Assessment of Serum Resistin and Plasma Calprotectin Levels as Biomarkers of Inflammation in Patients with Familial Mediterranean Fever Disease

Objective: While several inflammatory markers are known to increase in familial Mediterranean fever (FMF) disease cases, the need remains for diagnostic tests specific for FMF that monitor inflammatory activity. We aimed to investigate resistin and calprotectin levels during both attack and attack-free periods of FMF disease and evaluate their use as novel biomarkers of inflammation in patients with FMF. Materials and Methods: This cross-sectional study included 68 male patients diagnosed with FMF and 20 healthy individuals as controls. Blood samples were obtained from the patients in attack-free periods (at least 15 days after the last attack) and attack periods (in the first 24 hours). Serum resistin and plasma calprotectin levels was measured by ELISA method. Results: Resistin and calprotectin levels were significantly higher in patients during both attack (p =0.001, p <0.001) and attack-free periods (p =0.017, p =0.01) compared to the control group. Logistic regression analysis indicated that resistin levels were predictive for the diagnosis of FMF disease (OR: 1.21; 95% CI: 1.04–1.42; p =0.016). Resistin and calprotectin levels significantly correlated with C-reactive protein, erythrocyte sedimentation rate, fibrinogen, and white blood cells (0.301≤ r ≤ 0.505, p <0.05). Conclusion: Resistin and calprotectin levels were significantly higher in patients than controls, and resistin was predictive for monitoring inflammatory activity in patients with FMF.


INTRODUCTION
Familial Mediterranean fever (FMF) is a genetic disease resulting from Mediterranean FeVer (MEFV) gene variations. 2,3 This MEFV gene encodes the protein of pyrin, which plays an important role in inflammation and apoptosis. [2][3][4] The autoinflammatory disease FMF, characterized by shortlived recurrent episodes of peritonitis, pleuritis, arthritis, rash, and fever 1 , is commonly detected in Sephardic Jews, Armenians, Turks, Greeks, Arabs, and Italians in the Mediterranean region. 1,2 A diagnosis of FMF is currently based on clinical findings according to the Tel

ASSESSMENT OF SERUM RESISTIN AND PLASMA CALPROTECTIN LEVELS AS BIOMARKERS OF INFLAMMATION IN PATIENTS WITH FAMILIAL MEDITERRANEAN FEVER DISEASE
Hashomer and recently published Eurofever/PRINTO classification criteria which also includes genetic analysis. [1][2][3][4][5] While currently there is no definitive diagnosis for FMF, ethnicity, family history, and mutation in the MEFV gene support the diagnosis in patients with clinical findings. Other findings that contribute to an FMF diagnosis are high blood levels of acute-phase reactants (APR) during attack periods. The major mechanisms of pathogenesis for clinical manifestations include the over-activation of cytokine cascades. 3 Abnormal pyrin protein, which is proposed to result from MEFV gene mutations, is suggested to precipitate ineffective suppression of inflammation responsible for the inflammatory process. 3 Resistin is a regulatory cytokine that triggers the pro-inflammatory state by increasing the synthesis of cytokines such as TNF-α, IL-1β, and IL-6. Calprotectin is a heterodimer belonging to the family of S-100 calcium-binding proteins and has been studied as an inflammatory indicator in several diseases, including FMF. Difficulties lead to delays in diagnosis of FMF, with the main problems being atypical clinical presentations that do not fully meet the diagnostic criteria and overlap diseases. 6 Considering that the average delay in diagnosis of FMF is 7.3 years, one can speculate that new, reliable, faster parameters may be useful for a more expedient diagnosis. 7 In this study, we aimed to evaluate serum resistin and plasma calprotectin in patients during both attack and attack-free periods of FMF and investigate the role of serum resistin and plasma calprotectin levels in the diagnosis and activity of FMF disease.

Statistical analysis
The SPSS software program version 20.0 (SPSS Inc, Chicago, IL, USA) was used for statistical analysis. Continuous variables were examined with a Kolmogorov-Smirnov test for normal distribution. Differences between more than two groups were evaluated by variance analysis (one-way ANOVA). The differences between two groups were examined with the Mann-Whitney U test or Student's t-test. Pearson and Spearman correlation tests were used for correlation analysis. The efficiency of the variables for the diagnosis of FMF was evaluated by logistic regression analysis. A P <0.05 was considered statistically significant.

RESULTS
Demographic and clinical data of patient groups are presented in    Table 4).

DISCUSSION
The current study revealed that resistin and calprotectin levels were significantly higher in patients during attack and attack-free periods than in a control group. Calprotectin was also found to be useful in differentiating between attack and attack-free periods of FMF. Higher ESR, CRP, fibrinogen, white blood cell count, and serum amyloid A (SAA) are expected results in active FMF disease compared to the attack-free period. 8,9 However, in a systematic review investigating APR used for FMF diagnosis, Erer et al. reported that there was no effective APR to diagnose FMF disease. 9 The innate immune system in FMF patients is proposed to be disrupted, and the disease progresses with episodes of systemic inflammation. 10 The innate immune system activates the adaptive immune system by antigen-presenting cells. Thus, B and T cells respond and result as disease symptoms. 10 Cytokines IL-1β, IL-1α, TNF α, TNF β, and IL-6 play an important role in these mechanisms. 5 Also, IL-1β and NF-κB pathways are abnormally activated due to mutation in the C terminal B30.2 region of pyrin. 11 Resistin and calprotectin are effective over NF-κB, which plays a role in the centre of inflammation. In light of these mechanisms, our findings suggest that high resistin and calprotectin levels can be relevant diagnostic markers for FMF disease.
Resistin is a regulatory cytokine that triggers the pro-inflammatory state by increasing the synthesis of cytokines such as TNF-α, IL-1β, and IL-6. 12 Resistin targets toll-like receptor 4 (TLR4) or adenylyl cyclase-associated protein 1 (CAP1). Upon binding to TLR4 and CAP1, resistin can trigger various intracellular signal transduction pathways to induce inflammation. 13 Resistin is synthesised in bone marrow, trophoblastic cells, the pancreas, leukaemia cells, synovial tissue, adipose tissue, and mostly in human peripheral blood mononuclear cells. The serum resistin level increases in some inflammatory diseases. Rheumatoid arthritis has a strong correlation with serum resistin levels. 14 Kisacik et al. reported that resistin levels could be useful in diagnosing FMF patients with attacks, but it was not useful in the differential diagnosis of acute appendicitis. 15 In the present study, resistin studied in both attack and attack-free situations was significantly higher in FMF patients than in healthy individuals and was predictive of disease according to logistic regression analysis.
Calprotectin, a heterodimer belonging to the family of S-100 calcium-binding proteins, has been studied as   16 Altug Gucenmez et al. also noticed in a paediatric age group that faecal calprotectin levels of attack-free FMF patients were higher than those of healthy controls. 17 The authors suggested that there might be a subclinical intestinal inflammation related to autoinflammatory processes. Demirbas et al. evaluated faecal calprotectin in children with FMF during the non-attack period and also reported that faecal calprotectin was higher in FMF patients than in healthy children. 18 However, plasma calprotectin in patients with attacks has not yet been studied. We believe that our findings have taken steps forward by evaluating FMF patients during both attack and attack-free periods. In our study, logistic regression analysis indicated that high resistin levels were predictive for FMF disease. We think that the major advantage of resistin over common widely available conventional APR would be its usefulness for predicting both attack and attack-free FMF patients, while conventional APR is generally used in attack periods. In addition, ROC analysis revealed satisfactory sensitivity and specificity scores for resistin and calprotectin. There is a need for further prospective studies to evaluate resistin and calprotectin in different periods of FMF disease and their role in the differential diagnosis. An interesting result of our study was that calprotectin but not resistin was higher in attack periods than attack-free periods of FMF patients. This result may indicate that calprotectin is especially effective during attack period pathogenesis, or vice versa; resistin is more effective for identifying subclinical inflammation in attack-free periods. It was previously shown that neutrophil extracellular traps (NETs) play an important role in the pathogenesis of FMF. [19][20][21] During FMF attack, neutrophils release chromatin structures called NETs, which are decorated with bioactive IL-1β. 19 The calprotectin as a neutrophil related protein maybe one of the NET-associated proteins and quantitative analysis of these proteins reflect to NET formation and disease exacerbation. Logistic regression analysis, including both attack and attack-free samples, also supported the finding that resistin might be more effective for showing subclinical inflammation in attack-free periods. Subclinical inflammation is an insidious feature of FMF, and it is not known exactly whether this persistent inflammation is associated with any particular phenotype. Babaoglu et al. analysed the predictors of persistent subclinical inflammation in a comprehensive study and demonstrated the risk factors for persistent inflammation as male gender, history of exertional leg pain, inflammatory comorbidities, M694V homozygosity, colchicine resistance, and musculoskeletal attack dominance. 22 In this regard, we believe that future studies examining patients who have these risk factors would be valuable to evaluate the effectiveness of resistin, calprotectin, and other novel biomarkers. Another finding in our study was that resistin and calprotectin correlated with CRP, ESR, WBC, and fibrinogen. This correlation consolidates the potential of resistin and calprotectin as effective inflammatory biomarkers. There are other new candidate biomarkers for FMF in the literature. 8,[23][24][25][26][27][28] Pentraxin-3, omentin, serum CXC chemokine ligand 16, serum amyloid A, CD144 + and CD146 + as circulating endothelial microparticles, chitotriosidase, S10012A, and resolvin D1 have been investigated and had promising results. However, there is still a need for prospective large cohort studies before using these novel biomarkers in daily practice. This study has some limitations. First, the study population was small, and second, all study participants were male because the study was conducted at a tertiary military centre. The colchicine treatment may influence the results, as it has anti-inflammatory effects and can cause a decrease in pro-inflammatory cytokine levels. [2][3][4] Also, due to the cross-sectional design of our study, the samples reflect only one time period. We expect that these limitations do not weaken our results due to the efficient homogenization between the groups. In addition, our study population was young with no confounding co-morbid situations. Thus, our strict exclusion criteria render our research valuable.
In conclusion, we showed that resistin and calprotectin levels were significantly higher in patients with FMF than in healthy individuals. Further prospective, randomised, large studies are needed to elucidate the roles of resistin and calprotectin in the pathogenesis of FMF.