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Dry eye can damage the ocular surface and result in mild corneal epithelial defect to blinding corneal pannus formation and squamous metaplasia. Significant progress in the treatment of dry eye has been made in the last two decades; progressing from lubricating and hydrating the ocular surface with artificial tear to stimulating tear secretion; anti-inflammation and immune regulation. With the increase in knowledge regarding the pathophysiology of dry eye, we propose in this review the concept of ocular surface microenvironment. Various components of the microenvironment contribute to the homeostasis of ocular surface. Compromise in one or more components can result in homeostasis disruption of ocular surface leading to dry eye disease. Complete evaluation of the microenvironment component changes in dry eye patients will not only lead to appropriate diagnosis, but also guide in timely and effective clinical management. Successful treatment of dry eye should be aimed to restore the homeostasis of the ocular surface microenvironment.

There are several animal models illustrating dry eye pathophysiology. Current study would like to establish an ex vivo tissue culture model for characterizing dry eye. Human conjunctival explants were cultured under airlift or submerged conditions for up to 2 weeks, and only airlifted conjunctival cultures underwent increased epithelial stratification. Starting on day 4, the suprabasal cells displayed decreased K19 expression whereas K10 keratin became evident in airlift group. Pax6 nuclear expression attenuated already at 2 days, while its perinuclear and cytoplasmic expression gradually increased. MUC5AC and MUC19 expression dramatically decreased whereas the full thickness MUC4 and MUC16 expression pattern disappeared soon after initiating the airlift condition. Real time PCR showed K16, K10 and MUC16 gene up-regulated while K19, MUC5AC, MUC19 and MUC4 down-regulated on day 8 and day 14. On day 2 was the appearance of apoptotic epithelial and stromal cells appeared. The Wnt signaling pathway was transiently activated from day 2 to day 10. The inflammatory mediators IL-1β, TNF-α, and MMP-9 were detected in the conditioned media after 6 to 8 days. In conclusion, airlifted conjunctival tissue cultures demonstrated Wnt signaling pathway activation, coupled with squamous metaplasia, mucin pattern alteration, apoptosis and upregulation of proinflammatory cytokine expression. These changes mimic the pathohistological alterations described in dry eye. This correspondence suggests that insight into the pathophysiology of dry eye may be aided through the use of airlifted conjunctival tissue cultures.

Periostin is a non-structural matricellular protein. Little is known about periostin in human limbal stem cells (LSCs). This study was to explore the unique expression pattern and functional role of periostin in maintaining the properties of human LSCs. Fresh donor corneal tissues were used to make cryosections for evaluation of periostin expression on ex vivo tissues. Primary human limbal epithelial cells (HLECs) were generated from limbal explant culture. In vitro culture models for proliferation and epithelial regeneration were performed to explore functional role of periostin in LSCs. The mRNA expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), and the protein production and localization were detected by immunofluorescent staining and Western blot analysis. Periostin protein was found to be exclusively immunolocalized in the basal layer of human limbal epithelium. Periostin localization was well matched with nuclear factor p63, but not with corneal epithelial differentiation marker Keratin 3. Periostin transcripts was also highly expressed in limbal than corneal epithelium. In primary HLECs, periostin expression at mRNA and protein levels was significantly higher in 50% and 70% confluent cultures at exponential growth stage than in 100% confluent cultures at slow growth or quiescent condition. This expression pattern was similar to other stem/progenitor cell markers (p63, integrin β1 and TCF4). Periostin expression at transcripts, protein and immunoreactivity levels increased significantly during epithelial regeneration in wound healing process, especially in 16-24 hours at wound edge, which was accompanied by similar upregulation and activation of p63, integrin β1 and TCF4. Our findings demonstrated that periostin is exclusively produced by limbal basal epithelium and co-localized with p63, where limbal stem cells reside. Periostin promotes HLEC proliferation and regeneration with accompanied activation of stem/progenitor cell markers p63, integrin β1 and TCF4, suggesting its novel role in maintaining the phenotype and functional properties of LSC.

There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5-14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1 × 10(4) in a 35-mm dish (9.6 cm(2)) grew to confluence (about 1.87-2.41 × 10(6) cells) in 12-14 days, representing 187-241 fold expansion with over 7-8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction.

Amniotic membranes (AMs) are widely used as a corneal epithelial tissue carrier in reconstruction surgery. However, the engineered tissue transparency is low due to the translucent thick underlying AM stroma. To overcome this drawback, we developed an ultra-thin AM (UAM) by using collagenase IV to strip away from the epithelial denuded AM (DAM) some of the stroma. By thinning the stroma to about 30 μm, its moist and dry forms were rendered acellular, optically clear and its collagen framework became compacted and inerratic. Engineered rabbit corneal epithelial cell (RCEC) sheets generated through expansion of limbal epithelial cells on UAM were more transparent and thicker than those expanded on DAM. Moreover, ΔNp63 and ABCG2 gene expression was greater in tissue engineered cell sheets expanded on UAM than on DAM. Furthermore, 2 weeks after surgery, the cornea grafted with UAM based cell sheets showed higher transparency and more stratified epithelium than the cornea grafted with DAM based cell sheets. Taken together, tissue engineered corneal epithelium generated on UAM has a preferable outcome because the transplanted tissue is more transparent and better resembles the phenotype of the native tissue than that obtained by using DAM for this procedure. UAM preserves compact layer of the amniotic membrane and maybe an ideal substrate for corneal epithelial tissue engineering.

Ectodysplasin A (Eda), a member of the tumour necrosis factor superfamily, plays an important role in ectodermal organ development. An EDA mutation underlies the most common of ectodermal dysplasias, that is X‐linked hypohidrotic ectodermal dysplasia (XLHED) in humans. Even though it lacks a developmental function, the role of Eda during the postnatal stage remains elusive. In this study, we found tight junctional proteins ZO‐1 and claudin‐1 expression is largely reduced in epidermal, corneal and lung epithelia in Eda mutant Tabby mice at different postnatal ages. These declines are associated with tail ulceration, corneal pannus formation and lung infection. Furthermore, topical application of recombinant Eda protein markedly mitigated corneal barrier dysfunction. Using cultures of a human corneal epithelial cell line and Tabby mouse skin tissue explants, Eda up‐regulated expression of ZO‐1 and claudin‐1 through activation of the sonic hedgehog signalling pathway. We conclude that EDA gene expression contributes to the maintenance of epithelial barrier function. Such insight may help efforts to identify novel strategies for improving management of XLHED disease manifestations in a clinical setting.

Periostin is a non-structural matricellular protein. Little is known about periostin in human limbal stem cells (LSCs). This study was to explore the unique expression pattern and functional role of periostin in maintaining the properties of human LSCs. Fresh donor corneal tissues were used to make cryosections for evaluation of periostin expression on ex vivo tissues. Primary human limbal epithelial cells (HLECs) were generated from limbal explant culture. In vitro culture models for proliferation and epithelial regeneration were performed to explore functional role of periostin in LSCs. The mRNA expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), and the protein production and localization were detected by immunofluorescent staining and Western blot analysis. Periostin protein was found to be exclusively immunolocalized in the basal layer of human limbal epithelium. Periostin localization was well matched with nuclear factor p63, but not with corneal epithelial differentiation marker Keratin 3. Periostin transcripts was also highly expressed in limbal than corneal epithelium. In primary HLECs, periostin expression at mRNA and protein levels was significantly higher in 50% and 70% confluent cultures at exponential growth stage than in 100% confluent cultures at slow growth or quiescent condition. This expression pattern was similar to other stem/progenitor cell markers (p63, integrin beta 1 and TCF4). Periostin expression at transcripts, protein and immunoreactivity levels increased significantly during epithelial regeneration in wound healing process, especially in 16-24 hours at wound edge, which was accompanied by similar upregulation and activation of p63, integrin beta 1 and TCF4. Our findings demonstrated that periostin is exclusively produced by limbal basal epithelium and co-localized with p63, where limbal stem cells reside. Periostin promotes HLEC proliferation and regeneration with accompanied activation of stem/progenitor cell markers p63, integrin beta 1 and TCF4, suggesting its novel role in maintaining the phenotype and functional properties of LSC.

Sjögren disease (SjD) is an autoimmune disease driven by autoreactive CD4 T cells that leads to an immune-mediated loss of lacrimal glands. Meibomian glands are lipid-producing glands in the eyelids that help prevent tear evaporation. While the role of T cells in lacrimal gland-mediated destruction is well established, it is unknown whether pathogenic T cells can cause MG dysfunction (MGD). Herein, we investigated whether autoreactive CD4 T cells induce MGD and characterized the pathophysiologic mechanisms using an adoptive transfer model. T cells were isolated from CD25KO (CD4 ) or wild-type (CD4 ) mice, transferred into KO mice. Further, CD4 cells were co-adoptively transferred with WT regulatory T cells (CD4 +Tregs ). Our results demonstrate that CD4 recipients had MG dropout, CD4 IFN-γ infiltration, increased MHC II presentation within the periglandular area, MG fibrosis, and decreased lipid production and upregulation of pathways related to inflammation, including Type II interferon signaling. KO, CD4 , and CD4 +Tregs recipients exhibited minimal inflammation in the periglandular MG area. These results indicate that autoimmune CD4 T cells are sufficient to cause MGD, and healthy young regulatory T cells can prevent T-cell-mediated damage. Taken together, our findings provide mechanistic insights into the pathogenesis autoimmune SjD, and could impact how patients are managed in the clinic.