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The significance of α-defensins 1-3 in Behcet’s disease: a case-control study among Egyptian patients

Background: Behcet’s disease (BD) is associated with uveitis, oral ulcers, genital ulcers and arthritis. Enhanced activity of innate immunity components, such as neutrophils, natural killer (NK) cells and endothelial cells, is a prominent feature of BD. The aim of this study was to clarify the significance of α-defensins 1-3 in BD and detect their correlation with disease activity, severity and oral ulcer activity. Results: Significant differences were found between Behcet’s patients and controls as regards serum α-defensins 1-3 (median 10.1, IQR 4–45 μg/ml versus median 7.4, IQR 0.6–13.2 μg/ml, p = 0.001) and salivary α-defensins 1-3 (median 17.4, IQR 5.7–44 μg/ml versus median 8.6, IQR 3.3–11 μg/ml, p < 0.001). Receiver operating characteristic (ROC) showed a fair area under curve (AUC) (AUC = 0.743), with sensitivity of 40% and specificity 100% at cutoff value of 17.3 μg/ml for serum α-defensins 1-3, while salivary α-defensins 1-3 showed excellent AUC (AUC = 0.936), with sensitivity of 93.3% and specificity of 86.7% at cutoff value of 9.8 μg/ml. The levels of serum and salivary α-defensins 1-3 > cutoff value were significantly higher in active severe Behcet’s patients with active oral ulcers (p < 0.001). Conclusion: α-Defensins 1-3 may be involved in the pathogenesis of BD and could be valuable markers in the determination of disease activity, severity and oral ulcer activity.


Background
Behcet's disease (BD) is a systemic chronic disease characterised by oral ulcers, vasculitis, arthritis, genital ulcers and uveitis. It is more common in Turkey, China, Korea and Japan [1]. The innate immune system is suggested to participate in the pathogenesis of BD, as manifested by the expression of toll-like receptor expression in affected cells, increased mucocutaneous symptoms and neutrophil hyper-reactivity to streptococci or heat shock proteins [2]. It was found that BD neutrophils are more active and express more adhesion molecules than in healthy controls [3].
Initially, human alpha defensin peptides were found in neutrophils and are named human neutrophil peptides [5].
Human neutrophilic peptides 1-3 are encoded by two genes DEFA1 and DEFA3 found at chromosome 8, site 8p23.1. DEFA1 and DEFA3 encode similar peptides the difference in the first amino acid from alanine in HNP-1 to aspartic acid in HNP-3; HNP-2 is an N-terminally truncated iso-form without the first amino acid [6].
Defensins initially attracted interest due to their potent antimicrobial functions and highly conserved structure. Consequently, several immunomodulatory functions like chemotactic effects and the ability to induce the production of cytokines have been identified, suggesting that these peptides connect innate and adaptive immune responses [7]. Their importance for autoimmune disorders is supported by the recent finding of modulated defensin expression in patients with systemic lupus erythematosus [8].
Interestingly, high α-defensin levels have been reported in the bronchoalveolar lavage fluid and plasma of one Wegener's granulomatosis (WG) patient previously [9] and furthermore in other vasculitic diseases, like microscopic polyangiitis [10] and cutaneous vasculitis [11].
Defensins have a great role in the control of inflammatory and immunologic processes like complement activation, cytotoxicity, chemotaxis of immature dendritic cell, CD4+/CD45RA+ naïve T cell, CD8+ T cells and monocytes, cytokine induction and enhancement of humoral and cellular response [12]. Several studies found that one of the genes of chromosome 8 perhaps could be involved in the pathogenesis of BD [13].
Alpha defensins might be among the main effector molecules in BD lesions. Neutrophils and NK cells that invade the site of inflammation and secrete defensins in harmful amounts in response to stress signals may be involved in the pathogenesis of the disease. The examined skin biopsies from the forearm especially in the corium after pathergy reactions reveal an increase in neutrophils rich in HNP 1-3 [14].
BD is a disorder with oral tissue inflammation leading to ulcer development; the first line of defences with neutrophils can be expected to be enhanced in patients with BD. Various functions of neutrophils such as chemotaxis, phagocytosis and oxidative burst responses are increased in patients with BD, and over-reactive neutrophils have been suggested to participate to tissue damage [15].
As there is no universally recognised pathognomonic laboratory marker of BD [16], this study aimed to clarify the significance of serum as well as salivary α-defensins 1-3 in BD and their relation to disease activity and severity among Egyptian patients.

Study participants
This is a case-control study in a cohort of Egyptian patients and included 60 patients with BD with age ranging from 30 to 45 years who were diagnosed according to the international diagnostic criteria of BD [17]. Patients were recruited from the outpatient clinics of Rehabilitation, Rheumatology, and Internal Medicine, Mansoura University Hospital. They were compared to 60 age-and sexmatched healthy controls. A written informed consent was taken from all participants before the beginning of the study. The study was approved by the Institutional Research Board Mansoura University Faculty of Medicine code (R/16.03.118).

Exclusion criteria
-Oral mucosal disorders not caused by BD: ex.
Pemphigus vulgaris, mucous membrane pemphigoid, lichen plans and erythema multiform -Autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and spondyloarthropathies -Cancer -Infection

Data collection and clinical examination
Demographic and clinical data were taken from all patients including age, sex, disease duration, systemic clinical signs and symptoms, full musculoskeletal examination and intraoral examination.

Evaluation of BD
Disease activity was assessed by Behcet's Syndrome Activity Score (BSAS) [18]. BSAS has 10 questions, which consists of visual analogue scales for patient's level of discomfort with regard to oral ulcers, genital ulcers, skin lesions and current disease activity along with the number of oral ulcers, genital ulcers and skin lesions present, and records symptoms attributable to the gastrointestinal, vascular or eye involvement. The VAS questions are all scored 0-10, and the remaining are scored categorically, 0, 5 or 10 depending on the response, for a total score of 0-100; patients complete the BSAS at the time of the visit to the treating doctor, with no input from the physician.
Disease severity was estimated following Krause et al. study [19].
Severity score will be calculated as the sum of 1 point for each mild manifestation, 2 points for each moderate manifestation and 3 points for each severe manifestation.

Moderate
Arthritis, deep venous thrombosis of the legs, anterior uveitis, gastrointestinal bleeding Severe Posterior/panuveitis, retinal vasculitis, arterial thrombosis or major vein (vena cava, hepatic) thrombosis, aneurysms, neuro-Behçet, bowel perforation Oral ulcer activity was estimated by Mumcu composite index [20]. Mumcu et al. proposed a composite index to monitor the clinical manifestations associated with oral ulceration in BD patients.
This index evaluates the activity of the oral ulcer, ulcer pain and functional disability.
➢ Oral ulceration activity is recorded as the number of ulcers in the past 30 days. This is scored 0 point if there were no ulcers and scored 1 point for the presence of oral ulcers in the previous month.

Laboratory assessment
Serum and salivary samples were obtained from both patients and controls. Samples of unstimulated whole saliva were obtained during 15 min in the morning due to the circadian rhythm of saliva secretion. Salivary and serum α-defensins 1-3 were assayed using α-defensins 1-3 ELISA kit (PELOBIOTECH GmbH-Am Klopferspitz 19-82152 Planegg, Germany). IL-6 was assayed using ELISA Kit (Elabscience, Bethesda, MD, USA). Erythrocyte sedimentation rate (ESR), neutrophil count and Creactive protein were measured.

Statistical analysis
The collected data were revised, coded, tabulated and introduced to a PC using Statistical Package for Social Science (SPSS 20 for windows; SPSS Inc, Chicago, IL, 2011). The Student T test was taken to assess the statistical significance of the difference between two study groups means. The Mann-Whitney test (U test) was taken to assess the statistical significance of the difference of a non-parametric variable between two study groups. Differences were significant at the level p ≤ 0.05.

Discussion
In the present study, the α-defensins 1-3 serum level in BD patients was significantly higher than that in healthy controls. α-Defensins 1-3 have a crucial role in the regulation of immunologic and inflammatory processes such as immature dendritic cell chemotaxis, cytotoxicity, complement activation, induction of cytokines and stimulation of cellular and humeral response [12]. The present study detected higher αdefensins 1-3 salivary level in Behcet's patients as compared to controls. This can be explained by neutrophil migration via junctional epithelium surrounding the teeth to the oral cavity. In BD, there are inflamed oral mucosa, increased oral ulceration and neutrophil overactivity which secrete a pathological amount of α-defensins 1-3 [14].
Our results detected a significant correlation between α-defensins 1-3 serum level and arthritis, panuveitis, BD severity and activity.
α-Defensins induce the release of several cytokines such as IL-1, IL-8 and TNF-α; IL-8 and other inflammatory cytokines such as TNF-α were found to increase in BD [12]. The essential role of IL-8 is neutrophil chemotaxis which is the main origin of α-defensins 1-3, and this makes a positive feedback circuit between αdefensins and inflammatory cytokines, leading to a vicious cycle of inflammation in BD [21]. Several studies detected α-defensin accumulation in synovial cavity of rheumatoid arthritis patients, and the defensin level in synovial fluid was correlated significantly with joint destruction and erosion [22]. Thus, it is assumed that αdefensins may have a crucial role in aggravating or developing arthritis in BD.
Joong et al. [23] detected that high copy numbers of the DEFA1 gene and the associated increase in αdefensin-1 expression might initiate or increase the inflammatory response in the intestine of BD patients. Accordingly, high DEFA1 gene copy numbers may be involved in the increased susceptibility to intestinal pathology in patients with BD. Our results showed a positive significant correlation between α-defensins 1-3 serum level with a number of oral ulcers, oral ulcer activity score and α-defensins 1-3 salivary level. Saliva is considered as an ultra-filtrate of the blood, and elevated neutrophil count in the blood may lead to elevated α-defensins 1-3 salivary level [24]. The locally expressed or systemically secreted αdefensins 1-3 act as chemical barriers in the protection of oral mucosa with innate immune response [25].
The release of pathological amount of α-defensins 1-3 in saliva of BD patients from infiltrating neutrophils and those hyperactive and excessive neutrophils are blamable for increased oral ulcer activity and increased tissue damage [15]. We expected in our study that an increase in blood neutrophilic count and significant correlation between it and serum α-defensins 1-3 could be present, but we found no statistically significant difference in blood neutrophilic count between patients and controls and also there was no significant correlation between αdefensins 1-3 serum level and blood neutrophilic count. It seems that an increased serum level of α-defensins 1-3 is due to increased α-defensins 1-3 load in primary granules of neutrophils and increased neutrophil degranulation not due to increased number of neutrophils [26]. Elif Ugurel et al. [27] revealed that innate immunity is primarily involved in acute attacks of neuro-Behcet's disease (NBD) and treatment regimens attacking neutrophils might prove efficacy in the treatment of NBD and perhaps replace steroids that are already used for this disease.
In our results, serum and salivary α-defensins 1-3 levels were not significantly correlated with ESR and CRP; this goes with results of Katsantonis et al. [28], who found that ESR and CRP are not specific indicators for the process of inflammation and IL-8 is the most reliable indicator of disease activity and inflammation in Behcet's disease.
Serum and salivary α-defensins 1-3 were not significantly correlated with IL6; this could be explained by the study of Brook et al. [29] who found that αdefensins 1-3 act as molecular brake on macrophagedriven inflammation and inhibit the release of multiple cytokines (TNF-α, IL6, IL8 and IL1B) and are responsible for pathogen clearance and resolution of inflammation with minimal tissue damage; they prevent an excessive proinflammatory response that would create its own collateral damage while still acting as powerful antimicrobial peptides. This opens the way for developing similar peptide-based therapeutics that would act as effective combined antiinflammatory and antimicrobial agents.
This is the first study to measure both salivary and serum levels of α-defensins 1-3 and to construct ROC curve for both to discriminate between cases and controls, and we found that salivary α-defensins 1-3 were more sensitive and had excellent AUC. Future studies are needed to clarify the role of alpha defensins in bridging between innate and cellular immunity by measuring different cytokines such as IL8, TNF and IL1B.

Conclusion
-Alpha defensins are highly involved in the pathogenesis of Behcet's disease since it is correlated with activity and severity of the disease and with oral ulcer activity. It could be used as a biological marker for the assessment of treatment response. Future studies are suggested to examine if it could be used as a future target for therapy in Behcet's disease. -Salivary defensins are more sensitive and specific markers for the disease than serum defensins.