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

Abstract

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.

Figure 1. Induction of Th1-associated chemokines and IFN-γ occurs early in induction of dry eye disease

A-B. Gene expression of CXCL9, CXCL10, CXCL11, and IFN-γ in cornea (A) and conjunctiva (B) of non-stressed (NS), six hours (DS6H) of desiccating stress (DS), one day of DS (DS1), five (DS5), or ten days (DS10) of DS C57BL/6 mice. C. Merged pictures of laser scanning immunofluorescent confocal microscopy of cornea and conjunctiva sections immunostained for CXCL9 (green) and CXCL10 (green) of NS, DS1, or DS5 of C57BL/6 WT mice with propidium iodide (PI) (red) nuclear counter staining. Yellow indicates strong double positive staining. D-E. Gene expression of CXCL9, CXCL10, CXCL11, and IFN-γ in cornea (E) and conjunctiva (F) of NS, DS6H, DS1, DS5, or DS10 of RAG1KO mice. F. Merged pictures of laser scanning immunofluorescent confocal microscopy of cornea and conjunctiva immunostained for CXCL9 (green) and CXCL10 (green) of NS, DS1, or DS5 of RAG1KO mice with propidium iodide (red) nuclear counter staining. Scale bar −50 μm. Results represent the mean ± SD expression of 6-8 animals per time point.*, p < 0.05; **, p < 0.01; ***, p <0.001 compared to NS. White boxes indicate magnified area.

Figure 2. Desiccating stress increases disease severity in CD4⁺ T cell adoptive transfer recipients

A. Increased T cell infiltration in recipient mice with increased DS and chemokine expression. B. Decreased numbers of goblet cells in the conjunctiva of RAG1KO recipients that were treated with DS prior to adoptive transfer. C. Gene expression of MMP-3, MMP-9 and CXCL10 in the cornea of recipient mice. D. Gene expression of IFN-γ, IL-17A and IL-6 in conjunctiva with increasing lengths of DS-treated RAG1KO recipient mice. Results represent the mean ± SEM expression of 6-8 animals per time point.*, p < 0.05; **, p < 0.01; ***, p <0.001; ns, not significant.

Figure 3. Induction of Th1-associated chemokines requires IFN-γ

A-B. Gene expression of CXCL9, CXCL10, and CXCL11 in cornea (A) and conjunctiva (B) of NS, DS6H, DS1, DS5, or DS10 of IFN-γKO mice. Results represent the mean ± SD expression of 6-8 animals per time point.*, p < 0.05; **, p < 0.01; ***, p <0.001 compared to NS.

Figure 4. Th1-associated chemokines and IFN-γ expression in DS mice requires NK cells

Gene expression of CXCL9, CXCL10, CXCL11, and IFN-γ in non-stressed (NS), six hours (DS6H) of desiccating stress (DS), one day of DS (DS1), five days of DS (DS5), or ten days (DS10) of DS NK cell-depleted RAG1KO mice (○) or isotype-treated RAG1KO mice (■) in the cornea (A.) and conjunctiva (B.). Fold changes were determined by comparing to NS samples. P values were determined by comparing anti-NK1.1-treated to isotype-treated RAG1KO mice. Results represent the mean ± SD expression of 6-8 animals per time point.*, p < 0.05; **, p < 0.01; ***, p <0.001. *, compared to isotype controls. ##, p < 0.05 compared to NS animals of the same treatment.

Figure 5. NKG2D and its ligand, RAE-1, increases with exposure to DS and NKG2D neutralization decreases expression of Th1-associated chemokines and IFN-γ

A. Immunohistochemistry staining of NKG2D (red) with black arrows in the conjunctiva of NS and DS1 C57BL/6 mice B. Merged pictures of laser scanning immunofluorescent confocal microscopy of cornea and conjunctiva sections immunostained for RAE-1 (green) in conjunctiva of NS, DS1, or DS5 WT C57BL/6 mice. Counter stained with PI (red). Scale bar −20 μm. C. Gene expression of RAE-1 in cornea and conjunctiva of non-stressed (NS), one day (DS1) of desiccating stress (DS), five days of DS (DS5), or ten days (DS10) of DS WT C57BL/6 mice. D. Neutralization of NKG2D decreases conjunctival expression of CXCL9, CXCL10, CXCL11, and IFN-γ. E. Gene expression of Tbet and CD4 in the conjunctiva of non-stressed (NS), one day (DS1) of desiccating stress (DS), five days of DS (DS5), or ten days (DS10) of DS WT C57BL/6 mice. Results represent the mean ± SD expression of 6-8 animals per time point.*, p < 0.05; **, p < 0.01 compared to NS.