Environmental air pollutants including ozone cause severe lung injury and aggravate respiratory diseases such as asthma and COPD. Ozone is a major air pollutant causing pulmonary inflammation evolving into lung emphysema and/or fibrosis in mice exposed to ozone but the mechanisms are not well understood. Innate immune sensors such as nucleotide-oligomerization domain-like receptors (NLRs) are able to form cytosolic complexes named inflammasomes that mediate inflammatory cytokines production and immunological cell death. Among NLRs, NLRP3 was shown to be involved in establishment of lung inflammation to ozone. However, the role of NLRP6, another NLR in involved in inflammasome scaffold, like NLRP6 in ozone-induced lung pathologies is unknown.

Using a 6 weeks model of chronic ozone exposure in mice, we explored the role of the innate receptor NLRP6 in the context of immunological cell death, in ozone exposure-associated pulmonary inflammation.

We found that Nlrp6 deficiency dampens airway and lung inflammation with reduced neutrophils, eosinophils influx and production of associated chemokines/cytokines including Th2 response, remodeling factor production, collagen deposition and fibrosis in response to chronic ozone exposure. Interestingly, we observed increased NLRP6 expression in bronchial epithelial cells, pneumocytes and infiltrating macrophages in lung tissue and airway macrophages upon chronic ozone exposure. Mechanistically, we report that chronic ozone promotes NLRP6-mediated caspase-1- and caspase11-dependent gasdermin D activation in alveolar type 1 pneumocytes suggesting NLRP6 inflammasome drives lung inflammation. Moreover, gasdermin D deficiency leads to reduced pulmonary inflammation after chronic ozone exposure. Mice deficient for NLRP6 in alveolar epithelial cells display reduced inflammation with impaired caspase-1/11 and gasdermin D activation in lungs, which confirms NLRP6-driven inflammation in these cells. Finally, we found that caspase-3Casp3/8 and gasdermin E activation depends on NLRPlrp6.

In conclusion we provide the first evidence that NLRP6 is a key player in chronic ozone pulmonary inflammation associated with the induction of both gasdermin-D and gasdermin E-dependent pyroptosisimmunogenic cell death.