An Allergic Lung Microenvironment Suppresses Carbon Nanotube-Induced Inflammasome Activation via STAT6-Dependent Inhibition of Caspase-1

Abstract

BackgroundMulti-walled carbon nanotubes (MWCNTs) represent a human health risk as mice exposed by inhalation display pulmonary fibrosis. Production of IL-1β via inflammasome activation is a mechanism of MWCNT-induced acute inflammation and has been implicated in chronic fibrogenesis. Mice sensitized to allergens have elevated T-helper 2 (Th2) cytokines, IL-4 and IL-13, and are susceptible to MWCNT-induced airway fibrosis. We postulated that Th2 cytokines would modulate MWCNT-induced inflammasome activation and IL-1β release in vitro and in vivo during allergic inflammation.MethodsTHP-1 macrophages were primed with LPS, exposed to MWCNTs and/or IL-4 or IL-13 for 24 hours, and analyzed for indicators of inflammasome activation. C57BL6 mice were sensitized to house dust mite (HDM) allergen and MWCNTs were delivered to the lungs by oropharyngeal aspiration. Mice were euthanized 1 or 21 days post-MWCNT exposure and evaluated for lung inflammasome components and allergic inflammatory responses.ResultsPriming of THP-1 macrophages with LPS increased pro-IL-1β and subsequent exposure to MWCNTs induced IL-1β secretion. IL-4 or IL-13 decreased MWCNT-induced IL-1β secretion by THP-1 cells and reduced pro-caspase-1 but not pro-IL-1β. Treatment of THP-1 cells with STAT6 inhibitors, either Leflunomide or JAK I inhibitor, blocked suppression of caspase activity by IL-4 and IL-13. In vivo, MWCNTs alone caused neutrophilic infiltration into the lungs of mice 1 day post-exposure and increased IL-1β in bronchoalveolar lavage fluid (BALF) and pro-caspase-1 immuno-staining in macrophages and airway epithelium. HDM sensitization alone caused eosinophilic inflammation with increased IL-13. MWCNT exposure after HDM sensitization increased total cell numbers in BALF, but decreased numbers of neutrophils and IL-1β in BALF as well as reduced pro-caspase-1 in lung tissue. Despite reduced IL-1β mice exposed to MWCNTs after HDM developed more severe airway fibrosis by 21 days and had increased pro-fibrogenic cytokine mRNAs.ConclusionsThese data indicate that Th2 cytokines suppress MWCNT-induced inflammasome activation via STAT6-dependent down-regulation of pro-caspase-1 and suggest that suppression of inflammasome activation and IL-1β by an allergic lung microenvironment is a mechanism through which MWCNTs exacerbate allergen-induced airway fibrosis.

Multiwalled Carbon Nanotube Functionalization with High Molecular Weight Hyaluronan Significantly Reduces Pulmonary Injury

“…41 A significant increase in IL-1 β has already been reported after exposure to CNTs. 9,10,34,42,43 MWCNT exposure, similar to asbestos, can induce inflammasome activation in the lungs, leading to IL-1 β secretion. 42 We previously reported IL-1 β secretion from primary human bronchial epithelial cells after MWCNT exposure via activation of the inflammasome that contributed to increased profibrotic gene expression in human fibroblasts.…”

“…62 MWCNTs are associated with MCM development in the exposed lungs. 34,35,43,63,64 In order to evaluate the impact of HA functionalization in MW-induced MCM development, we analyzed mucus-producing goblet cells in lung sections and quantified chemical mediators involved in the MCM phenotype. Scoring criteria were based on published methodology and are detailed in Supporting Information methods.…”

In vivo activation of a T helper 2-driven innate immune response in lung fibrosis induced by multi-walled carbon nanotubes

“…MWCNT have been shown to stimulate allergy-like inflammation in mouse lung airways where expression of cytokines associated with Th2 functions, i.e., IL-4 and IL-13, was elevated (Rydman et al 2014). Allergen sensitization was found to induce the expression of IL-4 and IL-13, which appeared to suppress CNT-induced activation of inflammasomes (Shipkowski et al 2015). The function, cellular origin, and mechanism of induction of the cytokines associated with the allergic responses to CNT exposure remain to be defined.…”

STAT1-dependent and -independent pulmonary allergic and fibrogenic responses in mice after exposure to tangled versus rod-like multi-walled carbon nanotubes

“…Samples were diluted, loaded onto a Novex™ 4–12% SDS-PAGE gel (Invitrogen, Carlsbad, CA, #XP04122BOX), and separated by electrophoresis as described previously [43]. Briefly, samples were transferred to PVDF membranes, blocked, and incubated in primary antibody (1:1000 dilution).…”