The Abl and Arg Kinases Mediate Distinct Modes of Phagocytosis and Are 1 Required for Maximal ...

50 Pages·2012·1.69 MB·English

MCB Accepts, published online ahead of print on 4 June 2012
Mol. Cell. Biol. doi:10.1128/MCB.00086-12
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
1 The Abl and Arg Kinases Mediate Distinct Modes of Phagocytosis and Are
2 Required for Maximal Leishmania Infection
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4 Dawn M. Wetzel1,#, Diane McMahon-Pratt2, and Anthony J. Koleske3,4
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6 Departments of 1) Pediatrics, Yale School of Medicine, 2) Epidemiology of Microbial
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7 Diseases, Yale School of Public Health, and 3) Molecular Biophysics and Biochemistry o
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10 # corresponding author: Dawn M. Wetzel, Department of Pediatrics, Division of a
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11 Pediatric Infectious Diseases, 333 Cedar St; New Haven, CT 06520. Telephone 203- e
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12 785-5631. Fax [phone]. E-mail: [email protected]

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15 RUNNING TITLE: ABL/ARG MEDIATE PHAGOCYTOSIS AND LEISHMANIASIS //
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19 Abbreviations: BM = bone marrow, CR3 = complement receptor 3, dKO = double

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20 knockout, FcR = Fc receptor, IF = immunofluorescence, MФ = macrophage, min = ril
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21 minutes, PI = phagocytic index, vs = versus, WT = wild type, WTLM = wild type ,

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22 littermates. 1
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25 Leishmania, an obligate intracellular parasite, binds several receptors to trigger
26 engulfment by phagocytes, leading to cutaneous or visceral disease. These
27 receptors include complement receptor 3 (CR3), used by promastigotes, and the
28 Fc receptor (FcR), used by amastigotes. The mechanisms mediating uptake are
29 not well understood. Here we show that Abl family kinases mediate both
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30 phagocytosis and the uptake of L. amazonensis by macrophages (MФs). w
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31 Imatinib, an Abl/Arg kinase inhibitor, decreases opsonized polystyrene bead a
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32 phagocytosis and Leishmania uptake. Interestingly, phagocytosis of IgG-coated

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33 beads is decreased in Arg-deficient MФs, while that of C3bi-coated beads is m

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34 unaffected. Conversely, uptake of C3bi-coated beads is decreased in Abl- tp
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35 deficient MФs, but that of IgG-coated beads is unaffected. Consistent with these
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36 results, Abl-deficient MФs are inefficient at C3bi-opsonized promastigote uptake, a
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37 and Arg-deficient MФs are defective in IgG1-opsonized amastigote uptake. .o
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38 Finally, genetic loss of Abl or Arg reduces infection severity in murine cutaneous o
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39 leishmaniasis, and imatinib treatment results in smaller lesions with fewer
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40 parasites than controls. Our studies are the first to demonstrate that efficient 1
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41 phagocytosis and maximal Leishmania infection requires Abl family kinases. 0
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42 These results highlight Abl family kinase-mediated signaling pathways as
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43 potential therapeutic targets for leishmaniasis. u
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44 t
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45 Leishmania parasites cause cutaneous or visceral disease in 1 to 2 million
46 people a year in the developing world (17). Leishmania undergoes two life cycle
47 stages: the promastigote, found in the sand fly, and the amastigote, found in
48 mammalian hosts. When an infected sand fly bites a host, the injected promastigotes
49 must be engulfed by phagocytes to establish infection. The promastigotes then
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50 differentiate into amastigotes within the phagolysosome. If the amastigote finds itself w
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51 outside of a cell, it must be re-engulfed for continued infection (23). a
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52 Several MФ surface proteins permit Leishmania uptake. Promastigote

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53 internalization is mediated by the fibronectin receptor (integrin α5β1) (2), the mannose- m

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54 fucose receptor (63, 64), and complement receptors CR1 (10) and CR3 (38). p
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55 Promastigotes may interact directly with CR3 (49) but binding is facilitated by
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56 opsonization with C3bi, a complement component (40, 45). Both CR3 and the FcR s
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57 mediate amastigote uptake (16); interactions with the latter are facilitated by IgG o
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58 opsonization (35). The FcR subclass FcγR, which mediates IgG-mediated phagocytosis o
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59 (33), is most likely responsible for amastigote uptake by MФs. Indeed, internalization of p
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60 IgG-opsonized amastigotes via FcRγI and III sustains infection in murine cutaneous 12
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61 leishmaniasis (8, 24, 65). Adhesion of Leishmania to any of these receptors causes an 0
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62 actin-rich phagocytic cup to form and engulf the parasite (30). Our study explores the b
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63 requirement for actin regulatory proteins in efficient Leishmania internalization. u
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64 The Abl family kinases Abl and Arg translate signals from adhesion and growth
65 factor receptors into cytoskeletal structural changes (1, 43). Integrin engagement
66 stimulates Abl family kinases to interact with and phosphorylate activators of the actin-
67 polymerizing Arp2/3 complex in fibroblasts (5, 27, 28, 29, 34, 36, 61), causing dynamic
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68 cell edge protrusions resembling phagocytic intermediates. Interestingly, Abl family
69 kinases have been implicated in endocytosis (21, 58, 59), macropinocytosis (13), and
70 autophagy (66). They are also required for infectivity of viruses such as polyomavirus
71 (56) and poxviruses (46, 47). Their precise roles in phagocytosis are less clear,
72 although they are necessary for internalizing HIV (18) and bacteria such as Shigella (5),
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73 Salmonella (31) and mycobacteria (41). RNAi screens have implicated Abl family w
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74 kinases in the uptake of Pseudomonas (44) and Chlamydia (14). Imatinib, an Abl/Arg a
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75 inhibitor, decreases uptake of zymosan particles (a yeast cell wall derivative) (11).

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76 However, roles for Abl and Arg in the uptake of Leishmania or other parasites by MФs m

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77 have not been explored. tp
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78 We report that Abl and Arg play complementary, nonredundant roles in the
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79 phagocytosis of opsonized beads and Leishmania. Genetic loss of Abl function a
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80 significantly reduces complement-mediated phagocytosis, while Arg activity is required .o
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81 for efficient immunoglobulin-mediated phagocytosis. Consistent with this result, Abl o
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82 permits uptake of complement-opsonized L. amazonensis promastigotes, likely through
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83 CR3, while Arg mediates uptake of IgG-opsonized amastigotes, most likely via FcγRIII. il 1
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84 In addition, using imatinib or mice lacking either Abl or Arg, we show that Abl family 0
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85 kinases facilitate infection in murine cutaneous leishmaniasis. Our results implicate b
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86 cytoskeletal-based cell invasion pathways as promising drug targets to combat u
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87 leishmaniasis. t
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89 MATERIALS AND METHODS
90 Mice. C57BL/6 mice were purchased from Jackson Labs (Bar Harbor, ME). Abl
91 and Arg knockout mice (abl–/– and arg–/–) are in a mixed C57BL/6 X 129Sv/J genetic
92 background as described previously (25). Mice lacking Abl in hematopoetic and
93 endothelial cells, some in an arg–/–background (dKO), were generated by crossing mice
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94 carrying floxed Abl alleles (36) with mice carrying the Tie2-Cre allele (26) (generously w
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95 provided by Richard Flavell, Yale Immunobiology). The arg–/– and ablflox/flox/Tie2Cre+ a
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96 mice used in in vivo infection experiments (as well as dKO mice) were all backcrossed
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97 to C57BL/6 at least 4 times during generation. All experiments, and particularly the in

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98 vivo infection experiments using genetically manipulated mice, were performed with wild p
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99 type littermates (indicated as WTLM) to control for genetic background. The
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100 Institutional Animal Care and Use Committee at Yale University approved all
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101 experimental protocols. o
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102 Cell culture. For opsonized bead experiments using imatinib and CR3/FcR o
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103 blocking experiments, RAW 264.7 cells were grown in DMEM with 10% heat- p
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104 inactivated, endotoxin-free FBS (Invitrogen, Grand Island, NY). For other experiments, 1
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105 cells were harvested from the tibias and femurs of WTLM, abl–/–, arg–/–, and arg–/– 0
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106 ablflox/flox/Tie2Cre+ mice according to standard protocols (16) and differentiated into b
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107 bone marrow-derived (BM) primary MФs (WT/WTLM, abl–/–, arg–/–, or dKO) over 7 days u
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108 by growing them in DMEM/10% FBS/20% supernatant from L929 cells. Differentiation t
109 into BM MФs was confirmed by FACS using antibodies to F4/80 and C11b
110 (eBioscience, San Diego, CA); over 98% of differentiated cells were positive for both
111 markers.
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112 Parasite culture. L. amazonensis promastigotes (strain
113 MHOM/BR/767/LTB0016) were grown at 24°C in Schneider's Drosophila medium
114 supplemented with 15% heat-inactivated, endotoxin-free FBS and 10 mg/ml gentamicin
115 (24). For MФ invasion, promastigotes were incubated at stationary phase for 7 days to
116 maximize infective metacyclic promastigotes, which we defined as those isolated after
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117 purification at 3000 X g for 30 minutes through a step Percoll gradient (Sigma, St Louis, w
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118 MO). For experiments with L. amazonensis amastigotes, strain IFLA/BR/67/PH8 (kindly a
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119 provided by Norma Andrews, U Maryland), was grown axenically at 32°C in M199

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120 (Invitrogen) at pH 4.5 supplemented with 20% FBS, 1% penicillin-streptomycin, 0.1% m

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121 hemin (25mg/ml in 50% triethanolamine), 10 mM adenine, 5 mM L-glutamine, 0.25% tp
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122 glucose, 0.5% trypticase, and 40 mM sodium succinate (20). Where indicated,
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123 promastigotes and amastigotes were incubated in media containing DMSO or 3.3 μM a
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124 imatinib (LC Laboratories, Woburn, MA) to monitor for effects on parasite growth. Both o
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125 strains were passaged through mice to maintain virulence. o
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126 Immunoblotting. WT, abl–/–, arg–/–, or dKO MФs, were lysed in RIPA buffer (50 p
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127 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X100) (27) 1
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128 and protein concentration was assessed using BCA (Thermo Scientific, Waltham, MA). 0
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129 Lysates were run on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. b
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130 Abl was visualized with mouse anti 24-11 (Santa Cruz, Santa Cruz CA) at 1:1000. Arg u
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131 was visualized with a rabbit polyclonal antibody, as previously described (25) at 1:250. t
132 Immunoblotting with mouse anti-actin (Millipore, Billerica, MA) at 1:10,000 was
133 performed as a loading control.
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134 To measure relative Abl or Arg activation after receptor engagement, phorbol 12-
135 myristate 13-acetate (PMA, Sigma)-activated, M-CSF-starved WT, abl–/–, arg–/–, or dKO
136 MФs were added to dishes that were coated with C3bi or rabbit IgG1 (Sigma) for 15
137 minutes, and lysed with a buffer of 20mM Tris, 150 mM NaCl, 2 mM EDTA, 1% Triton X-
138 100 that contained protease and phosphatase inhibitors (61). Lysates of WT MФs
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139 allowed to spread on uncoated dishes were run to confirm that Crk was phosphorylated w
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140 in response to CR3 or FcR engagement. Equivalent micrograms of protein were loaded a
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141 onto 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes

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142 were probed with anti-phosphorylated Crk (Cell Signaling, Beverly, MA) at 1:1500, then m

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143 stripped and reprobed for Crk (Santa Cruz) at 1:500 to confirm protein levels. Relative tp
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144 levels of p-Crk, normalized to Crk, were compared among samples using Image J
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145 analysis software. Each experiment was performed 5 times. Statistical significance a
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146 was assessed with one-way ANOVAs. .o
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147 Phagocytosis assays. Primary MФs or RAW 264.7 cells, as indicated, were o
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148 plated at 2 X 105 - 5 X 105 cells per coverslip and incubated overnight in M-CSF starved p
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149 media or serum-free media, respectively. Experiments were performed at ~50% 1
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150 confluence. For drug experiments, MФs were preincubated in 3.3 μM imatinib or DMSO 20
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151 (Sigma) for 2 hours. Where indicated, RAW cells were incubated in M1/70 supernatant b
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152 (rat isotype IgG2b, Developmental Studies Hybridoma Bank, U. Iowa) at 1:10 for 20 u
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153 minutes to block CR3 or F16/32 (also rat IgG2b, eBioscience) at 1:50 to block FcγRII t
154 and III. An irrelevant antibody of the same isotype also did not affect uptake. For C3bi
155 experiments, 2 μM latex yellow-green beads (Sigma) were coated with human IgM
156 (Sigma) at 1:100 for 1 hour at 37°C, incubated in freshly isolated mouse serum diluted
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157 1:1 in PBS for 1 hour at 37°C and rinsed. For IgG experiments, beads were incubated
158 with rabbit IgG (Sigma) diluted 1:250 for 1 hour at 37°C and rinsed. Prior to C3bi bead
159 uptake, MФs were activated with PMA (Sigma) for 1 hour at 37°C. MФs were incubated
160 with a ratio of 10-15 particles per cell for 30 minutes at 37°C. Uptake was compared
161 between opsonized and unopsonized beads to monitor for the effectiveness of
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162 opsonization. Opsonization was confirmed by FACS analysis using a goat polyclonal w
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163 antibody to C3b (Pierce, Rockford, IL) and an A568-conjugated donkey-anti goat a
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164 secondary antibody (Invitrogen) or an A488-conjugated goat-anti rabbit secondary

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165 antibody for IgG (Invitrogen). After uptake, coverslips were rinsed and fixed with 3% m

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166 formaldehyde for 15 minutes. Coverslips were then blocked with 2% BSA without tp
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167 permeabilization. Coverslips with C3bi-coated beads were incubated with rabbit anti-
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168 human IgM (Sigma) at 1:250. All coverslips were labeled with A594-goat-anti-rabbit a
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169 secondary antibody (Invitrogen) at 1:250. Coverslips were permeabilized with 0.25% .o
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170 Triton for 10 minutes, blocked with 2% BSA, and stained with DAPI (Invitrogen). This o
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171 process, termed a two-color immunofluorescence assay (62), allowed us to distinguish
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172 the beads that were internalized from those that remained outside cells. Fluorescence 1
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173 was visualized on a Nikon Eclipse TE2000-5 inverted fluorescence microscope with a 0
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174 40X Nikon objective (1.0 aperture) by an observer blinded to experimental treatment. At
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175 least 10 randomly-selected fields were visualized for a total of at least 100 MФs and u
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176 over 100 beads per experimental determination. The mean phagocytic index, the t
177 number of particles internalized per 100 cells, was calculated for each experiment. The
178 phagocytic index for control cells was taken as the maximum (100%) value for each
179 experiment; the phagocytic index for the Abl kinase-deficient cells was determined and
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180 compared to the control. Means + standard errors were calculated, and each
181 experiment was performed at least three times. One-sample Student's t-tests were
182 performed to assess statistical significance. The representative images shown in
183 Figures 1 & 2 were taken with a Qimaging cooled CCD camera (mono, 12 bit) at room
184 temperature, acquired with Nikon Imaging software, and processed in Adobe
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185 Photoshop. w
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186 Zymosan particles (Sigma) were used to determine kinetics of the effect of a
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187 imatinib treatment. Primary MФs were incubated with 3.3 μm imatinib or DMSO, then d
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188 incubated with Oregon green-labeled zymosan at a ratio of 5 particles: MФ for up to 3

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189 hours prior to fixation and processing for two color IF. Fixed coverslips were incubated p
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190 with rabbit-anti zymosan (Sigma) and A594-goat-anti-rabbit secondary antibody
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191 (Invitrogen), both at 1:250 in PBS, prior to visualization and quantification of the PI. s
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192 For imaging and quantification of phagocytic cups in WT, abl–/–, arg–/–, or dKO o
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193 MФs, 4 μM Far-red beads (Bangs laboratories, Fishers, IL) were opsonized as above, o
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194 then added to cells for 10 minutes. Cells were fixed and phagocytic cups were labeled p
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195 with Oregon Green phalloidin (1:100, Invitrogen). For the Z-stack merges shown, 2
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196 phagocytic cups were imaged with a Nikon Ti-E Eclipse inverted spinning-disk confocal 0
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197 microscope with a 1 μm step size and photographed with a 14 bit Hamamatsu (C9100- b
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198 50) electron-multiplied, charge-coupled device (EMCCD). Images were converted to u
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199 TIFF format and the middle three layers of the phagocytic cup were merged using
200 ImageJ. For quantification, fifty cups per category from multiple experiments were
201 measured by an observer blinded to genotype using ImageJ and categories were
202 compared using one way ANOVA. Relative intensity of phagocytic cups was also
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203 measured to control for differences in cell thickness and did not decrease in knockout
204 macrophages.
205 Leishmania uptake assays: Primary mouse bone marrow-derived MФs were
206 employed for all assays except for phagocytic receptor blocking experiments, which
207 employed RAW cells. For imatinib assays, MФs were incubated with 3.3 μM imatinib for
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208 2 hours prior to the assay. For C3bi opsonization experiments, metacyclic
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209 promastigotes were incubated with freshly isolated mouse serum for 1 hour at 37°C, a
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210 and MФs were activated with PMA for 1 hour prior to experiments. Amastigotes were
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211 opsonized with anti-P8-proteoglycolipid complex (monoclonal antibody IgG1 (42)) for 1

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212 hour at 37°C. Opsonization was confirmed as above. Where indicated, RAW cells p
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213 were incubated in M1/70 supernatant (Developmental Studies Hybridoma Bank) at 1:10
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214 to block CR3 or F16/32 (eBioscience) at 1:50 to block FcγRII and III. Coverslips as
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215 containing primary MФs or RAW cells were incubated with metacyclic promastigotes at o
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216 a ratio of 10:1 or with amastigotes at a ratio of 2:1 for 90 minutes (unopsonized) or for o
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217 20 minutes (opsonized), unless otherwise indicated, and washed prior to fixation with p
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218 3% paraformaldehyde. External promastigotes were labeled with mouse anti-gp46 (48), 2
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219 while external amastigotes were labeled with mouse anti-P8. All coverslips were 0
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220 labeled with A594 goat anti-mouse secondary antibody (Invitrogen). After b
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221 permeabilization, promastigotes were relabeled with mouse anti-gp46, and amastigotes u
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222 were relabeled with mouse anti-P8 and A488 goat anti-mouse secondary antibody
223 (Invitrogen); coverslips were co-stained with DAPI. Uptake at 20 minutes was
224 compared between opsonized and unopsonized parasites to monitor opsonization
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