Serum GAS6, sAXL, IL-10, NO, and BCL-2 levels are decreased in patients with Behçet’s disease

Abstract

Purpose:

Behçet’s disease (BD) is an autoimmune chronic systemic inflammatory disease characterized by a versatile clinical spectrum. Growth arrest specific protein 6 (GAS6)/soluble AXL (sAXL) signaling pathway draws attention in the resolution of inflammation, and its deficiency is associated with chronic inflammatory, autoimmune diseases, as well as clearance of apoptotic cells by phagocytes – efferocytosis. In this study, it was aimed to investigate whether GAS6/sAXL, interleukin (IL)-10, nitric oxide (NO), and BCL-2 levels were associated with inflammation and efferocytosis contributes to the pathogenesis of BD.

Methods:

A total of 37 Behçet patients with ocular involvement and 30 healthy control subjects were included in this study. GAS6, sAXL, IL-10, NO, and BCL-2 levels were quantified using enzyme-linked immunosorbent assay (ELISA) method.

Results:

Serum GAS6, sAXL, IL-10, NO, and BCL-2 levels were significantly lower in patients with BD compared to the controls (P < 0.005, P < 0.001, P < 0.001, P < 0.001, and P < 0.001, respectively). In correlation analysis, research parameters decreased in patients with BD was significantly correlated with each other: GAS6–IL-10 (r = 0.585, P < 0.001), GAS6–BCL-2 (r = 0.541, P < 0.001), sAXL–BCL-2 (r = 0.696, P < 0.001), IL-10–NO (r = 0.717, P < 0.001), IL-10–BCL-2 (r = 0.759, P < 0.001), and NO–BCL-2 (r = 0.541, P < 0.001).

Conclusion:

In conclusion, decreased serum BCL-2 level may be an indicator of increased apoptosis in these patients and decreased levels of GAS6/sAXL, IL-10, and NO may indicate insufficient clearance of apoptotic bodies released as a result of increased apoptosis in BD patients.

Behçet’s disease (BD) is a complex multisystem inflammatory disorder of unknown etiology.[1] Growth arrest specific protein (GAS6) and soluble anexelecto (sAXL) play an essential role in the resolution of inflammation. Deficiencies in GAS6/sAXL pathway have been associated with chronic inflammatory and autoimmune diseases.[2] It has been suggested that the GAS6/AXL pathway is involved in immune system regulation through the clearance of apoptotic cells by phagocytosis, known as “efferocytosis,” which is necessary to maintain tissue homeostasis. sAXL levels in serum or plasma are altered in several diseases such as aortic aneurysm, heart failure, peripheral artery disease, chronic kidney disease, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and preeclampsia.[3,4]

When efferocytosis is impaired, it can lead to the development of chronic inflammatory diseases, and inflammation plays a crucial role in the resolution response.[5] Defective regulation of programmed cell death (apoptosis) may be involved in the development of BD, and B-Cell Leukemia/Lymphoma 2 (BCL-2) as an antiapoptotic marker is involved in the control of apoptosis.[6]

Interleukin (IL)-10 is an important anti-inflammatory regulator and is involved in sterile wound healing, autoimmunity, and homeostasis, protecting the host from ever-exuberant responses to pathogens and microbiota. It has been reported that IL-10 plays a role in the resolution of inflammation in BD and enhances efferocytosis by macrophages.[7] It is believed that IL-10 may also be an important factor in the regulation of nitric oxide (NO) mechanism.[8] NO is a key regulator of different physiological and inflammatory responses and has been previously reported to contribute to the development of vasculitis and endothelial cell (EC) dysfunction.[9] BD is characterized by vasculitis and EC dysfunction, and the role of NO has been demonstrated in BD.[10] The relationship of GAS6/AXL pathway with NO, IL-10, and BCL-2 has not been evaluated in patients with BD yet.

Therefore, the current research was planned to evaluate the GAS6/sAXL signaling pathway, which plays an important role in inflammatory processes and clearance of apoptotic cells in patients with BD.

Methods

Study population

A total of 37 Behçet patients who were admitted to the Uvea-Behçet unit of Erciyes University Ophthalmology Outpatient Clinic between May 2021 and January 2022 were included. All the patients were diagnosed according to the diagnostic criteria of International Study Group for BD.[11] The diagnosis of uveitis was made using the International Uveitis Study Group criteria.[12] The control group included 30 age- and sex-matched healthy volunteers. In addition, patients with cardiovascular disease, diabetes, hypertension, autoimmune, inflammatory or rheumatic disease, and previous intravitreal injection therapy were excluded. Because of their potential effects on the levels of the markers evaluated in this study, a detailed history of medication was obtained from both study groups, and patients receiving any systemic medication such as corticosteroids, nonsteroidal agents, and cytotoxic agents were excluded from the study.

Ethics approval and informed consent

Informed consent was obtained from all participants. This study was approved by the Erciyes University Clinical Research Ethics Committee on 02.17.2021 with decision no.: 2021/141.

Samples and biochemical analyses

Blood samples were collected in tubes without an anticoagulant agent (serum separator tubes, 8 mL, Vacuette) and centrifuged at 2000 ×g for 10 min. Routine tests were evaluated on the same day in serum samples and the remaining serum was kept frozen at -80°C for enzyme-linked immunosorbent assay (ELISA). The ELISA method was used to detect GAS6, sAXL, NO, IL-10, and BCL-2 in human serum following the manufacturer’s instructions (BT LAB; cat no.: E2029Hu, E7400Hu, E1510Hu, E0102Hu, and E1832Hu, respectively). Blood samples were collected from all the participants after an overnight fast and 30 min of supine rest in the morning hours (8:00–10:00 a.m.). None of the participants received any topical or systemic medications at least 4 weeks before blood sample collection.

Statistical analyses

Statistical analysis was performed with Statistical Package for the Social Sciences (SPSS) version 23.0 software. The conformity of data to the normal distribution was evaluated with the Shapiro–Wilk normality test and Q–Q graphs. The difference between the gender distribution of the two groups was evaluated with the Chi-square test. Mann–Whitney U test was used to compare continuous variables. Spearman correlation analysis was performed for correlation analysis. A P value below 0.05 was considered statistically significant.

Results

The BD group consisted of 23 (62.2%) women and 14 (37.8%) men with a mean age of 47.7 ± 2.0 years, while the control group consisted of 18 (60%) women and 12 (40%) men with a mean age of 42.1 ± 1.6 years. The age and gender distributions of the two groups were similar (P > 0.810 and P > 0.857, respectively). We found that GAS6, sAXL, IL-10, NO, and BCL-2 levels were significantly lower in patients with BD compared to the control subjects (P < 0.005, P < 0.001, P < 0.001, P < 0.001, and P < 0.001, respectively) [Table 1].

Table 1.

Demographic characteristics and research parameters in Behçet patients and control subjects

		Patients (n=37)		Healthy controls (n=30)		P
Age, years		47.7±2.0		42.1±1.6		0.810
Gender, n (%) female		23 (62.2%)		18 (60%)		0.857
GAS6 (ng/mL)		17.11 (10.42–27.81)		65.27 (12.49–109.20)		0.005
sAXL (ng/L)		449.42 (374.41–609.00)		1275.25 (702.75–1931.91)		<0.001
NO (μmol/L)		34.11 (21.67–72.69)		131 (61.00–212.76)		<0.001
IL-10 (pg/mL)		57.33 (44.32–86.86)		165.06 (61.96–228.17)		<0.001
BCL-2 (U/mL)		39.75 (20.79–77.58)		259.09 (73.67–403.62)		<0.001

GAS6=growth arrest specific protein 6, IL-10=interleukin-10, NO=nitric oxide, sAXL=soluble AXL, SD=standard deviation. Data presented as mean±SD, n (%), or median (25–75 percentile). Bold font indicates statistical significance

We next investigated the relationships between the levels of the research parameters in patients. Research parameters in patients with BD were significantly correlated with each other. GAS6–sAXL (r = 0.602, P < 0.001), GAS6–IL-10 (r = 0.585, P < 0.001), GAS6–NO (r = 0.611, P < 0.001), GAS6–BCL-2 (r = 0.541, P < 0.001), sAXL–IL-10 (r = 0.553, P < 0.001), sAXL–NO, (r = 0.623, P < 0.001), sAXL–BCL-2 (r = 0.696, P < 0.001), IL-10–NO (r = 0.717, P < 0.001), IL-10–BCL-2 (r = 0.759, P < 0.001), and NO–BCL-2 (r = 0.541, P < 0.001) [Table 2].

Table 2.

Correlation analysis in patients with BD

		rho		P
GAS6–sAXL		0.602		<0.001
GAS6–IL-10		0.585		<0.001
GAS6–NO		0.611		<0.001
GAS6–BCL-2		0.541		<0.001
sAXL–IL-10		0.553		<0.001
sAXL–NO		0.623		<0.001
sAXL–BCL-2		0.696		<0.001
IL-10–NO		0.717		<0.001
IL-10–BCL-2		0.759		<0.001
NO–BCL-2		0.675		<0.001

BD=Behçet’s disease, GAS6=growth arrest specific protein 6, IL-10=interleukin-10, NO=nitric oxide, sAXL=soluble AXL. Bold font indicates statistical significance

Discussion

Apoptosis occurs continuously in millions of cells, accompanied by the active removal of apoptotic debris to maintain tissue homeostasis. If an apoptotic cell is not cleared within a certain period, its breakdown and eventual expulsion of harmful contents become possible. As these released particles are considered autoantigens, they contribute to the stimulation of immune responses.[13,14] The process, known as efferocytosis, which involves the clearance of dead cells, is normally regulated throughout the lifespan of the human body. Due to signals released from dead cells and phagocytes, both professional cells (i.e., macrophages and dendritic cells) and nonprofessional cells (i.e., epithelial cells and fibroblasts) actively participate in the identification and engulfment of apoptotic corpses for their degradation. The engulfment of apoptotic cells by phagocytes before the release of immunogenic intracellular contents is considered an immunological event. Consequently, the disruption of this process may lead to numerous autoimmune disorders such as SLE, RA, and other diseases.[15]

In a study conducted on monocyte-derived macrophages obtained from patients with SLE, it has been reported that the noninflammatory phagocytosis of apoptotic cells is reduced, indicating inadequate efferocytosis. Therefore, it has been suggested that persistently uncleared apoptotic cells may serve as immunogenic and antigenic complexes for the induction of autoreactive lymphocytes and formation of immune complexes.[16]

In another study, anti-Tyro3 IgG associated with disease activity was investigated in SLE, RA, and primary Sjögren’s syndrome. It has been shown that the levels of this autoantibody are higher in SLE patients compared to healthy controls, RA, and primary Sjögren’s syndrome patients. In addition, it has been demonstrated that this autoantibody inhibits the efferocytosis of macrophages. Therefore, deficient phagocytosis, namely efferocytosis, has been indicated in SLE.[17]

Although the etiology of the disease is still unknown, autoinflammatory processes are thought to play an important role in the pathogenesis of BD disease.[1,18] Activated autoreactive lymphocytes are thought to be responsible for the development of inflammation in BD, and if these lymphocytes do not undergo proper apoptosis or efferocytosis, inflammation persists and recurs. Besides, many studies have demonstrated the role of dysregulation of apoptosis in the pathogenesis of BD.[19] Arimura et al.[20] investigated the apoptotic rate of bone marrow cells in patients with BD and reported that the apoptotic rate in BD patients was significantly higher than in normal controls. This situation may indicate insufficient clearance of apoptotic cells, pointing to efferocytosis. However, the underlying mechanisms involved in the pathogenic process of BD remain unclear.

GAS6/sAXL pathway is involved in abnormal clearance of apoptotic cells and, if it is impaired, accumulated apoptotic cell debris ensues, resulting in inflammation-associated pathologies or autoimmunity.[5]

In this study, first, we investigated serum GAS6 and sAXL levels to evaluate possible mechanisms in the pathogenesis of BD. We found that GAS6/sAXL signaling decreased in patients with BD and there was a significant correlation between GAS6 and sAXL levels. Similarly, recent studies have focused on efferocytosis which leads to autoimmune diseases such as RA, SLE, and BD.[15] Gheita et al.[21] showed that GAS6 levels were decreased in patients with BD and SLE. It has been reported that a decrease in GAS6 level is an indication that it is involved in apoptotic processes. In another study, it was shown that Mer, one of the Tyro-AXL- Mer (TAM) receptors, is required for the circadian phagocytosis of photoreceptor outer segments by retinal pigment epithelial cells, and deactivation of ProS and GAS6 leads to loss of Mer-dependent retinal pigment epithelial phagocytosis in mice.[22] However, AXL-deficient alveolar macrophages have been reported to exhibit reduced efferocytic capacity and increased morbidity to influenza virus infection associated with the accumulation of dead cells in the lung of mice lacking AXL expression.[23] Therefore, our observations suggested that decrease in GAS6 and sAXL levels may be associated with efferocytosis and could lead to the development or perpetuation of autoimmunity due to inadequate phagocytosis of inflammatory cells. Similarly, the observed correlation between GAS6 and sAXL suggested that GAS6 may act together with AXL, demonstrating its activity by binding to AXL, and they may play a collaborative role in the efferocytosis mechanism.

The production of IL-10, a major immune regulatory cytokine, by phagocytes during clearance of apoptotic cells is critical for maintaining cellular homeostasis and suppressing autoimmunity.[24] Then we evaluated that IL-10 levels in patient with BD and controls. It has been reported that IL-10 is also associated with autoimmune disorders accompanied by ocular symptoms such as ankylosing spondylitis and BD.[25] For instance, IL-10 supports the resolution and upregulation of inflammation in liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR) delta-dependently in efferocytic macrophages.[26] In our study, decreased IL-10 levels may indicate insufficient resolution of inflammation and decreased anti-inflammatory cytokine effect in patients with BD. We demonstrated a positive correlation between GAS6 and IL-10 levels. Previously, Goudarzi et al.[27] had investigated the effect of GAS6 on inflammatory cytokine response in glial cell cultures and reported that GAS6 promotes the expression of anti-inflammatory/pro-repair cytokine IL-10. It is stated that low GAS6/AXL indicates insufficient efferocytosis. In the present study, considering the anti-inflammatory effect of GAS6 and IL-10, their decreased levels together with the positive correlation between them may be an indication that they act together in the anti-inflammation mechanism or insufficient efferocytosis.

NO is a signaling molecule that plays a key role in the pathogenesis of inflammation. It provides anti-inflammatory effect under normal physiological conditions. Studies have reported that NO is an important player in the formation process of uveal inflammation. However, it exhibits a dual-pronged role in apoptosis, both proapoptotic and antiapoptotic.[13,28] In experimental autoimmune uveitis, NO has been shown to mediate apoptosis through the formation of peroxynitrite.[29] In our opinion, decrease in NO levels in patients with BD may be associated with inflammation and apoptotic process. NO levels may decrease due to the formation of peroxynitrite by reacting with increased superoxide anion and oxidative stress, which induces apoptosis in BD. In another study, Orem et al.[30] showed that nitrite, nitrate, and nitrite/nitrate levels decreased in the active group compared to the inactive and control groups.

Defective regulation of programmed cell death (apoptosis) may contribute to the development of BD, and the proto-oncogene BCL-2 is involved in the control of apoptosis in immune-competent cells. We evaluated the possible role of BCL-2, an antiapoptotic marker, in the pathogenesis of BD to elucidate the apoptotic mechanism. We found that BCL-2 levels were statistically significantly lower in BD patients compared to controls. Indeed, our results are consistent with the conducted studies. In a conducted study, the expression of BCL-2 in skin pathergy reactions (SPR) of BD was investigated, and it was reported that the majority of histopathologically positive leukocytes were stained with BCL-2.[31] In another study, an increase in BCL-2 expression was observed in peripheral blood lymphocytes of Behçet’s patients,[19] and it was demonstrated that the expression of BCL-2 in active skin lesions of Behçet’s patients was higher compared to cases with leukocytoclastic vasculitis.[32] BCL-2 levels were positively correlated with NO levels. The proto-oncogene BCL-2 is an important player in controlling apoptosis through antioxidant activity. One of the potential mechanisms exhibited by BCL-2 is its ability to counter the detrimental effects of cell damage caused by free radicals, thereby gaining its reputation as a well-known antioxidant.[32] Accordingly, NO and BCL-2 may act together and decrease depending on the antioxidant defense mechanism.

There are some limitations to our study: (1) Evidence suggests that GAS6/AXL plays a significant role in EC activation and is involved in the interactions among ECs, platelets, and leukocytes during inflammation.[33] However, we were unable to evaluate this since we lacked data for both the patient and control groups and (2) IL-1, IL-6, and tumor necrosis factor alpha (TNF-α) are major proinflammatory cytokines in Behçet’s patients. These cytokines have been present in the ocular fluids of patients with BD for over 20 years and are believed to be the principal inflammatory mediators leading to the development of the disease.[18] Due to the inadequacy of financial resources in our study, we were only able to assess IL-10 levels. We could not evaluate inflammatory cytokines and proapoptotic markers.

Conclusion

BCL-2 is an antiapoptotic marker, and its decrease may have increased apoptosis and thus contributed to the development of BD. Decreased levels of GAS6/AXL, IL-10, and NO and positive correlation of BCL-2 levels with other parameters may indicate impaired efferocytosis and play a role in the pathogenesis of the disease by causing accumulation of apoptotic cells in inflammatory foci. We have illustrated this hypothesis in Fig. 1. We thought that these factors may have important roles altogether in the pathophysiological mechanism, and therapeutic strategies aimed at improving efferocytosis might be beneficial in BD.

Figure 1.

Decreased serum BCL-2 level may be an indicator of increased apoptosis in these patients, and decreased levels of GAS6/AXL, IL-10, and NO may indicate insufficient clearance of apoptotic bodies in BD. BD = Behçet’s disease, GAS6 = growth arrest specific protein 6, IL-10 = interleukin-10, NO = nitric oxide, sAXL = soluble AXL

Author contribution

GB: concept and design, supervision, writing, drafting and revising the article, analysis and interpretation of data, critical revision for the intellectual content; MÖ: analysis and interpretation of data, drafting manuscript, writing; DGS: acquisition of data, drafting manuscript, critical revision for the intellectual content; KG: drafting manuscript, analysis and interpretation of data, writing; HA: acquisition of data; HS: acquisition of data, analysis and interpretation of data; KG: acquisition of data; CE: acquisition of data, drafting manuscript, critical revision for the intellectual content.

Informed consent

Informed consent was obtained from all human participants included in the study.

Financial support and sponsorship

This work was supported by the Coordinatorship of Scientific Research Projects of Erciyes University, Kayseri, Turkey (grant number: TSA-2021-11074).

Conflicts of interest

There are no conflicts of interest.