Dados do Trabalho

Título

Leishmania amazonensis infection impairs VLA-4 clustering and adhesion complex assembly at the base of J774 cells

Introdução

Cutaneous leishmaniasis is an infectious disease that can cause single or multiple disseminated cutaneous lesions. The mechanisms involved in the dissemination of Leishmania to different areas of the skin are poorly understood. Evidence suggests that Leishmania infection impairs Very Late Antigen-4 (VLA-4)-dependent phagocyte adhesion, which may be related to the dissemination mechanisms. VLA-4 is found in membrane microdomains known as lipid rafts. The disruption of these microdomains is a mechanism by which Leishmania evades the host immune response.

Objetivo (s)

We investigated the lipid raft-mediated VLA-4 mobilization through the cellular membrane, as well as integrin cluster formation with focal adhesion complex assembly at the cell base in infected cells.

Material e Métodos

We performed in vitro fluorescence assays using lineage macrophages (J774) incubated with or without Leishmania amazonensis. We used single or double immunofluorescence staining to label lipid rafts, VLA-4, talin, vinculin, and actin. Methyl-β-Cyclodextrin (MβCD) was used to investigate lipid raft-dependent mechanisms and latex beads were used for the adhesion assay to control for nonspecific effects of phagocytosis.

Resultados e Conclusão

J774 cells infected with L. amazonensis showed reduced adhesion (p=0.0030, One-way ANOVA), like cells treated with MβCD (p=0.0024, One-way ANOVA). L. amazonensis-infected macrophages and MβCD-treated cells both demonstrated decreased VLA-4 mobilization to the adhesion plane (p<0.0001, Kruskal-Wallis and p=0.0022, Kruskal-Wallis, respectively) and in integrin clustering (infected cells: p<0.0001; MβCD group: p=0.0004, Kruskal-Wallis). Lipid raft staining in infected cells did not show significant differences, but a decrease was observed in MβCD-treated cells (p=0.0303, Kruskal-Wallis). Also, infected cells showed talin depletion (p=0.0039, Mann-Whitney), as well as a decrease in the mobilization of adhesion complex proteins such as talin (p<0.0001, Mann-Whitney) and vinculin (p=0.0010, Mann-Whitney). Infected cells displayed limited cell-spreading (p=0.0007, Mann-Whitney). Our results suggest that Leishmania infection may interfere with the firm adhesion phase of the cell-spreading process, potentially contributing to the bloodstream dissemination of infected cells.

Palavras-chave

Leishmaniasis; Integrin; Focal adhesion; Cell adhesion

Agradecimentos

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