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Purpose: To evaluate the effects of dry eye on conjunctival immune cell number and transcriptional profiles with attention to mononuclear phagocytes., Methods: Expression profiling was performed by single-cell RNA sequencing on sorted conjunctival immune cells from non-stressed and C57BL/6 mice subjected to desiccating stress (DS). Monocle 3 modeled cell trajectory, scATAC-seq assessed chromatin accessibility and IPA identified canonical pathways. Inflammation and goblet cells were measured after depletion of MRC1 + MΦs with mannosylated clodronate liposomes., Results: Mononuclear phagocytes (monocytes, MΦs, DCs) comprised 72 % of immune cells and showed the greatest changes with DS. Distinct DS induced gene expression patterns were seen in phagocytes classified by expression of Ccr2 and [Timd4, Lyve1, Folr2 (TLR)]. Expression of phagocytosis/efferocytosis genes increased in TLF + CCR2 - MΦs. Monocytes showed the highest expression of Ace, Cx3cr1, Vegfa, Ifngr1,2, and Stat1 and TLF - CCR2 + cells expressed higher levels of inflammatory mediators (Il1a, Il1b, Il1rn, Nfkb1, Ccl5, MHCII, Cd80, Cxcl10, Icam1). A trajectory from monocyte precursors branched to terminate in regulatory MΦs or in mDCs via transitional MΦ and cDC clusters. Activated pathways in TLF + cells include phagocytosis, PPAR/RXRα activation, IL-10 signaling, alternate MΦ activation, while inflammatory pathways were suppressed. Depletion of MRC1 + MΦs increased IL-17 and IFN-γ expression and cytokine-expressing T cells, reduced IL-10 and worsened goblet loss., Conclusions: Dryness stimulates distinct gene expression patterns in conjunctival phagocytes, increasing expression of regulatory genes in TLF + cells regulated in part by RXRα, and inflammatory genes in CCR2 + cells. Regulatory MΦs depletion worsens DS induced inflammation and goblet cell loss., Competing Interests: Declaration of competing interest Patent BLG 22–028; “RXR Agonists in Eye Disorders”; BAYM. P0358WO was filed by Baylor College of Medicine on 1/29/23 and lists SCP and JA as coinventors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Lacrimal gland secretory dysfunction in Sjögren syndrome (SS) causes ocular surface desiccation that is associated with increased cytokine expression and number of antigen-presenting cells (APCs) in the conjunctiva. This study evaluated the hypothesis that desiccating stress (DS) alters the percentage and gene expression of myeloid cell populations in the conjunctiva., Methods: DS was induced by pharmacologic suppression of tear secretion and exposure to drafty low humidity environment. Bone marrow chimeras and adoptive transfer of CD45.1 + bone marrow cells to CD45.2 + C-C chemokine receptor 2 knockout (CCR2 -/- ) mice were used to track DS-induced myeloid cell recruitment to the conjunctiva. Flow cytometry evaluated myeloid cell populations in conjunctivae obtained from non-stressed eyes and those exposed to DS for 5 days. CD11b + myeloid lineage cells were gated on monocyte (Ly6C), macrophage (CD64, MHCII) and DC (CD11c, MHCII) lineage markers. NanoString immune arrays were performed on sorted MHCII + and MHCII - monocyte/macrophage cell populations., Results: DS significantly increased the recruitment of adoptively transferred MHCII positive and negative myeloid cells to the conjunctiva in a CCR2 dependent fashion. The percentage of resident conjunctival monocytes (Ly6C + CD64 - ) significantly decreased while CD64 + MHCII + macrophages increased over 5 days of DS (P<0.05 for both). Comparison of gene expression between the MHCII - monocyte and MHCII + populations in non-stressed conjunctiva revealed a ≥ 2 log 2 fold increase in 95 genes and decrease in 46 genes. Upregulated genes are associated with antigen presentation, cytokine/chemokine, M1 macrophage and NLRP3 inflammasome pathways. DS increased innate inflammatory genes in monocytes and MHCII + cells and increased M1 macrophage (Trem1, Ido1, Il12b, Stat5b) and decreased homeostasis (Mertk) and M2 macrophage (Arg1) genes in MHCII + cells., Conclusions: There are myeloid cell populations in the conjunctiva with distinct phenotype and gene expression patterns. DS recruits myeloid cells from the blood and significantly changes their phenotype in the conjunctiva. DS also alters expression of an array of innate inflammatory genes. Immature monocytes in the unstressed conjunctiva appear to cascade to MHCII + macrophages during DS, suggesting that DS promotes maturation of monocytes to antigen presenting cells with increased expression of inflammatory genes that may contribute to the pathogenesis of SS keratoconjunctivitis sicca., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alam, de Paiva and Pflugfelder.)

The conjunctiva is a goblet cell rich mucosal tissue. Goblet cells are supported by tear growth factors and IL-13 produced by resident immune cells. Goblet cell secretions are essential for maintaining tear stability and ocular surface homeostasis. In addition to producing tear stabilizing mucins, they also produce cytokines and retinoic acid that condition monocyte-derived phagocytic cells in the conjunctiva. Aqueous tear deficiency from lacrimal gland disease and systemic inflammatory conditions results in goblet cell loss that amplifies dry eye severity. Reduced goblet cell density is correlated with more severe conjunctival disease, increased IFN-γ expression and antigen presenting cell maturation. Sterile Alpha Motif (SAM) pointed domain epithelial specific transcription factor (Spdef) gene deficient mice that lack goblet cells have increased infiltration of monocytes and dendritic cells with greater IL-12 expression in the conjunctiva. Similar findings were observed in the conjunctiva of aged mice. Reduced retinoic acid receptor (RXRα) signaling also increases conjunctival monocyte infiltration, IFN-γ expression and goblet cell loss. Evidence suggests that dry eye therapies that suppress IFN-γ expression preserve conjunctival goblet cell number and function and should be considered in aqueous deficiency., Competing Interests: Declaration of competing interest None of the authors have any financial or personal relationships to disclose that would cause a conflict of interest regarding this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: The purpose of this study was to evaluate the potential of voclosporin (VOS) in preventing goblet cell (GC) loss and modulating interferon-gamma (IFN-γ) producing CD4+ T cells in the mouse desiccating stress (DS) dry eye model. Methods: Mice were subjected to DS and treated topically with vehicle, VOS, or cyclosporine A as a treatment control. Corneal barrier function was evaluated after 5 and conjunctival GC density after 10 days of desiccation. CD4+ T cells were isolated from ocular surface draining lymph nodes of dry eye donor mice and adoptively transferred into immune deficient RAG1-/- mice from which tears and conjunctiva were collected for the evaluation of inflammatory cytokines/chemokines and GC density. Results: Compared to the vehicle-treated group, VOS was significantly better in preserving corneal barrier function and preventing DS-induced conjunctival GC loss. CD4+ T cells from VOS treated dry eye donors caused less conjunctival GC loss than vehicle and suppressed expression of IFN-γ signature genes to a similar extent and transforming growth factor-beta to a greater extent than cyclosporine in adoptive transfer recipients. Conclusion: These findings suggest that VOS preserves corneal barrier function and conjunctival GCs and suppresses IFN-γ producing CD4+ T cells in experimental dry eye. [ABSTRACT FROM AUTHOR]

Corneal and conjunctival inflammation and dry eye develop in systemic vitamin A deficiency (VAD). The objective of this study was to investigate the lacrimal ocular surface retinoid axis, particularly immunomodulatory effects of retinoic acid (RA) and change in conjunctival myeloid cell number and phenotype in VAD. We discovered that ocular surface epithelial and myeloid cells express retinoid receptors. Both all trans- and 9-cis-RA suppressed production of dry eye relevant inflammatory mediators [interleukin(IL)-1β, IL-12, regulated upon activation, normal T cell expressed and secreted (RANTES)] by myeloid cells. Systemic VAD was associated with significant goblet cell loss and an increased number of CD45+ immune cells in the conjunctiva. MHCII−CD11b+ classical monocytes were significantly increased in the conjunctiva of VAD C57BL/6 and RXR-α mutated Pinkie strains. RNA seq revealed significantly increased expression of innate immune/inflammatory genes in the Pinkie conjunctiva. These findings indicate that retinoids are essential for maintaining a healthy, well-lubricated ocular surface and have immunomodulatory effects in the conjunctiva that are mediated in part via RXR-α signaling. Perturbation of the homeostatic retinoid axis could potentiate inflammation on the ocular surface. [ABSTRACT FROM AUTHOR]