Pub Date : 2024-10-01DOI: 10.1016/j.jtos.2024.10.003
Erin A. Hisey , Sydni Wong , Sangwan Park , Kevin Aguirre Gamarra , Sara A. Adelman , Kelly E. Knickelbein , Melinda Quan , Michelle H. Ferneding , Michelle McCorkell , Nicole Daley , Vanessa Ureno , Sophie Le , Monica Ardon , Liana Williams , Bryan Puentes , Morgan Bowman , Monica J. Motta , Hoang Quoc Hai Pham , Amber Wilkerson , Seher Yuksel , Brian C. Leonard
Purpose
There is an urgent need for animal models of meibomian gland dysfunction (MGD) and evaporative dry eye disease (EDED) to understand their pathophysiology and investigate novel therapeutics. This study sought to further define the acyl-CoA: wax alcohol acyltransferase 2 knockout (Awat2 KO) mouse as a model of EDED using a combination of novel clinical, biochemical, and biophysical endpoints.
Methods
Wildtype and Awat2 KO mice between 1 and 18 months of age were used. Ocular examinations and advanced imaging were performed. The lipidomic composition and in situ melting temperature of meibum were determined. qPCR was performed to define ocular surface gene and pro-inflammatory transcript expression. Dynamic contact angle goniometry was performed to assess the adherence capability of the ocular surface.
Results
Awat2 KO mice have mild, white, hyperreflective corneal opacities of the anterior stroma and significantly enlarged apical epithelial cells (P = 0.0004). In Awat2 KO meibum, wax esters were 9–10 times lower than in wildtype meibum. Additionally, meibum melting temperature increased from 32° to 47 °C (P < 0.0001), leading to impaired meibum secretion and dilation of the central duct. Awat2 KO corneal epithelia had significantly decreased mucin expression (Muc1 and Muc4, P = 0.0043) and increased interferon-γ production (P = 0.0303). Awat2 KO globes have a significantly shortened time of droplet adherence to their ocular surface (P = 0.0053), indicating a decreased tear film adherence capacity. Wildtype corneal epithelia does not express Awat2, indicating that the EDED phenotype is secondary to the loss of Awat2 from the meibomian glands.
Conclusions
Awat2 KO mice recapitulate many of features of human MGD and EDED, representing a model to test novel therapeutics.
目的：目前急需建立睑板腺功能障碍（MGD）和蒸发性干眼症（EDED）的动物模型，以了解其病理生理学并研究新型疗法。本研究试图结合新的临床、生化和生物物理终点，进一步确定酰基-CoA：蜡醇酰基转移酶 2 基因敲除（Awat2 KO）小鼠作为干眼症的模型：方法：使用 1 到 18 个月大的野生型和 Awat2 KO 小鼠。进行了眼部检查和高级成像。通过 qPCR 确定眼表基因和促炎转录物的表达。采用动态接触角测角法评估眼表的粘附能力：结果：Awat2 KO小鼠的前基质有轻度、白色、高反射性角膜不透明，角膜顶端上皮细胞明显增大（P=0.0004）。Awat2 KO 卵膜中的蜡酯含量比野生型卵膜低 9-10 倍。此外，咀嚼液的熔化温度从32°升至47°C（PConclusions：Awat2 KO小鼠再现了人类MGD和EDED的许多特征，是一种测试新型疗法的模型。
{"title":"Meibomian gland lipid alterations and ocular surface sequela in Awat2 knockout murine model of meibomian gland dysfunction and evaporative dry eye disease","authors":"Erin A. Hisey , Sydni Wong , Sangwan Park , Kevin Aguirre Gamarra , Sara A. Adelman , Kelly E. Knickelbein , Melinda Quan , Michelle H. Ferneding , Michelle McCorkell , Nicole Daley , Vanessa Ureno , Sophie Le , Monica Ardon , Liana Williams , Bryan Puentes , Morgan Bowman , Monica J. Motta , Hoang Quoc Hai Pham , Amber Wilkerson , Seher Yuksel , Brian C. Leonard","doi":"10.1016/j.jtos.2024.10.003","DOIUrl":"10.1016/j.jtos.2024.10.003","url":null,"abstract":"<div><h3>Purpose</h3><div>There is an urgent need for animal models of meibomian gland dysfunction (MGD) and evaporative dry eye disease (EDED) to understand their pathophysiology and investigate novel therapeutics. This study sought to further define the <em>acyl-CoA: wax alcohol acyltransferase 2</em> knockout (<em>Awat2</em> KO) mouse as a model of EDED using a combination of novel clinical, biochemical, and biophysical endpoints.</div></div><div><h3>Methods</h3><div>Wildtype and <em>Awat2</em> KO mice between 1 and 18 months of age were used. Ocular examinations and advanced imaging were performed. The lipidomic composition and <em>in situ</em> melting temperature of meibum were determined. qPCR was performed to define ocular surface gene and pro-inflammatory transcript expression. Dynamic contact angle goniometry was performed to assess the adherence capability of the ocular surface.</div></div><div><h3>Results</h3><div><em>Awat2</em> KO mice have mild, white, hyperreflective corneal opacities of the anterior stroma and significantly enlarged apical epithelial cells (<em>P</em> = 0.0004). In <em>Awat2</em> KO meibum, wax esters were 9–10 times lower than in wildtype meibum. Additionally, meibum melting temperature increased from 32° to 47 °C (<em>P</em> < 0.0001), leading to impaired meibum secretion and dilation of the central duct. <em>Awat2</em> KO corneal epithelia had significantly decreased mucin expression (<em>Muc1</em> and <em>Muc4</em>, <em>P</em> = 0.0043) and increased interferon-γ production (<em>P</em> = 0.0303). <em>Awat2</em> KO globes have a significantly shortened time of droplet adherence to their ocular surface (<em>P</em> = 0.0053), indicating a decreased tear film adherence capacity. Wildtype corneal epithelia does not express <em>Awat2</em>, indicating that the EDED phenotype is secondary to the loss of <em>Awat2</em> from the meibomian glands.</div></div><div><h3>Conclusions</h3><div><em>Awat2</em> KO mice recapitulate many of features of human MGD and EDED, representing a model to test novel therapeutics.</div></div>","PeriodicalId":54691,"journal":{"name":"Ocular Surface","volume":"34 ","pages":"Pages 489-503"},"PeriodicalIF":5.9,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142484786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.jtos.2024.10.004
Shinri Sato , Yoko Ogawa , Calvin W. Wong , Harrison L. Le , Richard W. Yee , Dan S. Gombos , Kazuno Negishi , Masatoshi Hirayama
Purpose
The topical administration of spironolactone, a mineralocorticoid receptor antagonist (MRA) improves dry eye symptoms in patients with ocular graft-versus-host disease (GVHD); however, the detailed mechanism remains unclear. This study aimed to investigate the effects of spironolactone eyedrops on the ocular surface using a chronic GVHD (cGVHD) mouse model and to determine the expression of the mineralocorticoid receptor (MR).
Methods
A cGVHD mouse model was established by allogeneic bone marrow transplantation (BMT) from B10.D2 mice to BALB/c mice. Subsequently, cGVHD mice were treated with either 0.005 % spironolactone or vehicle eyedrops. The eyelids, cornea and conjunctiva of the recipients were analyzed at 4-week intervals post-BMT in both groups.
Results
Signs of ocular GVHD, such as corneal epithelial damage, depletion of meibomian glands, and inflammatory cell infiltration onto the ocular surface, were significantly decreased in cGVHD mice treated with spironolactone eyedrops. The expression of the MR NR3C2 in the corneal and conjunctival epithelia was significantly increased in cGVHD mice. HSP47+NR3C2+ MR-expressing fibroblasts, CD45+NR3C2+ MR-expressing leukocytes, and CD4+NR3C2+ MR-expressing T cells infiltrated the ocular surface tissue of cGVHD mice significantly more than that of syngeneic controls.
Conclusions
MR expression is increased in epithelial cells, fibroblasts, and T cells in a murine cGVHD model, whereas MRA and spironolactone eyedrops could attenuate the severity of ocular GVHD. These findings suggest that MR signaling partially contributes to the development of ocular GVHD in this mouse model.
{"title":"Mineralocorticoid receptor expression and the effects of the mineralocorticoid receptor antagonist spironolactone in a murine model of graft-versus-host disease","authors":"Shinri Sato , Yoko Ogawa , Calvin W. Wong , Harrison L. Le , Richard W. Yee , Dan S. Gombos , Kazuno Negishi , Masatoshi Hirayama","doi":"10.1016/j.jtos.2024.10.004","DOIUrl":"10.1016/j.jtos.2024.10.004","url":null,"abstract":"<div><h3>Purpose</h3><div>The topical administration of spironolactone, a mineralocorticoid receptor antagonist (MRA) improves dry eye symptoms in patients with ocular graft-versus-host disease (GVHD); however, the detailed mechanism remains unclear. This study aimed to investigate the effects of spironolactone eyedrops on the ocular surface using a chronic GVHD (cGVHD) mouse model and to determine the expression of the mineralocorticoid receptor (MR).</div></div><div><h3>Methods</h3><div>A cGVHD mouse model was established by allogeneic bone marrow transplantation (BMT) from B10.D2 mice to BALB/c mice. Subsequently, cGVHD mice were treated with either 0.005 % spironolactone or vehicle eyedrops. The eyelids, cornea and conjunctiva of the recipients were analyzed at 4-week intervals post-BMT in both groups.</div></div><div><h3>Results</h3><div>Signs of ocular GVHD, such as corneal epithelial damage, depletion of meibomian glands, and inflammatory cell infiltration onto the ocular surface, were significantly decreased in cGVHD mice treated with spironolactone eyedrops. The expression of the MR NR3C2 in the corneal and conjunctival epithelia was significantly increased in cGVHD mice. HSP47<sup>+</sup>NR3C2<sup>+</sup> MR-expressing fibroblasts, CD45<sup>+</sup>NR3C2<sup>+</sup> MR-expressing leukocytes, and CD4<sup>+</sup>NR3C2<sup>+</sup> MR-expressing T cells infiltrated the ocular surface tissue of cGVHD mice significantly more than that of syngeneic controls.</div></div><div><h3>Conclusions</h3><div>MR expression is increased in epithelial cells, fibroblasts, and T cells in a murine cGVHD model, whereas MRA and spironolactone eyedrops could attenuate the severity of ocular GVHD. These findings suggest that MR signaling partially contributes to the development of ocular GVHD in this mouse model.</div></div>","PeriodicalId":54691,"journal":{"name":"Ocular Surface","volume":"34 ","pages":"Pages 477-488"},"PeriodicalIF":5.9,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142484787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.jtos.2024.09.008
Xiaozhao Zhang , Guangjin Chen , Yan He , Qingming Tang , Ying Yin , Ying Jie
Secretory glands, responsible for tears and saliva production, play essential roles in maintaining ocular and oral well-being. Disruptions in gland secretion can arise from various factors, including rhythm disturbances associated with sleep disorders. However, the underlying mechanisms governing these disruptions remain largely unexplored. We demonstrate that BMAL1, a core component of the circadian system, plays a critical role in regulating secretory gland secretion. Loss of BMAL1 induces vacuolation and atrophy phenotypes in acinar cells, subsequently leading to cell apoptosis and gland hypofunction, but does not cause Sjogren's syndrome, which is characterized by localized inflammatory cell infiltration. Mechanically, BMAL1 directly modulates the transcription of ITPR2 and ITPR3, thereby altering the secretion of Lactoferrin and Lysozyme. Restoration of ITPR2 and ITPR3 expression in Bmal1-deficient rats effectively alleviated the symptoms of lacrimal and parotid glands secretory dysfunction and significantly reduced dry mouth and dry eye conditions in rhythm-disordered rats. These findings highlight the essential role of BMAL1 in regulating salivary and lacrimal gland secretion and suggest a novel therapeutic approach for treating dry mouth and dry eyes associated with rhythm disorders.
{"title":"BMAL1 deficiency provokes dry mouth and eyes by down-regulating ITPR2/3","authors":"Xiaozhao Zhang , Guangjin Chen , Yan He , Qingming Tang , Ying Yin , Ying Jie","doi":"10.1016/j.jtos.2024.09.008","DOIUrl":"10.1016/j.jtos.2024.09.008","url":null,"abstract":"<div><div>Secretory glands, responsible for tears and saliva production, play essential roles in maintaining ocular and oral well-being. Disruptions in gland secretion can arise from various factors, including rhythm disturbances associated with sleep disorders. However, the underlying mechanisms governing these disruptions remain largely unexplored. We demonstrate that BMAL1, a core component of the circadian system, plays a critical role in regulating secretory gland secretion. Loss of BMAL1 induces vacuolation and atrophy phenotypes in acinar cells, subsequently leading to cell apoptosis and gland hypofunction, but does not cause Sjogren's syndrome, which is characterized by localized inflammatory cell infiltration. Mechanically, BMAL1 directly modulates the transcription of ITPR2 and ITPR3, thereby altering the secretion of Lactoferrin and Lysozyme. Restoration of ITPR2 and ITPR3 expression in <em>Bmal1</em>-deficient rats effectively alleviated the symptoms of lacrimal and parotid glands secretory dysfunction and significantly reduced dry mouth and dry eye conditions in rhythm-disordered rats. These findings highlight the essential role of BMAL1 in regulating salivary and lacrimal gland secretion and suggest a novel therapeutic approach for treating dry mouth and dry eyes associated with rhythm disorders.</div></div>","PeriodicalId":54691,"journal":{"name":"Ocular Surface","volume":"34 ","pages":"Pages 430-440"},"PeriodicalIF":5.9,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142335622","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.jtos.2024.09.006
Soojin Yi , Jeongho Kim , Mi Ju Kim , Che Gyem Yae , Ki Hean Kim , Hong Kyun Kim
Purpose
This study aimed to investigate the therapeutic potential of extracellular vesicles (EVs) derived from human amniotic epithelial cells (hAEC-EVs) for Dry Eye Disease (DED) treatment.
Methods
Highly purified EVs were isolated from the culture supernatants of hAECs, which obtained from term placenta and characterized. Proteomic contents were analyzed for assessing its biological function related to the therapeutic potentials for DED. Subsequently, we examined the therapeutic efficacy of hAEC-EVs on human corneal epithelial cells exposed to hyperosmotic stress and in an experimental DED mouse model induced by desiccation stress.
Results
Proteomic analysis of hAEC-EVs revealed proteins linked to cell proliferation and anti-inflammatory responses. We demonstrated efficient uptake of hAEC-EVs by ocular surface cells. Under DED conditions, EV treatment increased corneal epithelial cell proliferation and migration, and concurrently reducing inflammatory cytokines. In the DED mouse model, hAEC-EVs showed significant improvements in corneal staining score, tear secretion, corneal irregularity, and conjunctival goblet cell density. Additionally, hAEC-EVs exhibited a mitigating effect on ocular surface inflammation induced by desiccation.
Conclusions
These findings suggest that hAEC-EVs hold potential as a cell-free therapy for corneal epithelial defects and ocular surface diseases, presenting a promising treatment option for DED.
{"title":"Development of human amniotic epithelial cell-derived extracellular vesicles as cell-free therapy for dry eye disease","authors":"Soojin Yi , Jeongho Kim , Mi Ju Kim , Che Gyem Yae , Ki Hean Kim , Hong Kyun Kim","doi":"10.1016/j.jtos.2024.09.006","DOIUrl":"10.1016/j.jtos.2024.09.006","url":null,"abstract":"<div><h3>Purpose</h3><div>This study aimed to investigate the therapeutic potential of extracellular vesicles (EVs) derived from human amniotic epithelial cells (hAEC-EVs) for Dry Eye Disease (DED) treatment.</div></div><div><h3>Methods</h3><div>Highly purified EVs were isolated from the culture supernatants of hAECs, which obtained from term placenta and characterized. Proteomic contents were analyzed for assessing its biological function related to the therapeutic potentials for DED. Subsequently, we examined the therapeutic efficacy of hAEC-EVs on human corneal epithelial cells exposed to hyperosmotic stress and in an experimental DED mouse model induced by desiccation stress.</div></div><div><h3>Results</h3><div>Proteomic analysis of hAEC-EVs revealed proteins linked to cell proliferation and anti-inflammatory responses. We demonstrated efficient uptake of hAEC-EVs by ocular surface cells. Under DED conditions, EV treatment increased corneal epithelial cell proliferation and migration, and concurrently reducing inflammatory cytokines. In the DED mouse model, hAEC-EVs showed significant improvements in corneal staining score, tear secretion, corneal irregularity, and conjunctival goblet cell density. Additionally, hAEC-EVs exhibited a mitigating effect on ocular surface inflammation induced by desiccation.</div></div><div><h3>Conclusions</h3><div>These findings suggest that hAEC-EVs hold potential as a cell-free therapy for corneal epithelial defects and ocular surface diseases, presenting a promising treatment option for DED.</div></div>","PeriodicalId":54691,"journal":{"name":"Ocular Surface","volume":"34 ","pages":"Pages 370-380"},"PeriodicalIF":5.9,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142335623","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.jtos.2024.09.007
Jiayun Ge , Xiang Li , Yutong Xia , Zhitong Chen , Chen Xie , Yuan Zhao , Kuangqi Chen , Ye Shen , Jianping Tong
NLRP3 inflammasome is a cytosolic multiprotein complex formed in response to exogenous environmental stress and cellular damage. The three major components of the NLRP3 inflammasome are the innate immunoreceptor protein NLRP3, the adapter protein apoptosis-associated speck-like protein containing a C-terminal caspase activation and recruitment domain, and the inflammatory protease enzyme caspase-1. The integrated NLRP3 inflammasome triggers the activation of caspase-1, leading to GSDMD-dependent pyroptosis and facilitating the maturation and release of inflammatory cytokines, namely interleukin (IL)-18 and IL-1β. However, the inflammatory responses mediated by the NLRP3 inflammasome exhibit dual functions in innate immune defense and cellular homeostasis. Aberrant activation of the NLRP3 inflammasome matters in the etiology and pathophysiology of various corneal diseases. Corneal alkali burn can induce pyroptosis, neutrophil infiltration, and corneal angiogenesis via the activation of NLRP3 inflammasome. When various pathogens invade the cornea, NLRP3 inflammasome recognizes pathogen-associated molecular patterns or damage-associated molecular patterns to engage in pro-inflammatory and anti-inflammatory mechanisms. Moreover, chronic inflammation and proinflammatory cascades mediated by the NLRP3 inflammasome contribute to the pathogenesis of diabetic keratopathy. Furthermore, overproduction of reactive oxygen species, mitochondrial dysfunction, and toll-like receptor-mediated activation of nuclear factor kappa B drive the stimulation of NLRP3 inflammasome and participate in the progression of dry eye disease. However, there still exist controversies regarding the regulatory pathways of the NLRP3 inflammasome. In this review, we provide a comprehensive overview of recent advancements in the function of NLRP3 inflammasome in corneal diseases and its regulatory pathways primarily through studies using animal models. Furthermore, we explore prospects for pharmacologically targeting pathways associated with NLRP3.
{"title":"Recent advances in NLRP3 inflammasome in corneal diseases: Preclinical insights and therapeutic implications","authors":"Jiayun Ge , Xiang Li , Yutong Xia , Zhitong Chen , Chen Xie , Yuan Zhao , Kuangqi Chen , Ye Shen , Jianping Tong","doi":"10.1016/j.jtos.2024.09.007","DOIUrl":"10.1016/j.jtos.2024.09.007","url":null,"abstract":"<div><div>NLRP3 inflammasome is a cytosolic multiprotein complex formed in response to exogenous environmental stress and cellular damage. The three major components of the NLRP3 inflammasome are the innate immunoreceptor protein NLRP3, the adapter protein apoptosis-associated speck-like protein containing a C-terminal caspase activation and recruitment domain, and the inflammatory protease enzyme caspase-1. The integrated NLRP3 inflammasome triggers the activation of caspase-1, leading to GSDMD-dependent pyroptosis and facilitating the maturation and release of inflammatory cytokines, namely interleukin (IL)-18 and IL-1β. However, the inflammatory responses mediated by the NLRP3 inflammasome exhibit dual functions in innate immune defense and cellular homeostasis. Aberrant activation of the NLRP3 inflammasome matters in the etiology and pathophysiology of various corneal diseases. Corneal alkali burn can induce pyroptosis, neutrophil infiltration, and corneal angiogenesis via the activation of NLRP3 inflammasome. When various pathogens invade the cornea, NLRP3 inflammasome recognizes pathogen-associated molecular patterns or damage-associated molecular patterns to engage in pro-inflammatory and anti-inflammatory mechanisms. Moreover, chronic inflammation and proinflammatory cascades mediated by the NLRP3 inflammasome contribute to the pathogenesis of diabetic keratopathy. Furthermore, overproduction of reactive oxygen species, mitochondrial dysfunction, and toll-like receptor-mediated activation of nuclear factor kappa B drive the stimulation of NLRP3 inflammasome and participate in the progression of dry eye disease. However, there still exist controversies regarding the regulatory pathways of the NLRP3 inflammasome. In this review, we provide a comprehensive overview of recent advancements in the function of NLRP3 inflammasome in corneal diseases and its regulatory pathways primarily through studies using animal models. Furthermore, we explore prospects for pharmacologically targeting pathways associated with NLRP3.</div></div>","PeriodicalId":54691,"journal":{"name":"Ocular Surface","volume":"34 ","pages":"Pages 392-405"},"PeriodicalIF":5.9,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142368134","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-19DOI: 10.1016/j.jtos.2024.09.003
Jehan Alam , Ebru Yaman , Cintia S. de Paiva , De-Quan Li , Gerda Cristal Villalba Silva , Zhen Zuo , Stephen C. Pflugfelder
Purpose
To evaluate the effects of dry eye on conjunctival immune cell number and transcriptional profiles with attention to mononuclear phagocytes.
Methods
Expression profiling was performed by single-cell RNA sequencing on sorted conjunctival immune cells from non-stressed and C57BL/6 mice subjected to desiccating stress (DS). Monocle 3 modeled cell trajectory, scATAC-seq assessed chromatin accessibility and IPA identified canonical pathways. Inflammation and goblet cells were measured after depletion of MRC1+ MΦs with mannosylated clodronate liposomes.
Results
Mononuclear phagocytes (monocytes, MΦs, DCs) comprised 72 % of immune cells and showed the greatest changes with DS. Distinct DS induced gene expression patterns were seen in phagocytes classified by expression of Ccr2 and [Timd4, Lyve1, Folr2 (TLR)]. Expression of phagocytosis/efferocytosis genes increased in TLF+CCR2- MΦs. Monocytes showed the highest expression of Ace, Cx3cr1, Vegfa, Ifngr1,2, and Stat1 and TLF−CCR2+ cells expressed higher levels of inflammatory mediators (Il1a, Il1b, Il1rn, Nfkb1, Ccl5, MHCII, Cd80, Cxcl10, Icam1). A trajectory from monocyte precursors branched to terminate in regulatory MΦs or in mDCs via transitional MΦ and cDC clusters. Activated pathways in TLF+ cells include phagocytosis, PPAR/RXRα activation, IL-10 signaling, alternate MΦ activation, while inflammatory pathways were suppressed. Depletion of MRC1+ MΦs increased IL-17 and IFN-γ expression and cytokine-expressing T cells, reduced IL-10 and worsened goblet loss.
Conclusions
Dryness stimulates distinct gene expression patterns in conjunctival phagocytes, increasing expression of regulatory genes in TLF+ cells regulated in part by RXRα, and inflammatory genes in CCR2+ cells. Regulatory MΦs depletion worsens DS induced inflammation and goblet cell loss.
{"title":"Changes in conjunctival mononuclear phagocytes and suppressive activity of regulatory macrophages in desiccation induced dry eye","authors":"Jehan Alam , Ebru Yaman , Cintia S. de Paiva , De-Quan Li , Gerda Cristal Villalba Silva , Zhen Zuo , Stephen C. Pflugfelder","doi":"10.1016/j.jtos.2024.09.003","DOIUrl":"10.1016/j.jtos.2024.09.003","url":null,"abstract":"<div><h3>Purpose</h3><div>To evaluate the effects of dry eye on conjunctival immune cell number and transcriptional profiles with attention to mononuclear phagocytes.</div></div><div><h3>Methods</h3><div>Expression profiling was performed by single-cell RNA sequencing on sorted conjunctival immune cells from non-stressed and C57BL/6 mice subjected to desiccating stress (DS). Monocle 3 modeled cell trajectory, scATAC-seq assessed chromatin accessibility and IPA identified canonical pathways. Inflammation and goblet cells were measured after depletion of MRC1<sup>+</sup> MΦs with mannosylated clodronate liposomes.</div></div><div><h3>Results</h3><div>Mononuclear phagocytes (monocytes, MΦs, DCs) comprised 72 % of immune cells and showed the greatest changes with DS. Distinct DS induced gene expression patterns were seen in phagocytes classified by expression of Ccr2 and [Timd4, Lyve1, Folr2 (TLR)]. Expression of phagocytosis/efferocytosis genes increased in TLF<sup>+</sup>CCR2<sup>-</sup> MΦs. Monocytes showed the highest expression of Ace, Cx3cr1, Vegfa, Ifngr1,2, and Stat1 and TLF<sup>−</sup>CCR2<sup>+</sup> cells expressed higher levels of inflammatory mediators (Il1a, Il1b, Il1rn, Nfkb1, Ccl5, MHCII, Cd80, Cxcl10, Icam1). A trajectory from monocyte precursors branched to terminate in regulatory MΦs or in mDCs via transitional MΦ and cDC clusters. Activated pathways in TLF<sup>+</sup> cells include phagocytosis, PPAR/RXRα activation, IL-10 signaling, alternate MΦ activation, while inflammatory pathways were suppressed. Depletion of MRC1<sup>+</sup> MΦs increased IL-17 and IFN-γ expression and cytokine-expressing T cells, reduced IL-10 and worsened goblet loss.</div></div><div><h3>Conclusions</h3><div>Dryness stimulates distinct gene expression patterns in conjunctival phagocytes, increasing expression of regulatory genes in TLF<sup>+</sup> cells regulated in part by RXRα, and inflammatory genes in CCR2<sup>+</sup> cells. Regulatory MΦs depletion worsens DS induced inflammation and goblet cell loss.</div></div>","PeriodicalId":54691,"journal":{"name":"Ocular Surface","volume":"34 ","pages":"Pages 348-362"},"PeriodicalIF":5.9,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142305335","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-12DOI: 10.1016/j.jtos.2024.09.002
Nicole B. Kantor , Paula A. Sepulveda-Beltran , David Valdés-Arias , Elyana V.T. Locatelli , Lakshman Mulpuri , Araliya N. Gunawardene , Guillermo Amescua , Victor L. Perez , Rahul Tonk , Trent Wang , Anat Galor
Purpose
To evaluate the incidence of chronic cicatrizing conjunctivitis (CCC) and its associated risk factors in the context of chronic ocular graft-vs-host disease (coGVHD).
Methods
A retrospective chart review of individuals diagnosed with coGVHD following hematopoietic stem cell transplantation (HSCT) who were seen at the Bascom Palmer Eye Institute between May 2010 and November 2021 was performed. Data regarding baseline demographic characteristics, systemic co-morbidities, lid margin abnormalities, ocular cicatricial changes, transplant information, immunosuppressive therapy, and GVHD severity assessments were collected. The incidence of cicatricial conjunctivitis was estimated with Kaplan-Meier survival analysis. A Cox regression model was used to assess the contribution of demographic and systemic variables to the development of CCC.
Results
167 individuals were included (53.9 ± 14.7 years old; 60.5 % male). 65 individuals presented with features suggestive of CCC an average of 60.9 ± 53.8 months after HSCT, with 60-month and 120-month incidences of 29.3 % and 48.9 %, respectively. Multivariable analysis demonstrated that age younger than 50 at the time of the first eye visit was associated with a higher chance of CCC development (Hazard Ratio (HR): 2.14, 95 % Confidence Interval (CI): 1.16–3.97, p = 0.02).
Conclusion
Clinically detected cicatrizing conjunctivitis is an ocular manifestation of coGVHD, with an incidence that increases over time. Younger individuals may be at higher risk for CCC development.
{"title":"Epidemiology and risk factors for the development of cicatrizing conjunctivitis in chronic ocular graft-versus-host disease","authors":"Nicole B. Kantor , Paula A. Sepulveda-Beltran , David Valdés-Arias , Elyana V.T. Locatelli , Lakshman Mulpuri , Araliya N. Gunawardene , Guillermo Amescua , Victor L. Perez , Rahul Tonk , Trent Wang , Anat Galor","doi":"10.1016/j.jtos.2024.09.002","DOIUrl":"10.1016/j.jtos.2024.09.002","url":null,"abstract":"<div><h3>Purpose</h3><p>To evaluate the incidence of chronic cicatrizing conjunctivitis (CCC) and its associated risk factors in the context of chronic ocular graft-vs-host disease (coGVHD).</p></div><div><h3>Methods</h3><p>A retrospective chart review of individuals diagnosed with coGVHD following hematopoietic stem cell transplantation (HSCT) who were seen at the Bascom Palmer Eye Institute between May 2010 and November 2021 was performed. Data regarding baseline demographic characteristics, systemic co-morbidities, lid margin abnormalities, ocular cicatricial changes, transplant information, immunosuppressive therapy, and GVHD severity assessments were collected. The incidence of cicatricial conjunctivitis was estimated with Kaplan-Meier survival analysis. A Cox regression model was used to assess the contribution of demographic and systemic variables to the development of CCC.</p></div><div><h3>Results</h3><p>167 individuals were included (53.9 ± 14.7 years old; 60.5 % male). 65 individuals presented with features suggestive of CCC an average of 60.9 ± 53.8 months after HSCT, with 60-month and 120-month incidences of 29.3 % and 48.9 %, respectively. Multivariable analysis demonstrated that age younger than 50 at the time of the first eye visit was associated with a higher chance of CCC development (Hazard Ratio (HR): 2.14, 95 % Confidence Interval (CI): 1.16–3.97, p = 0.02).</p></div><div><h3>Conclusion</h3><p>Clinically detected cicatrizing conjunctivitis is an ocular manifestation of coGVHD, with an incidence that increases over time. Younger individuals may be at higher risk for CCC development.</p></div>","PeriodicalId":54691,"journal":{"name":"Ocular Surface","volume":"34 ","pages":"Pages 341-347"},"PeriodicalIF":5.9,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142232758","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-31DOI: 10.1016/j.jtos.2024.08.016
Anna-Lena Rau , Martin Schicht , Ingrid Zahn , Mohammad Javed Ali , Minas Theodore Coroneo , Friedrich Paulsen
Purpose
Our aim was to evaluate a potential role for the lacrimal drainage system (LDS) as a portal of entry and conduit for SARS-CoV-2 in human infection. We also investigate the mucosal surface area. The relatively long tear contact time in a closed system raises the possibility that this pathway may contribute to the initiation of systemic infection. We looked for expression of ACE2, the main receptor for SARS-CoV-2, as well as cofactors such as TMPRSS2 and other enzymes such as cathepsinB, CD147, elastase1, furin, neuropilin1, neuropilin2, TMPRSS11D and trypsin which also play a role in SARS-CoV-2 infection, in this system.
Methods
Human tissue samples of the draining tear ducts from body donors were analyzed by RT-PCR, Western blot and immunohistochemistry. It is not known whether the respective body donors were Sars-Cov-2 positive at any time; they were negative when they entered the institute. Besides, the draining LDS of body donors were measured to determine the mucosal surface in the lacrimal system.
Results
The expression of the main receptor studied, ACE2, cofactors such as TMPRSS2 and other enzymes such as cathepsinB, CD147, elastase1, furin, neuropilin1, neuropilin2, TMPRSS11D and trypsin were all detected at the gene and protein level. The average mucosal surface area of the lacrimal sac and nasolacrimal duct was calculated to be 110 mm2.
Conclusion
The results show the presence of all analyzed receptors in the efferent LDS. With an average tear passage time of 3 min and a relatively large mucosal surface area, the LDS could therefore be considered as a portal of entry and conduit for SARS-CoV-2. In addition, it represents a surface that should be taken into consideration in the administration of topically applied medication to the ocular surface.
{"title":"Detection of SARS-CoV-2 binding receptors and miscellaneous targets as well as mucosal surface area of the human lacrimal drainage system","authors":"Anna-Lena Rau , Martin Schicht , Ingrid Zahn , Mohammad Javed Ali , Minas Theodore Coroneo , Friedrich Paulsen","doi":"10.1016/j.jtos.2024.08.016","DOIUrl":"10.1016/j.jtos.2024.08.016","url":null,"abstract":"<div><h3>Purpose</h3><p>Our aim was to evaluate a potential role for the lacrimal drainage system (LDS) as a portal of entry and conduit for SARS-CoV-2 in human infection. We also investigate the mucosal surface area. The relatively long tear contact time in a closed system raises the possibility that this pathway may contribute to the initiation of systemic infection. We looked for expression of ACE2, the main receptor for SARS-CoV-2, as well as cofactors such as TMPRSS2 and other enzymes such as cathepsinB, CD147, elastase1, furin, neuropilin1, neuropilin2, TMPRSS11D and trypsin which also play a role in SARS-CoV-2 infection, in this system.</p></div><div><h3>Methods</h3><p>Human tissue samples of the draining tear ducts from body donors were analyzed by RT-PCR, Western blot and immunohistochemistry. It is not known whether the respective body donors were Sars-Cov-2 positive at any time; they were negative when they entered the institute. Besides, the draining LDS of body donors were measured to determine the mucosal surface in the lacrimal system.</p></div><div><h3>Results</h3><p>The expression of the main receptor studied, ACE2, cofactors such as TMPRSS2 and other enzymes such as cathepsinB, CD147, elastase1, furin, neuropilin1, neuropilin2, TMPRSS11D and trypsin were all detected at the gene and protein level. The average mucosal surface area of the lacrimal sac and nasolacrimal duct was calculated to be 110 mm<sup>2</sup>.</p></div><div><h3>Conclusion</h3><p>The results show the presence of all analyzed receptors in the efferent LDS. With an average tear passage time of 3 min and a relatively large mucosal surface area, the LDS could therefore be considered as a portal of entry and conduit for SARS-CoV-2. In addition, it represents a surface that should be taken into consideration in the administration of topically applied medication to the ocular surface.</p></div>","PeriodicalId":54691,"journal":{"name":"Ocular Surface","volume":"34 ","pages":"Pages 296-308"},"PeriodicalIF":5.9,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S154201242400096X/pdfft?md5=077e26d0d5c1c1c911787a4ebc91c667&pid=1-s2.0-S154201242400096X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142116709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-29DOI: 10.1016/j.jtos.2024.08.015
Lina Sprogyte, Mijeong Park, Lamia Nureen, Nicodemus Tedla, Alexander Richardson, Nick Di Girolamo
Purpose
Limbal stem cell deficiency (LSCD) secondary to ocular surface alkali burn is a blinding condition that features corneal conjunctivalization. Mechanistic insights into its pathophysiology are lacking. Here, we developed a mouse model that recapitulates human disease to comprehensively delineate the clinicopathological features of a conjunctivalized cornea.
Methods
LSCD was induced in the right eyes of 6-8-week-old C57BL/6 male and female mice (n = 151) by topical administration of 0.25N sodium hydroxide on the cornea. Uninjured left eyes served as controls. Clinical, histological, phenotypic, molecular, and immunological assessments were performed at multiple time-points over 6-months.
Results
Clinically, alkali burn caused persistent corneal opacity (p = 0.0014), increased punctate staining (p = 0.0002), and reduced epithelial thickness (p = 0.0082) compared to controls. Total LSCD was confirmed in corneal whole mounts by loss of K12 protein (p < 0.0001) and mRNA expression (p = 0.0090). Instead, K8+, K13+, K15+ and MUC5AC+ conjunctival epithelia prevailed. 20 % of injured corneas developed islands of K12+ epithelia, suggesting epithelial transdifferentiation. Squamous metaplasia was detected in 50 % of injured corneas. Goblet cell density peaked early post-injury but decreased over time (p = 0.0047). Intraepithelial corneal basal nerve density remained reduced even at 6-months post-injury (p = 0.0487).
Conclusions
We developed and comprehensively characterized a preclinical mouse model of alkali-induced LSCD. Understanding the pathophysiological processes that transpire on the ocular surface in LSCD is key to discovering, testing, and advancing biological and pharmacological interventions that can be dispensed prior to or in conjunction with stem cell therapy to rehabilitate the cornea and restore vision.
{"title":"Development and characterization of a preclinical mouse model of alkali-induced limbal stem cell deficiency","authors":"Lina Sprogyte, Mijeong Park, Lamia Nureen, Nicodemus Tedla, Alexander Richardson, Nick Di Girolamo","doi":"10.1016/j.jtos.2024.08.015","DOIUrl":"10.1016/j.jtos.2024.08.015","url":null,"abstract":"<div><h3>Purpose</h3><p>Limbal stem cell deficiency (LSCD) secondary to ocular surface alkali burn is a blinding condition that features corneal conjunctivalization. Mechanistic insights into its pathophysiology are lacking. Here, we developed a mouse model that recapitulates human disease to comprehensively delineate the clinicopathological features of a conjunctivalized cornea.</p></div><div><h3>Methods</h3><p>LSCD was induced in the right eyes of 6-8-week-old C57BL/6 male and female mice (<em>n = 151</em>) by topical administration of 0.25N sodium hydroxide on the cornea. Uninjured left eyes served as controls. Clinical, histological, phenotypic, molecular, and immunological assessments were performed at multiple time-points over 6-months.</p></div><div><h3>Results</h3><p>Clinically, alkali burn caused persistent corneal opacity (p = 0.0014), increased punctate staining (p = 0.0002), and reduced epithelial thickness (p = 0.0082) compared to controls. Total LSCD was confirmed in corneal whole mounts by loss of K12 protein (p < 0.0001) and mRNA expression (p = 0.0090). Instead, K8<sup>+</sup>, K13<sup>+</sup>, K15<sup>+</sup> and MUC5AC<sup>+</sup> conjunctival epithelia prevailed. 20 % of injured corneas developed islands of K12<sup>+</sup> epithelia, suggesting epithelial transdifferentiation. Squamous metaplasia was detected in 50 % of injured corneas. Goblet cell density peaked early post-injury but decreased over time (p = 0.0047). Intraepithelial corneal basal nerve density remained reduced even at 6-months post-injury (p = 0.0487).</p></div><div><h3>Conclusions</h3><p>We developed and comprehensively characterized a preclinical mouse model of alkali-induced LSCD. Understanding the pathophysiological processes that transpire on the ocular surface in LSCD is key to discovering, testing, and advancing biological and pharmacological interventions that can be dispensed prior to or in conjunction with stem cell therapy to rehabilitate the cornea and restore vision.</p></div>","PeriodicalId":54691,"journal":{"name":"Ocular Surface","volume":"34 ","pages":"Pages 329-340"},"PeriodicalIF":5.9,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1542012424000958/pdfft?md5=592d460a11f75adc137bf2afe15e6ac6&pid=1-s2.0-S1542012424000958-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142116710","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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