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
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
]. 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.
]. 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.
TNF-α, which functions as a proinflammatory cytokine, is greatly involved in the aggravation of BD, particularly the ocular symptoms [39
]. 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
]. 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
] as well as human uveitis [26
]. 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+
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
]. 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
]. 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.