Survival after bacterial challenges.

<p>(A) During the first septic assault, after CLP and prior to <i>P</i>. <i>aeruginosa</i> instillation (D5), 28% of CLP-operated mice (22/80) died. During this period, 100% of Sham-operated mice (n = 29) survived. (B) At D5 post CLP, mice were challenged with an intratracheal administration of <i>P</i>. <i>aeruginosa</i>. Since CLP mice died until 5 days, final survival data were given by subtracting lethality induced by CLP during the first assault. So we considered the remaining living mice as 100% for this second survival study. Either PBS (Sham n = 6, CLP n = 10) or <i>Pseudomonas aeruginosa</i> were instilled. For bacterial challenge, three doses were administered: 5.10⁶ CFU (Sham n = 6 and CLP n = 15), 2.10⁷ CFU (Sham n = 10 and CLP n = 17) and 10⁸ CFU (Sham n = 7 and CLP n = 20). Results are expressed as Kaplan Meier survival curves. p < 0.05 were considered statistically significant.</p

Immunological assay.

<p>Spleens were harvested at D5 and splenocytes were rapidly isolated in order to determine T cells characteristics (population, proliferation, and TNFα secretion). As compared to Sham-operated animals (n = 10), total T cells population (CD3+) in CLP mice (n = 8) decreased (A) among which Treg fraction (CD4⁺/CD25⁺) increased (B). In parallel, T cells proliferation, assessed by a stimulation with an anti-CD3⁺ antibody (200 000 splenocytes / anti CD3 coated wells (1<b>μ</b>g/ml)) and incorporation of [³H]-thymidine was diminished (C). Radioactivity counts reflect the lymphocytes proliferation. Results are expressed as proliferation ratios between counts per minute measured in stimulated (cpm stim) versus non-stimulated (cpm no stim) wells for the same experiment. Splenocytes ability to produce TNFα when stimulated for 24 hours with LPS (200 000 cells/well, LPS 1<b>μ</b>g/ml) was also decreased (D). *p<0.05 Mann Whitney U test, **p<0.01 Mann Whitney U test, °p<0.05 Wilcoxon paired test °°p<0.01, Wilcoxon paired test.</p

Bacterial dissemination.

<p>5 days after surgery (D5), mice were intratracheally instilled with 5.10⁶ CFU, 2.10⁷ CFU or 10⁸ CFU of <i>Pseudomonas aeruginosa</i> and their lungs, liver, spleen were harvested 2 (D7; A) or 7(D13; B) days later, grinded, diluted and cultured for the presence of <i>P</i>. <i>aeruginosa</i>. The results are expressed as log CFU/g tissue. *p<0.05 Mann Whitney U Test.</p

Theoretical potential inflammatory responses in mice double hit model.

<p>The observations made in this study show that pro and anti-inflammatory responses are concomitant in this model as in human. Despite immunoparalysis, intratracheal secondary infection due to <i>P</i>. <i>aeruginosa</i> reactivates slightly pro- and anti-inflammatory processes. Inflammatory response is represented by Red (pro) and blue (anti-inflammatory) lines. Dark lines represent initial inflammatory response and response after secondary infection whereas light lines represent the inflammatory response without secondary pulmonary infection.</p

Plasmatic cytokines assessment after bacterial challenges.

<p>Plasma was collected 1 (D6), 2 (D7) and 8 (D13) days after <i>P</i>. <i>aeruginosa</i> intratracheal instillation and TNFα, IL-6, sTNFr I and II and IL-10 were measured thanks to the Luminex technique (n≥4 for each groupe or dose). While no group, nor time or dose effect could be evidenced for TNFα (A), IL-6 and IL-10 levels were dose-dependent and decreased over time (B and C). No group effect but a dose-dependent decrease of sTNFr-I (D) at D13 and sTNFr-II (E) at D6, D7 and D13 were observed. sTNFr-I increased over time whereas sTNFr-II did not. Results are expressed as Log(pg/ml). Effects are considered significant when p<0.05.</p

Study protocol.

<p>Mice were Sham- or CLP-operated at D0. Five days after surgery (D5), mice were or were not intratracheally instilled with <i>P</i>. <i>aeruginosa</i>. Plasmatic cytokines were measured at 2h, 6h, D1, D2, D3, D5 and D13 and also at D6 and D7 only for instilled mice.</p

Plasmatic cytokines assessment after CLP.

<p>Plasma was collected 2 and 6 hours, 1 (D1), 2 (D2), 3 (D3), 5 (D5) and 13 (D13) days after CLP or Sham surgery with no secondary infection to measure TNFα, IL-6, sTNFr I and II and IL-10 concentrations thanks to the Luminex technique. At D0, before surgery, TNFα, IL-6 and IL-10 were undetectable. Both sTNFr were detectable in healthy mice. Pro-inflammatory cytokines TNFα (A) and IL-6 (B) are significantly increased as soon as 2h after sepsis induction, as anti-inflammatory mediators IL-10 (C) and sTNFr-I and II (D and E). TNFα was not detected anymore in Sham operated mice at D5 and D13. Results are expressed as Log(pg/ml). p values given are corrected p values according to the Bonferroni correction. *CLP <i>vs</i> Sham p<0.05, **CLP <i>vs</i> Sham p<0.01, ***CLP <i>vs</i> Sham p<0.001.</p

Study design flowchart.

<p>Mice were randomized into 2 groups: Sham or CLP. In each group, some mice were used to evaluate cytokines levels and immune status (dotted arrows) and were therefore not included in the survival evaluation after the first septic insult. Note that some mice were used for both cytokine assessment and immune status evaluation. The second stage started right after the intratracheal administration of <i>Pseudomonas aeruginosa</i>. Sham and CLP groups mice were randomized into 4 different groups according to the amount of bacteria they received: Sham- or CLP-PBS were instilled with PBS (vehicle) only; other groups were instilled with the different bacterial loads as follow: Sham- or CLP-5.10⁶ CFU, Sham- or CLP-2.10⁷ CFU and Sham- or CLP-10⁸ CFU. Systemic cytokine release and spleen and liver bacteria dissemination were evaluated in the same mice. These mice were not included in the survival evaluation of the second hit. “n” represent the animal number in each condition.</p