Dataset Information

Severity of Sjogren's Syndrome Keratoconjunctivitis Sicca Increases with Increased Percentage of Conjunctival Antigen-Presenting Cells.

ABSTRACT: This study investigated the relationship between clinical severity and percentage of conjunctival antigen-presenting cells (APCs) in Sjögren's syndrome (SS)-associated keratoconjunctivitis sicca (KCS). KCS clinical severity was based on symptom severity, tear volume, tear break-up time, and ocular surface dye staining. Conjunctival goblet cell density (GCD) was measured in periodic acid Schiff (PAS)-stained membranes. Conjunctival cells obtained by impression cytology were used for flow cytometry to measure percentages of CD45⁺HLA-DR⁺ APCs and mature CD11c⁺CD86⁺ dendritic cells (DCs). Compared to normal conjunctiva, the percentages of HLA-DR⁺ and CD11c⁺CD86⁺ cells were higher in the conjunctiva of the KCS group (p < 0.05). The percentage of CD45⁺HLA-DR⁺ cells positively correlated with clinical severity (r = 0.71, p < 0.05) and negatively correlated with GCD (r = -0.61, p < 0.05). Clinical severity also negatively correlated with GCD (r = -0.54, p < 0.05). These findings indicate that a higher percentage of APCs and mature DCs in the conjunctiva is associated with more severe KCS in SS. These APCs may contribute to the generation of the pathogenic Th1 cells that cause goblet cell loss in KCS.

SUBMITTER: Pflugfelder SC

PROVIDER: S-EPMC6165102 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Severity of Sjögren's Syndrome Keratoconjunctivitis Sicca Increases with Increased Percentage of Conjunctival Antigen-Presenting Cells.

Pflugfelder Stephen C SC Bian Fang F Gumus Koray K Farley William W Stern Michael E ME De Paiva Cintia S CS

International journal of molecular sciences 20180914 9

This study investigated the relationship between clinical severity and percentage of conjunctival antigen-presenting cells (APCs) in Sjögren's syndrome (SS)-associated keratoconjunctivitis sicca (KCS). KCS clinical severity was based on symptom severity, tear volume, tear break-up time, and ocular surface dye staining. Conjunctival goblet cell density (GCD) was measured in periodic acid Schiff (PAS)-stained membranes. Conjunctival cells obtained by impression cytology were used for flow cytometr ...[more]

PMID: 30223431

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Project description:Sjögren syndrome (SS) is an autoimmune condition that targets the salivary and lacrimal glands, with cardinal clinical signs of dry eye (keratoconjunctivitis sicca, KCS) and dry mouth. The conjunctiva of SS patients is often infiltrated by immune cells that participate in the induction and maintenance of local inflammation. The purpose of this study was to investigate immune-related molecular pathways activated in the conjunctiva of SS patients. Female SS patients (n=7) and controls (n=19) completed a series of oral, ocular surface exams. Symptom severity scores were evaluated using validated questionnaires (OSDI and SANDE). All patients fulfilled the ACR/EULAR criteria for SS and the criteria for KCS. Fluorescein and lissamine green dye staining evaluated tear-break-up time (TBUT), corneal and conjunctival disease, respectively. Impression cytology of the temporal bulbar conjunctiva was performed to collect cells lysed and subjected to gene expression analysis using the NanoString Immunology Panel. 53/594 differentially expressed genes (DEGs) were observed between SS and healthy controls; 49 DEGs were upregulated, and 4 were downregulated (TRAF5, TGFBI, KLRAP1, and CMKLRI). The top 10 DEGs in descending order were BST2, IFITM1, LAMP3, CXCL1, IL19, CFB, LY96, MX1, IL4R, CDKN1A. Twenty pathways had a global significance score greater or equal to 2. Spearman correlations showed that 29/49 upregulated DEGs correlated with either TBUT (inverse) or OSDI or conjunctival staining score (positive correlations). Venn diagrams identified that 26/29 DEGs correlated with TBUT, 5/26 DEGs correlated with OSDI, and 16/26 correlated with conjunctival staining scores. Five upregulated DEGs (CFB, CFI, IL1R1, IL2RG, IL4R) were uniquely negatively correlated with TBUT. These data indicate that the conjunctiva of SS patients exhibits a phenotype of immune activation, although some genes could be inhibitory. Some of the DEGs and pathways overlap with previous DEGs in salivary gland biopsies, but new DEGs were identified, and some of these correlated with symptoms and signs of dry eye. Our results indicate that gene analysis of conjunctiva imprints is a powerful tool to understand the pathogenesis of SS and develop new therapeutic targets.

Project description:BackgroundLacrimal 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.MethodsDS 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.ResultsDS 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 log2 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.ConclusionsThere 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.

Project description:Conjunctival epithelium forms a barrier between the ocular surface microbial flora and the ocular mucosa. In addition to secreting gel-forming mucins, goblet cells, located in the conjunctival epithelium, help maintain local immune homeostasis by secreting active TGFβ2 and promoting tolerogenic phenotype of dendritic cells in the vicinity. Although dendritic cell subsets, characteristic of mucosal tissues, are found in the conjunctiva, previous studies provided limited information about their location within the tissue. In this study, we examine immunostained conjunctiva explants to determine the location of CD11c-positive dendritic cells in the context of MUC5AC-positive goblet cells. Considering that conjunctival goblet cells are responsive to signaling induced by pathogen recognition receptors, we also assess if their responses to microbial product, flagellin, can contribute to the disruption of ocular mucosal homeostasis that promotes activation of dendritic cells and results in chronic ocular surface inflammation. We find that dendritic cells in the conjunctiva with an increased microbial colonization are located adjacent to goblet cells. While their cell bodies in the stromal layer are immediately below the epithelial layer, several extensions of dendritic cells are projected across the epithelium towards the ocular surface. Such trans-epithelial dendrites are not detectable in healthy ocular mucosa. In response to topically applied flagellin, increased proportion of CD11c-positive cells in the conjunctiva strongly express MHC class II relative to the untreated conjunctiva. This change is accompanied by reduced immunoreactivity to TGFβ-activating Thrombospondin-1 in the conjunctival epithelium. These findings are supported by in vitro observations in primary cultures of goblet cells that respond to the TLR5 stimulation with an increased expression of IL-6 and reduced level of active TGFβ. The observed changes in the conjunctiva after flagellin application correspond with the development of clinical signs of chronic ocular mucosal inflammation including corneal epitheliopathy. Collectively, these findings demonstrate the ability of ocular mucosal dendritic cells to extend trans-epithelial dendrites in response to increased microbial colonization at the ocular surface. Moreover, this study provides key insight into how goblet cell responses to microbial stimuli may contribute to the disruption of ocular mucosal homeostasis and chronic ocular mucosal inflammation.

Dataset Information

Severity of Sjogren's Syndrome Keratoconjunctivitis Sicca Increases with Increased Percentage of Conjunctival Antigen-Presenting Cells.

Publications

Severity of Sjögren's Syndrome Keratoconjunctivitis Sicca Increases with Increased Percentage of Conjunctival Antigen-Presenting Cells.

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