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CD25 knock-out (CD25KO) mice spontaneously develop Sjögren Syndrome (SS)-like inflammation. We investigated the role of commensal bacteria by comparing CD25KO mice housed in conventional or germ-free conditions. Germ-free CD25KO mice have greater corneal barrier dysfunction, lower goblet cell density, increased total lymphocytic infiltration score, increased expression of IFN-γ, IL-12 and higher a frequency of CD4IFN-γ cells than conventional mice. CD4 T cells isolated from female germ-free CD25KO mice adoptively transferred to naive immunodeficient RAG1KO recipients caused more severe Sjögren-like disease than CD4 T cells transferred from conventional CD25KO mice. Fecal transplant in germ-free CD25KO mice reversed the spontaneous dry eye phenotype and decreased the generation of pathogenic CD4IFN-γ cells. Our studies indicate that lack of commensal bacteria accelerates the onset and severity of dacryoadenitis and generates autoreactive CD4T cells with greater pathogenicity in the CD25KO model, suggesting that the commensal bacteria or their metabolites products have immunoregulatory properties that protect exocrine glands in the CD25KO SS model.

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Aging is a significant risk factor for dry eye. Here we used a murine aged mode to investigate the effects of aging on antigen presenting cells (APCs) and generation of pathogenic T helper (Th)1. Our results showed that APCs from aged mice accumulate at the conjunctiva, have higher levels of co-activation marker CD86 and lower aldehyde dehydrogenase activity. Using topical ovalbumin peptide as a surrogate antigen, we observed an increased number of antigen-loaded APCs in the draining cervical lymph nodes in the aged group and loss of tight junction protein occludin in the conjunctiva. Aged cervical lymph nodes APCs showed a greater generation of Th1 cells than young APCs in antigen-presentation assays in vitro . Aged lacrimal glands and draining nodes showed an accumulation of IFN-γ producing CD4 + T cells, while Th17 cells were present only in aged draining nodes. There was also an age-related increase in CD4 + CXCR3 + IFN-γ + cells in the conjunctiva, nodes and lacrimal glands while CD4 + CCR6 + IL-17A + cells increased in the draining nodes of aged mice. Adoptive transfer of aged CD4 + CXCR3 + donor cells into young, naive immunodeficient recipients caused greater goblet cell loss than young CD4 + CXCR3 + cells. Our results demonstrate that age-associated changes in APCs are critical for the pathogenesis of age-related dry eye.

Conjunctival goblet cell loss in ocular surface diseases is accompanied by increased number of interleukin-12 (IL-12)-producing antigen-presenting cells (APCs) and increased interferon-γ (IFN-γ) expression. This study tested the hypothesis that mouse conjunctival goblet cells produce biologically active retinoic acid (RA) that suppresses CD86 expression and IL-12 production by myeloid cells. We found that conditioned media from cultured conjunctival goblet cells (CjCM) suppressed stimulated CD86 expression, NF-κB p65 activation and IL-12 and IFN-γ production in unstimulated and lipopolysaccharide-stimulated cultured bone marrow-derived cells (BMDCs) containing a mixed population of APCs. Goblet cell-conditioned, ovalbumin-loaded APCs suppressed IFN-γ production and increased IL-13 production in co-cultured OTII cells. The goblet cell suppressive activity is due in part to their ability to synthesize RA from retinol. Conjunctival goblet cells had greater expression of aldehyde dehydrogenases Aldh1a1 and a3 and ALDEFLUOR activity than cornea epithelium lacking goblet cells. The conditioning activity was lost in goblet cells treated with an ALDH inhibitor, and a retinoid receptor alpha antagonist blocked the suppressive effects of CjCM on IL-12 production. Similar to RA, CjCM increased expression of suppressor of cytokine signaling 3 (SOCS3) in BMDCs. SOCS3 silencing reversed the IL-12-suppressive effects of CjCM. Our findings indicate that conjunctival goblet cells are capable of synthesizing RA from retinol secreted by the lacrimal gland into tears that can condition APCs. Evidence suggests goblet cell RA may function in maintaining conjunctival immune tolerance and loss of conjunctival goblet cells may contribute to increased Th1 priming in dry eye.

Aging is a complex process associated with dysregulation of the immune system and low levels of inflammation, often associated with the onset of many pathologies. The lacrimal gland (LG) plays a vital role in the maintenance of ocular physiology and changes related to aging directly affect eye diseases. The dysregulation of the immune system in aging leads to quantitative and qualitative changes in antibodies and cytokines. While there is a gradual decline of the immune system, there is an increase in autoimmunity, with a reciprocal pathway between low levels of inflammation and aging mechanisms. Elderly C57BL/6J mice spontaneously show LGs infiltration that is characterized by Th1 but not Th17 cells. The aging of the LG is related to functional alterations, reduced innervation and decreased secretory activities. Lymphocytic infiltration, destruction, and atrophy of glandular parenchyma, ductal dilatation, and secretion of inflammatory mediators modify the volume and composition of tears. Oxidative stress, the capacity to metabolize and eliminate toxic substances decreased in aging, is also associated with the reduction of LG functionality and the pathogenesis of autoimmune diseases. Although further studies are required for a better understanding of autoimmunity and aging of the LG, we described anatomic and immunology aspects that have been described so far.

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PurposeTo investigate IL-17 related mechanisms for developing dry eye disease in the Pinkie mouse strain with a loss of function RXRα mutation.MethodsMeasures 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.ResultsCompared 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+ γδ T cells in Pinkie. Tear concentration of the IL-17 inducer IL-23 is significantly higher in Pinkie. 9-cis RA treatment suppresses stimulated IL-17 production by γδ T and stimulatory activity of monocyte supernatant on γδ T cell IL-17 production. Compared to WT bone marrow chimeras, Pinkie chimeras have increased IL-17+ γδ T cells in the conjunctiva after desiccating stress and anti-IL-17 treatment suppresses dry eye induced corneal MMP-9 production/activity and conjunctival goblet cell loss.ConclusionThese 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.