The cornea encounters constant environmental insults, including ultra-violet light, osmolarity fluctuation, pathogens and injuries. Since vision is dependent on corneal avascularity and transparency, efficient and active coping mechanisms are required to maintain corneal integrity and function. The expression of immunoproteasome in normal cornea appears to contribute to the cytoprotective mechanisms in the cornea, as immunoproteasome deficiency compromised the survival stress response and led to greater cell death and slower wound healing.
We observed significantly more apoptosis of corneal epithelial cells from immunoproteasome KO mice both in vivo
(more frequent apoptotic cells in the stroma and endothelium) and in vitro
(greater caspase-3 activity). Our results are consistent with a recent study with Lmp7-deficient cells where enhanced caspase-3/7 activity was observed under cytokine-induced oxidative stress, or when treated with the apoptosis inducer etoposide 
. While it is possible that the immunproteasome deficiency of resident bone marrow-derived cells may contribute to the observed corneal phenotype in vivo
, CD11b-positive cells were not found in the outgrowths of our corneal explant cultures by CD11b immunostaining (data not shown). Accordingly we propose that the increased apoptosis of L7M1 corneal epithelial cells was a direct consequence of immunoproteasome deficiency.
Data from cells and tissues deficient in immunoproteasome also provide compelling evidence for a critical role in protecting from oxidative stress. A recent report from our lab showed cultured retinal pigment epithelial cells from L7M1 KO mice were more susceptible to peroxide-induced cell death compared with WT cells 
. In Lmp7-deficient cells, an increase in apoptosis was attributed to the compromised ability of immunoproteasome-deficient cells to process defective ribosomal products (DRiPs) that contain an abundance of oxidized and misfolded proteins 
. Tissues from Lmp2 knock-out mice showed an accumulation of oxidized proteins, suggesting that immunoproteasome's critical role in surviving an oxidative insult includes more efficient degradation of oxidized proteins 
. Since greater oxidative stress has been demonstrated in the superficial epithelium compared with basal layers of the cornea 
, the data from KO mice demonstrating a role for immunoproteasome in coping with oxidative damage suggest a possible explanation for the localized high immunoproteasome expression in the corneal epithelium.
In addition to its capacity to degrade oxidized protein substrates, immunoproteasome may also regulate stress-related pathways, such as NFκB signaling. NFκB signaling is activated by oxidants and ligands to toll-like and TNF receptors, and has been demonstrated to regulate both pro- and anti-apoptotic events 
. In the corneal epithelium, ultraviolet (UV) radiation is a major environmental insult that can up-regulate NFκB signaling, which is necessary for the stress-related response 
. NFκB signaling can be activated via (1) limited proteolysis of the precursors of NFκB family members (such as p100/p105), or (2) degradation of an inhibitory sequestering protein, IκB, which binds and prevents NFκB from entering the nucleus. Both types of proteolytic processing are accomplished by the proteasome. Studies on cultured lymphocytes from Lmp2 KO mice revealed defects in proteolytic processing of both NFκB precursors and the inhibitory regulator IκB, suggesting a direct contribution for the immunoproteasome in Lmp2 KO mice 
. An important regulatory mechanism built into the NFκB pathway is the induction of multiple inhibitors (i.e., IκBα, A20) that serve to attenuate or shut down signaling. Therefore, a potential consequence of altered signaling is the decreased production of negative feedback molecules that preventing chronic NFκB signaling and consequent overproduction of proteins, such as IL-6. Cells with immunoproteasome deficiency may therefore become more vulnerable to stress due to dysregulated NFκB signaling and the subsequent altered gene expression required for survival.
Dysregulated NFκB signaling in the immunoproteasome deficient cornea may also affect corneal wound healing via other signaling pathways, such as p38MAPK signaling, which is also essential for the corneal re-epithelialization process 
. The cross-talk of p38MAPK and NFκB is well-documented in various tissues and cells 
and occurs through the direct interaction of IκB kinase β(IKKβ), with p38MAPK 
. The p38MAPK pathway can also be activated by the accumulation of oxidized proteins. Importantly, the direct consequence of NFκB and p38MAPK activation is the upregulation of multiple cytokines, such as IL-6. Results from the current study show increased IL-6 cytokine production in immunoproteasome-deficient corneas, suggesting aberrant regulation of one or more pathways involved in the stress response. These results are consistent with reports from human patients with Nakajo-Nishimura syndrome (LMP7 mutation) that have abnormally high level of IL-6 in serum, cells (skin or B cells) and cultured fibroblasts 
. It was suggested that the observed hyperproduction of IL-6 was caused by the accumulation of ubiquitinated and oxidated proteins due to immunoproteasome dysfunction 
In addition to increased IL-6, we also observed a significantly higher level of IL-1α in the debrided L7M1 corneas. Both IL-1α and IL-6 were shown by immunostaining to be localized to the regenerating basal epithelial cells during injury 
. IL-1α is secreted by corneal epithelial cells as a key factor for stromal fibroblast activation, and also by stromal fibroblasts in an autocrine manner to stimulate their proliferation and differentiation. As an inflammatory cytokine, IL-6 is secreted by corneal epithelial cells and fibroblasts to promote the infiltration of inflammatory cells. Others have shown that IL-6 promoted epithelial wound healing of debrided corneas in rabbits 
, was essential for the corneal sterile inflammation and wound healing in mice 
, and facilitated the migration but not the proliferation of cultured epithelial cells 
, likely via a fibronectin-dependent mechanism 
. Therefore, IL-6 is required for optimal wound healing. However, the compromised corneal epithelial recovery in L7M1, despite the higher cytokines levels, suggests potential disruption in other processes downstream of IL-6 signaling or those involved in epithelial secretion of IL-6. Since our measurement of total content of IL-6 included both intracellular and secreted protein, future experiments are needed to distinguish the mechanism behind the discrepant results of higher IL-6 and delayed wound healing.
The high IL-6 production in the injured L7M1 cornea may negatively impact the re-establishment of epithelial barrier function. A recent study showed that high levels of IL-6 significantly reduced the expression of ZO-1, altered its localization pattern and disrupted the epithelial barrier function in a human epithelial cell line, HCE-2 
. These results may help explain the observed decreased ZO-1 staining and formation of the tight junction observed in the debrided L7M1 corneas.
In summary, we have shown substantial immunoproteasome expression in corneal epithelial cells. Immunoproteasome-deficient epithelial cells displayed more apoptosis in corneal explant cultures and in intact corneas, suggesting a potential contribution of the immunoproteasome in corneal homeostasis. We have also, for the first time, demonstrated that deficiency in immunoproteasome may also lead to delayed epithelial wound healing in the mechanical debridement mouse model. In light of these results and the recent discoveries that immunoproteasome provides cytoprotective effects in the retina and brain under stress and injury, a comprehensive study to investigate the novel roles of immunoproteasome in the cornea bears significant merit.