The Correlation of CD206, CD209, and Disease Severity in Behçet's Disease with Arthritis

The purpose of this study was to clarify the role of pattern recognition receptors in Behçet's disease (BD). The frequencies of several pattern recognition receptors (CD11b, CD11c, CD32, CD206, CD209, and dectin-1) were analyzed in patients with BD by flow cytometry, and cytokine levels, interleukin- (IL-) 18, IL-23, and IL-17A, were compared in plasma. The analysis was performed in active (n = 13) and inactive (n = 13) stages of BD patients. Rheumatoid arthritis patients (n = 19), as a disease control, and healthy control (HC) (n = 19) were enrolled. The frequencies of CD11b+ and CD32+ cells were significantly increased in active BD patients compared to HC. Disease severity score was correlated to CD11c+, CD206+, and CD209+ in whole leukocytes and CD11b+, CD11c+, CD206+, CD209+, and Dectin-1+ in granulocytes. The plasma levels of IL-17A were significantly different between HC and active BD. IL-18 showed significant difference between active and inactive BD patients. From this study, we concluded the expressions of several pattern recognition receptors were correlated to the joint symptoms of BD.


Introduction
In immune dysfunction of Behçet's disease (BD), innate immunity is regarded to become more significantly involved in the pathogenesis. The important function of innate immunity is the initiation of defense against infection, such as virus, bacteria, and fungus, and linking to the adaptive immune responses [1]. Pattern recognition receptors (PRR) are proteins expressed on the cells of the innate immune system [2]. PRR can recognize pathogen-associated molecular patterns [3]. Most classes of the human pathogens are recognized by c-type lectin receptors (CLR), which is one kind of PRR [4]. CLR includes the mannose receptor (CD206), primarily present on the surface of macrophages and dendritic cells (DC), and asialoglycoprotein receptor family which includes DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (CD209) and DC-associated C-type lectin-1 (Dectin-1) [5].
Several chronic inflammatory diseases, such as colitis, Crohn's disease, Kawasaki disease, and rheumatoid arthritis (RA), have been reported to be significantly associated with CD206, CD209, and Dectin-1 [6][7][8]. However, in BD, the correlation of CLR has not been published at all except mannose-binding lectin, one kind of soluble protein of CLR [9]. Therefore, in this study, the expression of CD206, CD209, and Dectin-1 was analyzed and compared between active and inactive BD patients with arthritis. The frequencies of CD11b, CD11c, and CD32 were also analyzed by combination with CD206, CD209, and Dectin-1.

BD Patients.
The patient population consisted of 13 patients with BD, who presented for the first time or were monitored at the Department of Rheumatology, Ajou University Hospital. Clinical characteristics and therapeutic histories of these patients are shown in Tables 1 and 2. According to the International Study Group for BD criteria, the presence of any two of the following symptoms, in 2 Mediators of Inflammation   NSAIDs  1st  2nd  1st  2nd  1st  2nd  1st  2nd  1st  2nd  1st  2nd  1st  2nd  1

Clinical Characteristics of the Patients.
Clinical characteristics of the 13 patients with BD are summarized in Table 1. Blood sampling was performed twice at active stage of disease with arthritis (1st) and inactive stage without symptoms after medication (2nd). The disease severity scores and erythrocyte sedimentation rate (ESR) were significantly downregulated at 2nd sampling compared to the 1st sampling ( < 0.001, 0.038 resp.). The levels of C-reactive protein (CRP) were also downregulated, but not significant. Table 2 shows the medication list at the time of blood sampling. Colchicine was used in 11 patients (84.6%), sulfasalazine in 6 patients (46.2%), and azathioprine in 3 patients (23.1%). And nonsteroidal anti-inflammatory drugs were used in 11 patients (84.6%).

Correlation between the Frequencies of Each PRR Marker in BD Patients.
The correlation analysis was also applied to each surface marker of PRR in whole leukocytes ( Table 5)

Discussion
This study has presented the significantly different expression levels of the frequencies for PRR between BD patients and HC. The frequencies of CD11b, CD11c, CD32, CD206, CD209, and Dectin-1 positive cells were differently expressed on whole leukocytes, granulocytes, monocytes, or lymphocytes among active BD, inactive BD, and HC. The frequencies of CD206 and CD209 showed significant correlation with plasma levels of IL-17A. Specifically, the frequencies of CD11b, CD11c, CD32, CD206, CD209, and Dectin-1 positive cells were correlated significantly to the disease severity score of BD patients.
It is well known PRR plays critical roles in pathogen recognition and linking to the generation of adaptive immunity [10]. Toll-like receptor, a family of PRR, has been revealed in BD [11][12][13][14]. However, C-type lectin receptors, another family of PRR, CD206, CD209, and Dectin-1, can be found in a few cases in BD patients until now. Downregulation/deficiency of C-type lectin domain family 12, member A (CLEC12A), a C-type lectin-like pattern recognition receptor, is associated with hyperinflammatory responses. Patients with severe forms of Behçet's disease underexpress CLEC12A with respect to patients with mild forms of the disease [15]. Mannose-binding lectin gene-2 polymorphisms and serum mannose-binding lectin levels are associated with the production of high levels of MBL in Behçet's disease. Dectin-1 is upregulated on immune cells such as macrophages and neutrophils in inflammatory bowel disease [16]. Dectin-1 plays a critical role in experimental autoimmune uveitis development by Freund's Complete Adjuvant/interphotoreceptor retinoid-binding protein mixture at the induction phase. Uveitis is one of the major symptoms of BD in patients. Anti-dectin-1 antibody treatment can prevent the development of experimental autoimmune uveitis [17]. In BD patients, the correlation with Dectin-1 was not reported. CD209 promote CD3-activated T cells to produce IL-2 and strengthen the interaction of T cell receptor-antigen presenting cell and enhance immune response [18]. CD209 was reported in secondary progressive multiple sclerosis [19] and Crohn's disease [20]; however, the correlation was not studied in BD. Our study showed the correlation of several PRR (CD11b, CD11c, CD32, CD206, CD209, and dectin-1) in the pathogenesis of BD.
The mean fluorescence intensity of CD11b on granulocytes was higher in BD patients with heart diastolic dysfunction than those of controls [21]. The mean fluorescence intensity of CD11b in CD8 bright cells was also higher in BD patients with uveitis than controls or BD without uveitis [22]. Similar to previous reports, our study showed that BD patients with arthritis had higher frequencies of CD11b+ cells than disease control RA patients and the HC. After improvement with treatment, the frequencies of CD11b+ cells of inactive BD were not changed.
Single nucleotide polymorphisms of CD11c have been suggested as susceptibility loci in Korean BD patients with cutaneous symptoms [23] and in Chinese Han population BD patients with eye symptoms [24]. The frequencies and protein expression levels of CD11c+ cells were significantly increased in the CD4+ and CD8+ cell populations of active BD patients with cutaneous symptoms compared to those in the HC. In BD patients with arthritis, from our study, CD11c+ cells showed similar frequencies to HC. Inactive BD patients showed downregulation of CD11c+ cells compared to active BD patients though the difference was not significant. This difference can be from the different immune responses between BD cutaneous symptoms and BD arthritic symptoms, even though 20% of enrolled patients have both cutaneous and arthritic symptoms in each report.   Until now, there were no papers regarding CD32 in BD patients. This is the first data describing CD32 in BD patients. In RA patients, CD32 on natural killer (NK) cells were significantly lower than HCs [25]. However, in our results the frequencies of CD32+ cells on whole leukocytes were higher in active BD patients with arthritis than HCs. In RA patients as a disease control, CD32 expressions were not downregulated as compared to HCs. Inactive BD patients were not different as compared to active BD patients with arthritis. It was reported that CD32 participated in the stimulation of plasmacytoid DC by immune complex combined with anti-DNA antibody in systemic lupus erythematosus [26]. CD32 seems to be involved in the induction of inflammatory responses, but still not enough for defining of the role in inflammation of BD.
The correlation of CD206 and BD has also been rarely reported. There is only one paper for CD206 in BD patients with cutaneous symptoms [27]. CD206 was highly expressed with significance in active BD patients with cutaneous symptoms (49.7 ± 35.2%) compared to inactive BD patients (4.7 ± 3.1%) ( = 0.007) and HCs (7.4 ± 0.8%) ( = 0.02). It is known that CD206 facilitates viral infections such as Dengue virus [28], hepatitis B virus [29], lymphadenopathy associated virus [30], and human immunodeficiency virus [31]. In collagen induced arthritis, CD206 was seen in the inflamed joints by micro-computed tomography imaging [32]. CD206 + macrophages were significantly induced in patients with mucosal healing after treatment with infliximab [33]. In the mixed lymphoid reaction, the antibodies against tumor necrosis factor induced CD206+ macrophages. CD206+ macrophages had an immunosuppressive phenotype showing inhibited proliferation of activated T cells [34]. In our results, CD206+ PBMC were downregulated after treatment compared to before treatment in BD patients.
The correlation of CD209 and BD has not been reported at all. Our present study has shown the frequencies of CD209+ cells were significantly decreased in whole leukocytes and monocytes populations after improvement compared to an active symptomatic state. In juvenile idiopathic arthritis, CD209+ cells were increased in peripheral blood and accumulated in synovial fluid [35]. CD209+ cells were also higher in RA synovium than osteoarthritis synovium [36]. CD209 + CD14 + cells were significantly increased in the lesion of lamina propria of Crohn's disease [37]. This subset might be involved in the pathogenesis of Crohn's disease [38].
Dectin-1 has been known to play a role in antifungal immunity [39] for the protection from Candida albicans infection [40] and is involved in the production of arachidonic acid metabolites [41] and DC maturation [42]. Dectin-1 recognizes -glucans and carbohydrates found in some bacteria and may also recognize other molecules such as endogenous ligand on T cells [43]. Dectin-1 expression is found on myeloid DC, monocytes, macrophages, and B cells [44]. In a genetically arthritis susceptible mouse model, Dectin-1 plays a role for the induction of arthritis [45]. In our experiment, the frequencies of Dectin-1+ cells in granulocytes populations were downregulated after treatment compared to active BD arthritis. Dectin-1 signaling increases the phagocytic activity of neutrophils through upregulation of CD11b expression [46] and increased the production of IL-17A. Dectin-1-dependent IL-17A production is mediated by CD11b+ neutrophils in an IL-23 dependent manner [47]. Our data has shown CD11b+Dectin-1+ granulocytes were downregulated after improvement of BD arthritis though the significance was not different. This is the first data for the Dectin-1 expression in BD.
CD11b+CD206+ macrophages play critical roles in the progression of hepatitis C virus induced inflammatory liver diseases [48]. In our results, the frequencies of CD11b+CD206+ in whole leukocytes and monocytes were downregulated with improvement of BD arthritis. Also, CD11b+CD32+ cell populations in whole leukocytes and monocytes were higher in BD patients compared to HC irrespective of arthritis activity.
With flow cytometric data, we can conclude the frequencies of CD11b+ and CD32+ cells show similar levels in active and inactive BD. CD11c+, CD206+, CD209+, and Dectin-1 were downregulated after improvement. This means higher levels of CD11b+ and CD32+ cells are correlated to the development of BD arthritis. CD11c+, CD206+, CD209+, and Dectin-1 are related to the regulation of BD symptom. For the therapeutic target, CD11c+, CD206+, CD209+, and Dectin-1 can be more reliable candidates for BD arthritis. This can be a direction for future experimental plans.
Several papers have shown higher levels of IL-18 in BD patients than HC [49,50]. IL-18 with IL-12 or IL-15 enhances the Th1 response, while IL-18 without IL-12 can stimulate Th2 responses [51]. IL-18 with IL-2 or IL-4 enhances the production of IL-4 and IL-13 [52]. In our data, inactive BD patients showed significantly higher IL-18 levels than active BD patients. In BD patients with arthritis, increased IL-18 in improved stage may have a role for Th2 shift function with IL-4 or IL-2, even though we did not show the levels of IL-4 or IL-2.
The serum levels of IL-23 are elevated more in BD patients with active uveitis than HC. BD patients with active uveitis significantly upregulated IL-23 as compared with BD patients without active uveitis [53]. In our data, the levels of IL-23 were higher in BD patients with arthritis than HC, though the level of IL-23 was not different between active and inactive BD patients. IL-17 was also significantly higher in active BD patients than HC. However, active and inactive BD patients with arthritis showed similar levels of IL-17. Several papers already reported BD patients with uveitis showed similar levels of IL-17A between active and inactive stage [54,55]. IL-18 gene polymorphism was reported by 5 papers in Turkish, Egyptian, and Korean. These 5 papers did not include the protein levels of IL-18. Oztas et al. reported the increased serum IL-18 protein expression compared to healthy control, but they did not show the comparison between active BD and inactive BD [56]. Musabak et al. reported the serum levels of IL-18 were increased in patients with inactive disease than active disease even though the difference was not significant [57]. They mentioned Th1 activation and subclinical inflammation persists during the inactive period of the disease. In our result, similarly to the previous reports, plasma IL-18 levels were higher in active BD than HC. Also, IL-18 in inactive BD was higher than active BD. According to Habibagahi et al., serum levels of IL-23 were higher in BD with uveitis [58]. IL-23 and IL-17 were higher in BD with active uveitis than BD with inactive uveitis [53]. Among this patients population, 33.3% of patients had arthritis. According to Emiroglu et al., serum IL-17A levels were not different between active BD and inactive BD [55]. This study included 57.9% articular involvement in cutaneous BD symptomatic patients. They enrolled different populations with and without symptoms instead of chasing the same patients. The expression was similar to our data. In our data, the average levels of IL-23 and IL-17 were similar between pretreatment and posttreatment; however, the changes of expression levels are diverse in each patient.
In conclusion, the CD11b+ and CD32+ circulating cells were significantly higher in active BD patients with arthritis than HC. CD11c+ and CD209+ cells were significantly decreased after improvement of arthritis in BD patients. Disease severity scores were significantly correlated to the frequencies of CD11b+, CD11c+, CD206+, CD209+, and Dectin-1+ cells. Plasma IL-17A expression levels were also correlated with the frequencies of CD206+ and CD209+ cells. CD206 in monocytes, CD209 in whole cells and monocytes, and CD11b+CD206+ in whole cells and monocytes can be disease activity markers for the improvement of BD arthritis.