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

Abstract

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-36γ 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-36γ by enzyme-linked immunosorbent assay, with or without sonication. In addition, conditioned medium was ultracentrifuged at 25,000 g and 100,000 g, to isolate microparticles and exosomes, respectively, and interleukin-36γ protein was assessed in each fraction by Western blot analysis. Interleukin-36γ 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-36γ protein was enhanced by adenosine 5'-triphosphate. Furthermore, extracellular interleukin-36γ protein detection was markedly enhanced by sonication to disrupt membrane-bound structures. Interleukin-36γ protein was detected by Western blot in microparticles and exosome fractions isolated by ultracentrifugation. Interleukin-36γ was induced and secreted from lung macrophages in response to Gram-negative and -positive bacterial stimulation. The results suggest that interleukin-36γ is secreted in a non-Golgi-dependent manner by lung macrophages in response to Gram-positive and -negative bacterial challenge.

IL-36 receptor deletion attenuates lung injury and decreases mortality in murine influenza pneumonia

“…22 BAL fluid and CM was centrifuged to remove cells and large debris at 3,000rpm for 10min and stored these samples at −80°C overnight. Each sample supernatant was centrifuged at 17,000rpm for 45min to obtain the MPs.…”

Regulation and function of interleukin‐36 cytokines

“…Lung IL‐36γ production is enhanced in response to Streptococcus pneumoniae and Klebsiella pneumoniae . IL‐36γ‐deficient mice infected with S. pneumoniae exhibited diminished lung bacterial clearance and increased bacterial dissemination and eventually higher mortality rate …”

Interleukin-36 cytokines may overcome microbial immune evasion strategies that inhibit interleukin-1 family signaling

“…Both in vitro and in vivo, this release is independent of caspase-1 (77,78). Extracellular IL-36g has been detected in culture medium from keratinocytes treated with ATP, the double-stranded RNA analog polyinosinic-polycytidylic acid [poly(I:C)], or the antimicrobial peptide LL-37 and from lung macrophages exposed to bacteria or bacterial components (24,26,31,79,80). Some of this released IL-36g appears to be present in extracellular vesicles (80,81); however, the purpose of such packaging is unknown.…”