Interleukin-17 receptor C is essential for the pro-inflammatory pathogenicity of granulocyte-macrophage-colony-stimulating factor-producing T helper cells in experimental autoimmune uveitis.

Autoimmune uveitis is an inflammatory disorder of the eye triggered by the responses of autoreactive T cells to ocular autoantigens. This study aims to understand the role of granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (ThGM) cells in the pathophysiology of mouse experimental autoimmune uveitis (EAU). We established an EAU model by immunizing mice with interphotoreceptor retinoid-binding protein (IRBP) 651-670. Splenic or eye-infiltrating ThGM cells were analyzed and enriched by flow cytometry according to the levels of an array of surface markers, transcription factors, and cytokines. Lentiviral transduction was conducted to silence or overexpress the target gene in differentiated ThGM cells. The adoptive transfer was applied to determine the pathogenicity of ThGM cells in vivo. We found that ThGM cells were present in the spleen and the eye after EAU induction. Both splenic and eye-infiltrating ThGM cells were phenotypically CD4+CCR7-CXCR3-CCR6-CCR10hi. Eye-infiltrating ThGM cells up-regulated interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and IL-17 receptor C (IL-17RC) relative to splenic ThGM cells. IL-17RC overexpression enabled interleukin-17A (IL-17A)-induced up-regulation of IL-1ß and IL-6 production in ThGM cells. Adoptive transfer of IL-17RC overexpressing ThGM cells exacerbated EAU severity, as evidenced by a higher histology score as well as increased pro-inflammatory cytokines and inflammatory cells in the eye. However, IL-17RC-silenced ThGM cells did not impact EAU. Therefore, for the first time, this study unveils the essential pro-inflammatory role of IL-17RC-expressing ThGM cells in EAU pathophysiology. We discovered a novel mechanism underlying the pathophysiology of autoimmune uveitis.

Chitosan Oligosaccharides Suppress Nuclear Factor-Kappa B Activation and Ameliorate Experimental Autoimmune Uveoretinitis in Mice.

We investigated the therapeutic potential and mechanism of chitosan oligosaccharides (COS) for experimental autoimmune uveoretinitis (EAU) in mice. EAU was induced in C57/BL6 mice by injection of human interphotoreceptor retinoid-binding protein (IRBP) peptides. At the same time, a high or low dose (20 or 10 mg/kg) of COS or phosphate-buffered saline (PBS) was given to mice daily after EAU induction. We found that mouse EAU is ameliorated by the high-dose COS treatment when compared with PBS treatment. In the retinas of high-dose COS-treated mice, the nuclear translocation of NF-κB subunit (p65) was suppressed, and the expression of several key EAU inflammatory mediators, IFN-γ, TNF-α, IL-1α, IL-4, IL-5, IL-6, IL-10, IL-17 and MCP-1 was lowered. These results suggest that COS may be a potential treatment for posterior uveitis.

Posttranslational modification of differentially expressed mitochondrial proteins in the retina during early experimental autoimmune uveitis.

PURPOSE: Posttranslational modification of proteins plays an important role in cellular functions and is a key event in signal transduction pathways leading to oxidative stress and DNA damage. In this study, we used matrix-assisted laser desorption/ionization- time of flight (MALDI-TOF) to investigate the posttranslational modifications of the differentially expressed proteins in the retinal mitochondria during early experimental autoimmune uveitis (EAU). METHODS: EAU was induced in 18 B10RIII mice with 25 µg of inter-photoreceptor retinoid-binding protein (IRBP) emulsified with complete Freund's adjuvant (CFA); 18 mice treated with CFA without IRBP served as controls. Retinas were removed from the experimental and control groups on day 7 post immunization; mitochondrial fractions were extracted and subjected to 2 dimentional-difference in gel electrophoresis (2D-DIGE); and the protein spots indicating differential expression were subjected to MALDI-TOF for protein identification and indication of any posttranslational modifications. RESULTS: Of the 13 proteins found to be differentially expressed by 2D-DIGE (including upregulated aconitase, mitochondrial heat shock protein (mtHsp) 70, lamin-1, syntaxin-binding protein, αA crystallin, ßB2 crystallin, along with downregulated guanine nucleotide-binding protein and ATP synthase) nine were found to undergo posttranslational modification. Oxidation was a common modification found to occur on aconitase, mtHsp 70, ATP synthase, lamin-1, ßB2-crystallin, guanine nucleotide-binding protein, and manganese superoxide dismutase (MnSOD). In addition, aconitase hydratase, mtHsp 70, guanine nucleotide-binding protein, ATP synthase, syntaxin-binding protein, ßB2-crystallin, and lamin-1 were also modified by carbamidomethylation. αA-crystallin had a pyro-glu modification. CONCLUSIONS: Several proteins present in the retinal mitochondria are posttranslationally modified during early EAU, indicating the presence of oxidative stress and mitochondrial DNA damage. The most common modifications are oxidation and carbamidomethylation. A better understanding of the proteins susceptible to posttranslational modifications in the mitochondria at the early stage of the disease may serve to advance therapeutic interventions to attenuate disease progression.

Coordinated gene expression of Th17- and Treg-associated molecules correlated with resolution of the monophasic experimental autoimmune uveitis.

PURPOSE: To investigate the role of T-cell-mediated immune response in a monophasic experimental autoimmune uveitis (EAU). METHODS: A monophasic EAU was induced in Lewis rats by immunization with interphotoreceptor retinoid-binding protein peptide. Optimized quantitative real-time RT-PCR was used for consecutive measurement of the relative expression of Th17-associated molecules, including interleukin 6 (IL-6), transforming growth factor-ß (TGF-ß), interleukin 23p19 (IL-23p19), interleukin 23p40 (IL-23p40), CD4, CD8, major histocompatibility complex I (MHC I), major histocompatibility complex II (MHC II), interleukin 17 (IL-17), interleukin 17F (IL-17F), interleukin 17 receptor A (IL-17RA), retinoic acid-related orphan receptor γt (RORγt) and Chemokine receptor 6 (CCR6), in addition to Treg-related forkhead box P3 (Foxp3), C-X-C chemokine receptor type 5 (CXCR5), and cluster of differentiation 25 (CD25) at the initiation, effector, and resolution phases of EAU and compared with those at 14 days post-immunization of control animals. Immunohistochemisty was used to examine IL-17 expression in retinas. Glial fibrillary acidic protein retinal astrocytes, Neuronal class III ß-Tubulin(Tuj1(+))retinal ganglion cells, and infiltrating CD11b(+) microglia were analyzed by fluorescent microscopy in a kinetic manner. RESULTS: Our results indicated well organized T-cell activity, measured by relative expression of multiple T-cell-related factors at the mRNA level, synchronized with the initiation of autoimmune inflammation, and thereafter resolution of the monophasic EAU. Immune balance was achieved several times through coordinated expression of Th17- and Treg-related factors. The expression pattern of these factors and results from immunochemistry with an IL-17 antibody indicated that there may be intensive crosstalk between infiltrating immune cells and the resident neural cells, which were significantly activated during the course of disease. CONCLUSIONS: T-cell-mediated immune response played a positive role in resolution of the monophasic EAU.

[Mouse corneal degeneration by bovine cornea-specific protein].

In a previous study, IgG deposits and changes were found in the corneal epithelial cells of BALB/c mice immunized by cornea-specific protein (CSP) contained in the bovine cornea. We observed the corneal changes in mice by immunoreaction to CSP for 6 months with slit-lamp microscopy, and found granular opacity under the central corneal epithelium. Histologically this opacity was composed of scattered deposits in the corneal substantia beneath the basal layer of the epithelium. The corneal epithelium in contact with the deposits became atrophied and flattened. The deposits were stained with PAS staining, and with ConA, WGA and RCA by the lectin enzyme histochemical method. Therefore, these results indicate that these deposits possess N-acetyl-D-glucosamine as a sugar residue. In the degenerated corneal epithelium, peroxidase-staining by the anti-CSP antibody enzyme histochemical method weakened and ConA-positive cells were obviously decreased. This suggests that the production of CSP decreased in the same lesion and that disorder of sugar metabolism developed at the corneal epithelium.

IFN-ß inhibits the increased expression of IL-9 during experimental autoimmune uveoretinitis.

PURPOSE: It has been shown that IL-9 plays a proinflammatory role in the pathogenesis of certain autoimmune diseases. This study was designed to investigate the possible role of IL-9 in the development of experimental autoimmune uveoretinitis (EAU) and the effect of IFN-ß on its expression. METHODS: EAU was induced in B10RIII mice by immunization with interphotoreceptor retinoid-binding protein peptide 161-180 (IRBP(161-180)). IFN-ß was administered subcutaneously to IRBP(161-180) immunized mice every other day from day one before immunization to the end of the study. Splenocytes and draining lymph node (DLN) cells from EAU mice or control mice or EAU mice treated with IFN-ß or PBS were stimulated with anti-CD3/CD28 or IRBP(161-180) for 3 days. Naïve T cells cultured under Th1 or Th17 polarizing conditions were incubated in the presence or absence of IFN-ß for 4 days. Effector/memory T cells were activated by anti-CD3/CD28 in the presence or absence of IFN-ß for 3 days. IFN-ß-treated monocytes were cocultured with naïve T cells or effector/memory T cells for 3 days. Culture supernatants were collected and IL-9 was detected by ELISA. RESULTS: IL-9 expression in splenocytes and DLN cells was increased in EAU mice during the inflammatory phase and returned back to lower levels during the recovery phase. IFN-ß in vivo treatment significantly inhibited EAU activity in association with a down-regulated expression of IL-9. In vitro polarized Th1 and Th17 cells both secreted IL-9 and the addition of IFN-ß suppressed production of IL-9 by both Th subsets. Beside its effect on polarized Th cells, IFN-ß also suppressed the secretion of IL-9 by effector/memory T cells. However, IFN-ß-treated monocytes had no effect on the production of IL-9 when cocultured with naïve or effector/memory T cells. CONCLUSION: IL-9 expression is increased during EAU which could be suppressed by IFN-ß.

Lacritin, a novel human tear glycoprotein, promotes sustained basal tearing and is well tolerated.

PURPOSE: Lacritin is a novel human tear glycoprotein that promotes basal tear peroxidase secretion by rat lacrimal acinar cells in vitro. This study investigates whether lacritin is prosecretory when added topically to the ocular surface of normal living rabbits, and if so, what is its efficacy and tolerability versus cyclosporine and artificial tears. METHODS: Purified recombinant human lacritin (1, 10, 50, or 100 µg/mL), inactive lacritin truncation mutant C-25 (10 µg/mL), cyclosporine (0.05%), or artificial tears were topically administered to eyes of normal New Zealand White rabbits either as a single dose or three times daily for 14 days with monitoring of basal tear production. Basal tearing under proparacaine anesthesia was repeatedly assessed throughout and 1 week after chronic treatment ceased. Eyes were examined weekly by slit-lamp biomicroscopy. RESULTS: Lacritin acutely increased basal tearing to 30% over vehicle at 240 minutes. Three times daily treatment with 10-100 µg/mL lacritin was well tolerated. Basal tearing became progressively elevated 4, 7, and 14 days later and was 50% over baseline (50 µg/mL lacritin) 1 week after treatment had ceased. Cyclosporine elevated tearing to a similar level on days 4 and 7 but had little or no effect on day 14 and had returned to baseline 1 week after ending treatment. C-25 and artificial tears had no effect. CONCLUSIONS: Lacritin acutely stimulates basal tear flow that is sustained for at least 240 minutes. Two weeks of lacritin treatment three times daily was well tolerated and progressively elevated the basal tear flow. One week after treatment ended, basal tearing was still 50% over baseline. In contrast, cyclosporine triggered mild to moderate corneal irritation and a temporary elevation in tearing.

Contact lens type, material, and deposits and giant papillary conjunctivitis.

Giant papillary conjunctivitis (GPC) is a condition commonly encountered in clinical practice. Much research has taken place aimed at more clearly understanding the pathogenesis of GPC. We review the current literature and discuss the association between GPC and contact lens type, material, and deposits.

Characterization of a potent uveitopathogenic site derived from rat phosducin.

Phosducin is a retinal and pineal phosphoprotein assumed to play an important role in visual phototransduction. Phosducin is also a uveitopathogenic retinal antigen, but its potency has been reported to be mild. During the course of studies aimed at identifying uveitopathogenic sites in phosducin, we found that rat phosducin possessed a potent uveitopathogenic site. In this study, we characterize the potent uveitopathogenic site by using synthetic peptides. Several synthetic peptides from this region plus adjuvants were injected into Lewis rats, and the uveitopathogenic core sequence was defined. We also determined the pivotal amino acid residues by using synthetic peptides with single residue substitution. Immunization with PDC(R)65-96 (amino acid residues 65 through 96 derived from rat phosducin) at doses of 0.83 nmol or more induced severe experimental autoimmune uveitis (EAU) in all rats within 12 days. Experimental autoimmune pinealitis (EAP) was also observed in all rats after immunization with 0.83 nmol or higher doses of the peptide. The lowest dose of the peptide to induce EAU and EAP was 0.24 nmol. The smallest peptide that induced EAU as severe as PDC(R)65-96 was PDC(R)77-87, which consisted of 11 amino acid residues (YELIHQDKEDE). The core sequence within the uveitopathogenic site was a pentapeptide (LIHQD), amino acid residues from 79 to 83. To determine the role of individual residues within PDC(R)77-87, we tested the uveitopathogenicity of analogues of PDC(R)75-85 and PDC(R)77-89, respectively, in which each of the residues from 77 to 87 was replaced by alanine (A). Analogous peptides bearing a single residue substitution at 80 (I-->A) and 82 (Q-->A), respectively, were not uveitopathogenic. Our findings demonstrated the presence of a potent uveitopathogenic site in PDC(R)65-96 whose potency in Lewis rats was comparable to that of S-antigen. The pivotal amino acid residues for uveitopathogenicity were the residues at 80 (I) and 82 (Q). The clinical and histological features of this EAU closely resembled those of the EAU induced by S-antigen and recoverin.