Surfactant Protein A Enhances the Degradation of LPS-Induced TLR4 in Primary Alveolar Macrophages Involving Rab7, β-arrestin2, and mTORC1

Abstract

Respiratory infections by Gram-negative bacteria are a major cause of global morbidity and mortality. Alveolar macrophages (AMs) play a central role in maintaining lung immune homeostasis and host defense by sensing pathogens via pattern recognition receptors (PRR). The PRR Toll-like receptor (TLR) 4 is a key sensor of lipopolysaccharide (LPS) from Gram-negative bacteria. Pulmonary surfactant is the natural microenvironment of AMs. Surfactant protein A (SP-A), a multifunctional host defense collectin, controls LPS-induced pro-inflammatory immune responses at the organismal and cellular level via distinct mechanisms. We found that SP-A post-transcriptionally restricts LPS-induced TLR4 protein expression in primary AMs from healthy humans, rats, wild-type and SP-A -/- mice by further decreasing cycloheximide-reduced TLR4 protein translation and enhances the co-localization of TLR4 with the late endosome/lysosome. Both effects as well as the SP-A-mediated inhibition of LPS-induced TNFα release are counteracted by pharmacological inhibition of the small GTPase Rab7. SP-A-enhanced Rab7 expression requires β-arrestin2 and, in β-arrestin2 -/- AMs and after intratracheal LPS challenge of β-arrestin2 -/- mice, SP-A fails to enhance TLR4/lysosome co-localization and degradation of LPS-induced TLR4. In SP-A -/- mice, TLR4 levels are increased after pulmonary LPS challenge. SP-A-induced activation of mechanistic target of rapamycin complex 1 (mTORC1) kinase requires β-arrestin2 and is critically involved in degradation of LPS-induced TLR4. The data suggest that SP-A post-translationally limits LPS-induced TLR4 expression in primary AMs by lysosomal degradation comprising Rab7, β-arrestin2, and mTORC1. This study may indicate a potential role of SP-A-based therapeutic interventions in unrestricted TLR4-driven immune responses to lower respiratory tract infections caused by Gram-negative bacteria.

Signaling Pathways That Mediate Alveolar Macrophage Activation by Surfactant Protein A and IL-4

“…Animal handling procedures to obtain BAL were reviewed and approved by the local ethics committee (both Complutense University of Madrid and Autonomous Community of Madrid), according to Directive 2010/63/EU of the European Parliament and the Spanish law RD53/2013 on protection of animals used for experimentation. In some experiments, AMs were obtained from BAL of SP-A-deficient mice as previously reported ( 4 , 24 ). Importantly, AMs from SP-A-deficient mice were normal in number, phenotype and ability to respond to IL-4 ( 4 ).…”

“…The sedimented cells were washed twice with PBS, and the cell pellet was resuspended in RPMI 1640 medium (10% heat inactivated FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin, supplemented with 2 mM glutamine). AMs were purified by adherence for 90 min at 37°C under a 95% air-5% CO 2 atmosphere in 150-mm culture dishes as reported previously ( 4 , 19 , 24 ). Adherent cells were 94.0 ± 1.1% viable (trypan blue exclusion test).…”

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“…TLR activation via SP‐A regulates the generation of cytokines and inflammatory mediators such as TNF and interleukin (IL)‐1. 27 SP‐A also inhibits the synthesis of IL‐8 in eosinophils. IL‐8, which is generated by macrophages and epithelial cells, causes acute lung injury (ALI) via SP‐A.…”

“…Equally, SP‐A interacts with toll‐like receptors (TLRs) 2 and 4 to modulate inflammatory responses triggered by viral exposures. TLR activation via SP‐A regulates the generation of cytokines and inflammatory mediators such as TNF and interleukin (IL)‐1 27 . SP‐A also inhibits the synthesis of IL‐8 in eosinophils.…”