Diazepam effects on immune cells actively involved during the development of experimental autoimmune encephalomyelitis

FERNÁNDEZ HURST, NICOLÁS; FALCÓN CRISTIAN R.; RUPIL LUCÍA L.; CERVI LAURA C.; MONFERRAN, CLARA G.; ROTH GERMAN A.

Medellín

Congreso; IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology (ALAI) - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología; 2015

Latin American Association of Immunology (ALAI)

Benzodiazepines (Bz) are classically known to be psychoactive drugs that share pharmacological properties of tranquilizers, such as anxiolytic, sedative, hypnotic, skeletal muscle relaxant and anticonvulsant effects. This action is mediated by the central benzodiazepine receptor (CBR), also known as GABAA receptor. Bz have an alternative site of binding named peripheral benzodiazepine receptor (PBR), also named Translocator protein 18 kDa (TSPO). This receptor is neither structurally related nor coupled to the GABAA receptor, and is abundantly expressed in endocrine organs, skin, heart, lung epithelium, bone narrow, liver, spleen and to a lesser extent in the central nervous system (CNS), where it is associated with glial cells. It is also expressed in immune cells and action of Bz through PBR have been related to some modulatory properties on immune system functions. On the other hand, experimental autoimmune encephalomyelitis (EAE) is an animal model that mimics many of the clinical and pathological features of the human disease, multiple sclerosis (MS). Both, MS and EAE, have innate and adaptive immune responses against CNS antigens leading to inflammatory pathology. EAE can be induced in genetically susceptible animals by a single injection of CNS antigens homogenized in an adequate adjuvant. Wistar rats develop a monophasic course of the disease (acute stage) 11-13 days post-induction (dpi) characterized by ataxia and hind limb paralysis associated with weight loss and fecal and urinary incontinence. We have previously demonstrated that rats challenged for EAE and treated with low doses (0.1 mg/kg/day) of Dz showed reduced incidence and clinical signs of the disease. Concomitantly, Dz diminished several immunological parameters of the disease such as delayed-type hypersensitivity (DTH) reaction, specific lymphocyte proliferation against the encephalitogenic myelin basic protein (MBP), anti-MBP antibody production and spinal cord histological lesions associated with the disease. However, the molecular mechanism and cells involved in the reduction of the autoreactive inflammatory responses remains unclear. The aim of the present work was to evaluate the direct effect of Dz on different immune cells that participate in EAE development. Dendritic cells (DC) are key to the beginning of antigen specific responses and the state of activation achieved by them defines the type of response that will be generated. During the development of EAE, immature DC take the encephalitogenic CNS antigens and migrate to closer lymphatic nodes to enable naïve T lymphocytes to initiate immune cell responses. We evaluated the effect of Dz on the activation of DC. DC were differentiated from bone narrow of C57BL/6 mice and treated with several concentrations of Dz (5-100 μM) in the presence or absence of lipopolysaccharide (LPS). Dz did not affect the viability of resting or stimulated DC at any dose assayed. However, it was observed a reduction in the percentage of cells expressing MHCII and CD40 when DC were stimulated with LPS in the presence of Dz. In addition, Dz also significantly reduced the production of pro-inflammatory cytokines such as IL-12, IL-6 and TNF-α (p<0.05) induced by LPS while increased the levels of IL-10, an anti-inflammatory cytokine. It was also analyzed the ability of differentially treated DC to initiate allogeneic responses. DC were incubated during 18 h with Dz (5, 10, 25 μM) in the presence or absence of LPS, washed and co-cultured with allogeneic splenocytes. The splenocytes cultured with DC treated with Dz and LPS were less capable to produce IL-17 and IFN-γ compared to those cultured with DC treated only with LPS (p<0.05). Macrophages (MΦ) have been described as one of the main cells involved in neuroinflammation at early times of EAE, being able to produce toxic mediators that affect neurons viability. Since Dz was capable of decreasing DC activation, it was expected that this benzodiazepine could also modulate MΦ maturation. First, we tested the ability of MΦ to release pro-inflammatory cytokines induced by LPS in presence of Dz. Dz and LPS treated MΦ showed a defective secretion of IL-12, TNF-α and IL-6 with respect to the LPS condition. Furthermore, MΦ incubated with Dz and LPS presented a lower inducible nitric oxide synthase (iNOS) expression and decreased nitric oxide (NO) production compared to the LPS treatment alone, suggesting a modulation in classic activation (M1). In addition, increased levels of the anti-inflammatory cytokines IL-10 and TGF-β (p<0.05) were observed when MΦ were treated with Dz. As expected, increased levels of arginase I expression were observed when MΦ were treated with Dz 5 μM, in the absence of LPS (p<0.05), suggesting that Dz may leads these cells to an alternative activation (M2) phenotype. Altogether, these results showed that Dz treatment impaired the classical maturation induced by LPS of two main innate cells (DC and MΦ) involved in the development of EAE. In addition to Dz modulation on DC and MΦ, it was also studied whether Dz was able to modulate lymphocyte proliferation and inflammatory cytokine production. For this, Wistar rats were sensitized with myelin in complete Freund?s adjuvant (CFA) to induce EAE and sacrificed during the acute phase of the disease. The mononuclear cells (MNC) were isolated from popliteal lymph nodes where the population of T lymphocytes was about 75 % of the total cells. It was evaluated the in vitro effect of Dz on cell viability, apoptosis induction, proliferation and cytokine production. Dz did not have a significant toxic nor pro-apoptotic effect at least up to 25 μM (p<0.05) on MNC, but it was observed that Dz reduces unspecific (Con A) and specific (MBP) proliferation (p<0.05) and the production of pro-inflammatory cytokines (IFN-γ and IL-17) in a dose dependent manner. In this work, we show that Dz acts as a potent immunosuppressor drug which decreases both, innate and adaptive immune responses, being an important tool to modulate immune cell functions in CNS inflammatory diseases.