Flavia L. Barbosa

Desiccating Stress–Induced Chemokine Expression in the Epithelium Is Dependent on Upregulation of NKG2D/RAE-1 and Release of IFN-γ in Experimental Dry Eye

The Th1-associated chemokines CXCL9, CXCL10, and CXCL11 coordinate migration of CXCR3+ Th1 cells. The objective of this study was to evaluate the role of the innate immune system in stimulating chemokine expression in an experimental model of dry eye and bridge the gap between innate and adaptive immunity. Desiccating stress (DS) induced very early (6 h) expression and production of Th1-associated chemokines in cornea and conjunctiva of C57BL/6 and RAG1 knockout (KO) mice, demonstrating that chemokine expression does not require innate T cells. We then demonstrated that activating the innate immune system prior to adoptive transfer of T cells to RAG1KO mice increased disease severity. Interestingly, lack of induction of chemokines CXCL9, CXCL10, and CXCL11 in IFN-γKO mice provided evidence that their expression requires IFN-γ for induction. Treatment of RAG1KO mice with anti-NK1.1 prevented the increase of CXCL9, CXCL10, and CXCL11 in response to DS, compared with isotype controls. Additionally, DS increased the expression of NKG2D in the conjunctiva. The expression of the NKG2D ligand, retinoic acid early inducible gene 1, also increased at the ocular surface at both the protein and gene levels. Neutralization of NKG2D at the ocular surface decreased the expression of CXCL9, CXCL10, CXCL11, and IFN-γ. In summary, upregulation of CXCL9, CXCL10, and CXCL11 expression in experimental dry eye is T cell–independent, requiring IFN-γ–producing NKG2D+ NK cells that are activated in response to DS-induced stress signals. This study provides insight into the events that trigger the initial immune response in dry eye pathology.

Altered balance of interleukin-13/interferon-gamma contributes to lacrimal gland destruction and secretory dysfunction in CD25 knockout model of Sjögren’s syndrome

IntroductionThe lacrimal gland (LG) of the CD25-/- model of Sjögren’s syndrome (SS) has high interleukin (IL)-17, IL-13 and interferon-gamma (IFN-γ) cytokines. The specific contribution of these cytokines to the onset and severity of dacryoadenitis in the CD25-/- mice has not been evaluated.MethodsCD25−/−IL-17A−/−, CD25−/−IL-17−/−IFN-γ−/− and CD25−/−IFN-γ−/− were used at 4, 8, 12, 16 weeks (W). Total lymphocytic infiltration was evaluated by histology and characterized by flow cytometry. Epidermal growth factor (EGF) concentration was measured in tears. Immunofluorescent staining evaluated expression of IFN-γ receptor (IFN-γR) and apoptosis. Real-time PCR evaluated inflammatory and T cell-related cytokines expression in LG. Caspase-3, -8, -9 activities was assayed in LG lysates. T helper cytokines were measured in serum by Luminex assay.ResultsThe greatest total LG infiltration at 8 W was seen in CD25−/−IL-17A−/− (95%), followed by CD25−/− (71%) and IL-17−/− (12%). Tear EGF concentration was in normal range in CD25−/− at 4 W and in very low levels in both CD25−/− and CD25−/−IL-17A−/−. CD25−/− had high levels of inflammatory cytokines transcripts in LG compared to IL-17−/− mice; however, CD25−/−IL-17A−/− had even higher IL-1β, IFN-γR, caspase-3, -8, -9 mRNA levels, greater immunoreactivity to IFN-γR in LG acini, greater number of apoptotic+ cells and greater caspases activities in the LG at 8 W. CD25−/−IL-17A−/− had lower IL-13 concentration and lower IL-13/IFN-γ ratio compared to CD25−/− in serum. CD25−/−IFN-γ−/− had lower number of apoptotic+ cells and decreased caspase-3 expression in LG. CD25−/−IL-17−/−IFN-γ−/− had lower total lymphocytic cell infiltration at 8 W (48%), CD4+T cell infiltration and expression of IFN-γR and apoptotic+ cells in the LG and increased tear EGF concentration in tears.ConclusionsIFN-γ is critical for LG destruction and secretory dysfunction in the CD25−/− model of SS. Altered balance between IFN-γ and IL-13 in the CD25−/−IL-17A−/− mice accelerates LG destruction by increasing glandular apoptosis and facilitating apoptosis through increased expression of IFN-γR by glandular epithelium and activation of caspases. Targeting both IFN-γ and IL-17 may be beneficial for treating the LG inflammation in SS.

Goblet Cells Contribute to Ocular Surface Immune Tolerance—Implications for Dry Eye Disease

Conjunctival goblet cell (GC) loss in dry eye is associated with ocular surface inflammation. This study investigated if conjunctival GCs contribute to ocular surface immune tolerance. Antigens applied to the ocular surface, imaged by confocal microscopy, passed into the conjunctival stroma through goblet cell associated passages (GAPs) in wild type C57BL/6 (WT), while ovalbumin (OVA) was retained in the epithelium of SAM pointed domain containing ETS transcription factor (Spdef) knockout mice (Spdef−/−) that lack GCs and are a novel model of dry eye. Stimulated GC degranulation increased antigen binding to GC mucins. Induction of tolerance to topically applied OVA measured by cutaneous delayed type hypersensitivity (DTH) was observed in WT, but not Spdef−/−. OTII CD4+ T cells primed by dendritic cells (DCs) from the conjunctival draining lymph nodes of Spdef−/− had greater IFN-γ production and lower Foxp3 positivity than those primed by WT DCs. These findings indicate that conjunctival GCs contribute to ocular surface immune tolerance by modulating antigen distribution and antigen specific immune response. GC loss may contribute to the abrogation of ocular surface immune tolerance that is observed in dry eye.

Interferon-gamma deficiency protects against aging-related goblet cell loss

Aging is a well-recognized risk factor for dry eye. Interferon-gamma (IFN-γ) has been implicated in conjunctival keratinization and goblet cell loss in dry eye. We investigated the role of IFN-γ in age-related dry eye by evaluating young (8 weeks) and aged (15 months; 15M) C57BL/6 (B6) and IFN-γKO mice. Age effects on the conjunctiva and cornea epithelium were assessed with PAS staining and corneal staining, respectively. Expression of T cell-related cytokines (IL-17A, IFN-γ), chemokines (CXCL10 and CCL20), in the ocular surface epithelium was evaluated by real time PCR. A significant decrease in filled goblet cells was noted in 15M B6 mice and this was significantly lower than age and sex-matched IFN-γKO mice. Aged male B6 had significantly higher IFN-γ, and CXCL10 mRNA in their conjunctiva than female B6 mice. Aged IFN-γKO females had significantly higher IL-17A mRNA in conjunctiva than IFN-γKO males and B6 mice. Corneal barrier dysfunction was observed in 15M female B6 and aged IFN-γKO mice of both sexes; however it was significantly higher in IFN-γKO compared to B6 mice. While there was a significant increase in IL 17A, and CCL20 in corneas of aged female B6 and IFN-γKO mice compared to males, these changes were more evident in aged female IFN-γKO group. Partial resistance of IFN-γKO mice to aging-induced goblet cell loss indicates IFN-γ is involved in the age-related decline in conjunctival goblet cells. Increased corneal IL-17A expression paralleled corneal barrier disruption in aging female of both strains. IFN-γ appears to suppress IL-17A on the ocular surface.

Goblet cell loss abrogates ocular surface immune tolerance

Intestinal epithelial cells condition tolerogenic properties in DCs. Aqueous-deficient dry eye is associated with goblet cell (GC) loss and increased IFN-γ expression in the conjunctiva. We hypothesized that loss of GCs reduces tolerance-inducing properties of antigen presenting cells (APCs) in the conjunctiva and draining nodes. Mice lacking the SAM pointed domain containing ETS transcription factor (Spdef) that is required for GC differentiation had an increased frequency of macrophages in the conjunctiva and CD11b+CD11c+ DCs in the conjunctiva and draining nodes, and these cells had greater IL-12 expression than WT mice. Conditioned media from cultured WT conjunctival GCs suppressed LPS-induced IL-12 production by conjunctival APCs. OVA antigen-specific OTII CD4+ T cells primed by Spdef-KO draining lymph node APCs showed greater proliferation, lower frequency of Foxp3+, increased frequency of IFN-γ+ and IL-17+ cells, and greater IFN-γ production than those primed by WT APCs. The immune tolerance to OVA antigen topically applied to the conjunctiva measured by cutaneous delayed type hypersensitivity (DTH) reaction, OVA-specific T cell proliferation, Foxp3 induction, and IFN-γ production observed in WT mice was lost in the Spdef-KO mice. We concluded that conjunctival GCs condition tolerogenic properties in APCs that suppress IL-12 production and Th1 polarization.