Date Published: September 10, 2013
Publisher: Public Library of Science
Author(s): Victoria E. McGilligan, Meredith S. Gregory-Ksander, Dayu Li, Jonathan E. Moore, Robin R. Hodges, Michael S. Gilmore, Tara C. B. Moore, Darlene A. Dartt, Suzanne Fleiszig.
The conjunctiva is a moist mucosal membrane that is constantly exposed to an array of potential pathogens and triggers of inflammation. The NACHT, leucine rich repeat (LRR), and pyrin domain-containing protein 3 (NLRP3) is a Nod-like receptor that can sense pathogens or other triggers, and is highly expressed in wet mucosal membranes. NLRP3 is a member of the multi-protein complex termed the NLRP3 inflammasome that activates the caspase 1 pathway, inducing the secretion of biologically active IL-1β, a major initiator and promoter of inflammation. The purpose of this study was to: (1) determine whether NLRP3 is expressed in the conjunctiva and (2) determine whether goblet cells specifically contribute to innate mediated inflammation via secretion of IL-1β. We report that the receptors known to be involved in the priming and activation of the NLRP3 inflammasome, the purinergic receptors P2X4 and P2X7 and the bacterial Toll-like receptor 2 are present and functional in conjunctival goblet cells. Toxin-containing Staphylococcus aureus (S. aureus), which activates the NLRP3 inflammasome, increased the expression of the inflammasome proteins NLRP3, ASC and pro- and mature caspase 1 in conjunctival goblet cells. The biologically active form of IL-1β was detected in goblet cell culture supernatants in response to S. aureus, which was reduced when the cells were treated with the caspase 1 inhibitor Z-YVAD. We conclude that the NLRP3 inflammasome components are present in conjunctival goblet cells. The NRLP3 inflammasome appears to be activated in conjunctival goblet cells by toxin-containing S. aureus via the caspase 1 pathway to secrete mature IL1-β. Thus goblet cells contribute to the innate immune response in the conjunctiva by activation of the NLRP3 inflammasome.
NOD Like Receptors (NLRs) have in recent years been evidenced to be guardians of the body. They are intracellular microbial and non-microbial sensors and include three main proteins; NLRP1, NLRP3 and NLRC4.  Each of which are known to form inflamamasomes, differing mainly in their activation stimuli. The assembly and activation of an elaborate multi-protein complex: nucleotide-binding oligomerization domain (NACHT), leucine rich repeat (LRR) domain, and pyrin domain-containing protein 3 (NLRP3) inflammasome is responsible for the recruitment of caspase 1 that processes IL-1β into a mature and biologically active form.  The NLRP3 inflammasome consists of the NLRP3 protein, which senses intracellular triggers resulting in oligomerization  that subsequently interacts with ASC (apoptosis-associated speck-like protein) through homotypic interactions of the pyrin domain.  ASC then interacts with pro caspase 1 resulting in cleavage and activation of caspase 1, which in turn cleaves pro IL-1β to its active form. , , .
We found that the NLRP3 inflammasome is present in rat conjunctival goblet cells and can be activated by S. aureus. The NLRP3 inflammasome appears to contribute to inflammation in the conjunctiva by activating the secretion of IL-1β via the caspase 1 pathway. NLRP3 was previously reported to be highly expressed in wet mucosal epithelium within the cytoplasm of cells.  Kummer and colleagues  speculated that the expression of NLRP3 in such sites allows rapid sensing of invading pathogens or other danger signals, thereby triggering an innate immune response. The ocular surface is exposed to the environment, yet to maintain visual clarity, it is required to balance inflammation with immune privilege. Previous work demonstrated that mouse eyes express high levels of NLRP3 mRNA compared to other body tissues.  An additional study also reported NLRP3 mRNA expression in the whole eyes of mice challenged with LPS, but not in unchallenged eyes . Benko et al previously reported NLRP3 mRNA was detected in human corneal epithelial cells, but was not detected at the protein level.  Our results show that NLRP3 protein, as well as the other constituent components of the NLRP3 inflammasome, are highly expressed in the goblet cells of the conjunctival epithelium.