© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 3130-3143 doi:10.1242/jcs.185595

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

ABSTRACT Several macrophage surface receptors allow Leishmania Leishmaniasis is a devastating disease that disfigures or kills nearly uptake. Promastigotes interact with multiple receptors, like the two million people each year. Establishment and persistence of complement receptor CR3 (Russell and Wright, 1988); binding is infection by the obligate intracellular parasite Leishmania requires enhanced by complement component fragment C3bi opsonization repeated uptake by macrophages and other phagocytes. Therefore, mediated by lipophosphoglycan (LPG) (Mosser et al., 1992; γ preventing uptake could be a novel therapeutic strategy for Puentes et al., 1988). The FcR subclass Fc R, which is required leishmaniasis. Amastigotes, the life cycle stage found in the for IgG-mediated phagocytosis, is primarily responsible for human host, bind Fc receptors and enter macrophages primarily amastigote uptake (Guy and Belosevic, 1993; Kima et al., 2000; through immunoglobulin-mediated phagocytosis. However, the host Woelbing et al., 2006), and IgG opsonization of amastigotes machinery that mediates amastigote uptake is poorly understood. We facilitates these interactions (Morehead et al., 2002). Leishmania have previously shown that the Arg (also known as Abl2) non- receptor binding causes actin-rich phagocytic cups to engulf the receptor facilitates L. amazonensis amastigote parasite (Lodge and Descoteaux, 2008); however, the signaling uptake by macrophages. Using small-molecule inhibitors and process directing cup formation is not well understood. primary macrophages lacking specific Src family kinases, we now The Abl family kinases Abl and Arg (also known as Abl1 demonstrate that the Hck, Fgr and Lyn kinases are also necessary for and Abl2, respectively) translate signals from growth factor and amastigote uptake by macrophages. Src-mediated Arg activation is adhesion receptors into cytoskeletal rearrangements (Bradley and required for efficient uptake. Interestingly, the dual Arg and Src kinase Koleske, 2009). Receptor engagement stimulates these kinases to inhibitor , which is approved to treat cancer, not only bind and phosphorylate Arp2/3 complex activators (Lapetina et al., decreases amastigote uptake, but also significantly reduces disease 2009; Miller et al., 2010), yielding dynamic cell edge protrusions severity and parasite burden in Leishmania-infected mice. Our results that resemble phagocytic intermediates. Abl and Arg also facilitate suggest that leishmaniasis could potentially be treated with host-cell- endocytosis (Jacob et al., 2009; Tanos and Pendergast, 2006, 2007), active agents such as kinase inhibitors. autophagy (Yogalingam and Pendergast, 2008), viral (Reeves et al., 2005, 2011; Swimm et al., 2010) and bacterial uptake (Burton et al., KEY WORDS: Leishmania, Phagocytosis, Kinase, Macrophage, 2003; Elwell et al., 2008; Ly and Casanova, 2009; Napier et al., Src, Abl 2011), and IgG-mediated phagocytosis (Greuber and Pendergast, 2012). We have previously reported that Abl and Arg allow INTRODUCTION complementary non-redundant processes during phagocytosis and The parasite Leishmania causes visceral or cutaneous disease in Leishmania uptake (Wetzel et al., 2012). Genetic loss of Arg over a million people every year. Drugs used to treat leishmaniasis prevents efficient IgG-mediated phagocytosis and amastigote have serious side effects, and parasites are developing resistance to uptake, whereas loss of Abl reduces C3bi-mediated phagocytosis them. The Leishmania life cycle has two main stages: promastigotes and L. amazonensis promastigote uptake. In addition, by using the in sand flies, and amastigotes in the mammalian host. If an infected Abl and Arg inhibitor imatinib and assessing mice lacking Abl or sandfly injects promastigotes into a host, the promastigotes must be Arg, we have shown that Abl family kinases mediate infection in engulfed by phagocytes to establish infection. Leishmania then murine cutaneous leishmaniasis (Wetzel et al., 2012). differentiates within the phagolysosome into the amastigote. If Src family kinases (SFKs) are non-receptor tyrosine kinases amastigotes are found outside of this acidic compartment, they must regulated by cell surface receptors that play roles in cell be re-engulfed to persist in the host (Kane and Mosser, 2000). morphogenesis. Src and Lyn directly bind the FcR (Wu et al., 2001), and macrophages lacking the SFKs Hck, Lyn and Fgr have substantial defects in IgG-mediated phagocytosis (Fitzer-Attas 1 Department of Pediatrics, Yale University, New Haven, CT 06520, USA. et al., 2000), and viral (Abram and Lowell, 2008; Bavagnoli et al., 2Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 3Department of Medicine, University of Massachusetts 2011; Cheng et al., 2015) and bacterial uptake (Hauck et al., 1998; Medical School, Worcester, MA 01605, USA. 4Department of Epidemiology of Paul et al., 2008; Van Langendonck et al., 1998). SFKs Microbial Disease, Yale School of Public Health, New Haven, CT 06520, USA. 5Department of Molecular Biochemistry and Biophysics, Yale University, CT 06520, phosphorylate and activate Arg (Mader et al., 2011; Plattner et al., USA. 6Department of Neuroscience, Yale University, New Haven, CT 06520, USA. 2004; Tanis et al., 2003), and this can be amplified by Arg *Present address: Departments of Pediatrics and Pharmacology, University of autophosphorylation on a distinct regulatory site (Bradley and Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA. Koleske, 2009). However, whether and how SFKs facilitate the uptake of Leishmania is not clear. Of note, if SFKs and Arg both ‡ Author for correspondence ([email protected]) were to mediate amastigote uptake, either within the same pathway, D.M.W., 0000-0002-3990-7043 or in different pathways, combining Arg and SFK inhibitors might show increased efficacy over Arg and Abl inhibitors for disrupting

Received 4 January 2016; Accepted 23 June 2016 the disease course of leishmaniasis. Journal of Cell Science

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Here, we provide evidence that host SFKs activate Arg to facilitate mice lacking Hck, Fgr and Lyn (Hu et al., 2004). We found that immunoglobulin-mediated phagocytosis and L. amazonensis these macrophages were defective in amastigote uptake (Fig. 2A), amastigote uptake. Using kinase inhibitors and macrophages which was decreased by 41±8% (mean±.s.e.m.) relative to lacking specific SFKs, we show that Hck, Fgr and Lyn also mediate controls. Internalization defects were also demonstrated for IgG- efficient amastigote uptake. SFKs signal through Arg to facilitate this opsonized beads (Fig. 2B), as seen previously (Fitzer-Attas et al., process. Finally, the combination Arg and SFK inhibitor bosutinib not 2000). No defects in the uptake of promastigotes (Fig. 2A) or only reduces amastigote uptake by macrophages but also significantly C3bi-opsonized beads (Fig. 2B) were observed in Hck−/− Fgr−/− ameliorates disease severity in Leishmania-infected mice. These Lyn−/− BMDMs. results suggest that leishmaniasis could be treated with drugs that inhibit these kinases or other host cell processes. Src family kinases lie upstream of Arg in a signaling pathway that governs amastigote uptake RESULTS Arg facilitates both immunoglobulin-mediated phagocytosis and SFKs are required for efficient amastigote but not amastigote uptake (Wetzel et al., 2012). In other biological systems, promastigote uptake Arg functions downstream of SFKs (Bradley and Koleske, 2009; We and others have demonstrated that IgG-opsonized amastigote Mader et al., 2011; Tegtmeyer and Backert, 2011). To determine uptake occurs primarily through an FcRγ-mediated process, whether a SFK–Arg signaling pathway governed amastigote uptake, whereas C3bi-opsonized promastigote uptake occurs primarily we first treated Hck−/− Fgr−/− Lyn−/− BMDMs with imatinib. The through a CR3-mediated process (Carter et al., 2009; Kima et al., IC50 of imatinib for Abl is 600 nM (Buchdunger et al., 1995) and its 2000; Mosser and Edelson, 1985; Russell and Wright, 1988; Ueno CC50 is ≥20 μM for a 2-h incubation with RAW 264.7 cells; hence, and Wilson, 2012). SFKs are known to facilitate immunoglobulin- 3.3 μM was selected as in Wetzel et al., 2012. Treating Hck−/− Fgr−/− mediated phagocytosis. To test whether they might mediate Lyn−/− BMDMs with imatinib did not cause additional defects in IgG- Leishmania uptake, we examined whether the SFK inhibitor mediated phagocytosis or amastigote uptake (Fig. 3A), suggesting SU6656 (which has an IC50 of 20–700 nM, depending on that SFKs and Abl family kinases might lie in the same pathway. the specific SFK; Blake et al., 2000) affected the uptake of To determine whether Abl or Arg was responsible for SFK-mediated L. amazonensis promastigotes or amastigotes. We used two-color processes, we used wild-type (WT), Ablflox/flox LysM Cre+, Arg−/− or immunofluorescence (Wetzel et al., 2003) to distinguish adherent Arg−/− Ablflox/flox LysM Cre+ BMDMs (henceforth referred to from internalized parasites and measured the phagocytic as double knockout, dKO) (Fig. 3B). We found that Ablflox/flox LysM index (number of particles internalized per 100 cells) in the Cre+ and dKO BMDMs had defects in C3bi-mediated phagocytosis presence of SU6656 or DMSO. We found that SU6656 inhibited and promastigote uptake, whereas Arg−/− and dKO BMDMs had IgG-opsonized bead phagocytosis by bone-marrow-derived defects in IgG-mediated phagocytosis and amastigote uptake. macrophages (BMDMs) (Fig. S1A) with an approximate IC50 of SU6656 inhibited IgG-mediated phagocytosis and amastigote flox/flox −/− 2.5 μM; its CC50 (cytotoxic concentration necessary to cause death uptake in WT and Abl LysM Cre+ BMDMs but not in Arg to 50% of viable cells) for BMDMs over the same incubation period or dKO BMDMs. was above our highest concentration of 10 μM. Treating BMDM We then used the small-molecule Abl family kinase activator with 2.5 μM SU6656 decreased the phagocytic index for IgG- DPH (Yang et al., 2011) to test whether activating Arg stimulated opsonized beads but not C3bi-opsonized beads (Fig. 1A). 2.5 μM phagocytosis and amastigote uptake. DPH is known to activate Arg SU6656 also decreased the phagocytic index for IgG-opsonized (Simpson et al., 2015) and Abl, with an EC50 of 250–400 nM (Yang amastigotes by 40±7% (mean±.s.e.m.) relative to controls, but did et al., 2011); its CC50 is ≥10 μM for BMDMs. DPH stimulated not affect the uptake of C3bi-opsonized promastigotes (Fig. 1B,C). phagocytosis in a dose-dependent manner (Fig. 3C). At low doses Amastigotes bound to SU6656-treated BMDMs at levels (250 nM), DPH rescued the IgG-opsonized bead and amastigote indistinguishable from controls (Fig. 1D), indicating that (Fig. 3D) uptake defects seen in Hck−/− Fgr−/− Lyn−/− BMDMs, decreased invasion did not simply result from reduced adhesion. returning uptake to 106±6% and 113±8% (mean±s.e.m.) of Similar results were found when the murine macrophage-like cell controls, respectively. DPH had no effect on amastigote uptake by line RAW 264.7 was used instead of BMDMs (Fig. 1E; Fig. S1B). Arg−/− BMDMs, suggesting that the drug was specifically activating Internalization defects were observed even over incubations of up to Arg (Fig. 3D). We then treated WT, Ablflox/flox LysM Cre+, Arg−/− 2 h, demonstrating that treated BMDMs did not overcome defects in and dKO BMDMs with DPH prior to phagocytosis or Leishmania phagocytosis even if a substantial amount of time elapsed uptake. DPH only increased C3bi-mediated phagocytosis and (Fig. S1C). Treating macrophages with another SFK inhibitor, promastigote uptake by BMDMs containing Abl (e.g. WT and −/− PP2 [IC50 for Src=1.4 μM (Blake et al., 1999); CC50 is substantially Arg BMDMs), whereas it only increased IgG-mediated above 20 μM (Beausejour et al., 2012)], also caused a defect in phagocytosis and amastigote uptake by BMDMs containing Arg amastigote uptake (Fig. 1F). (e.g. WT and Ablflox/flox LysM Cre+ BMDMs) (Fig. 3E). Taken together, these results are most consistent with a signaling pathway Hck, Fgr and Lyn are required for amastigote uptake in which SFKs act upstream of, and activate Arg but not Abl, during Both SU6656 and PP2 inhibit other kinases besides SFKs, such IgG-mediated phagocytosis and amastigote uptake. as Aurora kinases (Arai et al., 2012; Bain et al., 2003). Therefore, it was unclear whether the effects of these drugs resulted from The SFK and Arg inhibitor bosutinib decreases Leishmania SFK inhibition. There are nine SFKs, eight of which are uptake by macrophages expressed in macrophages. Previous data suggests that three We next reasoned that the dual specificity SFK and Arg kinase SFKs, namely Hck, Fgr, and Lyn, are especially important for inhibitor bosutinib might more effectively inhibit amastigote immunoglobulin-mediated phagocytosis (Fitzer-Attas et al., uptake. Bosutinib was recently approved by the US Food and 2000). Thus, we tested the role of these kinases in Drug Administration (FDA) for treatment of chronic myelogenous

L. amazonensis amastigote uptake by isolating BMDMs from leukemia and other cancers (Rusconi et al., 2014), and it inhibits Journal of Cell Science

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Fig. 1. SFKs are required for optimal IgG-mediated phagocytosis and amastigote uptake. Macrophages were treated with 2.5 μM SU6656 or DMSO for 2 h and incubated with C3bi- or IgG-coated beads (A) or L. amazonensis C3bi- coated promastigotes or IgG-coated amastigotes (B–F) for 30 min. Two-color immunofluorescence distinguished between intracellular (green) and extracellular (orange) beads or parasites. Nuclei are labeled with DAPI. (A) SU6656 decreases IgG-coated but not C3bi-coated bead uptake by BMDMs. Results are the mean±s.e.m. phagocytic index (PI) for BMDMs treated with 2.5 μM SU6656 normalized to the DMSO-treated phagocytic index (100%) for each experiment. (B) SU6656 decreases amastigote uptake but not promastigote uptake by BMDMs. Graph shows the normalized mean±s.e.m. SU6656-treated phagocytic index compared to the DMSO-treated phagocytic index. (C) Image of anti-P8 antibody IgG- opsonized amastigote uptake by BMDMs treated with DMSO (top) or SU6656 (bottom). Left panels, representative fields; right panels, enlarged view of boxed area. Scale bars: 10 μm (left); 5 μm (right). (D) SU6656 does not affect amastigote adhesion. Results are percentages of adhered amastigotes per 100 SU6656-treated BMDMs (adhesive index, AI) normalized to the DMSO-treated adhesive index from the experiment in B. (E) SU6656 decreases amastigote uptake by RAW 264.7 cells. Shown is the normalized mean± s.e.m. phagocytic index for SU6656-treated compared to DMSO-treated RAW 264.7 cells. (F) The SFK inhibitor PP2 decreases amastigote uptake. Shown is the normalized mean±s.e.m. phagocytic index for amastigote uptake by PP2-treated RAW 264.7 cells compared to DMSO-treated controls. *P<0.05; **P<0.01 (one-sample t-test); n=3 separate experiments.

purified Arg (Golas et al., 2003) and SFKs with an IC50 of ∼1nM Amastigote exposure activates SFK signaling through Arg (Boschelli et al., 2001; Remsing Rix et al., 2009). Low bosutinib Arg kinase activity is required for optimal IgG-mediated doses were sufficient to decrease IgG-opsonized bead uptake phagocytosis (Fig. 3; Greuber and Pendergast, 2012; Wetzel et al., (Fig. 4A), with an approximate IC50 of 0.5 μM to prevent uptake; its 2012). Incubation of RAW 264.7 cells with opsonized CC50 for the same incubation period as for BMDMs was >5 μM. We L. amazonensis amastigotes induced a significant increase in found that 1 μM bosutinib consistently inhibited BMDM and RAW phosphorylation of the Abl and Arg substrate CrkII (a splice 264.7 cell uptake of C3bi- or IgG-coated beads (Fig. 4B,C). It also variant encoded by the Crk ; denoted pCrk) (Kain and Klemke, inhibited Leishmania promastigote and amastigote uptake by 2001). This amastigote-induced increase in pCrk was significantly BMDMs by 49±3% and 46±5% (mean±s.e.m.), respectively reduced following treatment with the Abl and Arg inhibitor imatinib, (Fig. 4D,E). Treating RAW 264.7 cells with bosutinib and the SFK inhibitor SU6656, and the dual SFK and Arg inhibitor exposing them to parasites yielded similar results (Fig. 4F). bosutinib (Fig. 5A,B). To determine whether CrkII activation was Journal of Cell Science

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Fig. 2. Hck, Fgr and Lyn facilitate IgG-mediated − − − − phagocytosis and amastigote uptake. (A) Hck / Fgr / − − Lyn / BMDMs exhibit defects in amastigote uptake. BMDMs were incubated with opsonized promastigotes and amastigotes as described in Fig. 1. Graph shows the mean± s.e.m. phagocytic index (PI) for promastigotes and − − − − − − amastigotes for Hck / Fgr / Lyn / BMDMs, normalized to − − − − − − WT BMDMs. (B) Hck / Fgr / Lyn / BMDMs show defects in IgG-mediated phagocytosis. BMDMs were incubated with C3bi- or IgG-coated beads as in Fig. 1. Shown is the mean± s.e.m. phagocytic index for C3bi- or IgG-coated beads for − − − − − − Hck / Fgr / Lyn / BMDMs, normalized to WT BMDMs. *P<0.05 (one-sample t-test); n=3 separate experiments.

mediated through Abl or Arg during IgG-mediated amastigote 2007; Kovacs et al., 2014; Liberatore and Goff, 2009; Lin et al., binding and uptake, we incubated WT, Ablflox/flox LysM Cre+, Arg−/− 1994; Silberman et al., 2008; Zipfel et al., 2004). To determine and dKO (Arg−/−Ablflox/flox LysM Cre+) BMDMs with amastigotes whether the immunological effects of Abl, Arg or SFK inhibition with or without DPH. We found that pCrk levels increased were causing the healing seen in bosutinib-treated mice, we isolated significantly in WT and Ablflox/flox LysM Cre+ BMDMs incubated draining lymph nodes from infected DMSO and bosutinib-treated with amastigotes, but not in amastigote-treated Arg−/− or dKO mice and profiled cytokine secretion after stimulation with parasite BMDMs, indicating that CrkII activation during amastigote uptake lysate (Soong et al., 1996). Overall, there were decreased amounts of was specifically dependent upon Arg and not Abl (Fig. 5C,D). cytokines secreted in response to L. amazonensis by lymph nodes isolated from bosutinib-treated mice compared to control mice SFK and dual SFK and Arg inhibitors decrease lesion size and (Table 1, bosutinib); a change in the overall ratio of T-helper 1 to parasite burden in a murine model of cutaneous T-helper 2 cells (Th1/Th2) was not noted (Table S1). Bosutinib did leishmaniasis not affect cytokine release in response to concanavalin A (Con A) Knowing that inhibiting SFK and Arg significantly decreased stimulation in these same mice (Table 1, bosutinib), suggesting that parasite uptake in cultured cells, we next determined whether SFK this change was specific to L. amazonensis stimulation. and Arg inhibitors decreased the manifestations of cutaneous Consequently, the lower response to Leishmania antigen leishmaniasis in mice. Our previous work demonstrated that the Abl stimulation might reflect the reduced parasite load rather than an and Arg inhibitor imatinib decreased lesion size and parasite burden effect of bosutinib on the ongoing host response to infection. To in mice (Wetzel et al., 2012). To test the effects of SFK inhibition, evaluate this point, we examined the direct in vitro effect of we provided 2 mg/kg body weight of PP2 by intraperitoneal bosutinib on the response of lymph node cells to leishmanial injection three times weekly thoughout the course of infection. We antigens. Cytokine secretion did not decrease when infected found that PP2 significantly decreased the average lesion size in draining lymph nodes isolated from DMSO-treated infected mice mice infected with L. amazonensis (Fig. 6A), as well as the parasite were treated with 0.1% DMSO or bosutinib during culture burden (Fig. 6B) by >10 fold. To determine whether inhibiting both (Table S2), suggesting that bosutinib did not directly inhibit SFKs and Arg would also provide benefit, we provided 30 mg/kg cytokine secretion. Cytokine secretion did not significantly change body weight of bosutinib or vehicle (DMSO) in drinking water in lymph nodes isolated from imatinib-treated or PP2-treated mice throughout the course of infection. We found that bosutinib (Table 1, imatinib, PP2), with a few exceptions. Both imatinib (i.e. significantly decreased the average lesion size in L.-amazonensis- Abl family kinase inhibition alone) and PP2 decreased IL-1β levels, infected mice (Fig. 6C). A higher dose of bosutinib (150 mg/kg but based on previous studies, this decrease would not be expected body weight) did not cause additional decreases in lesion size to cause healing (Lima-Junior et al., 2013). PP2 also caused lower (Fig. S2). IFN-γ secretion at high antigenic stimulation (the opposite to what We first tested whether bosutinib impacted on lesions by directly would be expected if the change were contributing to healing in affecting parasite growth or survival. 10 μM bosutinib caused no PP2-treated mice; Soong et al., 2012) and decreased IL-17 secretion growth defects for promastigotes (Fig. S3A) or amastigotes with Leishmania antigenic stimulation. (Fig. S3B). Parasites could transition from promastigotes to Cytokines that attract immune cells to infection sites, termed amastigotes when cultures were grown in bosutinib, either chemokines, could also affect the outcome of leishmaniasis. We axenically (Fig. S3C) or in BMDMs (Fig. S3D). Finally, we next assessed whether Abl and Arg, SFK, or combined Abl and found no survival defects in amastigotes within bosutinib-treated SFK inhibition affected chemokine secretion. The production of previously infected BMDMs (Fig. S3E). chemokines implicated in Leishmania pathogenesis [IP-10, MIP- The reduced lesion size in bosutinib-treated mice could 1α (CCL3), MIP-2 (CXCL2), RANTES (CCL5), CXCL9 and potentially result from differences in the immune response to MIP-1β (CCL4)] did not change with bosutinib or imatinib infection. Of note, mice lacking SFKs and Abl family kinases have (Table S3A,B), suggesting that the Abl- and Arg-mediated significant immunological defects, including deficits in B cell and T differences in chemokines were not responsible for decreased cell development, proliferation and function, which could alter the lesions. PP2 caused decreased secretion of MIP-1α, MIP-2 and course of cutaneous leishmaniasis (Colucci et al., 1999; Gu et al., CXCL9 (Table S3C), but these changes are not known to decrease Journal of Cell Science

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− − − − − − Fig. 3. An SFK-Arg signaling pathway facilitates IgG-mediated phagocytosis and amastigote uptake. (A) Treating Hck / Fgr / Lyn / BMDMs with imatinib does not further decrease (left) IgG-mediated phagocytosis or (right) IgG-opsonized amastigote uptake. n=3 separate experiments. The left graph shows the mean± − − − − − − 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 with − − − − − − imatinib or DMSO (the latter normalized to 100%). The right graph shows the mean±s.e.m. phagocytic index for amastigotes for Hck / Fgr / Lyn / BMDMs incubated with imatinib or DMSO, compared to WT BMDMs incubated with imatinib or DMSO (the latter normalized to 100%). (B) Effects of SU6656 on BMDMs − − − − − − lacking Abl or Arg. Graphs show the mean±s.e.m. phagocytic index for WT, Ablflox/flox LysM Cre+ (referred to as Abl / ), Arg / ,orArg / Ablflox/flox LysM Cre+ (dKO) BMDMs incubated with DMSO or SU6656 and allowed to take up C3bi-coated beads, IgG-coated beads, C3bi-promastigotes or IgG-amastigotes. Data are normalized to WT DMSO-treated BMDMs (set at 100%). n=3 experiments. (C) The Arg activator DPH stimulates IgG-mediated phagocytosis. Shown is one representative experiment of two experiments demonstrating how increasing DPH doses affect the phagocytic index for IgG-coated beads. The maximally − − stimulating dose, 250 nM, was selected for the remaining experiments. (D) DPH rescues IgG-mediated phagocytosis (left) and amastigote uptake (right) in Hck / − − − − 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 or − − − − − − Hck / Fgr / Lyn / BMDMs treated with DMSO or DPH. The right graph shows the mean±s.e.m. phagocytic index for IgG-amastigotes incubated with WT − − − − − − BMDMs or Hck / Fgr / Lyn / BMDMs treated with DMSO or DPH. (E) Effects of DPH on BMDMs lacking Abl or Arg. Graphs show the mean±s.e.m. phagocytic − − − − index 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-incubated WT BMDMs (100%) for each condition. n=4

experiments. For all categories, *P<0.05; **P<0.01; n.s., not significant compared with DMSO-treated WT BMDMs (ANOVA). Journal of Cell Science

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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 each condition normalized to DMSO treatment (100%). A representative experiment of two experiments is shown. (B,C) Bosutinib decreases C3bi- or IgG-coated bead uptake. 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 BMDMs treated 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-opsonized promastigotes and IgG-opsonized amastigotes incubated with bosutinib-treated BMDMs (E) or RAW 264.7 cells (F), normalized to DMSO-treated cells. n=3 experiments. *P<0.05; **P<0.01 (by one-sample t-test). lesion size (Brandonisio et al., 2002; Lima-Junior et al., 2013; that the decreased cytokine secretion in bosutinib-treated mice Muller et al., 2003; Oghumu et al., 2010; Ritter and Korner, 2002; resulted from lower parasite burdens in these mice. Therefore, we Santiago et al., 2004). determined the parasite burdens in DMSO- and bosutinib-treated Given that bosutinib caused a significant inhibition of parasite mice at the end of the experiment (∼3 months of infection) by uptake, and uptake is required for parasite survival, we suspected limiting dilution. We found that the number of parasites contained Journal of Cell Science

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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 uncoated plates (−) or plates coated with anti-P8 antibody-opsonized amastigotes (+ amastigotes) for 15 min before processing for immunoblotting. (A) Representative immunoblot 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 shown in 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 LysM Cre+ (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-treated WT BMDMs (+ amastigotes), and DMSO- or DPH-treated − − − − Abl / , Arg / or dKO BMDMs (all +amastigotes). (D) Graph shows relative pCrk levels, normalized to total Crk levels, among the categories of BMDMs shown in 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 significant compared with amastigote-stimulated DMSO-treated WT BMDMs (ANOVA). within the infected foot was far lower in bosutinib-treated mice than DISCUSSION in DMSO-treated mice (Fig. 6D), with an average decrease of Previously, we have demonstrated that the host Arg non-receptor ∼50-fold. These results suggest that understanding the host cell tyrosine kinase facilitates L. amazonensis amastigote uptake by processes required for parasite uptake could provide new candidates macrophages (Wetzel et al., 2012). The results shown here are the for treating leishmaniasis. first to demonstrate that SFKs, and specifically Hck, Fgr and Lyn, Journal of Cell Science

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Fig. 6. SFKs, Abl and Arg permit efficient infection in a mouse model of cutaneous leishmaniasis. (A) PP2-treated mice have smaller lesions than untreated 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 DMSO by 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-treated mice contain fewer parasites than DMSO-treated mice (quantified by limiting dilution). Plotted is the mean±s.e.m. total lesion parasite 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 versus 179×103±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 to eight 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 days before infection and continuing until the mice were euthanized. Three experiments were performed; shown is a representative experiment containing five mice per group. *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 the end of the experiment in C; here there were 3.3×106±0.7×106 parasites in the DMSO-treated mice versus 61×103±21×103 parasites in the bosutinib-treated mice. **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. also facilitate amastigote uptake. SFKs activate Arg during efficient Leishmania amastigotes isolated from lesions are coated with IgG amastigote uptake. Finally, the small-molecule combination Arg and primarily bind to the FcR to stimulate uptake (Kima et al., and SFK inhibitor bosutinib decreases amastigote uptake by 2000). Based on their role during IgG-mediated phagocytosis, one macrophages and significantly reduces disease severity and might predict a role for SFKs during Leishmania uptake. However, parasite burden in Leishmania-infected mice. In summary, host a previous in vivo study in mice lacking the SFK Fyn failed to cell kinase inhibitors might provide novel exploratory candidates for demonstrate an effect of SFKs during Leishmania infection the treatment of leishmaniasis. (Yamakami et al., 2001), in retrospect, likely due to redundant Journal of Cell Science

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Table 1. Cytokine secretion in bosutinib-, imatinib- and PP2-treated mice. Cytokine secretion (pg) after Cytokine Treatment High stimulation Low stimulation Con A stimulation Bosutinib IFN-γ DMSO 23,861±5919 7476±2264 3511±239 Bosutinib 5404±989 6743±1636 3253±284 P-value 0.024 n.s. n.s. IL-1α DMSO 24.7±7.84 40.0±11.2 32.1±11.3 Bosutinib 58.3±23.0 64.7±21.4 58.6±18.9 P-value 0.053 n.s. n.s. IL-1β DMSO 49.1±5.0 75.6±17.2 62.0±14.5 Bosutinib 91.5±24.9 82.1±22.8 93.0±21.7 P-value n.s. n.s. n.s. IL-4 DMSO 1892±296 1558±326 843±273 Bosutinib 1036±232 537±104 712±244 P-value 0.052 0.0086 n.s. IL-10 DMSO 1063±223 892±114 938±220 Bosutinib 447±71.5 467±85.8 862±162 P-value 0.012 0.0099 n.s. IL-13 DMSO 1053±205 1043±118 416±203 Bosutinib 978±69.2 466±91.3 482±247 P-value n.s. 0.0030 n.s. IL-17 DMSO 225±20.0 314±55.6 303±27.7 Bosutinib 146±26.0 189±41.3 347±111 P-value 0.041 n.s. n.s. Imatinib IFN-γ DMSO 7151±3455 6165±2231 4504±1604 Imatinib 10,536±4505 11,689±4645 4678±473 P-value n.s. n.s. n.s. IL-1α DMSO 135±28.9 173±29.9 188±32.9 Imatinib 83.5±18.4 88.6±20.5 94.6±22.9 P-value n.s. n.s. n.s. IL-1β DMSO 116±23.3 85.2±29.3 119±24.8 Imatinib 37.2±14.2 40.2±12.1 67.0±20.4 P-value 0.035 n.s. 0.037 IL-4 DMSO 884±280 711±251 854±209 Imatinib 1474±293 681±189 1372±223 P-value n.s. n.s. n.s. IL-10 DMSO 559±121 364±118 468±56.8 Imatinib 904±152 629±194 647±88.7 P-value 0.097 n.s. n.s. IL-13 DMSO 159±35.5 121±29.6 237±62.1 Imatinib 413±127 123±17.5 569±261 P-value 0.079 n.s. n.s. IL-17 DMSO 90.1±18.5 111±36.8 210±93.8 Imatinib 208±63.5 257±88.1 213±86.7 P-value 0.080 n.s. n.s. PP2 IFN-γ DMSO 22,775±4233 9664±1583 11,896±2733 PP2 9087±2662 9258±2335 8692±2577 P-value 0.031 n.s. n.s. IL-1α DMSO 196±35.5 179±43.7 138±23.8 PP2 92.2±9.18 106±11.8 82.3±7.28 P-value n.s. n.s. 0.019 IL-1β DMSO 66.6±20.7 59.2±17.6 62.7±18.5 PP2 20.0±3.19 34.0±5.23 23.9±4.93 P-value 0.027 n.s. 0.040 IL-4 DMSO 974±291 420±119 366±86.2 PP2 652±226 1000±346 289±80.2 P-value n.s. n.s. n.s. IL-10 DMSO 1035±280 793±169 433±75.1 PP2 831±189 965±349 418±55.4 P-value n.s. n.s. n.s. IL-13 DMSO 517±143 428±114 787±216 PP2 189±46.4 430±157 972±410 P-value 0.10 n.s. n.s. Continued Journal of Cell Science

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Table 1. Continued Cytokine secretion (pg) after Cytokine Treatment High stimulation Low stimulation Con A stimulation IL-17 DMSO 263±40.1 305±51.7 364±37.5 PP2 103±10.5 160±23.1 240±60.8 P-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 lymph nodes 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 with Leishmania antigen stimulation is not significantly affected in imatinib-treated mice. Shown are profiles of draining lymph nodes isolated from eight DMSO versus imatinib-treated mice at the end of experiments described in Wetzel et al., 2012. PP2: cytokine production in DMSO versus PP2-treated mice during Leishmania antigen 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. For each 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 (low stimulation), or with Con A, as a positive control. Cytokine ELISAs were performed on harvested supernatants. P-values were determined by two-tailed t-tests and are listed if P<0.1. n.s., not significant. roles for multiple SFK members. Our work delineates specific roles As kinase inhibitors do not prevent 100% of activity in vivo, for Hck, Fgr and Lyn for stimulating amastigote uptake, both based targeting multiple signaling molecules in the same pathway could, on pharmacological inhibition and BMDMs from mice lacking in theory, yield a greater effect than inhibiting a single kinase. Thus, these three kinases. we turned to the potent combination SFK, Abl and Arg inhibitor Our work demonstrates that SFKs signal through Arg to facilitate bosutinib. Despite its effects on multiple host cell kinases, there are amastigote uptake, as modeled in Fig. 6E. We do not observe sufficiently few side effects that bosutinib was approved by the FDA additional deficits in IgG-mediated phagocytosis or amastigote to treat chronic myelogenous leukemia (Rusconi et al., 2014). We uptake when Hck−/− Fgr−/− Lyn−/− BMDMs are treated with find that bosutinib prevented C3bi- and IgG-mediated phagocytosis, imatinib or when Arg−/− BMDMs are treated with SU6656, as well as promastigote and amastigote uptake. indicating that SFKs and Arg lie in the same signaling pathway, as Our mouse studies also indicate that Leishmania survival and previously shown in other systems (Mader et al., 2011; Plattner pathogenesis depend on SFKs, Abl and Arg. We propose that the et al., 2004; Tanis et al., 2003). Another report has indicated that smaller lesions seen in these kinase-inhibited mice result at least partly macrophages treated with both imatinib and SU6656 had IgG- from uptake defects in macrophages. Consistent with this proposal, mediated phagocytosis defects beyond those seen with either drug fewer parasites are contained in lesions in bosutinib-treated mice than alone (Greuber and Pendergast, 2012), suggesting that SFKs, and in controls. Interestingly, bosutinib-treated mice had a larger decrease Arg and Abl might partly signal through different pathways. These in parasite burden than we saw with imatinib (Wetzel et al., 2012) or differences might have occurred if imatinib and SU6656 did not PP2 therapy. Thus, our results imply, although do not show directly, completely inhibit Abl, Arg and SFK activity, or if the kinases also that inhibiting multiple signaling required for parasite uptake provide a scaffolding function. In both cases, drug treatment alone might improve results over inhibiting a single kinase alone. might not fully prevent this IgG-mediated uptake pathway. Given that there are no direct effects of bosutinib treatment seen Furthermore, using BMDMs lacking Abl or Arg, we directly on parasite replication or survival within macrophages, the lesion demonstrate that a SFK–Arg pathway, and not a SFK–Abl pathway, size disparities in L. amazonensis infection could result from is employed during IgG-mediated phagocytosis and amastigote differences in cell entry and resulting parasite burden, or from uptake. These data are consistent with our previous report showing differences in the host inflammatory and/or immune response that Abl is required for C3bi-mediated phagocytosis and (Soong et al., 1995). The murine immune response to leishmaniasis promastigote uptake, whereas Arg is required for IgG-mediated is complicated and is dependent on the parasite species and mouse phagocytosis and amastigote uptake (Wetzel et al., 2012). We strain. In general, Th1 responses are protective, and Th2 responses suspect that the differential usage of Abl and Arg during are deleterious to the host (Jones et al., 1998). For example, phagocytosis and Leishmania uptake is due to their different inhibiting phosphoinositide 3-kinase (PI3K)γ, which can signal subcellular localization within macrophages. though SFKs, Abl and Arg (Bradley and Koleske, 2009), affects We find that activating Arg in Hck−/− Fgr−/− Lyn−/− BMDMs Leishmania uptake and impairs the Th2 response (Cummings et al., with low or sub-EC50 doses (Yang et al., 2011) of DPH rescues 2012). Mice lacking SFKs have defects in macrophage recruitment uptake. Ours is the first demonstration that activating Abl family (Park et al., 1999), the respiratory burst (Meng and Lowell, 1998), kinases facilitates phagocytosis. Interestingly, however, higher and B cell and T cell development and signaling (Lowell, 2011). doses of DPH inhibit phagocytosis, suggesting that either there is a Immunological defects in mice lacking Abl family kinases include point where overly active Arg is deleterious, or that the drug affects defects in B cell and T cell development and signaling, among other essential molecules at high concentrations. The action of SFKs others; drugs affecting SFK and Abl and Arg also decrease immune and Arg allows phosphorylation of their downstream mediator CrkII cell proliferation (Gu et al., 2007; Huang et al., 2008; Liberatore during FcR engagement by amastigotes. We also find that DPH and Goff, 2009; Silberman et al., 2008; Zipfel et al., 2004). All of increases CrkII phosphorylation in an Arg-dependent manner, these effects could contribute to the smaller lesions in Leishmania- consistent with previous observations (Yang et al., 2011). Clearly, infected bosutinib-treated mice. SFKs and Arg-independent mechanisms also contribute to IgG- We find that incubating cultured lymph nodes with bosutinib mediated phagocytosis and amastigote uptake, given that inhibiting does not directly affect cytokine secretion. Cytokine profiling of both kinases does not completely prevent these processes. Future lymph nodes isolated from infected bosutinib-treated mice studies will focus on uncovering novel signaling pathways demonstrated a decrease in many cytokines measured after permitting Leishmania uptake and further delineating the SFK– Leishmania antigen stimulation, but not after Con A stimulation.

Arg signaling pathway that occurs. These decreases are generally not statistically significant with Abl Journal of Cell Science

3139 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3130-3143 doi:10.1242/jcs.185595 and Arg, or SFK inhibition alone, and none of the changes seen with LysM Cre+ (termed dKO)] from a mixed background (C57BL/6X 129Sv/J) single inhibition are known to facilitate healing (Ehrlich et al., 2014; were backcrossed to C57BL/6 more than five times; littermates provided −/− −/− −/− Lima-Junior et al., 2013; Soong et al., 2012), with the possible controls. Hck, Fgr and Lyn triple-knockout mice (Hck Fgr Lyn ) exception of the decreased IL-17 secretion seen with PP2 (Sousa were as described in Hu et al. (2004). The Institutional Animal Care and Use et al., 2014). One potential explanation for the bosutinib-induced Committees at Yale University and UT Southwestern approved all protocols. cytokine decreases is that a lower parasite burden causes a diminished amount of cytokines to be secreted in response to L. amazonensis antigen. Another possibility is that Abl, Arg and Cell culture RAW 264.7 cells (ATCC, Manassas, VA) were grown in Dulbeco’s SFKs promote an immune response to L. amazonensis, which is modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine dampened by kinase inhibition, and does not reach statistical serum (FBS; Invitrogen, Grand Island, NY). For primary macrophage significance unless both families are inhibited. Unfortunately, it is experiments, cells were harvested from femurs and tibias of WT, Ablflox/flox difficult to distinguish between these possibilities in our system. LysM Cre+, Arg−/−, dKO, and Hck−/− Fgr−/− Lyn−/− mice and Even in bosutinib-treated mice, the ratios of IFN-γ to TH2 cytokines differentiated into bone-marrow-derived macrophages (BMDMs), which do not improve, suggesting that the differences in lesion size and was confirmed as in Wetzel et al. (2012). Cells were tested for Mycoplasma parasite burden are not fully explained by defective immune contamination (Uphoff and Drexler, 2013). responses to Leishmania infection. However, decreases in IL-10, like those described here, could facilitate lesion healing and Parasite culture decrease parasite burden during L. amazonensis murine infection L. amazonensis promastigotes (strain IFLA/BR/67/PH8, from Norma (Ji et al., 2005), contributing to the effects of bosutinib. W. Andrews, University of Maryland, College Park, MD) were grown in Chemokine profiling also does not reveal explanations for the Schneider’s Drosophila medium with 10 μg/ml gentamicin and 15% heat- smaller lesions seen with Abl and Arg, or SFK inhibitors. We inactivated FBS at 24°C (Wetzel et al., 2012). For uptake experiments, examined levels of IP-10, MIP-1α, MIP-2, RANTES, CXCL9 and promastigotes were incubated for 7 days to maximize infective metacyclics β (i.e. those isolated through a step Percoll gradient; Sigma, St Louis, MO). MIP-1 . Several changed upon PP2, but not bosutinib or imatinib Amastigotes were grown axenically (Wetzel et al., 2012). To assess for treatment, and, based on previous studies, these changes would not growth defects with bosutinib, medium containing 0.1% DMSO or 10 μM be expected to facilitate healing (Brandonisio et al., 2002; Lima- bosutinib (LC Laboratories, Woburn, MA) was used. Bosutinib remained in Junior et al., 2013; Muller et al., 2003; Oghumu et al., 2010; Ritter solution at neutral pH and has improved solubility at lower pH (Remsing Rix and Korner, 2002; Santiago et al., 2004). Of note, PP2 inhibits et al., 2009). Parasites were passed through mice to maintain virulence. multiple targets, not just SFKs (>50 targets were seen in Brandvold et al., 2012), and these off-target effects could contribute to its Phagocytosis assays chemokine and cytokine profile. More studies of chemokine RAW 264.7 cells or BMDMs were plated at ∼50% confluence and secretion after host manipulations like kinase inhibition might be incubated overnight in serum-free medium or M-CSF-starved medium, beneficial for unraveling the pathogenesis of leishmaniasis. respectively. Except where indicated, for drug experiments with IgG-coated μ Additionally, mice with defects in B cells or T cells can be disease beads, coverslips were preincubated in medium containing 3.3 M imatinib (LC Laboratories), 2.5 μM SU6656 (Sigma), 10 μM PP2 (Sigma), 1 μM resistant following L. amazonensis infection (Soong et al., 1997). bosutinib (LC Laboratories) or DMSO (0.1%) (Sigma) for 2 h. For C3bi- Indeed, the diminished swelling that we see in bosutinib-treated coated bead uptake, cells were preactivated with phorbol 12-myristate mice might actually be less than what might have been expected 13-acetate (PMA; Sigma), which added 0.1% DMSO to all PMA-treated from the parasite burden decrease alone. Using another Leishmania cells. Experiments were conducted as described previously (Wetzel et al., species might distinguish whether kinase-inhibition-induced 2012). Briefly, 2-μm latex yellow–green beads (Sigma) coated with human immune deficiencies are diminishing the effect we would IgM (cat. no. I-8260, Sigma) were incubated in fresh mouse serum for C3bi otherwise see from bosutinib. opsonization or rabbit anti-IgM (cat. no. 270A, Sigma) for IgG opsonization In summary, we have shown that SFKs signal through Arg to (confirmed as described previously; Wetzel et al., 2012). Cells were facilitate IgG-mediated phagocytosis and Leishmania uptake. SFKs, incubated with 10–15 beads/cell for 30 min at 37°C, fixed with 3% and Abl and Arg also govern the subsequent pathogenesis of formaldehyde for 15 min, blocked with 2% BSA without permeabilization, incubated with rabbit anti-human IgM plus Hoechst 33258 dye (Sigma) to leishmaniasis in the mouse model. These results strongly suggest that visualize nuclei, then incubated with Alexa-Fluor-594-conjugated goat anti- improving our understanding of cell entry by Leishmania could rabbit-IgG secondary antibody (cat. no. A11034, Invitrogen). This two- provide novel treatment strategies for leishmaniasis. Furthermore, they color immunofluorescence assay (Wetzel et al., 2003) allowed support the concept that drugs that target conserved host cell distinguishing of internalized and external beads. Coverslips were processes, rather than the infectious agents themselves, could be visualized using a 40×1.0 NA aperture Nikon objective on a Nikon used to treat a broad range of infectious diseases, including parasitic Eclipse TE2000-5 fluorescence microscope by a treatment-blinded diseases like leishmaniasis. Thus far, we have focused on clinically observer. Images were acquired with a Qimaging Cooled Charge-coupled available tyrosine kinase inhibitors with relatively benign side effect Device mono, 12-bit camera and Nikon Imaging software. At least ten profiles. In contrast, the agents currently used to treat leishmaniasis randomly selected fields were visualized for a total of over 100 macrophages have multiple toxic side effects, and resistance to these drugs is and beads per experiment. The mean phagocytic index (the number of particles internalized per 100 macrophages) for controls was set as the emerging. Host-cell-active agents could be combined with current maximum (100%) value for each experiment, and those for experimental antiparasitics, increasing their efficacy while lowering their toxicity. conditions were normalized to that value. Each experiment was performed Future work will continue to explore the potential use of cell entry three times and the mean±s.e.m. was calculated. A one-sample Student’s inhibitors for leishmaniasis. t-test or two-way ANOVA was used to determine statistical significance.

MATERIALS AND METHODS Leishmania uptake assays Mice BMDMs or RAW 264.7 cells were plated and treated with inhibitors, C57BL/6 mice were obtained from Jackson Labs (Bar Harbor, ME). Abl and DMSO or PMA as above. Metacyclic promastigotes were incubated in fresh flox/flox −/− −/− flox/flox Arg knockout mice [Abl LysM Cre+, Arg , and Arg Abl mouse serum for C3bi opsonization, and BMDMs or RAW 264.7 cells were Journal of Cell Science

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PMA-activated for 1 h. Amastigotes were coated with anti-P8- size was monitored with calipers weekly by an investigator blinded to proteoglycolipid complex (monoclonal antibody IgG1; Pan and condition, and the infected:uninfected size ratio was calculated (Champsi McMahon-Pratt, 1988). Coverslips with RAW cells or BMDMs were and McMahon-Pratt, 1988). The control and drug-treated groups were incubated with C3bi-opsonized promastigotes at a ratio of ten parasites to compared to swelling at time 0 by ANOVA. Parasite burdens in lesions were one cell or with IgG-opsonized amastigotes at two parasites to one cell for determined upon experimental termination between experimental weeks 12 30 min and fixed with 3% formaldehyde. External promastigotes were and 18 by limiting dilution (Soong et al., 1997). incubated with mouse anti-gp46 antibody; external amastigotes were Lymph nodes from infected DMSO versus drug-treated mice were incubated with mouse anti-P8 antibody as described previously (Wetzel harvested for cytokine and chemokine profiling (Soong et al., 1997, 1995, et al., 2012). All were incubated with Alexa-Fluor-568-conjugated donkey 1996). Cells were plated and stimulated with promastigote lysates or Con A anti-mouse-IgG secondary antibody (cat. no. A10037, Invitrogen). After (5 µg/ml; Sigma) (Wetzel et al., 2012). Supernatants were obtained after permeabilization, parasites were re-incubated with mouse anti-gp46 72 h. The cytokines IL-4, IL-10, IL-13, IL-17 or IFN-γ were assessed by (promastigotes) or mouse anti-P8 (amastigotes) antibodies and Alexa- ELISA (BD Biosciences, San Jose, CA). Background levels were assessed Fluor-488-conjugated donkey anti-mouse-IgG secondary antibody (cat. no. using unstimulated cell supernatants. Levels of the cytokines and A21202, Invitrogen) and DAPI (Sigma). At least ten random fields were chemokines IL-1β, IL-1α, IP-10, MIP-1α (CCL3), MIP-2 (CXCL2), selected containing at least 100 parasites and macrophages per experiment; RANTES (CCL5), and MIP-1β (CCL4) were profiled by using the custom each experiment was performed three times. The mean±s.e.m. of biological multiplex Luminex Platform (R&D Systems, Inc, Minneapolis, MN) replicates is shown. Images for analysis were collected as above. The through the UT Southwestern Microarray Core as described previously representative images shown in Figs 1 and 4 were visualized with a Zeiss (Gonzalez-Fajardo et al., 2015; Navas et al., 2014). CXCL9 levels were LSM 700 confocal microscope with a Zeiss 40×1.3 NA EC Plan-Neofluar assessed by ELISA (R&D Systems, Inc). oil objective (promastigotes) or a Zeiss 63×1.4 NA Plan-Apochromat objective (amastigotes), acquired with a Zeiss Axiocam 506 mono, 6 Acknowledgements megapixel monochrome camera and Zeiss Imaging software (Zeiss, Jena, We thank Norma W. Andrews for providing L. amazonensis strain IFLA/BR/67/PH8 Germany), and linearly processed in Adobe Photoshop (Adobe Systems, and the Koleske and McMahon-Pratt laboratories for helpful discussions. We thank San Jose, CA). A one-sample Student’s t-test or two-way ANOVA was used Margaret A. Phillips and her laboratory for the use of equipment and reagents, to determine statistical significance. James J. Collins for confocal microscope use, the parasitology group at UT Southwestern for valuable discussions, Indu Raman at the UT Southwestern Microarray Core for assistance with cytokine and chemokine profiling, and Emily Intra-macrophage assays T. Mamula, Imran Ullah, Hanspeter Niederstrasser, Meghan E. Kerrisk and Margaret For assays examining promastigote–amastigote transition within A. Cooper for technical and experimental assistance. macrophages or intracellular survival, 10 L. amazonensis promastigotes were added per WT BMDM cell (starved of G-CSF overnight) and plated in Competing interests triplicate wells at 50% confluence. After 4 h, the BMDMs were washed 5× The authors declare no competing or financial interests. with PBS, and DMSO or 2 μM bosutinib in medium containing G-CSF was added. The number of parasites per 100 BMDMs was counted at 24 h to Author contributions assess amastigote transition or 72 h to assess survival. D.M.W. designed and performed experiments, analyzed data and wrote the manuscript. E.L.R. performed experiments and collected and analyzed data. S.L. −/− −/− −/− Immunoblotting assisted with the Hck Fgr Lyn mouse studies and manuscript preparation. To measure phosphorylation of the downstream SFK, Abl and D.M.-P. and A.J.K. participated in experimental design and data analysis, discussed Arg effector CrkII (42 kDa), starved RAW 264.7 cells or WT, results and assisted with manuscript preparation. Ablflox/flox LysM Cre+, Arg−/− or dKO BMDMs were added for 15 min to uncoated dishes or dishes precoated for 1 h with anti-P8 antibody (Pan and Funding This work was supported by the National Institutes of Health [grant numbers McMahon-Pratt, 1988) IgG-opsonized amastigotes. Cells were lysed with a NRSA F32 AI094905 and K08 AI103036 to D.M.W.; R01 CA122142 to S.L.; R01 buffer of 20 mM Tris-HCl pH 7.2, 2 mM EDTA, 150 mM NaCl and 1% AI093775 to D.M.-P.; and R01 CA133346, R01 GM100411, and R01 NS089662 to Triton X-100 that contained protease and phosphatase inhibitors. For A.J.K.]; and funds from the University of Texas Southwestern Medical Center, representative images, equivalent protein amounts were loaded on 10% Department of Pediatrics (to D.M.W. and E.L.R.). Deposited in PMC for release SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with after 12 months. antibodies against phosphorylated CrkII (pCrkII) (Y221, cat. no. 3491S, Cell Signaling, Beverly, MA) at 1:1500, or CrkII (cat. no. sc-9004, Santa Supplementary information Cruz Biotechnology, Dallas, TX) at 1:500. For analysis, relative amounts of Supplementary information available online at pCrkII were compared with Image J analysis software and were normalized http://jcs.biologists.org/lookup/doi/10.1242/jcs.185595.supplemental to CrkII (membranes were stripped of pCrkII and reprobed for CrkII, as in Wetzel et al., 2012). Imaging was performed by using a phosphorimager References (ImageQuant LAS 4000, GE). Two-way ANOVA was used to determine Abram, C. L. and Lowell, C. A. (2008). The diverse functions of Src family kinases statistical significance. in macrophages. Front. Biosci. 13, 4426-4450. Arai, R., Tsuda, M., Watanabe, T., Ose, T., Obuse, C., Maenaka, K., Minami, A. and Ohba, Y. (2012). Simultaneous inhibition of Src and Aurora kinases by Murine infections SU6656 induces therapeutic synergy in human synovial sarcoma growth, invasion Four to eight female C57BL/6 mice per group were infected at between 6 and angiogenesis in vivo. Eur. J. Cancer 48, 2417-2430. and 8 weeks of age. Power calculations were performed as in Wetzel et al., Bain, J., McLauchlan, H., Elliott, M. and Cohen, P. (2003). The specificities of 2012. 1×106 metacyclic promastigotes in PBS were injected subcutaneously inhibitors: an update. Biochem. J. 371, 199-204. in the dorsal right hind foot. Imatinib experiments were performed as Bavagnoli, L., Dundon, W. G., Garbelli, A., Zecchin, B., Milani, A., Parakkal, G., Baldanti, F., Paolucci, S., Volmer, R., Tu, Y. et al. (2011). The PDZ-ligand described previously (Wetzel et al., 2012). For PP2-treated mice, two and Src-homology type 3 domains of epidemic avian influenza virus NS1 independent experiments were performed. Mice were provided 2 mg/kg protein modulate human Src kinase activity during viral infection. 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