Estudio de la función de SLAMF8 en macrófagos

Abstract

Macrophages (Mϕ) are innate immune cells and main sentinels responsible for defense against microorganisms. In addition to being essential in the elimination of microorganisms through phagocytosis and their microbicidal mechanisms, they are powerful immunomodulatory cells capable of producing cytokines and presenting antigens, key processes in the immune response developed by this cell type. Phagocytosis is a process initiated by pattern recognition receptors (PRRs) located in these cells, which is followed by a series of cellular events involved in the elimination of the phagocyted microorganism. These events would be the microbicidal mechanisms, which in turn are highly regulated to avoid damage to the host cell. The regulation of these mechanisms occurs through different activation pathways, which may be initiated by signal transduction mediated by the participation of other receptor molecules. The SLAM family consists of 9 membrane receptors. Initially described as adhesion molecules responsible for the immunological synapse in T and NK cells, they act as immunomodulators of diverse functions at the innate and adaptive level, control the differentiation and lineage of hematopoietic cells, and also regulate microbicidal mechanisms in Mϕ, neutrophils and dendritic cells (DC). SLAMF8 is one of the last described members of this family, its expression is induced by stimulation with Gram-negative and Gram-positive bacteria, LPS and IFNγ in neutrophils, Mϕ and DC. In addition, its expression has been described in B cells and a low percentage of T cells, with no ascribed function. Previous studies to this doctoral thesis have shown that SLAMF8 acts as a negative regulator of NADPH oxidase (NOX2) activation through PKCδ and probably on the p38 MAPK pathway in mouse Mϕ, as well as having a possible involvement in the inflammatory response by negatively regulating the migration of Mϕ, among other cells, to the inflammatory focus. Given the described role of this SLAM family member on the negative regulation of NOX2 and its possible function on the inflammatory response, we decided to analyze in depth the effect of SLAMF8 on the regulation of these mechanisms in Mϕ. First, we performed a study of NOX2 activation through direct and indirect PKC pathways in SLAMF8 mouse deficient Mϕ (SLAMF8-/-) compared with Mϕ from the wild-type strain (WT or SLAMF8+/+). For this study cells were stimulated with PMA and pure LPS, and we employed specific inhibitors for PKC-, p38- MAPK and PI3K-mediated activation pathways. The results confirmed that, in the absence of SLAMF8, the activation of NOX2 through PCK was increased in the p47phox and p40phox subunits, detecting also an increased activation of p38 and ERK1/2 MAPK. The study with inhibitors confirmed these results, and furthermore not only showed negative modulation of NOX2 by SLAMF8 through these pathways, but also that specific inhibition of PI3K equalized the production of reactive oxygen species (ROS) between samples. Moreover, with PI3K inhibitor the observed differences in NOX2 activation pathways disappeared, equalizing the level of activation between Mϕ SLAMF8-/- and WT. Furthermore, analysis on the mobilization of NOX2 cytosolic subunits and the reorganization of the actin cytoskeleton showed that, SLAMF8 also negatively regulates these processes, probably through its action on PI3K. These results indicated that SLAMF8 effects negatively regulate processes involved in the elimination of microorganisms, therefore we analyzed the consequence that this receptor could have on the progression of severe infections. For this purpose, we used an in vitro Salmonella typhimurium infectious model. The results showed that SLAMF8 was able to negatively regulate the microbicidal mechanisms of NOX2 and iNOS, and their activation pathways in Mϕ, confirming again by the use of inhibitors that, the absence of SLAMF8 affects the activation pathway on these mechanisms by PI3K. In this study, we also observed that SLAMF8 affected the modulation process of S. typhimurium on NOX2 and iNOS activation on the SCV (Salmonella-containing vacuole), analyzing the activation of the enzymes at different SCV maturation times: early, intermediate and late. In order to confirm the modification in the composition of the SCV at different stages of maturation, we analyzed the recruitment of Rab5, Rab7 and p47phox to the SCV by laser confocal microscopy. According to the results obtained, it was concluded that the absence of SLAMF8 generated a greater fusion of phagosomes with lysosomes and an altered progression of the SCV, which probably generated an impairment in the capacity of S. typhimurium to control the progression of the SCV, essential for the maintenance and proliferation of the bacteria. All these lead us to conclude that SLAMF8 is able to negatively modulate the activation of microbicidal mechanisms through its action on PI3K. We believe that the intervention negatively modulates the PKC and PI3K feedback loop, since PI3K inhibition completely eliminates the observed differences between SLAMF8-/- and WT Mϕ stimulated with bacteria, PKC agonists or pure LPS. This is consistent with the fact that Mϕ SLAMF8-/- Mϕ exhibit increased polarization and migration, mechanism also controlled via the PKC-PI3K feedback loop. Considering that some of the SLAM family receptors can modulate Mϕ activation through their association with Toll-like receptors (TLRs), the expression of TLRs involved in Salmonella clearance was analyzed. The results showed a slight significantly higher increase in the expression of tlr2 and tlr4 in the absence of SLAMF8. Although, we do not believe that this small difference is the cause of the observed phenotype, it could also contribute to it. We also analyzed the expression of Slamf9, a member of the family implicated in the elimination of this microorganism, since has been described a combined action with SLAMF8 in a model of sepsis with LPS, through modulation of the expression of TLR4. We found no differences in the expression of Slamf9 in the model of infection with S. typhimurium in vitro. On the other hand, we did observe that, in the absence of SLAMF8, the expression of the cytokine il-6 was increased in this model of infection, which indicated that SLAMF8 effectively modulates the inflammatory response in Mϕ and therefore its action is effective during infectious processes. Next, the role of SLAMF8 on the Src kinase and SHP-1 phosphatase activation pathway was analyzed due to its involvement in NOX2 activation processes and intervention in the activation mechanisms for the elimination of microorganisms. The results showed a greater activation of Src kinases and a detriment in the activation of SHP-1 phosphatase in the absence of SLAMF8, under the model of infection with S. typhimurium in vitro. It was observed that IFNγ treatment in SLAMF8-/- Mϕ already increases Src activation, and furthermore, that under S. typhimurium stimulation further increased Src kinase activation accompanied by a detriment in SHP-1. This fact may be due to the increase in ROS production described as an activator of Src and inhibitor of SHP1. Hence, in conclusion, SLAMF8 also modulates the priming or initiation of Mϕ activation, since we observed that treatment with LPS or IFNγ already shows differences in their activation. The role of SLAMF8 on NOX2 and iNOS microbicidal mechanisms activation in mouse Mϕ was confirmed by murine SLAMF8 overexpression experiments in the RAW264.7 cell line, in which a phenotype opposite to that observed in SLAMF8 deficient Mϕ was obtained. Subsequently, confirmation that SLAMF8 acts as regulator of microbicidal mechanisms was obtained by studying SLAMF8 on the microbicidal capacity of S. typhimurium in vivo. The results indicated that SLAMF8-deficient mice had a higher microbicidal capacity than WT mice, and that in the absence of SLAMF8 there is a reduction in the proliferative capacity of S. typhimurium. These results are probably produced by the greater induction of iNOS and NOX2 detected in SLAMF8-/- mice, which consequently, would generate less phagosome acidification, as already observed in SLAMF8-/- Mϕ, which would prevent accelerated acidification of the SCV, an effect used by the bacterium for its replication. Finally, given its function in mouse Mϕ, the role in human Mϕ was analyzed. First, it was determined by human SLAMF8 overexpression experiments in the THP-1 cell line that the effect of SLAMF8 on the regulation of NOX2 activation in human monocytes was similar to that observed in mouse Mϕ. Second, given the plasticity of functions associated with Mϕ, SLAMF8 expression was analyzed in human Mϕ M1 and M2 subtypes. The results showed that SLAMF8 was expressed in both Mϕ subtypes, possibly indicative of a possible involvement of SLAMF8 in the modulation of pro- and anti-inflammatory responses. In summary, this study performs an in-depth analysis of the role of SLAMF8 in Mϕ functionality. Thanks to the set of results obtained, it is confirmed that SLAMF8 is able to negatively regulate activation processes in vitro, through its negative regulating action on PI3K in the PKC and PI3K feedback loop. Furthermore, our results indicate that SLAMF8 is capable of negatively modulating microbicidal mechanisms both in vitro and in vivo, and that its function in human may be similar to that found in mouse. This raises the possibility of its use as a therapeutic target through its intervention for the treatment of severe or unresolved inflammatory states.