The IL-10 participation in the induced-endotoxins bacterial tolerance/immunosuppression

CORDOBA MORENO M; FONTANALS A; MAGLIOCO A; MARTIRE-GRECO D; RODRIGUEZ-RODRIGUES N; LANDONI V.I; FERNÁNDEZ G C; ISTURIZ M.A; REARTE B.

Medellin

Congreso; Immunocolombia; 2015

ALAI

Introduction: Septic processes constitute one of the major causes of death in intensive care units reaching a mortality rate of 30% in Europe as well as in countries such as the US. Even though sepsis presents a simultaneous induction of both an inflammatory and anti-inflammatory response, in early phases predominates a hyper-inflammatory state whereas during later phases an anti-inflammatory response becomes predominant. It is during this last state that more than 70% of sepsis deaths occur due to failure in controlling pathogens associated to an immunosuppression state. In Sepsis caused by Gram-negative bacteria, endotoxins, a normal constituent of the bacterial outer wall, also known as lipopolysaccharide (LPS), has been considered one of the principal agents causing the undesirable effects in this critical illness. The response to LPS involves a rapid secretion of both mediators proinflammatory as well as anti-inflammatory. Exposure of the host to repeated LPS dose induces a state of hyporesponsiveness to subsequent simulations, in a process known as LPS or endotoxins tolerance. Although it is recognized as a protective mechanism to avoid systemic inflammation, LPS tolerance has also been suggested as the main cause of the non-specific immunosuppression described in these patients. Numerous mediators have been proposed as causative factors of immunosuppression in sepsis such as anti-inflammatory cytokines (e.g.IL-10 and TGF-beta), as well as glucocorticoids among others. Recently we demonstrated that glucocorticoids play a substantial role in the LPS induced immunosuppression in murine models. Objective: The aim of this work is to evaluate the contribution of other agents involved in the induction and/or maintenance of the immunosuppression induced by LPS in a murine model such as the anti-inflammatory cytokines IL-10 participation and its potential relationship with glucocorticoids. Methods: Wild type (WT) and IL-10 deficient (KO) BALB/c mice were treated with different LPS doses (1, 5, 10 and 200ug/mouse) to assess their sensibility to endotoxin. Also, after the 200ug LPS challenge cytokines production (e.g. TNF-α and IL-10), and corticosterone levels were analyzed. Finally, we evaluated the effect of the treatment with exogenous glucocorticoids (e.g. dexamethasone (Dex)), before the (LPS 5ug and LPS 200ug/ mouse) LPS challenge in both mice strains. We also evaluated the possibility to establish an endotoxin tolerance/immunosuppression state in KO mice. For this, the mice were daily treated with increasing doses of LPS. Cytokines kinetics such as TNF-α and corticosterone levels in plasma were measured. In addition, tolerized/immunosuppressed mice were immunized with sheep red blood cells. The immune response analyzing the antibody title was evaluated on day 7 by means of flow cytometry and hemagglutination assay. Results: Clearly the KO mice were extremely more sensitive to the LPS challenge than the WT mice. Thus, the lethal dose for the KO mice was around to 5-10ug per mouse, whereas for the WT mice was 40 times larger (i.e. about 200ug per mouse) (mortality rate with LPS 10ug: wt 0/8; KO 8/8). In the WT mice a peak in IL-10 and TNF-α secretion was observed at 1.5 hours after the LPS 200ug inoculation and remained elevated up to 6 hours for IL-10, while the values of TNF-α returned to baseline levels after 3 hours . In the KO mice the production kinetics of TNF-α it behaved completely different to that observed in WT. Not only the secretion peak at 1.5 hours showed a significant almost 2 fold increase difference than in the WT mice (mean ± SEM TNF-α production 1.5 hours after LPS (pg/ml): WT= 2505±410, KO= 3788±60; p less than 0.05), but also the TNF-α level remained significantly elevated up to 6 hours after LPS stimulation (mean ± SEM TNF-α production 6 hours after LPS (pg/ml): WT= 731.8±98, KO= 3566±93; p less than 0.05). Regarding IL-10, non-detectable levels were observed in the KO mice. The corticosterone production peaked 3 hours after the LPS 200ug challenge. Levels were higher in the KO mice than in the WT (mean ± SEM corticosterone levels (pg/ml): WT= 1728±273, KO=3992±1390). We further observed that the Dex treatment induced refractoriness to a lethal dose of LPS (200ug) in WT mice but not in KO mice (mortality rate: WT = LPS: 4/4; Dex+LPS: 0/4. KO= LPS: 4/4; Dex+LPS: 4/4). However, when KO mice were treated with Dex and challenged with a sublethal dose of LPS (5ug) they survived (mortality rate: WT = LPS: 0/4; Dex+LPS: 0/4. KO= LPS: 4/4; Dex+LPS: 0/4). This LPS refractoriness induced by Dex correlated significantly with a low amount of TNF-α in plasma 1.5 hours after the LPS challenge (mean ± SEM TNF-α production (pg/ml): WT = LPS: 1661±209, Dex+LPS:609±180; p less than 0.05; KO= LPS: 2527±133, Dex+LPS:930±139; p less than 0.05). Due to the high sensitivity that KO mice showed to LPS, it was only possible to establish immunosuppression/tolerance when the treatment was started with a LPS dose much lower than in normal conditions. After the 3rd day of LPS challenge, the TNF-α kinetics was almost identical in both mice groups. The corticosterone levels were increased 3 hours after the LPS dose in both mice groups, being this most evident in KO mice (mean ± SEM corticosterone levels (pg/ml). TOL WT= 1216±338, TOL KO=4480±2216). Evaluations of the immune response showed that the primary response did not differ between WT and KO mice in basal conditions (mean ± SEM WT= 100±20%; KO 70.4±11%). Moreover, tolerized/immunosuppressed mice (IS) showed a significantly reduced response compared to their basals, but without differ between them (mean ±SEM WT IS = 0.4±0.2%; KO IS=0.9±0.5%.The secondary immune response on the other hand did show differences in basal conditions. The KO mice showed an increased antibodies title (mean ± SEM hemagglutination title: WT= 720±240, KO=5973±853 p less than 0.01). Conclusion: The KO mice showed an augmented sensitivity to a LPS challenge, which was reflected in the high and sustained levels of TNF-α in plasma over time. Despite this, it was possible to establish a state of endotoxin tolerance. This suggests that IL-10 is critical in controlling the levels of TNF-α, at least in the initial phase, most likely exerting an anti inflammatory and regulator effect of these cytokine levels induced by LPS. Furthermore, we observed that Dex treatment induced a partial protection in mice KO, suggesting that the refractory effect of Dexa could be manifested fully only in presence of IL-10. However, the partial effect together with the increase in levels of endogenous corticosterone both in KO basal and KO tolerant mice, propose an important role for the glucocorticoids in conditions in which IL-10 is absent, exerting a regulatory effect on TNF-α levels, thus avoiding lethality of endotoxin. Finally, the immune response assay revealed that the absence of IL-10 did not modified the immune status in basal conditions, nor changed the LPS induced immunosuppression state. However, the absence of IL-10 induced a substantial difference exacerbating the secondary immune response in basal conditions. These results indicate that IL 10 is involved in early events of the encounter with endotoxin to prevent an exacerbated inflammatory response and avoided thereby death. However, if possible reduce the initial inflammation, even in the absence of IL-10 can establish a tolerance and immunosuppression state similar to that observed in WT mice. In addition, the data demonstrate a relevant role of the IL-10 in secondary immune response modulation and besides, a possible relationship between glucocorticoids and IL-10. These facts should be further developed in the future.