The role of Mineralocorticoid Receptor pathway in Ocular Rosacea

The role of Mineralocorticoid Receptor pathway in Ocular Rosacea | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The role of Mineralocorticoid Receptor pathway in Ocular Rosacea Francine Behar-Cohen, Nilufer Yesilirmak, Danielle Rodrigues-Braz, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6589915/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Ocular rosacea (OR) is a chronic inflammatory disease of the ocular surface and the eyelids, favored by UV exposure. It can affect vision, damage the cornea and has a significant impact on quality of life. OR is linked to meibomian gland dysfunction (MGD), but is poorly understood, largely underdiagnosed and incurable. Human samples and an UVB-induced OR rat model bring evidence of the central role of mineralocorticoid receptor (MR) pathway overactivation in OR pathogenesis. In patients suffering from OR, MR was overexpressed in the meibomian glands. Signs of UVB-induced OR were exacerbated in a transgenic rat that overexpresses human MR through enhanced immune and inflammatory responses, oxidative stress and lipid dysmetabolism. Multi-omics cross-species analysis identified S100A9 as a key mediator of MR-associated pathogenicity. MR pathway antagonism appears as a potential therapeutic strategy. Health sciences/Diseases/Eye diseases/Eyelid diseases Health sciences/Molecular medicine Ocular Rosacea (OR) Meibomian gland dysfunction (MGD) Mineralocorticoid receptor (MR) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Rosacea, a prevalent chronic inflammatory skin disease, affects up to 22% of the global population 1 , 2 . Approximately three quarters of patients exhibit ocular surface involvement 3 characterized by inflammation and neurovascular dysfunction of the ocular surface and eyelids associated with Meibomian gland dysfunction (MGD) 4 . The structural and functional alteration of Meibomian glands (MGs) and the subsequent changes in the lipid composition of the tear film, result in corneal symptoms and alter the quality of life of patients with OR 5 . Eventually, neovascularization, loss of transparency and ulceration of the cornea can occur 3 . Despite its potentially blinding consequences, OR is often underdiagnosed and remains incurable, emphasizing the need for the identification of novel therapeutic targets. 6 The pathogenesis of OR is multifactorial and remains incompletely understood. It involves aberrant innate and adaptive immune responses, neurovascular dysregulation 7 – 9 , neurogenic inflammation 8 and fibrosis 10 . Activation of the toll-like receptor (TLR) 11 and transient receptor potential (TRP) ion channels 12 , 13 contribute to chronic inflammation of the ocular surface 14 . Environmental triggering factors, including ultraviolet (UV) exposure, psychosocial stress and glucocorticoids (GCs) 15 , 16 contribute to OR and to MGD, which is a major component of the disease 17 . However, the precise mechanism by which GCs, despite their anti-inflammatory effects, act on OR and contribute to MGD is unknown. Cortisol binds to the glucocorticoid receptor (GR) and to the mineralocorticoid receptor (MR) with similar high affinity. The metabolism of endogenous corticosteroids is controlled locally by hydroxysteroid dehydrogenase (11-βHSD) enzymes expressed in skin and eye tissues 18 , 19 . The low expression of 11-βHSD type 2 (11-βHSD2), which converts cortisol to inactive cortisone allows MR activation by GCs 20 , which could contribute to GC-induced OR. It has been demonstrated in previous studies that the oral administration of spironolactone, an MR antagonist, is an efficient treatment for skin rosacea 21 and that it can reduce its incidence 22 . Furthermore, the topical administration of MR antagonists has been demonstrated to improve GC-induced epidermal atrophy 23 and to enhance wound re-epithelialization in human skin treated with GC 24 . The administration of spironolactone drops increased the rate of corneal re-epithelialization in cases of wound healing delayed by glucocorticoid (GC) 25 , 26 . Furthermore, mounting evidence suggests that MR overactivation induces inflammation, oxidative stress, fibrosis, vascular dysfunction and lipid dysmetabolism, all of which are observed in OR. The subsequent investigation focused on elucidating the role of MR pathway overactivation in MGD and OR. 2. Results MR pathway is activated in eyelids from patients suffering from OR There was no significant difference in age between patients with OR and controls (67.7 ± 11, 72 ± 19 years, p = 0.54) and there were 3 men and 1 woman in both groups. In the OR group, two patients presented rhinophyma, while the two others only suffered from OR. Immunohistochemistry of GR, MR, 11βHSD1 and 11βHSD2 was performed on the eyelids. In all the examined OR samples, MR immunostaining (see Fig. 1a) was enhanced in the nuclei of conjunctival epithelial cells, meibocytes, sebocytes, and epithelial cells as compared to controls while GR signal localization and intensity did not differ (Fig. 1a). 11βHSD1 was expressed in the cytoplasm of conjunctival epithelial cells, meibocytes, epithelial cells of hair follicles and basal cells of epidermis, while 11βHSD2 exhibited diffused staining in both the cytoplasm and nuclei of these cells. In the eyelid of OR samples, 11βHSD1 immunostaining was enhanced, while 11βHSD2 immunostaining was reduced (Fig. 1b), indicating local increased availability of cortisol. The increase in the MR/GR and in the 11βHSD1/11βHSD2 ratio suggests that MR pathway is overactivation in the eyelids of patients with OR. Histological analysis of eyelid from OR patients revealed hyperplasia of the conjunctiva and the epidermis and infiltration of inflammatory cells in the conjunctiva, around MGs and beneath the epidermis (Fig. 1c). The acini appeared smaller and immature (Fig. 1c) and Sirius red that stains interstitial collagen indicated fibrosis within MG from patients suffering from OR (Fig. 1d and 1e). Overexpression of MR in P1.hMR rats favours MGD initiation. To investigate the contribution of MR overactivation to OR pathogenesis, we characterized the phenotype of the MG and ocular surface tissues from P1.hMR rats of 6 months of age 30 , 31 . It was first confirmed that the human NR3C2 transgene was expressed in the cornea, conjunctiva and MGs, similarly in male and female P1.hMR rats (Fig. 2 a). The total MR gene expression (hMR and rMR) exhibited a substantial increase in P1.hMR rats without altering endogenous rat MR expression (Fig. 2 a and Fig. S1 a). P1.hMR rats exhibited enhanced MR staining in both the nucleus and cytoplasm in cells in the conjunctiva, MG and cornea compared to WT rats, whereas no difference in GR immunostaining was observed in these tissues between both groups (Fig. 2 b and Fig. S1 b), confirming MR overexpression and increased MR/GR ratio in P1.hMR rats, similar to the human OR tissues. In the MG of P1.hMR rats, MR immunostaining demonstrated a 2.6-fold increase in MR protein expression compared to WT rats (Fig. 2 c). No gross histological differences in the MG, epidermis and conjunctiva were observed between P1.hMR and WT rats (Fig. 3 a), although incipient fibrosis was noted in the MG of P1.hMR rats (Fig. 3 b and 3 c). The expression of Ki67 and P63 in the basal layer of acini and ductal epithelium was diminished in the MG of P1.hMR rats as compared to WT (Fig. 3 d and 3 e), indicating impaired meibocyte renewal. The expression of two mitochondrial proteins, TOM20 and TIM23, was reduced in the MG acini of P1.hMR rats in comparison with WT rats (Fig. 3 f and 3 g), suggesting mitochondrial dysfunction. However, no significant consequences on MG lipid synthesis and no signs of hyperkeratinization were observed in transgenic rats (see Fig. S2a and S2b). Although a decline in MG stem cells and mitochondrial function was observed in P1.hMR rats, suggesting MGD initiation, no signs of OR was observed in 6-month old rats. MR overexpression induces molecular dysregulation in the MG of P1.hMR rats The transcriptome of the MG from P1.hMR rats of 6 months of age identified 30 differentially expressed genes (DEG) with 14 upregulated and 16 downregulated genes as compared to WT littermates (Fig. 4 a). Gene set enrichment analysis (GSEA) using REACTOME pathway database revealed pathways associated with the complement cascade, interferon signalling, keratinization, collagen and extracellular matrix organization (Fig. 4 b). HALLMARK gene set highlighted oxidative phosphorylation, angiogenesis, epithelial mesenchymal transition, adipogenesis and fatty acid metabolism pathways (Fig. 4 c). Interestingly, these pathways share a common signature with a human MGD published transcriptome 29 . Genes of particular importance in the pathogenesis of MGD include Vim, Pmel, Col3a1, Col1a1, Sdc4 and Fnln5 , which are associated with cell structure and the extracellular matrix; Cyp1b1, Cyp1a1, Pdha1, Dld, Crat and Acsm3 , which are linked to metabolism and energy production; and Kcnj8, Gas1, Wipf1 and Stc1 , which are related to signal transduction (Fig. 4 d and Fig. S3). These genes were validated by quantitative PCR (qPCR) using MG tissues from an independent batch of P1.hMR and WT rats (Fig. S3). Additional genes of interest, including Tlr3, Nos3, Cd36, Clu , and Ccl2 , identified in a published transcriptomic study on the skin of rosacea patients 30 , were also confirmed by qPCR in rat MG tissues (Fig. S3). MR overexpression exacerbates UVB-induced MGD in P1.hMR rats mimicking OR As UVB exposure is a triggering factor for OR, a rat model of OR and MGD was developed using a selective and short exposure of rat eyelids to high-dose UVB for five consecutive days 31 . It was applied in 3-month-old rats to evaluate whether MR overexpression aggravates the UV response. Following UVB exposure, the MG swelling and dropout and palpebral conjunctival hyperemia were exacerbated in P1.hMR rats as compared to WT (Fig. 5 a). Progressive fibrosis was observed in the MG from day 5 to 2 weeks after UVB exposure, with more extensive fibrosis in P1.hMR rats compared to WT rats (Fig. 5 b). Oxidative stress markers were exacerbated in P1.hMR rats as shown by a more pronounced staining of 4-hydroxynonenal (4-HNE), a marker for lipid peroxidation, and 3-nitrotyrosine (3-NT), a marker for nitrosative stress in the MG, epidermis and conjunctiva (Fig. 5 d-g). The transient inflammatory response observed on day 5 of UVB exposure was more severe in the transgenic rats, with increased infiltration of monocytes/macrophages and dendritic cell (IBA1 and ED1 positive) beneath the epidermis, within the connective tissue between MG acini and along the ducts in P1.hMR rats (Fig. 5 h and 5 i). MR overexpression aggravated UVB-induced MGD in P1.hMR rats. On day 5 of UVB exposure, markers of lipid synthesis dysregulation were significantly exacerbated in P1.hMR rats, with increased accumulation of intracellular lipid droplets in meibocytes (Fig. 6 a and 6 b) and elevated PPARγ expression in both meibocytes and ductal epithelial cells. The later then declined more severely by two weeks post-UVB in P1.hMR rats compared to WT (Fig. 6 c and 6 d). While keratinization did not differ between WT and P1.hMR rats (Fig. 6 e and 6 f), markers of meibocyte renewal declined more drastically in P1.hMR. The MG stem cell marker, P63, showed a continuous decrease up to two weeks (Fig. 6 g and 6 h), and the transient rise in KI67-positive proliferating meibocytes observed on day 5 was attenuated in P1.hMR rats (Fig. 6 i and 6 j), indicating impaired meibocytes renewal. Furthermore, TOM20 and TIM23 were reduced in P1.hMR rats on day 5 of UVB exposure compared to WT rats (Fig. 6 k and 6 l). MR overexpression exacerbated corneal signs of UVB-induced OR. No spontaneous morphological alterations of the cornea were detected in P1.hMR rats up to 6 months of age (Fig. S4a and S4b). At baseline and after 5 days of eyelid-targeted UVB exposure, no fluorescein staining was observed in either P1.hMR or WT rats, as the cornea was carefully shielded from direct irradiation during this period. However, by two weeks following UVB exposure, diffuse punctate fluorescein staining – indicative of punctuate keratitis, likely secondary to UVB-induced MGD – was observed, with more widespread staining in P1.hMR rats compared to WT (Fig. S4c and S4d). Corneal epithelial integrity was also more severely compromised in P1.hMR rats, as evidenced by greater disorganization of the cell junction proteins, E-cadherin and ZO-1 (Fig. S4e). Gene dysregulation in the MG in response to UVB radiation After 5 days of UVB exposure, 1,271 DEGs were found in the MG of WT rats (Table. S1), including 611 upregulated and 660 downregulated genes. In contrast, 2,040 DEGs were detected in the MG of P1.hMR rats (Table. S2), with 920 upregulated and 1,120 downregulated genes. Intersection analysis revealed 989 genes shared between P1.hMR and WT, while 1,031 genes were exclusively regulated in P1.hMR rats and 272 genes were specific to WT rats (Fig. 7 a). Among the shared DEGs, those associated with epithelial integrity, immune responses, collagen production and tissue remodelling showed greater fold changes in P1.hMR rats (Fig. 7 b). Gene ontology enrichment analysis of these common DEGs indicated involvement in biological processes such as cell cycle, inflammatory and humoral immune response, keratinization, extracellular matrix organization, angiogenesis, hormone metabolism, response to corticosteroid, unsaturated fatty acid metabolism, gland morphogenesis, Wnt signalling, and lipid metabolism (Fig. 7 c). Cellular component analysis highlighted enrichment in collagen and extracellular matrix regulation, keratin filaments, and cornified envelope pathways. KEGG pathway analysis further identified significant regulation of PI3K-Akt signalling, complement and coagulation cascades, TNF signalling, arachidonic acid metabolism, IL-17 signalling, and ECM-receptor interaction (Fig. 7 d). Transcriptomic regulation specific to MR overexpression in the UVB-induced OR model Enrichment analysis of UVB-induced DEGs exclusively identified in the MG of WT rats showed pathways related to cell cycle and proliferation, and collagen-extracellular matrix organisation (Fig. 7 e), indicating activation of repair mechanisms. In contrast, genes exclusively regulated in P1.hMR rats associated with inflammatory responses, unsaturated fatty acid and steroid hormone metabolism, extracellular matrix organization, neurotransmitter receptor and sodium ion transport (Fig. 7 f). These distinct transcriptomic changes in P1.hMR rats further support the role of MR overactivation in amplifying inflammation, neuroimmune responses, metabolic dysregulation and ion transport imbalance, all of which likely contribute to the exacerbation of OR. Shared gene regulations between UVB-induced MGD in P1.hMR rats and human MGD and skin rosacea The transcriptome of MGs from P1.hMR rats on day 5 of UVB exposure was compared to transcriptomic profiles of human MGs with MGD 29 (GSE17822) and of human skin with rosacea 32 (GSE65914). A notable overlap in gene regulation was observed between rat model and MGD patients, including genes involved in keratinization and immune responses (e.g., Cnfn, Sprr3, Serpina9, Serpinb2 and S100a9 ), extracellular matrix remodelling ( Mmp10 and Pdgfra ), and neuronal structure ( Tubb3 ) (Figures S5a. and Table S3) 29 . Furthermore, gene regulation in human rosacea skin exhibited substantial similarities with that of UVB-exposed P1.hMR rats, with 55 genes commonly regulated, including S100a9, Klk6, Klk13, Ccl27, Cxcl11, Mmp12, Mmp9, Sprr3 , and Tlr2 . (Fig. S5b. and Table S4) 33 . These cross-species comparisons provide further support for the involvement MR pathway activation in the pathogenesis of MGD and rosacea in human. S100a9 is a specific MR target, associated with MGD and skin rosacea Additional cross-analyses were performed between the DEGs specific to P1.hMR rats and the two human transcriptomic datasets 29 , 32 . A total of 21 genes specifically regulated by MR overaction in the P1.hMR rat model overlapped with genes deregulated in human rosacea skin (Fig. 8 a), while 41 MR-regulated genes in P1.hMR rats were shared with those found in human MGD (Fig. 8 b). Among these genes, CCL27, CCL20, CXCL11, TLR2, S100A9 and MMP12 were identified as an interaction network focus on immune cells chemotaxis, IL-17 and Toll-like receptor signaling, involved in rosacea and MGD (Fig. 8 c and Fig. 8 d) 34 – 36 . Further comparison between these two gene sets identified a single gene, S100A9 (Fig. 8 e), as a common marker of MR activation in both skin rosacea and MGD. S100A9 immunohistochemistry was further evaluated in human eyelid tissues. In control subjects, S100A9 was primarily located in the epithelium and in cells that may correspond to immune cells beneath the epidermis. In patients with OR, S100A9 expression was not only markedly increased in the epithelium and immune cells, but was also highly expressed in meibocytes in the MG (Fig. 8 f). These findings provide compelling evidence supporting the role of MR dysregulation in the pathophysiology of oculo-cutaneous rosacea and highlight S100A9 as a potential biomarker of MR pathway activation in both MGD and OR. 3. Discussion We provide herein a set of arguments that support the hypothesis that excessive activation of the MR pathway contributes directly to both the initiation and exacerbation of MGD and OR and that S100A9 is a biomarker of MR pathway activation and could be a therapeutic target or diagnostic marker in OR. MR overexpression was observed at the site of pathology in the eyelid and ocular surface tissues from patients with OR, associated with inflammatory cell infiltration and fibrosis in the MGs, consistent with pathological features reported in severe cutaneous rosacea 37 . Signs of abnormal acinar renewal and differentiation likely contribute to the development of MGD in OR patients 38 , 39 . In the posterior segment of the eye, it was shown that local MR pathway activation in retinal and choroidal cells induced low-grade inflammation, oxidative stress, fibrosis and neovascularization 40 . In systemic pathogenic mechanisms such as hypertension, wound healing, inflammation or metabolic diseases, a pathogenic role of MR hyperactivation has been demonstrated in vascular endothelial and smooth muscle cells. 41 But local pathogenic activation in specific organs or tissues has also been demonstrated, such as in adipose tissue, where it has been shown to contribute to adipocyte dysfunction and subsequent metabolic disorders 42 or in the heart, where MR-mediated damages were shown in cadiomyocytes using transgenic mouse models 43 . The generation of a transgenic rat overexpressing hMR (P1.hMR rats) provided further insight in the role of MR in the initiation and exacerbation of MGD and OR. In the 6-month-old transgenic rats, signs of MGD initiation including decline in meibocytes renewal and mitochondria dysfunctions were observed and the MG transcriptome identified pathways related to oxidative phosphorylation and peroxisome, which is consistent with the oxidative dysregulation observed in patients with MGD 44 . Upon UVB exposure, MR over-expression notably exacerbated the clinical, histological and transcriptomic manifestations of MGD and OR. The pathological features resemble the ones described in human tissues with OR, in which signs of MR overactivation have been evidenced. A greater number of genes related to inflammation, oxidative stress, epithelial integrity and tissue remodelling were regulated in the transgenic rats compared to in WT. More specifically, the upregulation of Sprr3 may drive aberrant keratinization, a key pathogenic mechanism in MGD 29 and regulate IL-33 which amplifies inflammation 45 and tissue remodelling 46 . In hMR-overexpressing rats, the higher Nos3 expression may lead to excessive nitric oxide (NO), contributing to vasodilation and the development of eyelid telangiectasia 47 . Among the DEGs uniquely regulated in P1.hMR rats after UVB exposure, several are involved in chemotaxis, IL-1 signalling, immune cell infiltration, fatty acid metabolism, and ion channel activity—pathways known to promote inflammation and clinical features of rosacea 48 . Notably, S100a9 , a direct MR target, was highly upregulated and is known to activate TLR4 and RAGE, amplifying inflammatory responses 49 . Additionally, the regulation of neurotransmitter receptor activity and sodium ion transport suggests MR involvement in neurovascular and neurogenic mechanisms, consistent with the role of neuropeptides and TRP channels in OR 50 , 51 . These findings support MR overactivation as a driver of immune, metabolic, and neurogenic dysregulation in OR. Intersection analysis revealed that nearly half of the genes commonly regulated in P1.hMR rats and in human MGD or cutaneous rosacea are associated with MR signalling, underscoring its central role in disease pathogenesis. Key genes such as CCL27, CCL20, CXCL11, TLR2, S100A9 , and MMP12 form an interaction network involved in chemotaxis, IL-17, and Toll-like receptor signalling—pathways known to drive rosacea-related inflammation 34 , 35 . Additionally, the MR-regulated gene SLC27A6, which mediates long-chain fatty acid uptake, may influence meibomian gland lipid composition, a hallmark of MGD 52 . Together, these findings implicate MR overactivation in immune, metabolic, epithelial, and neurogenic mechanisms that contribute to both MGD and oculo-cutaneous rosacea. The search for MR-specific downstream effectors identified S100A9 as a key mediator of MR-driven pathology in MGD and OR. S100A9 promotes inflammation via TLR4/MyD88 signalling, induces M1 macrophage polarization through TLR4/NF-κB 35,53 , enhances neutrophil chemotaxis, and drives keratinization 29 , 54 —all processes implicated in rosacea pathogenesis. In OR patient tissues, S100A9 expression was markedly increased in immune cells, the conjunctival epithelium, and MR-overexpressing meibocytes. Given its role in amplifying oxidative stress and inflammation 55 , 56 , S100A9 may directly contribute to MG damage and dysfunction. Notably, S100a9/S100A9 was the only gene both MR-specific in the rat OR model and associated with human OR pathology. Its expression is also downregulated by spironolactone, an MR antagonist, in a limbal stem cell deficiency model, relevant to corneal pathology in OR 28 —supporting its regulation by MR. Additionally, S100a9 is upregulated in MR-overexpressing mice with choroidal neuropathy, reinforcing its role as MR targets in ocular tissues 57 . As S100A9 is detectable in tears and correlates with MGD severity in dry eye disease 58 , it may serve as a non-invasive biomarker of MR activation in OR, offering a potential tool for patient stratification and MR-targeted therapies. This study has some limitations. While early alterations in the MGs of young P1.hMR rats were identified, the ocular surface phenotype in aged animals remains unexplored. Given the established role of MR in inflammaging, particularly in the heart 59 , it is plausible that age-related ocular changes may develop spontaneously in this model. Nonetheless, rodent models cannot fully recapitulate the human clinical spectrum of OR. To directly assess MR involvement in human disease, we have developed topical spironolactone eye drops, previously validated for safety; however, clinical efficacy data are not yet available. In conclusion, our findings demonstrate that MR pathway activation contributes to the pathogenesis of MGD and OR (Fig. 9 ). Accordingly, MR antagonism represents a promising therapeutic strategy, while S100A9 may serve as a biomarker to identify patients most likely to benefit from targeted intervention. Future clinical trials are warranted to confirm the therapeutic utility of MR blockade in MGD and OR using either systemic or local treatments. 4. Materials and Methods 4.1 Human eyelid samples collection Eight patients were operated in Cochin hospital in Paris by XM for lid surgery (ectropion or entropion) and the surgical pieces were immediately fixed in 10% formalin for 24 hours, dehydrated, embedded in paraffin wax and cut into 7 um thick slices and stained with hemalum-eosine, for histological analysis. All patients signed written consent for non-opposition to the use of surgical waste in research. Additional unused sections were used for specific staining relative to the present study (see below). We analyzed the tissue sections from 4 patients with OR (3 men and 1 woman, 70 years, 71 years, 52 years, 78 years) and 4 control subjects without ocular or skin rosacea (3 men and 1 woman 44years, 90 years, 78 years, 76 years). The collection and storage of human biological samples were approved by French ethics committee CCP Ile de France 1 (no. 2016-nov-14390). 4.2 Animal experiments All experiments were performed in accordance with the European communities Council Directive 86/609/EEC and French national regulations and approved by local ethics committee (# 23478-2020010317557546 v4, Charles Darwin). To study the role of MR in the pathology of OR, we generated P1.hMR rats on Sprague-Dawley genetic background. The human MR (hMR) gene is expressed under the control of the proximal P1 promoter of the NR3C2 as described previously 60 , 61 . Overexpression of NR3C2 expression was validated in ocular tissues in both male (WT: n = 3–6 rats; P1.hMR: n = 3–8 rats) and female (WT: n = 2–4 rats; P1.hMR: n = 6–8 rats) by transcriptomic and qPCR analysis. Other animals (WT: n = 4–6 rats; P1.hMR: n = 4–6 rats) were used for characterization in morphology and pathology. All animals were kept in pathogen-free conditions with free access to food and water and housed in temperature-controlled room with a 12-h light/12-h dark cycle. Anaesthesia was induced by intraperitoneal ketamine 100 mg/kg and Xylazine 10 mg/kg. Animal were euthanized by intraperitoneal injection of fatal dose of Euthasol® Vet. 4.3 UVB irradiation on rat eyelids The upper eyelids of rats were shaved and exposed to 1000 µW/cm2 of UVB (312 nm, Herolab, Wiesioch, Germany) for 5 min (300 mJ/ cm2) daily for 5 days, as described previously 31 . The cornea and the whole body were covered and protected. Lesions of eyelids were photographed under the microscopy from day 0 to day 5. Rats were euthanized after 5 days to evaluate the acute damages of ocular surface tissues and MR related pathology (WT: n = 5 rats; P1.hMR: n = 6 rats). To assess the prolonged effects of UVB irradiation on MGD on the cornea, a group of rats (WT: n = 6 rats; P1.hMR: n = 6 rats) were followed up until 2 weeks after UVB exposure. Corneal fluorescein staining was performed before, after 5 days of UVB and 2 weeks after UVB exposure Corneal defects were graded according to the criteria previously described 62 , 63 .. Rats were then euthanized, eyelids and corneas were dissected for morphological and immunohistochemical analysis. 4.4 Hematoxylin and eosin staining Eyelid samples from humans and rats were immediately fixed in 10% neutral-buffered formalin for 24 hours and 4% paraformaldehyde (PFA) for 2 hours, respectively, followed by graded dehydration and embedding in paraffin. Serial sections of 7 µm thickness were obtained using a microtome (LEICA). The paraffin-embedded sections of human and rat eyelids were deparaffinized in xylene and rehydrated through a descending ethanol series. The sections were then stained with hematoxylin reagent (RAL Diagnostics, #320550-2500, Bordeaux, France) for 15 minutes and rinsed with tap water to remove excess dye. Subsequently, the sections were immersed in eosin solution (RAL Diagnostics, #312730-0100, Bordeaux, France) for 4 minutes and rinsed again with tap water. After sequential baths in absolute ethanol and xylene, the tissue sections were mounted using Eukitt® mounting medium (O. Kindler GmbH, #200080, Freiburg, Germany). Morphological changes were observed under a light microscope (Olympus BX51, Rungis, France) equipped with a CCD camera (Olympus DP70). 4.5. Sirius red staining For collagen detection, human and rat eyelid sections were deparaffinized and rehydrated, then incubated with 1% Sirius Red in picric acid for 15 minutes, as previously described 64 . After sequential washes with acetic acid and absolute ethanol, the slides were mounted and examined under a microscope. Collagen content was quantified using Fiji ImageJ software (version 1.54b, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). A binary conversion of the red channel was performed to calculate the percentage of area occupied by collagen fibers. All quantifications were normalized to the analyzed tissue area. 4.6. Immunohistochemistry Paraffin-embedded rat and human eyelid sections were used for immunohistochemical staining as previously described 28 . Briefly, sections were deparaffinized and rehydrated, followed by antigen retrieval in citrate buffer (10 mM, pH 6.0) at 100°C for 20 minutes. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide. Sections were then blocked with TNB blocking buffer (AKOYA Biosciences, #FP1012, Massachusetts, USA) for 30 minutes at room temperature. Primary antibodies (MR, GR, HSD1, HSD2, Nitrotyrosine, 4-HNE, S100a9) were applied overnight at 4°C as reference listed in table 7. After washes with PBST, sections were incubated with biotinylated secondary antibodies in table 7. for 1 hour at room temperature. For GR and MR immunohistochemistry, the Tyramide Signal Amplification (TSA) biotin kit (AKOYA Biosciences, MA, USA) was used according to the manufacturer’s instructions to amplify the tyramide signal. For other primary antibodies, sections were processed with Vectastain Elite ABC reagent (Vector Laboratories, USA, #AK-5000) for 30 minutes. For S100A9, the signal was developed using the VIP Substrate Kit (Vector Laboratories, USA, #SK-4605) and stopped in PBS buffer. For other targets, the signal was visualized using 3,3’-diaminobenzidine (DAB) substrate (Vector Laboratories, USA, #SK-4105) and stopped in 50 mM TRIS buffer (pH 7.6) for 10 minutes. Tissue sections were mounted with Eukitt® mounting medium (O. Kindler GmbH, Freiburg, Germany). Negative controls were performed by omitting the primary antibody. For quantification, the positive staining area normalized to the same threshold for MR and GR, the mean grey value limited to the same threshold for staining intensity for other targets in MG were measured using ImageJ software (version 1.54b, National Institutes of Health, Bethesda, MD, USA). 4.7 Quantitative PCR in ocular surface tissues from rats RNA from various ocular surface tissues was isolated using the RNeasy Mini Kit (QIAGEN, Cat. No. 74106), followed by DNase I treatment (QIAGEN, Cat. No. 79254) according to the manufacturer’s protocol. Transcript levels were quantified by quantitative real-time PCR (qPCR) on the QuantStudio™ 5 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Green detection. Relative quantification was performed using the ΔΔCT method. Primer sequences for all analyzed genes are provided in Supplementary Table 5. Reference genes ( Hprt1 , Ubc , and 18S ) were used for normalization. 4.8 Immunofluorescence Rat ocular tissues were dissected, embedded in OCT compound, and frozen at − 80°C. Cryosections of 10 µm thickness were obtained using a cryostat (Leica). Immunofluorescence staining was performed as previously described 28 , rat eyelid and corneal cryosections were fixed in 4% PFA followed by washes with PBS. Sections were permeabilized with 0.1% Triton X-100 in PBS for 30 minutes and blocked with 5% goat serum in PBS for 30 minutes at room temperature. Tissues were incubated with primary antibodies overnight at 4°C. Secondary antibodies was applied the next day for 1 hour at room temperature. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; 1:5000) for 5 minutes, and slides were mounted using Gel Mount (Dako, Agilent, Les Ulis, France). Negative controls were performed by omitting the primary antibody. Sections were visualized using an Olympus fluorescence microscope (Olympus BX51). Primary and secondary antibodies, along with their dilutions, are listed in Supplementary Table 6. Quantification was performed using QuPath software. 4.9 Nile red staining Fresh-frozen rat MG sections were incubated with 1 µg/mL Nile red (Sigma-Aldrich, #72458, Missouri, USA) in PBS for 1 hour at 4°C. Tissues were then fixed in 4% paraformaldehyde (PFA) for 15 minutes, followed by DAPI staining and three washes with PBS. Stained sections were imaged using a confocal microscope (Zeiss LSM 710, Oberkochen, Germany). Strongly stained lipid droplets were quantified using Fiji ImageJ software by calculating the percentage of the highlighted area limited to threshold relative to the total analyzed area. 4.10 RNA Sequencing of Rat MG and Transcriptomic Analysis To characterize the pathology involved in P1.hMR phenotype, MGs were carefully dissected from 6-months-aged WT (n = 6 eyes) and P1.hMR male rats (n = 4 eyzs) for transcriptomic analysis. For UVB-induced MGD with ocular surface damages model of OR, 12-weeks-old Sprague-Dawley rats were irradiated by UVB on eyelids (WT: n = 6 eyes; P1.hMR: n = 10 eyes). Age- and sex matched control rats (WT: n = 4; P1.hMR: n = 5) were raised in the same condition without model. Animals were euthanized after 5 days of UVB irradiation. RNA extraction was conducted as described above. 1mg of the extracted RNA sample was sent for RNA sequencing at the iGenSeq transcriptomic platform located at the Brain and Spine Institute (ICM, Paris, France). RNA quality was checked by capillary electrophoresis and RNA integrity numbers (RIN) ranging from 8.8 to 9.3 was accepted for library generation. Quality of raw data was assessed using FastQC. STAR v2.5.3 was used to align reads on reference genome rn6 using standard options. Between 30 and 38 million reads were mapped. Quantification of gene and isoform abundances was done with rsem 1.2.28, and reproducibility of replicates was controlled with PCA representations. The transcriptomic data for rats were analyzed on the online platform of the Paris Brain institute (quby.icm-institute.org). First, differential gene expression analysis with DESeq2 method was utilized to identify the differentially expressed genes. Adjusted P values (pFDR) were calculated with the Benjamini-Hochberg procedure to control false discovery rate (FDR threshold set at 0.05, log2 fold-change threshold set at 0.5). Then, an enrichment analysis was performed (by GSEA and over-representation analysis) in different gene-set collections including Reactome gene sets (Reactome subset of Canonical pathways), Hallmark gene sets (H), Gene Ontology gene sets (C5: GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Gene Sets. Transcriptomic data for human meibomian gland dysfunction (MGD) were reanalyzed using the GEO2R online platform (GSE17822), with differentially expressed genes (DEGs) identified based on a significance threshold of P 1.5, P < 0.05) using GSE65914. An intersection analysis was performed to identify the most relevant DEGs across the three subtypes. Additional intersection analyses were conducted using the Xiantao Academic online platform ( https://www.xiantaozi.com/ ). 4.11 Statistics Analysis A study of the literature and an estimation using G-power software, version 3.1.9.6, were carried out to estimate the number of animals required. Data are expressed as mean ± SD. Statistical analysis was performed using the GraphPad Prism (GraphPad Software, version 9, San Diego, CA, USA). The unpaired t-test or Mann–Whitney test was used to compare two groups. The Kruskal–Walli’s test followed by Dunn’s test was used to compare more than two groups. The Friedman test was used to compare more than 2 matched groups. A p value less than 0.05 was considered statistically significant. Declarations Funding : This work was supported by INSERM, Agence National de la Recherche (grant n° ANR-21-CE18-0018), and RESTORE VISION grant under the EU Framework Horizon Europe Program (grant N°101080611). Acknowledgements The authors thank the Functional Exploration Center (CEF) and the Core Facilities of Histology, Imaging and Flow Cytometry (CHIC) at Centre de Recherche des Cordeliers, BioRender platform for figure modification https://BioRender.com. P1.hMR rat model generation services were provided by PHENOMIN-iCS (www.phenomin.fr). Authorship contribution F.B.-C. and M.Z. acquired the funding, conceived and directed the project, and designed and supervised the experiments. X.M operated the patients. L.Z. performed the microtome and histology of the human samples. N.Y. performed the prospective study in patients. L.Z., D.R.-B., E.G. and M.Z. performed all the in vivo experiments. L.Z. performed the histological, immunohistochemical and transcriptomic analysis of human and rat samples. L.Z. F.B.-C. and M.Z. interpreted the data. L.Z. and F.B.-C . constructed the figures. L.Z, and F.B.-C. wrote the original draft. F.B.-C., M.Z. N.Y. and J.L.B. reviewed the manuscript. L.Z. and F.B.-C. and M.Z. edited the manuscript. All authors read and approved the final version. Data availability statement All Data are available upon reasonable request. Raw RNAseq data will be made available on Gene Expression Omnibus GSE291177 (accessible on 08 July 2025). 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Le Menuet, D., Viengchareun, S., Muffat-Joly, M., Zennaro, M.-C. & Lombès, M. Expression and function of the human mineralocorticoid receptor: lessons from transgenic mouse models. Mol. Cell. Endocrinol. 217 , 127–136 (2004). Lin, Z. et al. A mouse dry eye model induced by topical administration of benzalkonium chloride. Mol. Vis. 17 , 257–264 (2011). Pauly, A. et al. New tools for the evaluation of toxic ocular surface changes in the rat. Invest. Ophthalmol. Vis. Sci. 48 , 5473–5483 (2007). Palacios-Ramirez, R. et al. Mineralocorticoid Receptor Antagonism Prevents the Synergistic Effect of Metabolic Challenge and Chronic Kidney Disease on Renal Fibrosis and Inflammation in Mice. Front. Physiol. 13 , 859812 (2022). Supplementary Figures Supplementary Figures S1-S5 are not available with this version. Additional Declarations There is NO Competing Interest. Supplementary Files SuplementaryTables14.xlsx Table S1-4 SupplementaryTable56.docx Table S 5 and 6,7 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6589915","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":456327096,"identity":"58b38f47-b95e-4ca8-a12a-11f285a7608b","order_by":0,"name":"Francine Behar-Cohen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYBAC9gYQaQDEEgxsQNIGiBkbD+DTwnMAVUsaSEsDEVoY4FoOg9n4tUgffvbgR8EdOQbp5mMPfu45b7e2/TDQlhqbaJxa+NLMDXsMnhkzyBxLN+x5djt525lEoJZjabkNOLTY8zCYSTMYHE5skMgxk+A5cDvZ7ABQC2PDYZxaeHjYv4G01DdI5H+T/HPgXLLZ+YeEtPCAbUlgkMhhk+Y5cMDO7AZBW3jKJHsMDhu2yRwzk5Y5kJxgdgNoSwIevwAdtk3ix5/D8vzSzc8k3xywszc7n/7wwYcaG5xa4IANSieCVSYQUo4M7ElRPApGwSgYBSMDAAAkhFxxQipVQwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8571-9513","institution":"Centre de Recherche des Cordeliers","correspondingAuthor":true,"prefix":"","firstName":"Francine","middleName":"","lastName":"Behar-Cohen","suffix":""},{"id":456327097,"identity":"d161caa0-1c84-41dd-a926-4b8209509455","order_by":1,"name":"Nilufer Yesilirmak","email":"","orcid":"","institution":"Centre de Recherche des Cordeliers","correspondingAuthor":false,"prefix":"","firstName":"Nilufer","middleName":"","lastName":"Yesilirmak","suffix":""},{"id":456327098,"identity":"267fa368-3d46-48e8-a41f-e9271ba858c6","order_by":2,"name":"Danielle Rodrigues-Braz","email":"","orcid":"","institution":"Centre de Recherche des Cordeliers","correspondingAuthor":false,"prefix":"","firstName":"Danielle","middleName":"","lastName":"Rodrigues-Braz","suffix":""},{"id":456327099,"identity":"f9256ae4-abc9-4f4e-9aaf-7d5a7bc5d4c9","order_by":3,"name":"Emmanuelle Gelize","email":"","orcid":"","institution":"Centre de Recherche des Cordeliers","correspondingAuthor":false,"prefix":"","firstName":"Emmanuelle","middleName":"","lastName":"Gelize","suffix":""},{"id":456327100,"identity":"1108f979-02a6-4ebf-bae1-27b76ef65c1e","order_by":4,"name":"Coralie LHEURE","email":"","orcid":"","institution":"Cochin Hospital AP-HP","correspondingAuthor":false,"prefix":"","firstName":"Coralie","middleName":"","lastName":"LHEURE","suffix":""},{"id":456327101,"identity":"26619c74-5d51-457e-be2b-87494810b2fc","order_by":5,"name":"Xavier Morel","email":"","orcid":"","institution":"Cochin Hospital AP-HP","correspondingAuthor":false,"prefix":"","firstName":"Xavier","middleName":"","lastName":"Morel","suffix":""},{"id":456327102,"identity":"5adcf206-96cb-4eba-8a0a-42f0d4d104ce","order_by":6,"name":"Jean-Louis Bourges","email":"","orcid":"https://orcid.org/0000-0002-7016-9819","institution":"Université Paris Cité - APHP; INSERM","correspondingAuthor":false,"prefix":"","firstName":"Jean-Louis","middleName":"","lastName":"Bourges","suffix":""},{"id":456327103,"identity":"a6466786-6ecf-44a6-b8c4-39116bb78f96","order_by":7,"name":"Frederic Jaisser","email":"","orcid":"https://orcid.org/0000-0001-9051-1901","institution":"INSERM, Sorbonne Université, USPC, Université Paris Descartes, Université Paris Diderot","correspondingAuthor":false,"prefix":"","firstName":"Frederic","middleName":"","lastName":"Jaisser","suffix":""},{"id":456327104,"identity":"6d2921fc-701e-4bcf-99dc-f6fd1bf295f2","order_by":8,"name":"Min Zhao","email":"","orcid":"","institution":"INSERM, U872, Team 17","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Zhao","suffix":""},{"id":456327105,"identity":"38c751c0-4ec4-4c96-aab1-957ad290ee64","order_by":9,"name":"Linxin ZHU","email":"","orcid":"","institution":"Centre de recherche des Cordeliers","correspondingAuthor":false,"prefix":"","firstName":"Linxin","middleName":"","lastName":"ZHU","suffix":""}],"badges":[],"createdAt":"2025-05-04 19:15:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6589915/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6589915/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88415711,"identity":"0c4d085d-8b11-4493-8260-e129531d09db","added_by":"auto","created_at":"2025-08-06 08:53:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":749028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMR upregulation in OR patients is associated with histological and clinical pathologies of MGD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Immunohistochemistry of MR and GR protein in eyelid tissues of patients with OR compared to control subjects. MR and GR protein levels in MGs determined by immunostaining and quantified by ImageJ (n = 4). \u003cstrong\u003eb.\u003c/strong\u003e Immunohistochemistry of 11βHSD1 and 11βHSD2 in eyelid tissues of patients with OR compared to control subjects.\u003cstrong\u003e c.\u003c/strong\u003e H\u0026amp;E staining of eyelid tissues from OR patients and control subjects. Infiltrating cells (arrows and asterisk).\u003cstrong\u003e d.\u003c/strong\u003e Sirius red staining for collagen fibers (arrows) in MGs. \u0026nbsp;\u003cstrong\u003ee.\u003c/strong\u003e Collagen staining area in MGs quantified by ImageJ based on the same threshold of positive Sirius red staining (n = 4).\u003c/p\u003e\n\u003cp\u003eCj, conjunctiva; Mg, meibomian gland; Du, ducts; Ep, epidermis; Hf, hair follicles; Sg, sebaceous gland. Scale bar: 100 µm. Data expressed as mean ± SEM. ns, p \u0026gt; 0.05 (Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/7b72ce280b928ff9c44b7b91.png"},{"id":88416592,"identity":"eb3a1660-19c7-4ee9-b180-dd03abd337f5","added_by":"auto","created_at":"2025-08-06 09:01:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":523929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMR is overexpressed in the eyelid and ocular surface tissues of P1.hMR rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. hMR, rMR and total MR expression in mRNA level in ocular surface tissues of P1.hMR and WT rats, including the cornea (CN), MGs, and conjunctiva (CJ). (n = 2-8 rats in each group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Immunohistochemistry of MR protein expression in the eyelid and ocular surface tissues of P1.hMR and WT rats.\u003cstrong\u003e c.\u003c/strong\u003e MR protein level in MGs determined by immunostaining and quantified by ImageJ (n = 4 rats).\u003c/p\u003e\n\u003cp\u003eCj, conjunctiva; Mg, meibomian gland; Du, ducts; Ep, epidermis; Hf, hair follicles; CoEp, corneal epithelium. Scale bar: 100 um. Data expressed as mean ± SD. ns, p \u0026gt; 0.05 (Multiple Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/a5a5cee9406f04109d451032.png"},{"id":88414327,"identity":"85678e74-9b9c-4164-9056-cf3a34869e47","added_by":"auto","created_at":"2025-08-06 08:45:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":594301,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP1.hMR rats overexpressing MR present early features of MGD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. H\u0026amp;E staining of eyelid tissues from WT and P1.hMR rats.\u003cstrong\u003e b.\u003c/strong\u003e Sirius-Red staining for collagen fibers (arrows) in the MG of WT and P1.hMR rats. \u003cstrong\u003e\u0026nbsp;c.\u003c/strong\u003e Collagen staining area in MGs quantified by ImageJ based on the same threshold of positive Sirius red staining (n = 4-5 rats).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed.\u003c/strong\u003e KI67 (red colour, left) and P63 (green colour, right) expression in the MG of WT and P1.hMR rats. Arrows indicate positively stained cells.\u003cstrong\u003e e.\u003c/strong\u003e KI67 and P63 expression levels in the MG determined by the ratio of positive immunosignal cells/ total DAPI-marked cells and quantified by QuPath (n = 6-8 rats). \u003cstrong\u003ef.\u003c/strong\u003e TIM23 (red colour, left) and TOM20 (green colour, right) expression in the MG of WT and P1.hMR rats. Arrows indicate positively stained cells. \u003cstrong\u003eg.\u003c/strong\u003eQuantification of fluorescence intensity of TIM23 and TOM20 by ImageJ (7-8 rats). Cj, conjunctiva; Mg, meibomian gland; Du, ducts; Ep, epidermis; Hf, hair follicles. Scale bar: 100 um. Data expressed as mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/a5565937660fc69cdf4c1e73.png"},{"id":88416596,"identity":"abfd4b58-45b2-4ce4-9aeb-53a774d6f1a3","added_by":"auto","created_at":"2025-08-06 09:01:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":402831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis of MG tissues from P1.hMR versus WT rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e The volcano plot displays genes differentially regulated by hMR overexpression in P1.hMR rats compared to WT rats. n = 3 in WT, n = 2 in P1.hMR rats. \u003cstrong\u003eb.\u003c/strong\u003e GSEA using the RACTOME database reveals 20 significantly regulated pathways. \u003cstrong\u003ec.\u003c/strong\u003e GSEA using the HALLMARK gene set identifies 11 significantly regulated pathways. \u003cstrong\u003ed.\u003c/strong\u003e Regulated HALLMARK pathways with highlighted genes associated with MGD and rosacea.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/4d2c558f4a29a7c6d6f69f7c.png"},{"id":88415715,"identity":"bf8d8bbb-4b2b-4130-8487-a7c5b6752aa3","added_by":"auto","created_at":"2025-08-06 08:53:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":667016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUVB-induced rat model mimicking OR is exacerbated by MR overexpression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Representative macroscopic images of the inner eyelid and MGs after 5 days of UVB eyelid irradiation. \u003cstrong\u003eb.\u003c/strong\u003e Sirius red staining for collagen fibers on day 5 of UVB and 2 weeks post-UVB exposure. \u003cstrong\u003ec.\u003c/strong\u003e Quantification of total collagen area in the MG using ImageJ (n = 4-6 rats). \u003cstrong\u003ed.\u003c/strong\u003e Immunohistochemistry of 4-HNE expression in eyelid tissues on day 5 of UVB. \u003cstrong\u003ee.\u003c/strong\u003e Quantification of 4-HNE immunostaining intensity by ImageJ (n = 5 rats)\u003cstrong\u003e f.\u003c/strong\u003e Immunohistochemistry of 3-NT expression in eyelid tissues on day 5 of UVB.\u003cstrong\u003e g.\u003c/strong\u003e Quantification of 3-NT immunostaining intensity by ImageJ (n = 4-5 rats)\u003cstrong\u003e h.\u003c/strong\u003e Infiltration of IBA1+ (green) and ED-1+ (red) cells in the eyelid tissues on day 5 and 2 weeks post-UVB. Arrows indicate infiltrating immune cells.\u003cstrong\u003e i.\u003c/strong\u003e Quantification of positive-stained cells per field (n = 4-8 rats). Cj, conjunctiva; Mg, meibomian gland; Ep, epidermis; Du, duct. Scale bar: 100 um. Data expressed as mean ± SEM. ns, p \u0026gt; 0.05 (Two-way ANOVA with Sidak’s multiple comparison in \u003cstrong\u003ec.\u003c/strong\u003eand \u003cstrong\u003eI.;\u003c/strong\u003e Mann-Whitney U test in \u003cstrong\u003ee.\u003c/strong\u003e and \u003cstrong\u003eg.\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/2acbb09fe42de7c97cd52640.png"},{"id":88416600,"identity":"7f51309e-e146-44bf-8b54-ca862d548148","added_by":"auto","created_at":"2025-08-06 09:01:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":629103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMR overexpression aggravates the UVB-induced MGD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Nile red staining (red color) in the MG on day 5 of UVB and 2 weeks post-UVB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Quantification of lipid droplet area in the MG using ImageJ (n = 4-8 rats).\u003cstrong\u003e c.\u003c/strong\u003e PPAR-\u003cem\u003eγ\u003c/em\u003e (green color) expression in the MG on day 5 of UVB and 2 weeks post-UVB. \u003cstrong\u003ed.\u003c/strong\u003ePPAR-\u003cem\u003eγ\u003c/em\u003e expression level in the MG determined by the ratio of positive immunosignal cells/ total DAPI-marked cells and quantified by QuPath (n = 4-8 rats).\u003cstrong\u003e e. \u003c/strong\u003eKRT1 (green color) expression in the MG on day 5 of UVB and 2 weeks post-UVB. \u003cstrong\u003ef.\u003c/strong\u003e Quantification of KRT1 immunofluorescence intensity by ImageJ (n = 8 rats in each group)\u003cstrong\u003e g.\u003c/strong\u003e P63 (green color) expression in the MG on day 5 of UVB and 2 weeks post-UVB. \u003cstrong\u003eh.\u003c/strong\u003e P63 expression level in the MG determined by the ratio of positive immunosignal cells/ total DAPI-marked cells and quantified by QuPath (n = 4-6 rats).\u003cstrong\u003ei.\u003c/strong\u003e KI67 (red color) and KRT14 (green color) immunostaining in the MG on day 5 of UVB and 2 weeks post-UVB. \u003cstrong\u003e\u0026nbsp;j.\u003c/strong\u003e KI67 expression level determined by the ratio of positive immunosignal cells/ total DAPI-marked cells and quantified by QuPath (n = 4-6 rats).\u003cstrong\u003e k.\u003c/strong\u003e TIM23 (red color, left) and TOM20 (green color, right) immunostaining in the MG on day 5 of UVB exposure.\u003cstrong\u003el.\u003c/strong\u003e Quantification of TIM23 and TOM20 immunofluorescence intensity by ImageJ (n = 6-8 rats). Mg, meibomian gland; Du, ducts. Arrows indicate stained cells or area. Scale bar: 100 um. Data expressed as mean ± SEM. ns, p \u0026gt; 0.05 (Two-way ANOVA with Sidak’s multiple comparison in \u003cstrong\u003eb.\u003c/strong\u003e \u003cstrong\u003ed. f. h. j.;\u003c/strong\u003e Mann-Whitney U test in \u003cstrong\u003el.\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/62dbb034c39a53d90da6a844.png"},{"id":88414333,"identity":"ab131e68-56e0-4af5-9c4d-3145938b9f22","added_by":"auto","created_at":"2025-08-06 08:45:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":603198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analyses of UVB-induced MGD in WT and P1.hMR rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Venn diagrams illustrating differentially expressed genes (DEGs) in the MG of WT and P1.hMR rats exposed to UVB. DEGs critically associated with MGD and rosacea are listed. \u003cstrong\u003eb.\u003c/strong\u003e Fold Change (FC) values of representative common DEGs in UVB-WT and UVB-P1.hMR rats. \u003cstrong\u003ec.\u003c/strong\u003e Enrichment analysis of common DEGs using GOBP database. \u003cstrong\u003ed.\u003c/strong\u003e Enrichment analysis of common DEGs using KEGG database. \u003cstrong\u003ee.\u003c/strong\u003e Enrichment analysis of DEGs exclusively identified in WT rats. \u003cstrong\u003ef.\u003c/strong\u003e Enrichment analysis of DEGs exclusively identified in P1.hMR rats. n = 3-5 rats in each group.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/0a13babae7f5e21355289b0c.png"},{"id":88415717,"identity":"edbe6fb3-1c6d-48fa-87d2-4b3f028f7a34","added_by":"auto","created_at":"2025-08-06 08:53:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":353203,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMulti-omics analyses identify S100A9/S100a9 as a key mediator of MR-exacerbated pathogenesis of MGD in Ocular Rosacea.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Venn diagram showing the intersection of DEGs in the skin of rosacea patients (GSE65914), UVB-induced MGD in P1.hMR rats (UVB P1.hMR), and UVB-induced MGD in WT rats (UVB WT). \u0026nbsp;Twenty-one genes specific to MR overactivation are commonly regulated in human skin rosacea. \u003cstrong\u003eb.\u003c/strong\u003e Venn diagram showing the overlap of DEGs in the MG from MGD patients (GSE17822), UVB-induced MGD in P1.hMR rats (UVB P1.hMR), and UVB-induced MGD in WT rats (UVB WT). Forty-one genes specific to MR overactivation are commonly regulated in human MGD. \u003cstrong\u003ec.\u003c/strong\u003e Network analysis reveals interaction among DEGs associated with rosacea, MGD, and MR overexpression. The highlighted strings indicate existing and potential interactions between genes. \u003cstrong\u003ed.\u003c/strong\u003e Gene Ontology Biological Process (GOBP) and KEGG pathway analysis of \u003cem\u003eS100A9 \u003c/em\u003eand its interacting genes. \u003cstrong\u003ee.\u003c/strong\u003eVenn diagram with the 4 databases identifies \u003cem\u003eS100A9\u003c/em\u003e/\u003cem\u003eS100a9 \u003c/em\u003eas a gene associated with MGD and rosacea and specifically linked to MR overexpression. \u003cstrong\u003ef.\u003c/strong\u003e Immunohistochemistry and quantification of S100A9 protein expression in human MGs tissues from OR patients and control subjects (n = 4). Arrows indicate positive staining.\u003c/p\u003e\n\u003cp\u003eCj, conjunctiva; Mg, meibomian gland; Ep, epidermis; Hf, hair follicles. Scale bar: 100 µm. Data expressed as mean ± SEM. (Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/5b4d63c893df98a6f9d11c78.png"},{"id":88414339,"identity":"c05483fa-caae-4662-bcc9-7ba6cd0f6185","added_by":"auto","created_at":"2025-08-06 08:45:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":258411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInvolvement of MR in MGD and OR\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/95ad7a69e233aed5be670f02.png"},{"id":88635088,"identity":"5b191116-d2fa-421a-9ba7-307f62db2f72","added_by":"auto","created_at":"2025-08-08 14:52:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6278250,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/779e8519-a4ed-429c-89c7-d9d108843471.pdf"},{"id":88414328,"identity":"9cf47eaf-e73f-4d34-a746-e992de900a8e","added_by":"auto","created_at":"2025-08-06 08:45:48","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4411576,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1-4\u003c/p\u003e","description":"","filename":"SuplementaryTables14.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/72fa665454079e3a730319f4.xlsx"},{"id":88414323,"identity":"3f596337-01b2-427b-a865-42920fb32e68","added_by":"auto","created_at":"2025-08-06 08:45:48","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27451,"visible":true,"origin":"","legend":"\u003cp\u003eTable S 5 and 6,7\u003c/p\u003e","description":"","filename":"SupplementaryTable56.docx","url":"https://assets-eu.researchsquare.com/files/rs-6589915/v1/4e16a104b520a1f6838b09d9.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The role of Mineralocorticoid Receptor pathway in Ocular Rosacea","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRosacea, a prevalent chronic inflammatory skin disease, affects up to 22% of the global population\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Approximately three quarters of patients exhibit ocular surface involvement\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e characterized by inflammation and neurovascular dysfunction of the ocular surface and eyelids associated with Meibomian gland dysfunction (MGD)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The structural and functional alteration of Meibomian glands (MGs) and the subsequent changes in the lipid composition of the tear film, result in corneal symptoms and alter the quality of life of patients with OR\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Eventually, neovascularization, loss of transparency and ulceration of the cornea can occur\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Despite its potentially blinding consequences, OR is often underdiagnosed and remains incurable, emphasizing the need for the identification of novel therapeutic targets.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe pathogenesis of OR is multifactorial and remains incompletely understood. It involves aberrant innate and adaptive immune responses, neurovascular dysregulation\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, neurogenic inflammation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and fibrosis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Activation of the toll-like receptor (TLR)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and transient receptor potential (TRP) ion channels\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e contribute to chronic inflammation of the ocular surface\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Environmental triggering factors, including ultraviolet (UV) exposure, psychosocial stress and glucocorticoids (GCs)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e contribute to OR and to MGD, which is a major component of the disease\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, the precise mechanism by which GCs, despite their anti-inflammatory effects, act on OR and contribute to MGD is unknown.\u003c/p\u003e \u003cp\u003eCortisol binds to the glucocorticoid receptor (GR) and to the mineralocorticoid receptor (MR) with similar high affinity. The metabolism of endogenous corticosteroids is controlled locally by hydroxysteroid dehydrogenase (11-βHSD) enzymes expressed in skin and eye tissues\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The low expression of 11-βHSD type 2 (11-βHSD2), which converts cortisol to inactive cortisone allows MR activation by GCs\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, which could contribute to GC-induced OR. It has been demonstrated in previous studies that the oral administration of spironolactone, an MR antagonist, is an efficient treatment for skin rosacea\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and that it can reduce its incidence\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Furthermore, the topical administration of MR antagonists has been demonstrated to improve GC-induced epidermal atrophy\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and to enhance wound re-epithelialization in human skin treated with GC\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The administration of spironolactone drops increased the rate of corneal re-epithelialization in cases of wound healing delayed by glucocorticoid (GC)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Furthermore, mounting evidence suggests that MR overactivation induces inflammation, oxidative stress, fibrosis, vascular dysfunction and lipid dysmetabolism, all of which are observed in OR.\u003c/p\u003e \u003cp\u003eThe subsequent investigation focused on elucidating the role of MR pathway overactivation in MGD and OR.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003eMR pathway is activated in eyelids from patients suffering from OR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no significant difference in age between patients with OR and controls (67.7\u0026thinsp;\u0026plusmn;\u0026thinsp;11, 72\u0026thinsp;\u0026plusmn;\u0026thinsp;19 years, p\u0026thinsp;=\u0026thinsp;0.54) and there were 3 men and 1 woman in both groups. In the OR group, two patients presented rhinophyma, while the two others only suffered from OR. Immunohistochemistry of GR, MR, 11\u0026beta;HSD1 and 11\u0026beta;HSD2 was performed on the eyelids. In all the examined OR samples, MR immunostaining (see Fig. 1a) was enhanced in the nuclei of conjunctival epithelial cells, meibocytes, sebocytes, and epithelial cells as compared to controls while GR signal localization and intensity did not differ (Fig. 1a). 11\u0026beta;HSD1 was expressed in the cytoplasm of conjunctival epithelial cells, meibocytes, epithelial cells of hair follicles and basal cells of epidermis, while 11\u0026beta;HSD2 exhibited diffused staining in both the cytoplasm and nuclei of these cells. In the eyelid of OR samples, 11\u0026beta;HSD1 immunostaining was enhanced, while 11\u0026beta;HSD2 immunostaining was reduced (Fig. 1b), indicating local increased availability of cortisol. The increase in the MR/GR and in the 11\u0026beta;HSD1/11\u0026beta;HSD2 ratio suggests that MR pathway is overactivation in the eyelids of patients with OR.\u003c/p\u003e\n\u003cp\u003eHistological analysis of eyelid from OR patients revealed hyperplasia of the conjunctiva and the epidermis and infiltration of inflammatory cells in the conjunctiva, around MGs and beneath the epidermis (Fig. 1c). The acini appeared smaller and immature (Fig. 1c) and Sirius red that stains interstitial collagen indicated fibrosis within MG from patients suffering from OR (Fig. 1d and 1e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression of MR in P1.hMR rats favours MGD initiation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the contribution of MR overactivation to OR pathogenesis, we characterized the phenotype of the MG and ocular surface tissues from P1.hMR rats of 6 months of age\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. It was first confirmed that the human \u003cem\u003eNR3C2\u003c/em\u003e transgene was expressed in the cornea, conjunctiva and MGs, similarly in male and female P1.hMR rats (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). The total MR gene expression (hMR and rMR) exhibited a substantial increase in P1.hMR rats without altering endogenous rat MR expression (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea and Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003ea). P1.hMR rats exhibited enhanced MR staining in both the nucleus and cytoplasm in cells in the conjunctiva, MG and cornea compared to WT rats, whereas no difference in GR immunostaining was observed in these tissues between both groups (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb and Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eb), confirming MR overexpression and increased MR/GR ratio in P1.hMR rats, similar to the human OR tissues. In the MG of P1.hMR rats, MR immunostaining demonstrated a 2.6-fold increase in MR protein expression compared to WT rats (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eNo gross histological differences in the MG, epidermis and conjunctiva were observed between P1.hMR and WT rats (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea), although incipient fibrosis was noted in the MG of P1.hMR rats (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). The expression of Ki67 and P63 in the basal layer of acini and ductal epithelium was diminished in the MG of P1.hMR rats as compared to WT (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee), indicating impaired meibocyte renewal. The expression of two mitochondrial proteins, TOM20 and TIM23, was reduced in the MG acini of P1.hMR rats in comparison with WT rats (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eg), suggesting mitochondrial dysfunction. However, no significant consequences on MG lipid synthesis and no signs of hyperkeratinization were observed in transgenic rats (see Fig. S2a and S2b). Although a decline in MG stem cells and mitochondrial function was observed in P1.hMR rats, suggesting MGD initiation, no signs of OR was observed in 6-month old rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMR overexpression induces molecular dysregulation in the MG of P1.hMR rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptome of the MG from P1.hMR rats of 6 months of age identified 30 differentially expressed genes (DEG) with 14 upregulated and 16 downregulated genes as compared to WT littermates (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). Gene set enrichment analysis (GSEA) using REACTOME pathway database revealed pathways associated with the complement cascade, interferon signalling, keratinization, collagen and extracellular matrix organization (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). HALLMARK gene set highlighted oxidative phosphorylation, angiogenesis, epithelial mesenchymal transition, adipogenesis and fatty acid metabolism pathways (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). Interestingly, these pathways share a common signature with a human MGD published transcriptome\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Genes of particular importance in the pathogenesis of MGD include \u003cem\u003eVim, Pmel, Col3a1, Col1a1, Sdc4 and Fnln5\u003c/em\u003e, which are associated with cell structure and the extracellular matrix; \u003cem\u003eCyp1b1, Cyp1a1, Pdha1, Dld, Crat\u003c/em\u003e and \u003cem\u003eAcsm3\u003c/em\u003e, which are linked to metabolism and energy production; and \u003cem\u003eKcnj8, Gas1, Wipf1\u003c/em\u003e and \u003cem\u003eStc1\u003c/em\u003e, which are related to signal transduction (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed and Fig. S3). These genes were validated by quantitative PCR (qPCR) using MG tissues from an independent batch of P1.hMR and WT rats (Fig. S3). Additional genes of interest, including \u003cem\u003eTlr3, Nos3, Cd36, Clu\u003c/em\u003e, and \u003cem\u003eCcl2\u003c/em\u003e, identified in a published transcriptomic study on the skin of rosacea patients\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, were also confirmed by qPCR in rat MG tissues (Fig. S3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMR overexpression exacerbates UVB-induced MGD in P1.hMR rats mimicking OR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs UVB exposure is a triggering factor for OR, a rat model of OR and MGD was developed using a selective and short exposure of rat eyelids to high-dose UVB for five consecutive days\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. It was applied in 3-month-old rats to evaluate whether MR overexpression aggravates the UV response. Following UVB exposure, the MG swelling and dropout and palpebral conjunctival hyperemia were exacerbated in P1.hMR rats as compared to WT (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Progressive fibrosis was observed in the MG from day 5 to 2 weeks after UVB exposure, with more extensive fibrosis in P1.hMR rats compared to WT rats (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). Oxidative stress markers were exacerbated in P1.hMR rats as shown by a more pronounced staining of 4-hydroxynonenal (4-HNE), a marker for lipid peroxidation, and 3-nitrotyrosine (3-NT), a marker for nitrosative stress in the MG, epidermis and conjunctiva (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed-g). The transient inflammatory response observed on day 5 of UVB exposure was more severe in the transgenic rats, with increased infiltration of monocytes/macrophages and dendritic cell (IBA1 and ED1 positive) beneath the epidermis, within the connective tissue between MG acini and along the ducts in P1.hMR rats (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eh and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ei).\u003c/p\u003e\n\u003cp\u003eMR overexpression aggravated UVB-induced MGD in P1.hMR rats. On day 5 of UVB exposure, markers of lipid synthesis dysregulation were significantly exacerbated in P1.hMR rats, with increased accumulation of intracellular lipid droplets in meibocytes (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb) and elevated PPAR\u0026gamma; expression in both meibocytes and ductal epithelial cells. The later then declined more severely by two weeks post-UVB in P1.hMR rats compared to WT (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed). While keratinization did not differ between WT and P1.hMR rats (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ee and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef), markers of meibocyte renewal declined more drastically in P1.hMR. The MG stem cell marker, P63, showed a continuous decrease up to two weeks (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eh), and the transient rise in KI67-positive proliferating meibocytes observed on day 5 was attenuated in P1.hMR rats (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ei and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ej), indicating impaired meibocytes renewal. Furthermore, TOM20 and TIM23 were reduced in P1.hMR rats on day 5 of UVB exposure compared to WT rats (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ek and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003el).\u003c/p\u003e\n\u003cp\u003eMR overexpression exacerbated corneal signs of UVB-induced OR. No spontaneous morphological alterations of the cornea were detected in P1.hMR rats up to 6 months of age (Fig. S4a and S4b). At baseline and after 5 days of eyelid-targeted UVB exposure, no fluorescein staining was observed in either P1.hMR or WT rats, as the cornea was carefully shielded from direct irradiation during this period. However, by two weeks following UVB exposure, diffuse punctate fluorescein staining \u0026ndash; indicative of punctuate keratitis, likely secondary to UVB-induced MGD \u0026ndash; was observed, with more widespread staining in P1.hMR rats compared to WT (Fig. S4c and S4d). Corneal epithelial integrity was also more severely compromised in P1.hMR rats, as evidenced by greater disorganization of the cell junction proteins, E-cadherin and ZO-1 (Fig. S4e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene dysregulation in the MG in response to UVB radiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 5 days of UVB exposure, 1,271 DEGs were found in the MG of WT rats (Table. S1), including 611 upregulated and 660 downregulated genes. In contrast, 2,040 DEGs were detected in the MG of P1.hMR rats (Table. S2), with 920 upregulated and 1,120 downregulated genes. Intersection analysis revealed 989 genes shared between P1.hMR and WT, while 1,031 genes were exclusively regulated in P1.hMR rats and 272 genes were specific to WT rats (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e\n\u003cp\u003eAmong the shared DEGs, those associated with epithelial integrity, immune responses, collagen production and tissue remodelling showed greater fold changes in P1.hMR rats (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb). Gene ontology enrichment analysis of these common DEGs indicated involvement in biological processes such as cell cycle, inflammatory and humoral immune response, keratinization, extracellular matrix organization, angiogenesis, hormone metabolism, response to corticosteroid, unsaturated fatty acid metabolism, gland morphogenesis, Wnt signalling, and lipid metabolism (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec). Cellular component analysis highlighted enrichment in collagen and extracellular matrix regulation, keratin filaments, and cornified envelope pathways. KEGG pathway analysis further identified significant regulation of PI3K-Akt signalling, complement and coagulation cascades, TNF signalling, arachidonic acid metabolism, IL-17 signalling, and ECM-receptor interaction (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptomic regulation specific to MR overexpression in the UVB-induced OR model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnrichment analysis of UVB-induced DEGs exclusively identified in the MG of WT rats showed pathways related to cell cycle and proliferation, and collagen-extracellular matrix organisation (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee), indicating activation of repair mechanisms. In contrast, genes exclusively regulated in P1.hMR rats associated with inflammatory responses, unsaturated fatty acid and steroid hormone metabolism, extracellular matrix organization, neurotransmitter receptor and sodium ion transport (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ef). These distinct transcriptomic changes in P1.hMR rats further support the role of MR overactivation in amplifying inflammation, neuroimmune responses, metabolic dysregulation and ion transport imbalance, all of which likely contribute to the exacerbation of OR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShared gene regulations between UVB-induced MGD in P1.hMR rats and human MGD and skin rosacea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptome of MGs from P1.hMR rats on day 5 of UVB exposure was compared to transcriptomic profiles of human MGs with MGD\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e (GSE17822) and of human skin with rosacea\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e (GSE65914). A notable overlap in gene regulation was observed between rat model and MGD patients, including genes involved in keratinization and immune responses (e.g., \u003cem\u003eCnfn, Sprr3, Serpina9, Serpinb2\u003c/em\u003e and \u003cem\u003eS100a9\u003c/em\u003e), extracellular matrix remodelling (\u003cem\u003eMmp10\u003c/em\u003e and \u003cem\u003ePdgfra\u003c/em\u003e), and neuronal structure (\u003cem\u003eTubb3\u003c/em\u003e) (Figures S5a. and Table S3)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Furthermore, gene regulation in human rosacea skin exhibited substantial similarities with that of UVB-exposed P1.hMR rats, with 55 genes commonly regulated, including \u003cem\u003eS100a9, Klk6, Klk13, Ccl27, Cxcl11, Mmp12, Mmp9, Sprr3\u003c/em\u003e, and \u003cem\u003eTlr2\u003c/em\u003e. (Fig. S5b. and Table S4)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. These cross-species comparisons provide further support for the involvement MR pathway activation in the pathogenesis of MGD and rosacea in human.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS100a9 is a specific MR target, associated with MGD and skin rosacea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional cross-analyses were performed between the DEGs specific to P1.hMR rats and the two human transcriptomic datasets\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. A total of 21 genes specifically regulated by MR overaction in the P1.hMR rat model overlapped with genes deregulated in human rosacea skin (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea), while 41 MR-regulated genes in P1.hMR rats were shared with those found in human MGD (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb). Among these genes, \u003cem\u003eCCL27, CCL20, CXCL11, TLR2, S100A9\u003c/em\u003e and \u003cem\u003eMMP12\u003c/em\u003e were identified as an interaction network focus on immune cells chemotaxis, IL-17 and Toll-like receptor signaling, involved in rosacea and MGD (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ec and Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ed)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Further comparison between these two gene sets identified a single gene, \u003cem\u003eS100A9\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ee), as a common marker of MR activation in both skin rosacea and MGD. S100A9 immunohistochemistry was further evaluated in human eyelid tissues. In control subjects, S100A9 was primarily located in the epithelium and in cells that may correspond to immune cells beneath the epidermis. In patients with OR, S100A9 expression was not only markedly increased in the epithelium and immune cells, but was also highly expressed in meibocytes in the MG (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ef). These findings provide compelling evidence supporting the role of MR dysregulation in the pathophysiology of oculo-cutaneous rosacea and highlight S100A9 as a potential biomarker of MR pathway activation in both MGD and OR.\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eWe provide herein a set of arguments that support the hypothesis that excessive activation of the MR pathway contributes directly to both the initiation and exacerbation of MGD and OR and that S100A9 is a biomarker of MR pathway activation and could be a therapeutic target or diagnostic marker in OR.\u003c/p\u003e \u003cp\u003eMR overexpression was observed at the site of pathology in the eyelid and ocular surface tissues from patients with OR, associated with inflammatory cell infiltration and fibrosis in the MGs, consistent with pathological features reported in severe cutaneous rosacea\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Signs of abnormal acinar renewal and differentiation likely contribute to the development of MGD in OR patients\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In the posterior segment of the eye, it was shown that local MR pathway activation in retinal and choroidal cells induced low-grade inflammation, oxidative stress, fibrosis and neovascularization\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In systemic pathogenic mechanisms such as hypertension, wound healing, inflammation or metabolic diseases, a pathogenic role of MR hyperactivation has been demonstrated in vascular endothelial and smooth muscle cells.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e But local pathogenic activation in specific organs or tissues has also been demonstrated, such as in adipose tissue, where it has been shown to contribute to adipocyte dysfunction and subsequent metabolic disorders\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e or in the heart, where MR-mediated damages were shown in cadiomyocytes using transgenic mouse models\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe generation of a transgenic rat overexpressing hMR (P1.hMR rats) provided further insight in the role of MR in the initiation and exacerbation of MGD and OR. In the 6-month-old transgenic rats, signs of MGD initiation including decline in meibocytes renewal and mitochondria dysfunctions were observed and the MG transcriptome identified pathways related to oxidative phosphorylation and peroxisome, which is consistent with the oxidative dysregulation observed in patients with MGD\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Upon UVB exposure, MR over-expression notably exacerbated the clinical, histological and transcriptomic manifestations of MGD and OR. The pathological features resemble the ones described in human tissues with OR, in which signs of MR overactivation have been evidenced. A greater number of genes related to inflammation, oxidative stress, epithelial integrity and tissue remodelling were regulated in the transgenic rats compared to in WT. More specifically, the upregulation of Sprr3 may drive aberrant keratinization, a key pathogenic mechanism in MGD\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and regulate IL-33 which amplifies inflammation\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e and tissue remodelling\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In hMR-overexpressing rats, the higher \u003cem\u003eNos3\u003c/em\u003e expression may lead to excessive nitric oxide (NO), contributing to vasodilation and the development of eyelid telangiectasia\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong the DEGs uniquely regulated in P1.hMR rats after UVB exposure, several are involved in chemotaxis, IL-1 signalling, immune cell infiltration, fatty acid metabolism, and ion channel activity\u0026mdash;pathways known to promote inflammation and clinical features of rosacea\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Notably, \u003cem\u003eS100a9\u003c/em\u003e, a direct MR target, was highly upregulated and is known to activate TLR4 and RAGE, amplifying inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Additionally, the regulation of neurotransmitter receptor activity and sodium ion transport suggests MR involvement in neurovascular and neurogenic mechanisms, consistent with the role of neuropeptides and TRP channels in OR\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. These findings support MR overactivation as a driver of immune, metabolic, and neurogenic dysregulation in OR.\u003c/p\u003e \u003cp\u003eIntersection analysis revealed that nearly half of the genes commonly regulated in P1.hMR rats and in human MGD or cutaneous rosacea are associated with MR signalling, underscoring its central role in disease pathogenesis. Key genes such as \u003cem\u003eCCL27, CCL20, CXCL11, TLR2, S100A9\u003c/em\u003e, and \u003cem\u003eMMP12\u003c/em\u003e form an interaction network involved in chemotaxis, IL-17, and Toll-like receptor signalling\u0026mdash;pathways known to drive rosacea-related inflammation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Additionally, the MR-regulated gene SLC27A6, which mediates long-chain fatty acid uptake, may influence meibomian gland lipid composition, a hallmark of MGD\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Together, these findings implicate MR overactivation in immune, metabolic, epithelial, and neurogenic mechanisms that contribute to both MGD and oculo-cutaneous rosacea.\u003c/p\u003e \u003cp\u003eThe search for MR-specific downstream effectors identified S100A9 as a key mediator of MR-driven pathology in MGD and OR. S100A9 promotes inflammation via TLR4/MyD88 signalling, induces M1 macrophage polarization through TLR4/NF-κB\u003csup\u003e35,53\u003c/sup\u003e, enhances neutrophil chemotaxis, and drives keratinization\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e\u0026mdash;all processes implicated in rosacea pathogenesis. In OR patient tissues, S100A9 expression was markedly increased in immune cells, the conjunctival epithelium, and MR-overexpressing meibocytes. Given its role in amplifying oxidative stress and inflammation\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, S100A9 may directly contribute to MG damage and dysfunction.\u003c/p\u003e \u003cp\u003eNotably, \u003cem\u003eS100a9/S100A9\u003c/em\u003e was the only gene both MR-specific in the rat OR model and associated with human OR pathology. Its expression is also downregulated by spironolactone, an MR antagonist, in a limbal stem cell deficiency model, relevant to corneal pathology in OR\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u0026mdash;supporting its regulation by MR. Additionally, \u003cem\u003eS100a9\u003c/em\u003e is upregulated in MR-overexpressing mice with choroidal neuropathy, reinforcing its role as MR targets in ocular tissues\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. As S100A9 is detectable in tears and correlates with MGD severity in dry eye disease\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, it may serve as a non-invasive biomarker of MR activation in OR, offering a potential tool for patient stratification and MR-targeted therapies.\u003c/p\u003e \u003cp\u003eThis study has some limitations. While early alterations in the MGs of young P1.hMR rats were identified, the ocular surface phenotype in aged animals remains unexplored. Given the established role of MR in inflammaging, particularly in the heart\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, it is plausible that age-related ocular changes may develop spontaneously in this model. Nonetheless, rodent models cannot fully recapitulate the human clinical spectrum of OR. To directly assess MR involvement in human disease, we have developed topical spironolactone eye drops, previously validated for safety; however, clinical efficacy data are not yet available.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings demonstrate that MR pathway activation contributes to the pathogenesis of MGD and OR (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Accordingly, MR antagonism represents a promising therapeutic strategy, while S100A9 may serve as a biomarker to identify patients most likely to benefit from targeted intervention. Future clinical trials are warranted to confirm the therapeutic utility of MR blockade in MGD and OR using either systemic or local treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Human eyelid samples collection\u003c/h2\u003e \u003cp\u003eEight patients were operated in Cochin hospital in Paris by XM for lid surgery (ectropion or entropion) and the surgical pieces were immediately fixed in 10% formalin for 24 hours, dehydrated, embedded in paraffin wax and cut into 7 um thick slices and stained with hemalum-eosine, for histological analysis. All patients signed written consent for non-opposition to the use of surgical waste in research. Additional unused sections were used for specific staining relative to the present study (see below). We analyzed the tissue sections from 4 patients with OR (3 men and 1 woman, 70 years, 71 years, 52 years, 78 years) and 4 control subjects without ocular or skin rosacea (3 men and 1 woman 44years, 90 years, 78 years, 76 years). The collection and storage of human biological samples were approved by French ethics committee CCP Ile de France 1 (no. 2016-nov-14390).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Animal experiments\u003c/h2\u003e \u003cp\u003eAll experiments were performed in accordance with the European communities Council Directive 86/609/EEC and French national regulations and approved by local ethics committee (# 23478-2020010317557546 v4, Charles Darwin). To study the role of MR in the pathology of OR, we generated P1.hMR rats on Sprague-Dawley genetic background. The human MR (hMR) gene is expressed under the control of the proximal P1 promoter of the \u003cem\u003eNR3C2\u003c/em\u003e as described previously\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Overexpression of \u003cem\u003eNR3C2\u003c/em\u003e expression was validated in ocular tissues in both male (WT: n\u0026thinsp;=\u0026thinsp;3\u0026ndash;6 rats; P1.hMR: n\u0026thinsp;=\u0026thinsp;3\u0026ndash;8 rats) and female (WT: n\u0026thinsp;=\u0026thinsp;2\u0026ndash;4 rats; P1.hMR: n\u0026thinsp;=\u0026thinsp;6\u0026ndash;8 rats) by transcriptomic and qPCR analysis. Other animals (WT: n\u0026thinsp;=\u0026thinsp;4\u0026ndash;6 rats; P1.hMR: n\u0026thinsp;=\u0026thinsp;4\u0026ndash;6 rats) were used for characterization in morphology and pathology. All animals were kept in pathogen-free conditions with free access to food and water and housed in temperature-controlled room with a 12-h light/12-h dark cycle. Anaesthesia was induced by intraperitoneal ketamine 100 mg/kg and Xylazine 10 mg/kg. Animal were euthanized by intraperitoneal injection of fatal dose of Euthasol\u0026reg; Vet.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e4.3 UVB irradiation on rat eyelids\u003c/h2\u003e \u003cp\u003eThe upper eyelids of rats were shaved and exposed to 1000 \u0026micro;W/cm2 of UVB (312 nm, Herolab, Wiesioch, Germany) for 5 min (300 mJ/ cm2) daily for 5 days, as described previously\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The cornea and the whole body were covered and protected. Lesions of eyelids were photographed under the microscopy from day 0 to day 5. Rats were euthanized after 5 days to evaluate the acute damages of ocular surface tissues and MR related pathology (WT: n\u0026thinsp;=\u0026thinsp;5 rats; P1.hMR: n\u0026thinsp;=\u0026thinsp;6 rats). To assess the prolonged effects of UVB irradiation on MGD on the cornea, a group of rats (WT: n\u0026thinsp;=\u0026thinsp;6 rats; P1.hMR: n\u0026thinsp;=\u0026thinsp;6 rats) were followed up until 2 weeks after UVB exposure. Corneal fluorescein staining was performed before, after 5 days of UVB and 2 weeks after UVB exposure Corneal defects were graded according to the criteria previously described\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e.. Rats were then euthanized, eyelids and corneas were dissected for morphological and immunohistochemical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Hematoxylin and eosin staining\u003c/h2\u003e \u003cp\u003eEyelid samples from humans and rats were immediately fixed in 10% neutral-buffered formalin for 24 hours and 4% paraformaldehyde (PFA) for 2 hours, respectively, followed by graded dehydration and embedding in paraffin. Serial sections of 7 \u0026micro;m thickness were obtained using a microtome (LEICA). The paraffin-embedded sections of human and rat eyelids were deparaffinized in xylene and rehydrated through a descending ethanol series. The sections were then stained with hematoxylin reagent (RAL Diagnostics, #320550-2500, Bordeaux, France) for 15 minutes and rinsed with tap water to remove excess dye. Subsequently, the sections were immersed in eosin solution (RAL Diagnostics, #312730-0100, Bordeaux, France) for 4 minutes and rinsed again with tap water. After sequential baths in absolute ethanol and xylene, the tissue sections were mounted using Eukitt\u0026reg; mounting medium (O. Kindler GmbH, #200080, Freiburg, Germany). Morphological changes were observed under a light microscope (Olympus BX51, Rungis, France) equipped with a CCD camera (Olympus DP70).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Sirius red staining\u003c/h2\u003e \u003cp\u003eFor collagen detection, human and rat eyelid sections were deparaffinized and rehydrated, then incubated with 1% Sirius Red in picric acid for 15 minutes, as previously described \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. After sequential washes with acetic acid and absolute ethanol, the slides were mounted and examined under a microscope. Collagen content was quantified using Fiji ImageJ software (version 1.54b, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). A binary conversion of the red channel was performed to calculate the percentage of area occupied by collagen fibers. All quantifications were normalized to the analyzed tissue area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.6. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin-embedded rat and human eyelid sections were used for immunohistochemical staining as previously described\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Briefly, sections were deparaffinized and rehydrated, followed by antigen retrieval in citrate buffer (10 mM, pH 6.0) at 100\u0026deg;C for 20 minutes. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide. Sections were then blocked with TNB blocking buffer (AKOYA Biosciences, #FP1012, Massachusetts, USA) for 30 minutes at room temperature. Primary antibodies (MR, GR, HSD1, HSD2, Nitrotyrosine, 4-HNE, S100a9) were applied overnight at 4\u0026deg;C as reference listed in table 7. After washes with PBST, sections were incubated with biotinylated secondary antibodies in table 7. for 1 hour at room temperature. For GR and MR immunohistochemistry, the Tyramide Signal Amplification (TSA) biotin kit (AKOYA Biosciences, MA, USA) was used according to the manufacturer\u0026rsquo;s instructions to amplify the tyramide signal. For other primary antibodies, sections were processed with Vectastain Elite ABC reagent (Vector Laboratories, USA, #AK-5000) for 30 minutes. For S100A9, the signal was developed using the VIP Substrate Kit (Vector Laboratories, USA, #SK-4605) and stopped in PBS buffer. For other targets, the signal was visualized using 3,3\u0026rsquo;-diaminobenzidine (DAB) substrate (Vector Laboratories, USA, #SK-4105) and stopped in 50 mM TRIS buffer (pH 7.6) for 10 minutes. Tissue sections were mounted with Eukitt\u0026reg; mounting medium (O. Kindler GmbH, Freiburg, Germany). Negative controls were performed by omitting the primary antibody. For quantification, the positive staining area normalized to the same threshold for MR and GR, the mean grey value limited to the same threshold for staining intensity for other targets in MG were measured using ImageJ software (version 1.54b, National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Quantitative PCR in ocular surface tissues from rats\u003c/h2\u003e \u003cp\u003eRNA from various ocular surface tissues was isolated using the RNeasy Mini Kit (QIAGEN, Cat. No. 74106), followed by DNase I treatment (QIAGEN, Cat. No. 79254) according to the manufacturer\u0026rsquo;s protocol. Transcript levels were quantified by quantitative real-time PCR (qPCR) on the QuantStudio\u0026trade; 5 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Green detection. Relative quantification was performed using the ΔΔCT method. Primer sequences for all analyzed genes are provided in Supplementary Table\u0026nbsp;5. Reference genes (\u003cem\u003eHprt1\u003c/em\u003e, \u003cem\u003eUbc\u003c/em\u003e, and \u003cem\u003e18S\u003c/em\u003e) were used for normalization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Immunofluorescence\u003c/h2\u003e \u003cp\u003eRat ocular tissues were dissected, embedded in OCT compound, and frozen at \u0026minus;\u0026thinsp;80\u0026deg;C. Cryosections of 10 \u0026micro;m thickness were obtained using a cryostat (Leica). Immunofluorescence staining was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, rat eyelid and corneal cryosections were fixed in 4% PFA followed by washes with PBS. Sections were permeabilized with 0.1% Triton X-100 in PBS for 30 minutes and blocked with 5% goat serum in PBS for 30 minutes at room temperature. Tissues were incubated with primary antibodies overnight at 4\u0026deg;C. Secondary antibodies was applied the next day for 1 hour at room temperature. Nuclei were counterstained with 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI; 1:5000) for 5 minutes, and slides were mounted using Gel Mount (Dako, Agilent, Les Ulis, France). Negative controls were performed by omitting the primary antibody. Sections were visualized using an Olympus fluorescence microscope (Olympus BX51). Primary and secondary antibodies, along with their dilutions, are listed in Supplementary Table\u0026nbsp;6. Quantification was performed using QuPath software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.9 Nile red staining\u003c/h2\u003e \u003cp\u003eFresh-frozen rat MG sections were incubated with 1 \u0026micro;g/mL Nile red (Sigma-Aldrich, #72458, Missouri, USA) in PBS for 1 hour at 4\u0026deg;C. Tissues were then fixed in 4% paraformaldehyde (PFA) for 15 minutes, followed by DAPI staining and three washes with PBS. Stained sections were imaged using a confocal microscope (Zeiss LSM 710, Oberkochen, Germany). Strongly stained lipid droplets were quantified using Fiji ImageJ software by calculating the percentage of the highlighted area limited to threshold relative to the total analyzed area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.10 RNA Sequencing of Rat MG and Transcriptomic Analysis\u003c/h2\u003e \u003cp\u003eTo characterize the pathology involved in P1.hMR phenotype, MGs were carefully dissected from 6-months-aged WT (n\u0026thinsp;=\u0026thinsp;6 eyes) and P1.hMR male rats (n\u0026thinsp;=\u0026thinsp;4 eyzs) for transcriptomic analysis. For UVB-induced MGD with ocular surface damages model of OR, 12-weeks-old Sprague-Dawley rats were irradiated by UVB on eyelids (WT: n\u0026thinsp;=\u0026thinsp;6 eyes; P1.hMR: n\u0026thinsp;=\u0026thinsp;10 eyes). Age- and sex matched control rats (WT: n\u0026thinsp;=\u0026thinsp;4; P1.hMR: n\u0026thinsp;=\u0026thinsp;5) were raised in the same condition without model. Animals were euthanized after 5 days of UVB irradiation. RNA extraction was conducted as described above. 1mg of the extracted RNA sample was sent for RNA sequencing at the iGenSeq transcriptomic platform located at the Brain and Spine Institute (ICM, Paris, France). RNA quality was checked by capillary electrophoresis and RNA integrity numbers (RIN) ranging from 8.8 to 9.3 was accepted for library generation. Quality of raw data was assessed using FastQC. STAR v2.5.3 was used to align reads on reference genome rn6 using standard options. Between 30 and 38\u0026nbsp;million reads were mapped. Quantification of gene and isoform abundances was done with rsem 1.2.28, and reproducibility of replicates was controlled with PCA representations.\u003c/p\u003e \u003cp\u003eThe transcriptomic data for rats were analyzed on the online platform of the Paris Brain institute (quby.icm-institute.org). First, differential gene expression analysis with DESeq2 method was utilized to identify the differentially expressed genes. Adjusted P values (pFDR) were calculated with the Benjamini-Hochberg procedure to control false discovery rate (FDR threshold set at 0.05, log2 fold-change threshold set at 0.5). Then, an enrichment analysis was performed (by GSEA and over-representation analysis) in different gene-set collections including Reactome gene sets (Reactome subset of Canonical pathways), Hallmark gene sets (H), Gene Ontology gene sets (C5: GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Gene Sets.\u003c/p\u003e \u003cp\u003eTranscriptomic data for human meibomian gland dysfunction (MGD) were reanalyzed using the GEO2R online platform (GSE17822), with differentially expressed genes (DEGs) identified based on a significance threshold of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. For human rosacea, DEGs were obtained by comparing control samples with three different subtypes (logFC\u0026thinsp;\u0026gt;\u0026thinsp;1.5, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) using GSE65914. An intersection analysis was performed to identify the most relevant DEGs across the three subtypes. Additional intersection analyses were conducted using the Xiantao Academic online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.xiantaozi.com/\u003c/span\u003e\u003cspan address=\"https://www.xiantaozi.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.11 Statistics Analysis\u003c/h2\u003e \u003cp\u003eA study of the literature and an estimation using G-power software, version 3.1.9.6, were carried out to estimate the number of animals required. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analysis was performed using the GraphPad Prism (GraphPad Software, version 9, San Diego, CA, USA). The unpaired t-test or Mann\u0026ndash;Whitney test was used to compare two groups. The Kruskal\u0026ndash;Walli\u0026rsquo;s test followed by Dunn\u0026rsquo;s test was used to compare more than two groups. The Friedman test was used to compare more than 2 matched groups. A p value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThis work was supported by INSERM, Agence National de la Recherche (grant n\u0026deg; ANR-21-CE18-0018), and RESTORE VISION grant under the EU Framework Horizon Europe Program (grant N\u0026deg;101080611).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Functional Exploration Center (CEF) and the Core Facilities of Histology, Imaging and Flow Cytometry (CHIC) at Centre de Recherche des Cordeliers, BioRender platform for figure modification https://BioRender.com.\u003c/p\u003e\n\u003cp\u003eP1.hMR rat model generation services were provided by PHENOMIN-iCS (www.phenomin.fr).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.B.-C. and M.Z.\u0026nbsp;\u003c/strong\u003eacquired the funding,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003econceived and directed the project, and designed and supervised the experiments.\u0026nbsp;\u003cstrong\u003eX.M\u0026nbsp;\u003c/strong\u003eoperated the patients. \u003cstrong\u003eL.Z.\u003c/strong\u003e performed the microtome and histology of the human samples. \u003cstrong\u003eN.Y.\u0026nbsp;\u003c/strong\u003eperformed the prospective study in patients. \u003cstrong\u003eL.Z., D.R.-B., E.G. and M.Z.\u0026nbsp;\u003c/strong\u003eperformed all the in vivo experiments. \u003cstrong\u003eL.Z.\u003c/strong\u003e performed the histological, immunohistochemical and transcriptomic analysis of human and rat samples. \u003cstrong\u003eL.Z.\u003c/strong\u003e \u003cstrong\u003eF.B.-C. and M.Z.\u003c/strong\u003e interpreted the data. \u003cstrong\u003eL.Z. and F.B.-C\u003c/strong\u003e. constructed the figures. \u003cstrong\u003eL.Z, and F.B.-C.\u0026nbsp;\u003c/strong\u003ewrote the original draft. \u003cstrong\u003eF.B.-C., M.Z. N.Y. and J.L.B.\u0026nbsp;\u003c/strong\u003ereviewed the manuscript.\u003cstrong\u003e\u0026nbsp;L.Z. and F.B.-C. and M.Z.\u0026nbsp;\u003c/strong\u003eedited the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll authors read and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll Data are available upon reasonable request. Raw RNAseq data will be made available on Gene Expression Omnibus GSE291177 (accessible on 08 July 2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGether, L., Overgaard, L. K., Egeberg, A. \u0026amp; Thyssen, J. P. Incidence and prevalence of rosacea: a systematic review and meta-analysis. \u003cem\u003eBr. J. 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It can affect vision, damage the cornea and has a significant impact on quality of life. OR is linked to meibomian gland dysfunction (MGD), but is poorly understood, largely underdiagnosed and incurable. Human samples and an UVB-induced OR rat model bring evidence of the central role of mineralocorticoid receptor (MR) pathway overactivation in OR pathogenesis. In patients suffering from OR, MR was overexpressed in the meibomian glands. Signs of UVB-induced OR were exacerbated in a transgenic rat that overexpresses human MR through enhanced immune and inflammatory responses, oxidative stress and lipid dysmetabolism. Multi-omics cross-species analysis identified S100A9 as a key mediator of MR-associated pathogenicity. MR pathway antagonism appears as a potential therapeutic strategy.\u003c/p\u003e","manuscriptTitle":"The role of Mineralocorticoid Receptor pathway in Ocular Rosacea","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-06 08:45:43","doi":"10.21203/rs.3.rs-6589915/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9502d29a-239b-417b-bb22-be8e88fbfe63","owner":[],"postedDate":"August 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":48503142,"name":"Health sciences/Diseases/Eye diseases/Eyelid diseases"},{"id":48503143,"name":"Health sciences/Molecular medicine"}],"tags":[],"updatedAt":"2025-08-06T08:45:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-06 08:45:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6589915","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6589915","identity":"rs-6589915","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}