Shintaro Horie

Inhibition of Th17 differentiation by anti-TNF-alpha therapy in uveitis patients with Behçet's disease

IntroductionThe purpose of this study was to determine whether anti-tumour necrosis factor alpha (anti-TNF-α) antibody, infliximab, can inhibit T helper 17 (Th17) differentiation in uveitis patients who have Behçets disease (BD).MethodsTo measure inflammatory cytokines, ocular fluid samples from BD patients being treated with infliximab were collected. Cluster of differentiation 4 (CD4)+ T cells from BD patients with active uveitis were co-cultured with anti-cluster of differentiation 3/cluster of differentiation 28 (CD3/CD28) antibodies in the presence of infliximab. For the induction of Th17 cells, CD4+ T cells from BD patients were co-cultured with anti-CD3/CD28, anti-interferon-gamma (anti-IFN-γ), anti-interleukin-4 (anti-IL-4), and recombinant proteins such as interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-23 (IL-23), and TNF-α. The BD T cells were co-cultured with infliximab, and the production of interleukin-17 (IL-17) was evaluated by ELISA and flow cytometry, and the expression of retinoid-acid receptor-related orphan receptor gamma t (RORγt) was also evaluated by flow cytometry. In addition, intraocular cells collected from mice with experimental autoimmune uveitis (EAU) were used for the assay with anti-TNF-α blocking antibody.ResultsOcular fluids from active uveitis patients who have BD contained significant amounts of inflammatory cytokines such as IFN-γ, IL-2, TNF-α, IL-6, and IL-17, while ocular fluids from infliximab patients did not contain any inflammatory cytokines. Activated CD4+ T cells from BD patients produced large amounts of TNF-α and IL-17, whereas T cells in the presence of infliximab failed to produce these cytokines. Polarized Th17 cell lines from BD patients produced large amounts of IL-17, and Th17 cells exposed to infliximab had significantly reduced IL-17 production. Polarized BD Th17 cells expressed large amounts of transcription factor RORγt. In contrast, in vitro-treated infliximab Th17 cells expressed less RORγt. Moreover, intraocular T cells from EAU mice had a high population of IL-17+ cells, and retinal antigen-specific T cells from EAU mice produced large amounts of IL-17 in the presence of retinal peptide. However, the EAU T cells produced less IL-17 if the T cells were treated with anti-TNF-α antibody.ConclusionsThese results indicate that anti-TNF-α therapy suppresses effector T-cell differentiation in BD patients with uveitis. Thus, suppression of effector T-cell differentiation by anti-TNF-α therapy may provide protection from severe ocular inflammation in BD.

Retinal Pigment Epithelium-Derived CTLA-2α Induces TGFβ-Producing T Regulatory Cells

T cells that encounter ocular pigment epithelium in vitro are inhibited from undergoing TCR-triggered activation, and instead acquire the capacity to suppress the activation of bystander T cells. Because retinal pigment epithelial (RPE) cells suppress T cell activation by releasing soluble inhibitory factors, we studied whether soluble factors also promote the generation of T regulatory (Treg) cells. We found that RPE converted CD4+ T cells into Treg cells by producing and secreting CTLA-2α, a cathepsin L (CathL) inhibitor. Mouse rCTLA-2α converted CD4+ T cells into Treg cells in vitro, and CTLA-2α small interfering RNA-transfected RPE cells failed to induce the Treg generation. RPE CTLA-2α induced CD4+CD25+Foxp3+ Treg cells that produced TGFβ in vitro. Moreover, CTLA-2α produced by RPE cells inhibited CathL activity in the T cells, and losing CathL activity led to differentiation to Treg cells in some populations of CD4+ T cells. In addition, T cells in the presence of CathL inhibitor increased the expression of Foxp3. The CTLA-2α effect on Treg cell induction occurred through TGFβ signaling, because CTLA-2α promoted activation of TGFβ in the eye. These results show that immunosuppressive factors derived from RPE cells participate in T cell suppression. The results are compatible with the hypothesis that the eye-derived Treg cells acquire functions that participate in the establishment of immune tolerance in the posterior segment of the eye.

Induction of Regulatory T Cells by Infliximab in Behcet's Disease

PURPOSE To determine whether infliximab induces the development of Foxp3+ T regulatory (Treg) cells in patients with Behçets disease. METHODS The subjects were patients with refractory uveitis caused by Behçets disease, Vogt-Koyanagi-Harada disease, or ocular toxoplasmosis and healthy volunteers. Purified CD4+ T cells were obtained from patients with uveitis who were treated with colchicine, cyclosporine, or infliximab. Flow cytometry was used to analyze the expression of Foxp3 on CD4+ T cells. RESULTS Foxp3 was expressed in a small percentage of CD4+ T cells (<5%) from healthy subjects and from patients with active uveitis caused by Behçets disease, Vogt-Koyanagi-Harada disease, or ocular toxoplasmosis. The percentage of Foxp3+ cells among CD4+ T cells was significantly decreased in patients with active uveitis compared with patients with inactive uveitis in the remission stage. Foxp3 was expressed at a similar level in CD4+ T cells from healthy donors, colchicine-treated patients, and cyclosporine-treated patients, whereas infliximab-treated patients expressed much higher percentages of Foxp3+ cells. Patients who had a high population of Foxp3+ cells during infliximab treatment did not experience any subsequent episodes of acute uveitis. On the other hand, patients with a low population of Foxp3+ cells did experience ocular inflammatory episodes. T cells exposed to infliximab in vitro greatly expressed Foxp3 and produced TGFβ, and the Treg cells significantly suppressed the activation of bystander T cells in vitro. CONCLUSIONS Anti-TNF-α therapy may be useful for patients with ocular complications of Behçets disease who have a decreased percentage of peripheral Treg cells.

T-cell suppression by programmed cell death 1 ligand 1 on retinal pigment epithelium during inflammatory conditions.

PURPOSE To determine whether retinal pigment epithelial (RPE) cells can inhibit in vitro T-cell activation during inflammatory conditions. METHODS Primary cultured RPE cells were established from normal C57BL/6 mice. Target bystander T cells were established from normal splenic T cells with anti-CD3 antibodies. T-cell activation was assessed for proliferation by both examining [(3)H]-thymidine incorporation and the production of interferon (IFN)gamma or IL-17, as determined by ELISA. Expression of programed cell death 1 ligand 1 (PD-L1) on RPE or recombinant mouse IFNgamma-pretreated RPE cells was evaluated using oligonucleotide microarray, RT-PCR, immune staining, and flow cytometry. Expression of programed cell death 1 (PD-1)(+) on target T cells was evaluated by flow cytometry. Anti-mouse PD-L1 or PD-L2 neutralizing antibodies or target T cells from PD-1 knockout donors were used for the assay. RESULTS IFNgamma-pretreated RPE greatly suppressed activation of bystander T cells, especially the IFNgamma production by the target T cells (Th1 cells, but not Th17 cells) via direct cell contact. By examining cell surface candidate molecules, IFNgamma-pretreated RPE expressed much higher levels of PD-L1 compared with the control nontreated RPE. Although primary RPE did not express the costimulatory molecule, expression of the molecule was induced on the surface of IFNgamma-pretreated RPE. PD-L1(+) RPE in the presence of IFNgamma selectively suppressed PD-1(+) T-cell activation. IFNgamma-pretreated RPE in the presence of anti-PD-L1 neutralizing antibodies, but not anti-PD-L2, failed to suppress T-cell production of IFNgamma. In addition, these RPE cells failed to suppress the production of IFNgamma by CD4(+) T cells from PD-1 null donors. CONCLUSIONS Suppression of T-cell activation was obtained in cultures only when RPE expressed negative costimulators. Therefore, the authors propose that in vitro, Th1 cytokine-exposed ocular resident cells can express this molecule and it is this expression that causes the suppression of the bystander Th1-type cells.

Evaluation of Characteristic Ocular Signs and Systemic Investigations in Ocular Sarcoidosis Patients

PurposeTo evaluate the diagnostic values of ocular signs and systemic investigations in ocular sarcoidosis, in a retrospective case–control study.MethodsSubjects were 67 consecutive uveitis patients with biopsy-proven sarcoidosis and 111 control patients with other clinical uveitis entities. The predictive values analyzed were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The five ocular signs for ocular sarcoidosis are (1) mutton fat keratic precipitates and iris nodules; (2) nodules at the trabecular meshwork and tent-shaped peripheral anterior synechia; (3) snowball vitreous opacities; (4) nodular periphlebitis, and (5) multiple chorioretinal lesions (active or atrophic) in the peripheral fundus. In addition, the results of the following five systemic investigations were considered: (1) negative tuberculin skin test; (2) elevated serum angiotensin-converting enzyme; (3) elevated serum lysozyme; (4) elevated serum γ-globulin; and (5) bilateral hilar lymphadenopathy on chest X-ray.ResultsThe incidence of all ocular signs and positive results for the systemic investigations were significantly higher in sarcoidosis patients than in controls (P < 0.001). The presence of two or three of the five ocular signs were indicative of a positive finding in the diagnostic parameters. The presence of two positive results among the five systemic investigations showed values higher than 0.800 for all diagnostic parameters.ConclusionsCombinations of the specified ocular signs and the results of systemic investigations can be used for the diagnosis of ocular sarcoidosis. Jpn J Ophthalmol 2007;51:121–126

Role of IL-22- and TNF-α-producing Th22 cells in uveitis patients with Behcet's disease.

Behçet’s disease is a systemic inflammatory disorder with recurrent episodes of oral ulceration, skin lesions, genital ulceration, and intraocular inflammation (uveitis). The intraocular inflammation is strictly associated with Th effector cells. IL-22 is a member of the IL-10 cytokine family that is involved in inflammatory processes. Recently, Th22 cells were identified as a Th cell population that produces IL-22 and TNF-α and are distinct from Th1, Th2, and Th17 cells. In this study, we established Th22-type T cell clones from ocular samples taken from Behçet’s disease patients with active uveitis. These clones produced large amounts of IL-22 and TNF-α but not the Th1 cytokine IFN-γ and the Th17 cytokine IL-17. CD4+ T cells from the peripheral blood of Behçet’s disease patients differentiated into Th22 cells in the presence of IL-6 and TNF-α in vitro. The polarized Th22 cell lines produced large amounts of IL-22, and the polarized Th1 and Th17 cells also produced IL-22. In the presence of anti–TNF-α– and anti–IL-6–blocking Abs, Behçet’s disease Th22-type T cells failed to produce IL-22. In addition, infliximab-pretreated Th22 cells and Th22-type ocular T cells produced less IL-22 and TNF-α. Moreover, IL-22–producing T cells were isolated from mice with experimental autoimmune uveitis, an animal model of Behçet’s disease, and the intraocular T cells from uveitis models produced large amounts of IL-22 in the presence of retinal Ags. Our results suggest that inflammatory cytokines IL-22 and TNF-α may play a key role in the ocular immune response in Behçet’s disease.

Human Corneal Endothelial Cells Expressing Programmed Death-Ligand 1 (PD-L1) Suppress PD-1+ T Helper 1 Cells by a Contact-Dependent Mechanism

PURPOSE This study was designed to determine whether human corneal endothelial (HCE) cells could regulate the activation of bystander T cells in vitro. METHODS HCE cell lines were established from primary HCE cells. Target-activated T cells were used allogeneic T cells and Jurkat T-cell lines. As an additional target, T-cell clones from uveitis patients were established from aqueous humor via a limiting dilution. T-cell activation was assessed for proliferation by [(3)H]-thymidine incorporation, carboxyfluorescein succinimidyl ester incorporation, or IFNgamma production. Expression of co-stimulatory molecules on IFNgamma-treated corneal endothelial and non-treated cells was evaluated by flow cytometry, RT-PCR, or immunohistochemistry. Expression of co-stimulatory receptors on target T cells was evaluated by flow cytometry. Blocking antibodies was used to abolish the HCE-inhibitory function. RESULTS HCE cells suppressed both in vitro proliferation and IFNgamma production by CD4(+) T cells via a cell contact-dependent mechanism. HCE constitutively expressed co-stimulatory molecules programmed death-ligand 1 (PD-L1) and PD-L2, and their expression was enhanced by IFNgamma. HCE efficiently inhibited the proliferation of Th1 cells that overexpressed PD-1 among various activated T-cell lines and clones established from patients with uveitis or corneal endotheliitis. A neutralizing mAb for PD-L1, but not PD-L2, blocked the suppressive effect of HCE on Th1 cells. CONCLUSIONS HCE can impair the effector functions and activation of Th1 infiltrating CD4(+) T cells via the PD-1/PD-L1 interaction. The data support the hypothesis that corneal endothelium may contribute to maintenance of the privileged immune status of the anterior chamber of the eye by inducing peripheral immune tolerance.

Diagnosis of bacterial endophthalmitis by broad-range quantitative PCR

Aim To measure the bacterial genome in ocular fluids and to analyse the clinical relevance of infectious endophthalmitis. Methods Nineteen ocular fluid samples (eight aqueous humour and 11 vitreous fluid samples) were collected from 19 patients with suspected bacterial endophthalmitis. Fifty ocular samples from uveitis patients were also collected along with 40 samples from patients without ocular inflammation and used as controls. Bacterial ribosomal DNA (16S rDNA) was measured by a quantitative PCR assay. Results Bacterial 16S rDNA was detected in patients with clinically suspected bacterial endophthalmitis (18/19, 95%). With the exception of one case, high copy numbers of bacterial DNA were detected (1.7×103–1.7×109 copies/ml) in these patients. There were 10 samples (53%) with positive bacterial cultures while there were nine samples (47%) with positive Gram-staining. Real-time PCR detected bacterial 16S rDNA in three (6%) of the 50 samples from the control uveitis patients. In addition, none of the samples from the control patients without intraocular inflammation were positive. Conclusions Quantitative broad-range PCR of bacterial 16S rDNA is a useful tool for diagnosing bacterial endophthalmitis.

Acquisition of T Regulatory Function in Cathepsin L-Inhibited T Cells by Eye-Derived CTLA-2α during Inflammatory Conditions

Pigment epithelium isolated from the eye possesses immunosuppressive properties such as regulatory T (Treg) cell induction; e.g., cultured retinal pigment epithelium (RPE) converts CD4+ T cells into Treg cells in vitro. RPE constitutively expresses a novel immunosuppressive factor, CTLA-2α, which is a cathepsin L (CathL) inhibitor, and this molecule acts via RPE to induce Treg cells. To clarify CTLA-2α’s role in the T cell response to RPE in ocular inflammation, we used the experimental autoimmune uveitis (EAU) animal model to examine this new immunosuppressive property of RPE. In EAU models, TGF-β, but not IFN-γ inflammatory cytokines, promotes the up-regulation of the expression of CTLA-2α in RPE. Similarly, CTLA-2α via RPE was able to promote TGF-β production by the CD4+ T cells. The RPE-exposed T cells (RPE-induced Treg cells) greatly produced TGF-β and suppressed bystander effector T cells. There was less expression of CathL by the RPE-exposed T cells, and CathL-inhibited T cells were able to acquire the Treg phenotype. Moreover, CathL-deficient mice spontaneously produced Treg cells, with the increase in T cells potentially providing protection against ocular inflammation. More importantly, CD4+ T cells from EAU in CathL knockout mice or rCTLA-2α from EAU animals were found to contain a high population of forkhead box p3+ T cells. In both EAU models, there was significant suppression of the ocular inflammation. These results indicate that RPE secretes CTLA-2α, thereby enabling the bystander T cells to be converted into Treg cells via TGF-β promotion.

Association of varicella zoster virus load in the aqueous humor with clinical manifestations of anterior uveitis in herpes zoster ophthalmicus and zoster sine herpete

Aim: To investigative whether clinical manifestations of anterior uveitis are associated with the viral load of varicella zoster virus (VZV) in the aqueous humor in patients with herpes zoster ophthalmicus (HZO) and zoster sine herpete (ZSH). Methods: After informed consent was given, an aliquot of aqueous humor was collected from patients with VZV anterior uveitis (n = 8). Genomic DNA of the human herpes viruses was measured in the aqueous humor by two PCR assays: a qualitative multiplex PCR and a quantitative real-time PCR. Results: All patients had unilateral acute anterior uveitis with high intraocular pressure, mutton fat keratic precipitates with some pigmentation, and trabecular meshwork pigmentation. Multiplex PCR demonstrated VZV genomic DNA in all of the samples, but not in other human herpes virus samples (human simplex virus types 1 and 2, Epstein–Barr virus, cytomegalovirus and human herpes virus types 6, 7 and 8). Real-time PCR revealed a high copy number of VZV DNA in the aqueous humor. After the initial onset of anterior uveitis, iris atrophy and distorted pupil with paralytic mydriasis developed. The intensity of iris atrophy and pupil distortion, but not ocular hypertension, correlated with the viral load of VZV in the aqueous humor. Conclusion: VZV viral load in the aqueous humor correlated significantly with damage to the iris (iris atrophy and pupil distortion) in patients with HZO and ZSH.