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

The conjunctiva contains a specialized population of lymphocytes that reside in the epithelium, named intraepithelial lymphocytes (IEL).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.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

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

Abstract Background IL-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. Methods Desiccating 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. Results CD25 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. Conclusion Our 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.</p

Flow cytometry analysis of freshly isolated cells from the ocular surface.

<p>Data is presented as mean ±standard deviation of 5–6 different experiments per group/time point. Lymphocytes were gated based on characteristic light-scatter properties (“gated cells”), subsequently gated based on forward scatter height vs. forward scatter area and propridium iodide live/dead exclusion (“live gated cells”). Data presented represents percentage of positive (⁺) or negative (^-) cells after background subtraction.</p><p>NS = non-stressed, DS5 = desiccating stress for 5 days, DS10 = desiccating stress for 10 days.</p

Adoptive transfer results.

<p><b>A</b> Mean± SD of IL-17 ELISPOTs showing IL-17- producing cells isolated from the ocular surface (OS) and CD4⁺ T cells isolated from spleen and cervical lymph nodes (CLN) in donor mice that received systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) antibody before (non-stressed, NS) and after 5 days of desiccating stress (DS5). Experiments were repeated two times with at least five mice per group per experiment. <b>B</b> Representative images of OGD corneal staining used to generate OGD intensity score in <b>C.</b> Bar charts show mean ± SD of three independent experiments with five mice for each group per experiment. <b>D-G-</b> Laser scanning immunofluorescent confocal microscopy of cornea immunostained for MMP-3 (in <b>D</b>) and MMP-9 (in <b>F</b>) in nude mice that received CD4⁺T cells isolated from donor mice treated with systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) after 5 days of desiccating stress (DS5). Bar graphs are mean±SD of fluorescence intensity measured in corneal epithelium for MMP-3 (E) and MMP-9 (G) of a total of two independent experiments with at least three mice per group per experiment. <b>H-K</b>-Gene expression analyses showing mean± SD (copies) of IL-17A (in <b>H</b>), CCL20 (in <b>I</b>), matrix metalloproteinases (MMP)-3 (in <b>J</b>) and MMP-9 (in <b>K</b>) mRNA transcripts in cornea epithelia of nude mice that received CD4⁺T cells isolated from donor mice that had received systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) after 5 days of desiccating stress (DS5). Data represents mean ± SD. Experiments were repeated two times with at least three mice per group per experiment.</p

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-β1, IL-17A, CC chemokine attractant ligand-20) and Th-1-associated cytokine transcripts (interferon-γ, 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