Divergence of the Response Induced by Xenogenic Immunization in the Sepsis Survival of Rats

<div><p>We have previously described that boosted natural xenoantibodies in rats cross-react to bacteria by targeting carbohydrate antigens. This type of immunization is associated with reduced survival after cecal ligation and puncture (CLP). In the present study, we investigated further this phenomenon by immunizing Lewis rats with three intraperitoneal injections, every other day, of hamster blood compared to saline-injected control animals. One day after the last injection, CLP was performed to produce a low-grade sepsis. Induction of xenoantibodies was associated with a reduction in animal survival after CLP relative to controls (45% vs. 90%, p<0.01). No bacterial blood load was observed after CLP in this model either with or without xenoantibody enhancement, indicating that the augmented mortality was not mediated by a direct effect of boosted xenoantibodies over blood bacteria. Nevertheless, the xenoimmunization produced a systemic inflammatory response in all rats. Additionally, a lack of weight gain at the time of CLP was present in animals that died after the procedure, which was not observed in surviving rats and controls. The cytokine profile at the time of CLP in animals that died after the procedure was characterized by an increase in the serum level of several cytokines, particularly adipokines. In contrast, the cytokine profile at CLP of xenoimmunized rats that survived the procedure was characterized by a reduction in the level of cytokines. In conclusion, this study failed to show a direct effect of boosted xenoantibodies over blood bacterial isolates as cause for the decreased survival after CLP. However, it evidenced that non-infectious systemic inflammation may lead to a pattern of augmented cytokines, particularly adipokines, which impairs survival after subsequent CLP. Therefore, the profile of cytokines existing before the infectious insult appears more crucial than that resulting from the condition for the outcome of sepsis.</p></div

Generation of rat anti-hamster xenoantibodies and their impact on sepsis survival after CLP.

<p>(<b><i>A</i></b>) Levels of anti-hamster IgM and IgG xenoantibodies in Lewis after three injections of hamster blood, with one injection administered every other day. Mean ± SEM of 5 animals per group. MFI: Mean fluorescence intensity. (<b><i>B</i></b>) Percent survival of Lewis rats with and without enhanced anti-hamster xenoantibodies submitted to CLP (n = 20 per group; *p<0.01). (<b><i>C</i></b>) Level of anti-hamster IgM and IgG xenoantibodies at baseline (day—5) and before CLP (day 0) in Lewis rats immunized with hamster blood and subsequently submitted to CLP that survived (Xeno-A; n = 9) or died (Xeno-D; n = 11) after the procedure. Values are the mean ± SEM.</p

Cytokine concentrations.

<p>Fold changes of 33 cytokines on days -5 (baseline), 0 (before CLP; black column) and 2 (two days after CLP; white column). (<b><i>A</i></b>) Control; (<b><i>B</i></b>) animals with increased anti-hamster xenoantibodies that survived CLP (Xeno-A); (<b><i>C</i></b>) animals with boosted anti-hamster xenoantibodies that died after CLP (Xeno-D). Mean ± SEM of 3 animals per group. (*p<0.05 compared to day -5;#p<0.05 compared to days -5 and 0).</p

Change in body weight.

<p>(<b><i>A</i></b>) Body weight before and after CLP (day 0) with or without enhancement of anti-hamster xenoantibodies (n = 20 per group from day -5 to 0, n = 18 in control from day 3 to 10, and n = 9 in Xeno group from day 6 to 10). (<b><i>B</i></b>) Body weight before CLP in animals with boosted anti-hamster xenoantibodies that survived (Xeno-A; n = 9) or died (Xeno-D; n = 11) after CLP. (*p<0.05 compared to control; #p<0.05 compared to Xeno-A).</p

Absolute leukocyte counts present in peripheral blood.

<p>Total leukocytes (<b><i>A</i></b>), neutrophils (<b><i>B</i></b>), lymphocytes (<b><i>C</i></b>) and monocytes (<b><i>D</i></b>), on days -5, 0 and 2 of CLP in animals with and without enhancement of anti-hamster xenoantibodies (n = 20 per group) and on days -5 and 0 in rats with boosted anti-hamster xenoantibodies that survived (Xeno-A; n = 9) or died (Xeno-D; n = 11) after CLP. (*p<0.05).</p

Clinical chemistry determinations in peripheral blood.

<p>ALT (<b><i>A</i></b>), AST (<b><i>B</i></b>), urea (<b><i>C</i></b>) and triglycerides (<b><i>D</i></b>), on days -5, 0 and 2 of CLP in animals with and without enhancement of anti-hamster xenoantibodies (n = 20 per group) and on days -5 and 0 in rats with boosted anti-hamster xenoantibodies that survived (Xeno-A; n = 9) or died (Xeno-D; n = 11) after CLP. (*p<0.05).</p

Final models of multivariable analysis adjusted by a propensity score showing differences between Body Mass Index groups.

<p>Final models of multivariable analysis adjusted by a propensity score showing differences between Body Mass Index groups.</p

Distribution of preoperative variables according Body Mass Index group.

<p>COPD = Chronic Obstructive Pulmonary Disease; NYHA = New York Heart Association classification; LVEF = Left ventricular ejection fraction; EuroSCORE = European system for cardiac operative risk evaluation. Results are expressed as mean ± standard deviation or percentage. Statistical results correspond to ANOVA <i>p</i> values. Bonferroni post hoc testing with statistical significant differences:</p><p>^A between Normal weight and overweight subgroup;</p><p>^B between Normal weight and obese subgroup;</p><p>^C between Normal weight and morbidly obese subgroup.</p><p>Distribution of preoperative variables according Body Mass Index group.</p

Kaplan-Meier survival curves comparing normal vs. overweight BMI groups.

<p>Kaplan-Meier survival curves comparing normal vs. overweight BMI groups.</p