Pepsin-mediated inflammation in laryngopharyngeal reflux via the ROS/NLRP3/IL-1ß signaling pathway.

BACKGROUND: Laryngopharyngeal reflux (LPR) is one of the most common disorders in otorhinolaryngology, affecting up to 10% of outpatients visiting otolaryngology departments. In addition, 50% of hoarseness cases are related to LPR. Pepsin reflux-induced aseptic inflammation is a major trigger of LPR; however, the underlying mechanisms are unclear. The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome has become an important bridge between stimulation and sterile inflammation and is activated by intracellular reactive oxygen species (ROS) in response to danger signals, leading to an inflammatory cascade. In this study, we aimed to determine whether pepsin causes LPR-associated inflammatory injury via mediating inflammasome activation and explore the potential mechanism. METHODS: We evaluated NLRP3 inflammasome expression and ROS in the laryngeal mucosa using immunofluorescence and immunohistochemistry. Laryngeal epithelial cells were exposed to pepsin and analyzed using flow cytometry, western blotting, and real-time quantitative PCR to determine ROS, NLRP3, and pro-inflammatorycytokine levels. RESULTS: Pepsin expression was positively correlated with ROS as well as caspase-1 and IL-1ß levels in laryngeal tissues. Intracellular ROS levels were elevated by increased pepsin concentrations, which were attenuated by apocynin (APO)-a ROS inhibitor-in vitro. Furthermore, pepsin significantly induced the mRNA and protein expression of thioredoxin-interacting protein, NLRP3, caspase-1, and IL-1ß in a dose-dependent manner. APO and the NLRP3 inhibitor, MCC950, inhibited NLRP3 inflammasome formation and suppressed laryngeal epithelial cell damage. CONCLUSION: Our findings verified that pepsin could regulate the NLRP3/IL-1ß signaling pathway through ROS activation and further induce inflammatory injury in LPR. Targeting the ROS/NLRP3 inflammasome signaling pathway may help treat patients with LPR disease.

Complement C1q synergizes with PTX3 in promoting NLRP3 inflammasome over-activation and pyroptosis in rheumatoid arthritis.

Excessive inflammatory cytokines play crucial roles in the pathogenesis of rheumatoid arthritis (RA), however, the underlying mechanism remains unclear. In this study, we demonstrated that pentaxin 3 (PTX3), an essential component of innate immunity, was elevated in RA and preferentially bound to CD14+ monocytes. C1q promoted the binding and resulted in increased cell proliferation, activation and caspase-1-related late apoptotic cells (7-AAD+annexin V+), as well as enhanced release of inflammatory cytokines including TNF-α, IL-1ß and IL-6. Serum from RA patients, compared with healthy controls, induced gasdermin D (GSDMD)-dependent pyroptosis in monocytes, and this ability was associated with disease activity. Moreover, PTX3 synergized with C1q to promote pyroptosis in RA-serum pre-incubated monocytes by coordinately enhancing NLRP3 inflammasome over-activation and inducing GSDMD cleavage, cell swelling with large bubbles, caspase-1-dependent cell death and inflammatory cytokine release including IL-6. On the other hand, IL-6 promoted PTX3 plus C1q-induced pyroptosis in both normal and RA serum pre-incubated monocytes. These findings collectively implicated an important role of IL-6 in driving PTX3 plus C1q-mediated pyroptosis in RA and shed lights on a potential new treatment strategy targeting pyroptosis-mediated persistent inflammatory cytokine release.