Diazepam Impairs Innate and Adaptive Immune Responses and Ameliorates Experimental Autoimmune Encephalomyelitis

Currently there is increasing attention on the modulatory effects of benzodiazepines on the immune system. Here, we evaluate how Diazepam (DZ) affects both innate and adaptive immunity. We observed that treatment with DZ and Lipopolysaccharide (LPS) on macrophages or dendritic cells (DCs) induced a defective secretion of IL-12, TNF-α, IL-6 and a lesser expression of classical activation markers as NO production and CD40 in comparison with LPS condition. More importantly, mice pre-treated with DZ and then challenged to LPS induced-septic shock showed reduced death. The DZ treatment shifted the LPS-induced pro-inflammatory cytokine production of peritoneal cells (PCs) to an anti-inflammatory profile commanded by IL-10. In agreement with this, DZ treatment prevented LPS-induced DC ability to initiate allogeneic Th1 and Th17 responses in vitro when compared with LPS-matured DC. Since these inflammatory responses are the key in the development of the experimental autoimmune encephalomyelitis (EAE), we treated EAE mice preventively with DZ. Mice that received DZ showed amelioration of clinical signs and immunological parameters of the disease. Additionally, DZ reduced the release of IFN-γ and IL-17 by splenocytes from untreated sick mice in vitro. For this reason, we decided to treat diseased mice therapeutically with DZ when they reached the clinical score of 1. Most importantly, this treatment ameliorated clinical signs, reduced the MOG-specific inflammatory cytokine production and prevented axonal damage. Altogether, these results indicate that DZ is a potent immunomodulator capable of controlling undesired innate and adaptive immune responses, both at the beginning of these responses and also once they have started.Fil: Falcón, Cristian Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Fernández Hurst, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Vivinetto, Ana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Lopez, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Zurita, Adolfo Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Gatti, Gerardo Alberto. Fundación Para El Progreso de la Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Cervi, Laura Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Monferran, Clara Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Roth, German Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

Fasciola hepatica Kunitz Type Molecule Decreases Dendritic Cell Activation and Their Ability to Induce Inflammatory Responses

Fil: Falcón, Cristian R.. Universidad Nacional de Villa María; Argentina.Fil: Masih, Diana. Universidad Nacional de Villa María; Argentina.Fil: Gatti, Gerardo. Universidad Nacional de Villa María; Argentina.Fil: Sanchez, María Cecilia. Universidad Nacional de Villa María; Argentina.Fil: Motrán, Claudia C.. Universidad Nacional de Villa María; Argentina.Fil: Cervi, Laura. Universidad Nacional de Villa María; Argentina

Fasciola hepatica Kunitz Type Molecule Decreases Dendritic Cell Activation and Their Ability to Induce Inflammatory Responses

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<i>Fasciola hepatica</i> Kunitz Type Molecule Decreases Dendritic Cell Activation and Their Ability to Induce Inflammatory Responses

<div><p>The complete repertoire of proteins with immunomodulatory activity in <i>Fasciola hepatica</i> (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L) induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite.</p></div

Reduced capacity of TE and F<10 KDa to prime allogeneic responses <i>in vitro</i>.

<p>(A) DC from BALB/c mice treated with medium, LPS (1 µg/ml), TE (80 µg/ml) or TE plus LPS for 18 h were co-cultured for 5 days with C57BL/6splenocytes at a 1∶10 ratio in the presence or absence of PMA. Allogeneic response was determined by IFN-γ production by ELISA. (B) DC from BALB/c mice, treated as described in A, without PMA, were cultured with a sorted EGFP negative cell population from splenocytes of Foxp3EGFP mice for 5 days. Dot plots show the CD4 vs CD25 profile of culture cells (upper panel) and the Foxp3 vs CD25 on the CD25 high gated cell population (lower panel). The numbers represent the percentage of Foxp3 positive and negative cells gated on the CD25 high cell population. (C) IL-10 was measured in the supernatant of allogeneic cultures as described in A in the presence of PMA for the last 18 h of culture, p<0.006 versus T/L-DC (ANOVA Test). (D) IL-27 mRNA was measured in DC treated with medium, LPS (1 µg/ml), TE (80 µg/ml) or TE plus LPS for 6 h by RT-PCR or IL-10 was detected in culture supernatant by ELISA after 18 h of culture, p<0.001 versus T/L-DC (ANOVA Test). (E). IFN-γ or IL-17 were detected in the supernatant of DC from BALB/c mice treated with medium, LPS, TE or TE plus LPS for 18 h, and cultured with splenocytes from C57BL/6 at a 1∶10 ratio for 5 days in the presence or absence of anti IL-10 R, anti-IL-27 or the control isotype Abs. Data are representative of three experiments, with p representing a significant difference in the Student's t-test. (F) IFN-γ was measured by ELISA in culture supernatants of splenocytes (C57BL/6) and DC (BALB/c mice), previously pulsed for 18 h with medium, or F<10 kDa (20 µg/ml) in the presence or absence of LPS (1 µg/ml) for 5 days. On day 4, the cells were stimulated with PMA for the last 18 h. (G) DC from BALB/c mice treated with medium or F<10 kDa (20 µg/ml) for 18 h were cultured with a sorted EGFP negative cell population from splenocytes of Foxp3EGFP in the presence or absence of anti- TGF β or anti IL-10R for 5 days. Dot plots show the Foxp3 <i>vs</i> CD25 profile of splenocytes gated on the CD4 population. (H) IFN-γ and IL-17 were detected in the supernatant of cultures as described in F, in the presence or absence of anti IL-27 or anti IL-10R blocking Abs. Data are representative of 2 experiments with similar results. The bar graphs shown one experiment representative of three with the mean ± SD. p represents a significant difference in the Student's t-test.</p

Kunitz type protease inhibitor present in FVII modulates TLR-induced DC activation (A).

<p>Elution profile of TE fractionated by Superdex G75. (B) IL-12p70, TNF and IL-6 production were detected by ELISA in the supernatants of DC cultured with medium, LPS (1 µg/mL), FVII (10 µg/ml) or FVII plus LPS (F/L) for 18 h. (C) FVII was resolved in 18% SDS-PAGE gel and stained with amido black (D) Comparison of the N-terminal sequences obtained for FVII and <i>F. hepatica</i> KTM. (E) DC were incubated with medium or rFh-KTM (20 µg/ml) in the presence or absence of LPS (1 µg/ml), and after 18 h the cytokine production was evaluated in the supernatants by ELISA. (F) IFN-γ and IL-17 were detected in the supernatant of DC from BALB/c mice treated with medium, LPS, rFh-KTM (10, 20 and 30 µg/ml) or rFh-KTM plus LPS for 18 h, and cultured with C57BL/6 splenocytes at a 1∶10 ratio for 5 days. Data are means ±SD of at least five wells and are representative results from two or three experiments. p represents a significant difference in the Student's t-test.</p