n TECHNICAL ADVANCE Technical Advance: Visualization of bone marrow monocyte mobilization using Cx3cr1gfp/+Flt3L2/2 reporter mouse by multiphoton intravital microscopy Maximilien Evrard,*,†,1 Shu Zhen Chong,* Sapna Devi,* Weng Keong Chew,* Bernett Lee,* Michael Poidinger,* Florent Ginhoux,* Suet Mien Tan,† and Lai Guan Ng*,1 *Singapore Immunology Network, Agency for Science, Technology and Research, Biopolis, Singapore; and †School of Biological Sciences, Nanyang Technological University, Singapore RECEIVED MAY 29, 2014; REVISED NOVEMBER 4, 2014; ACCEPTED NOVEMBER 22, 2014. DOI: 10.1189/jlb.1TA0514-274R
ABSTRACT Monocytes are innate immune cells that play critical roles in inflammation and immune defense. A better comprehension of how monocytes are mobilized and recruited is fundamental to understand their biologic role in disease and steady state. The BM represents a major “checkpoint” for monocyte homeostasis, as it is the primary site for their production and release. Our study determined that the Cx3cr1gfp/+ mouse strain is currently the most ideal model for the visualization of monocyte behavior in the BM by multiphoton intravital microscopy. However, we observed that DCs are also labeled with high levels of GFP and thus, interfere with the accuracy of monocyte tracking in vivo. Hence, we generated a Cx3cr1gfp/+Flt3L2/2 reporter mouse and showed that whereas monocyte numbers were not affected, DC numbers were reduced significantly, as DCs but not monocytes depend on Flt3 signaling for their development. We thus verified that mobilization of monocytes from the BM in Cx3cr1gfp/+Flt3L2/2 mice is intact in response to LPS. Collectively, our study demonstrates that the Cx3cr1gfp/+Flt3L2/2 reporter mouse model represents a powerful tool to visualize monocyte activities in BM and illustrates the potential of a Cx3cr1gfp/+-based, multifunctionality fluorescence reporter approach to dissect monocyte function in vivo. J. Leukoc. Biol. 97: 611–619; 2015.
Introduction Since its beginnings in the early 2000s, multiphoton intravital microscopy has shed new light on the understanding of immune Abbreviations: A*STAR = Agency for Science Technology and Research, BM = bone marrow, BRC = Biologic Resource Center, Ccr2rfp = B6.129(Cg)Ccr2tm2.1Ifc/J, Cx3cr1gfp = B6.129P-Cx3cr1tm1Litt/J, DC = dendritic cell, DT = diphtheria toxin, Flt3L = Fms-related tyrosine kinase 3 ligand, Flt3L2/2 = Flt3L-deficient (C57BL/6-flt3Ltm1Imx), MaFIA = macrophage Fas-induced apoptosis [C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J], RFP = red fluorescent protein, SHG = second harmonic generation, SIgN = Singapore Immunology Network The online version of this paper, found at www.jleukbio.org, includes supplemental information.
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processes in vivo [1]. From its origins as a tool used for the study of cellular interactions, occurring within lymphoid organs, the use of multiphoton intravital microscopy has now greatly expanded toward many other aspects of the immune response, such as host cell-pathogen interactions [2, 3], immune cell activation [4], and immune cell development [5]. In addition, this technique allows careful delineation of discrete cellular events in lymphoid and nonlymphoid organs, including the departure of DCs from the skin [6] and the egress of immune cells from the BM [7, 8]. Thus, multiphoton microscopy represents a powerful approach, as it has the capacity to provide visual dynamic insights that would not be achievable by other conventional immunologic techniques, including immunostaining of tissue sections and flow cytometry. Monocytes are versatile immune cells that play critical roles in host antimicrobial defense. Upon inflammation, circulating monocytes quickly infiltrate affected tissues and undergo rapid differentiation into macrophage- or DC-like cells [9, 10]. However, the uncontrolled activation of monocytes may be harmful for the host and has been linked with inflammatory diseases, such as atherosclerosis and cancer progression [11, 12]. Under physiologic conditions, the BM is the primary site for monocyte production, where they reside. However, inflammatory mediators, such as type 1 IFNs and TLR ligands, can quickly induce monocyte release from the BM to the bloodstream [13]. Thus, the mobilization of monocytes from BM represents a major checkpoint for monocyte homeostasis. Mechanisms governing monocyte trafficking are mostly described from cell populationbased assays, which allow quantitative investigation of cell numbers and localization in various body compartments. However, these assays lack the spatiotemporal resolution for examining subtle dynamic events. In the recent years, a number of intravital multiphoton imaging models have been established
1. Correspondence: SIgN, 8A, Biomedical Grove, #3 Immunos, Biopolis, 138648 Singapore. E-mails: [email protected] (L.G.N.); [email protected] (M.E.).
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to study the dynamics of cellular activities in the BM [7, 14–16]. Similar to most intravital imaging studies, these BM studies relied on the use of reporter mice expressing fluorescent proteins. However, these mice carry the caveat of marking more than 1 single cell population fluorescently [17, 18]. In this report, we aim to identify the most robust and reliable fluorescent reporter mice that can be used for in vivo imaging of BM monocytes. To achieve this goal, we have performed a comprehensive analysis of several fluorescent reporter mouse strains, in which monocytes are tagged with fluorescent proteins. To this end, we determined the Cx3cr1gfp reporter mouse to be most suitable for the imaging of BM monocytes by multiphoton microscopy. However, we found that high levels of GFP were also expressed by BM DCs in these mice, which compromised the ability to track monocyte activities specifically by multiphoton imaging. To overcome this issue, we generated a Cx3cr1gfp/+Flt3L2/2 reporter mouse, which displayed significantly reduced GFP+ DC frequency without affecting the frequency of GFP+ monocytes in the BM. Moreover, we observed that the monocyte response in this crossbred mouse appeared to be normal, as monocytes were able to egress from the BM in a similar manner as Cx3cr1gfp/+ control littermates in response to inflammatory stimuli, such as LPS. Together, our study introduces a novel approach that facilitates the in vivo tracking of monocyte activities in the BM and could ultimately help to understand better mechanisms involved in monocyte homeostasis.
EDTA. Femur cells were harvested and isolated by flushing. RBCs were lysed by use of 13 RBC lysis buffer solution from eBioscience (San Diego, CA, USA). Cells were stained with the following antibodies: F4/80 (clone Cl:A3-1; from AbD Serotec, Raleigh, NC, USA); CD45 (clone 30-F11; from BD PharMingen, Singapore; CD11c (clone N418), Ly6C (clone HK1.4), Ly6G (clone 1A8), and Siglec-H (clone 551; from BioLegend (San Diego, CA, USA); CD3e (clone 17A2), CD11b (clone M1/70), CD115 (clone AFS598), MHCII (IA-IE; clone M5/114.15.2), and NK1.1 (clone PK136; from eBioscience. Flow cytometry acquisition was performed on a LSR II (Becton Dickinson, Singapore) by use of FACSDiva software from BD Biosciences and was subsequently analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
In vivo MHCII staining Cx3cr1gfp/+Flt3L+/+ or Cx3cr1gfp/+Flt3L2/2 mice were injected i.v. with 4 mg of a PE-conjugated MHCII antibody (IA-IE; clone M5/114.15.2) where indicated. One hour after injection, mice were prepared for skull BM intravital imaging and were subsequently imaged for 5 min/field of view (450 3 450 mm). The fluorescence intensity of GFP and PE was detected by use of the 990 nm excitation wavelength. A minimum of 5 fields of view was imaged per mouse.
Image analysis Snapshots were stitched together by use of Fiji where indicated. Stitched images or snapshots were processed by use of Imaris software (Bitplane, Zurich, Switzerland) to adjust threshold values for the different fluorescence channels.
Cell morphology analysis MATERIALS AND METHODS
Mice Cx3cr1gfp, Ccr2rfp, and MaFIA mice were all obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Flt3L2/2 mice were from Taconic Biosciences (Hudson, NY, USA). All mice were maintained under specific pathogen-free facilities in the BRC of A*STAR (Biopolis, Singapore). All experiments involving mice were done under the approval of the Institutional Animal Care and Use Committee of the BRC.
Multiphoton intravital imaging of skull BM Skull BM imaging was performed as described previously with minor modifications [8]. In brief, mice were anesthetized by i.p. injection of ketamine (150 mg/kg) and xylazine (10 mg/kg). Mice were i.v. injected with 50 mg Evans blue in 50 ml sterile PBS to label the blood vessels. Mice were subsequently immobilized on a heated, custom-made stage to maintain the body temperature at 37°C. A small skin area on the skull was excised to expose the BM. The exposed skull was superfused with sterile PBS and covered with a glass coverslip. Imaging was conducted by use of a TriM Scope II microscope (LaVision BioTec, Bielefeld, Germany), equipped with a 203 1.4 numerical aperture water immersion objective lens and a Chameleon-pulsed infrared laser (titanium-sapphire and optical parametric oscillator; Coherent, Santa Clara, CA, USA). Excitation wavelengths (950 nm and 1100 nm) were used, respectively, for GFP and RFP detection.
Flow cytometry, antibodies, and reagents In brief, 100 ml blood was collected via an incision at the submandibular region into Eppendorf tubes containing 20 ml EDTA (0.5 M, pH = 8) to prevent clotting. Mice were anesthetized by i.p. injection of ketamine (150 mg/kg) and xylazine (10 mg/kg) and were subsequently perfused from the left ventricle of the heart with 10 mL cold PBS containing 2%
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CX3CR1-GFP+ objects were selected by use of the Imaris surface tool. Cell volume and sphericity were calculated by Imaris and subsequently plotted into Prism (GraphPad Software, La Jolla, CA, USA). Objects with a volume ,100 mm3 were excluded from analysis. Where indicated, GFP+ objects were further distinguished based on their MHCII-PE positivity.
LPS-induced BM mobilization For flow cytometry assays, mice were retro-orbitally injected with 10 ng LPS from Escherichia coli (serotype O55:B5) from Alexis Biochemicals (San Diego, CA, USA). Control mice were retro-orbitally injected with 100 ml sterile PBS. For multiphoton imaging experiments, mice were anesthetized by i.p. injection of ketamine (150 mg/kg) and xylazine (10 mg/kg). Mice were cannulated for the jugular vein, and 50 mg Evans blue in 50 ml sterile PBS was administered via the jugular vein to label blood vessels. After immobilization on the custom-made stage, mice were imaged for 30 min (baseline) and were subsequently injected with 10 ng LPS in 100 ml sterile PBS through the cannulum and imaged for an additional 3 h. Images were acquired every 20 s by use of a 4 mm z-step size with a depth of ;50 mm.
Cell tracking For LPS-induced BM mobilization, snapshots images were converted as time-lapse movies by use of Imaris software. Cell tracking was done semiautomatically by use of Imaris spot-tracking algorithms. Cells displaying “DC-like” features [defined as high cell volume (.1000 mm3), low sphericity (,0.60), and sessile after LPS administration] were excluded from analysis. The mean velocity and displacement length were calculated by Imaris.
Statistical analysis Statistical analyses were done by use of Prism. Student’s t-test (normal distribution) or 1-way ANOVA was performed. P , 0.05 was considered statistically significant.
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RESULTS AND DISCUSSION
Comparative analysis of Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA skull BMs To monitor immune cells in their native environment, one of the most popular approaches consists of the use of a “cell type” reporter animal that genetically expresses a fluorescent protein under the control of a specific promoter. In this system, we define 2 criteria that should be fulfilled to track accurately the cell of interest in a chosen organ; i.e., 1) the cell type of interest should constitute the majority of labeled cells, and 2) the cell type of interest should display the highest fluorescence intensity among labeled cells. For example, neutrophils constitute .70% of GFP+ cells, followed by monocytes, in the BM of Lyz2gfp reporter mice. However, neutrophils display the highest GFP intensity and were clearly distinguishable from monocytes [8]. Hence, we consider the Lyz2gfp reporter mouse as an ideal model for the tracking BM neutrophils based on the 2 previously enunciated criteria. Multiple fluorescent reporter animals are commonly used to study mononuclear phagocytes, with each model consisting of specific advantages and disadvantages [17]. We selected 3 different genetically encoded fluorescent reporter mouse strains—Cx3cr1gfp, Ccr2rfp, and MaFIA—that target genes known to play critical roles in monocyte trafficking and survival and are expressed by all monocytes [19–21]. In brief, CX3CR1 is essential for monocyte trafficking and survival of non-classical monocytes in the periphery [22]. Next, CCR2 is critical for the egress of classical monocytes from the BM [23]. Finally, MaFIA is a reporting/depleting system targeting Csf1R, a receptor essential for monocyte/macrophage development and survival [10, 17]. To identify the optimal reporter mouse for the study of monocyte behavior within the intact BM, we thus implemented the following strategy: we first determined the labeling pattern of each reporter mouse through flow cytometry; next, we imaged the BM of these mice by multiphoton microscopy and correlated these data with the flow cytometry data.
Imaging monocyte egress from the bone marrow
To characterize the various fluorescent-labeled BM immune cell populations in these mice, we used an antibody panel in conjunction with our gating strategy as shown (Supplemental Fig. 1). All 3 strains tested consisted of fluorescently labeled Ly6Chi and Ly6Clo monocyte subsets (Fig. 1A). However, a variety of other immune cell subsets were also labeled, and this degree varied based on the type of reporter strain used. We found that myeloid cells, such as neutrophils, macrophages, and DCs, were labeled in the MaFIA reporter mouse (Fig. 1A). On the other hand, macrophages, DCs, and NK, and/or T cells, but not neutrophils, possess varying levels of RFP fluorescence in the Ccr2rfp/+ reporter mouse (Fig. 1A). In contrast, the Cx3cr1gfp/+ reporter mouse labels DCs but has significantly less GFP expression in other immune cell subsets compared with the MaFIA and Ccr2rfp/+ animals (Fig. 1A). When we compared the number of each immune cell type in relation to all fluorescently labeled cells acquired, neutrophils but not monocytes were the major subset labeled in the MaFIA mouse BM, thus indicating that this reporter mouse is not suitable for monocyte tracking (Fig. 1B and Supplemental Table 1). In contrast, Ly6Chi monocytes represented an average of 60% of the labeled cells in Cx3cr1gfp/+ and Ccr2rfp/+ reporter animals (Fig. 1B and Supplemental Table 1). However, the Ccr2rfp/+ substantially differed from the Cx3cr1gfp/+ reporter animals in term of the fluorescence intensity among labeled cells. We noted that myeloid cells, T lymphocytes, and/or NK cells express similar levels of RFP as monocytes in Ccr2rfp/+ mice. On the other hand, a high level of GFP expression was detected specifically, only in myeloid populations in Cx3cr1gfp/+ mice. In particular, Ly6Clo monocytes and DCs express the highest levels of GFP expression, followed by Ly6Chi monocytes. However, despite their low numbers in the BM in Cx3cr1gfp/+ mice, we found DCs to express similar levels of GFP in this mouse that may interfere with the direct tracking of monocytes. Of note, we found that, 20–25% of labeled cells in Cx3cr1gfp/+ and Ccr2rfp/+ reporter mice could not be determined by our multicolor antibody panel (Fig 1B). Whereas these
Figure 1. Phenotypic analysis of BM cells in Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA reporter mice. (A) Histograms demonstrate the relative GFP/RFP intensity in different leukocyte populations among Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA reporter mice. (B) Pie charts represent the proportion of specific leukocyte populations among the GFP/RFP+ fraction in Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA reporter animals. The relative percentages are shown in Supplemental Table 1. (A and B) The depicted leukocyte populations were gated as shown in Supplemental Fig. 1 (n = 4–5 mice/reporter strain).
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undetermined cells expressed intermediate to high levels of RFP in Ccr2rfp/+ animals, their levels of GFP in Cx3cr1gfp/+ animals were relatively much lower. As such, unlike with Ccr2rfp/+ mice, these undetermined GFP+ cells in Cx3cr1gfp/+ animals would likely fall below the detection limit of the multiphoton imaging. These results further illustrate the incompatibility of the use of Ccr2rfp/+ animals but highlight the efficiency of the Cx3cr1gfp/+ mice model for the purpose of BM monocyte-tracking studies. To validate further the suitability of each strain for in vivo imaging, we examined each of the Cx3cr1gfp, Ccr2rfp, and MaFIA reporter mice by multiphoton intravital fluorescence microscopy. We used the skull calvarium BM imaging model, originally established by von Andrian’s group [24], which is widely used for direct observation of individual cells within the BM of living animals [8, 14, 16]. For our analysis, we acquired stitched images to obtain a comprehensive view of the skull BM architecture (Fig. 2A and B). We used Evans blue to highlight BM blood sinusoidal vessels [25] and the SHG signal for the visualization of the bone/collagen structures. Of note, the BM parenchyma that contains the hematopoietic cells is characterized by the absence of the SHG signal (Fig. 2B and C). We found that in the MaFIA reporter mouse, almost all of the parenchyma space contained GFP+ cells (Fig. 2B). At a higher magnification, GFP+ cells presented heterogeneous morphology, with the majority of these
cells displaying a low cell volume and a spherical morphology, which is consistent with the fact that the majority of the cells labeled in this system consists of neutrophils (Figs. 1B and 2C). On the other hand, the Ccr2rfp/+ reporter mouse exhibited fewer labeled cells than the MaFIA mouse (Fig. 2B). However, the RFP+ cells displayed heterogeneous morphology—from large and elliptic to small and spherical shapes, as well as ,1 mm diameter objects (Fig. 2B and C). This apparent morphologic heterogeneity observed is likely a result of the labeling of multiple cell populations, as shown in our flow cytometry data (Fig. 1A). Finally, the Cx3cr1gfp/+ reporter mouse showed much less labeled cells in the BM compared with the Ccr2rfp/+ animals (Fig. 2B). Two major types of morphology could be observed: large and elliptic cells with protruding dendrites, as well as small and spherical cells (Fig. 2B and C). These data are in agreement with our flow cytometry data, whereby monocytes and DCs represent 2 immune cell subsets that are predominantly labeled and display the highest GFP intensity among CX3CR1+ cells (Fig. 1B and Supplemental Table 1). Thus far, we have analyzed the BM of 3 common strains used to study mononuclear phagocytes, namely Cx3cr1gfp, Ccr2rfp, and MaFIA, by flow cytometry and multiphoton imaging. Among these 3 reporters, we established that the Cx3cr1gfp reporter mouse appeared to be the most ideal reporter for the tracking of
Figure 2. Visualization of the skull BM in Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA reporter mice via multiphoton intravital microscopy. (A) An overview of the steps involved during intravital imaging of the skull calvarium BM. Skin was first removed carefully to expose the BM. Images were acquired via stitching snapshots of the BM to form a final image of 800 3 1200 mm or as a single snapshot sized at 450 3 450 mm. To label sinusoidal vessels, mice were i.v. injected with Evans blue (n = 3–5/ reporter strain). (B) A representative stitched image of the skull BM in Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA reporter mice. Original scale bars, 100 mm. (C) (Upper) snapshot of the skull BM in the 3-strain mouse, whereby single plane images were acquired at different depths ranging from 0 to 36 mm along the z-axis. Original scale bars, 50 mm. (Lower) In addition, magnified single-plane images at z-positions 12 mm and 24 mm are shown. Original scale bars, 20 mm. GFP/RFP+ cells, Green; sinusoidal blood vessels, red; and bone/collagen structures (through SHG), blue.
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monocytes in the BM. As shown, monocytes in these mice account for the majority of GFP-labeled cells and expressed the highest fluorescence intensity among labeled cells in the BM. Whereas we demonstrated the suitability of the Cx3cr1gfp reporter mouse as a tool for visualization of BM monocytes, our study also revealed that DCs are labeled with similar levels of GFP expression as monocytes. In addition, our data suggest the presence of large and elliptic GFP+ cells that are localized in close proximity with monocytes in the BM and may thus interfere with the accuracy of monocyte tracking in vivo.
CX3CR1+ cells carrying a dendritic morphology are DCs and share a similar location with monocytes After selecting Cx3cr1gfp/+ as the most suitable reporter animal, we next sought to confirm the identity of the GFP bright, large, and elliptic cells, located in the BM parenchyma. Our flow cytometry results suggest that these cells are DCs (Fig. 1A). Furthermore, BM DCs have been reported previously to be CX3CR1+MHCII+CD11c+ [26]. Thus, to identify DCs in situ, we administered a fluorescent MHCII antibody to Cx3cr1gfp/+ animals (Fig. 3A). We found that CX3CR1+MHCII+ cells were generally larger with protruding dendrites, whereas CX3CR1+MHCII2 cells were generally smaller and spherical
Imaging monocyte egress from the bone marrow
(Fig. 3A and Supplemental Video 1). To confirm these observations, we devised an image analysis strategy to correlate cellular morphology and staining for MHCII expression (Fig. 3B). We first selected cells based on GFP expression, followed by their positivity for MHCII; finally, we plotted MHCII+ and MHCII2 cells according to morphologic criteria—cell volume and sphericity. We defined cells with a high cell volume and low sphericity as having a DC-like morphology, whereas cells that consist of a low cell volume and high sphericity have a “monocyte-like” morphology (Fig. 3B). With the use of these morphologic criteria, we found that the majority of CX3CR1+MHCII+ cells had a DC-like morphology, whereas the majority of CX3CR1+MHCII2 cells had a monocyte-like morphology (Fig. 3C and D). Together, this provides further evidence that morphologic criteria can be used as a plausible and inexpensive approach to discriminate DCs from monocytes in the BM of Cx3cr1gfp/+ animals.
Flt3L-deficiency reduces DC numbers and improves the identification of monocytes in the BM of the Cx3cr1gfp/+ reporter mouse To this end, our results indicated that DCs can be discriminated from monocytes morphologically. To track monocyte activities in
Figure 3. CX3CR1+ cells that possess a DC-like morphology in the BM of Cx3cr1gfp/+ mice are DCs. Cx3cr1gfp/+ reporter mice were i.v. administered with PE-conjugated MHCII antibody and Evans blue to detect for MHCII+ cells and blood vessels, respectively. (A) Snapshots sized at 450 3 450 mm of Cx3cr1gfp/+ reporter mice, demonstrating the individual fluorescent channels detecting for CX3CR1+ cells, MHCII+ cells, and blood sinusoids, with a final merged image of all channels. (Lower) Magnified region of the individual snapshots. Original scale bars, 20 mm. (B) An outline of the process involved during image analysis. Objects were first “gated” for a GFP+ signal. Following this, CX3CR1+ objects were separated based on MHCII expression, CX3CR1+MHCII+ and CX3CR1+MHCII2 objects were subsequently analyzed for cell volume and sphericity, represented on a scatter plot. Objects with a volume .1000 mm3 and sphericity ,0.65 were considered to display a DC-like morphology, whereas monocyte-like cells possessed volumes of ,2000 mm3 and sphericity .0.65. MFI, Mean fluorescence intensity. (C) Percentage of cells, displaying a DC-like or monocyte-like morphology, are shown in red and green, respectively. (D) Scatter plot, demonstrating a merged view of the total amount of CX3CR1+MHCII+ and CX3CR1+MHCII2 cells present in Cx3cr1gfp/+ reporter mice. Data are pooled from 4 fields of view from the same mouse (n = 3 mice).
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the BM accurately and efficiently, we aim to deplete specifically the DC population from the BM in the Cx3cr1gfp/+ reporter mouse. It has been reported previously that CX3CR1+ DCs can be depleted by administration of DT in CD11c-DTR mice, which express DTR under the CD11c promoter (also termed integrin a X) [14, 26]. However, this system lacks specificity for DC depletion, as other CD11c-expressing cells have also been shown to be affected upon DT administration [27, 28]. In addition, DTinduced cell depletion is known to result from the induction of cell death, which consequently, may lead to neutrophilia and monocytosis [29]. Thus, this approach precluded the use of these mice to the study of monocyte mobilization from the BM under physiologic conditions. Flt3 signaling plays a central, nonredundant role in DC homeostasis, as the lack of Flt3L or its receptor Flt3 affects DC proliferation, differentiation, and maintenance of DC numbers [30]. We thus took advantage of this property and generated
a crossbred Cx3cr1gfp/+Flt3L2/2 reporter mouse. We first performed intravital imaging on the skull BM. From the acquired stitched images, we performed a semiautomated quantification of GFP+ cells that were separated according to their morphology— DC-like and monocyte-like (Fig. 4A). Accordingly, with the use of these parameters, we found a significant reduction in cells harboring a DC-like morphology in Cx3cr1gfp/+Flt3L2/2 mice compared with Cx3cr1gfp/+Flt3L+/+ control mice (Fig. 4B). To validate our imaging data further, we analyzed the frequency of DCs, monocytes, and macrophage Cx3cr1gfp/+Flt3L2/2 animals compared with Cx3cr1gfp/+Flt3L+/+ controls via flow cytometry (Fig. 4C and D). Notably, the absence of Flt3L is known to affect general hematopoiesis [31]. Consequently, we found a decrease in BM cellularity in Cx3cr1gfp/+Flt3L2/2 mice. To ensure an unbiased comparison of the cellular distribution in Flt3L2/2 and wild-type animals, we expressed the cell count in each individual cell types as a fraction of the
Figure 4. DC but not monocyte BM development is impaired in the Cx3cr1gfp/+Ftl3L2/2 reporter mouse. (A) Representative stitched images of the skull BMof Cx3cr1gfp/+Flt3L+/+ andCx3cr1gfp/+Flt3L2/2 reporter mice. Mice were i.v. administered with Evans blue to label sinusoidal vessels (n = 3/strain). Original scale bars (left panel), 100 mm. A white box on the stitched images magnifies the region indicated, whereby single plane images for z = 12 and 24 mm are illustrated. Original scale bars (right panel), 20 mm. DC-like cells, Magenta (arrowheads); monocyte-like cells, green; and BM sinusoids, gray. (B) CX3CR1+ cells were distinguished according to their morphology; i.e., cells with a volume .1000 mm3 and sphericity ,0.65 and volume ,2000 mm3 and sphericity .0.65 were considered to display a DC-like or monocyte-like morphology, respectively. The frequencies of DC-like or monocyte-like cells present in Cx3cr1gfp/+Flt3L+/+ and Cx3cr1gfp/+Flt3L2/2 reporter mice were analyzed and represented as a pie chart (n = 3 mice/reporter strain). A typical example of flow cytometry plots identifying (C) BM DCs (CX3CR1+CD11chiMHCIIhi) and (D) monocytes (CX3CR1+CD11b+CD115+Ly6Chi or Ly6Clo, according to the subset). Comparison of percentages of (E) DCs, (G) Ly6Chi monocytes (Mo), (F) macrophages, and (H) Ly6Clo monocytes in Cx3cr1gfp/+Flt3L+/+ and Cx3cr1gfp/+Flt3L2/2 mice. Cell percentages are expressed as a ratio of the cell count over the total GFP + cell number. (C–H) Cell populations were gated accordingly by use of the strategy shown in Supplemental Fig. 1 (n = 5 mice/reporter strain). Data are shown as mean 6 SEM and are pooled from 2 different experiments. n.s., Nonsignificant; *P , 0.05 (unpaired t-test).
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total GFP+ cells. Based on this evaluation, we showed that DC frequency was significantly less in Flt3L2/2 animals, whereas no difference was observed in Csf1R-dependent macrophages and monocytes (Fig. 4D and F–H), as reported previously [32, 33]. Of note, we detected a modest but significant decrease of DC-like cells by imaging (Fig. 4B) compared with a sharp decrease of DCs by flow cytometry (1.53- vs. 2.43-fold decrease; Fig. 4B and E). Conceivably, this discrepancy could be attributed to a population of CX3CR1+ BM macrophages [14], which may possess a DC-like morphology, as detected by multiphoton imaging but are not affected by the loss of Flt3L. In summary, we showed that Flt3L-deficiency leads to the reduction of DC-like morphology-bearing CX3CR1+ cells in the BM, and this reduction was correlated with a significant reduction in BM DC frequency, thereby facilitating the tracking of BM monocyte behavior in Cx3cr1gfp/+Flt3L2/2 animals. Moreover, analysis of our imaging data also suggests that macrophages in the BM possess a DC-like morphology, indicating that the morphologic criteria that we applied here can be used specifically to “segment” out monocytes in Cx3cr1gfp/+ mice.
Imaging monocyte egress from the bone marrow
Flt3L2/2 in the Cx3cr1gfp/+ reporter mouse does not impair monocyte mobilization from the BM Among monocytes, Ly6Chi monocytes are short lived, proinflammatory cells that egress the BM through a CCR2-dependent mechanism upon an inflammatory challenge [23]. Thus far, we have shown that the Cx3cr1gfp/+Flt3L2/2 reporter mouse, which had significantly reduced DC numbers, appears to be the most ideal system for the visualization of monocytes in the BM. As our objective is to track BM monocyte movements in these mice, we have to ensure that the absence of Flt3L does not affect the migratory behavior of monocytes in the BM. To this end, we subjected Cx3cr1gfp/+ reporter mice to subclinical doses of LPS, which is known to mobilize monocytes to the circulation [34], and followed medullar and circulating Ly6Chi monocyte numbers over time. In this set of experiments, we defined Ly6Chi monocytes as Lin2CD11b+SSCloLy6G2Ly6ChiCX3CR1+ (Fig. 5A and B). We observed that Ly6Chi monocyte numbers peaked in the blood, 4 h after LPS treatment, whereas their BM numbers decreased significantly within 2 h (Fig. 5A). We next compared the ability of Cx3cr1gfp/+Flt3L2/2 animals to egress from the BM after LPS challenge with their control Cx3cr1gfp/+Flt3L+/+
Figure 5. Monocyte mobilization by LPS is not affected by the loss of Flt3L. (A) Kinetics of Ly6Chi monocytes in blood and BM after i.v. injection of LPS (10 ng) in Cx3cr1gfp/+Flt3L+/+ mice (n = 4 mice/ group/time-point). Data are shown as mean 6 SD and are representative of 2 independent experiments. **P , 0.01 (unpaired t-test). (B) Comparison of the frequencies of Ly6Chi monocytes (expressed as a ratio of the cell count over the total GFP+ cell number) in the blood or BM, 2 h after LPS administration in Cx3cr1gfp/+Flt3L+/+ and Cx3cr1gfp/+Flt3L2/2 mice. In both tissues, Ly6Chi monocytes were considered Lin2 (CD3, NK1.1, Siglec-H) CD11b+SSCloLy6G2CX3CR1+ (n = 6–10 mice/group/condition). Data are shown as mean 6 SEM and are pooled from 2 different experiments. ****P , 0.0001 (unpaired t-test). (C) (Left) Maximum z-projected images of the skull BM in the Cx3cr1gfp/+ mouse at baseline. White dotted areas represent the BM parenchyma. (Right) Thirty-minute duration tracks of individual GFP+ monocyte-like cells at baseline and 1 and 2 h after LPS treatment in Cx3cr1gfp/+Flt3L+/+ and Cx3cr1gfp/+Flt3L2/2 mice. Red dotted areas represent the BM parenchyma. Original scale bars, 20 mm. GFP+ cells, Green; sinusoidal blood vessels, red, (D) The mean velocity and (E) displacement of GFP+ monocyte-like cells were measured at baseline and 1 and 2 h after LPS treatment in Cx3cr1gfp/+Flt3L+/+ and Cx3cr1gfp/+Flt3L2/2 mice (n = 3 mice/group/treatment). ****P , 0.0001 (1-way ANOVA). (F) Mean velocity of GFP+ DC-like cells at baseline and 1 and 2 h after LPS treatment in Cx3cr1gfp/+Flt3L+/+ mice (n = 3 mice/treatment). (G) A representative end-point image illustrating the tracks of GFP+ DC-like from baseline to 2 h after LPS treatment. Original scale bar, 20 mm.
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littermates. We found that similar to the control littermates, Ly6Chi monocytes showed increased blood counts and decreased BM counts within 2 h after LPS treatment (Fig. 5B). Collectively, these data provide evidence that the lack of Flt3L did not impair the ability of Ly6Chi monocytes to egress from the BM to the bloodstream in response to proinflammatory stimuli, such as LPS. To validate this finding further, we performed intravital imaging to study the cellular behavior of monocytes in the BM during LPS challenge. These studies showed evidences that in Cx3cr1gfp/+Flt3L2/2 and Cx3cr1gfp/+Flt3L+/+ animals, monocytes in the BM parenchyma remained mostly sessile at baseline and until 1 h after LPS administration. However, they steadily increased their motility from 1 h after LPS treatment and gradually egressed from the parenchymal space into the sinusoidal vessels within 2 h (Fig. 5C and D and Supplemental Videos 2 and 3). Furthermore, this increase in cell motility was accompanied with increased cellular displacement, in agreement with monocyte departure from the BM parenchyma (Fig. 5E). Our tracking analysis indicated that BM monocytes, in Cx3cr1gfp/+Flt3L2/2 and Cx3cr1gfp/+Flt3L+/+ mice, displayed similar motility patterns in terms of their velocity and displacement, 1 h after LPS administration. Interestingly, we observed that monocytes in the BM of Cx3cr1gfp/+Flt3L2/2 mice exhibited a slight but significant increase in their velocity and displacement compared with control mice at 2 h after LPS injection. Despite this difference, we believe that the overall functional mobilization of BM monocytes is not altered in Cx3cr1gfp/+Flt3L2/2 mice, as there is no detectable difference in the number of circulating monocytes compared with Cx3cr1gfp/+Flt3L+/+ mice in response to LPS (Fig. 5B). Of note, our morphologic identification method allowed us to follow the behavior of BM DC-like cells over time. These cells were sessile during baseline conditions. However, unlike monocytes, DC-like cells did not increase their motility following LPS treatment (Fig. 5F and G). In summary, we present a novel approach that allows the study of monocyte mobilization from the BM through a combinatorial use of the Cx3cr1gfp/+Flt3L2/2 reporter mouse, multiphoton intravital microscopy, and subsequent image analysis, based on cell morphology. Our current study also provides a significant conceptual advance by illustrating the use of a Cx3cr1gfp/+-based, dual functionality fluorescence reporter line for improving the specificity of monocyte imaging in the BM. More importantly, these results obtained by multiphoton imaging support and complement our flow cytometry data by providing in situ information that includes morphologic identification of DCs and monocytes and the migratory status of these cells. Our current report proposes the use of the Cx3cr1gfp/+Flt3L2/2 mouse as a tool for in vivo tracking and analysis of the cellular behavior of monocytes in the BM. The control of monocyte release from the BM represents a major checkpoint in monocyte homeostasis, and this process has a significant impact on the number of circulating monocytes. Thus, it is imperative to characterize the spatiotemporal regulation of monocyte mobilization from the BM to understand monocyte function better. Whereas we still do not fully understand whether the loss of DCs will have an impact on overall monocyte function, our study demonstrates the suitability of the Cx3cr1gfp/+Flt3L2/2 mouse 618
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for the in vivo tracking of monocytes in the BM. Lastly, we envision that this Cx3cr1gfp/+-based, combined approach would also be beneficial toward understanding the dynamic behavior and development of other mononuclear phagocytes in different organs.
AUTHORSHIP M.E. designed the research, performed experiments, analyzed results, and wrote the manuscript. S.Z.C. and S.D. performed experiments and wrote the manuscript. W.K.C. generated the Cx3cr1gfp/+Flt3L2/2 mouse. B.L. analyzed results. M.P. contributed intellectually. F.G. provided the Flt3L2/2 mouse, contributed intellectually, and prepared the manuscript. S.M.T. contributed intellectually and prepared the manuscript. L.G.N. designed the research and wrote the manuscript.
ACKNOWLEDGMENTS This research was funded by SIgN, A*STAR (Singapore). The authors thank the flow cytometry facility of SIgN for its technical help and support.
DISCLOSURES
The authors declare no conflicts of interest.
REFERENCES
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KEY WORDS: dendritic cells macrophages trafficking live imaging •
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ABSTRACT Monocytes are innate immune cells that play critical roles in inflammation and immune defense. A better comprehension of how monocytes are mobilized and recruited is fundamental to understand their biologic role in disease and steady state. The BM represents a major “checkpoint” for monocyte homeostasis, as it is the primary site for their production and release. Our study determined that the Cx3cr1gfp/+ mouse strain is currently the most ideal model for the visualization of monocyte behavior in the BM by multiphoton intravital microscopy. However, we observed that DCs are also labeled with high levels of GFP and thus, interfere with the accuracy of monocyte tracking in vivo. Hence, we generated a Cx3cr1gfp/+Flt3L2/2 reporter mouse and showed that whereas monocyte numbers were not affected, DC numbers were reduced significantly, as DCs but not monocytes depend on Flt3 signaling for their development. We thus verified that mobilization of monocytes from the BM in Cx3cr1gfp/+Flt3L2/2 mice is intact in response to LPS. Collectively, our study demonstrates that the Cx3cr1gfp/+Flt3L2/2 reporter mouse model represents a powerful tool to visualize monocyte activities in BM and illustrates the potential of a Cx3cr1gfp/+-based, multifunctionality fluorescence reporter approach to dissect monocyte function in vivo. J. Leukoc. Biol. 97: 611–619; 2015.
Introduction Since its beginnings in the early 2000s, multiphoton intravital microscopy has shed new light on the understanding of immune Abbreviations: A*STAR = Agency for Science Technology and Research, BM = bone marrow, BRC = Biologic Resource Center, Ccr2rfp = B6.129(Cg)Ccr2tm2.1Ifc/J, Cx3cr1gfp = B6.129P-Cx3cr1tm1Litt/J, DC = dendritic cell, DT = diphtheria toxin, Flt3L = Fms-related tyrosine kinase 3 ligand, Flt3L2/2 = Flt3L-deficient (C57BL/6-flt3Ltm1Imx), MaFIA = macrophage Fas-induced apoptosis [C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J], RFP = red fluorescent protein, SHG = second harmonic generation, SIgN = Singapore Immunology Network The online version of this paper, found at www.jleukbio.org, includes supplemental information.
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processes in vivo [1]. From its origins as a tool used for the study of cellular interactions, occurring within lymphoid organs, the use of multiphoton intravital microscopy has now greatly expanded toward many other aspects of the immune response, such as host cell-pathogen interactions [2, 3], immune cell activation [4], and immune cell development [5]. In addition, this technique allows careful delineation of discrete cellular events in lymphoid and nonlymphoid organs, including the departure of DCs from the skin [6] and the egress of immune cells from the BM [7, 8]. Thus, multiphoton microscopy represents a powerful approach, as it has the capacity to provide visual dynamic insights that would not be achievable by other conventional immunologic techniques, including immunostaining of tissue sections and flow cytometry. Monocytes are versatile immune cells that play critical roles in host antimicrobial defense. Upon inflammation, circulating monocytes quickly infiltrate affected tissues and undergo rapid differentiation into macrophage- or DC-like cells [9, 10]. However, the uncontrolled activation of monocytes may be harmful for the host and has been linked with inflammatory diseases, such as atherosclerosis and cancer progression [11, 12]. Under physiologic conditions, the BM is the primary site for monocyte production, where they reside. However, inflammatory mediators, such as type 1 IFNs and TLR ligands, can quickly induce monocyte release from the BM to the bloodstream [13]. Thus, the mobilization of monocytes from BM represents a major checkpoint for monocyte homeostasis. Mechanisms governing monocyte trafficking are mostly described from cell populationbased assays, which allow quantitative investigation of cell numbers and localization in various body compartments. However, these assays lack the spatiotemporal resolution for examining subtle dynamic events. In the recent years, a number of intravital multiphoton imaging models have been established
1. Correspondence: SIgN, 8A, Biomedical Grove, #3 Immunos, Biopolis, 138648 Singapore. E-mails: [email protected] (L.G.N.); [email protected] (M.E.).
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to study the dynamics of cellular activities in the BM [7, 14–16]. Similar to most intravital imaging studies, these BM studies relied on the use of reporter mice expressing fluorescent proteins. However, these mice carry the caveat of marking more than 1 single cell population fluorescently [17, 18]. In this report, we aim to identify the most robust and reliable fluorescent reporter mice that can be used for in vivo imaging of BM monocytes. To achieve this goal, we have performed a comprehensive analysis of several fluorescent reporter mouse strains, in which monocytes are tagged with fluorescent proteins. To this end, we determined the Cx3cr1gfp reporter mouse to be most suitable for the imaging of BM monocytes by multiphoton microscopy. However, we found that high levels of GFP were also expressed by BM DCs in these mice, which compromised the ability to track monocyte activities specifically by multiphoton imaging. To overcome this issue, we generated a Cx3cr1gfp/+Flt3L2/2 reporter mouse, which displayed significantly reduced GFP+ DC frequency without affecting the frequency of GFP+ monocytes in the BM. Moreover, we observed that the monocyte response in this crossbred mouse appeared to be normal, as monocytes were able to egress from the BM in a similar manner as Cx3cr1gfp/+ control littermates in response to inflammatory stimuli, such as LPS. Together, our study introduces a novel approach that facilitates the in vivo tracking of monocyte activities in the BM and could ultimately help to understand better mechanisms involved in monocyte homeostasis.
EDTA. Femur cells were harvested and isolated by flushing. RBCs were lysed by use of 13 RBC lysis buffer solution from eBioscience (San Diego, CA, USA). Cells were stained with the following antibodies: F4/80 (clone Cl:A3-1; from AbD Serotec, Raleigh, NC, USA); CD45 (clone 30-F11; from BD PharMingen, Singapore; CD11c (clone N418), Ly6C (clone HK1.4), Ly6G (clone 1A8), and Siglec-H (clone 551; from BioLegend (San Diego, CA, USA); CD3e (clone 17A2), CD11b (clone M1/70), CD115 (clone AFS598), MHCII (IA-IE; clone M5/114.15.2), and NK1.1 (clone PK136; from eBioscience. Flow cytometry acquisition was performed on a LSR II (Becton Dickinson, Singapore) by use of FACSDiva software from BD Biosciences and was subsequently analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
In vivo MHCII staining Cx3cr1gfp/+Flt3L+/+ or Cx3cr1gfp/+Flt3L2/2 mice were injected i.v. with 4 mg of a PE-conjugated MHCII antibody (IA-IE; clone M5/114.15.2) where indicated. One hour after injection, mice were prepared for skull BM intravital imaging and were subsequently imaged for 5 min/field of view (450 3 450 mm). The fluorescence intensity of GFP and PE was detected by use of the 990 nm excitation wavelength. A minimum of 5 fields of view was imaged per mouse.
Image analysis Snapshots were stitched together by use of Fiji where indicated. Stitched images or snapshots were processed by use of Imaris software (Bitplane, Zurich, Switzerland) to adjust threshold values for the different fluorescence channels.
Cell morphology analysis MATERIALS AND METHODS
Mice Cx3cr1gfp, Ccr2rfp, and MaFIA mice were all obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Flt3L2/2 mice were from Taconic Biosciences (Hudson, NY, USA). All mice were maintained under specific pathogen-free facilities in the BRC of A*STAR (Biopolis, Singapore). All experiments involving mice were done under the approval of the Institutional Animal Care and Use Committee of the BRC.
Multiphoton intravital imaging of skull BM Skull BM imaging was performed as described previously with minor modifications [8]. In brief, mice were anesthetized by i.p. injection of ketamine (150 mg/kg) and xylazine (10 mg/kg). Mice were i.v. injected with 50 mg Evans blue in 50 ml sterile PBS to label the blood vessels. Mice were subsequently immobilized on a heated, custom-made stage to maintain the body temperature at 37°C. A small skin area on the skull was excised to expose the BM. The exposed skull was superfused with sterile PBS and covered with a glass coverslip. Imaging was conducted by use of a TriM Scope II microscope (LaVision BioTec, Bielefeld, Germany), equipped with a 203 1.4 numerical aperture water immersion objective lens and a Chameleon-pulsed infrared laser (titanium-sapphire and optical parametric oscillator; Coherent, Santa Clara, CA, USA). Excitation wavelengths (950 nm and 1100 nm) were used, respectively, for GFP and RFP detection.
Flow cytometry, antibodies, and reagents In brief, 100 ml blood was collected via an incision at the submandibular region into Eppendorf tubes containing 20 ml EDTA (0.5 M, pH = 8) to prevent clotting. Mice were anesthetized by i.p. injection of ketamine (150 mg/kg) and xylazine (10 mg/kg) and were subsequently perfused from the left ventricle of the heart with 10 mL cold PBS containing 2%
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CX3CR1-GFP+ objects were selected by use of the Imaris surface tool. Cell volume and sphericity were calculated by Imaris and subsequently plotted into Prism (GraphPad Software, La Jolla, CA, USA). Objects with a volume ,100 mm3 were excluded from analysis. Where indicated, GFP+ objects were further distinguished based on their MHCII-PE positivity.
LPS-induced BM mobilization For flow cytometry assays, mice were retro-orbitally injected with 10 ng LPS from Escherichia coli (serotype O55:B5) from Alexis Biochemicals (San Diego, CA, USA). Control mice were retro-orbitally injected with 100 ml sterile PBS. For multiphoton imaging experiments, mice were anesthetized by i.p. injection of ketamine (150 mg/kg) and xylazine (10 mg/kg). Mice were cannulated for the jugular vein, and 50 mg Evans blue in 50 ml sterile PBS was administered via the jugular vein to label blood vessels. After immobilization on the custom-made stage, mice were imaged for 30 min (baseline) and were subsequently injected with 10 ng LPS in 100 ml sterile PBS through the cannulum and imaged for an additional 3 h. Images were acquired every 20 s by use of a 4 mm z-step size with a depth of ;50 mm.
Cell tracking For LPS-induced BM mobilization, snapshots images were converted as time-lapse movies by use of Imaris software. Cell tracking was done semiautomatically by use of Imaris spot-tracking algorithms. Cells displaying “DC-like” features [defined as high cell volume (.1000 mm3), low sphericity (,0.60), and sessile after LPS administration] were excluded from analysis. The mean velocity and displacement length were calculated by Imaris.
Statistical analysis Statistical analyses were done by use of Prism. Student’s t-test (normal distribution) or 1-way ANOVA was performed. P , 0.05 was considered statistically significant.
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TECHNICAL ADVANCE Evrard et al.
RESULTS AND DISCUSSION
Comparative analysis of Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA skull BMs To monitor immune cells in their native environment, one of the most popular approaches consists of the use of a “cell type” reporter animal that genetically expresses a fluorescent protein under the control of a specific promoter. In this system, we define 2 criteria that should be fulfilled to track accurately the cell of interest in a chosen organ; i.e., 1) the cell type of interest should constitute the majority of labeled cells, and 2) the cell type of interest should display the highest fluorescence intensity among labeled cells. For example, neutrophils constitute .70% of GFP+ cells, followed by monocytes, in the BM of Lyz2gfp reporter mice. However, neutrophils display the highest GFP intensity and were clearly distinguishable from monocytes [8]. Hence, we consider the Lyz2gfp reporter mouse as an ideal model for the tracking BM neutrophils based on the 2 previously enunciated criteria. Multiple fluorescent reporter animals are commonly used to study mononuclear phagocytes, with each model consisting of specific advantages and disadvantages [17]. We selected 3 different genetically encoded fluorescent reporter mouse strains—Cx3cr1gfp, Ccr2rfp, and MaFIA—that target genes known to play critical roles in monocyte trafficking and survival and are expressed by all monocytes [19–21]. In brief, CX3CR1 is essential for monocyte trafficking and survival of non-classical monocytes in the periphery [22]. Next, CCR2 is critical for the egress of classical monocytes from the BM [23]. Finally, MaFIA is a reporting/depleting system targeting Csf1R, a receptor essential for monocyte/macrophage development and survival [10, 17]. To identify the optimal reporter mouse for the study of monocyte behavior within the intact BM, we thus implemented the following strategy: we first determined the labeling pattern of each reporter mouse through flow cytometry; next, we imaged the BM of these mice by multiphoton microscopy and correlated these data with the flow cytometry data.
Imaging monocyte egress from the bone marrow
To characterize the various fluorescent-labeled BM immune cell populations in these mice, we used an antibody panel in conjunction with our gating strategy as shown (Supplemental Fig. 1). All 3 strains tested consisted of fluorescently labeled Ly6Chi and Ly6Clo monocyte subsets (Fig. 1A). However, a variety of other immune cell subsets were also labeled, and this degree varied based on the type of reporter strain used. We found that myeloid cells, such as neutrophils, macrophages, and DCs, were labeled in the MaFIA reporter mouse (Fig. 1A). On the other hand, macrophages, DCs, and NK, and/or T cells, but not neutrophils, possess varying levels of RFP fluorescence in the Ccr2rfp/+ reporter mouse (Fig. 1A). In contrast, the Cx3cr1gfp/+ reporter mouse labels DCs but has significantly less GFP expression in other immune cell subsets compared with the MaFIA and Ccr2rfp/+ animals (Fig. 1A). When we compared the number of each immune cell type in relation to all fluorescently labeled cells acquired, neutrophils but not monocytes were the major subset labeled in the MaFIA mouse BM, thus indicating that this reporter mouse is not suitable for monocyte tracking (Fig. 1B and Supplemental Table 1). In contrast, Ly6Chi monocytes represented an average of 60% of the labeled cells in Cx3cr1gfp/+ and Ccr2rfp/+ reporter animals (Fig. 1B and Supplemental Table 1). However, the Ccr2rfp/+ substantially differed from the Cx3cr1gfp/+ reporter animals in term of the fluorescence intensity among labeled cells. We noted that myeloid cells, T lymphocytes, and/or NK cells express similar levels of RFP as monocytes in Ccr2rfp/+ mice. On the other hand, a high level of GFP expression was detected specifically, only in myeloid populations in Cx3cr1gfp/+ mice. In particular, Ly6Clo monocytes and DCs express the highest levels of GFP expression, followed by Ly6Chi monocytes. However, despite their low numbers in the BM in Cx3cr1gfp/+ mice, we found DCs to express similar levels of GFP in this mouse that may interfere with the direct tracking of monocytes. Of note, we found that, 20–25% of labeled cells in Cx3cr1gfp/+ and Ccr2rfp/+ reporter mice could not be determined by our multicolor antibody panel (Fig 1B). Whereas these
Figure 1. Phenotypic analysis of BM cells in Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA reporter mice. (A) Histograms demonstrate the relative GFP/RFP intensity in different leukocyte populations among Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA reporter mice. (B) Pie charts represent the proportion of specific leukocyte populations among the GFP/RFP+ fraction in Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA reporter animals. The relative percentages are shown in Supplemental Table 1. (A and B) The depicted leukocyte populations were gated as shown in Supplemental Fig. 1 (n = 4–5 mice/reporter strain).
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undetermined cells expressed intermediate to high levels of RFP in Ccr2rfp/+ animals, their levels of GFP in Cx3cr1gfp/+ animals were relatively much lower. As such, unlike with Ccr2rfp/+ mice, these undetermined GFP+ cells in Cx3cr1gfp/+ animals would likely fall below the detection limit of the multiphoton imaging. These results further illustrate the incompatibility of the use of Ccr2rfp/+ animals but highlight the efficiency of the Cx3cr1gfp/+ mice model for the purpose of BM monocyte-tracking studies. To validate further the suitability of each strain for in vivo imaging, we examined each of the Cx3cr1gfp, Ccr2rfp, and MaFIA reporter mice by multiphoton intravital fluorescence microscopy. We used the skull calvarium BM imaging model, originally established by von Andrian’s group [24], which is widely used for direct observation of individual cells within the BM of living animals [8, 14, 16]. For our analysis, we acquired stitched images to obtain a comprehensive view of the skull BM architecture (Fig. 2A and B). We used Evans blue to highlight BM blood sinusoidal vessels [25] and the SHG signal for the visualization of the bone/collagen structures. Of note, the BM parenchyma that contains the hematopoietic cells is characterized by the absence of the SHG signal (Fig. 2B and C). We found that in the MaFIA reporter mouse, almost all of the parenchyma space contained GFP+ cells (Fig. 2B). At a higher magnification, GFP+ cells presented heterogeneous morphology, with the majority of these
cells displaying a low cell volume and a spherical morphology, which is consistent with the fact that the majority of the cells labeled in this system consists of neutrophils (Figs. 1B and 2C). On the other hand, the Ccr2rfp/+ reporter mouse exhibited fewer labeled cells than the MaFIA mouse (Fig. 2B). However, the RFP+ cells displayed heterogeneous morphology—from large and elliptic to small and spherical shapes, as well as ,1 mm diameter objects (Fig. 2B and C). This apparent morphologic heterogeneity observed is likely a result of the labeling of multiple cell populations, as shown in our flow cytometry data (Fig. 1A). Finally, the Cx3cr1gfp/+ reporter mouse showed much less labeled cells in the BM compared with the Ccr2rfp/+ animals (Fig. 2B). Two major types of morphology could be observed: large and elliptic cells with protruding dendrites, as well as small and spherical cells (Fig. 2B and C). These data are in agreement with our flow cytometry data, whereby monocytes and DCs represent 2 immune cell subsets that are predominantly labeled and display the highest GFP intensity among CX3CR1+ cells (Fig. 1B and Supplemental Table 1). Thus far, we have analyzed the BM of 3 common strains used to study mononuclear phagocytes, namely Cx3cr1gfp, Ccr2rfp, and MaFIA, by flow cytometry and multiphoton imaging. Among these 3 reporters, we established that the Cx3cr1gfp reporter mouse appeared to be the most ideal reporter for the tracking of
Figure 2. Visualization of the skull BM in Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA reporter mice via multiphoton intravital microscopy. (A) An overview of the steps involved during intravital imaging of the skull calvarium BM. Skin was first removed carefully to expose the BM. Images were acquired via stitching snapshots of the BM to form a final image of 800 3 1200 mm or as a single snapshot sized at 450 3 450 mm. To label sinusoidal vessels, mice were i.v. injected with Evans blue (n = 3–5/ reporter strain). (B) A representative stitched image of the skull BM in Cx3cr1gfp/+, Ccr2rfp/+, and MaFIA reporter mice. Original scale bars, 100 mm. (C) (Upper) snapshot of the skull BM in the 3-strain mouse, whereby single plane images were acquired at different depths ranging from 0 to 36 mm along the z-axis. Original scale bars, 50 mm. (Lower) In addition, magnified single-plane images at z-positions 12 mm and 24 mm are shown. Original scale bars, 20 mm. GFP/RFP+ cells, Green; sinusoidal blood vessels, red; and bone/collagen structures (through SHG), blue.
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monocytes in the BM. As shown, monocytes in these mice account for the majority of GFP-labeled cells and expressed the highest fluorescence intensity among labeled cells in the BM. Whereas we demonstrated the suitability of the Cx3cr1gfp reporter mouse as a tool for visualization of BM monocytes, our study also revealed that DCs are labeled with similar levels of GFP expression as monocytes. In addition, our data suggest the presence of large and elliptic GFP+ cells that are localized in close proximity with monocytes in the BM and may thus interfere with the accuracy of monocyte tracking in vivo.
CX3CR1+ cells carrying a dendritic morphology are DCs and share a similar location with monocytes After selecting Cx3cr1gfp/+ as the most suitable reporter animal, we next sought to confirm the identity of the GFP bright, large, and elliptic cells, located in the BM parenchyma. Our flow cytometry results suggest that these cells are DCs (Fig. 1A). Furthermore, BM DCs have been reported previously to be CX3CR1+MHCII+CD11c+ [26]. Thus, to identify DCs in situ, we administered a fluorescent MHCII antibody to Cx3cr1gfp/+ animals (Fig. 3A). We found that CX3CR1+MHCII+ cells were generally larger with protruding dendrites, whereas CX3CR1+MHCII2 cells were generally smaller and spherical
Imaging monocyte egress from the bone marrow
(Fig. 3A and Supplemental Video 1). To confirm these observations, we devised an image analysis strategy to correlate cellular morphology and staining for MHCII expression (Fig. 3B). We first selected cells based on GFP expression, followed by their positivity for MHCII; finally, we plotted MHCII+ and MHCII2 cells according to morphologic criteria—cell volume and sphericity. We defined cells with a high cell volume and low sphericity as having a DC-like morphology, whereas cells that consist of a low cell volume and high sphericity have a “monocyte-like” morphology (Fig. 3B). With the use of these morphologic criteria, we found that the majority of CX3CR1+MHCII+ cells had a DC-like morphology, whereas the majority of CX3CR1+MHCII2 cells had a monocyte-like morphology (Fig. 3C and D). Together, this provides further evidence that morphologic criteria can be used as a plausible and inexpensive approach to discriminate DCs from monocytes in the BM of Cx3cr1gfp/+ animals.
Flt3L-deficiency reduces DC numbers and improves the identification of monocytes in the BM of the Cx3cr1gfp/+ reporter mouse To this end, our results indicated that DCs can be discriminated from monocytes morphologically. To track monocyte activities in
Figure 3. CX3CR1+ cells that possess a DC-like morphology in the BM of Cx3cr1gfp/+ mice are DCs. Cx3cr1gfp/+ reporter mice were i.v. administered with PE-conjugated MHCII antibody and Evans blue to detect for MHCII+ cells and blood vessels, respectively. (A) Snapshots sized at 450 3 450 mm of Cx3cr1gfp/+ reporter mice, demonstrating the individual fluorescent channels detecting for CX3CR1+ cells, MHCII+ cells, and blood sinusoids, with a final merged image of all channels. (Lower) Magnified region of the individual snapshots. Original scale bars, 20 mm. (B) An outline of the process involved during image analysis. Objects were first “gated” for a GFP+ signal. Following this, CX3CR1+ objects were separated based on MHCII expression, CX3CR1+MHCII+ and CX3CR1+MHCII2 objects were subsequently analyzed for cell volume and sphericity, represented on a scatter plot. Objects with a volume .1000 mm3 and sphericity ,0.65 were considered to display a DC-like morphology, whereas monocyte-like cells possessed volumes of ,2000 mm3 and sphericity .0.65. MFI, Mean fluorescence intensity. (C) Percentage of cells, displaying a DC-like or monocyte-like morphology, are shown in red and green, respectively. (D) Scatter plot, demonstrating a merged view of the total amount of CX3CR1+MHCII+ and CX3CR1+MHCII2 cells present in Cx3cr1gfp/+ reporter mice. Data are pooled from 4 fields of view from the same mouse (n = 3 mice).
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the BM accurately and efficiently, we aim to deplete specifically the DC population from the BM in the Cx3cr1gfp/+ reporter mouse. It has been reported previously that CX3CR1+ DCs can be depleted by administration of DT in CD11c-DTR mice, which express DTR under the CD11c promoter (also termed integrin a X) [14, 26]. However, this system lacks specificity for DC depletion, as other CD11c-expressing cells have also been shown to be affected upon DT administration [27, 28]. In addition, DTinduced cell depletion is known to result from the induction of cell death, which consequently, may lead to neutrophilia and monocytosis [29]. Thus, this approach precluded the use of these mice to the study of monocyte mobilization from the BM under physiologic conditions. Flt3 signaling plays a central, nonredundant role in DC homeostasis, as the lack of Flt3L or its receptor Flt3 affects DC proliferation, differentiation, and maintenance of DC numbers [30]. We thus took advantage of this property and generated
a crossbred Cx3cr1gfp/+Flt3L2/2 reporter mouse. We first performed intravital imaging on the skull BM. From the acquired stitched images, we performed a semiautomated quantification of GFP+ cells that were separated according to their morphology— DC-like and monocyte-like (Fig. 4A). Accordingly, with the use of these parameters, we found a significant reduction in cells harboring a DC-like morphology in Cx3cr1gfp/+Flt3L2/2 mice compared with Cx3cr1gfp/+Flt3L+/+ control mice (Fig. 4B). To validate our imaging data further, we analyzed the frequency of DCs, monocytes, and macrophage Cx3cr1gfp/+Flt3L2/2 animals compared with Cx3cr1gfp/+Flt3L+/+ controls via flow cytometry (Fig. 4C and D). Notably, the absence of Flt3L is known to affect general hematopoiesis [31]. Consequently, we found a decrease in BM cellularity in Cx3cr1gfp/+Flt3L2/2 mice. To ensure an unbiased comparison of the cellular distribution in Flt3L2/2 and wild-type animals, we expressed the cell count in each individual cell types as a fraction of the
Figure 4. DC but not monocyte BM development is impaired in the Cx3cr1gfp/+Ftl3L2/2 reporter mouse. (A) Representative stitched images of the skull BMof Cx3cr1gfp/+Flt3L+/+ andCx3cr1gfp/+Flt3L2/2 reporter mice. Mice were i.v. administered with Evans blue to label sinusoidal vessels (n = 3/strain). Original scale bars (left panel), 100 mm. A white box on the stitched images magnifies the region indicated, whereby single plane images for z = 12 and 24 mm are illustrated. Original scale bars (right panel), 20 mm. DC-like cells, Magenta (arrowheads); monocyte-like cells, green; and BM sinusoids, gray. (B) CX3CR1+ cells were distinguished according to their morphology; i.e., cells with a volume .1000 mm3 and sphericity ,0.65 and volume ,2000 mm3 and sphericity .0.65 were considered to display a DC-like or monocyte-like morphology, respectively. The frequencies of DC-like or monocyte-like cells present in Cx3cr1gfp/+Flt3L+/+ and Cx3cr1gfp/+Flt3L2/2 reporter mice were analyzed and represented as a pie chart (n = 3 mice/reporter strain). A typical example of flow cytometry plots identifying (C) BM DCs (CX3CR1+CD11chiMHCIIhi) and (D) monocytes (CX3CR1+CD11b+CD115+Ly6Chi or Ly6Clo, according to the subset). Comparison of percentages of (E) DCs, (G) Ly6Chi monocytes (Mo), (F) macrophages, and (H) Ly6Clo monocytes in Cx3cr1gfp/+Flt3L+/+ and Cx3cr1gfp/+Flt3L2/2 mice. Cell percentages are expressed as a ratio of the cell count over the total GFP + cell number. (C–H) Cell populations were gated accordingly by use of the strategy shown in Supplemental Fig. 1 (n = 5 mice/reporter strain). Data are shown as mean 6 SEM and are pooled from 2 different experiments. n.s., Nonsignificant; *P , 0.05 (unpaired t-test).
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total GFP+ cells. Based on this evaluation, we showed that DC frequency was significantly less in Flt3L2/2 animals, whereas no difference was observed in Csf1R-dependent macrophages and monocytes (Fig. 4D and F–H), as reported previously [32, 33]. Of note, we detected a modest but significant decrease of DC-like cells by imaging (Fig. 4B) compared with a sharp decrease of DCs by flow cytometry (1.53- vs. 2.43-fold decrease; Fig. 4B and E). Conceivably, this discrepancy could be attributed to a population of CX3CR1+ BM macrophages [14], which may possess a DC-like morphology, as detected by multiphoton imaging but are not affected by the loss of Flt3L. In summary, we showed that Flt3L-deficiency leads to the reduction of DC-like morphology-bearing CX3CR1+ cells in the BM, and this reduction was correlated with a significant reduction in BM DC frequency, thereby facilitating the tracking of BM monocyte behavior in Cx3cr1gfp/+Flt3L2/2 animals. Moreover, analysis of our imaging data also suggests that macrophages in the BM possess a DC-like morphology, indicating that the morphologic criteria that we applied here can be used specifically to “segment” out monocytes in Cx3cr1gfp/+ mice.
Imaging monocyte egress from the bone marrow
Flt3L2/2 in the Cx3cr1gfp/+ reporter mouse does not impair monocyte mobilization from the BM Among monocytes, Ly6Chi monocytes are short lived, proinflammatory cells that egress the BM through a CCR2-dependent mechanism upon an inflammatory challenge [23]. Thus far, we have shown that the Cx3cr1gfp/+Flt3L2/2 reporter mouse, which had significantly reduced DC numbers, appears to be the most ideal system for the visualization of monocytes in the BM. As our objective is to track BM monocyte movements in these mice, we have to ensure that the absence of Flt3L does not affect the migratory behavior of monocytes in the BM. To this end, we subjected Cx3cr1gfp/+ reporter mice to subclinical doses of LPS, which is known to mobilize monocytes to the circulation [34], and followed medullar and circulating Ly6Chi monocyte numbers over time. In this set of experiments, we defined Ly6Chi monocytes as Lin2CD11b+SSCloLy6G2Ly6ChiCX3CR1+ (Fig. 5A and B). We observed that Ly6Chi monocyte numbers peaked in the blood, 4 h after LPS treatment, whereas their BM numbers decreased significantly within 2 h (Fig. 5A). We next compared the ability of Cx3cr1gfp/+Flt3L2/2 animals to egress from the BM after LPS challenge with their control Cx3cr1gfp/+Flt3L+/+
Figure 5. Monocyte mobilization by LPS is not affected by the loss of Flt3L. (A) Kinetics of Ly6Chi monocytes in blood and BM after i.v. injection of LPS (10 ng) in Cx3cr1gfp/+Flt3L+/+ mice (n = 4 mice/ group/time-point). Data are shown as mean 6 SD and are representative of 2 independent experiments. **P , 0.01 (unpaired t-test). (B) Comparison of the frequencies of Ly6Chi monocytes (expressed as a ratio of the cell count over the total GFP+ cell number) in the blood or BM, 2 h after LPS administration in Cx3cr1gfp/+Flt3L+/+ and Cx3cr1gfp/+Flt3L2/2 mice. In both tissues, Ly6Chi monocytes were considered Lin2 (CD3, NK1.1, Siglec-H) CD11b+SSCloLy6G2CX3CR1+ (n = 6–10 mice/group/condition). Data are shown as mean 6 SEM and are pooled from 2 different experiments. ****P , 0.0001 (unpaired t-test). (C) (Left) Maximum z-projected images of the skull BM in the Cx3cr1gfp/+ mouse at baseline. White dotted areas represent the BM parenchyma. (Right) Thirty-minute duration tracks of individual GFP+ monocyte-like cells at baseline and 1 and 2 h after LPS treatment in Cx3cr1gfp/+Flt3L+/+ and Cx3cr1gfp/+Flt3L2/2 mice. Red dotted areas represent the BM parenchyma. Original scale bars, 20 mm. GFP+ cells, Green; sinusoidal blood vessels, red, (D) The mean velocity and (E) displacement of GFP+ monocyte-like cells were measured at baseline and 1 and 2 h after LPS treatment in Cx3cr1gfp/+Flt3L+/+ and Cx3cr1gfp/+Flt3L2/2 mice (n = 3 mice/group/treatment). ****P , 0.0001 (1-way ANOVA). (F) Mean velocity of GFP+ DC-like cells at baseline and 1 and 2 h after LPS treatment in Cx3cr1gfp/+Flt3L+/+ mice (n = 3 mice/treatment). (G) A representative end-point image illustrating the tracks of GFP+ DC-like from baseline to 2 h after LPS treatment. Original scale bar, 20 mm.
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littermates. We found that similar to the control littermates, Ly6Chi monocytes showed increased blood counts and decreased BM counts within 2 h after LPS treatment (Fig. 5B). Collectively, these data provide evidence that the lack of Flt3L did not impair the ability of Ly6Chi monocytes to egress from the BM to the bloodstream in response to proinflammatory stimuli, such as LPS. To validate this finding further, we performed intravital imaging to study the cellular behavior of monocytes in the BM during LPS challenge. These studies showed evidences that in Cx3cr1gfp/+Flt3L2/2 and Cx3cr1gfp/+Flt3L+/+ animals, monocytes in the BM parenchyma remained mostly sessile at baseline and until 1 h after LPS administration. However, they steadily increased their motility from 1 h after LPS treatment and gradually egressed from the parenchymal space into the sinusoidal vessels within 2 h (Fig. 5C and D and Supplemental Videos 2 and 3). Furthermore, this increase in cell motility was accompanied with increased cellular displacement, in agreement with monocyte departure from the BM parenchyma (Fig. 5E). Our tracking analysis indicated that BM monocytes, in Cx3cr1gfp/+Flt3L2/2 and Cx3cr1gfp/+Flt3L+/+ mice, displayed similar motility patterns in terms of their velocity and displacement, 1 h after LPS administration. Interestingly, we observed that monocytes in the BM of Cx3cr1gfp/+Flt3L2/2 mice exhibited a slight but significant increase in their velocity and displacement compared with control mice at 2 h after LPS injection. Despite this difference, we believe that the overall functional mobilization of BM monocytes is not altered in Cx3cr1gfp/+Flt3L2/2 mice, as there is no detectable difference in the number of circulating monocytes compared with Cx3cr1gfp/+Flt3L+/+ mice in response to LPS (Fig. 5B). Of note, our morphologic identification method allowed us to follow the behavior of BM DC-like cells over time. These cells were sessile during baseline conditions. However, unlike monocytes, DC-like cells did not increase their motility following LPS treatment (Fig. 5F and G). In summary, we present a novel approach that allows the study of monocyte mobilization from the BM through a combinatorial use of the Cx3cr1gfp/+Flt3L2/2 reporter mouse, multiphoton intravital microscopy, and subsequent image analysis, based on cell morphology. Our current study also provides a significant conceptual advance by illustrating the use of a Cx3cr1gfp/+-based, dual functionality fluorescence reporter line for improving the specificity of monocyte imaging in the BM. More importantly, these results obtained by multiphoton imaging support and complement our flow cytometry data by providing in situ information that includes morphologic identification of DCs and monocytes and the migratory status of these cells. Our current report proposes the use of the Cx3cr1gfp/+Flt3L2/2 mouse as a tool for in vivo tracking and analysis of the cellular behavior of monocytes in the BM. The control of monocyte release from the BM represents a major checkpoint in monocyte homeostasis, and this process has a significant impact on the number of circulating monocytes. Thus, it is imperative to characterize the spatiotemporal regulation of monocyte mobilization from the BM to understand monocyte function better. Whereas we still do not fully understand whether the loss of DCs will have an impact on overall monocyte function, our study demonstrates the suitability of the Cx3cr1gfp/+Flt3L2/2 mouse 618
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for the in vivo tracking of monocytes in the BM. Lastly, we envision that this Cx3cr1gfp/+-based, combined approach would also be beneficial toward understanding the dynamic behavior and development of other mononuclear phagocytes in different organs.
AUTHORSHIP M.E. designed the research, performed experiments, analyzed results, and wrote the manuscript. S.Z.C. and S.D. performed experiments and wrote the manuscript. W.K.C. generated the Cx3cr1gfp/+Flt3L2/2 mouse. B.L. analyzed results. M.P. contributed intellectually. F.G. provided the Flt3L2/2 mouse, contributed intellectually, and prepared the manuscript. S.M.T. contributed intellectually and prepared the manuscript. L.G.N. designed the research and wrote the manuscript.
ACKNOWLEDGMENTS This research was funded by SIgN, A*STAR (Singapore). The authors thank the flow cytometry facility of SIgN for its technical help and support.
DISCLOSURES
The authors declare no conflicts of interest.
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KEY WORDS: dendritic cells macrophages trafficking live imaging •
Volume 97, March 2015
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Journal of Leukocyte Biology 619
