Staphylococcus aureus Activates the NLRP3 Inflammasome in Human and Rat Conjunctival Goblet Cells

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

Age and Age-related Diseases: Role of Inflammation Triggers and Cytokines

Cytokine dysregulation is believed to play a key role in the remodeling of the immune system at older age, with evidence pointing to an inability to fine-control systemic inflammation, which seems to be a marker of unsuccessful aging. This reshaping of cytokine expression pattern, with a progressive tendency toward a pro-inflammatory phenotype has been called “inflamm-aging.” Despite research there is no clear understanding about the causes of “inflamm-aging” that underpin most major age-related diseases, including atherosclerosis, diabetes, Alzheimer’s disease, rheumatoid arthritis, cancer, and aging itself. While inflammation is part of the normal repair response for healing, and essential in keeping us safe from bacterial and viral infections and noxious environmental agents, not all inflammation is good. When inflammation becomes prolonged and persists, it can become damaging and destructive. Several common molecular pathways have been identified that are associated with both aging and low-grade inflammation. The age-related change in redox balance, the increase in age-related senescent cells, the senescence-associated secretory phenotype (SASP) and the decline in effective autophagy that can trigger the inflammasome, suggest that it may be possible to delay age-related diseases and aging itself by suppressing pro-inflammatory molecular mechanisms or improving the timely resolution of inflammation. Conversely there may be learning from molecular or genetic pathways from long-lived cohorts who exemplify good quality aging. Here, we will discuss some of the current ideas and highlight molecular pathways that appear to contribute to the immune imbalance and the cytokine dysregulation, which is associated with “inflammageing” or parainflammation. Evidence of these findings will be drawn from research in cardiovascular disease, cancer, neurological inflammation and rheumatoid arthritis

Attitudes towards Diagnostic Tests and Therapies for Dry Eye Disease

Aim: The purpose of this study was to survey the attitudes of optometrists and ophthalmologists, located in a number of different countries, towards diagnostic tests and therapies for dry eye disease. Methods: A web-based questionnaire was used to survey attitudes using forced-choice questions and Likert scales. Results: Sixty-one respondents (23 ophthalmologists and 38 optometrists) reported a wide range of patient dry eye symptoms. A large variation in use of diagnostic tests was noted. Patient symptoms and fluorescein staining were reported to be significantly more valuable and more frequently performed than any other test. Artificial tear supplements and improved lid hygiene were the preferred therapeutic options selected by the entire group. The results demonstrated a wide variation in attitudes in relation to satisfaction with the range of available diagnostic and therapeutic options. Conclusions: This study indicates that the interest for the issue of dry eye is relatively limited amongst eye professionals, as demonstrated by the poor participation in the questionnaire

Purinergic receptors P2X4, P2X7, and TLR2 are expressed in the rat conjunctiva and rat goblet cell cultures.

<p>All three receptors were identified by red immunofluorescent staining. UEA stains goblet cell secretory products green, allowing the identification of goblet cells within the conjunctiva. Rabbit isotype controls were negative. Arrows indicate location of goblet cells.</p

Active caspase 1 expression in rat goblet cells treated with <i>S. aureus</i> and ATP.

<p>Primary cultures of rat goblet cells were incubated with <i>S. aureus</i> (MOI 20 or 60) for 6 h. Cultures were treated for an additional 2 h with ATP (5 mM) or buffer alone. The FLICA reagent, which detects only active caspase 1, was added followed by the nuclear Hoescht stain and viewed by immunofluorescence microscopy. Representative micrographs are shown in <b>A</b>. The total number of nuclei in four fields of view and the number of cells with staining green (indicative of active caspase 1) were counted. Data is expressed as mean ± SEM from 3 independent experiments, and are shown in <b>B</b>. <b>*</b> indicates significance of <i>p</i><0.05 compared to no addition, which was set to 1. Magnification 40×.</p

Effect of Inhibition of Caspase 1 on IL-1β secretion in response to <i>S. aureus</i> and ATP.

<p>Primary cultures of rat goblet cells were treated with or without the caspase 1 inhibitor Z-YVAD for 1 h and then incubated with <i>S. aureus</i> (MOI 20 or 60) for 6 h. Cultures were treated for an additional 2 h with ATP (5 mM) or buffer alone. Culture supernatant was removed and analyzed for IL-1β by ELISA. Data is expressed as mean ± SEM from 3 independent experiments. <b>*</b> indicates significance of <i>p</i><0.05 compared to no addition. # indicates significance of <i>p</i><0.05 compared to no inhibitor.</p

Purinergic receptors P2X4, P2X7, and TLR2 are expressed in the rat conjunctiva and rat goblet cell cultures.

<p>All three receptors were identified by red immunofluorescent staining. UEA stains goblet cell secretory products green, allowing the identification of goblet cells within the conjunctiva. Rabbit isotype controls were negative. Arrows indicate location of goblet cells.</p