Functionally distinct IFN-γ+ IL-17A+ Th cells in experimental autoimmune uveitis: T-cell heterogeneity, migration, and steroid response.

Immunopathogenic roles for both Th1 (CD4+ IFN-γ+ ) and Th17 (CD4+ IL-17A+ ) cells have been demonstrated in experimental autoimmune uveitis (EAU). However, the role for Th17/Th1 (CD4+ T cells co-expressing IFN-γ and IL-17A) cells in EAU is not yet understood. Using interphotoreceptor retinoid-binding protein peptide-induced EAU in mice, we found increased levels of Th17/Th1 cells in EAU retinae (mean 9.6 ± 4.2%) and draining LNs (mean 8.4 ± 3.9%; p = 0.01) relative to controls. Topical dexamethasone treatment effectively reduced EAU severity and decreased retinal Th1 cells (p = 0.01), but had no impact on retinal Th17/Th1 or Th17 cells compared to saline controls. Using in vitro migration assays with mouse CNS endothelium, we demonstrated that Th17/Th1 cells were significantly increased within the migrated population relative to controls (mean 15.6 ± 9.5% vs. 1.9 ± 1.5%; p = 0.01). Chemokine receptor profiles of Th17/Th1 cells (CXCR3 and CCR6) did not change throughout the transendothelial migration process and were unaffected by dexamethasone treatment. These findings support a role for Th17/Th1 cells in EAU and their resistance to steroid inhibition suggests the importance of targeting both Th17 and Th17/Th1 cells for improving therapy.

Small-molecule antagonist of VLA-4 (GW559090) attenuated neuro-inflammation by targeting Th17 cell trafficking across the blood-retinal barrier in experimental autoimmune uveitis.

BACKGROUND: The integrin VLA-4 (α4ß1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4ß1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets. METHODS: Mice (female; B10.RIII or C57Bl/6; aged 6-8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry. RESULTS: There was a significant reduction in clinical and histological scores in GW10- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers. CONCLUSIONS: This α4ß1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4ß1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.

Profibrotic phenotype of conjunctival fibroblasts from mucous membrane pemphigoid.

Ocular mucous membrane pemphigoid is an immunobullous disease in which excessive conjunctival fibrosis causes blindness, and the pathogenesis of scarring is incompletely understood. To establish whether profibrotic fibroblasts with an altered phenotype exist in ocular mucous membrane pemphigoid, we compared the functional characteristics of pemphigoid conjunctival fibroblasts to normal conjunctival fibroblasts with respect to cell division; migration; collagen contraction; matrix metalloproteinase, secretion of collagen and chemokines; and myofibroblast differentiation. We found that pemphigoid fibroblasts showed increased cell division (P = 0.01), increased migration in serum-free medium (72 ± 18 migrated cells versus 33 ± 11, P = 0.04), increased collagen contraction in the presence of 10 ng/ml tumor necrosis factor-α, increased collagen type I secretion (P = 0.03), increased secretion of matrix metalloproteinase-3 (P = 0.03), and increased secretion of eotaxin in response to interleukin-13 (P = 0.04). Differences between pemphigoid and normal conjunctival fibroblasts with respect to collagen contraction and MMP secretion in the presence of interleukin-13 were also observed. Together, these findings indicate that pemphigoid conjunctival fibroblasts have a profibrotic phenotype that is maintained in vitro. No differences between pemphigoid fibroblasts obtained from acutely inflamed versus clinically uninflamed conjunctiva were observed. Developing effective antifibrotic therapies will require understanding of the mechanisms that both induce and maintain the profibrotic phenotype.

Conjunctival interleukin-13 expression in mucous membrane pemphigoid and functional effects of interleukin-13 on conjunctival fibroblasts in vitro.

Interleukin-13 (IL-13) is the dominant effector cytokine of fibrosis in pulmonary and liver disease. Excessive conjunctival fibrosis in the immunobullous disease ocular mucous membrane pemphigoid (MMP) causes blindness; the pathogenesis of scarring in this disease is incompletely understood. To determine whether IL-13 is involved in conjunctival fibrosis in MMP, we studied the expression of IL-13 in ocular MMP patients before and after systemic immunosuppression and examined the effects of IL-13 on normal human conjunctival fibroblasts. We found high stromal cell expression of IL-13 in active ocular MMP by immunohistochemistry; 80% of these cells were CD3-positive T cells. Following immunosuppression, in clinically uninflamed, treated, ocular MMP patients, the number of IL-13 positive cells was significantly reduced, but this was still fourfold greater than in normal conjunctiva. IL-13 stimulated collagen lattice contraction and migration, and decreased production of mmp-3 and mmp-10 by human conjunctival fibroblasts. The addition of T cell culture supernatant to IL-13 synergistically augmented fibroblast migration. IL-13 also up-regulated surface expression of HLA-DR, CD80, CD40, and CD154 by conjunctival fibroblasts, suggesting a potential mechanism for fibroblast-T cell cross talk, via which fibroblasts may actively engage in perpetuating chronic inflammation and continued fibrosis. Together, these findings suggest that IL-13 is involved in conjunctival fibrosis in MMP, and that IL-13 has both profibrotic and pro-inflammatory effects on human conjunctival fibroblasts.

Cytokine responses by conjunctival epithelial cells: an in vitro model of ocular inflammation.

OBJECTIVES: We examined the differential secretion of cytokines by a conjunctival epithelial cell line in response to proinflammatory cytokines to identify the potential contributions during ocular surface inflammation. METHODS: A conjunctival epithelial cell line was exposed to IFN-gamma, TNF-alpha, IL-4, or IL-13, and cytokine production was determined in supernatants at different times after exposure. Cell apoptosis was measured by flow cytometry. RESULTS: TNF-alpha induced the greatest effect on cytokine secretion, which was time-dependent. TNF-alpha-stimulated secretion of IL-12p40 was significantly increased by 30 min; GM-CSF, MCP-1, IL-6, IL-7, IL-8, and RANTES were significantly increased by 2 h, and IFN-gamma and IL-1alpha by 24 h. After 48 h, TNF-alpha also induced a significant increase in IL-1beta, IL-3, and IP-10 secretion. IFN-gamma significantly enhanced IP-10 and RANTES secretion after 48 h of exposure. Following IL-4 treatment there was a significant increase in eotaxin-1 after 24h, and IL-12p40 and IL-3 after 48 h. IL-13 significantly increased the secretion of eotaxin-1 after 24 h, and IL-8 after 48 h. CONCLUSION: Our results suggest that conjunctival epithelial cells are an important source of cytokines and chemokines that are regulated by proinflammatory cytokines and may play an important role in ocular surface inflammation.

Clinical Remission of Sight-Threatening Non-Infectious Uveitis Is Characterized by an Upregulation of Peripheral T-Regulatory Cell Polarized Towards T-bet and TIGIT.

Background: Non-infectious uveitis can cause chronic relapsing and remitting ocular inflammation, which may require high dose systemic immunosuppression to prevent severe sight loss. It has been classically described as an autoimmune disease, mediated by pro-inflammatory Th1 and Th17 T-cell subsets. Studies suggest that natural immunosuppressive CD4+CD25+FoxP3+ T-regulatory cells (Tregs) are involved in resolution of inflammation and may be involved in the maintenance of clinical remission. Objective: To investigate whether there is a peripheral blood immunoregulatory phenotype associated with clinical remission of sight-threatening non-infectious uveitis by comparing peripheral blood levels of Treg, Th1, and Th17, and associated DNA methylation and cytokine levels in patients with active uveitic disease, control subjects and patients (with previously active disease) in clinical remission induced by immunosuppressive drugs. Methods: Isolated peripheral blood mononuclear cells (PBMC) from peripheral blood samples from prospectively recruited subjects were analyzed by flow cytometry for CD3, CD4, FoxP3, TIGIT, T-bet, and related orphan receptor γt. Epigenetic DNA methylation levels of FOXP3 Treg-specific demethylated region (TSDR), FOXP3 promoter, TBX21, RORC2, and TIGIT loci were determined in cryopreserved PBMC using a next-generation sequencing approach. Related cytokines were measured in blood sera. Functional suppressive capacity of Treg was assessed using T-cell proliferation assays. Results: Fifty patients with uveitis (intermediate, posterior, and panuveitis) and 10 control subjects were recruited. The frequency of CD4+CD25+FoxP3+ Treg, TIGIT+ Treg, and T-bet+ Treg and the ratio of Treg to Th1 were significantly higher in remission patients compared with patients with active uveitic disease; and TIGIT+ Tregs were a significant predictor of clinical remission. Treg from patients in clinical remission demonstrated a high level of in vitro suppressive function compared with Treg from control subjects and from patients with untreated active disease. PBMC from patients in clinical remission had significantly lower methylation levels at the FOXP3 TSDR, FOXP3 promoter, and TIGIT loci and higher levels at RORC loci than those with active disease. Clinical remission was also associated with significantly higher serum levels of transforming growth factor ß and IL-10, which positively correlated with Treg levels, and lower serum levels of IFNγ, IL-17A, and IL-22 compared with patients with active disease. Conclusion: Clinical remission of sight-threatening non-infectious uveitis has an immunoregulatory phenotype characterized by upregulation of peripheral Treg, polarized toward T-bet and TIGIT. These findings may assist with individualized therapy of uveitis, by informing whether drug therapy has induced phenotypically stable Treg associated with long-term clinical remission.

Tear Cytokine Levels in Contact Lens Wearers With Acanthamoeba Keratitis.

PURPOSE: To determine differences in key tear film cytokines between mild and severe cases of acanthamoeba keratitis (AK) and control contact lens (CL) wearers. METHODS: This was a prospective study of CL wearers with AK attending Moorfields Eye Hospital and control CL wearers from the Institute of Optometry, London. Basal tear specimens were collected by 10-µL capillary tubes (BLAUBRAND intraMark, Wertheim, Germany), and tear protein levels were measured with a multiplex magnetic bead array (Luminex 100; Luminex Corporation, Austin, TX) for cytokines interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-17A, IL-17E, IL-17F, IL-22, and interferon gamma and with enzyme-linked immunosorbent assay (Abcam, Cambridge, United Kingdom) for CXCL2. Severe cases of AK were defined as having active infection for over 12 months and at least 1 severe inflammatory event. RESULTS: One hundred and thirty-two tear samples were collected from a total of 61 cases (15 severe and 46 mild-moderate) and 22 controls. IL-8, part of the Toll-like receptor 4 cytokine cascade, was found to be expressed at a detectable level more often in cases of AK than in control CL wearers (P = 0.003) and in higher concentrations in severe cases than in milder forms of the disease (z = -2.35). IL-22, part of the IL-10 family, and a proinflammatory Th17 cytokine, was detected more often in severe cases than in milder forms of AK (P < 0.02). CONCLUSIONS: Profiling patients with AK during disease shows differences in cytokine levels between severe and milder disease that may inform clinical management. The Toll-like receptor 4 and IL-10/Th17 inflammatory pathways should be included in further investigations of this disease.

Multiplex cytokine detection versus ELISA for aqueous humor: IL-5, IL-10, and IFNgamma profiles in uveitis.

PURPOSE: The purpose of this study was to determine levels of IL-2, -4, -5, -10, TNF-alpha, and IFN-gamma in aqueous humor (AH) from patients with active panuveitis, anterior uveitis (AU), and noninflammatory controls by using a flow cytometric mutiplex array (CBA) and to compare with results from ELISA. METHODS: Pooled normal AH was spiked with six cytokines at decreasing concentrations for evaluating the CBA. AH was also obtained from 10 controls (cataract patients) and 36 patients with active uveitis. Cell-free supernatants were added to a cocktail of capture beads and detector antibodies or to antibody-coated wells for CBA and ELISA determination, respectively. RESULTS: CBA demonstrated greater sensitivity for detecting IL-4, IL-10, and TNF-alpha than with ELISA. Increased IFN-gamma was detected in both AU and panuveitis groups compared with controls (P < 0.01). IL-10 was higher in the panuveitis group on steroids (P < 0.01). IL-5 was detected in the control (P < 0.01) and AU groups (P < 0.05) but was undetectable in the panuveitis group (n = 10). Correlations between IFN-gamma and IL-10 were found in all uveitis groups (P < 0.01) but not in controls, whereas TNF-alpha correlations with IL-4/IFN-gamma were obtained in controls but not in the uveitis groups (P < 0.01). CONCLUSIONS: It was possible to measure cytokines titrated into normal AH specimens by CBA, and a greater number of cytokines were detected with increased sensitivity than with ELISA. Elevated IFN-gamma in active uveitis and decreased IL-5 in posterior uveitis suggest Th1 polarity is more marked, with greater uveal tract involvement. The increased IL-10 in the steroid treated group suggests glucocorticoid-induced IL-10 upregulation.

Statin Modulation of Human T-Cell Proliferation, IL-1ß and IL-17 Production, and IFN-γ T Cell Expression: Synergy with Conventional Immunosuppressive Agents.

HMG-CoA reductase inhibitors (statins) have been demonstrated to be immunomodulatory for human immune-mediated disease and in experimental models. The aim of this study was to compare statin-mediated immunosuppressive effects on human T-cell responses in vitro with those of conventional immunosuppressives (dexamethasone, cyclosporin A (CsA), mycophenolate, and rapamycin). Statins (atorvastatin, lovastatin, and simvastatin) were investigated for their modulatory effects on human PBMC viability, cytokine profiles, and T-cell proliferation. At concentrations that inhibited anti-CD3/28-stimulated T-cell proliferation (P < 0.01), simvastatin significantly decreased intracellular CD4(+) T-cell expression of IFN-γ (P < 0.01) to levels similar to those induced by conventional immunosuppressives. Atorvastatin and lovastatin also decreased IFN-γ expression, although to a lesser degree (P < 0.05). All three statins reduced levels of IL-17 production (P < 0.01). However, in response to anti-CD3/28 stimulation, simvastatin significantly upregulated IL-1ß production (P < 0.05). The profile of cytokines produced in response to anti-CD3/28 stimulation was similar when both atorvastatin and dexamethasone were added as compared with dexamethasone alone, suggesting that atorvastatin can synergise with dexamethasone with respect to immunomodulation of cytokines. This data supports the hypothesis of selective statin-mediated immunomodulatory effects on human immune cells.

IL6 and the human limbal stem cell niche: a mediator of epithelial-stromal interaction.

The corneal epithelium is maintained by the limbal epithelial stem cells (LESCs). In this study, an in vitro model is proposed for the investigation of cell-cell interactions involving LESC maintenance. Imaging of the limbal niche demonstrated close spatial arrangement between basal limbal epithelial cells within putative LESC niche structures and fibroblasts in the stroma. Interactions of the human limbal epithelial (HLE) cells and mitotically active human limbal fibroblasts (HLF) were studied for the first time in a serum-free in vitro model that simulated aspects of the limbal niche microenvironment. HLE cocultured in a ratio 3:1 with HLF exhibited enhanced expression of the putative stem cell markers ABCG2 and p63α and holoclones were preserved as shown by colony-forming efficiency assays, clonal analysis, and colony characterisation. Interleukin 6 (IL6) was found to be up-regulated in the 3.1SF system when compared to the HLE culture with growth-arrested fibroblasts and serum (gold standard system, GS). IL6 caused a time-dependent phosphorylation of STAT3 in HLE cells. STAT3 and IL6 inhibition in 3.1SF cultures significantly reduced HLE colony-forming efficiency, suggesting a previously undetected STAT3-mediated involvement of IL6 in the maintenance of HLE cells in a progenitor-like state.

Tumor necrosis factor-alpha in ocular mucous membrane pemphigoid and its effect on conjunctival fibroblasts.

PURPOSE: First, to determine whether tumor necrosis factor-(TNF)-alpha is expressed in the conjunctiva of ocular mucous membrane pemphigoid (MMP) and the consequences of systemic immunosuppressive treatment on this expression. Second, to investigate the in vitro effects of TNFalpha on human conjunctival fibroblasts. METHODS: The expression of TNFalpha in conjunctival tissues of patients with actively inflamed ocular MMP (n = 10), patients with clinically noninflamed ocular MMP after systemic immunosuppressive treatment (n = 10), and normal subjects (n = 10) was studied by immunohistochemistry. The effect of TNFalpha on functional assays of human conjunctival fibroblast activity were investigated, including migration, collagen lattice contraction, matrix metalloproteinase (mmp), and tissue inhibitor of matrix metalloproteinase (timp) secretion, proliferation, and surface expression of HLA-DR, ICAM, CD80, CD86, CD40, CD40-ligand. RESULTS: In active ocular MMP, TNFalpha is expressed by a large number of stromal infiltrating cells (234 cells/mm(2)), and although the level of stromal TNFalpha expression is significantly reduced after immunosuppressive treatment (90 cells/mm(2)), these levels are still significantly elevated compared with normal conjunctiva (10 cells/mm(2), P < 0.05). TNFalpha stimulates increased migration by conjunctival fibroblasts (P < 0.001), increased production of mmp-9 (P = 0.01), decreased production of timp-2 (P = 0.01) and timp-4 (P = 0.04), and upregulated expression of CD40 and ICAM (P = 0.04). No significant effects of TNFalpha on fibroblast proliferation or collagen lattice contraction were detected. CONCLUSIONS: Increased conjunctival expression of TNFalpha in ocular MMP suggests that systemic TNFalpha antagonists are likely to be effective in controlling severe disease unresponsive to conventional systemic immunosuppression. Residual TNFalpha expression persists in clinically noninflamed disease. TNFalpha appears to have profibrotic and proinflammatory effects on human conjunctival fibroblasts.

Ocular anti-allergic compounds selectively inhibit human mast cell cytokines in vitro and conjunctival cell infiltration in vivo.

BACKGROUND: Conjunctival mast cells (MCs) are important effector cells in seasonal allergic conjunctivitis, via histamine and cytokine secretion. Several new anti-allergic eye drops stabilize MCs and block histamine receptors, but their anti-inflammatory effects are unclear. OBJECTIVE: Anti-allergic drugs were compared for their anti-inflammatory effects in an in vitro model of human MC activation and in an experimental murine model of allergic conjunctivitis. METHODS: Human cord blood stem cell-derived (CBMC) and conjunctival biopsy-derived MCs were stimulated via FcepsilonRI, degranulation and histamine release were assayed at 1 h and cytokine secretion at 24 h using multiplex arrays. Mice sensitized to short ragweed pollen were given anti-allergics topically before allergen challenge, and conjunctival immuno-staining was performed at 24 h. RESULTS: After a 1 h stimulation, 80% of the CBMC had degranulated and secreted histamine (27.9+/-4.7 ng/10(6) cells; P<0.05). Pre-treatment by all drugs significantly reduced histamine and TNF-alpha, whereas IL-5, IL-8, IL-10 and TNF-beta profiles were differentially decreased. For conjunctival biopsy-derived cultures (n=11), FcepsilonR1 stimulation increased histamine, TNF-alpha, TNF-beta, IL-5 and IL-8 levels and the production of IL-5, IL-6 (P<0.05), histamine and IL-8 (P<0.01) was inhibited by epinastine. In vivo, epinastine and olopatadine pre-treatment significantly reduced the clinical scores and eosinophil numbers (n=6; P<0.05) while epinastine also reduced neutrophils (P<0.02). CONCLUSION: Differential effects on MC cytokine inhibition were observed, with epinastine inhibiting MC secretion of IL-5, IL-8, IL-10 and conjunctival neutrophil infiltration. The anti-allergic drugs have anti-histamine and mast-cell stabilizing properties but might differ in clinical improvement depending on the individual and the cytokines involved.

Normalized CD8+ but not CD4+ lymphocyte IL-2 expression is associated with early treatment with highly active antiretroviral therapy.

CD4+ and CD8+ lymphocyte cytokine production in patients with HIV/AIDS and Controls, in response to stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin was assessed using single cell flow cytometric methods. Sixty-eight patients with HIV were divided into those on no antiretroviral therapy and those on highly active antiretroviral therapy (HAART). Patients on HAART were analyzed further on the basis of gender, ethnicity, viral load (> or 100 or <100 cells/mm(3)) and CD4 count (>200 or <200 cells/mm(3)). Interferon gamma (IFNgamma) expression by CD4+ and CD8+ lymphocytes was elevated in HIV-infected groups as compared to Controls. This elevation was statistically significant for patients on HAART but not for those not on HAART. The most significant difference was seen when the CD4+ count reached >200 cells/mm(3) (p=0.018 for CD4+ IFNgamma production and p=0.004 for CD8+ IFNgamma production). CD4+ interleukin-2 (IL-2) expression was significantly lower in HIV patients as compared to Controls but did not significantly improve however good the response to HAART. IL-2 expression by CD8+ lymphocytes was also lower in HIV patients as compared to Controls. IL-2 expression by CD8+ lymphocytes significantly improved in all patients on HAART as compared to HIV patients on no HAART. IL-2 expression was not significantly different from that of the Controls when the HIV viral load was less than 50 copies/ml. These results demonstrate improvements in both CD4+ and CD8+ responsiveness with HAART. IFNgamma production was elevated in response to HAART and was maximal only with significant CD4 count recovery. In contrast, normalization of IL-2 production by CD8+ lymphocytes was seen early in patients receiving HAART even when there was only a small increase in CD4+ lymphocyte numbers.

Cytokines and chemokines in uveitis: is there a correlation with clinical phenotype?

Uveitis is a general term for intraocular inflammation and includes a large number of clinical phenotypes. As a group of disorders, it is responsible for 10% of all registered blind patients under the age of 65 years. Immune-mediated uveitis may be associated with a systemic disease or may be localized to the eye. The pro-inflammatory cytokines interleukin (IL)-1beta, IL-2, IL-6, interferon-gamma and tumor necrosis factor-alpha have all been detected within the ocular fluids or tissues in the inflamed eye together with others, such as IL-4, IL-5, IL-10 and transforming growth factor-beta. The chemokines IL-8, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and fractalkine are also thought to be involved in the associated inflammatory response. There have been a number of studies in recent years investigating cytokine profiles in different forms of uveitis with a view to determining what cytokines are important in the inflamed eye. This review attempts to present the current state of knowledge from in vitro and in vivo research on the inflammatory cytokines in intraocular inflammatory diseases.