Tissue inhibitor of metalloproteinases-1 (TIMP-1) exerts pleiotropic effects on cells including conferring metastatic properties to cancer cells. As for metastatic cells, recent paradigms of leukocyte migration attribute important roles to the amoeboid migration mode of dendritic cells (DCs) for rapid locomotion in tissues. However, the role of TIMP-1 in immune cell migration and in the context of infection has not been addressed. We report that, upon challenge with the obligate intracellular parasite Toxoplasma gondii, primary DCs secrete TIMP-1 with implications for their migratory properties. Using a short hairpin RNA (shRNA) gene silencing approach, we demonstrate that secreted TIMP-1 and its ligand CD63 are required for the onset of hypermotility in DCs challenged with T. gondii. Further, gene silencing and antibody blockade of the β1-integrin CD29 (ITGB1) inhibited DC hypermotility, indicating that signal transduction occurred via ITGB1. Finally, gene silencing of the ITGB1-associated focal adhesion kinase (FAK, also known as PTK2), as well as pharmacological antagonism of FAK and associated kinases SRC and PI3K, abrogated hypermotility. The present study identifies a TIMP-1–CD63–ITGB1–FAK signaling axis in primary DCs, which T. gondii hijacks to drive high-speed amoeboid migration of the vehicle cells that facilitate its systemic dissemination.
Resident dendritic cells (DCs) in the intestinal lamina propria are among the first leukocytes to encounter pathogens. The obligate intracellular parasite Toxoplasma gondii infects virtually all warm-blooded vertebrates, with an estimated 30% of humans chronically infected (Pappas et al., 2009). From the point of entry in the intestinal tract, T. gondii rapidly achieves systemic dissemination and establishes chronic infection of the central nervous system. Reactivated and acute infection in immunosuppressed or immunocompromised individuals can lead to lethal toxoplasmic encephalitis (Montoya and Liesenfeld, 2004).
Upon T. gondii infection, DCs play a determinant role in pathogenesis by driving protective T helper 1 (Th1) responses through interleukin-12 (IL-12) production (Liu et al., 2006). Paradoxically, the T. gondii tachyzoite stage exploits the inherent migratory ability of DCs for dissemination via a ‘Trojan horse’ mechanism (Lambert et al., 2006, 2009b; Courret et al., 2006; Bierly et al., 2008). Within minutes of active invasion by tachyzoites, DCs adopt a hypermigratory phenotype (reviewed in Weidner and Barragan, 2014), which mediates rapid systemic dissemination of the parasite in mice (Lambert et al., 2006; Kanatani et al., 2017). This dramatic migratory activation requires the discharge of parasitic secretory organelles into the host cell cytoplasm (Weidner et al., 2013) and is triggered by GABAA receptor-mediated (Fuks et al., 2012) activation of voltage-dependent calcium channels (VDCC) on the host DC plasma membrane (Kanatani et al., 2017). Tachyzoite-infected DCs undergo rapid cytoskeletal reorganization, express enhanced locomotion on 2-dimensional (2D) surfaces (termed hypermotility) (Weidner et al., 2013), enhanced migration in 3D collagen matrix (Kanatani et al., 2015) and enhanced transmigration in vitro (Lambert et al., 2006).
After encountering a foreign antigen, DCs undergo a complex maturation process, encompassing upregulation of co-stimulatory molecules and MHC class II. A shift in chemokine receptor expression facilitates homing to secondary lymphoid organs (Ohl et al., 2004). Another hallmark of DC maturation is the transition from mesenchymal to amoeboid (MAT) migration mode, which enables high-speed locomotion through interstitial tissues and across biological barriers (Alvarez et al., 2008; Lämmermann et al., 2008; Friedl and Wolf, 2003). In contrast to mesenchymal migration, amoeboid migration is not dependent on firm adhesion to and degradation of extracellular matrix (ECM). Instead, the protrusive flow of the actin cytoskeleton primarily drives locomotion (Lämmermann et al., 2008). It was recently reported that T. gondii tachyzoites induce MAT in infected DCs but the signaling behind this dramatic migratory activation remained unknown (Weidner et al., 2013; Kanatani et al., 2015; Olafsson et al., 2018).
The tissue inhibitors of matrix metalloproteinases (TIMPs) comprise four members (TIMP-1, -2, -3 and -4), which tightly regulate the turnover of ECM proteins as inhibitors of matrix metalloproteinases (MMPs) (reviewed in Visse and Nagase, 2003). However, numerous MMP-independent roles for the TIMPs have been identified, including pro-metastatic roles (reviewed in Stetler-Stevenson, 2008). Specifically, previous studies identified CD63 as a receptor for TIMP-1 in human breast epithelial cells (Jung et al., 2006). CD63, a ubiquitously expressed tetraspanin, is localized at the plasma membrane and within the endosomal system. Like other tetraspanins, CD63 interacts with numerous proteins, including integrins (Radford et al., 1996). Upon binding TIMP-1, CD63 activates the β1-subunit of integrins (ITGB1) (Jung et al., 2006; Lee et al., 2014). In normal and cancer cells, activated ITGB1 recruits focal adhesion kinase (FAK), which auto-phosphorylates Tyr-397 and subsequently recruits SRC kinase (Ando et al., 2018) and phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) (Toricelli et al., 2013), leading to MAPK activation and cytoskeletal rearrangements (Jung et al., 2006; Ando et al., 2018). Mounting evidence suggests TIMP-1–CD63 signaling functions as a key regulator of cancer cell survival (Jung et al., 2006), metastasis (Forte et al., 2017) and stem cell migration (Wilk et al., 2013; Lee et al., 2014). However, the role of TIMP-1–CD63 signaling in leukocyte biology remains enigmatic.
Previously, we reported that autocrine TIMP-1 secretion upon Toxoplasma challenge impedes the ability of DCs to degrade ECM (Olafsson et al., 2018). Here, in an infection model with the tachyzoite stage of T. gondii, we address the impact of TIMP-1 in the migratory activation of DCs and define a TIMP-1–CD63–ITGB1–FAK signaling axis in primary DCs.
Toxoplasma-challenged DCs secrete TIMP-1, and Timp-1 gene silencing inhibits DC hypermotility
We recently reported that in Toxoplasma-challenged DCs, autocrine TIMP-1 secretion reduced degradation of the ECM (Olafsson et al., 2018). Because TIMP-1 has also been associated with the invasive properties of metastatic cancer cells, we investigated the role of TIMP-1 in the hypermigratory phenotype exhibited by tachyzoite-infected DCs derived from C57BL/6 mice. Shortly after challenge with Toxoplasma, DCs upregulated Timp-1 mRNA expression (Fig. 1A) and secreted TIMP-1 protein (Fig. 1B). To investigate whether secreted TIMP-1 impacted on Toxoplasma-induced DC hypermotility, we applied a shRNA gene silencing approach. First, the transduction and knockdown efficiency of lentivirus targeting Timp-1 mRNA (shTIMP-1) and control virus (shLuc) was quantified. ShTIMP-1-transduced DCs exhibited significantly reduced levels of Timp-1 mRNA expression (Fig. 1C) and secreted TIMP-1 protein (Fig. 1D). Next, the motility of Toxoplasma-challenged transduced DCs was analyzed (Fig. 1E,F). Importantly, transduction with shTIMP-1 significantly inhibited the motility and mean velocities of Toxoplasma-infected DCs, while the baseline motility and mean velocities of non-challenged DCs were non-significantly affected by shTIMP-1 transduction (Fig. 1F,G). Further, recombinant mouse TIMP-1 (rmTIMP-1) partially restored hypermotility in Toxoplasma-challenged TIMP-1-silenced DCs (Fig. 1F,H). In contrast, treatment with rmTIMP-1 impacted non-significantly on the motility of mock-treated DCs or shLuc-transduced unchallenged (Fig. S1A,B) and Toxoplasma-challenged DCs (Fig. S1A,C). The finding that TIMP-1 modulates the hypermigratory phenotype of Toxoplasma-infected DCs prompted us to investigate the signaling pathways that mediate this infection-related enhanced migration of DCs.
Silencing of the Cd63 gene suppresses DC hypermotility
Because CD63 is the only described non-MMP binding partner for TIMP-1 (Jung et al., 2006) and the CD63–TIMP-1 complex has been associated with cytoskeletal rearrangements (Lee et al., 2014), we investigated the putative implication of CD63 in DC hypermotility. First, we assessed Cd63 mRNA and CD63 protein expression in unchallenged and Toxoplasma-infected DCs. Cd63 mRNA expression was slightly downregulated upon infection (Fig. S2A) and CD63 antibodies stained unchallenged and Toxoplasma-infected DCs (Fig. 2A,B). Flow cytometry analyses detected non-significant differences in total and plasma membrane CD63 protein expression between unchallenged DCs, Toxoplasma-infected DCs and bystander DCs (Fig. 2C; Fig. S2B,C). Next, we silenced Cd63 mRNA expression to investigate its role in hypermotility. DCs transduced with shCD63 (Fig. S2D) exhibited significantly reduced Cd63 mRNA (Fig. 2D) and protein expression (Fig. 2E; Fig. S2E). Consonant with motility data from Timp-1-silenced DCs (Fig. 1), gene silencing of Cd63 significantly reduced the motility and the mean velocities of infected DCs, which reached velocities non-significantly different from unchallenged DCs (Fig. 2F,G). Taken together, these data situate CD63 as a component in the signaling cascade that facilitates hypermotility and support the involvement of TIMP-1–CD63 signaling in the migratory activation of Toxoplasma-infected DCs.
ITGB1 signaling is implicated in DC hypermotility
Because the TIMP-1–CD63 complex interacts with and activates ITGB1 integrin signaling (Jung et al., 2006; Lee et al., 2014), we examined ITGB1 signaling in hypermotility. Antibody staining indicated ITGB1 protein expression in both unchallenged DCs, Toxoplasma-infected DCs and bystander DCs (Fig. 3A,B), with overall similar expression levels to those determined using flow cytometry (Fig. 3C; Fig. S3A). Similar results were obtained when ITGB1 levels were examined in cells gated for CD63 expression (Fig. S3B). To assess the role of ITGB1 signaling in Toxoplasma-induced hypermotility, we blocked ITGB1 activation with two separate monoclonal antibodies (Sangaletti et al., 2008). First, we tested the ITGB1-blocking efficiency of the antibodies by examining their impact on DC adhesion to fibronectin (the principal ECM ligand of ITGB1). DC binding to fibronectin was significantly reduced in the presence of ITGB1-blocking antibodies, while non-significant effects were exerted by the isotype control (Fig. S3C,D). Next, we investigated the impact of ITGB1-blockade on Toxoplasma-induced DC hypermotility. A significant decrease in the mean velocity of Toxoplasma-infected DCs was observed upon ITGB1 blockade, while non-significant effects were recorded for baseline motility or by treatment with isotype control (Fig. 3D,E). Finally, Itgb1 mRNA expression was silenced, with a significant reduction in ITGB1 mRNA (Fig. 3F) and protein expression (Fig. 3G; Fig. S3E) in shITGB1-transduced cells. The motility and mean velocities of Toxoplasma-infected DCs transduced with shITGB1 were significantly inhibited, while the baseline motility and mean velocities of non-challenged DCs were non-significantly affected by shITGB1 treatment (Fig. 3H,I; Fig. S3F,H). Because ITGB1 binds to fibronectin, we additionally assessed the motility of shITGB1-transduced DCs in presence of fibronectin (Fig. S3G,H). Consonant with data in collagen, the motility and mean velocities of shITGB1-transduced Toxoplasma-infected DCs were significantly inhibited (Fig. S3I). Taken together, these data support a role for ITGB1 signaling in Toxoplasma-induced hypermotility. Further, as ITGB1 activation is necessary for TIMP-1–CD63 signal transduction, the data reinforce the involvement of TIMP-1–CD63 signaling in Toxoplasma-induced DC hypermotility.
FAK (Ptk2) silencing impedes DC hypermotility
Upon ITGB1 activation, FAK auto-phosphorylates Tyr-397 and recruits the kinases SRC and PI3K (Ando et al., 2018; Toricelli et al., 2013). Because FAK is a central node in the ITGB1 signaling cascade, we hypothesized that FAK, and substrate kinases SRC and PI3K, impacted on hypermotility. First, we assessed total protein expression and phosphorylation of FAK and SRC in DCs challenged with Toxoplasma. We detected non-significant differences in total protein expression of both kinases when compared with uninfected cells, while the relative phosphorylated protein forms were significantly elevated shortly after challenge with Toxoplasma (Fig. 4A,B). Next, we assessed the impact of pharmacological antagonism of FAK, SRC, PI3K and phosphatase and tensin homolog (PTEN, a PI3K substrate phosphatase) on hypermotility (Fig. S4B). First, non-significant effects were observed on Toxoplasma invasion and replication in the presence of inhibitors (Fig. S4A). Antagonism of PTEN had non-significant effects on DC hypermotility. In sharp contrast, antagonism of FAK, SRC and PI3K phosphorylation significantly inhibited the hypermotility of Toxoplasma-infected DCs, with non-significant effects on the baseline motility of unchallenged DCs (Fig. 4C,D).
As gene silencing confers minimal off-target effects compared with pharmacological inhibition, we investigated the motility of DCs transduced with lentivirus targeting Ptk2 mRNA (shFAK) (Fig. S4C). Ptk2 mRNA and FAK protein expression were significantly reduced in shFAK-transduced DCs (Fig. 4E,F). Importantly, motility and mean velocities were significantly reduced in shFAK-transduced Toxoplasma-infected DCs, while non-significant effects were observed on the baseline motility of unchallenged DCs (Fig. 4G,H). Taken together, these data demonstrate that FAK signaling plays a determinant role in Toxoplasma-induced DC hypermotility. Because FAK is phosphorylated upon challenge with Toxoplasma and is necessary for ITGB1 signal transduction, these data support the activation of TIMP-1–CD63 signaling via ITGB1 in DCs upon Toxoplasma infection.
Because T. gondii tachyzoites utilize infected DCs for dissemination, and TIMP-1 is highly upregulated in parasitized DCs, we investigated putative roles for TIMP-1 in DC migration. We report a novel role of TIMP-1 as a signaling molecule for the induction of amoeboid high-speed migration in infected DCs and extend the TIMP-1–CD63–ITGB1–FAK signaling axis from cancer cell migration to leukocyte migration.
We demonstrate that silencing of the genes encoding TIMP-1 and its receptor CD63 inhibit the hypermigratory phenotype exhibited by parasitized DCs. Further, we show that inhibition of TIMP-1 and/or CD63 downstream signaling elements (ITGB1, FAK, SRC and PI3K) inhibits hypermotility. CD63 is the only known non-protease ligand for TIMP-1, with an impact on intracellular signaling (Jung et al., 2006). Additionally, a ligand–receptor interaction linked to apoptosis has been described for TIMP-1 and pro-MMP-9–CD44 (Lambert et al., 2009a). However, we previously showed that Mmp9 mRNA expression is strongly downregulated in Toxoplasma-challenged DCs (Olafsson et al., 2018) and it is therefore unlikely that this signaling significantly impacts on hypermotility. Further, several ligand–receptor functions have been described for TIMP-2, -3 and -4 (Stetler-Stevenson, 2008). However, in sharp contrast to the elevated mRNA expression and protein secretion of TIMP-1, we previously reported downregulation of Timp-2, -3 and -4 mRNA in DCs upon challenge with T. gondii (Olafsson et al., 2018). Taken together, our data outline a primary role for the TIMP-1–CD63 signaling axis in the motility of Toxoplasma-infected DCs.
Toxoplasma-induced DC hypermigration is initiated by autocrine γ-aminobutyric acid (GABA) secretion from infected DCs (Fuks et al., 2012), which activates ionotropic GABAA receptor channels, triggering VDCC activation and the influx of extracellular Ca2+ (Kanatani et al., 2017). Ca2+ acts as a ubiquitous secondary messenger and governs TIMP-1 exocytosis (Dranoff et al., 2013). Thus, oscillating Ca2+ in Toxoplasma-infected DCs (Kanatani et al., 2017) plausibly stimulates the secretion of TIMP-1. Because DCs display the hypermigratory phenotype throughout infection (Lambert et al., 2006) and TIMP-1 secretion by infected DCs is maintained up to 24 h post infection (Olafsson et al., 2018), TIMP-1 is a prime candidate for a feedback loop that maintains hypermotility of infected DCs over time. Yet, the mechanisms by which T. gondii upregulates Timp-1 gene expression of the infected host cell await further investigation.
Upon T. gondii infection, dramatic morphological changes take place in DCs, with rounding-up (amoeboid) morphology, loss of podosomes, decreased adhesion with redistribution of integrins, and high-velocity locomotion on 2D and in 3D confinements (Weidner et al., 2013; Kanatani et al., 2015; Olafsson et al., 2018). All this is consistent with the cell undergoing a mesenchymal to amoeboid transition (MAT), reminiscent of that described in cancer cells and rapidly moving T lymphocytes (Friedl and Wolf, 2010). Further, amoeboid cell motility is characterized by non-proteolytic migration and reduced dependence on integrin-ECM binding (Lämmermann et al., 2008), which is consistent with the reduced adhesion of Toxoplasma-infected DCs to ECM components (Weidner et al., 2013; Kanatani et al., 2015). Jointly, because an inhibitory effect on hypermotility was observed upon antibody blockade or gene silencing of Itgb1, this indicates that hypermotility-related ITGB1 signaling is likely more dependent on CD63 activation than on tactile adhesion-dependent interactions with ECM components. Additionally, a dysregulation of focal adhesion-related integrin signaling was reported in Toxoplasma-infected monocytic cells (Cook et al., 2018). With the dual effects of TIMP-1 in mind, namely inhibition of MMP-dependent ECM remodeling and promoting locomotion through CD63–ITGB1 signaling, the elevated TIMP-1 secretion by Toxoplasma-infected DCs is compatible with the amoeboid migration mode of leukocytes (Lämmermann et al., 2008). In this context, our data show that T. gondii infection induces amoeboid hypermotility in DCs, with dependence on secreted TIMP-1. Conversely, treatment with rmTIMP-1 significantly rescued hypermotility in TIMP-1-silenced infected DCs but was insufficient to induce hypermotility in naïve DCs. This may indicate that additional early activation of the DC occurs upon challenge with Toxoplasma (Kanatani et al., 2017), which is also in line with the redistribution and altered post-translational modifications of CD63 upon DC maturation (Engering et al., 2003) and the redistribution of integrins in Toxoplasma-infected DCs (Weidner et al., 2013). Nonetheless, in the absence of TIMP-1 or CD63–ITGB1–FAK signaling, amoeboid high-speed migration did not take place, situating TIMP-1 as a prime mediator of hypermotility in Toxoplasma-infected DCs.
We demonstrate that gene silencing of Ptk2 and pharmacological antagonism of FAK inhibit DC hypermotility. Additionally, FAK and SRC phosphorylation was rapidly amplified in Toxoplasma-infected DCs. Upon integrin activation, FAK auto-phosphorylates Tyr-397, then recruits and phosphorylates SRC and PI3K, leading to reorganization of the actin cytoskeleton (Xing et al., 1994; Chen et al., 1996). Because FAK is a central node in bridging signaling cues to the actin cytoskeleton via integrins (Guan, 1997), it likely represents a key step in the cytoskeletal changes and migratory activation of Toxoplasma-infected DCs. Importantly, the rapid onset of hypermotility is well in line with FAK and SRC phosphorylation shortly after parasite invasion and likely precedes transcriptional regulation of hypermotility (Weidner et al., 2013).
We report that the TIMP-1–CD63–ITGB1–FAK signaling axis can be activated in primary DCs with an impact on their migratory properties. To our knowledge, the data also provide the first evidence of an intracellular pathogen manipulating this signaling axis to promote its dissemination. Based on the data at hand, we propose a model for the migratory activation (Fig. 5): T. gondii elicits TIMP-1 secretion by parasitized DCs, with TIMP-1 binding to CD63 then triggering ITGB1–FAK signaling, which promotes the protrusive flow of actin and amoeboid motility.
In line with the paradox of matrix proteolysis in infectious diseases (Elkington et al., 2005), TIMP-1 dysregulation may have implications for immune cell responses and inflammation during toxoplasmosis. From the perspective of the host, under conditions of T. gondii infection, TIMP-1 upregulation may confer enhanced dissemination in shuttling DCs, and also reduce tissue pathology, which may be especially advantageous in dampening the effects of encephalitic infection. In Toxoplasma-infected mice, systemic TIMP-1 is elevated (Tomasik et al., 2016) and reduced parasitic loads in the central nervous system have been reported in TIMP-1-deficient mice (Clark et al., 2011). Further, elevated serum TIMP-1 is associated with severe Plasmodium falciparum malaria (Dietmann et al., 2008), and elevated TIMP-1 in cerebrospinal fluid is associated with viral and bacterial meningitis (Kolb et al., 1998; Tsai et al., 2011). Because MMPs can have a role in immunity but can also contribute to immunopathology in bacterial, viral and parasitic infections (Elkington et al., 2005), our data highlight possible pathophysiological effects of TIMP-1: pro-disseminatory effects by activating shuttling leukocytes, while reducing tissue proteolysis (Olafsson et al., 2018) and thereby dampening inflammation systemically or locally in the perivascular microenvironment. Along these lines, TIMP-1 upregulation is consistently associated with cancer progression and pro-metastatic effects (Jackson et al., 2017), mainly, but not exclusively, mediated by TIMP-1–CD63–ITGB1 activation of FAK signaling (Grunwald et al., 2016). However, this pathway has remained unexplored in immune cells and in the context of infection. This work also highlights possible conserved signaling mechanisms between metastasizing cells and immune cells, to be taken into consideration when designing cancer therapies that target TIMP-1–CD63–ITGB1–FAK signaling.
MATERIALS AND METHODS
The Regional Animal Research Ethical Board, Stockholm, Sweden, approved experimental procedures and protocols involving extraction of cells from mice (N135/15, N78/16), following proceedings described in EU legislation (Council Directive 2010/63/EU).
Cells and parasites
Murine bone marrow-derived DCs (DCs) were generated as previously described (Fuks et al., 2012). Briefly, cells from bone marrow of 6–10-week-old male or female C57BL/6 mice (Charles River) were cultivated in RPMI 1640 with 10% fetal bovine serum (FBS), gentamicin (20 μg/ml), glutamine (2 mM) and HEPES (0.01 M), referred to as complete medium (CM; all reagents from Life Technologies), and supplemented with 10 ng/ml recombinant mouse GM-CSF (Peprotech). Loosely adherent cells were harvested after 6 or 10 days of maturation. Toxoplasma gondii tachyzoites of the wild-type and RFP-expressing Prugniaud strain (Pru and Pru-RFP, type II) (Pepper et al., 2008) or GFP-expressing Ptg strain (Ptg-GFP, type II) (Kim et al., 2001; Hitziger et al., 2005) lines were maintained by serial 2-day passages in human foreskin fibroblast (HFF-1 SCRC-1041, American Type Culture Collection) monolayers. The hypermigratory phenotype induced in DCs by type II strains has been previously characterized (Lambert et al., 2009b).
The following soluble reagents were used in functional assays; rmTIMP-1 (1 µg/ml, R&D systems), LEAF-purified anti-CD29 (clone HMβ1-1, 102209, BioLegend), LEAF-purified IgG isotype control (clone HTK888, 400969, BioLegend), purified NA/LE anti-CD29 (clone Ha2/5, 555002, BD Pharmingen), PF-573228 (PF-228), Wortmanin, SrcI1, SF-1670. All inhibitors were acquired from Tocris and used at 1 µM. Blocking antibodies and isotype control were used at 10 µg/ml.
Motility assays were performed as previously described (Weidner et al., 2013) with slight modification as in Olafsson et al. (2018). Briefly, DCs were cultured in 96-well plates in CM with and without freshly egressed T. gondii tachyzoites (Pru-RFP or Ptg-GFP, MOI 3, 4 h) and soluble reagents (as indicated). Bovine collagen I (1 mg/ml, Life Technologies) or fibronectin (100 µg/ml, Life Technologies), as indicated, were then added and live cell imaging was performed for 1 h, 1 frame/min, at 10× magnification (Z1 Observer with Zen 2 Blue v. 4.0.3, Zeiss). Time-lapse images were consolidated into stacks and motility data was obtained from 30 cells/condition (Manual Tracking, ImageJ) yielding mean velocities (Chemotaxis and migration tool v2.0, Ibidi). Infected cells were defined by RFP or GFP cell co-localization. Transduced cells were defined by eGFP, tGFP or mRuby reporter expression.
T. gondii tachyzoite (Pru-RFP, MOI 1) replication in DCs was assessed 24 h post-infection by vacuole counts as previously described (Dellacasa-Lindberg et al., 2011) and analyzed using immunocytochemistry (100 vacuoles were quantified per condition).
Lab-tek chambers (VWR) were coated with 5 μg/cm2 fibronectin (Gibco) for 2 h at 37°C. Wells were washed twice with RPMI (Thermo Fisher scientific) and the cell suspension was allowed to attach at 37°C, 5% CO2 for 15 min. Unbound cells were removed by washing twice with RPMI and once with PBS. Attached cells were fixed (4% PFA), stained (DAPI) and 5 fields of view per condition were imaged with a 10× objective on a Z1 Observer (Zeiss) microscope with Zen 2 Blue software (v. 4.0.3, Zeiss). DAPI stained nuclei were quantified with Cellprofiler v2.1.1(Broad Institute).
Polymerase chain reaction (PCR)
DCs were cultured in CM with and without freshly egressed T. gondii tachyzoites (Pru-RFP, MOI 3) for 1, 2, 3 or 4 h. Total RNA was extracted using TRIzol reagent (Ambion). First-strand cDNA was synthesized with Superscript III Reverse Transcriptase (Invitrogen). Real-time quantitative PCR (qPCR) was performed using SYBR Green PCR master mix (Kapa biosystems), forward and reverse primers (200 nM) and cDNA (100 ng) with a Rotor Gene 6000 system (Corbett). qPCR was run for 45 cycles. Products were analyzed with RG-6000 application software (v1.7, Corbett). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB) were used as housekeeping genes to generate ΔCt values. 2−ΔCt values were used to calculate relative knockdown efficiency and 2−ΔΔCt values were used to calculate expression fold-change upon infection. All primers (Invitrogen) were designed using Get-prime or Primer-BLAST software (Table S1).
Supernatants from DCs cultured in CM with and without freshly egressed T. gondii tachyzoites (Pru-RFP, MOI 3) were collected 1, 2, 3 or 4 h post infection. Supernatants from mock-treated and shRNA-transduced DCs challenged with freshly egressed T. gondii tachyzoites (Pru-RFP, MOI 3) were collected 5 days post transduction and 4 h post infection. The samples were analyzed using ELISA (Mouse TIMP-1 Quantikine ELISA Kit, R&D Systems) according to the manufacturer's instructions. Results were normalized to a CM control.
DCs were cultured in CM with and without freshly egressed T. gondii tachyzoites (Ptg-GFP, MOI 3, 4 h). Following Fc receptor blockade (1:100; clone 2.4G2, 101301, BD Pharmingen), cells were stained with anti-CD11c–PE–Cy7 (clone N418, 25-0114-82, eBiosciences). After fixation (PFA 4%) cells were directly stained (plasma membrane CD63 and ITGB1 expression) with anti-CD63–APC (clone NVG-2, 17-0631-82, eBiosciences) and/or anti-ITGB1–Pacific Blue (clone HMB1-1, 102224, BioLegend), rat isotype control–APC (clone eBR2a, 12-4321-42, eBiosciences) or hamster isotype control–Pacific Blue (clone HTK888, 400925, BioLegend). Alternatively, cells were permeabilized (IntraPrep Permeabilization kit, Beckman Coulter) before CD63 or ITGB1 staining (total CD63 and ITGB1 expression). All antibodies were used at a dilution of 1:100. Samples were run on the LSRFortessa Cell Analyzer (BD Biosciences). Data was analyzed in FlowJo (Tree Star).
DCs were plated on poly-L-lysine (Sigma-Aldrich)-coated glass coverslips and challenged with freshly egressed T. gondii tachyzoites (Pru, MOI 2, 4 h). After fixation (4% PFA, Sigma-Aldrich) and blockade (5% BSA, Sigma-Aldrich) cells were incubated with anti-CD63 (clone 446703, MAB5417, R&D Systems) or anti-ITGB1 (MAB1997, Millipore). Cells were then permeabilized (0.2% Triton X-100, Sigma-Aldrich) and stained with phalloidin Alexa Fluor 488 (Invitrogen) and anti-SAG-1 (clone P30/3, MA1-83499, Invitrogen). Primary antibodies were used at 1:1000. Anti-mouse Alexa Fluor 350 (A21049, Invitrogen) and anti-rat Alexa Fluor 594 (A21471, Invitrogen) were used as secondary antibodies at 1:1000. Coverslips were mounted with Lab Vision PermaFluo Aqueous Mounting Medium (Thermo Scientific). Micrographs were generated using a 63× objective on a laser scanning confocal microscope (LSM 800, Zeiss). Maximum intensity projections (MIP) were produced in ImageJ from 5 µm thick z-stacks generated using a 63× objective of a laser scanning confocal microscope (LSM 800, Zeiss).
DCs were cultured in CM with and without freshly egressed T. gondii tachyzoites (Pru-RFP, MOI 3). Cells were homogenized in RIPA buffer (150 mM NaCl, 50 mM Tris, 0.1% Triton, 0.5% deoxycholic acid, 0.1% SDS) with cOmplete mini protease and phosphatase inhibitors (Roche). Proteins were separated using 8% SDS-PAGE (Life Technologies), blotted onto a PVDF membrane (Millipore) and blocked (5% BSA, Sigma-Aldrich) followed by incubation with anti-FAK (3285, Cell Signaling Technology), anti-Tyr-397-FAK (3283, Cell Signaling Technology), anti-SRC (2108, Cell Signaling Technology), anti-Tyr-416-SRC (clone D49G4, 6943, Cell Signaling Technology) or anti-GAPDH (ABS16, Millipore) followed by anti-rabbit or anti-mouse HRP (7074 and 7076, Cell Signaling Technology). All antibodies were used at 1:1000, except anti-GAPDH (1:3000). Proteins were revealed by mean of enhanced chemiluminescence (GE Healthcare) in a BioRad ChemiDoc XRS+. Densitometry analysis was performed using ImageJ (NIH, MD, USA).
Lentiviral vector production and in vitro transduction
Self-complementary hairpin DNA oligos targeting Timp-1 (shTIMP-1) and Cd63 (shCD63) mRNA and a non-related sequence (luciferase, Luc) were chemically synthesized (DNA Technology, Denmark), aligned and ligated in a self-inactivating lentiviral vector (pLL3.7, 11795, Addgene) containing a CMV-driven eGFP (shTIMP-1, shLuc) or mRuby (shCD63, shLuc) reporter and a U6 promoter upstream of the cloning restriction sites (HpaI and XhoI) (Table S2). Restriction enzyme analysis and direct DNA sequencing confirmed the correct insertion of short hairpin RNA (shRNA) sequences. shRNA targeting Ptk2 (shFAK, TRCN0000023485, Sigma-Aldrich) or Itgb1 (shITGB1, TRCN0000066645, Genscript) mRNA were on self-inactivating lentiviral vectors (pLKO.1 and pLL3.7, respectively) with CMV-driven tGFP (pLKO.1) or eGFP (pLL3.7) reporter expression (Table S2). Transfer plasmid (shRNA targeting TIMP-1, CD63, ITGB1, FAK or Luc) was co-transfected with psPAX2 (12260, Addgene) packaging vector and pCMV-VSVg (8454, Addgene) envelope vector into Lenti-X 293T cells (Clontech) using Lipofectamine 2000 (Invitrogen). The resulting supernatant was harvested 24 h and 48 h post-transfection. Recovered lentiviral particles were centrifuged to eliminate cell debris, filtered through 0.45-mm cellulose acetate filters and concentrated by means of ultracentrifugation at 50,000 g. Titers were determined by transducing Lenti-X 293T cells with serial dilutions of concentrated lentivirus. DCs (3 days post-bone marrow extraction) were transduced by means of spinoculation at 1000 g for 30 min in the presence of hexadimethrine bromide (Polybrene, 8 µg/ml; Sigma-Aldrich). 5–7 days post-transduction, reporter (eGFP, mRuby or tGFP) expression was verified using epifluorescence microscopy before cells were used in experiments. The transduction efficiency of cells used in all assays, defined as the number of reporter-expressing cells by ocular inspection related to the total numbers of cells in five representative fields of view, was consistently >40% (Kanatani et al., 2017; Olafsson et al., 2018). mRNA knockdown efficiency was quantified 5–7 days post transduction using qPCR. Knockdown of TIMP-1, FAK, CD63 and ITGB1 protein was quantified from: supernatants from shTIMP-1-transduced T. gondii-challenged DCs (Pru-RFP, MOI 3, 4 h) using ELISA; cell lysates of shFAK-transduced DCs using western blotting; and from DCs transduced with shCD63 (5 days post-transduction) or shITGB1 (7 days post-transduction) lentivirus using FACS.
All statistics were performed with Prism (v7, GraphPad). Normality was tested by the Shapiro–Wilks test. In all statistical tests P≥0.05 were defined as non-significant and P<0.05 were defined as significant.
We thank all members of the Barragan lab for critical input.
Conceptualization: E.B.O., A.B.; Methodology: E.B.O., E.C.R., M.V.-G.; Validation: A.B.; Formal analysis: E.B.O., E.C.R.; Investigation: E.B.O., E.C.R.; Resources: M.V.-G.; Data curation: E.B.O.; Writing - original draft: E.B.O., A.B.; Visualization: E.B.O., E.C.R.; Project administration: A.B.; Funding acquisition: A.B.
The research was supported by grants from Vetenskapsrådet (Swedish Research Council) (K2014-56X-15133-11-6 and 2018-02411 to A.B.) and the European Community ERA-NET NEURON network (VR/2014-7533 to A.B.).
The authors declare no competing or financial interests.