Conjunctival Inflammatory Gene Expression Profiling in Dry Eye Disease: Correlations With HLA-DRA and HLA-DRB1

Purpose: In several multicenter clinical trials, HLA-DR was found to be a potential biomarker of dry eye disease (DED)'s severity and prognosis. Given the fact that HLA-DR receptor is a heterodimer consisting in an alpha and a beta chain, we intended to investigate the correlation of inflammatory targets with the corresponding transcripts, HLA-DRA and HLA-DRB1, to characterize specific targets closely related to HLA-DR expressed in conjunctival cells from patients suffering from DED of various etiologies.Methods: A prospective study was conducted in 88 patients with different forms of DED. Ocular symptom scores, ocular-staining grades, tear breakup time (TBUT) and Schirmer test were evaluated. Superficial conjunctival cells were collected by impression cytology and total RNAs were extracted for analyses using the new NanoString® nCounter technology based on an inflammatory human code set containing 249 inflammatory genes.Results: Two hundred transcripts were reliably detected in conjunctival specimens at various levels ranging from 1 to 222,546 RNA copies. Overall, from the 88 samples, 21 target genes showed a highly significant correlation (R > 0.8) with HLA-DRA and HLA-DRB1, HLA-DRA and B1 presenting the highest correlation (R = 0.9). These selected targets belonged to eight family groups, namely interferon and interferon-stimulated genes, tumor necrosis factor superfamily and related factors, Toll-like receptors and related factors, complement system factors, chemokines/cytokines, the RIPK enzyme family, and transduction signals such as the STAT and MAPK families.Conclusions: We have identified a profile of 21 transcripts correlated with HLA-DR expression, suggesting closely regulated signaling pathways and possible direct or indirect interactions between them. The NanoString® nCounter technology in conjunctival imprints could constitute a reliable tool in the future for wider screening of inflammatory biomarkers in DED, usable in very small samples. Broader combinations of biomarkers associated with HLA-DR could be analyzed to develop new diagnostic approaches, identify tighter pathophysiological gene signatures and personalize DED therapies more efficiently

modulation of inflammation related genes in the cornea of a mouse model of dry eye upon treatment with cyclosporine eye drops

ABSTRACTPurpose/Aim: Inflammation is recognized as playing an etiological role in dry eye disease. This study aimed to assess the efficacy of various topical cyclosporine A (CsA) formulations on co..

Explorations physiopathologiques de la maladie de l’œil sec à travers la recherche de biomarqueurs

Dry eye disease (DED) or keratoconjunctivitis sicca (KCS) is a multifactorial pathology of the anterior segment of the eye affecting the ocular surface (OS). This thesis work aimed to decipher and explore the cellular and molecular mechanisms underlying the initiation and progression of the disease, which could lead to the identification of candidate biomarkers. This was undertaken through targeted or untargeted molecular omics approaches, using human samples of patient OS and experimental models of MOS. This research program was structured into 3 main parts. Firstly, omics approaches and pre-analytical considerations of OS from human samples. Then, clinical phenotyping with specific imaging for precise classification of patients. Finally, the identification of candidate biomarkers for dry eye. This multimodal approach ensured the application of a rational methodology for the dissection of molecular networks from rare samples such as conjunctival imprints or Schrimer strips. The identified molecular effectors highlight the role of epithelial cells in OS homeostasis through the cross-talk between oxidative stress and inflammation, mediated by the IFNs and TNF-alpha pathways, among others. In addition, clinical phenotyping through anatomical characteristics through precision or cellular imaging of the OS has made it possible to provide new thinking to reconsider the refined classification of patients into subgroups.La maladie de l’œil sec (MOS) ou la kératoconjonctivite sèche (KCS) est une pathologie multifactorielle du segment antérieur de l’œil altérant la surface oculaire (SO). Ce travail de thèse a eu pour but de décrypter et d’explorer les mécanismes cellulaires et moléculaires qui sous-tendent l'initiation et la progression de la maladie, pouvant conduire à l’identification des biomarqueurs candidats. Cela a été entrepris à travers des approches moléculaires omiques ciblées ou non ciblées, à partir de prélèvements humains de la SO de patients et de modèles expérimentaux de la MOS. Ce programme de recherche s’est articulé selon 3 parties principales. En premier lieu, les approches omiques et les considérations pré-analytiques de la SO à partir des prélèvements humains. Ensuite, un phénotypage clinique avec imagerie spécifique pour une classification précise des patients. Enfin, l’identification des biomarqueurs candidats pour l’œil sec. Cette approche multimodale a permis d’assurer l’application d'une méthodologie rationnelle pour la dissection des réseaux moléculaires à partir des prélèvements rares comme les empreintes conjonctivales ou les bandelettes de Schrimer. Les effecteurs moléculaires identifiés mettent en lumière le rôle des cellules épithéliales dans l’homéostasie de la SO par les relations croisées entre le stress oxydant et l’inflammation, médiés par les voies des IFNs et du TNF-alpha, entres autres. De plus, le phénotypage clinique à travers des caractéristiques anatomiques par l’imagerie de précision ou cellulaire de la SO, a permis d’apporter une réflexion nouvelle pour reconsidérer la classification affinée des patients en sous-groupes

Comparison of Two Experimental Mouse Dry Eye Models through Inflammatory Gene Set Enrichment Analysis Based on a Multiplexed Transcriptomic Approach

The goal of this study was to explore the specific signaling pathways related to inflammation in two experimental mouse dry eye (EDE) models. Female C57BL/6 mice housed for 10 days in a controlled desiccative environment were either treated with scopolamine (EDE-1; n = 18) or subjected to extraorbital lacrimal gland excision bilaterally (EDE-2; n = 10). Non-induced mice (n = 20) served as healthy controls. A corneal fluorescein staining (CFS) scoring was used at baseline through to day (D) 10 to evaluate epitheliopathy. At D10, corneas and conjunctivas were collected for multiplexed transcriptomic analysis with the NanoString® mouse inflammatory CodeSet. Both EDE-1 and EDE-2 mice presented a change in corneal integrity, with a significant increase in CFS scores at D10. More gene transcripts were identified in EDE-2 compared with EDE-1 (116 vs. 96, respectively), and only a few were common to both models, 13 for the cornea and 6 for the conjunctiva. The gene functional annotation analysis revealed that the same inflammatory pathways were involved in both models. Comparative profiling of gene expression in the two EDE models leads to the identification of various targets and signaling pathways, which can be extrapolated to and confirmed in human disease

The Dual Effect of Rho-Kinase Inhibition on Trabecular Meshwork Cells Cytoskeleton and Extracellular Matrix in an In Vitro Model of Glaucoma

International audienceThe trabecular meshwork (TM) is the main site of drainage of the aqueous humor, and itsdysfunction leads to intraocular pressure elevation, which is one of the main risk factors of glaucoma.We aimed to compare the effects on cytoskeleton organization and extracellular matrix (ECM) oflatanoprost (LT) and a Rho-kinase inhibitor (ROCKi) on a transforming growth factor beta2 (TGF- 2)-induced glaucoma-like model developed from primary culture of human TM cells (pHTMC). TheTGF- 2 stimulated pHTMC were grown and incubated with LT or a ROCKi (Y-27632) for 24 h.The expression of alpha-smooth muscle actin ( SMA) and fibronectin (FN), and phosphorylationof the myosin light chain (MLC-P) and Cofilin (Cofilin-P) were evaluated using immunofluorescenceand Western blot. The architectural modifications were studied in a MatrigelTM 3D culture.TGF- 2 increased the expression of  SMA and FN in pHTMC and modified the cytoskeleton withcross-linked actin network formation. LT did not alter the expression of  SMA but decreased FNdeposition. The ROCKi decreased TGF- 2-induced  SMA and FN expression, as well as MLC-P andCofilin-P, and stimulated the cells to recover a basal cytoskeletal arrangement. In the preliminary3D study, pHTMC organized in a mesh conformation showed the widening of the TM under theeffect of Y-27632. By simultaneously modifying the organization of the cytoskeleton and the ECM,with fibronectin deposition and overexpression, TGF- 2 reproduced the trabecular degenerationdescribed in glaucoma. The ROCKi was able to reverse the TGF- 2-induced cytoskeletal and ECMrearrangements. LT loosened the extracellular matrix but had no action on the stress fibers

Profiling tear film enzymes reveals major metabolic pathways involved in the homeostasis of the ocular surface

Abstract The ocular surface (OS) enzymes are of great interest due to their potential for novel ocular drug development. We aimed first to profile and classify the enzymes of the OS to describe major biological processes and pathways that are involved in the maintenance of homeostasis. Second, we aimed to compare the enzymatic profiles between the two most common tear collection methods, capillary tubes (CT) and Schirmer strips (ScS). A comprehensive tear proteomic dataset was generated by pooling all enzymes identified from nine tear proteomic analyses of healthy subjects using mass spectrometry. In these studies, tear fluid was collected using CT (n = 4), ScS (n = 4) or both collection methods (n = 1). Classification and functional analysis of the enzymes was performed using a combination of bioinformatic tools. The dataset generated identified 1010 enzymes. The most representative classes were hydrolases (EC 3) and transferases (EC 2). Phosphotransferases, esterases and peptidases were the most represented subclasses. A large portion of the identified enzymes was common to both collection methods (n = 499). More enzymes were specifically detected in the ScS-extracted proteome. The major pathways in which the identified enzymes participate are related to the immune system and protein, carbohydrate and lipid metabolism. Metabolic processes for nucleosides, cellular amides, sugars and sulfur compounds constituted the most enriched biological processes. Knowledge of these molecules highly susceptible to pharmacological manipulation might help to predict the metabolism of ophthalmic medications and develop novel prodrug strategies as well as new drug delivery systems. Combining such extensive knowledge of the OS enzymes with new analytical approaches and techniques might create new prospects for understanding, predicting and manipulating the metabolism of ocular pharmaceuticals. Our study reports new, essential data on OS enzymes while also comparing the enzyme profiles obtained via the two most popular methods of tear collection, capillary tubes and Schirmer strips

Proteomic Analysis of Tears and Conjunctival Cells Collected with Schirmer Strips Using timsTOF Pro: Preanalytical Considerations

International audienceThis study aimed to investigate the human proteome profile of samples collected from whole (W) Schirmer strips (ScS) and their two parts—the bulb (B) and the rest of the strip (R)—with a comprehensive proteomic approach using a trapped ion mobility mass spectrometer, the timsTOF Pro. Eight ScS were collected from two healthy subjects at four different visits to be separated into three batches, i.e., 4W, 4B, and 4R. In total, 1582 proteins were identified in the W, B, and R batches. Among all identified proteins, binding proteins (43.4%) and those with catalytic activity (42.2%) constituted more than 80% of the molecular functions. The most represented biological processes were cellular processes (31.2%), metabolic processes (20.8%), and biological regulation (13.1%). Enzymes were the most represented protein class (41%), consisting mainly of hydrolases (47.5%), oxidoreductases (22.1%), and transferases (16.7%). The bulb (B), which is in contact with the conjunctiva, might collect both tear and cell proteins and therefore promote the identification of more proteins. Processing B and R separately before mass spectrometry (MS) analysis, combined with the high data acquisition speed and the addition of ion-mobility-based separation in the timsTOF Pro, can bring a new dimension to biomarker investigations of a limited sample such as tear fluid

Identification of new Omega-3 very long chain poly-unsaturated fatty acids in meibomian gland secretions

International audienc