Solherny B. Pangelinan

IL-17 disrupts corneal barrier following desiccating stress.

T helper (Th)-17 is a recently identified subtype of Th response that has been implicated in host defense and autoimmunity. We investigated whether there is evidence for a Th-17 response in human and experimental murine dry eye (DE). Gene expression in the human DE conjunctiva showed increased levels of the Th-17 inducers, interleukin (IL)-23, IL-17A, and interferon-gamma (IFN-γ). In the murine model, we found that desiccating stress increased matrix metalloproteinase-9, Th-17-associated genes (IL-6, IL-23, transforming growth factor-β1 and -2, IL-23R, IL-17R, IL-17A, retinoid-related orphan receptor-γt, and CC chemokine attractant ligand-20) and IFN-γ in cornea and conjunctiva. Furthermore, we found a significantly increased concentration of IL-17 in tears and number of IL-17-producing cells on the ocular surface. Antibody neutralization of IL-17 ameliorated experimental DE-induced corneal epithelial barrier dysfunction and decreased the expression of matrix metalloproteinases 3 and 9. Taken together, these findings suggest that IL-17 has a role in corneal epithelial barrier disruption in DE.

Age-related T-cell cytokine profile parallels corneal disease severity in Sjögren’s syndrome-like keratoconjunctivitis sicca in CD25KO mice

OBJECTIVES IL-2ralpha (CD25)(-/-) mice develop autoimmunity and lymphoproliferative disorders, including SS-like disease. The objective of this study was to evaluate the severity of corneal epithelial disease and T-cell cytokine profile in the ocular surface tissues of CD25KO mice. METHODS CD25KO mice were evaluated at 8, 12 and 16 weeks of age. Corneal epithelial smoothness and corneal permeability were measured. Phenotype of infiltrating lymphocytes was evaluated by immunohistochemistry. Th-1, -2 and -17 associated factors were measured by real-time PCR in cornea and conjunctiva and by Luminex immunobead assay in tears. RESULTS Compared with 8-week-old wild-type (WT) mice, CD25KO mice of the same age had significantly greater corneal irregularity and a significant increase in the number of CD4(+) and CD8(+) T cells infiltrating the conjunctiva. CD25KO mice had significantly higher levels of IL-6, TGF-beta1, IL-23R, IL-17A, IL-17F, IL-21, CCL20, IL-10, GATA-3 and IFN-gamma mRNA transcripts in their cornea and conjunctiva than WT mice at 8 weeks. IL-17A and IL-17F mRNA transcripts peaked at 12 weeks, whereas IFN-gamma spiked at 16 weeks in CD25KO mice. Increased expression of IL-17A and IL-17F at 12 weeks in CD25KO mice was accompanied by a worsening of corneal surface parameters and an increase of CD4(+) T cell infiltrating the cornea. CONCLUSIONS Disruption of IL-2 signalling in CD25KO mice results in age-dependent SS-like autoimmune lacrimal-keratoconjunctivitis. A mix of Th-1 and Th-17 cytokines was detected. The peak severity of corneal epithelial disease corresponded to the peak of IL-17 expression.

Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13

Although the effects of the interleukin 13 (IL-13) on goblet cell (GC) hyperplasia have been studied in the gut and respiratory tracts, its effect on regulating conjunctival GC has not been explored. The purpose of this study was to determine the major IL-13-producing cell type and the role of IL-13 in GC homeostasis in normal murine conjunctiva. Using isolating techniques, we identified natural killer (NK)/natural killer T (NKT) cells as the main producers of IL-13. We also observed that IL-13 knockout (KO) and signal transducer and activator of transcription 6 knockout (STAT6KO) mice had a lower number of periodic acid Schiff (PAS)+GCs. We observed that desiccating stress (DS) decreases NK population, GCs, and IL-13, whereas it increases interferon-γ (IFN-γ) mRNA in conjunctiva. Cyclosporine A treatment during DS maintained the number of NK/NKT cells in the conjunctiva, increased IL-13 mRNA in NK+ cells, and decreased IFN-γ and IL-17A mRNA transcripts in NK+ and NK− populations. C57BL/6 mice chronically depleted of NK/NKT cells, as well as NKT cell-deficient RAG1KO and CD1dKO mice, had fewer filled GCs than their wild-type counterparts. NK depletion in CD1dKO mice had no further effect on the number of PAS+ cells. Taken together, these findings indicate that NKT cells are major sources of IL-13 in the conjunctival mucosa that regulates GC homeostasis.

NK Cells Promote Th-17 Mediated Corneal Barrier Disruption in Dry Eye

Background The conjunctiva contains a specialized population of lymphocytes that reside in the epithelium, named intraepithelial lymphocytes (IEL). Methodology/Principal Findings Here we characterized the IEL population prior to and after experimental desiccating stress (DS) for 5 or 10 days (DS5, DS10) and evaluated the effect of NK depletion on DS. The frequency of IELs in normal murine conjunctiva was CD3+CD103+ (∼22%), CD3+γδ+ (∼9.6%), CD3+NK+ (2%), CD3−NK+ (∼4.4%), CD3+CD8α (∼0.9%), and CD4 (∼0.6%). Systemic depletion of NK cells prior and during DS led to a decrease in the frequency of total and activated DCs, a decrease in T helper-17+ cells in the cervical lymph nodes and generation of less pathogenic CD4+T cells. B6.nude recipient mice of adoptively transferred CD4+T cells isolated from NK-depleted DS5 donor mice showed significantly less corneal barrier disruption, lower levels of IL-17A, CCL20 and MMP-3 in the cornea epithelia compared to recipients of control CD4+T cells. Conclusions/Significance Taken together, these results show that the NK IELs are involved in the acute immune response to desiccation-induced dry eye by activating DC, which in turn coordinate generation of the pathogenic Th-17 response.

Essential Role for c-Jun N-Terminal Kinase 2 in Corneal Epithelial Response to Desiccating Stress

OBJECTIVE To investigate the protective effects of c-Jun N-terminal kinase (JNK)-1 and -2 gene knockout (KO) on the corneal epithelial response to desiccating stress. METHODS The C57BL/6, JNK1KO, and JNK2KO mice were subjected to desiccating stress (DS) for 5 days. The effects of DS on the corneal epithelium were evaluated by measuring corneal smoothness and permeability. Expression of matrix metalloproteinases (MMP)-1, MMP-9, and cornified envelope protein precursors (small proline-rich protein [SPRR]-1a, SPRR-2a, and involucrin) in the corneal epithelia was evaluated by immunostaining and real-time polymerase chain reaction. Collagenase and gelatinase activity in corneal sections as measured with in situ fluorescent assays. RESULTS The JNK2KO mice had smoother corneal surfaces and less corneal barrier disruption in response to DS than JNK1KO mice and C57BL/6 wild-type control mice. The DS increased levels of MMP-1, MMP-9, SPRR-1a, SPRR-2a, involucrin immunoreactivity, and mRNA transcripts in the corneal epithelium of JNK1KO and C57BL/6 mice, but not in JNK2KO mice. Knockout of JNK2 prevented DS-induced increase in gelatinase and collagenase activity in the cornea. CONCLUSION The JNK2 protein appears to have an essential role in desiccation-induced corneal epithelial disease by stimulating production of MMP-1, MMP-9, and cornified envelope precursors. Clinical Relevance The JNK2 protein could be a novel therapeutic target in dry eye disease.

Pharmacological Cholinergic Blockade Stimulates Inflammatory Cytokine Production and Lymphocytic Infiltration in the Mouse Lacrimal Gland

PURPOSE To investigate the effects of cholinergic blockade on inflammatory cell infiltration and cytokine production in the mouse lacrimal gland (LG). METHODS C57BL/6 mice were untreated (UT) or received subcutaneous injections of either scopolamine hydrobromide (SCOP; 0.5 mg/0.2 mL) or saline (SAL) four times daily for 2 or 5 days (2D, 5D). This was followed by a 7-day rest period in separate groups. Tear volume (cotton thread) and tear epidermal growth factor (EGF, by ELISA) concentrations were measured. Extraorbital LGs were surgically excised and sectioned or lysed for gene expression analysis. Immunohistochemistry evaluated immunophenotype of infiltrating cells. Expression of EGF and T helper (Th)-1, -2, and -17-associated cytokines in LGs was evaluated by real-time PCR. Goblet cell density was evaluated in periodic acid Schiff-stained conjunctival sections. RESULTS Tear volume and EGF protein levels were significantly reduced in SCOP5D mice compared with controls, indicating that cholinergic blockade decreased LG secretory function. LGs of SCOP2D and SCOP5D mice showed an increased density of CD4(+), CD11c+, CD11b+, and myeloperoxidase+ cells compared with UT controls. At day 5, these cells were significantly elevated compared with SAL-treated counterparts. Elevated levels of IL-17A, IL-17R, IFN-γ, IL-12Rβ1, IL-2, IL-13, IL-6, IL-1β, and TNF-α transcripts were noted in SCOP2D mice and IFN-γ, TGF-β1, and IL-18R transcripts in SCOP5D mice. CONCLUSIONS Pharmacological blockade of lacrimal secretion induced a significant CD4(+) infiltration in the LG, mimicking Sjögrens syndrome. The mRNA expression profile revealed elevations of a mix of inflammatory cytokines and Th-1-associated factors.

Spontaneous Autoimmune Dacryoadenitis in Aged CD25KO Mice

To investigate time-related immunopathological changes in the lacrimal glands (LGs) of CD25KO mice, we examined LGs of C57BL/6 (wild-type) and CD25KO mice at 8, 12, and 16 weeks of age. T cell infiltration was quantified by flow cytometry, and gland function by tear peroxidase activity and epidermal growth factor mRNA expression. T helper (Th)-1, -2 and -17-associated cytokine expression was evaluated by real-time PCR. Epithelial apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and activated caspase-3 staining. Eight-week-old CD25KO mice demonstrated significantly increased numbers of CD4 and CD8 T cells infiltrating the LGs. This peaked at 12 weeks of age. No peroxidase secretion was detected, and epidermal growth factor mRNA expression was barely detected in CD25KO mice. Ductal epithelial apoptosis was noted in CD25KO mice. Young CD25KO LGs had higher Th-17- (interleukin [IL]-23R, transforming growth factor-beta1, IL-17A, CC chemokine attractant ligand-20) and Th-1-associated cytokine transcripts (interferon-gamma, T-bet, IL-12, IL-2, IL-18) than young wild-type LGs. There was also a significant time-related decrease in IL-17A and CC chemokine attractant ligand-20 in CD25KO LGs. Taken together, autoimmune LG infiltration with loss of LG function was observed in CD25KO mice as early as 8 weeks of age. Time-related switch from Th-17 to Th-1 inflammation was noted in CD25KO mice.

Cleavage of functional IL-2 receptor alpha chain (CD25) from murine corneal and conjunctival epithelia by MMP-9

BackgroundIL-2 has classically been considered a cytokine that regulates T cell proliferation and differentiation, signaling through its heterotrimeric receptor (IL-2R) consisting of α (CD25), β (CD122), γ chains (CD132). Expression of IL-2R has also been detected in mucosal epithelial cells. Soluble IL-2Rα (CD25) has been reported as an inflammatory marker. We evaluated the expression of CD25 and CD122 in the ocular surface epithelium and investigated the mechanism of proteolytic cleavage of CD25 from these cells.MethodsDesiccating stress (DS) was used as an inducer of matrix metalloproteinase 9 (MMP-9). DS was created by subjecting C57BL/6 and MMP-9 knockout (BKO) mice and their wild-type littermates (WT) mice to a low humidity and drafty environment for 5 days (DS5). A separate group of C57BL/6 mice was subjected to DS5 and treatment with topical 0.025% doxycycline, a MMP inhibitor, administered QID. The expression of CD25 and CD122 was evaluated in cryosections by dual-label laser scanning confocal microscopy. Western blot was used to measure relative levels of CD25 in epithelial lysates. Gelatinase activity was evaluated by in situ zymography. Soluble CD25 in tear fluid was measured by an immunobead assay.ResultsCD25 and CD122 were abundantly expressed in cornea (all layers) and conjunctiva epithelia (apical and subapical layers) in nonstressed control mice. After desiccating stress, we found that immunoreactivity to CD25, but not CD122, decreased by the ocular surface epithelia and concentration of soluble CD25 in tears increased as MMP-9 staining increased. CD25 was preserved in C57BL/6 mice topically treated with an MMP-9 inhibitor and in MMP-9 knock-out mice. MMP-9 treatment of human cultured corneal epithelial cells decreased levels of CD25 protein in a concentration dependent fashion.ConclusionOur results indicate that functional IL-2R is produced by the ocular surface epithelia and that CD25 is proteolytic cleaved to its soluble form by MMP-9, which increases in desiccating stress. These findings provide new insight into IL-2 signaling in mucosal epithelia.