoglu Hatice 3
Haznedaroglu Seminur 2
Goker Berna 2
Ozturk Mehmet Akif 2
1 https://ror.org/054xkpr46 grid.25769.3f 0000 0001 2169 7132 Faculty of Medicine, Department of Internal Medicine, Gazi University, Ankara, Turkey
2 https://ror.org/054xkpr46 grid.25769.3f 0000 0001 2169 7132 Faculty of Medicine, Department of Rheumatology, Gazi University, Ankara, Turkey
3 https://ror.org/054xkpr46 grid.25769.3f 0000 0001 2169 7132 Faculty of Medicine, Department of Medical Biochemistry, Gazi University, Ankara, Türkiye Turkey
12 9 2024
12 9 2024
2024
14 2135116 5 2024
10 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
In a previous study, it has been shown that the population of Th17 lymphocytes was increased in patients with FMF. IL-21 and IL-23 play significant roles in the production and differentiation of Th17 cells. In this study, we aimed to evaluate serum levels of IL-21 and IL-23 in FMF patients both at diagnosis and after treatment, and to compare these levels with those of healthy controls. Twenty-seven newly diagnosed patients with FMF in attack-free periods and twenty-seven healthy volunteers enrolled in the study. The groups were comparable with respect to age and gender. IL-21 and IL-23 levels in serum samples from patients at the time of diagnosis, in remission after treatment, and from the control groups were analysed using the ELISA method. There was no significant difference between the cytokine levels of the patient group at the time of diagnosis and the cytokine levels of the control group (for IL-21, p: 0.28 and for IL-23, p: 0.56). Similarly, there was no significant difference between the patients’ cytokine levels at the time of diagnosis and after treatment (for IL-21, p: 0.99 and for IL-23, p: 0.08). Interleukin levels at the time of diagnosis did not differ among patient groups based on the presence of clinical findings or the M694V genotype. Our results suggest that IL-21 and IL-23 do not play a role in the pathogenesis of the disease. However, while interpreting these findings, it should be considered that patients with active episodes were excluded and cytokine levels were not measured in tissue samples.

Keywords

Familial mediterranean fever
Interleukin-21
Interleukin-23
T helper 17
Subject terms

Rheumatic diseases
Interleukins
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Monogenic autoinflammatory syndromes describe lifelong and recurrent hereditary diseases with common autoinflammatory symptoms and signs such as fever, abdominal pain, arthralgia, diarrhea, and rash1. Familial Mediterranean Fever (FMF) is the most common monogenic autoinflammatory syndrome and can lead to serosal, synovial, and cutaneous inflammation, which is characterized by recurrent episodes of fever, typically lasting 12 to 72 h2. The disease is caused by pathogenic variations in the MEFV gene, located on the short arm of chromosome 16 (16p13.3), which encodes the pyrin protein. The most common mutations are M694V, V726A, M694I, M680I, and E148Q3,4. FMF treatment typically begins with colchicine, a drug that binds to microtubules, exhibits antimitotic activity, and provides various anti-inflammatory effects. For patients who develop resistance to colchicine, alternative treatment options include IL-1 inhibitors (such as anakinra, rilonacept, and canakinumab) or tocilizumab, which targets the IL-6 receptor5,6.

T helper 17 (Th17) is a member of the CD4 + T lymphocyte family that has interleukin-21 (IL-21) and interleukin-23 (IL-23) receptors and secrete IL-6, IL-17, IL-21, IL-22, and TNF-α; some of which also have major roles in FMF pathogenesis7. On the other hand, IL-23 is secreted by antigen-presenting cells, mainly activated macrophages and dendritic cells8. Both cytokines have important proinflammatory effects such as stimulating cell-mediated immune response and IFN-γ production as well as increasing the proliferation of lymphocytes, the cytotoxic effects of NK cells, and the protease and degrading enzyme secretion of fibroblasts8,9.

IL-1 and IL-6, which have a major role in the FMF pathogenesis, stimulate the production of Th17; not surprisingly, significantly increased levels of the Th17 population have been demonstrated in FMF patients10. The disruption of Th17 regulation via IL-21 and IL-23, leading to a proinflammatory state11–13.

Both IL-21 and IL-23 have been shown to play a significant role in the pathogenesis of autoimmune and inflammatory diseases such as RA, SLE, psoriasis, and IBD14,15. However, whether both cytokines contribute to the pathogenesis of FMF remains to be elucidated. Moreover, IL-23 has also become a treatment target in different diseases such as IBD or psoriasis with various agents16,17.

The purpose of this study is to determine the possible contribution of IL-21 and IL-23 to FMF pathogenesis in newly diagnosed patients by evaluating cytokine levels before and after treatment and comparing them to healthy controls.

Materials and methods

Twenty-seven newly diagnosed FMF patients and 27 healthy volunteers were included in the study. All participants were ≥ 18 years old. The study was performed in the Gazi University Faculty of Medicine, Department of Rheumatology. The diagnosis was made clinically and according to the Tel Hashomer criteria by an expert rheumatologist. The patients in the active attack period of FMF were excluded. Additionally, those with concomitant infection, high or low white blood cell count, autoimmune and/or autoinflammatory disease (RA, Sjögren Syndrome, IBD, SLE, Psoriasis, etc.) were excluded from the study. The treatment decisions and follow-up of the patients were made by the rheumatologist in routine clinical practice.

Peripheral serum samples obtained from patients at the time of diagnosis, in remission after treatment, and stored under appropriate conditions (-20 C) were used for the measurement of IL-21 and IL-23. Measurement of cytokines was performed by the ELISA method and using the “Human IL-21 Kit” and “Human IL-23 Kit” (eBioscience, San Diego, USA). The samples were analyzed in duplicate. The results were reported in terms of pg/ml. Just one patient’s serum sample obtained at the time of diagnosis could not be analysed due to technical reasons.

The erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), white blood cell (WBC) from peripheral blood samples, obtained simultaneously for cytokine tests were analyzed via routine laboratory methods. Also, protein tests in spot urine samples were performed simultaneously. CRP elevation was defined as ≥ 12 mg/L that was twice and above the upper limit of normal values (0–6 mg/L). Patients were questioned about attack characteristics which were fever, peritonitis, pleuritis, arthritis, and skin lesions. FMF gene mutation analysis of all patients was performed by scanning the related 12 mutations with the FMF sequence kits (Qiagen) by means of the polymerase chain reaction (PCR) method.

SPSS (Statistical Package for Social Sciences) version 17 was used to evaluate the data obtained from the study, and create the tables. Median and minimum-maximum values were used for the presentation of the continuous variables obtained by measurement, whereas frequency and percent values were used for the presentation of categorical variables. Mann-Whitney U test was used to compare continuous variables of the two groups whereas Kruskal-Wallis test was used to compare continuous variables of more than two groups. Spearman’s rank correlation test was applied for the correlation of continuous variables. Wilcoxon Signed Rank Test was used to ascertain whether there is a difference between the patients’ measurements before and after treatment. p < 0.05 was adopted for significance level at all statistical analyses.

Gazi University Clinical Research Ethics Committee’s approval was obtained. All research was performed in accordance with the Declaration of Helsinki and informed consent was obtained from all participants.

Results

The demographic features of the patient group and the control group were comparable in terms of age and sex. Peritonitis was identified as the most common clinical feature during attacks in these patients (n = 23). The most common result of genetic analysis demonstrated the M694V homozygous genotype (n = 7) (Table 1).

Table 1 Patient characteristics and mutation analyses.

Table A	Patients (n = 27)	Controls (n = 27)	P	
Age (mean ± SD)	34.03 ± 2,30	35.3 ± 1.73	ns	
Sex (male/female)	11/16	11/16	ns	
Table B	n	%		
Clinical Findings				
Peritonitis	 23	 85.2		
 Fever (≥ 38 C)	 17	 63		
 Arthritis	 16	 59.3		
 Pleurit	 13	 48.1		
 Skin Lesions	 5	 18.5		
Mutation Analyses				
M694V/M694V	7			
M694V/ -	6			
M694V/M680I	3			
M680I/M680I	2			
M680I/V726A	2			
V726A/V726A	1			
M694V/V726A	1			
M694V/A744S	1			
M694V/E148Q	1			
M680I/-	1			
-/ -	2			

There was no significant difference between patients’ serum IL-21 levels (median 138 pg / ml, min-max: 18-7680) and IL-23 (median 30 pg / ml, min-max: 6-106) levels at the time of diagnosis, and those of the control group (Table 2). 

Table 2 Interleukin Levels of Controls and Patient Group before and after treatment.

Patient Group		Control Group	p	
Before Treatment				
IL-21 pg/ml	138 (18–7680)	84 (12–892)	ns	
IL-23 pg/ml	30 (6–106)	26 (6–676)		
After Treatment				
IL-21 pg/ml	120 (12–7496)			
IL-23 pg/ml	22 (3–88)			
p	ns			

The treatments were started as soon as the patients were diagnosed. It was determined that there was a median 13 months (min-max:3–29) between the samples collected at the time of diagnosis and after treatment. Patients received colchicine treatment of 1–4 tablets per day. All patients were under colchicine treatment when the post-treatment samples obtained, and no other agent except for colchicine was administered for FMF treatment.

There was no significant difference between cytokine levels of patients at the time of diagnosis and after treatment (for IL-21 median: 120 pg/ml, min-max: 12-7496; for IL-23 median: 22 pg/ml, min-max: 3–88) (p = 0.99 and p = 0.08, respectively).

As expected, there was a significant decrease in patients’ ESR and CRP levels after treatment as compared to those at the time of diagnosis (p = 0.003 and p = 0.007, respectively). However, there was no significant correlation between ESR and interleukin levels. In addition, there was no significant difference between cytokine levels of patients with increased CRP levels (n = 12) and patients with no increased CRP levels. Similarly, it was also noted that the presence of the M694V mutation, whether the genotype was homozygous (n = 7; median: 204 pg/ml [18–288] for IL-21 and median: 28 pg/ml [6–38] for IL-23) or heterozygous (n = 11; median: 96 pg/ml [40-1196] for IL-21 and median: 34 pg/ml [6–82] for IL-23), had no significant effect on either interleukin level. There was no significant association between the presence of clinical findings such as fever, pleuritis and arthritis, and interleukin levels at the time of diagnosis.

Discussion

In our study, we observed no significant difference between serum IL-21 and IL-23 levels in FMF patients compared to healthy controls. Furthermore, the cytokine levels did not change significantly after treatment. While our findings suggest that IL-21 and IL-23 may not play a substantial role in the pathogenesis of FMF, several factors warrant consideration.

Although inflammasome formation in FMF pathogenesis arises from the innate immune system, the immunologic process following caspase-1 activation also impacts the acquired immun system18. IL-1 and IL-6 are critical in FMF pathogenesis and stimulate Th17 differentiation and proliferation via STAT3 and IRF4 7,19. This explains the increased Th17 population observed in FMF patients10. Activated Th17 cells enhance the secretion of IL-21, their primary effector molecule. Elevated IL-21 levels further induce Th17 cell proliferation in an autocrine manner via STAT3 20. Additionally, IL-23 contributes to Th17 differentiation, proliferation, and stability through the STAT3 pathway21,22.

Despite evidence suggesting that IL-21 and IL-23 might contribute to FMF pathogenesis, our study did not reveal significant differences in cytokine levels. We assessed cytokine levels from serum samples obtained from peripheral blood. Recent data have shown that the cytokine levels might differ between serum and tissue samples23. For instance, in the study of Ibrahim JN et al., IL-17 and IL-22 levels were evaluated in PBMC cultures from FMF patients during both remission and active episodes and compared to healthy controls. Although cytokine levels in blood did not vary significantly across groups, active episode patients produced substantially higher levels of IL-17 in cell cultures than those in remission or healthy controls. Additionally, patients in the attack-free period had significantly higher IL-22 levels, which have anti-inflammatory effects, compared to those in active episodes23. Thus, the lack of difference in IL-21 and IL-23 serum levels between healthy individuals and FMF patients may not reflect tissue levels of these cytokines or their potential contribution to pathogenesis. Another possible explanation for our results is that while the Th17 cell population increases during recurrent episodes, this increase may not necessarily be accompanied by elevated release of effector cytokines during remission.

FMF is characterized by episodes of acute illness followed by periods of remission. The findings of exaggerated autoinflammatory response during episodes are well established2. However, subclinical inflammation may persist during remission periods, potentially accelerating disease complications24,25. Several studies have demonstrated that levels of various cytokines associated with disease etiology, such as TNF-α, IL-1, IL-8, IL-17, and IL-18, particularly acute phase reactants and IL-6, are elevated in patients in remission compared to healthy controls23,26,27. In our study, we excluded patients experiencing an acute episode to avoid the potential ‘peak’ effect of an active episode on the cytokine levels under investigation. Nevertheless, high ESR and CRP levels in patients significantly decreased after colchicine treatment, which may be related to the subclinical inflammation as previously discussed.

Several studies have explored the relationship between different FMF gene mutations and cytokine levels that might influence the disease process28,29. For example, research examining the Th17 population in cell cultures from FMF patients demonstrated that individuals homozygous for M694V secreted 1.5 times more IL-17 than other patients, regardless of their attack or remission status10. However, our study found no significant difference in cytokine levels among patient groups based on the presence of the M694V mutation.

To our knowledge, our study is the first to investigate IL-21 and IL-23 levels in newly diagnosed FMF patients both before and after treatment and to compare them with healthy individuals. We believe our findings are valuable for reflecting daily clinical practice and the newly diagnosed FMF patient population starting treatment with colchicine. Sharing these results, even though they show no significant differences, may help improve future study designs and resource management. However, our study had important limitations.

In a rare disease like FMF, especially in adults, it is challenging to recruit a large number of newly diagnosed participants in a single-center study. The small number of patients, especially in subgroups based on gene mutations or clinical findings, was a significant limitation. The disease severity score increases with an earlier age of onset or in the presence of amyloidosis30. It is possible that the results may differ in younger patients or those with higher disease severity scores due to complications such as amyloidosis. A better understanding of the pathogenesis and identification of different patient subgroups may lead to more effective studies in the future.

Our results may be evaluated as suggesting that IL-21 and IL-23 do not have a role in the pathophysiology of FMF. However, while interpreting these findings, it should be taken into consideration that patients with active episodes were excluded and cytokine levels were not measured in tissue samples.

Author contributions

M.H. did statistical analysis and wrote the main manuscript text. MAO and AT made the study design. OTP and HP analysed the interleukin levels. All authors reviewed the manuscript.

Data availability

All data generated or analysed during this study were included in this article. Further inquiries can be directed to the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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