The Abl and Arg Kinases Mediate Distinct Modes of Phagocytosis and Are Required for Maximal Leishmania Infection

Abstract

Leishmania, an obligate intracellular parasite, binds several receptors to trigger engulfment by phagocytes, leading to cutaneous or visceral disease. These receptors include complement receptor 3 (CR3), used by promastigotes, and the Fc receptor (FcR), used by amastigotes. The mechanisms mediating uptake are not well understood. Here we show that Abl family kinases mediate both phagocytosis and the uptake of Leishmania amazonensis by macrophages (Ms). Imatinib, an Abl/Arg kinase inhibitor, decreases opsonized polystyrene bead phagocytosis and Leishmania uptake. Interestingly, phagocytosis of IgG-coated beads is decreased in Arg-deficient Ms, while that of C3bi-coated beads is unaffected. Conversely, uptake of C3bi-coated beads is decreased in Abl-deficient Ms, but that of IgG-coated beads is unaffected. Consistent with these results, Abl-deficient Ms are inefficient at C3bi-opsonized promastigote uptake, and Arg-deficient Ms are defective in IgG1-opsonized amastigote uptake. Finally, genetic loss of Abl or Arg reduces infection severity in murine cutaneous leishmaniasis, and imatinib treatment results in smaller lesions with fewer parasites than in controls. Our studies are the first to demonstrate that efficient phagocytosis and maximal Leishmania infection require Abl family kinases. These results highlight Abl family kinase-mediated signaling pathways as potential therapeutic targets for leishmaniasis. L eishmania parasites cause cutaneous or visceral disease in 1 to 2 million people a year in the developing world (17). Leishmania undergoes two life cycle stages: (i) the promastigote, found in the sand fly, and (ii) the amastigote, found in mammalian hosts. When an infected sand fly bites a host, the injected promastigotes must be engulfed by phagocytes to establish infection. The promastigotes then differentiate into amastigotes within the phagolysosome. If the amastigote finds itself outside a cell, it must be reengulfed for continued infection (23).Several M surface proteins permit Leishmania uptake. Promastigote internalization is mediated by the fibronectin receptor (integrin ␣5␤1) (2), the mannose-fucose receptor (63, 64), and complement receptors CR1 (10) and CR3 (38). Promastigotes may interact directly with CR3 (49), but binding is facilitated by opsonization with C3bi, a complement component (22,37,40,45). Both CR3 and the Fc receptor (FcR) mediate amastigote uptake (16); interactions with the latter are facilitated by IgG opsonization (35). The FcR subclass Fc␥R, which mediates IgG-mediated phagocytosis (33), is most likely responsible for amastigote uptake by Ms. Indeed, internalization of IgG-opsonized amastigotes via FcR␥I and -III sustains infection in murine cutaneous leishmaniasis (8,24,65). Adhesion of Leishmania to any of these receptors causes an actin-rich phagocytic cup to form and engulf the parasite (30). Our study explores the requirement for actin regulatory proteins in efficient Leishmania internalization.The Abl family kinases Abl and Arg translate signals from adhesion and growth...

Multifunctional Abl kinases in health and disease

“…Consistent with a role for Abl kinases in membrane trafficking events, Abl kinases were recently shown to control the inward trafficking of caveolin-1, in part, through regulation of actin stress fibers by diaphanous-1 (DIAPH1), a member of the formin family of actin regulatory proteins (Echarri et al, 2012) (see poster). Further, Abl kinases have been shown to regulate phagocytosis of antibodycoated pathogens in macrophages (Greuber and Pendergast, 2012;Wetzel et al, 2012) (see poster). Bone marrow-derived macrophages from mice that lack both Abl1 and Abl2 display impaired phagocytosis, and treatment of macrophages with pharmacological inhibitors of the Abl kinases impairs FcγR-mediated phagocytosis in part by decreased Abl-mediated phosphorylation of the tyrosineprotein kinase Syk (Greuber and Pendergast, 2012).…”

“…Bone marrow-derived macrophages from mice that lack both Abl1 and Abl2 display impaired phagocytosis, and treatment of macrophages with pharmacological inhibitors of the Abl kinases impairs FcγR-mediated phagocytosis in part by decreased Abl-mediated phosphorylation of the tyrosineprotein kinase Syk (Greuber and Pendergast, 2012). Inhibition of the Abl kinases also decreases phagocytosis of opsonized polystyrene beads and reduces the uptake of Leishmania amazonensis by macrophages (Wetzel et al, 2012). Several pathogens rely on the activity of the endogenous Abl kinases to enter into host cells (see Box 2).…”

“…Several pathogens rely on the activity of the endogenous Abl kinases to enter into host cells (see Box 2). Although loss of ABL1 reduces complement-mediated phagocytosis, ABL2 activity is required for immunoglobulinmediated phagocytosis (Wetzel et al, 2012). Further, Abl kinases are required for human immunodeficiency virus type 1 (HIV-1) infection by regulating virus-induced lipid mixing or hemifusion of the plasma membrane, which is required for pore formation and virus entry (Harmon et al, 2010).…”

The Src kinases Hck, Fgr, and Lyn activate Abl2/Arg to facilitate IgG-mediated phagocytosis andLeishmaniainfection

“…Abl and Arg also facilitate endocytosis (Jacob et al, 2009;Pendergast, 2006, 2007), autophagy (Yogalingam and Pendergast, 2008), viral (Reeves et al, 2005(Reeves et al, , 2011Swimm et al, 2010) and bacterial uptake (Burton et al, 2003;Elwell et al, 2008;Ly and Casanova, 2009;Napier et al, 2011), and IgG-mediated phagocytosis (Greuber and Pendergast, 2012). We have previously reported that Abl and Arg allow complementary non-redundant processes during phagocytosis and Leishmania uptake (Wetzel et al, 2012). Genetic loss of Arg prevents efficient IgG-mediated phagocytosis and amastigote uptake, whereas loss of Abl reduces C3bi-mediated phagocytosis and L. amazonensis promastigote uptake.…”

“…Genetic loss of Arg prevents efficient IgG-mediated phagocytosis and amastigote uptake, whereas loss of Abl reduces C3bi-mediated phagocytosis and L. amazonensis promastigote uptake. In addition, by using the Abl and Arg inhibitor imatinib and assessing mice lacking Abl or Arg, we have shown that Abl family kinases mediate infection in murine cutaneous leishmaniasis (Wetzel et al, 2012).…”