Effect of anti-inflammatory dendrimers on the pathogenesis of multiple sclerosis

Abstract

Inflammation is required for efficient clearance of infections and repair of injured tissue. However, dysregulated inflammation contributes to the pathogenesis of major peripheral and CNS diseases. Multiple sclerosis (MS), one of the most common neurological disorders in young adults, is an autoimmune disease affecting the CNS with an important inflammatory component that leads to demyelination and diffuse neurodegeneration. During the last years, a significant number of new treatments have been approved by the regulatory agencies. However, none of them is able to reverse the disease progression. They have limited long-term effectiveness and produce significant side-effects. Therefore, new therapies are required for treating MS and those that have an inflammatory component in their pathophysiology. Currently, one of the most active research fields is the quest for new treatments is nanomedicine. Within this field, dendrimers and, within them, phosphorous dendrimers are one of the most promising nanoparticles for biomedical applications. Their stability, multifunctionality and possibility to be synthesized under GMP conditions make them suitable candidates to be studied in different pathologies including autoimmune diseases such as MS and rheumatoid arthritis. This doctoral thesis focuses on the study of the anti-inflammatory properties of two new neutral phosphorous dendrimers (G3bis and G4bis) on MS pathogenesis. G3bis and G4bis did not show toxicity on astrocytes, cortical neurons and peritoneal macrophages in mice and neither on human monocytes under the conditions studied. However, both dendrimers reduced the proliferation of mouse thymus and spleen lymphocytes, whereas no effect on human lymphocytes was observed. In the initial phases of MS, peripheral and resident macrophages participate in the induction and development of the disease, playing a dual role in the pathogenesis depending on the phenotype: M1 population with an acute pro-inflammatory phenotype and M2 population with an anti-inflammatory role. Both phosphorous dendrimers reduced LPS-induced CD86 and iNOS expression, and decreased nitrite levels, as well as, pro-inflammatory cytokines release such as TNF-α and IL-1β from both mouse peritoneal macrophages and human monocytes. These results suggest that the dendrimers inhibited macrophage M1 polarization. Moreover, both dendrimers enhanced secretion of IL-4 and increased CD163 expression as markers for the M2 population. Taken together, these results indicate that G3bis and G4bis phosphorous dendrimers switch polarization of mouse peritoneal macrophages from a M1 pro-inflammatory phenotype to a M2 anti-inflammatory phenotype. In addition, while G3bis and G4bis produced a generalized reduction in the secretion of all cytokines studied from mouse lymphocytes, probably due to the reduction of the proliferation rate, in the case of human lymphocytes only the secretion of the main pro-inflammatory cytokines was reduced. The molecular mechanism of action beneath the anti-inflammatory effect of G3bis and G4bis seems to be related to an inhibition of the NFkB pathway activation, by preventing NFkB translocation from cytosol to nucleus. Later studies found that the dendrimers might act inhibiting the proteasome activity, and avoiding IkB degradation. After identifying the mechanism of action of the phosphorous dendrimers, we studied whether these nanoparticles also displayed an anti-inflammatory profile in two models of inflammation in vivo. On the one hand, we studied the effect of phosphorous dendrimers on a sub-chronic model of inflammation, the mouse air pouch. Our results suggested that G3bis and G4bis showed a marked anti-inflammatory activity through preventing macrophages polarization to M1 state and returning the balance M1/M2 toward a non-inflammatory situation. On the other hand, we also evaluated the effect of G3bis and G4bis on a murine model of multiple sclerosis, the EAE. The induction of EAE caused an increase in the disability score up to 2.5-3 that was prevented by both phosphorous dendrimers with a similar extension than the gold standard treatment fingolimod. Finally, we studied the pharmacokinetic and biodistribution of the phosphorous dendrimers. The results showed G3bis-8Pt followed a two-compartment model and accumulated in reticuloendothelial organs. Taking together, our findings suggest that G3 and G4 phosphorous dendrimers present a good anti-inflammatory profile that might be useful for the treatment of autoimmune and inflammatory diseases. However, it is still necessary to evaluate the long-term toxicity as well as to determine its effect on other cell types involved in the pathogenesis of those diseases.