Pseudomonas aeruginosa lung infection and respiratory muscle weakness: Role of cytokines in diaphragm muscle dysfunction.

摘要

The primary muscle of respiration is the diaphragm. Diaphragm muscle dysfunction and ventilatory pump failure are well documented phenomena in animal models of sepsis. However, the primary cellular mechanisms underlying respiratory muscle dysfunction in sepsis are poorly understood. In addition, most investigations of respiratory muscle dysfunction in sepsis have been performed in models involving high doses of bacterial endotoxin and these investigations have been criticized on the basis of questionable relevance to human sepsis. Therefore, the objective in the first study of this thesis was to study respiratory muscle dysfunction in a more clinically relevant animal model, namely, the Pseudomonas aeruginosa pulmonary infection model. Remote inflammatory processes in different diseases, such as cancer, arthritis, sepsis, and cystic fibrosis are known to contribute to muscle wasting and weakness through more widespread systemic effects. In keeping with the above notion, we hypothesized that sustained P. aeruginosa lung infection would cause diaphragmatic and limb muscle weakness. In this thesis, we demonstrate for the first time that persistent pulmonary infection with P. aeruginosa induces significant dose- and time-dependent contractile dysfunction of the diaphragm. By comparison, prototypical slow- and fast-twitch hindlimb muscles were not influenced by pulmonary P. aeruginosa infection.;P. aeruginosa lung infection is a major cause of morbidity and mortality among cystic fibrosis (CF) patients and many patients with CF have weak peripheral and respiratory muscles. Although the role of pro-inflammatory cytokines has been extensively studied within the lungs of CF patients, the involvement of these cytokines in skeletal muscle dysfunction in animal models of CF or in human CF patients has not been studied. Therefore, in the third study of this thesis we have used mice sharing the same genetic defect as CF patients (Cftr knockout mice), in combination with our model of P. aeruginosa lung infection, to address several fundamental questions related to muscle function in CF. Our first objective in this portion of the thesis was to determine if diaphragmatic skeletal muscle cells express the CFTR mRNA. Our second objective was to ascertain whether intrinsic differences between CF and wild-type muscle cells could be detected in vitro, which might differentially affect the regulation of pro-inflammatory mediators in the setting of infection/inflammation. Our third objective was to evaluate possible differences in the ability of respiratory muscles to generate force prior to and after P. aeruginosa lung infection in Cftr knockout mice, as compared to their wild-type littermates. Finally, we aimed to determine if the absence of CFTR expression would predispose to muscle dysfunction triggered by up-regulation of intra-diaphragmatic pro-inflammatory gene expression. Our major results indicate that: First, in vitro stimulation with pro-inflammatory cytokines (TNF-α, IL-1α, and IFN-γ) and LPS (extracted from Pseudomonas aeruginosa) triggered increased expression of pro-inflammatory mediators (iNOS, RANTES, MIP-1α, MIP-1β, MIP-2 and KC) in both Cftr -/- and wild-type diaphragmatic myotubes, but the magnitude of cytokine/chemokine upregulation was significantly greater in CF than in wild-type diaphragm muscle cells. Second, CF mice are more vulnerable to diaphragm contractile dysfunction and increased intra-diaphragmatic pro-inflammatory gene expression (TNF-α, IL-1α, IL-β, IL-18, RANTES, MIP-1α, and MIP-2) after pulmonary P. aeruginosa infection in comparison with wild-type mice.;In the final study of this thesis, we sought to test the hypothesis that increased diaphragm muscle activation would lead to increased production of intra-diaphragmatic cytokine expression, since this could possibly explain the greater susceptibility of the diaphragm to express pro-inflammatory cytokines in response to pulmonary P. aeruginosa infection as compared with the hindlimb muscle. To test this hypothesis, we subjected rats to inspiratory resistive loading (IRL), corresponding to 45-50% of the maximum inspiratory pressure, and described that mRNA levels of IL-1β, IL-6, and to a lesser extent, IL-4, IL-10, TNF-α, and IFN-γ were all significantly increased in a time-dependent fashion in the diaphragm but not hindlimb muscle (gastrocnemius) of loaded animals. In addition, elevated protein levels of IL-1β and IL-6 in response to loading were confirmed with immunoblotting and immunostaining. We also detected significant IL-6 protein to be localized inside diaphragmatic muscle fibers of loaded animals. We conclude that increased diaphragm muscle activity during resistive loading induces upregulation of pro-inflammatory cytokine gene expression in the diaphragm, which could also provide an explanation for the greater cytokine expression observed in the diaphragms of animals with P. aeruginosa lung infection.;Because skeletal muscles can express a variety of immune modulating molecules such as cytokines, chemokines, adhesion molecules, and major histocompatibility molecules, the objective of the second study in this thesis was to study the possible role of pro-inflammatory cytokines in diaphragm muscle dysfunction in our animal model. Our results indicate for the first time that intra-diaphragmatic pro-inflammatory cytokine gene expression (TNF-α, IL-1α, IL-1β, IL-6, and IL-18) is highly up-regulated in infected animals and the magnitude of such upregulation is dependent upon the dose of P. aeruginosa lung infection. Parallel to the absence of muscle contractile dysfunction in hindlimb muscle under the same conditions, P. aeruginosa infection did not alter the levels of pro-inflammatory gene expression within the hindlimb muscle. To further address the involvement of muscle-derived pro-inflammatory cytokines in diaphragmatic contractile dysfunction, we have employed recombinant adenovirus (Ad) as a vehicle for systemic delivery of the anti-inflammatory cytokine IL-10, in order to shift the balance between pro- and anti-inflammatory cytokines within the diaphragm toward a more anti-inflammatory profile. We report here that systemic delivery of Ad-IL-10 suppresses pro-inflammatory gene expression and improves force generating capacity of the diaphragm in P. aeruginosa infected animals. This finding emphasizes the role of anti-inflammatory cytokines as beneficial immune modulators in respiratory muscle failure caused by pro-inflammatory cytokines.