Desiccating stress worsens alkali burn injury by magnifying caspase-8-induced imbalance of NLRP3 and NLRP6.

To the Editor: Alkali burn (AB), one of the most devastating types of ocular trauma, often develops secondary dry eye, which, in turn, worsens ocular surface disorder. The corneal epithelium is the first defensive line of the eye involved in pathogen-associated molecular pattern recognition that leads to robust innate immune responses. However, how damage-associated molecular pattern recognition of eye injuries initiates the innate immune response of the corneal epithelium remains unclear. In the present study, we used AB and AB-concomitant desiccating stress (DS) models to explore the potential functions and mechanisms of innate immune response in certain diseases (see text in this article’s Online Repository at www.jacionline. org). We observed that tear volume rapidly decreased after AB injury and led to severe secondary dry eye, which further significantly delayed corneal wound healing and worsened the ocular surface disorder. Nucleotide-binding oligomerization domain–like receptors (NLRs) are cytoplasmic pattern recognition receptors initiating innate immune response during cellular stress. In the present study, we therefore explored the involvement and potential roles of NLRs in the innate immunity of AB and secondary dry eye after AB injury. NLRP3 is the most versatile and importantly also the most clinically implicated NLR in many inflammatory diseases. Our data showed that both the mRNA and protein levels of NLRP3 and adaptor apoptosis-associated speck-like protein containing a CARD (ASC) were significantly higher in the cornea of AB mice than in the cornea of the untreated group (Fig 1, A, B, and E). The activity of caspase-1, the other component of the NLRP3 inflammasome, was also significantly elevated after AB injury (Fig 1, C). Furthermore, DS synergized AB injury to further increase NLRP3 and ASC production. Once NLRP3 was inhibited (for the choice of optimal doses of inhibitors, see the Methods and Results sections and Fig E1 in this article’s Online Repository at www.jacionline.org), ASC expression and caspase-1 activation were subsequently suppressed (Fig 1, B, C, and E). Blocking NLRP3 effectively relieved AB-induced opacification (Fig 1, H) and significantly promoted corneal wound healing (Fig 1, I). Furthermore, NLRP3 inhibition also markedly reduced the dry eye-worsen the ocular surface disorder after AB injury and eliminated corneal perforation (Fig 1,G). These results indicated that the activation of NLRP3 inflammasome mediates AB-induced ocular surface injury and DS-worsened AB damage. Thus, the processing of IL-1b and IL-18 was significantly suppressed after NLRP3 inhibition (Fig 1, J), suggesting that NLRP3 drives ocular surface disorder by promoting the processing of active IL-1b and IL-18. NLRP6 is a newly found NLR, described by recent studies that NLRP6 serves to inhibit the proliferation of intestinal epithelial cells following and its deficiency leads to defective wound healing. In contrast to NLRP3, the expression of NLRP6 mRNA and protein was downregulated in both AB and AB combined with DS groups compared with the untreated group (Fig 1,D and E). We further explored the influence of NLRP6 using NLRP6 siRNA to inhibit local NLRP6 production. Our findings demonstrated that NLRP6 inhibition significantly exacerbated the severity of ABand DS-induced ocular surface damage, including an increased corneal perforation rate, enhanced corneal transparency, and delayed corneal wound healing (Fig 1, G-I), indicating that NLRP6 negatively regulated the inflammation to dampen the damage of these diseases. Our study further disclosed that the production of IL-1b had no significant alternation after blocking NLRP6, but the processing of IL-18 was suppressed (Fig 1, K). In the present study, our findings first discovered that NLRP6 was significantly upregulated after NLRP3 inhibition (Fig 1, D and E). However, NLRP6 had no effect on NLRP3 production (Fig 1, A and F). These results indicate that ABand DSinduced NLRP3 upregulation participates in the downregulation of NLRP6 production. Thus, targetedly inhibiting ABand DSinduced NLRP3 markedly reduced corneal opacification and corneal perforation rate and promoted corneal wound healing via reversing the imbalance of NLRP3 and NLRP6. Yamada et al observed that antagonism of IL-1 after AB injury markedly reduces corneal inflammation and enhances corneal transparency. Caspase-8 is a classical cysteine-aspartic acid protease in apoptotic signals whose new paradigm of nonapoptotic role in the innate immunity has been disclosed by us. Our previous study demonstrated that caspase-8 activation is involved in the innate immune response of retina by promoting the processing of IL-1b. Therefore, we hypothesized that the increased IL-1b production in AB may also be due to the participation of caspase-8 activation. Our data showed that AB injury significantly activated caspase-8 compared with untreated corneas. Furthermore, we disclosed that DS synergized AB, inducing a higher caspase-8 activity (Fig 2, A; see Fig E2 in this article’s Online Repository at www.jacionline.org). Inhibiting caspase-8 significantly attenuated AB-induced corneal pacification as well as DS-worsened AB injury, promoted the corneal wound healing, and resulted in no corneal perforation, indicating that caspase-8 signaling was actively involved in ABand DS-induced ocular surface disorder (Fig 2, B-D). Blocking caspase-8 activation significantly reduced the processing of IL-1b and IL-18 (Fig 2, E-G), suggesting that caspase-8 signaling drove the processing of IL-1b and IL-18 to accelerate ocular surface disorder induced by AB and worsened by DS and targetedly blocking caspase-8 relieved the damage. We then determined the correlation of caspase-8 and NLRs. Notably, inhibiting caspase-8 signaling suppressed the production of NLRP3, ASC, and caspase-1 in both AB and AB combined DS groups (Fig 2, H, I, K, and L). However, NRLP6 was upregulated after caspase-8 inhibition compared with noninhibition (Fig 2, J and K). Thus, NLRP3 and NLRP6 had no effect on the activation of caspase-8 (Fig 2, A). Together, these results revealed a previously unidentified correlation of NLRP3/NLRP6 and caspase-8 in AB and AB with DS. Caspase-8 signaling is upstream of NLRP3 and NLRP6. Caspase-8 activation induced by AB and DS leads to the upregulation of NLRP3 and downregulation of NLRP6, which further regulates the processing of IL-1b and IL-18 involved in AB and DS-worsened AB injury.