Synergistic Costimulatory Effect of Chlamydia pneumoniae with Carbon Nanoparticles on NLRP3 Inﬂammasome-Mediated Interleukin-1 (cid:2) Secretion in Macrophages

The obligate intracellular bacterium Chlamydia pneumoniae is not only a causative agent of community-acquired pneumonia but is also associated with a more serious chronic disease, asthma, which might be exacerbated by air pollution containing carbon nanoparticles. Although a detailed mechanism of exacerbation remains unknown, the proinﬂammatory cytokine interleu-kin-1 (cid:2) (IL-1 (cid:2) ) is a critical player in the pathogenesis of asthma. C. pneumoniae induces IL-1 (cid:2) in macrophages via NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inﬂammasome activation and Toll-like receptor 2/4 (TLR2/4) stimulation. Carbon nanoparticles, such as carbon nanotubes (CNTs), can also evoke the NLRP3 inﬂammasome to trigger IL-1 (cid:2) secretion from lipopolysaccharide-primed macrophages. This study assessed whether costimulation of C. pneumoniae with CNTs synergistically enhanced IL-1 (cid:2) secretion from macrophages, and determined the molecular mechanism involved. Enhanced IL-1 (cid:2) secretion from C. pneumoniae -infected macrophages by CNTs was dose and time dependent. Transmission electron microscopy revealed that C. pneumoniae and CNTs were engulfed concurrently by macrophages. Inhibitors of actin polymerization or caspase-1, a component of the inﬂammasome, signiﬁcantly blocked IL-1 (cid:2) secretion. Gene silencing using small interfering RNA (siRNA) targeting the NLRP3 gene also abolished IL-1 (cid:2) secretion. Other inhibitors (K (cid:3) efﬂux inhibitor, cathepsin B inhibitor, and reactive oxygen species-generating inhibitor) also blocked IL-1 (cid:2) secretion. Taken together, these ﬁndings demonstrated that CNTs synergistically enhanced IL-1 (cid:2) secretion from C. pneumoniae -infected macrophages via the NLRP3 inﬂammasome and caspase-1 activation, providing novel insight into our understanding of how C. pneumoniae infection can exacerbate asthma. = -AGC CAC GCT AAT GAT CGA CT-3 = ; reverse, 5 = -CAG GCT CAG AAT GCT CAT CA-3 = ) (26) and the GAPDH gene (24). Statistical analysis. Statistical analysis was performed using an un-paired Student’s t test. A P value of (cid:9) 0.05 was considered signiﬁcant. (cid:2) secretionalso occurred in a dose-dependent manner, dependent upon the CNT concentra-tion.Theresultsarerepresentativeofthreeindependentexperiments. Tubulinwasusedasaninternalcontrol.sup,desalinizedculturesupernatantsofTHP-1cells;cell,theremainingcells;Cpn, C. pneumoniae ; CNT, carbon nanotube; LPS, lipopolysaccharide; IL-1 (cid:2) , interleukin-1 (cid:2) . upstream sig- nals: K (cid:7) efﬂux, lysosomal degradation, and ROS production. Cells stimulated with or without Chlamydia pneumoniae in the presence of absence of CNTs were incubated with either 70 mM KCl, 10 (cid:4) M CA-074 Me (a cathepsin B inhibitor), or ROS inhibitors (5 mM NAC or 10 (cid:4) M DPI) for 24 h, and then IL-1 (cid:2) secretion from macrophages was detected. Treatment with KCl (A), CA-074Me(B),orDPI(C),butnotNAC,blockedIL-1 (cid:2) secretion.Resultsare representative of three independent experiments. C. pneumoniae nanotube; interleukin-1 lipopolysaccharide;