Introduction
Behçet's disease (BD) is a serious sight-threatening clinical entity of uveitis that can be accompanied by recurrent oral aphthous ulcers, genital ulcers, and skin lesions. Patients with BD have recurrent episodes of uveoretinitis, which can cause irreversible damage to the neural retina and optic nerve, leading to vision loss [
1]. Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine that plays a significant role in the immune response in BD. Previous studies have suggested that BD is predominated by a T helper 1 (Th1) immune response. Increased levels of Th1-associated cytokines, such as interferon-γ (IFN-γ), interleukin-12 (IL-12), and TNF-α have been found in BD patients [
2,
3]. Recently, several investigators reported that active BD was characterized by increased levels of IL-17 as compared to BD in remission or control healthy donors [
4‐
6]. Importantly, recent genetic surveys including genome-wide association studies (GWAS) have identified IL23R-IL12RB2 and IL10 as BD susceptibility loci, suggesting that BD is predominated by Th1/Th17-type immune responses [
7,
8]. Therefore, Th17 cells, in addition to Th1 cells, should be instrumental in the pathogenesis of BD and uveitis.
A new anti-TNF-α monoclonal antibody, infliximab, greatly suppresses ocular inflammation in uveitis patients with BD [
9‐
16]. The antibody neutralizes membrane-bound TNF-α and soluble TNF-α and suppresses TNF-α production by macrophages and lymphocytes. An alternative inhibition mechanism of infliximab is the promotion of regulatory T cells that acquire suppressive functions in the periphery including the eye [
17]. Thus, infliximab is extremely effective in the suppression of intraocular inflammation in BD. However, the suppression mechanisms of infliximab remain unknown. We suspect that other factor(s) are involved in the mechanisms.
The present study showed that the production of IL-17 by stimulated CD4+ T cells, which is associated with active ocular inflammation in BD patients, is significantly elevated in BD patients with active uveitis. In addition, the production of IL-17 by polarized Th17 cell lines exposed to infliximab in vitro or fresh CD4+ T cells from BD patients being treated with infliximab was greatly reduced, and the Th17 transcription factor RORγt in T cells was also reduced. Moreover, TNF-α promoted Th17 differentiation in BD CD4+ T cells. These data suggest that the inhibition of Th17 differentiation by anti-TNF-α therapy may protect BD patients from severe ocular inflammation.
Materials and methods
Subjects
Behçet's disease (BD) was diagnosed based on the criteria of the BD Research Committee of the Ministry of Health and Welfare of Japan [
18]. Subjects were uveitis patients with BD at Tokyo Medical and Dental University Hospital between 2009 and 2011. The subjects did not have severe active systemic inflammation at the time of study participation. After informed consent was obtained, samples of aqueous humor and vitreous fluid were collected from patients with uveitis, either active (n = 6) or inactive (n = 4), associated with BD. At the time of aqueous humor sampling, the uveitis patients had active intraocular inflammation, but they were not being treated with systemic therapy. In patients with uveitis who were undergoing vitreous surgery, vitreous fluid samples were collected during the surgery. At the time of surgery, none of the patients were receiving systemic corticosteroids, and uveitis was active in two BD patients who developed retinal detachment associated with a macular hole and inactive in two patients who developed vitreous hemorrhages. We also collected ocular fluid samples from BD patients who were being treated with infliximab (all aqueous humor, n = 8). The controls consisted of the aqueous humor of patients with age-related cataracts (n = 3) and the vitreous fluid of patients with idiopathic macular holes (n = 3) obtained during surgery. These control patients had no clinical history of uveitis or systemic diseases.
About 0.1 ml of aqueous humor was drawn into tuberculin syringes. After the aqueous humor samples were centrifuged at 3000 rpm for five minutes and the vitreous fluid (about 0.5 ml) was centrifuged at 10000 rpm for five minutes, the supernatants were collected and stored in separate tubes at -80°C until use. The research followed the tenets of the Declaration of Helsinki, and the study was approved by the Institutional Ethics Committees of Tokyo Medical and Dental University.
Isolation of purified T cells and induction of Th17 cell lines
Peripheral blood mononuclear cells (PBMCs) were obtained from the BD patients or a sarcoidosis patient (control) with active uveitis and healthy donors. Freshly purified T cells were enriched for CD4+ cells using MACS cell isolation kits (Miltenyi Biotec, Auburn, CA, USA > 93% CD4+) and applied to flow cytometric analysis or in vitro assays.
For the induction of human Th17 cells, purified CD4+ T cells from BD patients or healthy donors were co-cultured with anti-human CD3 antibody (2 μg/ml, BD PharMingen, San Diego, CA, USA), anti-human CD28 antibody (2 μg/ml, BD PharMingen), anti-human IFN-γ antibody (5 μg/ml, R&D Systems, Minneapolis, MN, USA), anti-human IL-4 antibody (5 μg/ml, R&D Systems), and recombinant human proteins such as IL-1β (20 ng/ml, Peprotech, Rocky Hill, NJ, USA), IL-6 (20 ng/ml, R&D Systems), IL-23 (20 ng/ml, R&D Systems), and TNF-α (20 ng/ml, R&D Systems). After five days of culture, the harvested T cells that produced large amounts of IL-17 were used for assays.
ELISA or cytometric beads array (CBA) assay for cytokines
Purified CD4+ T cells were co-cultured with recombinant human IL-2 (100 U/ml), anti-human CD3 antibody (2 μg/ml), and anti-human CD28 antibody (2 μg/ml) in the presence (or absence) of infliximab (10 μg/ml) for 48 hours. As the control antibodies (Abs) for infliximab, anti-human TNF-α Abs (10 μg/ml, R&D Systems) and anti-human IL-6 monoclonal Abs (10 μg/ml, R&D Systems) were also used.
The concentration of cytokines in supernatants of the T-cell cultures (stimulated T cells in the presence of infliximab) was measured by a CBA assay kit that included IL-4, IL-6, IL-10, TNF-α, or IFN-γ. The assay was performed according to the manufacturer's instructions (Human Th1/Th2 Cytokine Kit, BD PharMingen). The concentration of IL-17 (R&D Systems) in the supernatants of the T-cell cultures was also measured by ELISA. Collected ocular fluids (aqueous humor or vitreous fluids) from uveitis patients (active uveitis, inactive uveitis without treatment, or infliximab treatment) and controls were also measured by CBA (IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ) or ELISA (IL-17).
Flow cytometric analysis
Flow cytometric analysis of Th17 cell lines derived from BD patients or healthy donors was performed using phycoerythrin (PE)-labeled anti-human IL-17 monoclonal antibodies (R&D Systems). T cells were pre-cultured with GolgiPlug (BD Biosciences, San Jose, CA, USA), ionomycin (0.5 μg/ml; Sigma-Aldrich Co., St. Louis, MO, USA), and phorbol-12-myristate-13-acetate (PMA, 40 ng/ml; Merck Chemical, Darmstadt, Germany) for five hours before intracellular staining. After permeabilization, Th17 cells were stained with PE-labeled anti-human IL-17 antibodies and fluorescein isothiocyanate (FITC)-labeled anti-human CD4 Abs. PE-conjugated goat immunoglobulin G (IgG; R&D Systems) was used as the isotype control. Cells (1 × 106) were stained for 40 minutes at room temperature in the dark. These T cells, Th17 cells and infliximab-exposed Th17 cells were also stained with PE-labeled anti-human RORγt abs (eBioscience, San Diego, CA, USA) and FITC-labeled anti-human CD4 Abs. Fresh CD4 T cells from an active BD patient, a BD patient with infliximab treatment, and a healthy donor were also stained with anti-human RORγt Abs. PE-conjugated rat IgG (eBioscience) was used as the isotype control. After permeabilization, cells (1 × 106) were stained for 30 minutes at 4°C in the dark.
Induction of experimental autoimmune uveitis (EAU) in mice and use of spleen cells and intraocular cells
Normal mice were subcutaneously immunized in the neck with 200 μg of interphotoreceptor retinoid-binding protein peptide (IRBP
1-20) emulsified in complete Freund's adjuvant (Difco, Detroit, MI, USA) and containing Mycobacterium tuberculosis strain H37Ra (Difco), as previously described [
19]. Funduscopic examinations were performed on days 14 and 21, as previously described [
20‐
22]. Inflammation was evaluated based on the fundus findings, and spleen cells and intraocular cells were used for
in vitro assays.
Statistical evaluation
Each experiment was repeated at least twice with similar results. Parametric data were analyzed with the Student's t test. Nonparametric data were analyzed with the Mann-Whitney U test. Values were considered statistically significant at P < 0.05.
Discussion
In the present study, we showed that ocular fluids from BD patients who have active uveitis contained significant amounts of inflammatory cytokines including TNF-α, whereas ocular fluids from patients being treated with infliximab as well as ocular fluids from control noninflammatory patients did not contain any inflammatory cytokines. Moreover, ocular fluids from BD patients who have active uveitis contained significant amounts of inflammatory cytokine IL-17. The levels of IL-17 in ocular fluids from inactive uveitis patients were very low or undetectable, and ocular fluids from patients who were being treated with infliximab as well as ocular fluids from control noninflammatory patients did not contain inflammatory cytokines. Activated CD4
+ T cells obtained from BD patients produced large amounts of TNF-α
in vitro, whereas T cells in the presence of infliximab did not produce inflammatory cytokines. Thus, infliximab, which is a chimeric antibody against human TNF-α, neutralized TNF-α-producing cells. We also demonstrated that CD4
+ T cells exposed to infliximab
in vitro failed to produce IL-17, suggesting that TNF-α is required for Th17 differentiation in BD. To confirm this result, both human and mouse recombinant TNF-α proteins were used. CD4
+ BD T cells exposed to rTNF-α
in vitro promoted IL-17 production, and murine Th17 cells greatly expressed intracellular IL-17 when the T cells were co-cultured with mouse rTNF-α. In addition, fresh T cells from a uveitis patient with BD expressed high levels of the RORγt transcription factor, whereas fresh T cells from a patient being treated with infliximab expressed low levels of RORγt. Similarly, Th17 cell lines from a BD uveitis patient expressed high levels of the Th17-related transcription factor. In contrast, the expression of RORγt was suppressed in Th17 cells cultured with infliximab. These results suggest that anti-TNF-α blockade may prevent the differentiation of Th17 cells. We suspect that these CD4
+ T cells exposed to infliximab may convert into T regulatory (Treg) cells, as described in our previous report [
17]. In fact, CD4
+ T cells can convert into Th17 cells in the presence of IL-6 and TGFβ, but the CD4
+ T cells can also convert into Treg cells through the TGFβ signal in the absence of IL-6 [
23,
34].
As well as soluble TNF-α. infliximab neutralizes membrane-binding TNF-α in addition to suppressing TNF-α production by antigen-presenting cells. When infliximab has been used to treat patients with active rheumatoid arthritis, the population of Treg cells that express forkhead box P3 (Foxp3) has increased [
35,
36]. We previously found that infliximab-induced Treg cells inducibly express Foxp3 through the TGFβ signal and that the Treg cells may provide protection from the inflammatory conditions, which is consistent with the results of Nadkarni
et al. [
36]. Importantly, infliximab-induced Treg cells from BD patients suppressed the activation of target T cells. The infliximab-induced Treg cells produced significant amounts of TGFβ1. Thus, peripherally induced Treg cells may work as an alternative inhibition mechanism for infliximab. It is assumed that treatment with anti-TNF-α antibody promotes the conversion of inflammatory Th17 cells (RORγt+) to Treg cells (Foxp3+) to establish homeostasis in BD patients. In other words, the Th17/Treg balance might be important for the pathogenesis of inflammation in BD, as recently reported by Hamzaoui
et al. [
37,
38].
TNF-α, which functions as a proinflammatory cytokine, is greatly involved in the aggravation of BD, particularly the ocular symptoms [
39‐
41]. TNF-α and other proinflammatory cytokines are produced by monocytes [
39] and T lymphocytes [
40] in BD uveitis patients, and these inflammatory cytokines are critical for the formation of inflammatory lesions, such as ocular lesions. As shown in the current experiments, infliximab significantly suppressed IFN-γ, IL-6 and IL-17 inflammatory cytokines in addition to TNF-α produced by activated CD4
+ T cells from BD patients who have active uveitis. Ocular fluids from BD patients being treated with infliximab did not contain these inflammatory cytokines. These results imply that Th1/Th17 cells, which play a significant role in the immune responses in BD, may disappear in the peripheral lesions including ocular lesions after infliximab therapy.
Th17 cells constitute a third subset of effector helper T cells. The effector functions of Th17 cells are distinct from those of Th1 and Th2 cells [
23,
24]. Th17 cells and IL-17 play a critical role in the pathogenic mechanisms of intraocular inflammation in an animal model of human uveitis in BD [
28‐
30] as well as human uveitis [
26,
27]. Anti-mouse IL-17-blocking antibodies [
42] as well as anti-TNF-α antibody [
33] suppress intraocular inflammation in experimental uveitis models. In the present study, we showed that fresh intraocular T cells from immunized EAU donors had a large population of IL-17
+ cells, suggesting that CD4
+IL-17
+ Th17-type T cells may be associated with the pathogenic mechanisms of intraocular inflammation. We also showed that retinal antigen-specific CD4
+ T cells from EAU produced large amounts of IL-17 in the presence of retinal peptide. Importantly, retinal antigen-specific CD4
+ EAU T cells produced less IL-17 if the T cells were treated with anti-TNF-α blocking antibody. Thus, anti-TNF-α blockade provides protection from intraocular inflammation by Th17-type helper T cells in animal models of BD.
Recently, several investigators reported that active BD was characterized by increased levels of IL-17 as compared to BD in remission or control healthy donors [
4‐
6]. Chi
et al. reported that IL-23 mRNA in PBMCs, IL-23 in serum, and IL-17 production in supernatants of PBMCs were all markedly increased in BD patients who have active uveitis [
6]. Significantly upregulated IL-17-producing T cells were also found in BD patients. They concluded that IL-23 and IL-17 are associated with active ocular inflammation in BD patients. Recent genetic surveys including GWAS have identified IL23R-IL12RB2 and IL10 as BD susceptibility loci [
7,
8]. These recent reports suggest that BD including ocular inflammation is predominated by Th1/Th17-type immune responses. Thus, blocking Th17 differentiation may be a very important treatment strategy in BD.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SS was the principal investigator, designed and performed experiments, and wrote the manuscript. YK performed EAU induction and in vitro experiments. AI performed flow cytometry. YY performed EAU induction. SH performed in vitro experiments. MM designed and conceptualized the study and drafted and edited the manuscript. All authors have read and approved the manuscript for publication.