Elucidating Iron Metabolism through Molecular Imaging

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

First, this contribution is well written; the quality of the English is excellent for presumably non-native English-speaking authors.

I was really excited by reading the title, which suggested the authors were going to review the what molecular imaging had recently discovered about iron metabolism.  However, this was not done.  

Also note the title needs to be rewritten.  "Molecular Imaging in Iron Metabolism" suggests molecular imaging is involved in iron metabolism.  The title should be something like "Using Molecular Imaging to Elucidate Iron Metabolism".

Sections 1.1 and 1.2 on the role of iron in the body and molecular mechanisms of iron metabolism are unnecessary.  These subjects have been reviewed innumerable times.  Besides, the authors made several errors in these sections, which really lead one to question whether they are experts in the area.  For example in line 87, iron ions do not bind to the heme moiety; heme means the combination of iron and appropriate porphyrin.  In line 92, catalase does not cycle between Fe²⁺ and Fe³⁺, but also involves higher oxidation states of iron.  Figure 1 (once I increased the image size so that it could actually be read) indicates that Fe²⁺ is bound to transferrin; this is incorrect - it binds as Fe³⁺.  

The rest of the review describes some techniques that can or have been used to image iron.  A few instances of prior use to probe Fe metabolism are cited, but the results are not described in any depth.  If there was the expected depth here, then section 1.2 might be valuable.  Primarily after the basic introduction of the technique, the manuscript suggests that the technique could have future uses.  A detailed examination of what imaging techniques have been used to probe iron metabolism and what has been learned has not been presented.  If the prior results were discussed in more detail, this would aid in being able to describe the future potential of the techniques.

How can you write a review on molecular imaging without a figure of a single image?????????????????????????????????????

Author Response

Reviewer #1

Comment 1: Also note the title needs to be rewritten. "Molecular Imaging in Iron Metabolism" suggests molecular imaging is involved in iron metabolism.  The title should be something like "Using Molecular Imaging to Elucidate Iron Metabolism".

Response: Thanks a lot for highlighting this concern. We have revised the title of this article to "Elucidating Iron Metabolism through Molecular Imaging" to more accurately reflect the theme of our review.

 

Comment 2: Sections 1.1 and 1.2 on the role of iron in the body and molecular mechanisms of iron metabolism are unnecessary.  These subjects have been reviewed innumerable times.  Besides, the authors made several errors in these sections, which really lead one to question whether they are experts in the area.  For example in line 87, iron ions do not bind to the heme moiety; heme means the combination of iron and appropriate porphyrin.  In line 92, catalase does not cycle between Fe2+ and Fe3+, but also involves higher oxidation states of iron.  Figure 1 (once I increased the image size so that it could actually be read) indicates that Fe2+ is bound to transferrin; this is incorrect - it binds as Fe3+.  

Response: Thank you for your detailed review and valuable feedback on my manuscript. I understand your concerns regarding Sections 1.1 and 1.2, which cover extensively discussed topics. However, I believe these foundational segments are crucial to ensure all readers, especially those new to the field, fully grasp the importance of iron metabolism and its molecular mechanisms. This background information provides an essential framework for understanding the application of molecular imaging techniques in iron metabolism research. Nonetheless, we have reviewed these sections to ensure they are concise yet informative.

Simultaneously, we have carefully revised the manuscript and corrected the inaccuracies. In the original line 87, we have amended the text to reflect that "iron influences the catalytic efficiency of CYPs through its role at the heme center; heme, formed by the complex of iron with porphyrin, is pivotal in ensuring CYPs can effectively metabolize a vast array of endogenous and exogenous substances." In line 92, we corrected the statement to "iron also plays a role in other critical enzymes such as catalase, facilitating the transfer of electrons and protons by transitioning between different oxidation states (including Fe²⁺, Fe³⁺, and higher oxidation states), thus supporting cellular energy metabolism and respiration." We deeply apologize for the mistake regarding Fe2+ binding to transferrin in Figure 1. We have made the necessary corrections to the figure, thoroughly revising the iron metabolism pathway and redrawing the diagram to accurately represent the process.( Figure 1)

We understand these errors might have affected your trust in our expertise. Rest assured, we have meticulously reviewed the entire article to eliminate any potential misleading information or inaccuracies. Thank you once again!

 

Figure 1 Iron homeostasis is highly regulated at both systemic and whole body. ①Non-heme iron is converted from Fe3+ to Fe2+ under the action of DCYTB. ②Tf carries Fe3+ into the cell via endocytosis through TfR1, where it is converted to Fe2+ under the action of STEAP3. ③Heme iron releases Fe2+ in the lysosome through the action of HO. ④Excess Fe2+ is stored. ⑤Ferritin nega-tively regulates the expression of FPN1 to modulate the concentration of Fe2+. ⑥The IRP/IRE system regulates the mRNA stability and translation of key proteins in iron metabolism.

Note: DCYTB: Duodenal cytochromeb; DMT1: Divalent metal transporter 1; HCP1: Heme carrier protein 1; SLC46A1: Solute carrier family 46, member 1; HO: Heme oxygenase; LIP: Labile iron pool; FPN: Ferroportin; Tf: Transferrin; TfR: Transferrin receptor; STEAP3: Six-Transmembrane Epithelial Antigen of Prostate 3; CP: Ceruloplasmin; HEPH: Hephaestin；IRP: Iron Regulatory Proteins; IRE: Iron Responsive Elements.

 

 

Comment 3: The rest of the review describes some techniques that can or have been used to image iron.  A few instances of prior use to probe Fe metabolism are cited, but the results are not described in any depth.  If there was the expected depth here, then section 1.2 might be valuable.  Primarily after the basic introduction of the technique, the manuscript suggests that the technique could have future uses.  A detailed examination of what imaging techniques have been used to probe iron metabolism and what has been learned has not been presented.  If the prior results were discussed in more detail, this would aid in being able to describe the future potential of the techniques.

Response: Thank you very much for your meticulous review and valuable suggestions. We have thoroughly revised and enhanced our manuscript to more accurately articulate the application of various molecular imaging techniques in iron metabolism research. We have elaborately described the working principles of each imaging technique and the types of information they can provide, such as iron ion concentration, ferritin expression levels, and molecular structures, ensuring that readers can understand the unique value and application scope of each technique. For example, as stated in our manuscript:" T2 relaxation time quantifies the rate at which the MRI signal decays to a predefined fraction of its original magnitude, whereas T2* relaxation time exhibits heightened sensitivity to inhomogeneities within the magnetic field. In the presence of iron accumulation, the T2 and T2* relaxation times (especially the T2* relaxation time) are shortened, thus affecting the imaging results, which can lead to a signal loss on T2-weighted spin-echo/fast spin-echo and T2*-weighted gradient-echo images, causing phase distortion of the signal, manifested as low signal intensity and darkening of image brightness” (In line 252-259); “Fluorescence imaging plays a vital role in the study of iron metabolism. It relies on specially designed exogenous fluorescent probes to label iron and its related forms, such as Fe2+, Fe3+, iron-binding proteins, Tf, heme, cytochrome P450, and catalase [61–65], generating imaging signals by enhancing image contrast, which helps researchers to detect and analyze the content and distribution of iron in organisms”（In line 348-352）“Using Raman spectroscopy to detect the vibrational motion of chemical bonds, researchers can identify unique chemical signatures of iron-binding proteins in cellular and tissue samples while targeting predetermined vibrational peaks to generate subcellular chemical profiles further”（In line 440-443）and so on.

Following the overview of instances where molecular imaging has been applied in iron metabolism, we delved deeply into how these techniques precisely reveal the dynamic changes of iron ions across various physiological and pathological states. We emphasized the significant role of these imaging techniques in elucidating the normal and aberrant processes of iron metabolism, ensuring that this section is cohesively linked with Section 1.2, thereby forming a logical continuity. For instance, in discussing MPI, we state, “Pathological conditions such as inflammation, ulcers, or tumors in the gastrointestinal tract can disrupt the normal process of iron absorption, thereby affecting systemic iron homeostasis. More directly, gastrointestinal bleeding leads to the immediate loss of iron stored in Hb, potentially resulting in iron-deficiency anemia. Therefore, accurate diagnosis and monitoring of such bleeding are essential. Recent research [100] has demonstrated using MPI technology combined with specially designed long-circulating SPIO tracers to quantitatively detect gastrointestinal bleeding with high precision in a mouse model. This technique tracks the distribution of SPIO tracers within the body, effectively revealing the accumulation of these tracers in the lower gastrointestinal tract, thereby precisely quantifying the extent of bleeding.” (In line 565-574), and so on.

Furthermore, we have updated the discussion on the latest research advancements in various imaging technologies, including their limitations, strengths, and weaknesses, and we have looked forward to the future direction of these technologies. We particularly highlighted how these advancements could offer new perspectives and potential impacts for the diagnosis and treatment of iron metabolism-related diseases, showcasing the immense potential of molecular imaging techniques in future medical research and clinical applications. For example, in fluorescence imaging, we state, “the design challenges of fluorescent probes, particularly in optimizing their specificity, stability, and biocompatibility, remain key factors limiting the widespread application of this technology. Moreover, their safety and clinical efficacy must undergo rigorous evaluation to facilitate the clinical application of these probes. Future technological advancements are anticipated to enhance diagnostic precision and therapeutic outcomes for iron metabolism disorders by developing novel fluorescent markers, integrating multimodal imaging techniques, and establishing personalized imaging strategies, all while minimizing treatment-related risks and offering personalized and precise treatment options for patients.” (In line 406-413), and so on.

Once again, we appreciate your review comments, which are invaluable in enhancing the quality of our paper.

 

 

Comment 4: How can you write a review on molecular imaging without a figure of a single image?????????????????????????????????????

Response: We greatly appreciate your valuable comments and suggestions, which are crucial for enhancing the quality of our manuscript. We have taken your feedback regarding the absence of illustrative figures seriously and understand that this omission could limit readers' understanding of the application of molecular imaging techniques in iron metabolism research. Indeed, this was an oversight. Initially, our focus was more on discussing theories and mechanisms, which led us to neglect the demonstration of these technologies' practical applications and imaging results. We have improved the manuscript by adding result images from MRI, optical imaging, Raman imaging, and MPI to visually demonstrate the application of these techniques in iron metabolism studies. (Figure2~ Figure5, Figure6, Figure7)

 

Figure 2 A. Gradient echo images of the liver at echo times of 1.1, 4.6, 9.9, and 13.4 ms. Reprinted with permission from[57], Copyright ©2008, Informa Healthcare USA, Inc.  B. Examples of quantitative MRI maps of a single subject. PD, Proton density. Reprinted from reference [58] with permission from Elsevier.  C. From top to bottom: Gd-enhanced T1-weighted image, R1 map, and R2* map in a representative subject with a meningioma brain tumor (white arrow), Reproduced with permission from Yu E et al. [59], Copyright © 2023, The Author(s)

 

 

Figure 3 Ratiometric fluorescence imaging is employed to map the dynamic changes in cellular iron levels accurately. FIP-1 is a peroxisome-based FRET (Förster Resonance Energy Transfer) probe that operates through the Fe(II)-induced cleavage of peroxides, linking the fluorophores luciferase and Cy3. This cleavage reduces FRET, enabling the probe to selectively detect and significantly reflect the changes in the labile iron pool within living cells in an aqueous buffer solution. Reprinted with permission from [67], Copyright © 2016 American Chemical Society.

 

Figure 4 In the MPP+-induced Parkinson's disease model, the hyperspectral signal of iron deposition is mapped in red on the cell image after 6 hours of FAC (ferric ammonium citrate) exposure. Representative images of points a, b, c, d, and e are enlarged images (4x magnification) from the area enclosed by the yellow box in (a) and (b). Scale bar (a and b): 40 μm. Scale bars (a, b, c, d, and e): 10 μm. Reproduced with permission from Oh ES et al. [69], Copyright © 2014, The Author(s).

 

 

Figure 5 A. Raman spectrum peak at ~1300 cm−1 identifies the iron-bound state of Tf in intact cells. Reprinted from reference [75] with permission from Elsevier.  B. Illustration of the Raman spectra for transferrin samples, differentiating between the iron-saturated (holo-Tf) and iron-free (apo-Tf) states at a 75 μg/mL concentration. Reprinted with permission from [67], Copyright © 2016, Society of Photo-Optical Instrumentation Engineers (SPIE).

 

 

Figure 7 Quantitative comparison of MPI and fluorescence signal variations with tissue depth. Six pairs of equivalent SPIO and fluorescent probes were placed in capillaries and embedded in sliced porcine muscle phantoms at various depths. Figures A-B represent the MPI/CT images with maximum intensity projection, illustrating that the SPIO signal remains constant at different tissue depths, whereas the corresponding fluorescence probe signals significantly weaken as the depth increases. Reprinted with permission from [90], Copyright © 2016, Ivyspring International Publisher.

 

Figure 8 A. Representative bioluminescence image of the MDA-MB-231-luc xenografted breast tumor, confirmed using bioluminescence imaging (IVIS Lumina) to verify the presence of the tumor. B. Three-dimensional MPI maximum intensity projection image showing the lower abdomen of a mouse four weeks post-tumor implantation (field of view: 4 × 4 × 5.8 cm). The image was acquired 6 hours after the custom long-circulating SPIO tracer LS-008 injection, combined with CT imaging. Reprinted with permission from [98], Copyright © 2017, American Chemical Society.  C. MPI imaging of GI bleeding in mice (integrated with X-ray anatomy), employed for evaluating gastrointestinal bleeding in a mouse model. Reprinted with permission from [100], Copyright ©2017, American Chemical Society.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a very useful appropriately referenced review that provides a nice summary of molecular imaging modalities available for the assessment of iron metabolism.

Comments are minor.

1. On line 38, the authors should refer to "advances in the understanding of" rather than "advancements in".

2. The acronym SPIO is used in Table 1, but has not yet been defined in the manuscript.

3. On line 312, "lack of radiation" is more correct than "zero radiation".

4. There appears to be a typographic error on line 354 the phrase "Iron levels in cancer cell types [52]." appears to be misplaced.

Author Response

Comment 1: On line 38, the authors should refer to "advances in the understanding of" rather than "advancements in".

Response: Thanks a lot for your valuable suggestions. We are more than happy to take your advice and we will change “advancements” to “advances”.

 

Comment 2: The acronym SPIO is used in Table 1, but has not yet been defined in the manuscript.

Response: We are grateful for your detailed suggestions. We are very sorry that we did not pay attention to these details. In Table 1, we have offered the definition of SPIO, which is delineated as superparamagnetic iron oxide.

 

Comment 3: On line 312, "lack of radiation" is more correct than "zero radiation".

Response: Many thanks for your well-meaning suggestions. We agree to change "zero radiation" to "lack of radiation" to accurately express the advantages of optical imaging.

 

Comment 4: There appears to be a typographic error on line 354 the phrase "Iron levels in cancer cell types [52]." appears to be misplaced.

Response: Many thanks to the reviewer for your careful review of our manuscript and valuable comments. We have carefully checked the content of line 354 and found that there is a typographical error. We have modified "which can help to identify cancer cell types with high basal unbalanced iron levels. Iron levels in cancer cell types" for “which helps identify cancer cell types with higher basal labile iron levels. "

Reviewer 3 Report

Comments and Suggestions for Authors

The title of this review implies that it will be more informative than it actually is. In the introduction, the cursory review of human iron metabolism leaves out hephaestin, ceruloplasmin, iron regulatory proteins and translational regulation of IRE containing transcripts. It does not make clear that DMT1 is a transmembrane transporter, seeming to imply that it helps guide iron through the cytosol. It uses the phrase "controlling negligible" when they likely mean "regulatory' but it's hard to know. An oddity of the review is that the only figures are incomplete cartoons about iron metabolism proteins, whereas there are no figures that illustrate MRI results, optical imaging, Raman imaging or MPI. There are references, but a good review would leave the reader with a better sense of how and when these techniques are useful, and how the images actually appear. 

There are some misstatements or odd usages;

1. Line 244, the liver is the organ in which iron is stored under normal physiological conditions. 

2. Line 354- incomplete sentence- meaning unclear

3. line3 492, IRE-dependent regulatory mechanisms- need to explain better

Overall, the review needs to distinguish methods already in use from those in development, and it needs to explain and illustrate the output of these methods better, and the settings in which they expect the techniques discussed to be useful. 

 

Comments on the Quality of English Language

some clarifications discussed in general review

Author Response

Comment 1: The title of this review implies that it will be more informative than it actually is. In the introduction, the cursory review of human iron metabolism leaves out hephaestin, ceruloplasmin, iron regulatory proteins and translational regulation of IRE containing transcripts.

 

Response: Many thanks to the reviewers for examining our manuscript so seriously and for making such reliable suggestions. We have revised the title of this article to "Elucidating Iron Metabolism through Molecular Imaging" to more accurately reflect the theme of our review. Furthermore, we have enriched our overview of iron metabolism with comprehensive additions, including aspects such as hepcidin, ceruloplasmin, iron regulatory proteins, and the translational regulation of IRE-containing transcripts. Based on these additions, we have also updated the mechanistic diagrams related to the regulation of iron metabolism.

 

 

Figure 1 Iron homeostasis is highly regulated at both systemic and whole body. ①Non-heme iron is converted from Fe3+ to Fe2+ under the action of DCYTB. ②Tf carries Fe3+ into the cell via endocytosis through TfR1, where it is converted to Fe2+ under the action of STEAP3. ③Heme iron releases Fe2+ in the lysosome through the action of HO. ④Excess Fe2+ is stored. ⑤Ferritin negatively regulates the expression of FPN1 to modulate the concentration of Fe2+. ⑥The IRP/IRE system regulates the mRNA stability and translation of key proteins in iron metabolism.

Note: DCYTB: Duodenal cytochromeb; DMT1: Divalent metal transporter 1; HCP1: Heme carrier protein 1; SLC46A1: Solute carrier family 46, member 1; HO: Heme oxygenase; LIP: Labile iron pool; FPN: Ferroportin; Tf: Transferrin; TfR: Transferrin receptor; STEAP3: Six-Transmembrane Epithelial Antigen of Prostate 3; CP: Ceruloplasmin; HEPH: Hephaestin；IRP: Iron Regulatory Proteins; IRE: Iron Responsive Elements.

 

 

 

Comment 2: It does not make clear that DMT1 is a transmembrane transporter, seeming to imply that it helps guide iron through the cytosol. It uses the phrase "controlling negligible" when they likely mean "regulatory' but it's hard to know.

Response: We appreciate your valuable advice to us. We concur that DMT1 is a transmembrane transporter protein, serving as a multi-metal transporter, which primarily functions in the transport of iron, facilitating the guidance of iron through the cytosolic sol. We have amended "controlling negligible" to "regulating" to more accurately depict the pivotal role of DMT1 in the regulation of iron metabolism.

 

Comment 3: An oddity of the review is that the only figures are incomplete cartoons about iron metabolism proteins, whereas there are no figures that illustrate MRI results, optical imaging, Raman imaging or MPI. There are references, but a good review would leave the reader with a better sense of how and when these techniques are useful, and how the images actually appear.

Response: We greatly appreciate your valuable comments and suggestions, which are crucial for enhancing the quality of our manuscript. We have taken your feedback regarding the absence of illustrative figures seriously and understand that this omission could limit readers' understanding of the application of molecular imaging techniques in iron metabolism research. Indeed, this was an oversight. Initially, our focus was more on discussing theories and mechanisms, which led us to neglect the demonstration of these technologies' practical applications and imaging results. We have made several improvements in our manuscript to address these issues:

Firstly, we have provided a more comprehensive explanation of the molecular mechanisms of iron metabolism and, based on this, enriched the mechanism diagrams relating to the regulation of iron metabolism. Secondly, we have included figures illustrating MRI, optical imaging, Raman imaging, and MPI results, offering a visual demonstration of these technologies' applications in iron metabolism research. Lastly, we have added several case studies, especially those offering a visual experience of the images, to enhance the practicality and depth of our article. (Figure 2~Figure 8)

We believe that these improvements will make the revised article more comprehensive and specific, effectively addressing your concerns and enabling readers to gain a clearer understanding of the application and value of molecular imaging technologies in iron metabolism research.

 

Figure 2 A. Gradient echo images of the liver at echo times of 1.1, 4.6, 9.9, and 13.4 ms. Reprinted with permission from[57], Copyright ©2008, Informa Healthcare USA, Inc.  B. Examples of quantitative MRI maps of a single subject. PD, Proton density. Reprinted from reference [58] with permission from Elsevier.  C. From top to bottom: Gd-enhanced T1-weighted image, R1 map, and R2* map in a representative subject with a meningioma brain tumor (white arrow), Reproduced with permission from Yu E et al. [59], Copyright © 2023, The Author(s)

 

Figure 3 Ratiometric fluorescence imaging is employed to map the dynamic changes in cellular iron levels accurately. FIP-1 is a peroxisome-based FRET (Förster Resonance Energy Transfer) probe that operates through the Fe(II)-induced cleavage of peroxides, linking the fluorophores luciferase and Cy3. This cleavage reduces FRET, enabling the probe to selectively detect and significantly reflect the changes in the labile iron pool within living cells in an aqueous buffer solution. Reprinted with permission from [67], Copyright © 2016 American Chemical Society.

 

Figure 4 In the MPP+-induced Parkinson's disease model, the hyperspectral signal of iron deposition is mapped in red on the cell image after 6 hours of FAC (ferric ammonium citrate) exposure. Representative images of points a, b, c, d, and e are enlarged images (4x magnification) from the area enclosed by the yellow box in (a) and (b). Scale bar (a and b): 40 μm. Scale bars (a, b, c, d, and e): 10 μm. Reproduced with permission from Oh ES et al. [69], Copyright © 2014, The Author(s).

 

 

Figure 5 A. Raman spectrum peak at ~1300 cm−1 identifies the iron-bound state of Tf in intact cells. Reprinted from reference [75] with permission from Elsevier.  B. Illustration of the Raman spectra for transferrin samples, differentiating between the iron-saturated (holo-Tf) and iron-free (apo-Tf) states at a 75 μg/mL concentration. Reprinted with permission from [67], Copyright © 2016, Society of Photo-Optical Instrumentation Engineers (SPIE).

 

Figure 6 A. Schematic diagram of field-free point (FFP) magnetic particle imaging. The setup includes a magnetic field gradient generated by NdFeB permanent magnets and the control of the FFP movement in the x and y directions by electromagnets, along with the movement of an animal bed in the z direction to acquire the maximum intensity projection of three-dimensional MPI images.  B. MPI scanner. Reprinted from reference [89] with permission from Elsevier.

 

 

Figure 7 Quantitative comparison of MPI and fluorescence signal variations with tissue depth. Six pairs of equivalent SPIO and fluorescent probes were placed in capillaries and embedded in sliced porcine muscle phantoms at various depths. Figures A-B represent the MPI/CT images with maximum intensity projection, illustrating that the SPIO signal remains constant at different tissue depths, whereas the corresponding fluorescence probe signals significantly weaken as the depth increases. Reprinted with permission from [90], Copyright © 2016, Ivyspring International Publisher.

 

Figure 8 A. Representative bioluminescence image of the MDA-MB-231-luc xenografted breast tumor, confirmed using bioluminescence imaging (IVIS Lumina) to verify the presence of the tumor. B. Three-dimensional MPI maximum intensity projection image showing the lower abdomen of a mouse four weeks post-tumor implantation (field of view: 4 × 4 × 5.8 cm). The image was acquired 6 hours after the custom long-circulating SPIO tracer LS-008 injection, combined with CT imaging. Reprinted with permission from [98], Copyright © 2017, American Chemical Society.  C. MPI imaging of GI bleeding in mice (integrated with X-ray anatomy), employed for evaluating gastrointestinal bleeding in a mouse model. Reprinted with permission from [100], Copyright ©2017, American Chemical Society.

 

Comment 4: Line 244, the liver is the organ in which iron is stored under normal physiological conditions. 

Response: Thanks a lot for your kind advice sincerely. The core essence of the content is, "The liver, by synthesizing ferritin and transferrin, serves not only as the primary storage site for iron but also regulates the systemic distribution of iron." "As the principal organ where iron reserves continue to accumulate with an increase in systemic iron levels, the liver is typically the first to be affected by iron overload." This modification has been incorporated into our review. Thanks again for your friendly advice.

 

 

Comment 5: Line 354- incomplete sentence- meaning unclear

Response: Many thanks to the reviewer for your careful review of our manuscript and valuable comments. We have carefully checked the content of line 354 and found that there is a typographical error. We have modified "which can help to identify cancer cell types with high basal unbalanced iron levels. Iron levels in cancer cell types" for “which helps identify cancer cell types with higher basal labile iron levels. "

 

Comment 6: line 492, IRE-dependent regulatory mechanisms- need to explain better

Response: Thank you very much for your meticulous advice. Based on our understanding, the impact of SPIO treatment on macrophage polarization, particularly its role in immunoregulation and disease progression, appears to be closely associated with the activation of IRE-dependent regulatory mechanisms [1–3]. This mechanism finely controls intracellular iron levels through the interaction of Iron Regulatory Proteins (IRPs) with IRE sequences. IRPs are capable of responding to changes in cellular iron levels, regulating the expression of key iron metabolism proteins, such as transferrin receptors and ferritin, by binding to or dissociating from IRE sequences, thereby maintaining iron homeostasis [4]. SPIO treatment may indirectly affect IRP activity and its binding to IREs by adjusting intracellular iron concentrations, not only altering the polarization state of macrophages but also potentially modulating the immune response by promoting the production of the anti-inflammatory cytokine IL-10. We further explain the above in the manuscript. And we believe that SPIO treatment and its potential impact on IRE-dependent regulatory mechanisms offer a new perspective for understanding the role of iron metabolism in immunoregulation and disease progression, and provide possible targets for the development of new therapeutic strategies. Thank you again!

 

Comment 7: Overall, the review needs to distinguish methods already in use from those in development, and it needs to explain and illustrate the output of these methods better, and the settings in which they expect the techniques discussed to be useful.

Response: Thank you for your thorough review and valuable feedback on our manuscript. Your comments have not only helped us identify areas for improvement but also provided us with directions for enhancement. In response to your concerns, we have made the following revisions and additions:

Regarding the distinction between technologies in use and those under development, we have emphasized the research stages of MRI, MPI, and optical imaging technologies. For instance, MRI is highlighted as a mature technology widely applied in clinical and research fields, whereas MPI and optical imaging are still in the laboratory development or early exploration stages. Concurrently, we have updated the latest research progress for each technology.

In explaining and illustrating the outputs of these methods, we provided more detailed descriptions of each technology, clarifying their working principles and the types of information they can provide, such as iron ion concentration, ferritin expression levels, and molecular structure. Additionally, we included several research case studies to demonstrate how these technologies are utilized in addressing specific scientific questions.

When discussing the application contexts of these technologies, we listed the advantages and disadvantages of each molecular imaging technique and discussed their applicability and limitations in various research and clinical settings. This includes considerations of sensitivity, specificity, cost-effectiveness, and potential for clinical translation. Furthermore, we discussed the future directions of technological development and the potential impact of these technologies on the diagnosis and treatment of iron metabolism diseases.

 

References

[1] Jin R, Liu L, Zhu W, et al. Iron oxide nanoparticles promote macrophage autophagy and inflammatory response through activation of toll-like Receptor-4 signaling. Biomaterials 2019; 203. doi:10.1016/j.biomaterials.2019.02.026

[2] Rojas JM, Sanz-Ortega L, Mulens-Arias V, et al. Superparamagnetic iron oxide nanoparticle uptake alters M2 macrophage phenotype, iron metabolism, migration and invasion. Nanomedicine Nanotechnol Biol Med 2016; 12: 1127–1138. doi:10.1016/j.nano.2015.11.020

[3] Ding H, Zhang Y, Mao Y, et al. Modulation of macrophage polarization by iron-based nanoparticles. Med Rev 2021 2023; 3: 105–122. doi:10.1515/mr-2023-0002

[4] Zhou ZD, Tan E-K. Iron regulatory protein (IRP)-iron responsive element (IRE) signaling pathway in human neurodegenerative diseases. Mol Neurodegener 2017; 12: 75. doi:10.1186/s13024-017-0218-4

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is massively improved in quality.  It is essentially a new manuscript.  The authors have addressed the reviewers' concerns.  However, the manuscript needs one more round of careful proofing - for example, the caption to Figure 1 contain a "Fe2+" rather than "Fe²⁺".

The font size should be enlarged if possible for most wording in Figure 1.

 

Author Response

Comment 1：The manuscript is massively improved in quality.  It is essentially a new manuscript.  The authors have addressed the reviewers' concerns.  However, the manuscript needs one more round of careful proofing - for example, the caption to Figure 1 contain a "Fe²⁺" rather than "Fe2+". The font size should be enlarged if possible for most wording in Figure 1.

 

Response: We sincerely appreciate your meticulous review and valuable comments. Regarding your latest feedback, we have noted the issue you pointed out with the "Fe2+" annotation in Figure 1. Indeed, the correct chemical symbol "Fe²⁺" should be used. We have already made this correction to ensure that all chemical symbols are accurate. Simultaneously, concerning the font size of most text in Figure 1, we have adjusted the graphics to ensure that all text is clear and readable while maintaining the figure's neatness and professionalism. We thank you once again for your suggestions.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

There are still some mistakes in discussion of iron metabolism. The most significant is using HCP as heme carrier protein, when this report from over 20 years ago has been discredited. There are one or two other such lapses. They improved the contents of the main paper enough. A paper of this type should not contain basic factual errors, and they need to check carefully and revise some of their statements. It might help if they incorporated some more recent reviews that no longer rely on discredited facts and proteins. 

Comments on the Quality of English Language

It's ok. 

Author Response

Comment 1：There are still some mistakes in discussion of iron metabolism. The most significant is using HCP as heme carrier protein, when this report from over 20 years ago has been discredited. There are one or two other such lapses. They improved the contents of the main paper enough. A paper of this type should not contain basic factual errors, and they need to check carefully and revise some of their statements. It might help if they incorporated some more recent reviews that no longer rely on discredited facts and proteins.

 

Response: Thank you very much for your detailed review and valuable feedback. We are particularly grateful for your important corrections regarding the discussion of iron metabolism. We have carefully reviewed and revised our review on iron metabolism, especially the sections concerning the mechanisms of heme iron absorption. Following your advice, we have consulted some of the most recent review articles to ensure that our manuscript does not contain discredited facts and data. We have updated our manuscript accordingly, incorporating the latest literature to provide an accurate and up-to-date overview of the topic——“Although the mechanisms of non-heme iron absorption have been well characterized, the mechanisms by which heme iron is absorbed from the intestine remain less understood. Heme Carrier Protein 1 (HCP 1) (SLC 46 A1) was initially identified as an intestinal importer of heme; however, it was later shown to be a high-affinity, pH-dependent folate transporter [16]. The transporter directly associated with heme iron absorption has not yet been unequivocally identified [17], but it is known that the absorption process of heme iron is a distinct mechanism, crucial for the systemic regulation and cellular utilization of iron, supporting the synthesis of key proteins such as Hb and iron-sulfur cluster enzymes. In the body, the reticuloendothelial system (RES) macrophages degrade aged or damaged red blood cells (RBCs), and iron (Fe²⁺) is released by the catalysis of heme decomposition by heme oxygenase (HO). This iron is then transported into the bloodstream by specific membrane iron transporters, where it can be stored by ferritin or released via FPN1 into the bloodstream, subsequently carried throughout the body by Tf to meet cellular functional demands and maintain iron homeostasis.”

Again, thank you for your constructive comments. We believe that these revisions have made our manuscript more rigorous and reflective of the current state of knowledge in the field of iron metabolism.

 

References：

[16] Shayeghi M, Latunde-Dada GO, Oakhill JS, et al. Identification of an intestinal heme transporter. Cell 2005; 122: 789–801. doi:10.1016/j.cell.2005.06.025

[17] Dutt S, Hamza I, Bartnikas TB. Molecular Mechanisms of Iron and Heme Metabolism. Annu Rev Nutr 2022; 42: 311–335. doi:10.1146/annurev-nutr-062320-112625