IL-36γ is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components

Interleukin-36 is a family of novel interleukin-1-like proinflammatory cytokines that are highly expressed in epithelial tissues and several myeloid-derived cell types. Like those of classic interleukin-1 cytokines, the secretion mechanisms of interleukin-36 are not well understood. Interleukin-36g secretion in dermal epithelial cells requires adenosine 5'-triphosphate, which suggests a nonclassical mechanism of secretion. In this study, murine pulmonary macrophages and human alveolar macrophages were treated with recombinant pathogen-associated molecular patterns ( intact bacteria: Klebsiella pneumoniae or Streptococcus pneumoniae). Cell lysates were analyzed for messenger ribonucleic acid by quantitative real-time polymerase chain reaction, and conditioned medium was analyzed for interleukin-36 gamma by enzyme-linked immunosorbent assay, with or without sonication. In addition, conditioned medium was ultra-centrifuged at 25,000 g and 100,000 g, to isolate microparticles and exosomes, respectively, and interleukin-36 gamma protein was assessed in each fraction by Western blot analysis. Interleukin-36 gamma mRNA was induced in both murine and human lung macrophages by a variety of pathogen-associated molecular patterns, as well as heat-killed and live Klebsiella pneumoniae and Streptococcus pneumoniae, and induction occurred in a myeloid differentiation response gene 88-dependent manner. Secretion of interleukin-36 gamma protein was enhanced by adenosine 5'-triphosphate. Furthermore, extracellular interleukin-36 gamma protein detection was markedly enhanced by sonication to disrupt membrane-bound structures. Interleukin-36 gamma protein was detected by Western blot in microparticles and exosome fractions isolated by ultracentrifugation. Interleukin-36 gamma was induced and secreted from lung macrophages in response to Gram-negative and -positive bacterial stimulation. The results suggest that interleukin-36 gamma is secreted in a non-Golgi-dependent manner by lung macrophages in response to Gram-positive and -negative bacterial challenge.