Abstract
Breathing allows a multitude of airborne microbes and microbial compounds to access the lung. Constant exposure of the pulmonary microenvironment to immunogenic particles illustrates the need for proper control mechanisms ensuring the differentiation between threatening and harmless encounters. Discrimination between live and dead bacteria has been suggested to be such a mechanism. In this study, we performed infection studies of murine precision cut lung slices (PCLS) with live or heat-killed P. aeruginosa, in order to investigate the role of viability for induction of an innate immune response. We demonstrate that PCLS induce a robust transcriptomic rewiring upon infection with live but not heat-killed P. aeruginosa. Using mutants of the P. aeruginosa clinical isolate CHA, we show that the viability status of P. aeruginosa is assessed in PCLS by TLR5-independent sensing of flagellin and recognition of the type three secretion system. We further demonstrate that enhanced cytokine expression towards live P. aeruginosa is mediated by uptake of viable but not heat-killed bacteria. Finally, by using a combined approach of receptor blockage and genetically modified PCLS we report a redundant involvement of MARCO and CD200R1 in the uptake of live P. aeruginosa in PCLS. Altogether, our results show that PCLS adapt the extent of cytokine expression to the viability status of P. aeruginosa by specifically internalizing live bacteria.
Highlights
At the onset of infection, host cell defense depends on the rapid initiation of an innate immune response based on the recognition of a broad variety of pathogen associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) on innate immune cells [1, 2]
This study was conducted with murine precision cut lung slices (PCLS) as an ex vivo lung model system to investigate the regulation of immune responses as a complex interplay between pulmonary cells organized in their natural architecture rather than studying an isolated pulmonary cell type
We provide evidence for four major findings: (I) PCLS discriminate between live and dead P. aeruginosa infection. (II) Cytokine induction in PCLS depends on the flagellar-filament protein fliC and the T3SS, with special emphasis on the needle protein pscF. (III) PCLS internalize live P. aeruginosa. (IV) MARCO and CD200R1 are redundantly involved in the internalization of live P. aeruginosa and IL6 induction
Summary
At the onset of infection, host cell defense depends on the rapid initiation of an innate immune response based on the recognition of a broad variety of pathogen associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) on innate immune cells [1, 2]. Upon host cell contact or calcium-depletion, P. aeruginosa transcribes and assembles the T3SS needle complex and the translocation pore [19,20,21]. Another important virulence factor of P. aeruginosa is the flagellum that enables the bacterium to swim [22]. Internalization of bacteria, with contribution of the receptors CD200R1 and MARCO, is important for live/dead discrimination
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