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

MARLINA CóRDOBA MORENO; ADRIANA FONTANALS; ANDREA MAGLIOCO; DAIANA MARTIRE-GRECO; NAHUEL RODRIGUEZ-RODRIGUES; VERóNICA LANDONI; GABRIELA FERNANDEZ; MARTíN ISTURIZ; BáRBARA REARTE

Congreso; XI Congreso de la Asociación Latinoamericana de Inmunología - ALAI.; 2015

Introduction:Septic processes constitute one of the major causes of death in intensive careunits reaching a mortality rate of 30% in Europe as well as in countries suchas the US. Even though sepsis presents a simultaneous induction of both aninflammatory and anti-inflammatory response, in early phases predominates a hyper-inflammatorystate whereas during later phases an anti-inflammatory response becomespredominant. It is during this last state that more than 70% of sepsis deathsoccur due to failure in controlling pathogens associated to animmunosuppression state. In Sepsis caused by Gram-negative bacteria,endotoxins, a normal constituent of the bacterial outer wall, also known aslipopolysaccharide (LPS), has been considered one of the principal agentscausing the undesirable effects in this critical illness. The response to LPSinvolves a rapid secretion of both mediators proinflammatory as well asanti-inflammatory. Exposure of the host to repeated LPS dose induces a state ofhyporesponsiveness to subsequent simulations, in a process known as LPS orendotoxins tolerance. Although it is recognized as a protective mechanism toavoid systemic inflammation, LPS tolerance has also been suggested as the maincause of the non-specific immunosuppression described in these patients.Numerous mediators have been proposed as causative factors of immunosuppressionin sepsis such as anti-inflammatory cytokines (e.g.IL-10 and TGF-beta), as wellas glucocorticoids among others. Recently we demonstrated that glucocorticoidsplay a substantial role in the LPS induced immunosuppression in murine models.Objective: The aim of this work is to evaluate the contribution of other agentsinvolved in the induction and/or maintenance of the immunosuppression inducedby LPS in a murine model such as the anti-inflammatory cytokines IL-10 participationand 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, afterthe 200ug LPS challenge cytokines production (e.g. TNF-á and IL-10), andcorticosterone levels were analyzed. Finally, we evaluated the effect of thetreatment with exogenous glucocorticoids (e.g. dexamethasone (Dex)), before the(LPS 5ug and LPS 200ug/ mouse) LPS challenge in both mice strains. We alsoevaluated the possibility to establish an endotoxin tolerance/immunosuppressionstate in KO mice. For this, the mice were daily treated with increasing dosesof LPS. Cytokines kinetics such as TNF-á and corticosterone levels in plasmawere measured. In addition, tolerized/immunosuppressed mice were immunized withsheep red blood cells. The immune response analyzing the antibody title wasevaluated on day 7 by means of flow cytometry and hemagglutination assay.Results: Clearly the KO mice were extremely more sensitive to the LPS challengethan the WT mice. Thus, the lethal dose for the KO mice was around to 5-10ugper mouse, whereas for the WT mice was 40 times larger (i.e. about 200ug permouse) (mortality rate with LPS 10ug: wt 0/8; KO 8/8). In the WT mice a peak inIL-10 and TNF-á secretion was observed at 1.5 hours after the LPS 200uginoculation and remained elevated up to 6 hours for IL-10, while the values ofTNF-á returned to baseline levels after 3 hours . In the KO mice the productionkinetics of TNF-á it behaved completely different to that observed in WT. Notonly the secretion peak at 1.5 hours showed a significant almost 2 foldincrease difference than in the WT mice (mean ± SEM TNF-á production 1.5 hoursafter LPS (pg/ml): WT= 2505±410, KO= 3788±60; p<0.05), but also the TNF-álevel remained significantly elevated up to 6 hours after LPS stimulation (mean± SEM TNF-á production 6 hours after LPS (pg/ml): WT= 731.8±98, KO= 3566±93;p<0.05). Regarding IL-10, non-detectable levels were observed in the KOmice. The corticosterone production peaked 3 hours after the LPS 200ugchallenge. Levels were higher in the KO mice than in the WT (mean ± SEMcorticosterone levels (pg/ml): WT= 1728±273, KO=3992±1390). We further observedthat 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 andchallenged 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 refractorinessinduced by Dex correlated significantly with a low amount of TNF-á in plasma1.5 hours after the LPS challenge (mean ± SEM TNF-á production (pg/ml): WT =LPS: 1661±209, Dex+LPS:609±180; p < 0.05; KO= LPS: 2527±133,Dex+LPS:930±139; p<0.05). Due to the high sensitivity that KO mice showed toLPS, it was only possible to establish immunosuppression/tolerance when thetreatment was started with a LPS dose much lower than in normal conditions.After the 3rd day of LPS challenge, the TNF-á kinetics was almost identical inboth mice groups. The corticosterone levels were increased 3 hours after theLPS dose in both mice groups, being this most evident in KO mice (mean ± SEMcorticosterone levels (pg/ml). TOL WT= 1216±338, TOL KO=4480±2216). Evaluationsof the immune response showed that the primary response did not differ betweenWT and KO mice in basal conditions (mean ± SEM WT= 100±20%; KO 70.4±11%).Moreover, tolerized/immunosuppressed mice (IS) showed a significantly reducedresponse compared to their basals, but without differ between them (mean ±SEMWT IS = 0.4±0.2%; KO IS=0.9±0.5%.The secondary immune response on the otherhand did show differences in basal conditions. The KO mice showed an increasedantibodies title (mean ± SEM hemagglutination title: WT= 720±240, KO=5973±853p<0.01). Conclusion: The KO mice showed an augmented sensitivity to a LPSchallenge, which was reflected in the high and sustained levels of TNF-á inplasma over time. Despite this, it was possible to establish a state ofendotoxin tolerance. This suggests that IL-10 is critical in controlling thelevels of TNF-á, at least in the initial phase, most likely exerting an antiinflammatory and regulator effect of these cytokine levels induced by LPS.Furthermore, we observed that Dex treatment induced a partial protection inmice KO, suggesting that the refractory effect of Dexa could be manifestedfully only in presence of IL-10. However, the partial effect together with theincrease in levels of endogenous corticosterone both in KO basal and KOtolerant mice, propose an important role for the glucocorticoids in conditionsin which IL-10 is absent, exerting a regulatory effect on TNF-á levels, thusavoiding lethality of endotoxin. Finally, the immune response assay revealedthat the absence of IL-10 did not modified the immune status in basalconditions, nor changed the LPS induced immunosuppression state. However, theabsence of IL-10 induced a substantial difference exacerbating the secondaryimmune response in basal conditions. These results indicate that IL 10 isinvolved in early events of the encounter with endotoxin to prevent anexacerbated inflammatory response and avoided thereby death. However, ifpossible reduce the initial inflammation, even in the absence of IL-10 canestablish a tolerance and immunosuppression state similar to that observed inWT mice. In addition, the data demonstrate a relevant role of the IL-10 insecondary immune response modulation and besides, a possible relationshipbetween glucocorticoids and IL-10. These facts should be further developed inthe future.