Inflammatory cytokines in whole blood stimulated <i>ex vivo</i> by <i>S</i>. <i>aureus</i> in various conditions.

<p>Interleukin (IL)-8 and tumor necrosis factor (TNF)-α mean levels (SD) in the supernatant of whole blood from either spontaneously breathing rabbits or animals subjected to mechanical ventilation. Atorvastatin was added to the medium (“statin”) or given to the rabbit prior to blood sampling (“Premed”). Values are presented as means (SD). 2-way ANOVA test was used for all intergroup comparisons with Tukey’s adjustment for multiple comparisons. One missing [IL-8] and one missing [TNF-α] value in the Whole blood+MRSA and in the Whole blood+MRSA+statin groups. MRSA: methicillin-resistant <i>S</i>. <i>aureus</i>; SB: spontaneously breathing; MV: mechanical ventilation; SD: standard deviation.</p

Pulmonary and systemic bacterial burden of <i>S</i>. <i>aureus</i>.

<p>Pulmonary bacterial clearance expressed as the median (IQR) ratio between the amounts of bacteria recovered from the lung culture and the initial <i>Staphylococcus aureus</i> inoculum 24 hours after bacterial challenge in either spontaneously breathing or mechanically ventilated animals. Pulmonary-to-systemic bacterial translocation expressed as the spleen positive cultures rate 24 hours after bacteria instillation according to the experimental group in either spontaneously breathing or mechanically ventilated animals with <i>Staphylococcus aureus</i> pneumonia. Values are presented as percentages <i>(right)</i>. Kruskall-Wallis test with Dunn correction for multiple comparisons or Fisher exact test was used as appropriate for intergroup comparisons. CFU: colony forming unit; MRSA: methicillin-resistant S. aureus; LNZ: linezolid; SB: spontaneously breathing; MV: mechanical ventilation; IQR: interquartile range.</p

Time course of serum inflammatory cytokine concentrations during experiment.

<p>Serum concentrations of interleukin (IL)-8 and tumor necrosis factor (TNF)-α expressed as % of the mean baseline value measured by enzyme-linked immune-sorbent assay at baseline (100%; 24 hours before bacterial airway challenge), just before intra-tracheal instillation of 10⁹ CFU of <i>S</i>. <i>aureus</i> (H0), and then 8 (H8) and 24 hours (H24) after instillation, in the spontaneously breathing (A) and the mechanically ventilated animals (B). Concentrations measured in non-infected controls are shown in panel C. Variables were compared at each time-point with Mann-Whitney U test and p values were adjusted for multiple comparisons. (A): (<i>left</i>) * denotes p = 0.014 between rabbits treated with atorvastatin vs. untreated animals, and ** denotes p = 0.027 between rabbits treated with LNZ+atorvastatin vs. untreated animals; (<i>right</i>) * denotes p = 0.049 between rabbits treated with LNZ vs. untreated animals, and ** denotes p = 0.006 between rabbits treated with LNZ+atorvastatin vs. untreated animals. (B): (<i>left</i>) * denotes p = 0.004 between rabbits treated with atorvastatin vs. untreated animals, ** denotes p = 0.003 between rabbits treated with LNZ vs. untreated animals, and *** denotes p = 0.014 between rabbits treated with LNZ+atorvastatin vs. untreated animals; (<i>right</i>) * denotes p = 0.034 between rabbits treated with atorvastatin vs. untreated animals, ** denotes p = 0.013 between rabbits treated with LNZ vs. untreated animals, and *** denotes p = 0.002 between rabbits treated with LNZ+atorvastatin vs. untreated animals. CFU: colony forming unit; MRSA: methicillin-resistant <i>S</i>. <i>aureus</i>; LNZ: linezolid; SB: spontaneously breathing; MV: mechanical ventilation. CFU: colony forming unit; MRSA: methicilline-resistant S. aureus; LNZ: linezolid; SB: spontaneously breathing; MV: mechanical ventilation; IQR: interquartile range.</p

Toll-like receptor 2 gene expression.

<p>TLR2 gene expression was measured in both lung (<i>left</i>) and spleen (<i>right</i>) homogenates in either spontaneously breathing rabbits (SB) or animals subjected to mechanical ventilation (MV) with or without atorvastatin pre-treatment (n = 5 in each group). All values are reported as fold increases compared with SB rabbits. Results are expressed as mean±standard deviation. Kolmogorov-Smirnov test was used for all intergroup comparisons.</p

Inflammatory cytokines in the lung and in the spleen of rabbits in various experimental conditions.

<p>Inflammatory cytokines (interleukin [IL]-8 and tumor necrosis factor [TNF]-α) concentrations in lung (A) and spleen (B) homogenates 24 hours after tracheal instillation of saline (controls) 10⁹ CFU of <i>Staphylococcus aureus</i> in either spontaneously breathing rabbits or animals subjected to mechanical ventilation with various treatment combinations. Values are presented as means (SD). 2-way ANOVA test was used for all intergroup comparisons with Tukey’s adjustment for multiple comparisons. CFU: colony forming unit; MRSA: methicilline-resistant S. aureus; LNZ: linezolid; SB: spontaneously breathing; MV: mechanical ventilation; SD: standard deviation.</p

Lung injury main features according to the experimental condition.

<p>Histologic score ranging from 0 to 3, based on the degree of polymorphonuklear infiltration, haemorrhage, and oedema in the interstitial and alveolar spaces, following tracheal instillation of saline (controls) or 9 log₁₀CFU of <i>Staphylococcus aureus</i> in either spontaneously breathing rabbits or animals subjected to mechanical ventilation with various treatment combinations. Mean values (SD) are shown (A) as well as representative light microphotographs of rabbit lungs in various conditions fixed at the same transpulmonary pressure (hematoxylin and eosin x400) (B). Two-tailed Mann-Whitney U test was used for all intergroup comparisons. CFU: colony forming unit; MRSA: methicilline-resistant S. aureus; LNZ: linezolid; SB: spontaneously breathing; MV: mechanical ventilation; SD: standard deviation.</p

Concentration-time profile for linezolid in rabbits submitted to various experimental conditions.

<p>Two-tailed Mann-Whitney U test was used at each time-point for all intergroup comparisons. MRSA: methicillin-resistant <i>S</i>. <i>aureus</i>; LNZ: linezolid; SB: spontaneously breathing; MV: mechanical ventilation.</p

Histologic score according to the experimental condition.

<p>Histologic score ranging from 0 to 3, based on the degree of polymorphonuclear infiltration, haemorrhage, and oedema in the interstitial and alveolar spaces, following tracheal instillation of saline (controls) in either spontaneously breathing (SB, n = 5) rabbits or animals subjected to mechanical ventilation (MV, n = 5). Infected animals were challenged with 10⁹ CFU of <i>Staphylococcus aureus</i>. After bacterial challenge animals were kept SB or under MV for 8 hours (<i>n</i> = 5 and 6, respectively) and 24 hours (n = 8 and 11, respectively). The histologic score was higher under MV. ** denotes <i>p</i><0.05 between SB and MV animals; ns: not significant. CFU: colony forming unit. Controls SB versus (vs) MV p = 0.002; Mann-Whitney U test, two-tailed. <i>S</i>. <i>aureus</i> pneumonia SB vs MV 8 hours p = 0.011; Mann-Whitney U test, two-tailed. <i>S</i>. <i>aureus</i> pneumonia SB vs MV 24 hours p = 0.216; Mann-Whitney U test, two-tailed.</p

Inflammatory cytokines in the lung.

<p>Inflammatory cytokines (interleukin [IL]-8 and tumor necrosis factor [TNF]-α) in lung homogenates and in broncho-alveolar lavage fluid (BALF) 8 to 24 hours after tracheal instillation of saline (controls) in either spontaneously breathing (SB, n = 5) rabbits or animals subjected to mechanical ventilation (MV, n = 5), or 8 hours (n = 5) or 24 hours (n = 10) after challenge with 10⁹ CFU of <i>Staphylococcus aureus</i> (+<i>S</i>. <i>aureus</i> H8 and H24). IL-8 levels were increased in lung homogenates and in BALF under MV in controls, but we did not find any significant difference between SB and MV groups after bacterial challenge. Similarly, TNF-α levels were significantly increased in lung homogenates from animals under MV than in controls, but not after bacterial challenge. ** denotes <i>p</i><0.05; ns: not significant; ND: concentration below the limits of detection of the assay. CFU: colony forming unit. (A) Controls SB versus (vs) MV <i>p</i> = 0.333, Mann-Whitney U test, two-tailed. <i>S</i>. <i>aureus</i> H8 SB vs MV p = 0.397, Mann-Whitney U test, two-tailed. <i>S</i>. <i>aureus</i> H24 SB vs MV p = 0.395, Mann-Whitney U test, two-tailed. (B) Controls SB vs MV <i>p</i> = 0.004, Mann-Whitney U test, two-tailed. <i>S</i>. <i>aureus</i> H8 SB vs MV p = 0.413, Mann-Whitney U test, two-tailed. <i>S</i>. <i>aureus</i> H24 SB vs MV p = 0.881, Mann-Whitney U test, two-tailed. (C) Controls SB vs MV <i>p</i> = 0.024, Mann-Whitney U test, two-tailed. <i>S</i>. <i>aureus</i> H8 SB vs MV p = 0.114, Mann-Whitney U test, two-tailed. <i>S</i>. <i>aureus</i> H24 SB vs MV p = 0.700, Mann-Whitney U test, two-tailed. (D) <i>S</i>. <i>aureus</i> H8 SB vs MV p = 0.829, Mann-Whitney U test, two-tailed. <i>S</i>. <i>aureus</i> H24 SB vs MV p = 0.700, Mann-Whitney U test, two-tailed.</p

Pulmonary and systemic bacterial burden of <i>Staphylococcus aureus</i>.

<p><b>(4A)</b><b><i>S</i></b>. <b><i>aureus</i></b><b>lung concentrations 8 and 24 hours after bacteria instillation.</b> Pulmonary bacterial burden according to the experimental group in either spontaneously breathing (SB + <i>S</i>. <i>aureus</i>) or mechanically ventilated (MV + <i>S</i>. <i>aureus</i>) animals with <i>Staphylococcus aureus</i> pneumonia 8 hours after inoculation (n = 5 and 6, respectively) and 24 hours after inoculation (n = 8 and 11 respectively). The bacterial burden was higher in the MV group both after 8 hours (5.00±1.03 versus [vs.] 5.87±0.38 log₁₀CFU/g, <i>p</i> = 0.028) and 24 hours of MV (4.96±1.31 vs. 6.13±0.63 log₁₀CFU/g, <i>p</i> = 0.002). *denotes <i>p</i>≤0.05. CFU: colony forming unit. (4B) <i>S</i>. <i>aureus</i> spleen concentrations 8 and 24 hours after bacteria instillation. Spleen bacterial burden according to the experimental group in either spontaneously breathing (SB + <i>S</i>. <i>aureus</i>) or mechanically ventilated (MV + <i>S</i>. <i>aureus</i>) animals with <i>Staphylococcus aureus</i> pneumonia (n = 4 and 5, respectively*). Interestingly, MV appears to promote early pulmonary-to-systemic translocation during <i>S</i>. <i>aureus</i> pneumonia, as indicated by the quantitative spleen cultures, as an indirect marker of bacteraemia, which were positive only in ventilated animals after 8 hours, even though there was no longer any difference after 24 hours. *spleen was lost in 1 animal from each group. Lung <i>S</i>. <i>aureus</i> concentrations SB versus (vs) MV 8 hours <i>p</i> = 0.050; Mann-Whitney U test, two-tailed. Lung <i>S</i>. <i>aureus</i> concentrations SB vs MV 24 hours <i>p</i> = 0.007; Mann-Whitney U test, two-tailed. Spleen <i>S</i>. <i>aureus</i> concentrations SB vs MV 8 hours <i>p</i> = 0.430; Mann-Whitney U test, two-tailed. Spleen <i>S</i>. <i>aureus</i> concentrations SB vs MV 24 hours <i>p</i> = 0.110; Mann-Whitney U test, two-tailed.</p