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Intracellular Galectin-9 Controls Dendritic Cell Function by Maintaining Plasma Membrane Rigidity

Summary Endogenous extracellular Galectins constitute a novel mechanism of membrane protein organization at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organization and function. By combining functional, super-resolution and atomic force microscopy experiments to analyze membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure by modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organization. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type-lectin receptor-mediated pathogen uptake by DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionarily conserved.


INTRODUCTION
Dendritic cells (DCs) constitute the major group of antigen-presenting cells that constantly patrol the body for microbes and are essential for linking innate and adaptive immune responses (Banchereau and Steinman, 1998). DCs are equipped with a diverse membrane receptor repertoire to take up pathogens including Toll-like receptors, scavenger receptors, and C-type lectins, such as the mannose receptor and the dendritic cell-specific intercellular adhesion molecule grabbing non-integrin receptor (DC-SIGN) (Banchereau et al., 2000;Savina and Amigorena, 2007;Buschow et al., 2012;Heinsbroek et al., 2008;. Engagement of these receptors with their ligand is accompanied by cytoskeletal changes, which allow for the capture and engulfment of phagocytic targets (Sano et al., 2003;Baranov et al., 2016). Actin polymerization is instrumental in forming a nascent phagosome for pathogen engulfment, and actin-driven mechanical forces enable pathogen internalization (May et al., 2000). In addition, phagocytosis is dependent on plasma membrane organization and loss of membrane structure results in impaired pathogen recognition, defective migration, and compromised immunological synapse formation (Alvarez et al., 2008;Heuze et al., 2013;Buschow et al., 2012). Galectins, a family of ß-galactoside-binding proteins, have been recently identified as a novel mechanism of membrane organization (Lajoie et al., 2009;Elola et al., 2015;Nabi et al., 2015) due to their ability to interact with and cross-link specific carbohydrate structures. As such, Galectins can simultaneously interact with multiple glycoconjugates, thereby regulating the dynamics of glycosylated-binding partners, limiting receptor internalization, and establishing membrane microdomains (Elola et al., 2015). Notably, Galectins are also abundantly expressed intracellularly, although their cytosolic functions are not well characterized (Hsu et al., 2015a;Johannes et al., 2018;Liu et al., 2002;Liu and Rabinovich, 2005). Recently, Galectins have been discovered as novel regulators of several immune processes, such as T cell homeostasis, inflammation, and immune disorders (Sundblad et al., 2017;Thiemann and Baum, 2016;de Oliveira et al., 2015;Rabinovich and Toscano, 2009).
Galectin-9 was first discovered as an eosinophil chemoattractant, and to date, most studies have focused on studying Galectin-9 in inflammation or infection processes (Jost et al., 2013;Curciarello et al., 2014;Hsu et al., 2015b). Extracellular Galectin-9 has been implicated in inhibiting T cell immunity by promoting T cell  (NT). Surface only (A) and total (B) Galectin-9 and Galectin-3 knockdown were confirmed by flow cytometry 48 h after transfection. Red population, NT siRNA; blue population, gal9 and gal3 siRNAs transfected moDCs; black population, isotype control. Numbers in inset indicate geometrical mean fluorescence intensity. (C) NT, gal9, and/or gal3 siRNA-transfected cells were challenged with zymosan for 60 min, after which cells were fixed, stained, and the phagocytic index calculated. Graphs show representative results for one donor. Each dot represents apoptosis and differentiation into regulatory T cells (Anderson et al., 2007;Bi et al., 2008;Zhu et al., 2005). Furthermore, extracellular Galectin-9 acts as a suppressor of B cell signaling by binding to the B cell receptor (Cao et al., 2018;Giovannone et al., 2018). Although these studies indicate that Galectin-9 plays an inhibitory role on lymphocytes, its function in myeloid cells remains poorly understood. Moreover, Galectin-9 is also highly expressed intracellularly, and although implicated in protein-protein interactions and mRNA splicing (Liu et al., 2002;Sundblad et al., 2017;Heusschen et al., 2013), the function of cytosolic Galectin-9 in the immune system continues to be ill defined.
Here, we demonstrate that intracellular Galectin-9 is essential for sustaining cortical actin cytoskeleton rigidity and phagocytosis in DCs. Our work indicates a novel evolutionary conserved mechanism by which intracellular Galectins stabilize plasma membrane structure by actin cytoskeleton reorganization.

Galectins Are Essential in Governing Human Dendritic Cell Function
The role of Galectins in the initiation of the immune response is poorly understood, and although Galectin-3 has been implicated in macrophage-mediated uptake in mice (Sano et al., 2003), few studies have been performed to elucidate Galectin function in DCs (de Kivit et al., 2017;Leskela et al., 2015;Hsu et al., 2015b;Dai et al., 2005). To address this question, we generated DCs lacking Galectin-3 and/or Galectin-9 by electroporating human monocyte-derived dendritic cells (moDCs) with either a specific galectin small interfering RNA (siRNA) (gal3 and/or gal9) or a non-targeting (NT) siRNA control before challenge them with fluorescein isothiocyanate (FITC)-labeled zymosan particles, a fungal cell wall extract (de la Rosa et al., 2005). Subsequent immunolabeling without permeabilization using an antibody directed against FITC allowed for selective labeling of membrane-bound particles. Galectin-9 and Galectin-3 protein knockdowns were confirmed by flow cytometry showing that both proteins were depleted to a similar extent (70%, Figures 1A and 1B). The efficiency of Galectin knockdown was comparable between cells transfected with a single siRNA or with dual siRNA, and Galectin-9 knockdown (Gal-9 KD) did not affect Galectin-3 expression, or vice versa ( Figure S1). Depletion of Galectin-9 impaired particle uptake to a greater extent when compared with that observed upon Galectin-3 knockdown ( Figures 1C, 1D, and 1E). Moreover, there was no additive effect of knocking down both Galectin-9 and Galectin-3 ( Figure 1E). Taken together, these data demonstrate that Galectins are required for phagocytosis by DCs, and indicates that Galectin-9 is a major player in this process.

Galectin-9 Is Essential for Phagocytosis by Dendritic Cells
We previously identified Galectin-9 as part of the DC-SIGN-mediated, a phagocytic receptor present in immature DCs, phagosomes, although no functional studies were performed to assess the role of Galectin-9 in DC function (Buschow et al., 2012;Manzo et al., 2012;Liu et al., 2017;Cambi et al., 2003;. Co-immunoprecipitation experiments revealed DC-SIGN association with Galectin-9 in DCs, demonstrating their molecular interaction (Figure 2A). To examine whether this interaction occurs in the cytosolic compartment and/or at the extracellular matrix, co-immunoprecipitations were performed on lactose-treated moDCs and in the presence of lactose to prevent unspecific binding of Galectin-9 to DC-SIGN during cell lysis. Lactose impairs cell surface glycan-based interactions mediated by Galectins by competing for their major ligands, which dissociates Galectins from the cell surface (Lajoie et al., 2007;Cambi et al., 2009). As shown, addition of lactose successfully removed Galectin-9 from the surface of moDCs ( Figure S2A). Nonetheless, Galectin-9 was found to still bind DC-SIGN, albeit to a lesser extent than in the untreated control ( Figure 2B). These data indicate that Galectin-9 binds to DC-SIGN both extraand intracellularly. To investigate the role of Galectin-9 in DC-SIGN-mediated phagocytosis, Gal-9 KD and NT control (referred to as wild-type [WT]) DCs were challenged with zymosan particles. Galectin-9 protein knockdown (90%) was confirmed by flow cytometry ( Figure S2B) and western blotting ( Figure S2C). No  (D). Results show the mean G SEM for four independent donors. Unpaired Student's t test was conducted between NT and gal9 siRNA-and between NT and gal3 siRNA-transfected cells. *p < 0.05, **p < 0.005, ***p < 0.0001. See also Figure S1. . Galectin-9 Is Required for Optimal Phagocytic Capacity in DCs (A) moDCs were lysed and whole-cell extract prepared for incubation with anti-DC-SIGN antibody (H200) or isotype control (total rabbit IgG). Immunoprecipitated (IP) complexes were resolved and probed with DC-SIGN-and Galectin-9specific antibodies. Graph shows quantification of Galectin-9 content of each sample using ImageJ. (B) moDCs were treated with 35 mM lactose for 48 h before being lysed as per (A). IP complexes were resolved and probed with DC-SIGN-and Galectin-9 specific antibodies. Graph shows quantification of Galectin-9 content of each sample using ImageJ. (C) NT or gal9 siRNA-transfected cells were challenged with zymosan for the indicated time points. After this time, cells were fixed, stained, and phagocytic indexes calculated for each time point. Graphs show representative results for one donor. Each dot represents phagocytic index obtained for each microscopic field, each of which contained 10-20 cells.
(D) Quantification and statistical analysis of experiments shown in (C). Twenty frames were analyzed for each donor and transfection. Results show the mean value G SEM for four independent donors. Unpaired Student's t test was conducted between NT and gal9 siRNA-transfected cells for all time points. significant differences in zymosan binding were observed between NT and gal9 siRNA-transfected DCs ( Figure S2D), implying that Galectin-9 is not required for particle binding. To study the involvement of Galectin-9 in particle uptake, the phagocytic index was calculated for each of the conditions and specified time points (Figures 2C, 2D, and S2E). Gal-9 KD resulted in impaired zymosan internalization 60 min after challenging moDCs ( Figure 2D). Quantification of the number of particles internalized per cell revealed that the impaired uptake upon Gal-9 KD is likely due to a decrease in the number of zymosan particles internalized per cell rather than a decrease in the total amount of cells able to uptake particles ( Figure 2E). Gal-9 KD did not alter DC-SIGN membrane expression or receptor internalization excluding that the uptake defect was due to deficient receptor surface levels ( Figure S3). Next, WT and Gal-9 KD moDCs were incubated with a DC-SIGN-blocking antibody (clone AZN-D1) or isotype control before challenging them with zymosan particles. AZN-D1 does not induce DC-SIGN signaling and has a modified Fc region that cannot be recognized by the Fc receptors expressed on DCs Tacken et al., 2005). As expected, blocking DC-SIGN resulted in defective zymosan uptake by NT-transfected moDCs, although zymosan uptake was unaffected by the addition of isotype controls ( Figures S4A and S4B). Analysis performed on multiple donors confirmed our observations, and zymosan uptake was significantly impaired upon DC-SIGN blocking, indicating that DC-SIGN is the major receptor for zymosan in DCs ( Figure S4C). These results demonstrate that Galectin-9 is an essential component in DC-SIGN receptor-mediated uptake by DCs.
Zymosan uptake experiments were also performed with murine bone marrow-derived dendritic cells (BMDCs) from WT and galectin-9-deficient (galectin9 À/À ) mice. In line with human DCs, lack of Galectin-9 in murine DCs resulted in defective phagocytic capacity, suggesting an evolutionarily conserved role for Galectin-9 in phagocytosis ( Figures 3A and 3B). To investigate the effect of Galectin-9 in DC-mediated immunity against fungal pathogens, galectin-9-null mice were immunized with heat-inactivated Candida albicans and DC function analyzed ( Figure 3C). Although we were not able to quantify phagocytosis in vivo, murine DCs lacking Galectin-9 displayed a significant decrease in cytokine secretion upon infection, indicating poor initiation of a proper immune response ( Figures 3D-3G).

Intracellular Galectin-9 Controls Plasma Membrane Structure in Dendritic Cells
To examine whether the extra-or the intracellular pool of Galectin-9 was responsible for the defect in phagocytosis, moDCs were treated with lactose to remove extracellular galectins from the cell surface before being challenged with zymosan particles. Although lactose treatment effectively reduced the surface levels of , no effects on zymosan uptake were observed compared with untreated cells ( Figure 4C). Lactose was also added during zymosan incubation, and no differences in the phagocytic index of moDCs treated with lactose were observed, regardless of whether lactose was present during zymosan incubation ( Figure S5). This indicates that the intracellular pool of Galectin-9 is responsible for particle uptake by moDCs and that particle binding and internalization are independent of Galectin-9-mediated interactions at the cell surface. These findings led us to hypothesize that Galectin-9 may interact with specific cytoskeleton components, which could alter the stability and/or the formation of phagosomes. To address this, Gal-9 KD and WT DCs were analyzed for their uptake ability upon treatment with cytochalasin D (cytD), which blocks actin polymerization by its binding to actin filaments. Addition of cytD resulted in a significant decrease of particle uptake in WT cells in contrast to Gal-9 KD moDCs that were not affected by cytD after challenging cells for 60 min ( Figure 5A). Earlier time points were also analyzed, but DCs were still unable to take up any particles due to the inhibition of the actin cytoskeleton polymerization ( Figures S6A and S6B). We excluded that this was due to a difference in particle binding between WT and Gal-9 KD cells (L. Querol Cano, unpublished data) or cell viability, which was not affected upon treatment with cytD ( Figure S6C). Similar results were obtained when DCs were treated with increasing concentrations of cytD, confirming gal9 siRNA-transfected cells to be less sensitive to actin disruption than their WT counterparts ( Figure S6D). To corroborate an impairment in the actin cytoskeleton upon Galectin-9 depletion, levels of F-actin were measured in WT and Gal-9 KD cells moDCs. A decrease of approximately 20% in the total levels of F-actin was seen in moDCs depleted for Galectin-9, confirming a specific effect for this lectin in the actin cytoskeleton arrangement ( Figures 5B and 5C). Continued (E) NT or gal9 siRNA-transfected cells were challenged as per (C) and the number of internalized zymosan particles quantified for each frame. Ten to twenty frames were analyzed for each condition. Data represent mean percentage of DCs that had internalized the specified number of particles of one representative donor out of three independent experiments. For statistical analysis two-way ANOVA followed by a Bonferroni post hoc test was applied. n.s.: p > 0.05; *p < 0.05, **p < 0.005. See also Figures S2-S4. Figure 3. Galectin-9 Function in DC Is Conserved between Mouse and Human and Alters Dendritic Cell-Mediated Immune Responses In Vivo (A) Bone marrow-derived dendritic cells (BMDCs) obtained from either wild-type (control) or galectin-9-null (galectin-9 À/À ) mice were seeded on coverslips, challenged with zymosan for 60 min, and the phagocytic index calculated. Results show the mean phagocytic index value G SEM for three independent mice. Unpaired Student's t test was conducted between wild-type and galectin-9 À/À mice. *p < 0.05. (B) Representative images from results shown in (A). Scale bar, 10 mm.
(C-G) (C) Scheme depicting work protocol to assess anti-fungal immunity in galectin-9-null animals. Four wild-type (WT) and galectin-9 À/À mice were injected with heat-inactivated Candida albicans. Three hours after injection, lymph nodes and spleen were removed, single-cell suspensions made, and cells seeded onto 96-well plates. Spleen cell suspensions were re-stimulated Experiments performed with galectin-9 À/À murine BMDCs confirmed this defect in cellular actin content ( Figures 5D and 5E). Furthermore, confocal imaging showed that the percentage of F-actin-positive phagosomes was reduced upon Gal-9 KD in moDCs challenged with zymosan particles by approximately 40% (Figures 5F and 5G) in line with our previous observation ( Figure 5B). These data suggest that depletion of Galectin-9 leads to reduced actin filament formation both under basal conditions and around phagosomes. To further verify Galectin-9 involvement in directly controlling the actin cytoskeleton, super-resolution laser scanning microscopy was performed on ventral plasma membrane sheets of moDCs. These studies demonstrated that Galectin-9 closely associates with the cortical actin cytoskeleton under basal conditions ( Figure 6).
To unravel the mechanism underlying Galectin-9 function in plasma membrane integrity and structure, we exploited atomic force microscopy (AFM) to analyze the cellular stiffness of moDCs transfected with either NT or gal9 siRNA. Nanomechanical probing of the cells was achieved by obtaining a series of force-distance curves on selected points of the cell surface. A sharp non-functionalized cantilever with a radius of approximately 35 nm was brought into contact with a flat area of a single DC attached to a glass coverslip applying a mechanical force ( Figure 7A). The use of a combined bright-field AFM setup allowed for accurately positioning the cantilever over specific areas of interest on the cell surface (Figure 7B). Analysis of the approach force-distance curves obtained for each point of interest allowed calculation of the cytoskeletal stiffness using the linearized Sneddon equation ( Figure 7C). For this purpose, minimum and maximum fit boundaries (shown in blue) were defined respectively as 10% and 70% of the maximum force after baseline correction ( Figure 7C). The portion of the curves that was used for fitting with the linearized model is shown in purple, and the separation distance that corresponds to this fit region is in the range of 200-600 nm ( Figure 7C). Given that the thickness of the lipid bilayer is approximately 4 nm (Yokokawa et al., 2008), it is plausible to presume that the underlying cytoskeletal structures such as cortical actin and peripheral cytoplasm were also probed in our experiments. The mechanical characterization performed on DCs shows that moDCs lacking Galectin-9 have a decreased cytoskeletal rigidity compared with their WT counterparts ( Figure 7D), in line with the defect in their cytoskeleton previously observed.
As it is well known that actin polymerization is mediated by small GTPases of the Rho family including Rac1 (Caron and Hall, 1998;May et al., 2000;Swanson, 2008;Norman et al., 1996), we investigated the effect of Galectin-9 depletion on Rac1 activation by specifically measuring its GTP-bound fraction using a G-LISA colorimetric assay. Incubation of control moDCs with zymosan particles resulted in a fast induction of Rac1-GTP activity already after 5-min stimulation ( Figures 8A and 8B), which was sustained in time ( Figures  8A and 8C). Depletion of Galectin-9 abrogated Rac1 induction, and no increase in its GTP-bound form could be observed upon zymosan stimulation in moDCs transfected with gal9 siRNA at any of the time points analyzed ( Figures 8A-8C). The recruitment of total Rac1 to nascent phagocytic cups was also impaired upon Gal-9 KD ( Figures 8D-8F), suggesting that Galectin-9 promotes both Rac1 recruitment and activity on phagosomes.
Taken together, intracellular Galectin-9 controls plasma membrane structure via modulating Rac1 activity and actin polymerization, which underlies Galectin-9 requirement for phagocytosis in DCs.

DISCUSSION
Galectins have gained increasing interest for their role as extracellular organizers of plasma membrane components via glycan-mediated interactions. Nonetheless, their mechanism of action remains poorly understood, and in particular, their intracellular functions are ill-defined (Buschow et al., 2012). Here, we identified a previously unrecognized function for intracellular Galectin-9 in actin cytoskeleton reorganization and report a novel, functional interaction between Galectin-9 and the C-type lectin receptor DC-SIGN at the cytosol of DCs. Several members of the Galectin family are expressed in the cytosol, and some, such as Galectin-1 or Galectin-3 are predominantly intracellular proteins (Liu et al., 2002; . Continued with C. albicans, and 24 h after seeding cytokine secretion was measured in supernatants by ELISA. Tumor necrosis factor (TNF)-a production in spleen (D) or lymph node (E) samples. Interleukin (IL)-12 production in spleen (F) or lymph node (G) samples. Graph shows the mean value G SEM for four animals. Unpaired Student's t test was conducted between WT and Gal9 KO cells. *p < 0.05. Wilson et al., 1989;Clerch et al., 1988;Hubert et al., 1995). Very little is known regarding the localization and function of cytoplasmic Galectin-9, although it has been implicated in protein folding and signal transduction (John and Mishra, 2016;Vasta et al., 2012). Our study now demonstrates that the large intracellular pool of Galectin-9 is responsible for the phagocytic capacity in DCs by modulating plasma Galectin-9 and Galectin-3 expression was confirmed by flow cytometry. Red population, untreated cells; blue population, lactose-treated cells; black population, isotype control. Numbers in inset indicate gMFI. Panels depict representative results for one donor, and graphs show the mean phagocytic index G SEM for three independent donors. Unpaired Student's t test was conducted between untreated control and lactose-treated cells. (C) Control or lactose-treated cells were challenged with zymosan for 60 min. Cells were then fixed, stained, and the phagocytic index calculated. Graphs show the mean G SEM for three independent donors. Unpaired Student's t test was conducted between untreated control and lactose-treated cells. n.s.: p > 0.05, *p < 0.05. See also Figure S5. membrane structure, revealing a novel function for Galectins in cytoskeleton remodeling. This was observed in both human and murine cells, which indicates Galectin-9 as an evolutionarily conserved lectin required for maintaining the cortical cytoskeleton structure and function in DCs. Our data support a model in which Galectin-9 is essential for DC-SIGN-mediated phagocytosis, by (1) maintaining plasma membrane and cortical actin stiffness and (2) controlling receptor function (Figure 9). We identified that the underlying mechanism involves Galectin-9-dependent activation and recruitment of Rac1-GTP upon particle incubation, which triggers actin polymerization and the subsequent formation of phagocytic cups.
In line with this, our AFM studies demonstrate that Galectin-9-depleted cells have a less rigid plasma membrane and cortical cytoskeleton, rendering them unable to adequately modify their structure upon Actin Galectin-9 Merged  (C) Representative force-distance curves obtained on gal9 siRNA-transfected (black) and NT siRNA (red) moDCs and curve-fitting approach to determine the Young's modulus of elasticity. Blue lines correspond to upper-and lower-fit boundaries (70% and 10% of the maximum force, respectively); purple lines show fitted portion of the curves used to calculate Young's modulus.
(D) Young's modulus of elasticity was calculated by fitting the force-distance curves indicated in (C). Data represent mean average Young's modulus of elasticity G SEM of three independent donors, and each data point shows the average value for three different locations for each moDC. Ten to thirty cells were analyzed for each donor in each independent experiment. Unpaired Student's t test was conducted between NT and gal9 siRNAtransfected cells. ***p < 0.001. After this time, cells were lysed, total protein was quantified, and Rac1-GTP activation was determined. Results are expressed as fold increase Rac1-GTP levels and relative to the unstimulated corresponding sample. Data represent mean average Rac1-GTP fold induction G SEM of three independent donors. Unpaired Student's t test was conducted between NT and gal9 siRNA-transfected cells.
(B and C) Each symbol represents one independent donor, and lines connect paired NT and gal9 siRNA-transfected moDCs after stimulation with zymosan for either 5 min (B) or 30 min (C).
particle engulfment. Moreover, inhibition of actin polymerization did not affect the phagocytic ability of Gal-9 KD cells in contrast to WT cells. Although actin executes a pivotal function in phagocytosis, little is known regarding the mechanisms that govern F-actin recruitment to a nascent phagosome due to the lack of high-resolution data (Baranov et al., 2016). Our data support a new concept in which intracellular Galectin-9 is required for actin polymerization, directly controlling plasma membrane rigidity, by enhancing the activity of the actin-binding protein Rac1. In line with this, Galectin-1 has been recently shown to re-activate F-actin protein levels (Quinta et al., 2016). Similarly, intracellular Galectin-3 has been proved to enhance phagocytosis in macrophages by its interaction with F-actin in the phagocytic cups (Sano et al., 2003;Serizawa et al., 2015). Intracellular ligands have been proposed to bind Galectins through protein-protein interactions independent of carbohydrate-mediated recognition, although whether both proteins interact directly or through an intermediary molecule is not known (de Oliveira et al., 2015;Johannes et al., 2018;Shimura et al., 2004). Whether Galectin-9 function in our study is carbohydrate independent remains to be elucidated, but knockdown of Galectin-9 in the presence of other Galectins was sufficient to induce defects in actin polymerization and cellular rigidity in DCs. DC-SIGN is known to interact with actin and Lsp-1, an F-actin-interacting protein through its cytoplasmic tail (Smith et al., 2007), which likely allows for extracellular particle binding, plasma membrane deformation, and actin polymerization to occur simultaneously. Moreover, DC-SIGN signaling results in enhanced RhoA-GTPase activity (den Dunnen et al., 2009;Hodges et al., 2007). Our data now support that depletion of intracellular Galectin-9 is sufficient to disrupt the cytosolic complex of DC-SIGN with actin-binding proteins, ultimately impairing cytoskeleton reorganization and causing a reduction in the cellular phagocytic capacity. We identified Galectin-9 as an integral component of the actin cytoskeleton as well as of the intracellular DC-SIGN-associated complex, and it is conceivable that Galectin-9 exerts its effects on the cytoskeleton remodeling by directly linking F-actin filaments, actin remodeling proteins, and DC-SIGN in a multi-protein complex ( Figure 9). Alternatively, Galectin-9 may connect DC-SIGN with other plasma membrane receptors known to associate with actin, such as CD44, which has been previously shown to interact with Galectin-9, as a component of DC-SIGN-directed phagosomes in DCs (Wu et al., 2014;Buschow et al., 2012).
Aside of its direct effects on DC-SIGN, we expect Galectin-9 depletion to have additional effects on the function of other lectin receptors involved in phagocytosis (mannose receptor, complement receptor 3, TLR2) (Sung et al., 1983;Xia et al., 1999). To date, extracellular Galectin-9 has been previously shown to interact with CD44, glucose transporter-2, immunoglobulin E, and Tim-3, a T cell type 1 membrane protein, known to be involved in T cell apoptosis and phagocytosis of apoptotic cells (Wu et al., 2014;Zhu et al., 2005). All these interactions, though, are carbohydrate dependent and mediated via glycan-lectin associations. To the best of our knowledge, no intracellular binding partners have been previously reported for Galectin-9.
The intracellular functions of Galectin-9 and particularly its role in phagocytosis have not been previously addressed, and our work is in line with Galectin-3 and Galectin-1 function in particle uptake, highlighting the broad importance of intracellular Galectins in enhancing cellular uptake (Farnworth et al., 2008;Sano et al., 2003;Barrionuevo et al., 2007;Caberoy et al., 2012;Linden et al., 2013;Quattroni et al., 2012). Furthermore, our studies with primary DCs demonstrate that disruption of glycan interactions solely alters particle uptake but not their binding to the cell membrane, which is in agreement with previous findings (Sano et al., 2003).
In summary, our work demonstrates a novel role for intracellular Galectin-9 in the regulation of the phagocytic activity through reorganization of the actin cytoskeleton that underlies plasma membrane rigidity in Figure 8. Continued (D) NT or gal9 siRNA-transfected moDCs were challenged with zymosan_FITC for 5 min before being stained for F-actin (magenta) and Rac1 (blue) and imaged with super-resolution microscopy. A representative confocal image out of 9 images is shown. Scale bar, 10 mm. Arrows indicate overlap between Rac1 and F-actin signal on phagocytic cups. (E) Magnification of representative phagocytic cups in NT and gal9 siRNA-transfected moDCs treated as in (D).
(F) Number of Rac1-and/or F-actin-positive phagocytic cups found on moDCs treated as in (D). Nine images containing between 20 and 30 cells were analyzed for each condition. Data represent mean average number of phagocytic cups G SEM. Unpaired Student's t test was conducted between NT and gal9 siRNA-transfected cells. *p < 0.05; **p < 0.01; n.s p > 0.05.
DCs. Given the plethora of cellular biological processes Galectin-9 is involved in, this novel intracellular Galectin-9 mechanism of action contributes to the general understanding of plasma membrane structure and their implications in cell function.

Limitations of the Study
In this study we identified Galectin-9 as novel regulator of the actin cytoskeleton and plasma membrane structure in DCs. We have also defined and characterized the interaction and functional relationship between Galectin-9 and the phagocytic receptor DC-SIGN. Further work could be performed to confirm that both proteins interact intracellularly. Given the cytosolic localization and function of Galectin-9, a further in-depth characterization of the role Galectin-9 in governing the intracellular signaling pathway downstream of DC-SIGN would also be pertinent.

METHODS
All methods can be found in the accompanying Transparent Methods supplemental file.

Infection model and cytokine measurements
Wild-type C57BL/6J (Charles River) and galectin-9 null mice were maintained under specific pathogen-free conditions at the Central Animal Laboratory (Nijmegen, the Netherlands). Drinking water and food were provided ad libitum. The experiments were performed according to guidelines for animal care of the Nijmegen Animal Experiments Committee and in accordance with the ethical standards described in the declaration of Helsinki. Endotoxin-free Candida albicans was prepared as described previously (van Spriel et al., 1999). Yeast cells were heat-inactivated by incubating them at 65 °C for 90 minutes. Animals were injected with 12x10 6 heat-inactivated Candida albicans in a volume of 200 µl PBS intravenously and 3x10 6 in 50 µl PBS subcutaneously. Three hours after injection, spleen and popliteal lymph nodes were isolated and meshed to obtain single cell suspension. Spleen and lymph node cells were meshed through a 100 µm cell strainer by using a syringe plunger. Cell suspension was spun at 400xg for 5 min and resuspended in 3 ml of 1x ammonium chloride solution for the lysis of erythrocytes. After 3 min of incubation at room temperature cells were washed with 20 ml of PBS 2 times. To analyse cytokine secretion, 380x10 3 lymph node cells or 1x10 6 spleen cells were seeded into 96-well plates for 24 h after which supernatants were collected and stored at -20 °C. Spleen samples were re-challenged with 1x10 6 C. albicans prior to being seeded for 24 h and supernatants collected.
The levels of IL-12 and TNFα in the supernatants of spleen and lymph node homogenates were determined using a commercial ELISA kits (ThermoFisher Scientific). Standard curves were run at the same time and were used to calculate the concentration of cytokines in the samples.

Antibodies and reagents
The following primary antibodies were used for Western Blotting: rabbit anti-DC-SIGN (H200, with ImageJ software. Twenty to thirty frames were analysed for each condition and the phagocytic index ((total number of particles -total number of membrane-bound particles)/number of cells) and the binding index (number or bound particles/number of cells) calculated for each frame.

Flow cytometry
Single cell suspensions were stained with specific antibodies or isotype control as negative control for 30 min at 4 °C. The following antibodies were used: mouse-anti DC-SIGN (clone AZN-D1, ) at 5 μg/ml; goat anti-Galectin-9 (AF2045, R&D systems) at 20 μg/ml. Before staining, moDCs and U-937 cells were incubated with 2 % human serum for 10 min on ice to block non- and results analysed using FlowJo version X software (Tree Star, Ashland, Oregon).

Confocal microscopy
gal9 or non-targeting siRNA -transfected moDCs were collected at day 5 and blocked with PBA + 1 % human serum prior to being incubated with primary antibody for 30 min on ice. Cells were then washed and incubated for further 30 min with the corresponding secondary antibody followed by a 60 min incubation at 12 ° C. Cells were then washed extensively, fixed in 2 % PFA and allowed to attach on poly-L-lysine-coated 12 mm glass coverslips ( Ventral plasma membrane staining and super resolution imaging 5x10 5 moDCs were seeded onto 25 mm glass coverslips (Electron Microscopy Sciences, Hatfield, Pennsylvania) in phenol red free RPMI containing 10 % FBS, 1 mM ultra-glutamine, IL-4 (500 U/ml) and GM-CSF (800 U/ml) in a humidified, 5 % CO2-containing atmosphere for 48 hours. After this time, ventral plasma membranes were prepared by sonication using a Sartorius Labsonic P sonicator with cycle set at 1 and amplitude at 20 % output. First, the sonicator tip was placed in a glass beaker containing 100 ml prewarmed hypotonic PHEM buffer (6 mM PIPES, 5 mM HEPES, 0.4 mM Mg2SO4, 2 mM EGTA). Next, coverslips were held 1-2 cm below the sonicator tip at a 45 degrees angle in the hypotonic PHEM solution and cells were sonicated for approximately 1 second. Directly after sonication coverslips were transferred to a pre-warmed PBS solution containing 4 % paraformaldehyde and 0.05 % glutaraldehyde and incubated for 30 min at room temperature. For Galectin-9 and actin staining, coverslips were subsequently incubated with anti-Galectin-9 antibody (AF23045, R&D systems) for 2 h at room temperature, washed and incubated for 1 h with the secondary antibody donkey-anti-goat Alexa 568 (A11057) at 1:200 (v/v) and Alexa Fluor 488-labelled phalloidin at 1:100 (v/v) followed by DAPI staining (Sigma-Aldrich) at 1:3000 (v/v) for 10 min and at room temperature. Cells were then washed twice with PBS, one time with milliQ H2O, embedded onto glass slides using Mowiol (Calbiochem) and stored at 4 °C until imaging. Confocal images were obtained in a sequential manner using a commercial Zeiss LSM880 confocal scanning microscope equipped with an Airyscan Unit, 405 and 561 nm diode lasers, argon (458, 488, 514 nm) lasers and a 633 nm laser and a 63× Plan Apochromat (1.4 NA) oil immersion objective. Images were obtained using the ZEN software (Zeiss Microscopy, Breda, Netherlands) and processed with the ZEN Airyscan processing toolbox and Fiji software.

Atomic force microscopy
6x10 5 moDCs transfected with either Non-Targeting or gal9 siRNA were seeded in phenol-red free RPMI supplemented as before in a 40 mm glass bottom dish (GWST-5040, WillCo, Amsterdam, Netherlands) for 48 hours. After this time, mechanical probing of cells was performed with a Catalyst BioScope (Bruker, Kalkar, Germany) atomic force microscope coupled to a confocal microscope (TCS SP5II, Leica) using the "point and shoot" feature of the Nanoscope software (Bruker). Silicon nitride cantilevers with nominal spring constants of 0.06 N/m (S-NL type D, Bruker) were used without any tip modification. The system was calibrated first in air and then in cell-free medium at 37 °C prior to each experiment by measuring the deflection sensitivity on a glass surface, which enabled determination of the cantilever spring constant using the thermal noise method (te Riet et al., 2011). Before the sample was placed, the xy movement of the sample stage was calibrated using the NanoScope software. An optical image of the cells was captured after the tip position was registered, which allowed for selection of the point (or region) of interest on the optical image. Two force − distance curves were sequentially acquired from each point selected on the membranes of stretched cells and three independent points were probed for each cell. The forward (approach) and reverse (retraction) velocities were kept constant at 1 μm/s, ramping the cantilever by 4 μm with a 3 nN threshold in a closed z loop. After baseline correction, approach curves were analysed for determination of Young's modulus of elasticity using Sneddon's conical indenter model (Sneddon, 1965) for which Poisson's ratio was set as 0.5 (Lin et al., 2007) and the half angle of the indenter as 18°. Contact point-independent linearised Sneddon equation was used for fitting the approach curves (van Helvert and Friedl, 2016). The region on the approach curve through which the model was fit was determined via setting the lower and upper boundaries that corresponded to approximately 10 % and 70 % of the difference between the maximum and minimum forces exerted, respectively. Two curves per point and 2-3 points per cell were averaged to obtain Young's modulus of elasticity per cell. Approximately 10 to 30 cells were analysed for each condition and experiment.

Rac1-GTPase activation assay
To assess GTP-bound Rac1 levels, colorimetric G-LISA activity assay kit (BK128-S, Cytoskeleton, Denver, Colorado) was used according to manufacturer's instructions. Day 5 moDCs were stimulated with zymosan particles (Z4250, Sigma-Aldrich) for 5, 15 or 30 min prior to being lysed in icecold lysis buffer, snap-frozen in liquid nitrogen and stored at -80 °C. Protein concentrations were determined using the micro BCA protein assay kit (23235, ThermoFisher scientific) and 50 μg of total protein were subsequently used for the G-LISA assay. Rac1-GTP levels were determined using the Rac1-GTP binding 96-well plates. Absorption of the wells at 490 nm was determined with an iMark microplate reader (BioRad).

Statistical analysis
All data was processed using Excel 2013 (Microsoft) and GraphPad Prism 5 software. All Image processing was performed on ImageJ software and statistical analysis was done using Prism5. The