In vivo tracking of transplanted macrophages with near infrared fluorescent dye reveals temporal distribution and specific homing in the liver that can be perturbed by clodronate liposomes.

Macrophages play an indispensable role in both innate and acquired immunity, while the persistence of activated macrophages can sometimes be harmful to the host, resulting in multi-organ damage. Macrophages develop from monocytes in the circulation. However, little is known about the organ affinity of macrophages in the normal state. Using in vivo imaging with XenoLight DiR®, we observed that macrophages showed strong affinity for the liver, spleen and lung, and weak affinity for the gut and bone marrow, but little or no affinity for the kidney and skin. We also found that administered macrophages were still alive 168 hours after injection. On the other hand, treatment with clodronate liposomes, which are readily taken up by macrophages via phagocytosis, strongly reduced the number of macrophages in the liver, spleen and lung.

Introduction

Macrophages are a type of myeloid cell that play an indispensable role in both innate and acquired immunity [1]. Macrophage phenotypes and functions can vary with different external stimuli, and macrophages are divided into two major classifications: classically activated, i.e. inflammatory, and alternatively activated, i.e. anti-inflammatory macrophages [1]. Persistence of activated macrophages can sometimes be harmful to the host [2, 3]. Hemophagocytic lymphohistiocytosis (HLH), or macrophage activation syndrome (MAS), is a hyperinflammatory state with a high mortality rate and a therapeutic challenge characterized by inappropriate survival of histiocytes and cytotoxic T cells (CTLs), leading to a cytokine storm, hemophagocytosis and multi-organ damage [4]. Monocytes in the circulation, originating from hematopoietic precursors in the bone marrow, lodge in certain organs and eventually develop into tissue macrophages [5]. Thus, the analysis of the mechanism of macrophage homing could lead to new treatment options. However, methods to track macrophages in vivo have not been established to date.

Clodronate is a biphosphanate clinically approved for human use as a bone resorption inhibitor and antihypercalcemic agent. Clodronate is metabolized to a toxic adenosine 5’-triphosphanate inside mammalian cell, resulting in cell death by apoptosis [6]. Therefore, efficient delivery of clodronate into the cytoplasm could enhance their therapeutic effect. It is known that clodronate liposomes are readily taken up mainly by macrophages and polymorphonuclear leukocyte via phagocytosis and strongly attenuate the number of macrophages retained in the cytoplasm [7, 8]. Additionally, activated macrophages are more sensitive to clodronate than resting macrophages [8].

Here, we evaluated the organ affinity and fate of transplanted macrophages using in vivo imaging with XenoLight DiR®. We also report that treatment with clodronate liposomes efficiently attenuates the number of macrophages in the liver, spleen and lung, for which macrophages possess strong affinities.

Materials and methods

Mice

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Nagoya University (Protocol Number: 24298). All procedures was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

Male 6- to 8-week-old male C57BL/6J mice were obtained from Chubu Kagaku Shizai (Nagoya, Japan). Animals were maintained at constant ambient temperature (22±1°C) under a 12-h light/dark cycle (lights on between 9:00 and 21:00), with food and water available ad libitum.

Preparation of peritoneal macrophages

Thioglycolate-recruited macrophages were isolated from C57BL/6J mice by peritoneal lavage 3 days after intraperitoneal injection of 1 mL 2% sterile thioglycolate medium as described elsewhere with slight modification [9, 10]. Briefly, 20 g of dehydrated Brewer thioglycolate medium powder (Kanto Chemical Co. Inc., Tokyo, Japan) was dissolved in 1000 mL deionized water and autoclaved for 20 minutes at 15 pounds of pressure (121°C). The autoclaved medium was kept in the dark under sterile conditions at room temperature for at least 3 months before use. Obtained peritoneal macrophages were purified using CD11b microbeads and the AutoMACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). The cell viabilities were evaluated by trypan blue exclusion.

In vitro assay for the effects of liposomal clodronate on macrophages

A murine macrophage cell line, RAW264.7, was purchased from American Type Culture Collection (Manassas, VA) and was cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). Liposomal clodronate (lipo-CL2MDP) was kindly gifted by Dr N. van Rooijen [11, 12]. Liposomal PBS (lipo-PBS) was used as a control. RAW264.7 cells treated with the indicated concentrations of lipo-CL2MDP or lipo-PBS were seeded at a density of 10,000 cells/well in a 96-well plate and incubated for 48 hours at 37°C. After the indicated time, the viability of the cells was assessed by a colorimetric assay (TetraColor One®; Seikagaku Co., Tokyo, Japan) as described elsewhere [10, 13, 14]. Briefly, 10 μL of TetraColor One® was added to each well, and the mixture was incubated for an additional 4 hours to measure the viability of cells. Absorbance at 450 nm was monitored and the IC50 values of the cells were calculated.

Labeling protocol

Macrophages were labeled as described previously with slight modifications [15]. The commercially available lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (XenoLight DiR®, Caliper, Hopkinton, MA) was used in all experiments. The substance can be solved in ethanol, and its excitation and emission spectra lie in the near-infrared (NIR) (excitation spectrum, 745 nm; emission spectrum, 800 nm). For the following experiments, macrophages (1×106/mL of medium) were transfected with 2 mL of DiR®-labeling solution (19.7 μmol/L final concentration) for 5 min, washed 3 times with phosphate-buffered saline (PBS), and resuspended in medium. Because of some variability in the labeling intensity between different preparations of primary murine macrophages, cells from the same labeling reaction were used for every set of in vivo experiments.

Imaging protocol

For the assessment of macrophage distribution and localization in mice, animals were studied in a Xenogen IVIS 200 imager (Caliper) under isoflurane anesthesia at different time points, as described elsewhere [16]. Labeled macrophages (2×106/mouse) were injected via the tail vein, and 200 μL of lipo-CL2MDP or lipo-PBS was administered 3 hours after macrophage injection. Hair of mice was removed with depilatory cream since fluorescence is absorbed by the black hair. Image acquisition times were 1 second. Seven days after drug administration, mice were euthanized. The major organs were harvested to detect the fluorescence signal. The fluorescence signal intensities in different organs were analyzed by Xenogen Living Image® software, Version 3.1 (WaveMetrix, USA).

Immunohistochemistry

Immunostaining of mouse liver, spleen and lung specimens was performed as previously described with slight modification [10, 17]. Briefly, after fixation with 10% formaldehyde, samples were embedded in paraffin, cut into 3.5 mm sections, mounted on glass slides coated with silane, deparaffinized in xylene and rehydrated through a series of ethanol solutions. The avidin–biotin complex method was used. For antigen retrieval, the sections were autoclaved at 98°C for 45 minutes in diluted immunosaver® (1:200). Then, specimens were incubated with normal rabbit serum (Dako; diluted to 1:75), and thereafter with a monoclonal antibody for F4/80 (CI:A3-1, Novus, Littleton, CO) (1:100) for 15 minutes using intermittent microwave irradiation [18, 19]. Sections were then incubated with biotin-labeled rabbit anti-mouse IgG serum (diluted to 1:300; Dako) and a streptavidin–biotin detection kit (Ultra Tech HRP kit®, Beckman Coulter, Brea CA) sequentially. Finally, sections were stained with diaminobenzidin solution.

Statistical analysis

Statistical significance of group differences was evaluated by Student’s t-test between two groups using Excel software (Microsoft, Redmond, WA). Statistical differences between groups were considered significant at P < 0.05.

Results

Kinetics of in vivo imaging of macrophages

To label macrophages, we incubated macrophages with lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (XenoLight DiR®) for 5 min, washed 3 times with phosphate-buffered saline (PBS), and resuspended in medium. Neither morphological changes nor inviable cells in the macphages treated with XenoLight DiR® were observed compared to intact macrophages, suggesting no harmful impact of XenoLight DiR® on macrophages. Accordingly, fluorescence signal from macrophages was strongly detectable even 7 days after the injection into mice, suggesting that labeled macrophages were still viable in vivo (Fig 1A). In control mice with no labeled macrophages, minimal fluorescence signal was observed (Fig 1A). In vivo imaging showed that labeled macrophages gathered in the center of the body even 3 hours after the injection and stayed there for 7 days. Quantitative analysis showed that the difference between the intensity of fluorescence signal from mice which received 5×106 labeled macrophages and that from mice with 2×106 labeled macrophages was clearly distinguishable (Fig 1B), suggesting that fluorescence intensity paralleled the number of macrophages. It also showed that fluorescence intensity in the group of 5×106 labeled macrophages decreased only by 29.3% 168 hours after administration (Fig 1B).

Fig 1

In vivo imaging of macrophages.

(A) Macrophages labeled with/without XenoLight DiR® were injected into mice via tail vein. The time course of total fluorescence signal intensities was evaluated for 7 days. Scale bar, 10 mm. (B) Different numbers (2×106/body or 5×106/body) of macrophages labeled with XenoLight DiR® were injected into mice. The total fluorescence signal intensities were monitored for 7 days. (C) Seven days after the injection of macrophages labeled with XenoLight DiR®, mice were euthanized. The major organs were harvested to detect the fluorescence signal, showing that labeled macrophages homed specifically in the liver, spleen and lung. Scale bar, 10 mm.

In vivo homing patterns of macrophages

To obtain additional anatomic patterns of macrophage homing, mice were euthanized 7 days after the administration of labeled macrophages. The major organs were harvested to detect the fluorescence signal. As shown in Fig 1C, we observed strong signals in the liver, spleen and lung, and weak signals in the gut and bone marrow, but few signals in the kidney or skin. None or minimal signal was detected in the controls (Fig 1C, upper columns).

Effects of liposomal clodronate on macrophage viability in vitro and in vivo

The proliferation of mouse macrophage-like RAW264.7 cells after liposomal clodronate (lipo-CL2MDP) treatment (48 hours) was evaluated using a colorimetric assay (Fig 2A). Liposomal PBS (lipo-PBS) was used as a control. Lipo-CL2MDP inhibited the proliferation of RAW264.7 cells in a dose-dependent manner. Lipo-CL2MDP decreased the viability of RAW264.7 cells by 75% at a concentration of 100 μM while lipo-PBS exerted no inhibitory effect on the proliferation of RAW264.7 cells. Next, we tested the effect of lipo-CL2MDP on macrophages in vivo. Labeled macrophages (2×106/mouse) were injected via the tail vein, and 200 μL of lipo-CL2MDP or lipo-PBS was administered 3 hours after macrophage injection (Fig 2B). The difference in signal intensities between the control mice and lipo-CL2MDP-treated mice became apparent only 3 hours after lipo-CL2MDP administration (Fig 2B). Since then, the signal intensity in the mice that received lipo-CL2MDP decreased continuously and reached a plateau at the 24-hour time point (Fig 2B). Seven days following drug administration, mice were euthanized. The major organs were harvested to detect the fluorescence signal. Strong signals were detectable in the liver, spleen, and lung from control mice as observed above, while weak signals and minimal signal were detectable in the liver, spleen, and lung, respectively, from the lipo-CL2MDP-injected mice (Fig 2C). Immunohistochemical staining with F4/80 visualized macrophages (brown cells) scattered within the liver, spleen and lung from control mice (Fig 2D, first row panels). By contrast, rare macrophages were identified in these organs from lipo-CL2MDP-injected mice (Fig 2D, third row panels). However, no histological differences were observed between the liver, spleen and lung from control mice and those from lipo-CL2MDP-injected mice (Fig 2D, second and fourth panels).

Fig 2

Effect of liposomal clodronate on macrophages in vitro and in vivo.

(A) The viability of mouse macrophage-like RAW264.7 cells after liposomal clodronate (lipo-CL2MDP) treatment (48 hours) was evaluated using a colorimetric assay. Liposomal PBS (lipo-PBS) was used as a control. The percentage viability was calculated as follows: (OD value in the presence of each concentration of lipo-CL2MDP or lipo-PBS/OD value with no additive) ×100. The results reflect the mean ± SD of three independent determinations (representative experiment of three performed). The asterisk denotes statistical significance (*P < .05). (B) The effects of lipo-CL2MDP on macrophages injected into mice were analyzed by an image analyzer. Lipo-CL2MDP or lipo-PBS was administered into mice 3 hours after the injection of macrophages labeled with XenoLight DiR®. The time course of total fluorescence signal intensities was evaluated for 168 hours. The asterisk denotes statistical significance (*P < .05). (C) Lipo-CL2MDP or lipo-PBS was administered into mice 3 hours after the injection of macrophages labeled with XenoLight DiR®. The fluorescence signal intensities in different organs were compared after 7 days. Labeled macrophages home specifically in the liver, spleen and lung (upper panel). However, in the subgroup treated with lipo-CL2MDP (lower panel), fluorescence signal was detected slightly (liver and spleen) and minimally (lung). Scale bar, 10 mm. (D) Pathological assessment of macrophages after lipo-CL2MDP treatment. Liver (left panels), spleen (middle panels) and lung (right panels) specimens were stained with H&E (top panels in each subgroup) and the macrophages were detected with the anti-mouse F4/80 monoclonal antibody (bottom panels in each subgroup). Representative images of three mice are shown. Original magnification, ×400. Scale bar, 80 μm.

Discussion

Here we show that XenoLight DiR® is useful for in vivo bioluminescent tracking of macrophages. Total fluorescence signal from macrophages decreased only by 29.3% 168 hours after administration (Fig 1A), suggesting that labeled macrophages were still viable in vivo and the fluorescence of XenoLight DiR® did not fade for a long period. Similarly, Eisenblätter et al. has reported that macrophages labeled with XenoLight DiR® were viable, and successfully tracked when injected intravenously, at the site of inflammation using a cutaneous granuloma mouse model [15].

Previous cell tracking studies showed that most types of cells were initially trapped in the lung, which is enriched with small capillaries, and then migrated to specific organs [20, 21]. Similarly, macrophages were trapped in the lung, but stayed there even 7 days after intravenous injection. Thus, we concluded that Thioglycolate-recruited macrophages possess a specific affinity for the lung, in addition to the liver and spleen (Fig 1C). Thioglycolate-recruited macrophages are activated macrophages expressing immature myeloid markers: Ly6C and ER-MP58 [22]. Their homing patterns are somewhat different those of macrophages established from bone marrow progenitor cells. Eisenblätter et al. has reported that macrophages established from bone marrow progenitor cells mainly distributed to the lungs, bone marrow and liver [15]. It is well known that proinflammatory chemokines, such as CCL2, recruit monocytes and macrophages to the inflammatory site [14]. However, the mechanism for the strong affinities of macrophages for these organs has not been fully evaluated to date, even though the lung, liver and spleen include a large number of macrophages. It can be assumed that these organs possess a common mechanism for attracting macrophages.

Haemophagocytic lymphohistiocytosis (HLH), or macrophage activation syndrome (MAS), is a life-threatening condition that causes multi-organ damage, especially liver dysfunction with splenomegaly, and lung involvement [4, 23]. This specific organ damage is of unknown etiology, but can be explained by the high affinities of macrophages for these organs, as described above. A corticosteroid, such as dexamethasone, is a key drug in the treatment of HLH/MAS [24]. However, liposome-encapsulated drugs enhance the anti-macrophage efficacy to a greater extent, compared to intact drugs, because macrophages ingest the drug by phagocytosis [11]. It has been reported that clodronate was converted into adenosine 5’- triphosphate inside cells, which was toxic to macrophages, and then induced cell death by apoptosis [6]. We have reported that dexamethasone palmitate emulsion (DP) treatment clinically ameliorated macrophage-rich graft versus host disease (GVHD) and hemophagocytic syndrome after stem cell transplantation [10, 25]. Here, we showed that macrophages were systemically depleted 24 hours after liposomal clodronate injection (Fig 2C). Similarly, it has been reported that liposomal clodronate efficiently depleted macrophages in large animals, including dogs [26]. As described above, activated macrophages are more sensitive to clodronate than resting macrophages [8]. However, little is known to date about the clinical effects of liposomal clodronate on HLH/MAS. This evidence and results suggest that liposomal clodronate can be applied to treat HLH/MAS.

8 Oct 2019

PONE-D-19-27394

In vivo bioluminescent tracking of transplanted macrophages reveals temporal distribution and specific homing in the liver that can be perturbed by clodronate liposomes

PLOS ONE

Dear Dr. Nakayama,

Thank you for submitting your manuscript to PLOS ONE. Your manuscript requires some relevant revisions before it can be sent out for peer review process. Specifically:

- Figure 1A: Authors need to insert a scalebar in all panels describing the bioluminescence in mice.

- Figure 1C: Authors need to insert a scalebar in all panels depicting the bioluminescence in organs.

- Figure 2C: Authors need to resize all panels depicting the bioluminescence in organs, so that the panels will be displayed with identical sizes.

Figure 2D: Authors must insert a scalebar for each depicted panel in this figure.

Figure legends must be revised in accordance to these new modifications.

We would appreciate receiving your revised manuscript by Nov 22 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Fabrizio Mattei, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

1. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

26 Nov 2019

Our responses to each of the comments are as follows. The modifications to the manuscript are tracked in yellow.

- Figure 1A: Authors need to insert a scalebar in all panels describing the bioluminescence in mice.

We have insert a scalebar in all panels.

- Figure 1C: Authors need to insert a scalebar in all panels depicting the bioluminescence in organs.

We have insert a scalebar in all panels.

- Figure 2C: Authors need to resize all panels depicting the bioluminescence in organs, so that the panels will be displayed with identical sizes.

We have made the heights of all panels equal, as the editor suggested.

- Figure 2D: Authors must insert a scalebar for each depicted panel in this figure.

We have insert a scalebar in all panels.

- Figure legends must be revised in accordance to these new modifications.

We have mentioned scale bars in the figure legends.

Submitted filename: response to comments 11-26.rtf

Click here for additional data file.

7 Apr 2020

PONE-D-19-27394R1

In vivo bioluminescent tracking of transplanted macrophages reveals temporal distribution and specific homing in the liver that can be perturbed by clodronate liposomes

PLOS ONE

Dear Dr. Nakayama,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.In particular, Authors must carefully and thoroughly address all the points raised by the Reviewer 1. Furthermore, the use of liposomal clodronate is not sufficiently motivated in the text. For this reason, Auhtors must extensively clarify in their manuscript why they studied liposomal clodronate raather than other types of liposomal products.

We would appreciate receiving your revised manuscript by May 22 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Fabrizio Mattei, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors report the localization of fluorescent labelled macrophages within the lungs, spleen and liver and the impact of liposomal clodronate upon maintenance of fluorescence. They demonstrated that labelled macs accumulated flourescence in the above organs, but not kidney or skin. Clodronate is biphosphonate with a liposome and action is not mentioned within the article.

Issues:

1. thioglycollate recruited macrophages are differentially activated through the recruitment process. The old literature (80s) spoke about the type of thiogycollate used and differential ability to phagocytose bacterial. The authors didn't mention the impact/activation of the type of recruitment upon subsequent homing of cells to the various locations. Some mention of cell surface receptors for endothelial or tissue ligands may be useful. Some of the differences are discussed in old literature, again such as J Immunol November 1, 2003, 171 (9) 4816-4823

2. There should be discussion of liposomal clodronate and the induction of apoptosis. It is not mentioned.

3. there is not discussion of statistical analysis of the findings.

4. the authors keep discussing the possibility of liposomal steroids, but did not study it. Liposomal clodronate has a putative role in autoimmune and transplant related conditions where on may want to remove tissue macrophage damage. It was not mentioned within the context of this manuscript. the fact that inert liposomes did not appear to alter macrophages and clodronate loaded liposomes was associated with loss of macrophage associated fluorescence suggests that this may be a way to gain targeted drug delivery, but it is very round about.

Reviewer #2: Nakayama et al presents a new method to track macrophages in vivo using bioluminescence and probe the method of depletion using clodronate liposomes.

Actually the article checks a macrophage tracking method in vivo. Regarding the methodology used, the necessary controls are considered to validate the technique and the materials and methods are adequate to verify that the method works correctly. Perhaps it would be interesting to test it on immunodeficient mice or another animal species to see the possibilities of the technique. The Editor should assess whether it is of sufficient scientific quality to be published on Plos One.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Carlos Alfaro

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

21 May 2020

response to reviewers

Academic Editor: The use of liposomal clodronate is not sufficiently motivated in the text. For this reason, Authors must extensively clarify in their manuscript why they studied liposomal clodronate rather than other types of liposomal products.

As the academic editor suggested, we have added descriptions about advantages of liposomal clodronate to deplete macrophages (page 3, line56-57 and page 4, line 62-63).

Reviewer #1: The authors report the localization of fluorescent labelled macrophages within the lungs, spleen and liver and the impact of liposomal clodronate upon maintenance of fluorescence. They demonstrated that labelled macs accumulated fluorescence in the above organs, but not kidney or skin. Clodronate is bisphosphonate with a liposome and action is not mentioned within the article.

We are grateful for the useful comments and suggestions that have helped us to improve our paper. We have taken all these comments and suggestions into account in the revised version of our manuscript.

1. thioglycolate recruited macrophages are differentially activated through the recruitment process. The old literature (80s) spoke about the type of thioglycolate used and differential ability to phagocytose bacterial. The authors didn't mention the impact/activation of the type of recruitment upon subsequent homing of cells to the various locations. Some mention of cell surface receptors for endothelial or tissue ligands may be useful. Some of the differences are discussed in old literature, again such as J Immunol November 1, 2003, 171 (9) 4816-4823

As the reviewer suggested, we have added the description about difference of homing patterns between thioglycolate-recruited macrophages and macrophages established by other means (page 14, line 232-234 and page 15, line 247).

2. There should be discussion of liposomal clodronate and the induction of apoptosis. It is not mentioned.

As the reviewer suggested, we have added the description about apoptotic cell death of macrophages induced by clodronate (page 14, line 232-234 and page 15, line 247).

3. there is not discussion of statistical analysis of the findings.

As the reviewer suggested, we have added descriptions and asteriks which denotes statistical significance in the Material and Method and Results sections, and in the Figure 2, respectively.

4. the authors keep discussing the possibility of liposomal steroids, but did not study it. Liposomal clodronate has a putative role in autoimmune and transplant related conditions where on may want to remove tissue macrophage damage. It was not mentioned within the context of this manuscript. the fact that inert liposomes did not appear to alter macrophages and clodronate loaded liposomes was associated with loss of macrophage associated fluorescence suggests that this may be a way to gain targeted drug delivery, but it is very round about.

We are very sorry for the errors we made in the page 15, line 238 . Liposomal steroid should have been liposomal clodronate. We have corrected the errors. Additionally, we have changed the description about clodronate (page 3, line 56-58) since it was confusing and roundabout as the reviewer pointed out.

Reviewer #2: Nakayama et al presents a new method to track macrophages in vivo using bioluminescence and probe the method of depletion using clodronate liposomes.

Actually, the article checks a macrophage tracking method in vivo. Regarding the methodology used, the necessary controls are considered to validate the technique and the materials and methods are adequate to verify that the method works correctly. Perhaps it would be interesting to test it on immunodeficient mice or another animal species to see the possibilities of the technique. The Editor should assess whether it is of sufficient scientific quality to be published on Plos One.

We thank the reviewer for helpful suggestions to increase the quality of our manuscript.

The reviewer pointed out that we should have used appropriate controls in the tracking experiments. It is very difficult to pick up "appropriate controls". However, we have tracked down the fluorescence -labeled mesenchymal stromal cells in vivo after intravenous injection, showing that mesenchymal stromal cells transiently lodged in the lung and then moved to liver and spleen (now preparing for submission). The in vivo behavior of macrophages apparently differs from that of mesenchymal stromal cells. Thus, we concluded that the strong affinities for the lung, liver and spleen were specific to macrophages.

The reviewer also proposed that we should test it on immunodeficient mice or another animal species to see the possibilities of the technique. We had similar ideas as well, but we gave up to perform experiments since chemokine systems are different between mice and humans (e.g. mouse CCL2 is 58% identical to human CCL2). Thus, we were afraid that we might not obtain accurate data when human cells were injected into immunodeficient mice. Additionally, we don’t have an image analyzer for large animals

Submitted filename: Response to reviewers.rtf

Click here for additional data file.

1 Oct 2020

PONE-D-19-27394R2

In vivo bioluminescent tracking of transplanted macrophages reveals temporal distribution and specific homing in the liver that can be perturbed by clodronate liposomes

PLOS ONE

Dear Dr. Nakayama,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

As you can see from the comments, the reviewers felt that the scientific soundness of this study should be improved before the acceptance of this work. One reviewer raised the important issue on quantitative analysis, which needs to be addressed. To help me expedite processing, please explicitly address the questions raised by the reviewers in your cover letter and also point out the changes made in the manuscript. I will go back to the reviewers for further input and advice before any final decision on possible publication is made.

Please submit your revised manuscript by Nov 15 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Bing Xu, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors have made an effort to answer the questions raised by the reviewers and are generally acceptable and correct. The article has improved significantly with the changes introduced.

Reviewer #3: The paper by Nakayama and colleagues aim to image the biodistribution of Raw macrophages and see the effect of Clodronate liposomes on their depletion using optical imaging. Since the authors use the XenolighDIR fluorescent dye they are imaging near infrared fluorescence and not bioluminescence so the title is misleading.

Moreover since the perform direct labelling and the dye bound to cell membranes, there is no proof that cells retain it after 24h so for their analysis at 168h the authors should provide more evidence that they are looking at macrophages in the different organs and not at the free dye. Moreover the paper lacks quantitative analysis. The author should report the fluorescence signal in the organ in relation to weight of the organ so that a comparison of the prevalence of the cells can be claimed and statistical analysis performed.

Finally A. Taylor and colleagues ( doi: 10.1155/2018/2514796) injected bioluminescent RAW macrophages in animals and, although they also see signals in the liver , they were able to see it in the brain area too. So it would be nice to add this organ to the analysis.

I am afraid that in this form the paper is more appropriate as a technical note and not a research article.

Reviewer #4: In this manuscript, the authors studied the biodistribution of implanted macrophages in mice model. The results revealed that the macrophages exhibited organ-specific accumulation. This result provides new knowledge for immunologist. Moreover, the authors developed a method using clodronate liposomes to remove the macrophages accumulated in the liver, spleen and lung. This way could potentially address diseases like Haemophagocytic lymphohistiocytosis (HLH), or macrophage activation syndrome (MAS) which are relevant to the high affinities of macrophages. This work is technically sound. The experiment groups and the controls are well designed. The authors have fully address the concerns raised by previous reviewers. Thus, I recommend the acceptance of this manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Carlos Alfaro

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

27 Oct 2020

Our responses to each of the comments are as follows. The modifications to the manuscript are tracked in yellow.

Reviewer #3: The paper by Nakayama and colleagues aim to image the biodistribution of Raw macrophages and see the effect of Clodronate liposomes on their depletion using optical imaging.

Query: Since the authors use the XenolighDIR fluorescent dye they are imaging near infrared fluorescence and not bioluminescence so the title is misleading.

Answer: As pointed out by the Reviewer 3, the term “bioluminescence” was inappropriate. Thus, we changed the title as “In vivo tracking of transplanted macrophages with near infrared fluorescent dye reveals temporal distribution and specific homing in the liver that can be perturbed by clodronate liposomes”.

Query: Moreover since the perform direct labelling and the dye bound to cell membranes, there is no proof that cells retain it after 24h so for their analysis at 168h the authors should provide more evidence that they are looking at macrophages in the different organs and not at the free dye.

Answer: The product sheet of Xenolight DIR tells that the two long 18-carbon chains insert into the cell membrane, resulting in specific and stable cell staining with negligible dye transfer between cells (https://www.perkinelmer.com/product/dir-125964).

There was virtually no free dye in the cell suspension, because we washed macrophages labeled with Xenolight DIR three times with PBS before we injected as reported previously [J Nucl Med 2009; 50: 1676-1682]. It has been reported that primary macrophages retained the Xenoligh DIR fluorescent dye over 7 days [J Nucl Med 2009; 50: 1676-1682].

In Fig2, fluorescence signal intensities were much decreased in the liver, spleen and lung of mice treated with Lipo-CL2MDP, where the number of macrophages was much decreased as well, suggesting that Xenolight DIR inserted into the cell membrane of macrophages were not trasferred to other cells.

We had wanted to identify cells with fluorescence as macrophages, but it was practically impossible because (1) the resolution of in vivo imaging system at our facility was too low to detect a single cell and (2) microscopic images of organs, immunostained with F4/80, could not be overlaid with fluorescence images obtained from in vivo imaging.

Query: Moreover the paper lacks quantitative analysis. The author should report the fluorescence signal in the organ in relation to weight of the organ so that a comparison of the prevalence of the cells can be claimed and statistical analysis performed.

Answer:

The intensities of fluorescence signal were expressed as photons/sec/unit area, not total photons of each organ. Thus, thickness, not weight, of organs may correlates with signal intensities. However, there is little and ignorable difference of thickness among organs, except for femors.

Each organ has unique structure (e.g. bones in the femurs, alveolars in the lung, and blood in the heart), which can attenuate the signal intensities to some extent. Statisitical analysis can not be performed among subgroups in different backgrounds. Thus, these were reasons why we analyzed trends, not statistical significances, in the macrophage accumulation as shown in Figure 1C and 2C. On the other hand, we performed statistical analysis between mice treated with clodronate liposomes and mice treated without clodronate liposomes to confirm the effect of clodronate liposomes on the macrophage accumulation since both subgroups had similar backgrounds.

Query: Finally A. Taylor and colleagues ( doi: 10.1155/2018/2514796) injected bioluminescent RAW macrophages in animals and, although they also see signals in the liver , they were able to see it in the brain area too. So it would be nice to add this organ to the analysis.

Answer: We did not analyze the intensity of fluorescence signal in the brain because minimal signal intensity was observed in the head region as shown Figure 1A after primary macrophages were administrated via tail vein. In the Contrast Media & Molecular Imaging paper (doi.org/10.1155/2018/2514796), the authors injected the luciferase gene-transduced RAW 264.7 cells via the left cardiac ventricle, suggesting that RAW 264.7 cells were directly delivered to the brain via large arteries. RAW 264.7 is a macrophage-like leukemia cell transformed by virus (https://www.atcc.org/products/all/tib-71.aspx). Thus, we believe that the route of administration and the cell type are likely cause of failure for us to detect the signal in the head.

We hope that the Editor finds that our re-revised manuscript adequately addresses the comments made by the reviewer #3 and that the manuscript is now ready for publication in the PLOS One.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

4 Nov 2020

In vivo tracking of transplanted macrophages with near infrared fluorescent dye reveals temporal distribution and specific homing in the liver that can be perturbed by clodronate liposomes

PONE-D-19-27394R3

Dear Dr. Nakayama,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Bing Xu, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

24 Nov 2020

PONE-D-19-27394R3

In vivo tracking of transplanted macrophages with near infrared fluorescent dye reveals temporal distribution and specific homing in the liver that can be perturbed by clodronate liposomes

Dear Dr. Nakayama:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Bing Xu

Academic Editor

PLOS ONE