Efecto del vih-1 en la desregulación de los linfoctios b. Papel de dendrímeros carbosilano en la respuesta inflamatoria, como agentes transfectantes y en la polarización de macrófagos de tipo m2

Resumen

This work is divided into two main subjects: ¿the effect of HIV-1 on the deregulation of B lymphocytes¿, and ¿the role of carbosilane dendrimers in inflammatory response, as transfecting agents and, on the polarization of M2-type macrophages¿. PART I: B cells are a critical component of the adaptive immune system, due to its capacity of producing highly specific antibodies. During HIV infection, patients with AIDS could exhibit hyperimmunoglobulinemia, increased expression of cell-activation markers, depletion of memory B cells, polyclonal B-cell hyperactivity, and altered differentiation of naïve B cells that could result in impaired immunoglobulin class switch recombination (CSR), and thus production of nonspecific immunoglobulin (Ig) G, IgE and IgA antibodies. All of these processes finally provoke the exhaustion of B-cell and defective responses against opportunistic pathogens. However, little is know about the molecular mechanism responsible of the B-cell deregulation and whether it is due to a direct effect of HIV on B cells. In this study we have evaluated the effects of a direct exposition of B cells to HIV-1 particles in order to identify and describe the deregulation of B cells by HIV-1 evaluating CSR, AID protein expression, AID-related miRNA expression and Ig production in an environment free of T lymphocytes. We showed that HIV-1 particles deregulate human primary B cells, increasing their survival, proliferation, modifying their phenotype and function on cultured B-cell. Moreover, expression level of AID mRNA in human primary B cells was highly increased and its subsequent IgM/IgE; IgM/IgA and IgM/IgG class switch was detected in vitro. Finally, the results indicate that the mechanism by which HIV-1 deregulates B cells is through the BCR/SYK signaling pathway, promoting the mobilization of BCR in the membrane, which leads to the activation of JNK. In summary, in this study we have demonstrated that direct contact between HIV-1 particles and B-cell was sufficient to induce a deregulation of B-cell. The model in vitro developed in this study, which is independent of CD4 T cells and CD40L, can be useful to study the mechanisms of B-cell deregulation in the context of HIV infection. In addition, these results may highlight a possible relation between HIV-1 infection and B cells hyperactivation, loss of memory B cells or hyperglobulinemia. By all that, these results contribute to the better understanding of the general immune deregulation observed in HIV-1 patients, and allow us to lay the groundwork for development of better anti-HIV vaccines. PART II: New objectives of nanomedicine consist in developing and characterizing nanoparticles as new preventive treatment, therapeutic or diagnostic tools with the aim to improve current treatments. The major advantage of carbosilane dendrimers is based on their regular structure and skeletons and surfaces easy to modify. Moreover, carbosilane dendrimers can be used as molecules that have an effect per se in the treatment of HIV-1, in autoimmune diseases and inflammation, as well as molecules that can shuttle nucleic acids and drugs to the cell interior. We have studied the ability of cationic dendrimers 2G-NN16 and 2G-03NN24 to transfect siRNA-Nef in CD4 T lymphocytes, in the context of HIV-1. The results have shown that both dendrimers are able to form stable complexes with siRNA and protect them against RNase. Both dendrimers facilitate CD4 T lymphocytes transfection with siRNA. The 2G-03NN24 dendrimer is better transfectant than 2G-NN16. Our results indicate that 2G-03NN24 could protect its cargo better than 2G-NN16. We have also studied the effect of 5 carbosilane dendrimers (anionic dendrimer 2G-S16 and cationic dendrimers 2G-NN16, 1G-03NN12, 2G-03NN24 and 3G03NN48) on M1 macrophages. Dendrimers did not induce the release of TNF-¿, IL-12p40, CCL3, CCL4, IL-1ß and IL-6. Especially, 2G-NN16 decreased the expression of several genes implicated in the pro-inflamnmatory function of M1 macrophages, suppressed the expression of TNF-¿ and IL-12p40. These data indicate that 2G-NN16 has a slightly non-inflammatory effect, which could be beneficial in HIV therapy because local inflammation allows higher cell activation, which facilitates the HIV-1 infection. An additional effect of the 2G-03NN24 is the decreased expression of the CCR2 co-receptor which is involved in macrophage infection by HIV-1. The results obtained with different dendrimers in M1 open promising lines of research, suggesting that they could be used as safe biological agents without promoting inflammation and that they could be useful for the treatment of several medical conditions. Since most of the tumor-associated macrophages (TAM) are similar to M2, we have also studied the role of carbosilane dendrimers on M2 macrophages to determine if they are able to induce a switch on the macrophages phenotype, to evaluate the potential application of dendrimers in tumor immunotherapy. Tumor microenvironment favours the escape from immunosurveillance, promoting anti-inflammatory responses and inhibiting pro-inflammatory ones. The 2G-03NN24 dendrimer decreases the production of IL-10 by the LPS-stimulated M2 macrophages and also switches the M2 genetic phenotype to a M1 phenotype. Furthermore, this dendrimer decreases the activity of STAT3 by diminishing its phosphorylation through the PDGFR and EGFR receptors pathways. In vivo, dendrimer-treated tumors show that the TAM over-express iNOS, a protein typically expressed by the M1, that possess anti-tumor properties. In addition, other changes induced by the dendrimer would create a more favorable microenvironment within tumors as seen in preliminary in vivo studies. Results indicate that 2G-03NN24 dendrimer might be used for the therapy of tumors through its ability to suppress polarization of M2 macrophages, generally associated with tumor proliferation. These results are very encouraging showing that the 2G-03NN24 dendrimer can be a new anti-tumor compound.