Participação da via de 5-Lipoxigenase no desenvolvimento da doença do Enxerto-Versus-Hospedeiro (GVHD) em camundongos: Novas possibilidade

Abstract

Unfortunately, graft-versus-host disease (GVHD) remains the major limitation of bone marrow transplant to treat patient with malignant and non-malignant hematological diseases. The first-line treatment for GVHD are steroids such as methylprednisolone or prednisone because of the broad anti- inflammatory and lymphocytotoxic effects of these drugs. However, steroid-refractory GVHD patients are at high risk of related complications from the disease or death and there is no standard treatment strategy for these patients. Thus, new therapies are necessary. The leukotriene B4 (LTB4) is a pro- inflammatory mediator produced from arachidonic acid metabolism by 5-lipoxygenase (5-LO) catalytic action and is associated with the development of various inflammatory diseases including intestinal GVHD in humans. However, the role of LTB4 in the GVHD pathophysiology is incompletely understood. In this report we evaluated the participation of the 5-LO/LTB4 axis in pathogenesis of GVHD by transplant of 5-LO deficient leukocytes and the effect of its pharmacological inhibition by zileuton, a drug usually employed to treat asthmatic patients. To test our hypothesis that 5-LO participates to GVHD, we induced the disease in mice by the transplant of 1x107 bone marrow cells plus 3x107 splenocytes from WT or 5LO-/- mice to B6D2F1 mice. Mice that received syngeneic (B6D2F1) bone marrow cells and splenocytes not develop disease and were considered the control group. After transplant, intestinal and hepatic mRNA expression of 5-LO was evaluated by Real Time PCR. The levels of LTB4 were evaluated in the blood, liver and intestine by enzymatic immune assay. To pharmacological inhibition of 5-LO, GVHD was induced and recipient mice treated by zileuton (30mg/kg, orally, 12h/12h). Mice were monitored every 2 days for survival and GVHD clinical signs. The liver and intestine were also subjected to histopathology analysis, ELISA, NAG and flow cytometry. The leukocytes recruitment and adhesion were assessed by intravital of mesenteric venules. The chimerism, the accumulation of inflammatory cells in the lymphoid organs and the GVL reaction were evaluated by FACS analysis in the spleen and bone marrow. Although we did not observe an increase of 5-LO mRNA in the jejunum ileum and liver in mice subjected to GVHD at onset mortality, the transplant of semi-allogenic 5-LO deficient splenocytes reduced the serum and hepatic level of LTB4 and the intestinal and liver injury. These results were associated with inhibition of proinflammatory leukocytes and decreased levels of cytokines and chemokines. In addition, treatment with zileuton also effectively ammeliorates the disease by reduction of liver and serum LTB4 and diminution of the inflammatory response in the intestine and liver. Importantly, mice that received 5LO-deficient cells or zileuton-treated mice had prolonged survival and reduced GVHD clinical scores. Moreover, the transplant of 5LO-/- leukocytes or zileuton-treated mice did not interfere in the chimerism or in the GVL response. Taken together, these data provide evidence that 5-LO/LTB4 axis orchestrates the GVHD development, describing a potential therapeutic application of zileuton to GVHD treatment.