Ayano Imai

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.

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.

Comparison of the ocular characteristics of anterior uveitis caused by herpes simplex virus, varicella-zoster virus, and cytomegalovirus

PurposeTo compare the clinical characteristics of anterior uveitis (AU) caused by herpes simplex virus (HSV), varicella-zoster virus (VZV), or cytomegalovirus (CMV).MethodsThe medical records were reviewed of 46 patients whose diagnoses were based on their clinical characteristics [e.g., unilateral involvement, presence of keratic precipitates (KPs), and elevation of intraocular pressure (IOP)] and on PCR detection of herpes virus DNA in the aqueous humor. The demographics, chief complaints, and clinical characteristics of the three types of herpetic AU were compared.ResultsOf the 46 patients with AU, eight had HSV-AU, 20 had VZV-AU, and 18 had CMV-AU. HSV-AU and VZV-AU shared common features, i.e., a relatively acute disease process and the presence of large KPs. Among the three groups of patients, the characteristic features of those with VZV-AU were severe intraocular inflammation, as shown by severe aqueous flare, highest viral load in the aqueous humor, and presence of segmental iris atrophy. In comparison, patients with CMV-AU had the mildest intraocular inflammation, lowest corneal endothelial cell density, and highest IOP.ConclusionsAlthough the AU caused by each of the three types of herpes viruses has a number of common features, each disease also has distinct features that should facilitate an accurate diagnosis.

Immunosuppressive properties of regulatory T cells generated by incubation of peripheral blood mononuclear cells with supernatants of human RPE cells.

PURPOSE To determine whether supernatants of human retinal pigment epithelium (RPE) cells can convert CD4⁺ T cells into regulatory T cells (Tregs) under Treg-induction conditions in vitro and in vivo. METHODS Peripheral blood mononuclear cells were cocultured with supernatants from TGFβ2-pretreated human RPE lines on anti-CD3-coated plates. Cells were then separated with a CD4⁺CD25⁺ Treg isolation kit and cultured with supernatants from RPE, anti-CD3/CD28 antibodies, high-dose IL-2, and TGFβ2. By flow cytometry sorting, CD25⁺CD45RA⁻ Tregs were separated. Expressions of CD25(high), Foxp3, CD152, and TNFRSF 18 on Tregs were analyzed by flow cytometry. Cytokine production by Tregs was measured by ELISA. Proliferation of target T cells was assessed by [³H]thymidine incorporation or CFSE incorporation. In addition, mouse RPE-induced Tregs were used for the in vitro assay and in vivo experimental autoimmune uveitis (EAU) models. RESULTS Human RPE-induced Tregs expressed higher levels of the Treg markers CD25(high), Foxp3, CD152, and TNFRSF 18. In addition, RPE-induced Tregs included significant numbers of CD4⁺CD25(high)Foxp3(high)CD45RA⁻ active effector Tregs that significantly suppressed the activation of Th1/Th17 cell lines, indicating that they have immunosuppressive properties. Furthermore, CD4⁺CD25(low)Foxp3(low)CD45RA⁻ nonsuppressing cytokine-secreting T cells were removed from the in vitro-manipulated Treg population. Administration of mouse RPE-induced Tregs significantly suppressed ocular inflammation in mice with EAU. In addition, the Tregs suppressed retinal antigen-specific T cells in vitro. CONCLUSIONS It is hoped that through the data provided in this study that Tregs might become useful as individualized therapeutic agents for ocular autoimmune diseases.

Mature Dendritic Cell Suppression by IL-1 Receptor Antagonist on Retinal Pigment Epithelium Cells

PURPOSE To determine whether retinal pigment epithelial (RPE) cells can inhibit mature dendritic cells (mDCs). METHODS Cultured RPE cells were established from C57BL/6 mice. DCs were established from bone marrow cells of normal mice, and mDCc were induced by culture in medium containing granulocyte macrophage-colony-stimulating factor (GM-CSF) and IL-4 in the presence of lipopolysaccharide and TNF-α. Activation of mDCs was assessed by a proliferation assay and ELISA to measure the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-12p40). Expression of major histocompatibility complex (MHC) class II, CD11c, and costimulatory molecules such as CD80, CD86, programmed cell death 1 ligand 1 (PD-L1), and PD-L2 on mDCs or RPE-exposed mDCs was evaluated by immune staining and flow cytometry. Production of IL-1 receptor antagonist (IL-1Ra) by RPE cells was evaluated by oligonucleotide microarray or ELISA. Anti-IL-1Ra neutralizing antibodies or RPE cells from IL-1Ra knockout donors were used for the assay. RESULTS Cultured RPE cells greatly suppressed the activation of mDCs, especially the production of pro-inflammatory cytokines, and the expression of cell-surface molecules. Moreover, RPE cells significantly suppressed mixed lymphocyte reactions by mDCs. In an examination of immunoregulatory candidate molecules, RPE cells expressed much higher levels of IL-1Ra as compared with control cells, and RPE cells pretreated with recombinant TNF-α and/or IL-1β produced high levels of IL-1Ra. RPE cells in the presence of anti-IL-1Ra antibodies, but not other candidate factors, failed to suppress activation by mDCs. In addition, RPE cells from IL-1Ra null donors failed to suppress mDC activation. CONCLUSIONS Our results suggest that ocular resident cells can produce pro-inflammatory cytokine antagonist that suppresses antigen-presenting cell activation.

Suppression of IL-22-producing T helper 22 cells by RPE cells via PD-L1/PD-1 interactions.

PURPOSE To determine whether RPE cells can suppress a novel T helper subset, the Th22 cells, via costimulatory interactions. METHODS Primary RPE cells were established from normal C57BL/6 mice. The target CD4(+) Th22 cells from spleen cells in wild-type control or knockout donors were used. T cell activation was assessed by examining BrdU incorporation (proliferation) and cytokine production. Expression of costimulatory molecules on RPE cells and expression of costimulatory receptors on target Th22 cells were evaluated by flow cytometry. Neutralizing antibodies were used to abolish the suppression function. In addition, human RPE cells and target Th22 cells induced from human CD4(+) cells were also used in similar experiments. RESULTS Cultured RPE cells significantly suppressed activation of target Th22 cells (e.g., T cell proliferation and IL-22 production). Moreover, human RPE cells suppressed Th22 cell lines and T cell clones established from active uveitis patients. Although cultured RPE cells expressed various costimulatory molecules including programmed cell death 1 ligand 1 (PD-L1), only PD-L1 on the RPE cells was actually delivered to the target Th22 cells. Th22 cells greatly express programmed cell death 1 (PD-1), and RPE cells failed to suppress IL-22 expression by target Th22 cells from PD-1 knockout donors. In addition, if neutralizing antibodies for PD-L1 were cocultured with RPE cells, Th22 suppression was impaired. CONCLUSIONS RPE cells express PD-L1 costimulatory molecules and suppress bystander Th22-type PD-1(+) bystander T cells through negative costimulatory interactions.

Inhibitory effect of corneal endothelial cells on IL-17-producing Th17 cells.

Aim To determine whether cultured corneal endothelial (CE) cells suppress interleukin 17 (IL-17)-producing effector T cells in vitro. Methods CE cell lines established from a normal mouse were used. Target bystander T cells were established from normal splenic T cells with anti-CD3 antibodies. Production of IL-17 by target T cells was evaluated by ELISA, flow cytometry and quantitative PCR. To abolish the CE-inhibitory function, transforming growth factor β (TGFβ)-small interfering RNA-transfected CE cells or transwell membrane inserts, which block cell-to-cell contact, were used. Results Cultured CE cells greatly suppressed the activation of bystander target cells (pan-T, CD4 T, CD8 T, and B cells) in vitro, particularly inflammatory cytokine production by CD4 cells. Cultured CE cells significantly suppressed IL-17-producing T cells and fully suppressed polarised T helper 17 (Th17) cell lines that are induced by Th17-associated differentiation factors. However, CE cells failed to suppress Th17 cells if the CE cell lines were pretreated with TGFβ small interfering RNA or if direct contact with T cells was blocked with transwell membrane inserts. Conclusion CE cells impair the effector functions and activation of IL-17-producing helper T cells in a cell-contact-dependent mechanism. Thus, corneal endothelium may contribute to the maintenance of the privileged immune status in the eye by inducing peripheral immune tolerance.

Development of Extranodal NK/T-cell Lymphoma Nasal Type in Cerebrum Following Epstein-Barr Virus-positive Uveitis

A 74-year-old woman developed bilateral uveitis with high Epstein-Barr virus (EBV) DNA load in the vitreous fluid without lymphoma cells. Four years after the onset, T2-weighted contrast-enhanced MRI revealed hyperintense lesions in the right occipital and parietal lobe. A biopsy resulted in the diagnosis of extranodal NK/T-cell lymphoma nasal type (ENKL). The repeat region of LMP1, an EBV gene, detected in the brain lesion was identical to that detected in the vitreous fluid. ENKL of the central nervous system is quite rare, and the pathogenesis has not been determined. The lymphoma in this case might have been closely associated with the EBV-positive uveitis.

Presumed toxoplasmic central retinal artery occlusion and multifocal retinitis with perivascular sheathing

Central retinal artery occlusion (CRAO) and multifocal retinitis with perivascular sheathing are rare in ocular toxoplasmosis. We report a case of toxoplasmic CRAO and multifocal retinitis with perivascular sheathing. A healthy 83-year-old male developed left panuveitis. Funduscopic examination of the left eye showed a swollen optic disc and sheathing of the retinal artery with a dense vitreous haze and a white retinal lesion. Serum anti-toxoplasma antibodies were positive in a latex agglutination assay. Vitrectomy was performed to improve visualization of the retinal lesions and for examination of causative microorganisms. A postoperative fundus examination revealed CRAO with optic disc involvement and multifocal retinitis with perivascular sheathing. Qualitative multiplex polymerase chain reaction detected the Toxoplasma gondii B1 gene in ocular fluid from both the aqueous and vitreous humor. The presumed diagnosis of ocular toxoplasmosis was made and treatment was started with prednisone and acetylspiramycin with subsequent improvement. Two months later, the patient developed active retinochoroiditis in the left eye. After 6 weeks of anti-toxoplasma therapy, the disease involuted. Retinal vascular occlusions and multifocal retinitis with perivascular sheathing are rare in toxoplasmosis. This is the first case report of toxoplasmic CRAO and multifocal retinitis with perivascular sheathing. The diagnosis of ocular toxoplasmosis should be considered in patients with retinal artery occlusions and multifocal retinitis with perivascular sheathing associated with inflammation.