Age-related changes in ocular mucosal tolerance: Lessons learned from gut and respiratory tract immunity

The ocular surface is the part of the visual system directly exposed to the environment, and it comprises the cornea, the first refractive tissue layer and its surrounding structures. The ocular surface has evolved to keep the cornea smooth and wet, a prerequisite for proper sight, and also protected. To this aim, the ocular surface is a bona fide mucosal niche with an immune system capable of fighting against dangerous pathogens. However, due to the potential harmful effects of uncontrolled inflammation, the ocular surface has several mechanisms to keep the immune response in check. Specifically, the ocular surface is maintained inflammation-free and functional by a particular form of peripheral tolerance known as mucosal tolerance, markedly different from the immune privilege of intraocular structures. Remarkably, conjunctival tolerance is akin to the oral and respiratory tolerance mechanisms found in the gut and airways, respectively. And also similarly, this form of immunoregulation in the eye is affected by ageing just as it is in the digestive and respiratory tracts. With ageing comes an increased prevalence of immune-based ocular surface disorders, which could be related to an age-related impairment of conjunctival tolerance. The purpose of this review was to summarize the present knowledge of ocular mucosal tolerance and how it is affected by the ageing process in the light of the current literature on mucosal immunoregulation of the gut and airways.Fil: Galletti, Jeremías Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de Paiva, Cintia S.. Baylor College of Medicine; Estados Unido

IL-17 producing lymphocytes cause dry eye and corneal disease with aging in RXRα mutant mouse

PURPOSE: To investigate IL-17 related mechanisms for developing dry eye disease in the Pinkie mouse strain with a loss of function RXRα mutation. METHODS: Measures of dry eye disease were assessed in the cornea and conjunctiva. Expression profiling was performed by single-cell RNA sequencing (scRNA-seq) to compare gene expression in conjunctival immune cells. Conjunctival immune cells were immunophenotyped by flow cytometry and confocal microscopy. The activity of RXRα ligand 9-cis retinoic acid (RA) was evaluated in cultured monocytes and γδ T cells. RESULTS: Compared to wild type (WT) C57BL/6, Pinkie has increased signs of dry eye disease, including decreased tear volume, corneal barrier disruption, corneal/conjunctival cornification and goblet cell loss, and corneal vascularization, opacification, and ulceration with aging. ScRNA-seq of conjunctival immune cells identified γδ T cells as the predominant IL-17 expressing population in both strains and there is a 4-fold increased percentage of γδ T cells in Pinkie. Compared to WT, IL-17a, and IL-17f significantly increased in Pinkie with conventional T cells and γδ T cells as the major producers. Flow cytometry revealed an increased number of IL-17 CONCLUSION: These findings indicate that RXRα suppresses generation of dry eye disease-inducing IL-17 producing lymphocytes s in the conjunctiva and identifies RXRα as a potential therapeutic target in dry eye

Patterned expression of neurotrophic factors and receptors in human limbal and corneal regions

PURPOSE: To evaluate the expression patterns of neurotrophic factors (NTFs) and their receptors in the human cornea with the intention of exploring the role of NTFs in maintaining corneal epithelial stem cells in the limbus. METHODS: Fresh human corneoscleral tissues were prepared for frozen sections. Immunofluorescent staining was performed with primary antibodies against six members of three NTF families and their six receptors. To confirm the specificity of NTF primary antibodies, neutralization experiments with their corresponding peptides and western blot analysis were performed. RESULTS: Based on spatial and differential immuno-localization, three patterns of NTF expression were potentially involved in epithelial-mesenchymal interaction on the ocular surface: (1) the epithelial type: nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF); (2) the paracrine type: neurotrophin (NT)-3 and NT-4/5; and (3) the reciprocal type: brain-derived neurotrophic factor (BDNF). The stem cell-enriched basal cells of the limbal epithelium expressed three unique staining patterns for NTFs: (1) exclusively positive for NGF, GDNF, and their corresponding receptors, TrkA and GDNF family receptor alpha (GFR)-1; (2) relatively high levels of BDNF; and (3) negative for NT-3 and NT-4. Additionally, the neurotrophin common low-affinity receptor, p75NTR, was mainly expressed by the basal layer of the entire corneal and limbal epithelia, and TrkB and TrkC were evenly expressed by the entire corneal and limbal epithelia. BDNF, p75NTR, TrkB, and TrkC are also abundantly expressed by limbal stroma cells. No specific immunoreactivity to ciliary neurotrophic factor (CNTF) and its receptor, CNTFR, was detected in cornea tissue in situ. CONCLUSIONS: Our findings revealed patterned expression of NTFs and their receptors in the human ocular surface, suggesting that they may play a vital role in maintaining corneal epithelial stem cells in the limbus. NGF, GDNF, GFR-1, TrkA, and BDNF may serve as new limbal basal cell markers defining the corneal epithelial stem cell phenotype.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000250807800002&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Biochemistry & Molecular BiologyOphthalmologySCI(E)42ARTICLE217-191934-19411

Expression of Th-1 Chemokines and Chemokine Receptors on the Ocular Surface of C57BL/6 Mice: Effects of Desiccating Stress

PURPOSE. To evaluate the effects of desiccating ocular surface stress on the expression of chemokines and their receptors by the corneal epithelium and conjunctiva of C57BL/6 and BALB/c mice. METHODS. Experimental dry eye was created in C57BL/6 and BALB/c mice. The concentrations of macrophage inflammatory protein 1␣ (MIP-1␣), MIP-1␤, monokine induced by interferon (MIG)-␥, and interferon-␥-inducible protein (IP)-10 in the corneal epithelia and conjunctiva were measured by a multiplex immunobead assay. Expression of MIP-1␣; MIP-1␤; regulated on activation, normal T-cell expressed and secreted (RANTES), MIG, IP-10; monocyte chemoattractant protein (MCP)-3; eotaxin-1; CCR5; CXCR3; and CCR3 in the cornea and conjunctiva were evaluated by real-time PCR and immunostaining. RESULTS. Desiccating stress significantly increased concentrations of MIP-1␣, MIP-1␤, IP-10, and MIG proteins in the corneal epithelium and conjunctiva of C57BL/6 mice. Furthermore, it increased levels of MIP-1␣, MIP-1␤, and CCR5 transcripts in the cornea and conjunctiva and RANTES, MIG, IP-10, and CXCR3 transcripts in the conjunctiva of C57BL/6 mice. In contrast, levels of MCP-3, eotaxin-1, and CCR3 transcripts increased in both tissues of BALB/c mice. In situ immunodetection of chemokines and their receptors was similar to their pattern of gene expression. CONCLUSIONS. Specific patterns of Th-1 and -2 chemokines and their receptors are induced in the mouse ocular surface by desiccating stress in a strain-related fashion. Desiccating stress potently stimulates the expression of Th-1 cell-attracting chemokines and chemokine receptors on the ocular surface of C57BL/6 mice. (Invest Ophthalmol Vis Sci

IL-13 Stimulates Proliferation and Expression of Mucin and Immunomodulatory Genes in Cultured Conjunctival Goblet Cells

Citation: Tukler Henriksson J, Coursey TG, Corry DB, De Paiva CS, Pflugfelder SC. IL-13 stimulates proliferation and expression of mucin and immunomodulatory genes in cultured conjunctival goblet cells. Invest Ophthalmol Vis Sci. 2015;56:4186-4197. DOI:10.1167/iovs.14-15496 PURPOSE. To investigate the effects of IL-13 on goblet cell proliferation, differentiation, and expression of mucin and immunomodulatory genes. METHODS. Explants were excised from the conjunctiva of young C57BL/6 mice. Cultures received 200 lL per week of either Keratinocyte media (KSFM) or KSFM supplemented with 10 ng/mL IL-13 and were incubated for 3 (D3), 7 (D7), or 14 (D14) days. Subsequently, cell proliferation was assessed or cultures were immunostained, collected for dot blot, or for reverse transcription (RT) and quantitative real-time PCR (qPCR) or for RT-PCR gene array. RESULTS. The cultured conjunctival epithelium expressed goblet cell associated keratin 7 and mucins MUC5AC and MUC2 and when stimulated with IL-13 showed increased proliferation at D3 and D7 (P < 0.05) compared with control. MUC5AC expression was increased in the IL-13-treated group at D3 and D14 (P < 0.05). IL-13-treated cultures showed increased chemokine ligand 26 (CCL26), chloride channel calcium activated channel 3 (CLCA3), fas ligand (FasL), and Relm-b at D7. All conjunctival cultures expressed MUC2, and its expression was decreased at D3 (P < 0.05) and increased at D14 (P < 0.05) with IL-13 treatment. CONCLUSIONS. This study demonstrated that conjunctival goblet cells are IL-13 responsive cells that produce factors known to maintain epithelial barrier, stimulate mucin production, and modulate immune response in nonocular mucosa when treated with IL-13. The functional significance of IL-13-stimulated factors remains to be determined. Keywords: conjunctiva, goblet cells, interleukin-13, cell culture T he conjunctiva covers two-thirds of the ocular surface and functions as a support tissue for cornea. 2,3 Conjunctival goblet cells are surrounded by lymphocytes and dendritic cells and their density has been found to change in certain ocular surface immune/inflammatory conditions. 4 Goblet cell density has been reported to decrease in aqueous tear deficiency, a condition where T helper 1 (Th1) and Th17 cells infiltrate the conjunctiva, and increase in atopic keratoconjunctivitis and vernal keratoconjunctivitis, predominantly Th2-mediated diseases. 5-8 The mucus stimulating activity of the Th2 cytokine IL-13 has been reported to have a defensive role in the intestines by eliminating helminthic parasites and in the airways by protecting from particles or allergens. 9,10 However, excessive IL-13 expression is associated with goblet cell hyperplasia and mucous hypersecretion, both in the gut and in the airways where it can result in airway obstruction. The purpose of the present study was to investigate whether the Th2 cytokine IL-13 can modulate proliferation, differentiation, and expression of mucin and immunomodulatory gene

Disruption of TGF-β Signaling Improves Ocular Surface Epithelial Disease in Experimental Autoimmune Keratoconjunctivitis Sicca

TGF-β is a pleiotropic cytokine that can have pro- or anti-inflammatory effects depending on the context. Elevated levels of bioactive TGF-β1 in tears and elevated TGF-β1mRNA transcripts in conjunctiva and minor salivary glands of human Sjögren's Syndrome patients has also been reported. The purpose of this study was to evaluate the response to desiccating stress (DS), an experimental model of dry eye, in dominant-negative TGF-β type II receptor (CD4-DNTGFβRII) mice. These mice have a truncated TGF-β receptor in CD4(+) T cells, rendering them unresponsive to TGF-β.DS was induced by subcutaneous injection of scopolamine and exposure to a drafty low humidity environment in CD4-DNTGFβRII and wild-type (WT) mice, aged 14 weeks, for 5 days. Nonstressed (NS) mice served as controls. Parameters of ocular surface disease included corneal smoothness, corneal barrier function and conjunctival goblet cell density. NS CD4-DNTGFβRII at 14 weeks of age mice exhibited a spontaneous dry eye phenotype; however, DS improved their corneal barrier function and corneal surface irregularity, increased their number of PAS+ GC, and lowered CD4(+) T cell infiltration in conjunctiva. In contrast to WT, CD4-DNTGFβRII mice did not generate a Th-17 and Th-1 response, and they failed to upregulate MMP-9, IL-23, IL-17A, RORγT, IFN-γ and T-bet mRNA transcripts in conjunctiva. RAG1KO recipients of adoptively transferred CD4+T cells isolated from DS5 CD4-DNTGFβRII showed milder dry eye phenotype and less conjunctival inflammation than recipients of WT control.Our results showed that disruption of TGF-β signaling in CD4(+) T cells causes paradoxical improvement of dry eye disease in mice subjected to desiccating stress

Aqueous Tear Deficiency Increases Conjunctival Interferon-c (IFN-c) Expression and Goblet Cell Loss

PURPOSE. To investigate the hypothesis that increased interferon-c (IFN-c) expression is associated with conjunctival goblet cell loss in subjects with tear dysfunction. METHODS. Goblet cell density (GCD) was measured in impression cytology from the temporal bulbar conjunctiva, and gene expression was measured in cytology samples from the nasal bulbar conjunctiva obtained from 68 subjects, including normal control, meibomian gland disease (MGD), non-Sjögren syndrome (non-SSATD)-, and Sjögren syndrome (SSATD)-associated aqueous tear deficiency. Gene expression was evaluated by real-time PCR. Tear meniscus height (TMH) was measured by optical coherence tomography. Fluorescein and lissamine green dye staining evaluated corneal and conjunctival disease, respectively. Between-group mean differences and correlation coefficients were calculated. RESULTS. Compared to control, IFN-c expression was significantly higher in both ATD groups, and its receptor was higher in SSATD. Expression of IL-13 and its receptor was similar in all groups. Goblet cell density was lower in the SSATD group; expression of MUC5AC mucin was lower and cornified envelope precursor small proline-rich region (SPRR)-2G higher in both ATD groups. Interferon-c transcript number was inversely correlated with GCD (r ¼ À0.37, P < 0.04) and TMH (r ¼ À0.37, P ¼ 0.02), and directly correlated with lissamine green staining (r ¼ 0.51, P < 0.001) and SPRR-2G expression (r ¼ 0.32, P < 0.05). CONCLUSIONS. Interferon-c expression in the conjunctiva was higher in aqueous deficiency and correlated with goblet cell loss and severity of conjunctival disease. These results support findings of animal and culture studies showing that IFN-c reduces conjunctival goblet cell number and mucin production