RESEARCH ARTICLE

The Src kinases Hck, Fgr and Lyn activate Arg to facilitateIgG-mediated phagocytosis and Leishmania infectionDawn M. Wetzel1,*,‡, Emma L. Rhodes2, Shaoguang Li3, Diane McMahon-Pratt4 and Anthony J. Koleske5,6

ABSTRACTLeishmaniasis is a devastating disease that disfigures or kills nearlytwo million people each year. Establishment and persistence ofinfection by the obligate intracellular parasite Leishmania requiresrepeated uptake by macrophages and other phagocytes. Therefore,preventing uptake could be a novel therapeutic strategy forleishmaniasis. Amastigotes, the life cycle stage found in thehuman host, bind Fc receptors and enter macrophages primarilythrough immunoglobulin-mediated phagocytosis. However, the hostmachinery that mediates amastigote uptake is poorly understood. Wehave previously shown that the Arg (also known as Abl2) non-receptor tyrosine kinase facilitates L. amazonensis amastigoteuptake by macrophages. Using small-molecule inhibitors andprimary macrophages lacking specific Src family kinases, we nowdemonstrate that the Hck, Fgr and Lyn kinases are also necessary foramastigote uptake by macrophages. Src-mediated Arg activation isrequired for efficient uptake. Interestingly, the dual Arg and Src kinaseinhibitor bosutinib, which is approved to treat cancer, not onlydecreases amastigote uptake, but also significantly reduces diseaseseverity and parasite burden in Leishmania-infectedmice. Our resultssuggest that leishmaniasis could potentially be treated with host-cell-active agents such as kinase inhibitors.

KEY WORDS: Leishmania, Phagocytosis, Kinase, Macrophage,Src, Abl

INTRODUCTIONThe parasite Leishmania causes visceral or cutaneous disease inover a million people every year. Drugs used to treat leishmaniasishave serious side effects, and parasites are developing resistance tothem. The Leishmania life cycle has two main stages: promastigotesin sand flies, and amastigotes in the mammalian host. If an infectedsandfly injects promastigotes into a host, the promastigotes must beengulfed by phagocytes to establish infection. Leishmania thendifferentiates within the phagolysosome into the amastigote. Ifamastigotes are found outside of this acidic compartment, they mustbe re-engulfed to persist in the host (Kane and Mosser, 2000).

Several macrophage surface protein receptors allow Leishmaniauptake. Promastigotes interact with multiple receptors, like thecomplement receptor CR3 (Russell and Wright, 1988); binding isenhanced by complement component fragment C3bi opsonizationmediated by lipophosphoglycan (LPG) (Mosser et al., 1992;Puentes et al., 1988). The FcR subclass FcγR, which is requiredfor IgG-mediated phagocytosis, is primarily responsible foramastigote uptake (Guy and Belosevic, 1993; Kima et al., 2000;Woelbing et al., 2006), and IgG opsonization of amastigotesfacilitates these interactions (Morehead et al., 2002). Leishmaniareceptor binding causes actin-rich phagocytic cups to engulf theparasite (Lodge and Descoteaux, 2008); however, the signalingprocess directing cup formation is not well understood.

The Abl family kinases Abl and Arg (also known as Abl1and Abl2, respectively) translate signals from growth factor andadhesion receptors into cytoskeletal rearrangements (Bradley andKoleske, 2009). Receptor engagement stimulates these kinases tobind and phosphorylate Arp2/3 complex activators (Lapetina et al.,2009; Miller et al., 2010), yielding dynamic cell edge protrusionsthat resemble phagocytic intermediates. Abl and Arg also facilitateendocytosis (Jacob et al., 2009; Tanos and Pendergast, 2006, 2007),autophagy (Yogalingam and Pendergast, 2008), viral (Reeves et al.,2005, 2011; Swimm 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 allowcomplementary non-redundant processes during phagocytosis andLeishmania uptake (Wetzel et al., 2012). Genetic loss of Argprevents efficient IgG-mediated phagocytosis and amastigoteuptake, whereas loss of Abl reduces C3bi-mediated phagocytosisand L. amazonensis promastigote uptake. In addition, by using theAbl and Arg inhibitor imatinib and assessing mice lacking Abl orArg, we have shown that Abl family kinases mediate infection inmurine cutaneous leishmaniasis (Wetzel et al., 2012).

Src family kinases (SFKs) are non-receptor tyrosine kinasesregulated by cell surface receptors that play roles in cellmorphogenesis. Src and Lyn directly bind the FcR (Wu et al.,2001), and macrophages lacking the SFKs Hck, Lyn and Fgr havesubstantial defects in IgG-mediated phagocytosis (Fitzer-Attaset al., 2000), and viral (Abram and Lowell, 2008; Bavagnoli et al.,2011; Cheng et al., 2015) and bacterial uptake (Hauck et al., 1998;Paul et al., 2008; Van Langendonck et al., 1998). SFKsphosphorylate and activate Arg (Mader et al., 2011; Plattner et al.,2004; Tanis et al., 2003), and this can be amplified by Argautophosphorylation on a distinct regulatory site (Bradley andKoleske, 2009). However, whether and how SFKs facilitate theuptake of Leishmania is not clear. Of note, if SFKs and Arg bothwere to mediate amastigote uptake, either within the same pathway,or in different pathways, combining Arg and SFK inhibitors mightshow increased efficacy over Arg and Abl inhibitors for disruptingthe disease course of leishmaniasis.Received 4 January 2016; Accepted 23 June 2016

1Department of Pediatrics, Yale University, New Haven, CT 06520, USA.2Department of Pediatrics, University of Texas Southwestern Medical Center,Dallas, TX 75390, USA. 3Department of Medicine, University of MassachusettsMedical School, Worcester, MA 01605, USA. 4Department of Epidemiology ofMicrobial Disease, Yale School of Public Health, New Haven, CT 06520, USA.5Department of Molecular Biochemistry and Biophysics, Yale University, CT 06520,USA. 6Department of Neuroscience, Yale University, New Haven, CT 06520, USA.*Present address: Departments of Pediatrics and Pharmacology, University ofTexas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390,USA.

‡Author for correspondence (dawn.wetzel@utsouthwestern.edu)

D.M.W., 0000-0002-3990-7043

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Here, we provide evidence that host SFKs activate Arg to facilitateimmunoglobulin-mediated phagocytosis and L. amazonensisamastigote uptake. Using kinase inhibitors and macrophageslacking specific SFKs, we show that Hck, Fgr and Lyn also mediateefficient amastigote uptake. SFKs signal through Arg to facilitate thisprocess. Finally, the combinationArg and SFK inhibitor bosutinib notonly reduces amastigote uptake bymacrophages but also significantlyameliorates disease severity in Leishmania-infected mice. Theseresults suggest that leishmaniasis could be treated with drugs thatinhibit these kinases or other host cell processes.

RESULTSSFKs are required for efficient amastigote but notpromastigote uptakeWe and others have demonstrated that IgG-opsonized amastigoteuptake occurs primarily through an FcRγ-mediated process,whereas C3bi-opsonized promastigote uptake occurs primarilythrough a CR3-mediated process (Carter et al., 2009; Kima et al.,2000; Mosser and Edelson, 1985; Russell and Wright, 1988; Uenoand Wilson, 2012). SFKs are known to facilitate immunoglobulin-mediated phagocytosis. To test whether they might mediateLeishmania uptake, we examined whether the SFK inhibitorSU6656 (which has an IC50 of 20–700 nM, depending onthe specific SFK; Blake et al., 2000) affected the uptake ofL. amazonensis promastigotes or amastigotes. We used two-colorimmunofluorescence (Wetzel et al., 2003) to distinguish adherentfrom internalized parasites and measured the phagocyticindex (number of particles internalized per 100 cells) in thepresence of SU6656 or DMSO. We found that SU6656 inhibitedIgG-opsonized bead phagocytosis by bone-marrow-derivedmacrophages (BMDMs) (Fig. S1A) with an approximate IC50 of2.5 μM; its CC50 (cytotoxic concentration necessary to cause deathto 50% of viable cells) for BMDMs over the same incubation periodwas above our highest concentration of 10 μM. Treating BMDMwith 2.5 μM SU6656 decreased the phagocytic index for IgG-opsonized beads but not C3bi-opsonized beads (Fig. 1A). 2.5 μMSU6656 also decreased the phagocytic index for IgG-opsonizedamastigotes by 40±7% (mean±.s.e.m.) relative to controls, but didnot affect the uptake of C3bi-opsonized promastigotes (Fig. 1B,C).Amastigotes bound to SU6656-treated BMDMs at levelsindistinguishable from controls (Fig. 1D), indicating thatdecreased invasion did not simply result from reduced adhesion.Similar results were found when the murine macrophage-like cellline RAW 264.7 was used instead of BMDMs (Fig. 1E; Fig. S1B).Internalization defects were observed even over incubations of up to2 h, demonstrating that treated BMDMs did not overcome defects inphagocytosis even if a substantial amount of time elapsed(Fig. S1C). Treating macrophages with another SFK inhibitor,PP2 [IC50 for Src=1.4 μM (Blake et al., 1999); CC50 is substantiallyabove 20 μM (Beausejour et al., 2012)], also caused a defect inamastigote uptake (Fig. 1F).

Hck, Fgr and Lyn are required for amastigote uptakeBoth SU6656 and PP2 inhibit other kinases besides SFKs, suchas Aurora kinases (Arai et al., 2012; Bain et al., 2003). Therefore,it was unclear whether the effects of these drugs resulted fromSFK inhibition. There are nine SFKs, eight of which areexpressed in macrophages. Previous data suggests that threeSFKs, namely Hck, Fgr, and Lyn, are especially important forimmunoglobulin-mediated phagocytosis (Fitzer-Attas et al.,2000). Thus, we tested the role of these kinases inL. amazonensis amastigote uptake by isolating BMDMs from

mice lacking Hck, Fgr and Lyn (Hu et al., 2004). We found thatthese macrophages were defective in amastigote uptake (Fig. 2A),which was decreased by 41±8% (mean±.s.e.m.) relative tocontrols. Internalization defects were also demonstrated for IgG-opsonized beads (Fig. 2B), as seen previously (Fitzer-Attas et al.,2000). No defects in the uptake of promastigotes (Fig. 2A) orC3bi-opsonized beads (Fig. 2B) were observed in Hck−/− Fgr−/−

Lyn−/− BMDMs.

Src family kinases lie upstream of Arg in a signaling pathwaythat governs amastigote uptakeArg facilitates both immunoglobulin-mediated phagocytosis andamastigote uptake (Wetzel et al., 2012). In other biological systems,Arg functions downstream of SFKs (Bradley and Koleske, 2009;Mader et al., 2011; Tegtmeyer and Backert, 2011). To determinewhether a SFK–Arg signaling pathway governed amastigote uptake,we first treated Hck−/− Fgr−/− Lyn−/− BMDMs with imatinib. TheIC50 of imatinib for Abl is 600 nM (Buchdunger et al., 1995) and itsCC50 is ≥20 μM for a 2-h incubation with RAW 264.7 cells; hence,3.3 μMwas selected as inWetzel et al., 2012. TreatingHck−/− Fgr−/−

Lyn−/−BMDMswith imatinib did not cause additional defects in IgG-mediated phagocytosis or amastigote uptake (Fig. 3A), suggestingthat SFKs and Abl family kinases might lie in the same pathway.To determine whether Abl or Arg was responsible for SFK-mediatedprocesses, we used wild-type (WT), Ablflox/flox LysM Cre+, Arg−/− orArg−/− Ablflox/flox LysM Cre+ BMDMs (henceforth referred toas double knockout, dKO) (Fig. 3B). We found that Ablflox/flox LysMCre+ and dKO BMDMs had defects in C3bi-mediated phagocytosisand promastigote uptake, whereas Arg−/− and dKO BMDMs haddefects in IgG-mediated phagocytosis and amastigote uptake.SU6656 inhibited IgG-mediated phagocytosis and amastigoteuptake in WT and Ablflox/flox LysM Cre+ BMDMs but not in Arg−/−

or dKO BMDMs.We then used the small-molecule Abl family kinase activator

DPH (Yang et al., 2011) to test whether activating Arg stimulatedphagocytosis and amastigote uptake. DPH is known to activate Arg(Simpson et al., 2015) and Abl, with an EC50 of 250–400 nM (Yanget al., 2011); its CC50 is ≥10 μM for BMDMs. DPH stimulatedphagocytosis in a dose-dependent manner (Fig. 3C). At low doses(250 nM), DPH rescued the IgG-opsonized bead and amastigote(Fig. 3D) uptake defects seen in Hck−/− Fgr−/− Lyn−/− BMDMs,returning uptake to 106±6% and 113±8% (mean±s.e.m.) ofcontrols, respectively. DPH had no effect on amastigote uptake byArg−/−BMDMs, suggesting that the drugwas specifically activatingArg (Fig. 3D). We then treated WT, Ablflox/flox LysM Cre+, Arg−/−

and dKO BMDMs with DPH prior to phagocytosis or Leishmaniauptake. DPH only increased C3bi-mediated phagocytosis andpromastigote uptake by BMDMs containing Abl (e.g. WT andArg−/− BMDMs), whereas it only increased IgG-mediatedphagocytosis and amastigote uptake by BMDMs containing Arg(e.g. WT and Ablflox/flox LysM Cre+ BMDMs) (Fig. 3E). Takentogether, these results are most consistent with a signaling pathwayin which SFKs act upstream of, and activate Arg but not Abl, duringIgG-mediated phagocytosis and amastigote uptake.

The SFK and Arg inhibitor bosutinib decreases Leishmaniauptake by macrophagesWe next reasoned that the dual specificity SFK and Arg kinaseinhibitor bosutinib might more effectively inhibit amastigoteuptake. Bosutinib was recently approved by the US Food andDrug Administration (FDA) for treatment of chronic myelogenousleukemia and other cancers (Rusconi et al., 2014), and it inhibits

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purified Arg (Golas et al., 2003) and SFKs with an IC50 of ∼1 nM(Boschelli et al., 2001; Remsing Rix et al., 2009). Low bosutinibdoses were sufficient to decrease IgG-opsonized bead uptake(Fig. 4A), with an approximate IC50 of 0.5 μM to prevent uptake; itsCC50 for the same incubation period as for BMDMswas >5 μM.Wefound that 1 μM bosutinib consistently inhibited BMDM and RAW264.7 cell uptake of C3bi- or IgG-coated beads (Fig. 4B,C). It alsoinhibited Leishmania promastigote and amastigote uptake byBMDMs by 49±3% and 46±5% (mean±s.e.m.), respectively(Fig. 4D,E). Treating RAW 264.7 cells with bosutinib andexposing them to parasites yielded similar results (Fig. 4F).

Amastigote exposure activates SFK signaling through ArgArg kinase activity is required for optimal IgG-mediatedphagocytosis (Fig. 3; Greuber and Pendergast, 2012; Wetzel et al.,2012). Incubation of RAW 264.7 cells with opsonizedL. amazonensis amastigotes induced a significant increase inphosphorylation of the Abl and Arg substrate CrkII (a splicevariant encoded by the Crk gene; denoted pCrk) (Kain and Klemke,2001). This amastigote-induced increase in pCrk was significantlyreduced following treatment with the Abl and Arg inhibitor imatinib,the SFK inhibitor SU6656, and the dual SFK and Arg inhibitorbosutinib (Fig. 5A,B). To determine whether CrkII activation was

Fig. 1. SFKs are required for optimal IgG-mediatedphagocytosis and amastigote uptake.Macrophages weretreated with 2.5 μM SU6656 or DMSO for 2 h and incubatedwith C3bi- or IgG-coated beads (A) or L. amazonensis C3bi-coated promastigotes or IgG-coated amastigotes (B–F) for30 min. Two-color immunofluorescence distinguishedbetween intracellular (green) and extracellular (orange)beads or parasites. Nuclei are labeled with DAPI.(A) SU6656 decreases IgG-coated but not C3bi-coatedbead uptake by BMDMs. Results are the mean±s.e.m.phagocytic index (PI) for BMDMs treated with 2.5 μMSU6656 normalized to the DMSO-treated phagocytic index(100%) for each experiment. (B) SU6656 decreasesamastigote uptake but not promastigote uptake by BMDMs.Graph shows the normalized mean±s.e.m. SU6656-treatedphagocytic index compared to the DMSO-treatedphagocytic index. (C) Image of anti-P8 antibody IgG-opsonized amastigote uptake by BMDMs treated withDMSO (top) or SU6656 (bottom). Left panels, representativefields; right panels, enlarged view of boxed area. Scale bars:10 μm (left); 5 μm (right). (D) SU6656 does not affectamastigote adhesion. Results are percentages of adheredamastigotes per 100 SU6656-treated BMDMs (adhesiveindex, AI) normalized to the DMSO-treated adhesive indexfrom the experiment in B. (E) SU6656 decreases amastigoteuptake by RAW264.7 cells. Shown is the normalized mean±s.e.m. phagocytic index for SU6656-treated compared toDMSO-treated RAW 264.7 cells. (F) The SFK inhibitor PP2decreases amastigote uptake. Shown is the normalizedmean±s.e.m. phagocytic index for amastigote uptake byPP2-treated RAW 264.7 cells compared to DMSO-treatedcontrols. *P<0.05; **P<0.01 (one-sample t-test); n=3separate experiments.

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mediated through Abl or Arg during IgG-mediated amastigotebinding and uptake, we incubatedWT, Ablflox/flox LysM Cre+, Arg−/−

and dKO (Arg−/−Ablflox/flox LysM Cre+) BMDMs with amastigoteswith or without DPH. We found that pCrk levels increasedsignificantly in WT and Ablflox/flox LysM Cre+ BMDMs incubatedwith amastigotes, but not in amastigote-treated Arg−/− or dKOBMDMs, indicating that CrkII activation during amastigote uptakewas specifically dependent upon Arg and not Abl (Fig. 5C,D).

SFKanddual SFKandArg inhibitors decrease lesion size andparasite burden in a murine model of cutaneousleishmaniasisKnowing that inhibiting SFK and Arg significantly decreasedparasite uptake in cultured cells, we next determined whether SFKand Arg inhibitors decreased the manifestations of cutaneousleishmaniasis in mice. Our previous work demonstrated that the Abland Arg inhibitor imatinib decreased lesion size and parasite burdenin mice (Wetzel et al., 2012). To test the effects of SFK inhibition,we provided 2 mg/kg body weight of PP2 by intraperitonealinjection three times weekly thoughout the course of infection. Wefound that PP2 significantly decreased the average lesion size inmice infected with L. amazonensis (Fig. 6A), as well as the parasiteburden (Fig. 6B) by >10 fold. To determine whether inhibiting bothSFKs and Arg would also provide benefit, we provided 30 mg/kgbody weight of bosutinib or vehicle (DMSO) in drinking waterthroughout the course of infection. We found that bosutinibsignificantly decreased the average lesion size in L.-amazonensis-infected mice (Fig. 6C). A higher dose of bosutinib (150 mg/kgbody weight) did not cause additional decreases in lesion size(Fig. S2).We first tested whether bosutinib impacted on lesions by directly

affecting parasite growth or survival. 10 μM bosutinib caused nogrowth defects for promastigotes (Fig. S3A) or amastigotes(Fig. S3B). Parasites could transition from promastigotes toamastigotes when cultures were grown in bosutinib, eitheraxenically (Fig. S3C) or in BMDMs (Fig. S3D). Finally, wefound no survival defects in amastigotes within bosutinib-treatedpreviously infected BMDMs (Fig. S3E).The reduced lesion size in bosutinib-treated mice could

potentially result from differences in the immune response toinfection. Of note, mice lacking SFKs and Abl family kinases havesignificant immunological defects, including deficits in B cell and Tcell development, proliferation and function, which could alter thecourse of cutaneous leishmaniasis (Colucci et al., 1999; Gu et al.,

2007; Kovacs et al., 2014; Liberatore and Goff, 2009; Lin et al.,1994; Silberman et al., 2008; Zipfel et al., 2004). To determinewhether the immunological effects of Abl, Arg or SFK inhibitionwere causing the healing seen in bosutinib-treated mice, we isolateddraining lymph nodes from infected DMSO and bosutinib-treatedmice and profiled cytokine secretion after stimulation with parasitelysate (Soong et al., 1996). Overall, therewere decreased amounts ofcytokines secreted in response to L. amazonensis by lymph nodesisolated from bosutinib-treated mice compared to control mice(Table 1, bosutinib); a change in the overall ratio of T-helper 1 toT-helper 2 cells (Th1/Th2) was not noted (Table S1). Bosutinib didnot affect cytokine release in response to concanavalin A (Con A)stimulation in these same mice (Table 1, bosutinib), suggesting thatthis change was specific to L. amazonensis stimulation.Consequently, the lower response to Leishmania antigenstimulation might reflect the reduced parasite load rather than aneffect of bosutinib on the ongoing host response to infection. Toevaluate this point, we examined the direct in vitro effect ofbosutinib on the response of lymph node cells to leishmanialantigens. Cytokine secretion did not decrease when infecteddraining lymph nodes isolated from DMSO-treated infected micewere treated with 0.1% DMSO or bosutinib during culture(Table S2), suggesting that bosutinib did not directly inhibitcytokine secretion. Cytokine secretion did not significantly changein lymph nodes isolated from imatinib-treated or PP2-treated mice(Table 1, imatinib, PP2), with a few exceptions. Both imatinib (i.e.Abl family kinase inhibition alone) and PP2 decreased IL-1β levels,but based on previous studies, this decrease would not be expectedto cause healing (Lima-Junior et al., 2013). PP2 also caused lowerIFN-γ secretion at high antigenic stimulation (the opposite to whatwould be expected if the change were contributing to healing inPP2-treated mice; Soong et al., 2012) and decreased IL-17 secretionwith Leishmania antigenic stimulation.

Cytokines that attract immune cells to infection sites, termedchemokines, could also affect the outcome of leishmaniasis. Wenext assessed whether Abl and Arg, SFK, or combined Abl andSFK inhibition affected chemokine secretion. The production ofchemokines implicated in Leishmania pathogenesis [IP-10, MIP-1α (CCL3), MIP-2 (CXCL2), RANTES (CCL5), CXCL9 andMIP-1β (CCL4)] did not change with bosutinib or imatinib(Table S3A,B), suggesting that the Abl- and Arg-mediateddifferences in chemokines were not responsible for decreasedlesions. PP2 caused decreased secretion of MIP-1α, MIP-2 andCXCL9 (Table S3C), but these changes are not known to decrease

Fig. 2. Hck, Fgr and Lyn facilitate IgG-mediatedphagocytosis and amastigote uptake. (A) Hck−/− Fgr−/−

Lyn−/− BMDMs exhibit defects in amastigote uptake.BMDMs were incubated with opsonized promastigotes andamastigotes as described in Fig. 1. Graph shows themean±s.e.m. phagocytic index (PI) for promastigotes andamastigotes forHck−/− Fgr−/− Lyn−/−BMDMs, normalized toWTBMDMs. (B)Hck−/− Fgr−/− Lyn−/−BMDMs show defectsin IgG-mediated phagocytosis. BMDMswere incubated withC3bi- or IgG-coated beads as in Fig. 1. Shown is the mean±s.e.m. phagocytic index for C3bi- or IgG-coated beads forHck−/− Fgr−/− Lyn−/− BMDMs, normalized to WT BMDMs.*P<0.05 (one-sample t-test); n=3 separate experiments.

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Fig. 3. AnSFK-Argsignalingpathway facilitates IgG-mediatedphagocytosis andamastigote uptake. (A) TreatingHck−/−Fgr−/− Lyn−/−BMDMswith imatinibdoes not further decrease (left) IgG-mediated phagocytosis or (right) IgG-opsonized amastigote uptake. n=3 separate experiments. The left graph shows themean±s.e.m. phagocytic index (PI) for IgG-coated beads for Hck−/− Fgr−/− Lyn−/− BMDMs incubated with imatinib or DMSO, compared to WT BMDMs incubated withimatinib or DMSO (the latter normalized to 100%). The right graph shows the mean±s.e.m. phagocytic index for amastigotes for Hck−/− Fgr−/− Lyn−/− BMDMsincubated with imatinib or DMSO, compared to WT BMDMs incubated with imatinib or DMSO (the latter normalized to 100%). (B) Effects of SU6656 on BMDMslacking Abl or Arg. Graphs show the mean±s.e.m. phagocytic index for WT, Ablflox/flox LysM Cre+ (referred to as Abl−/−), Arg−/−, or Arg−/−Ablflox/flox LysM Cre+(dKO) BMDMs incubated with DMSO or SU6656 and allowed to take upC3bi-coated beads, IgG-coated beads, C3bi-promastigotes or IgG-amastigotes. Data arenormalized to WT DMSO-treated BMDMs (set at 100%). n=3 experiments. (C) The Arg activator DPH stimulates IgG-mediated phagocytosis. Shown is onerepresentative experiment of two experiments demonstrating how increasing DPH doses affect the phagocytic index for IgG-coated beads. The maximallystimulating dose, 250 nM, was selected for the remaining experiments. (D) DPH rescues IgG-mediated phagocytosis (left) and amastigote uptake (right) inHck−/−

Fgr−/− Lyn−/− BMDMs. n=3 separate experiments. The left graph shows the mean±s.e.m. phagocytic index for IgG-coated beads incubated with WT BMDMs orHck−/− Fgr−/− Lyn−/− BMDMs treated with DMSO or DPH. The right graph shows the mean±s.e.m. phagocytic index for IgG-amastigotes incubated with WTBMDMs orHck−/− Fgr−/− Lyn−/−BMDMs treated with DMSO or DPH. (E) Effects of DPH on BMDMs lacking Abl or Arg. Graphs show themean±s.e.m. phagocyticindex for WT, Ablflox/flox LysM Cre+ (referred to as Abl−/−), Arg−/− or dKO BMDMs incubated with DMSO or DPH and allowed to take up C3bi-coated beads, IgG-coated beads, C3bi-promastigotes or IgG-amastigotes. Data are normalized to phagocytic index for DMSO-incubatedWTBMDMs (100%) for each condition. n=4experiments. For all categories, *P<0.05; **P<0.01; n.s., not significant compared with DMSO-treated WT BMDMs (ANOVA).

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lesion size (Brandonisio et al., 2002; Lima-Junior et al., 2013;Muller et al., 2003; Oghumu et al., 2010; Ritter and Korner, 2002;Santiago et al., 2004).Given that bosutinib caused a significant inhibition of parasite

uptake, and uptake is required for parasite survival, we suspected

that the decreased cytokine secretion in bosutinib-treated miceresulted from lower parasite burdens in these mice. Therefore, wedetermined the parasite burdens in DMSO- and bosutinib-treatedmice at the end of the experiment (∼3 months of infection) bylimiting dilution. We found that the number of parasites contained

Fig. 4. The SFK, Abl and Arg inhibitor bosutinib decreases phagocytosis and Leishmania uptake. (A) Effects of bosutinib on IgG-mediated phagocytosis.After treatment with the listed bosutinib doses or 0.1% DMSO, BMDMs were incubated with IgG-coated beads. Shown is the phagocytic index (PI) for eachcondition normalized to DMSO treatment (100%). A representative experiment of two experiments is shown. (B,C) Bosutinib decreases C3bi- or IgG-coated beaduptake. Shown is the mean±s.e.m. phagocytic index for C3bi- or IgG-opsonized beads incubated with bosutinib-treated BMDMs (B) or RAW 264.7 cells (C),normalized to DMSO-treated cells. n=3 experiments. (D) Bosutinib decreases promastigote uptake. Images of C3bi-coated promastigote uptake by WT BMDMstreated with DMSO (top) or bosutinib (bottom) are shown. Left panels, wide-field images; right panels, enlarged views of the boxed areas. Scale bars: 20 μm (left);10 μm (right). (E,F) Bosutinib decreases L. amazonensis promastigote and amastigote uptake. Shown is the mean±s.e.m. phagocytic index for C3bi-opsonizedpromastigotes and IgG-opsonized amastigotes incubated with bosutinib-treated BMDMs (E) or RAW 264.7 cells (F), normalized to DMSO-treated cells. n=3experiments. *P<0.05; **P<0.01 (by one-sample t-test).

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within the infected foot was far lower in bosutinib-treated mice thanin DMSO-treated mice (Fig. 6D), with an average decrease of∼50-fold. These results suggest that understanding the host cellprocesses required for parasite uptake could provide new candidatesfor treating leishmaniasis.

DISCUSSIONPreviously, we have demonstrated that the host Arg non-receptortyrosine kinase facilitates L. amazonensis amastigote uptake bymacrophages (Wetzel et al., 2012). The results shown here are thefirst to demonstrate that SFKs, and specifically Hck, Fgr and Lyn,

Fig. 5. SFKs and Arg phosphorylate downstream effectors during amastigote uptake. (A,B) Phosphorylation of the SFK, Abl and Arg substrate CrkII (pCrk)induced upon amastigote uptake is decreased in imatinib-, SU6656- or bosutinib-treated macrophages. RAW 264.7 cells were allowed to adhere to uncoatedplates (−) or plates coated with anti-P8 antibody-opsonized amastigotes (+ amastigotes) for 15 min before processing for immunoblotting. (A) Representativeimmunoblot of pCrk (top) and total CrkII (bottom) in DMSO-treated RAW 264.7 cells [with (+) or without (–) amastigotes] and amastigote-stimulated imatinib-,SU6656- or bosutinib-treated RAW 264.7 cells. (B) Graph presents the relative pCrk levels, normalized to CrkII levels, among RAW 264.7 cell categories shownin A. Levels of pCrk are normalized to amastigote-exposed DMSO-treated RAW 264.7 cells (100%). n=3 experiments. *P<0.05 compared with amastigote-stimulated DMSO-treated RAW 264.7 cells (ANOVA). (C,D) pCrk is induced upon amastigote uptake, is increased by DPH and is Arg-dependent. BMDMs [WT,Ablflox/flox LysMCre+ (Abl−/−), Arg−/− or dKO] were adhered to plates as above before processing for immunoblotting. (C) Representative immunoblot of pCrk (top)and total CrkII (bottom) in DMSO-treated BMDMs (− amastigotes), DMSO-, DPH- and bosutinib-treatedWTBMDMs (+ amastigotes), and DMSO- or DPH-treatedAbl−/−, Arg−/− or dKO BMDMs (all +amastigotes). (D) Graph shows relative pCrk levels, normalized to total Crk levels, among the categories of BMDMs shownin C. Levels of pCrk for each category are normalized to the level in amastigote-exposed DMSO-treated BMDMs. n=5 experiments. *P≤0.05; n.s., not significantcompared with amastigote-stimulated DMSO-treated WT BMDMs (ANOVA).

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also facilitate amastigote uptake. SFKs activate Arg during efficientamastigote uptake. Finally, the small-molecule combination Argand SFK inhibitor bosutinib decreases amastigote uptake bymacrophages and significantly reduces disease severity andparasite burden in Leishmania-infected mice. In summary, hostcell kinase inhibitors might provide novel exploratory candidates forthe treatment of leishmaniasis.

Leishmania amastigotes isolated from lesions are coated with IgGand primarily bind to the FcR to stimulate uptake (Kima et al.,2000). Based on their role during IgG-mediated phagocytosis, onemight predict a role for SFKs during Leishmania uptake. However,a previous in vivo study in mice lacking the SFK Fyn failed todemonstrate an effect of SFKs during Leishmania infection(Yamakami et al., 2001), in retrospect, likely due to redundant

Fig. 6. SFKs, Abl and Arg permit efficient infection in a mouse model of cutaneous leishmaniasis. (A) PP2-treated mice have smaller lesions thanuntreated mice. Four or five C57BL/6 mice per category were injected with 1×106 L. amazonensis promastigotes and given 2 mg/kg body weight of PP2 or DMSOby intraperitoneal injection three times weekly, starting 7 days before infection and continuing until the mice were euthanized. Two experiments were performed;shown is one experiment containing 5 mice per group. Results represent the mean±s.e.m. foot size increase over the uninfected foot (normalized to 1). *P<0.05(ANOVA). (B) Lesions in PP2-treatedmice contain fewer parasites than DMSO-treatedmice (quantified by limiting dilution). Plotted is themean±s.e.m. total lesionparasite burden in millions at the end of the experiment in A; in this example, there were 3.7×106±1.5×106 parasites in the DMSO-treated mice versus179×103±77×103 parasites in the PP2-treated mice. *P=0.042 (two-tailed t-test). (C) Bosutinib-treated mice have smaller lesions than untreated mice. Four toeight C57BL/6 mice per category were infected as in A and treated with 30 mg/kg body weight/day of bosutinib or DMSO in their drinking water, starting 4 daysbefore infection and continuing until the micewere euthanized. Three experiments were performed; shown is a representative experiment containing five mice pergroup. *P<0.05 (ANOVA). (D) Lesions in bosutinib-treated mice contain fewer parasites than DMSO-treated mice. Plotted is the parasite burden in millions at theend of the experiment in C; here therewere 3.3×106±0.7×106 parasites in the DMSO-treatedmice versus 61×103±21×103 parasites in the bosutinib-treatedmice.**P=0.0023 (two-tailed t-test). (E) Relationship between FcR signaling, SFKs and Arg during Leishmania amastigote uptake. Upon FcRγ ligation by amastigotes,Hck, Fgr and Lyn are activated. These SFKs phosphorylate and activate Arg kinase, which phosphorylates and activates CrkII, leading to actin polymerization.

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Table 1. Cytokine secretion in bosutinib-, imatinib- and PP2-treated mice.

Cytokine Treatment

Cytokine secretion (pg) after

High stimulation Low stimulation Con A stimulation

BosutinibIFN-γ DMSO 23,861±5919 7476±2264 3511±239

Bosutinib 5404±989 6743±1636 3253±284P-value 0.024 n.s. n.s.

IL-1α DMSO 24.7±7.84 40.0±11.2 32.1±11.3Bosutinib 58.3±23.0 64.7±21.4 58.6±18.9P-value 0.053 n.s. n.s.

IL-1β DMSO 49.1±5.0 75.6±17.2 62.0±14.5Bosutinib 91.5±24.9 82.1±22.8 93.0±21.7P-value n.s. n.s. n.s.

IL-4 DMSO 1892±296 1558±326 843±273Bosutinib 1036±232 537±104 712±244P-value 0.052 0.0086 n.s.

IL-10 DMSO 1063±223 892±114 938±220Bosutinib 447±71.5 467±85.8 862±162P-value 0.012 0.0099 n.s.

IL-13 DMSO 1053±205 1043±118 416±203Bosutinib 978±69.2 466±91.3 482±247P-value n.s. 0.0030 n.s.

IL-17 DMSO 225±20.0 314±55.6 303±27.7Bosutinib 146±26.0 189±41.3 347±111P-value 0.041 n.s. n.s.

ImatinibIFN-γ DMSO 7151±3455 6165±2231 4504±1604

Imatinib 10,536±4505 11,689±4645 4678±473P-value n.s. n.s. n.s.

IL-1α DMSO 135±28.9 173±29.9 188±32.9Imatinib 83.5±18.4 88.6±20.5 94.6±22.9P-value n.s. n.s. n.s.

IL-1β DMSO 116±23.3 85.2±29.3 119±24.8Imatinib 37.2±14.2 40.2±12.1 67.0±20.4P-value 0.035 n.s. 0.037

IL-4 DMSO 884±280 711±251 854±209Imatinib 1474±293 681±189 1372±223P-value n.s. n.s. n.s.

IL-10 DMSO 559±121 364±118 468±56.8Imatinib 904±152 629±194 647±88.7P-value 0.097 n.s. n.s.

IL-13 DMSO 159±35.5 121±29.6 237±62.1Imatinib 413±127 123±17.5 569±261P-value 0.079 n.s. n.s.

IL-17 DMSO 90.1±18.5 111±36.8 210±93.8Imatinib 208±63.5 257±88.1 213±86.7P-value 0.080 n.s. n.s.

PP2IFN-γ DMSO 22,775±4233 9664±1583 11,896±2733

PP2 9087±2662 9258±2335 8692±2577P-value 0.031 n.s. n.s.

IL-1α DMSO 196±35.5 179±43.7 138±23.8PP2 92.2±9.18 106±11.8 82.3±7.28P-value n.s. n.s. 0.019

IL-1β DMSO 66.6±20.7 59.2±17.6 62.7±18.5PP2 20.0±3.19 34.0±5.23 23.9±4.93P-value 0.027 n.s. 0.040

IL-4 DMSO 974±291 420±119 366±86.2PP2 652±226 1000±346 289±80.2P-value n.s. n.s. n.s.

IL-10 DMSO 1035±280 793±169 433±75.1PP2 831±189 965±349 418±55.4P-value n.s. n.s. n.s.

IL-13 DMSO 517±143 428±114 787±216PP2 189±46.4 430±157 972±410P-value 0.10 n.s. n.s.

Continued

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roles for multiple SFK members. Our work delineates specific rolesfor Hck, Fgr and Lyn for stimulating amastigote uptake, both basedon pharmacological inhibition and BMDMs from mice lackingthese three kinases.Our work demonstrates that SFKs signal through Arg to facilitate

amastigote uptake, as modeled in Fig. 6E. We do not observeadditional deficits in IgG-mediated phagocytosis or amastigoteuptake when Hck−/− Fgr−/− Lyn−/− BMDMs are treated withimatinib or when Arg−/− BMDMs are treated with SU6656,indicating that SFKs and Arg lie in the same signaling pathway, aspreviously shown in other systems (Mader et al., 2011; Plattneret al., 2004; Tanis et al., 2003). Another report has indicated thatmacrophages treated with both imatinib and SU6656 had IgG-mediated phagocytosis defects beyond those seen with either drugalone (Greuber and Pendergast, 2012), suggesting that SFKs, andArg and Abl might partly signal through different pathways. Thesedifferences might have occurred if imatinib and SU6656 did notcompletely inhibit Abl, Arg and SFK activity, or if the kinases alsoprovide a scaffolding function. In both cases, drug treatment alonemight not fully prevent this IgG-mediated uptake pathway.Furthermore, using BMDMs lacking Abl or Arg, we directlydemonstrate that a SFK–Arg pathway, and not a SFK–Abl pathway,is employed during IgG-mediated phagocytosis and amastigoteuptake. These data are consistent with our previous report showingthat Abl is required for C3bi-mediated phagocytosis andpromastigote uptake, whereas Arg is required for IgG-mediatedphagocytosis and amastigote uptake (Wetzel et al., 2012). Wesuspect that the differential usage of Abl and Arg duringphagocytosis and Leishmania uptake is due to their differentsubcellular localization within macrophages.We find that activating Arg in Hck−/− Fgr−/− Lyn−/− BMDMs

with low or sub-EC50 doses (Yang et al., 2011) of DPH rescuesuptake. Ours is the first demonstration that activating Abl familykinases facilitates phagocytosis. Interestingly, however, higherdoses of DPH inhibit phagocytosis, suggesting that either there is apoint where overly active Arg is deleterious, or that the drug affectsother essential molecules at high concentrations. The action of SFKsand Arg allows phosphorylation of their downstreammediator CrkIIduring FcR engagement by amastigotes. We also find that DPHincreases CrkII phosphorylation in an Arg-dependent manner,consistent with previous observations (Yang et al., 2011). Clearly,SFKs and Arg-independent mechanisms also contribute to IgG-mediated phagocytosis and amastigote uptake, given that inhibitingboth kinases does not completely prevent these processes. Futurestudies will focus on uncovering novel signaling pathwayspermitting Leishmania uptake and further delineating the SFK–Arg signaling pathway that occurs.

As kinase inhibitors do not prevent 100% of activity in vivo,targeting multiple signaling molecules in the same pathway could,in theory, yield a greater effect than inhibiting a single kinase. Thus,we turned to the potent combination SFK, Abl and Arg inhibitorbosutinib. Despite its effects on multiple host cell kinases, there aresufficiently few side effects that bosutinib was approved by the FDAto treat chronic myelogenous leukemia (Rusconi et al., 2014). Wefind that bosutinib prevented C3bi- and IgG-mediated phagocytosis,as well as promastigote and amastigote uptake.

Our mouse studies also indicate that Leishmania survival andpathogenesis depend on SFKs, Abl and Arg. We propose that thesmaller lesions seen in these kinase-inhibitedmice result at least partlyfrom uptake defects in macrophages. Consistent with this proposal,fewer parasites are contained in lesions in bosutinib-treated mice thanin controls. Interestingly, bosutinib-treated mice had a larger decreasein parasite burden than we saw with imatinib (Wetzel et al., 2012) orPP2 therapy. Thus, our results imply, although do not show directly,that inhibiting multiple signaling proteins required for parasite uptakemight improve results over inhibiting a single kinase alone.

Given that there are no direct effects of bosutinib treatment seenon parasite replication or survival within macrophages, the lesionsize disparities in L. amazonensis infection could result fromdifferences in cell entry and resulting parasite burden, or fromdifferences in the host inflammatory and/or immune response(Soong et al., 1995). Themurine immune response to leishmaniasisis complicated and is dependent on the parasite species and mousestrain. In general, Th1 responses are protective, and Th2 responsesare deleterious to the host (Jones et al., 1998). For example,inhibiting phosphoinositide 3-kinase (PI3K)γ, which can signalthough SFKs, Abl and Arg (Bradley and Koleske, 2009), affectsLeishmania uptake and impairs the Th2 response (Cummings et al.,2012). Mice lacking SFKs have defects in macrophage recruitment(Park et al., 1999), the respiratory burst (Meng and Lowell, 1998),and B cell and T cell development and signaling (Lowell, 2011).Immunological defects in mice lacking Abl family kinases includedefects in B cell and T cell development and signaling, amongothers; drugs affecting SFK andAbl andArg also decrease immunecell proliferation (Gu et al., 2007; Huang et al., 2008; Liberatoreand Goff, 2009; Silberman et al., 2008; Zipfel et al., 2004). All ofthese effects could contribute to the smaller lesions in Leishmania-infected bosutinib-treated mice.

We find that incubating cultured lymph nodes with bosutinibdoes not directly affect cytokine secretion. Cytokine profiling oflymph nodes isolated from infected bosutinib-treated micedemonstrated a decrease in many cytokines measured afterLeishmania antigen stimulation, but not after Con A stimulation.These decreases are generally not statistically significant with Abl

Table 1. Continued

Cytokine Treatment

Cytokine secretion (pg) after

High stimulation Low stimulation Con A stimulation

IL-17 DMSO 263±40.1 305±51.7 364±37.5PP2 103±10.5 160±23.1 240±60.8P-value 0.0093 0.037 0.090

Bosutinib: bosutinib-treated mice have mildly decreased cytokine secretion during Leishmania antigen stimulation. Shown are cytokine profiles of draining lymphnodes isolated from eight DMSO versus bosutinib-treated mice at the end of the experiments in Fig. 6C,D. Imatinib: except for IL1-β, cytokine secretion withLeishmania antigen stimulation is not significantly affected in imatinib-treated mice. Shown are profiles of draining lymph nodes isolated from eight DMSO versusimatinib-treated mice at the end of experiments described in Wetzel et al., 2012. PP2: cytokine production in DMSO versus PP2-treated mice during Leishmaniaantigen stimulation. Shown are profiles of draining lymph nodes isolated from eight DMSO versus PP2-treated mice at the end of experiments in Fig. 6A,B. Foreach category, a total of 5×106 cells were cultured for 72 h with medium (unstimulated), or lysates of 2.5×106 parasites (high stimulation) or 5×105 parasites (lowstimulation), or with Con A, as a positive control. Cytokine ELISAs were performed on harvested supernatants.P-values were determined by two-tailed t-tests andare listed if P<0.1. n.s., not significant.

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and Arg, or SFK inhibition alone, and none of the changes seen withsingle inhibition are known to facilitate healing (Ehrlich et al., 2014;Lima-Junior et al., 2013; Soong et al., 2012), with the possibleexception of the decreased IL-17 secretion seen with PP2 (Sousaet al., 2014). One potential explanation for the bosutinib-inducedcytokine decreases is that a lower parasite burden causes adiminished amount of cytokines to be secreted in response toL. amazonensis antigen. Another possibility is that Abl, Arg andSFKs promote an immune response to L. amazonensis, which isdampened by kinase inhibition, and does not reach statisticalsignificance unless both families are inhibited. Unfortunately, it isdifficult to distinguish between these possibilities in our system.Even in bosutinib-treated mice, the ratios of IFN-γ to TH2 cytokinesdo not improve, suggesting that the differences in lesion size andparasite burden are not fully explained by defective immuneresponses to Leishmania infection. However, decreases in IL-10,like those described here, could facilitate lesion healing anddecrease parasite burden during L. amazonensis murine infection(Ji et al., 2005), contributing to the effects of bosutinib.Chemokine profiling also does not reveal explanations for the

smaller lesions seen with Abl and Arg, or SFK inhibitors. Weexamined levels of IP-10, MIP-1α, MIP-2, RANTES, CXCL9 andMIP-1β. Several changed upon PP2, but not bosutinib or imatinibtreatment, and, based on previous studies, these changes would notbe expected to facilitate healing (Brandonisio et al., 2002; Lima-Junior et al., 2013; Muller et al., 2003; Oghumu et al., 2010; Ritterand Korner, 2002; Santiago et al., 2004). Of note, PP2 inhibitsmultiple targets, not just SFKs (>50 targets were seen in Brandvoldet al., 2012), and these off-target effects could contribute to itschemokine and cytokine profile. More studies of chemokinesecretion after host manipulations like kinase inhibition might bebeneficial for unraveling the pathogenesis of leishmaniasis.Additionally, micewith defects in B cells or T cells can be disease

resistant following L. amazonensis infection (Soong et al., 1997).Indeed, the diminished swelling that we see in bosutinib-treatedmice might actually be less than what might have been expectedfrom the parasite burden decrease alone. Using another Leishmaniaspecies might distinguish whether kinase-inhibition-inducedimmune deficiencies are diminishing the effect we wouldotherwise see from bosutinib.In summary, we have shown that SFKs signal through Arg to

facilitate IgG-mediated phagocytosis and Leishmania uptake. SFKs,and Abl and Arg also govern the subsequent pathogenesis ofleishmaniasis in the mouse model. These results strongly suggest thatimproving our understanding of cell entry by Leishmania couldprovidenovel treatment strategies for leishmaniasis. Furthermore, theysupport the concept that drugs that target conserved host cellprocesses, rather than the infectious agents themselves, could beused to treat a broad range of infectious diseases, including parasiticdiseases like leishmaniasis. Thus far, we have focused on clinicallyavailable tyrosine kinase inhibitors with relatively benign side effectprofiles. In contrast, the agents currently used to treat leishmaniasishave multiple toxic side effects, and resistance to these drugs isemerging. Host-cell-active agents could be combined with currentantiparasitics, increasing their efficacy while lowering their toxicity.Future work will continue to explore the potential use of cell entryinhibitors for leishmaniasis.

MATERIALS AND METHODSMiceC57BL/6micewere obtained from Jackson Labs (Bar Harbor, ME). Abl andArg knockout mice [Ablflox/flox LysM Cre+, Arg−/−, and Arg−/−Ablflox/flox

LysM Cre+ (termed dKO)] from a mixed background (C57BL/6X 129Sv/J)were backcrossed to C57BL/6 more than five times; littermates providedcontrols. Hck, Fgr and Lyn triple-knockout mice (Hck−/− Fgr−/− Lyn−/−)were as described in Hu et al. (2004). The Institutional Animal Care and UseCommittees at Yale University and UT Southwestern approved allprotocols.

Cell cultureRAW 264.7 cells (ATCC, Manassas, VA) were grown in Dulbeco’smodified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovineserum (FBS; Invitrogen, Grand Island, NY). For primary macrophageexperiments, cells were harvested from femurs and tibias of WT, Ablflox/flox

LysM Cre+, Arg−/−, dKO, and Hck−/− Fgr−/− Lyn−/− mice anddifferentiated into bone-marrow-derived macrophages (BMDMs), whichwas confirmed as in Wetzel et al. (2012). Cells were tested forMycoplasmacontamination (Uphoff and Drexler, 2013).

Parasite cultureL. amazonensis promastigotes (strain IFLA/BR/67/PH8, from NormaW. Andrews, University of Maryland, College Park, MD) were grown inSchneider’s Drosophila medium with 10 μg/ml gentamicin and 15% heat-inactivated FBS at 24°C (Wetzel et al., 2012). For uptake experiments,promastigotes were incubated for 7 days to maximize infective metacyclics(i.e. those isolated through a step Percoll gradient; Sigma, St Louis, MO).Amastigotes were grown axenically (Wetzel et al., 2012). To assess forgrowth defects with bosutinib, medium containing 0.1% DMSO or 10 μMbosutinib (LC Laboratories, Woburn, MA) was used. Bosutinib remained insolution at neutral pH and has improved solubility at lower pH (Remsing Rixet al., 2009). Parasites were passed through mice to maintain virulence.

Phagocytosis assaysRAW 264.7 cells or BMDMs were plated at ∼50% confluence andincubated overnight in serum-free medium or M-CSF-starved medium,respectively. Except where indicated, for drug experiments with IgG-coatedbeads, coverslips were preincubated in medium containing 3.3 μM imatinib(LC Laboratories), 2.5 μM SU6656 (Sigma), 10 μM PP2 (Sigma), 1 μMbosutinib (LC Laboratories) or DMSO (0.1%) (Sigma) for 2 h. For C3bi-coated bead uptake, cells were preactivated with phorbol 12-myristate13-acetate (PMA; Sigma), which added 0.1% DMSO to all PMA-treatedcells. Experiments were conducted as described previously (Wetzel et al.,2012). Briefly, 2-μm latex yellow–green beads (Sigma) coated with humanIgM (cat. no. I-8260, Sigma) were incubated in fresh mouse serum for C3biopsonization or rabbit anti-IgM (cat. no. 270A, Sigma) for IgG opsonization(confirmed as described previously; Wetzel et al., 2012). Cells wereincubated with 10–15 beads/cell for 30 min at 37°C, fixed with 3%formaldehyde for 15 min, blocked with 2% BSAwithout permeabilization,incubated with rabbit anti-human IgM plus Hoechst 33258 dye (Sigma) tovisualize nuclei, then incubated with Alexa-Fluor-594-conjugated goat anti-rabbit-IgG secondary antibody (cat. no. A11034, Invitrogen). This two-color immunofluorescence assay (Wetzel et al., 2003) alloweddistinguishing of internalized and external beads. Coverslips werevisualized using a 40×1.0 NA aperture Nikon objective on a NikonEclipse TE2000-5 fluorescence microscope by a treatment-blindedobserver. Images were acquired with a Qimaging Cooled Charge-coupledDevice mono, 12-bit camera and Nikon Imaging software. At least tenrandomly selected fields were visualized for a total of over 100 macrophagesand beads per experiment. The mean phagocytic index (the number ofparticles internalized per 100 macrophages) for controls was set as themaximum (100%) value for each experiment, and those for experimentalconditions were normalized to that value. Each experiment was performedthree times and the mean±s.e.m. was calculated. A one-sample Student’st-test or two-way ANOVA was used to determine statistical significance.

Leishmania uptake assaysBMDMs or RAW 264.7 cells were plated and treated with inhibitors,DMSO or PMA as above. Metacyclic promastigotes were incubated in freshmouse serum for C3bi opsonization, and BMDMs or RAW 264.7 cells were

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PMA-activated for 1 h. Amastigotes were coated with anti-P8-proteoglycolipid complex (monoclonal antibody IgG1; Pan andMcMahon-Pratt, 1988). Coverslips with RAW cells or BMDMs wereincubated with C3bi-opsonized promastigotes at a ratio of ten parasites toone cell or with IgG-opsonized amastigotes at two parasites to one cell for30 min and fixed with 3% formaldehyde. External promastigotes wereincubated with mouse anti-gp46 antibody; external amastigotes wereincubated with mouse anti-P8 antibody as described previously (Wetzelet al., 2012). All were incubated with Alexa-Fluor-568-conjugated donkeyanti-mouse-IgG secondary antibody (cat. no. A10037, Invitrogen). Afterpermeabilization, parasites were re-incubated with mouse anti-gp46(promastigotes) or mouse anti-P8 (amastigotes) antibodies and Alexa-Fluor-488-conjugated donkey anti-mouse-IgG secondary antibody (cat. no.A21202, Invitrogen) and DAPI (Sigma). At least ten random fields wereselected containing at least 100 parasites and macrophages per experiment;each experiment was performed three times. The mean±s.e.m. of biologicalreplicates is shown. Images for analysis were collected as above. Therepresentative images shown in Figs 1 and 4 were visualized with a ZeissLSM 700 confocal microscope with a Zeiss 40×1.3 NA EC Plan-Neofluaroil objective (promastigotes) or a Zeiss 63×1.4 NA Plan-Apochromatobjective (amastigotes), acquired with a Zeiss Axiocam 506 mono, 6megapixel monochrome camera and Zeiss Imaging software (Zeiss, Jena,Germany), and linearly processed in Adobe Photoshop (Adobe Systems,San Jose, CA). A one-sample Student’s t-test or two-way ANOVAwas usedto determine statistical significance.

Intra-macrophage assaysFor assays examining promastigote–amastigote transition withinmacrophages or intracellular survival, 10 L. amazonensis promastigoteswere added per WT BMDM cell (starved of G-CSF overnight) and plated intriplicate wells at 50% confluence. After 4 h, the BMDMs were washed 5×with PBS, and DMSO or 2 μM bosutinib in medium containing G-CSF wasadded. The number of parasites per 100 BMDMs was counted at 24 h toassess amastigote transition or 72 h to assess survival.

ImmunoblottingTo measure phosphorylation of the downstream SFK, Abl andArg effector CrkII (42 kDa), starved RAW 264.7 cells or WT,Ablflox/flox LysM Cre+, Arg−/− or dKO BMDMs were added for 15 min touncoated dishes or dishes precoated for 1 h with anti-P8 antibody (Pan andMcMahon-Pratt, 1988) IgG-opsonized amastigotes. Cells were lysed with abuffer of 20 mM Tris-HCl pH 7.2, 2 mM EDTA, 150 mM NaCl and 1%Triton X-100 that contained protease and phosphatase inhibitors. Forrepresentative images, equivalent protein amounts were loaded on 10%SDS-PAGE gels, transferred to nitrocellulose membranes, and probed withantibodies against phosphorylated CrkII (pCrkII) (Y221, cat. no. 3491S,Cell Signaling, Beverly, MA) at 1:1500, or CrkII (cat. no. sc-9004, SantaCruz Biotechnology, Dallas, TX) at 1:500. For analysis, relative amounts ofpCrkII were compared with Image J analysis software and were normalizedto CrkII (membranes were stripped of pCrkII and reprobed for CrkII, as inWetzel et al., 2012). Imaging was performed by using a phosphorimager(ImageQuant LAS 4000, GE). Two-way ANOVA was used to determinestatistical significance.

Murine infectionsFour to eight female C57BL/6 mice per group were infected at between 6and 8 weeks of age. Power calculations were performed as in Wetzel et al.,2012. 1×106 metacyclic promastigotes in PBSwere injected subcutaneouslyin the dorsal right hind foot. Imatinib experiments were performed asdescribed previously (Wetzel et al., 2012). For PP2-treated mice, twoindependent experiments were performed. Mice were provided 2 mg/kgbody weight of PP2 or 5% DMSO (the diluent) by intraperitoneal injectionthree times a week, starting a week before infection and continuing throughuntil the mice were euthanized. For bosutinib-treated mice, threeindependent experiments were conducted. Mice were provided 30 mg/kgbody weight/day of bosutinib or <0.5% DMSO (the diluent) in drinkingwater starting 4 days before infection and continuing through until the micewere euthanized. Bottle weight was obtained to monitor water intake. Lesion

size was monitored with calipers weekly by an investigator blinded tocondition, and the infected:uninfected size ratio was calculated (Champsiand McMahon-Pratt, 1988). The control and drug-treated groups werecompared to swelling at time 0 by ANOVA. Parasite burdens in lesions weredetermined upon experimental termination between experimental weeks 12and 18 by limiting dilution (Soong et al., 1997).

Lymph nodes from infected DMSO versus drug-treated mice wereharvested for cytokine and chemokine profiling (Soong et al., 1997, 1995,1996). Cells were plated and stimulated with promastigote lysates or Con A(5 µg/ml; Sigma) (Wetzel et al., 2012). Supernatants were obtained after72 h. The cytokines IL-4, IL-10, IL-13, IL-17 or IFN-γ were assessed byELISA (BD Biosciences, San Jose, CA). Background levels were assessedusing unstimulated cell supernatants. Levels of the cytokines andchemokines IL-1β, IL-1α, IP-10, MIP-1α (CCL3), MIP-2 (CXCL2),RANTES (CCL5), and MIP-1β (CCL4) were profiled by using the custommultiplex Luminex Platform (R&D Systems, Inc, Minneapolis, MN)through the UT Southwestern Microarray Core as described previously(Gonzalez-Fajardo et al., 2015; Navas et al., 2014). CXCL9 levels wereassessed by ELISA (R&D Systems, Inc).

AcknowledgementsWe thank Norma W. Andrews for providing L. amazonensis strain IFLA/BR/67/PH8and the Koleske and McMahon-Pratt laboratories for helpful discussions. We thankMargaret A. Phillips and her laboratory for the use of equipment and reagents,James J. Collins for confocal microscope use, the parasitology group at UTSouthwestern for valuable discussions, Indu Raman at the UT SouthwesternMicroarray Core for assistance with cytokine and chemokine profiling, and EmilyT. Mamula, ImranUllah, Hanspeter Niederstrasser, Meghan E. Kerrisk andMargaretA. Cooper for technical and experimental assistance.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsD.M.W. designed and performed experiments, analyzed data and wrote themanuscript. E.L.R. performed experiments and collected and analyzed data. S.L.assisted with the Hck−/− Fgr−/− Lyn−/− mouse studies and manuscript preparation.D.M.-P. and A.J.K. participated in experimental design and data analysis, discussedresults and assisted with manuscript preparation.

FundingThis work was supported by the National Institutes of Health [grant numbersNRSA F32 AI094905 and K08 AI103036 to D.M.W.; R01 CA122142 to S.L.; R01AI093775 to D.M.-P.; and R01 CA133346, R01 GM100411, and R01 NS089662 toA.J.K.]; and funds from the University of Texas Southwestern Medical Center,Department of Pediatrics (to D.M.W. and E.L.R.). Deposited in PMC for releaseafter 12 months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.185595.supplemental

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