Background
Long term outcomes of lung transplantation are impacted by the occurrence of chronic
lung allograft dysfunction (CLAD). Recent evidence suggests a role for the lung microbiome
in the occurrence of CLAD, but the exact mechanisms are not well defined. We hypothesize
that the lung microbiome inhibits epithelial autophagic clearance of pro-fibrotic
proteins in an IL-33 dependent manner, thereby augmenting fibrogenesis and risk for
CLAD.
Methods
Autopsy derived CLAD and non-CLAD lungs were collected. IL-33, P62 and LC3 immunofluorescence
was performed and assessed using confocal microscopy. Pseudomonas aeruginosa (PsA), Streptococcus Pneumoniae (SP), Prevotella Melaninogenica (PM), recombinant IL-33 or PsA-lipopolysaccharide was co-cultured with primary human
bronchial epithelial cells (PBEC) and lung fibroblasts in the presence or absence
of IL-33 blockade. Western blot analysis and quantitative reverse transcription (qRT)
PCR was performed to evaluate IL-33 expression, autophagy, cytokines and fibroblast
differentiation markers. These experiments were repeated after siRNA silencing and
upregulation (plasmid vector) of Beclin-1.
Results
Human CLAD lungs demonstrated markedly increased expression of IL-33 and reduced basal
autophagy compared to non-CLAD lungs. Exposure of co-cultured PBECs to PsA, SP induced
IL-33, and inhibited PBEC autophagy, while PM elicited no significant response. Further,
PsA exposure increased myofibroblast differentiation and collagen formation. IL-33
blockade in these co-cultures recovered Beclin-1, cellular autophagy and attenuated
myofibroblast activation in a Beclin-1 dependent manner.
Conclusion
CLAD is associated with increased airway IL-33 expression and reduced basal autophagy.
PsA induces a fibrogenic response by inhibiting airway epithelial autophagy in an
IL-33 dependent manner.
KEYWORDS
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Published online: October 05, 2022
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