Evaluation of 3-(3-chloro-phenyl)-5-(4-pyridyl)-4,5-dihydroisoxazole as a Novel Anti-Inflammatory Drug Candidate

BACKGROUND: 3-(3-chloro-phenyl)-5-(4-pyridyl)-4,5-dihydroisoxazole (DIC) is a five-membered heterocyclic compound containing a N-O bond. The anti-inflammatory effects of this compound were studied both in vitro and in vivo. PRINCIPAL FINDINGS: DIC effectively decreased TNF-α and IL-6 release from LPS-stimulated macrophages in a dose dependent manner. DIC diminished the levels of COX-2 with subsequent inhibition of PGE(2) production. DIC also compromised HMGB1 translocation from the nucleus to the cytoplasm. Moreover, DIC prevented the nuclear translocation of NF-κB and inhibited the MAPK pathway. In vivo, DIC inhibited migration of neutrophils to the peritoneal cavity of mice. CONCLUSIONS: This study presents the potential utilization of a synthetic compound, as a lead for the development of novel anti-inflammatory drugs

Downregulation of Microparticle Release and Pro-Inflammatory Properties of Activated Human Polymorphonuclear Neutrophils by LMW Fucoidan

International audienceExposition of neutrophils (polymorphonuclear neutrophils, PMNs) to bacterial products triggers exacerbated activation of these cells, increasing their harmful effects on host tissues. We evaluated the possibility of interfering with the classic immune innate responses of human PMNs exposed to bacterial endotoxin (lipopolysaccharide, LPS), and further stimulated with bacterial formyl peptide (N-formyl-methionine-leucine-phenylalanine, fMLP). We showed that the low- molecular-weight fucoidan (LMW-Fuc), a polysaccharide extracted from brown algae, attenuated the exacerbated activation induced by fMLP on LPS-primed PMNs, in vitro, impairing chemotaxis, NET formation, and the pro-survival and pro-oxidative effects. LMW-Fuc also inhibited the activation of canonical signaling pathways, AKT, bad, p47phox and MLC, activated by the exposition of PMN to bacterial products. The activation of PMN by sequential exposure to LPS and fMLP induced the release of L-selectin+ microparticles, which were able to trigger extracellular reactive oxygen species production by fresh PMNs and macrophages. Furthermore, we observed that LMW-Fuc inhibited microparticle release from activated PMN. In vivo experiments showed that circulating PMN-derived microparticles could be detected in mice exposed to bacterial products (LPS/fMLP), being downregulated in animals treated with LMW-Fuc. The data highlight the autocrine and paracrine role of pro-inflammatory microparticles derived from activated PMN and demonstrate the anti-inflammatory effects of LMW-Fuc on these cells

Murine IL-17+ Vγ4 T lymphocytes accumulate in the lungs and play a protective role during severe sepsis

Submitted by sandra infurna ([email protected]) on 2016-04-18T16:50:53Z No. of bitstreams: 1 richard_valente_etal_IOC_2015.pdf: 1511614 bytes, checksum: 53c0a3196037eb84c301fc62a641a380 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-04-18T17:07:27Z (GMT) No. of bitstreams: 1 richard_valente_etal_IOC_2015.pdf: 1511614 bytes, checksum: 53c0a3196037eb84c301fc62a641a380 (MD5)Made available in DSpace on 2016-04-18T17:07:27Z (GMT). No. of bitstreams: 1 richard_valente_etal_IOC_2015.pdf: 1511614 bytes, checksum: 53c0a3196037eb84c301fc62a641a380 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Farmanguinhos. Departamento de Farmacologia. Laboratório de Farmacologia Aplicada.. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Nacional de Ciência e Tecnologia de Inovação em Doenças Negligenciadas (INCT-IDN). Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Departamento de Farmacologia. Laboratório de Farmacologia Aplicada. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Departamento de Farmacologia. Laboratório de Farmacologia Aplicada. Rio de Janeiro, RJ, Brasil / Mount Sinai School of Medicine. New York City, NY, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.Faculdade de Medicina de Petrópolis. Laboratório de Imunologia. Petrópolis, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Departamento de Farmacologia. Laboratório de Farmacologia Aplicada. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Nacional de Ciência e Tecnologia de Inovação em Doenças Negligenciadas (INCT-IDN). Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Ciências Biomédicas. Laboratório de Inflamação, Estresse Oxidativo e Câncer. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Departamento de Farmacologia. Laboratório de Farmacologia Aplicada. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Nacional de Ciência e Tecnologia de Inovação em Doenças Negligenciadas (INCT-IDN). Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.Background: Lung inflammation is a major consequence of the systemic inflammatory response caused by severe sepsis. Increased migration of γδ T lymphocytes into the lungs has been previously demonstrated during experimental sepsis; however, the involvement of the γδ T cell subtype Vγ4 has not been previously described. Methods: Severe sepsis was induced by cecal ligation and puncture (CLP; 9 punctures, 21G needle) in male C57BL/ 6 mice. γδ and Vγ4 T lymphocyte depletion was performed by 3A10 and UC3-10A6 mAb i.p. administration, respectively. Lung infiltrating T lymphocytes, IL-17 production and mortality rate were evaluated. Results: Severe sepsis induced by CLP in C57BL/6 mice led to an intense lung inflammatory response, marked by the accumulation of γδ T lymphocytes (comprising the Vγ4 subtype). γδ T lymphocytes present in the lungs of CLP mice were likely to be originated from peripheral lymphoid organs and migrated towards CCL2, CCL3 and CCL5, which were highly produced in response to CLP-induced sepsis. Increased expression of CD25 by Vγ4 T lymphocytes was observed in spleen earlier than that by αβ T cells, suggesting the early activation of Vγ4 T cells. The Vγ4 T lymphocyte subset predominated among the IL-17+ cell populations present in the lungs of CLP mice (unlike Vγ1 and αβ T lymphocytes) and was strongly biased toward IL-17 rather than toward IFN-γ production. Accordingly, the in vivo administration of anti-Vγ4 mAb abrogated CLP-induced IL-17 production in mouse lungs. Furthermore, anti-Vγ4 mAb treatment accelerated mortality rate in severe septic mice, demonstrating that Vγ4 T lymphocyte play a beneficial role in host defense. Conclusions: Overall, our findings provide evidence that early-activated Vγ4 T lymphocytes are the main responsible cells for IL-17 production in inflamed lungs during the course of sepsis and delay mortality of septic mice

Effect of DIC on LPS-induced TNF-α and IL-6 production.

<p><b>A</b> and <b>B</b>, following pretreatment with Polymyxin B (Pol B, 15 µg/mL), vehicle (DMSO 0.25%) or DIC (10−200 µM) for 2 h, the cells were treated with LPS (100 ng/mL) for 4 h (A) or 24 h (B). Negative control (CTRL −): cell medium only; Positive control (CTRL +): cells stimulated with LPS, only. TNF-α and IL-6 levels were assayed by ELISA. Values represent means ± SD of three independent experiments. NS, non-significant <i>vs</i> CTRL +; * p<0.05 <i>vs</i> vehicle; ** non-significant <i>vs</i> vehicle. Significances between treated groups were determined using unpaired t-test.</p

Effect of DIC on LPS-induced PGE₂ production.

<p>(<b>A</b>) RAW 264.7 macrophages were pretreated with DIC 200 µM for 2 h prior to addition of LPS (1 µg/mL) for 24 h and then PGE₂ levels were determined by EIA. The values shown are means ± SD of three independent experiments. NS, non-significant <i>vs</i> CTRL+; * p<0.05 <i>vs</i> vehicle; ** non-significant <i>vs</i> vehicle. Significances between treated groups were determined using unpaired t-test. (<b>B</b>) Protein levels of COX-2 were determined by western blot analysis of cellular protein extract (upper panel). A representative immunoblot out of three independent experiments were shown.</p

Chemical structure of 3-(3-chloro-phenyl)-5-(4-pyridyl)-4,5-dihydroisoxazole, DIC.

<p>Chemical structure of 3-(3-chloro-phenyl)-5-(4-pyridyl)-4,5-dihydroisoxazole, DIC.</p

Effect of DIC on macrophage viability.

<p>RAW 264.7 macrophages were treated with DIC (from 10 µM to 500 µM) for 24 h. Cell viabilities were determined by LDH release (<b>A</b>) and MTT assay (<b>B</b>). Values represent means ± SD of three independent experiments. * Significant differences (p>0.05) between treated and untreated cells (250–500 µM), using unpaired t-test.</p

Effect of DIC on the MAPK pathway.

<p>RAW 264.7 macrophages were pretreated with 200 µM of DIC for 2 h prior to addition of LPS (1 µg/mL) for 15 min, and then the whole cell lysate was analyzed by western blot using antibodies against the phosphorylated (activated) and unphosphorylated MAPK. The data shown are representative of three independent experiments.</p