Kupffer Cells as a Target for Immunotherapy
Round 1
Reviewer 1 Report
Takashi Sakai and Wen-Ling Lin present a quality and well-written review manuscript focusing on Kupffer cells as a target for immunotherapy.
Authors summarize the outlook and the issue of immunotherapy using the Kupffer cells transplantation.
Authors suggest that Kupffer cells (KCs) are the resident macrophages in the liver. They have a high phagocytic competency, allowing them to remove the foreign medium, for example, viruses and bacteria, apoptotic cells, and cellular debris. Accordingly, KCs in the liver sinusoids have a crucial role as gatekeepers in the hepatic immune system. KCs play a central role in immunity and tissue injury and repair in the liver. KCs generate various inflammatory mediators containing cytokines, prostaglandins, and reactive oxygen species mainly through NADPH-oxidase or inducible NO-synthase (iNOS) activities . Thre is no doubt that KCs dysfunction contributes to the pathogenesis of nonalcoholic fatty liver diseases (NAFLD). Nevertheless, the role of KCs in regulating liver metabolism and the incidence of metabolic disease remains unknowable. Thus, if KCs were transplanted efficaciously in the liver with engraftment and long-term survival, main issues could be addressed regarding their characters in microbial clearance, antigen presentation, and tissue inflammation or repair. Some studies have displayed that KCs transplantation is helpful. On the other hand, the kinetics of how the administered KCs settle in the liver remains unclear.
Finally, authors conclude that the regulation of KCs activation should be crucial in progressive phases such as NASH and HCC. It is expected to establish novel therapeutic strategies for the in vivo modulation of KCs by chemicals, diet or exercising, or the ex vivo methods using KCs transplantation.
Overall, the manuscript is valuable for the scientific community and should be accepted for publication.
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Other comments:
1) Please check for minor typos throughout the manuscript.
2) Authors are kindly encouraged to cite this article that describes various aspects of immunotherapy that might be relevant also for targeting Kupffer cells. DOI: 10.3390/cancers14041078
Author Response
Dear Reviewer 1,
Thank you very much for the careful review.
Changes in the revised manuscript are underlined.
Our responses are shown below.
Thank you very much for your kind comments. According to your suggestions, we revised our manuscript.
1) We had our English proofread by AJE (www.aje.com).
2) We added new sentences with a citation (p6, lines 13-19).
Author Response File: 
Author Response.docx
Reviewer 2 Report
Kupffer cells (KCs) or liver resident macrophages are critical for proper functioning of the hepatic immune system. Although, there has been a significant progress in the understanding of characteristics and functioning of these cells in the recent years, the exact mechanism by which KCs modulate liver functions remains unknown. Moreover, regulation of the fine balance between the M1/M2 differentiation state of KCs is crucial for prevention of inflammatory liver disorders and maintenance of liver homeostasis. Therefore, KCs are an ideal candidate for immunotherapy for liver diseases like non-alcoholic fatty liver diseases or hepato-cellular carcinoma
In this article, the authors review the current knowledge of usage of KCs as immune-therapeutic agents. I would like to suggest the following revisions:
1. The article has multiple missing references. For example:-
a. Lines 20-23 (Macrophages have high plasticity that serve multiple functions, including phagocytosis……..)
b. Lines 29-31 (KCs in the liver sinusoids 29 have a crucial role as gatekeepers in the hepatic immune system. KCs play a central role in immunity and tissue injury and repair in the liver.)
c. Lines 43-48(Nutrients absorbed from the small intestine flow through the portal vein or lymph vessels to the liver, where they are metabolized as an energy source)
d. Lines 56-57, states many studies reported the involvement of KCs in those diseases but only two references have been cited. Include more references or remove the word many
e. Lines 76-78 (Considering the possibility of lymphatic mediation, we investigated the spleen and lymph nodes to confirm the presence of transplanted KCs……….)
f. Lines 86-89 (present heterogeneously in their volume, phagocytotic activity, and enzymatic activities even in the normal physiological situation. The smaller inactive cells predominantly……….)
g. Lines 91-92 (In the pathogenesis of NAFLD, accumulated lipids and their metabolites activate KCs)
h. Lines 102-108
i. Lines 127-131
2. Can the authors explain this in more detail - In KCs, direct entry through the surface of the liver is unlikely. What characteristics of liver surface or KCs make is unlikely for KCs to enter via liver surface.
3. The authors must proofread the manuscript again. The writing is complicated in many sections. For example, Line 36, incidence of liver disease remains unknown instead of unknowable. Line 86, KCs present heterogeneity instead of heterogeneously
4. Role of Nucling in determining the differentiation state of KCs in more detail in the main text of the article and not in the discussion section.
Author Response
Dear Reviewer 2,
Thank you very much for the careful review.
Changes in the revised manuscript are underlined.
Our responses are shown below.
Thank you very much for your kind comments. According to your suggestions, we revised our manuscript.
1) According to the comment, we added new references. Additional references are No. 1,3,14-16,22,26-32, and 35-39.
2) Q. from reviewer; Can the authors explain this in more detail? In KCs, direct entry through the surface of the liver is unlikely. What characteristics of liver surface or KCs make is unlikely for KCs to enter via liver surface?
Ans. To answer the question, we added the sentences below on page 2, lines 33-37.
According to Nishida et al., the critical size of molecules that can be absorbed from the liver surface covered by the capsule is estimated to be about 70,000. This is slightly larger than the 6.6K molecular weight of albumin. This suggests that it would be very difficult for KCs to pass directly through the liver surface.
3) According to the comment, we proofread it by AJE (www.aje.com) and revised it.
4) Reviewer's comment: Role of Nucling in determining the differentiation state of KCs in more detail in the main text of the article and not in the discussion section.
Ans. According to the comment, we transferred the Nucling section with minor revisions in the main text (p2-3, “5 Nucling/NF-kB signal and KC activation.”).
Author Response File: Author Response.docx
Reviewer 3 Report
The authors summarized the outlook and the issue of immunotherapy using the KCs transplantation. The manuscript is clear and consistent. However, some concepts need to reseen before a final decision.
“Migration Pathway of Intraperitoneally Administered KCs to the Liver. The route of translocation of intraperitoneally administered KCs to the liver is not clear. In the intraperitoneal administration of drugs, there are four possible routes by which administered drugs reach the liver. 1) diffusion and absorption from the wall-side intraper itoneal capillary bed via the portal vein, 2) absorption from the visceral side intraperitoneal capillary bed via the body circulatory system, 3) via the lymphatic vascular system, and 4) direct absorption from the liver surface. In KCs, direct entry through the surface of the liver is unlikely”
Comment: When innoculated KCs enter in an injuried hepatic parenchyma, they can be differenciatted in some kinds of phenotypes by activity of hepatic star cells (HSC). The author should describe the loop of immunomodulation between KCs and HSCs.
In liver, the biggest problem of the chronic inflammatory is foward for cicatrization whose process is characterized for fibrosis. Cirrosis means fibrosis. M2 macrophages are responsible for immunosupression with high expression of IL-10 and TGF-b that stimulate SCs to produce collagen fibers. In discussion of manuscript, the authors describe “The activated KCs release pro-inflammatory cytokines that promote NAFLD development. Progressive NAFLD further stimulates KCs to be activated, helping to progress to hepatitis, cirrhosis, and HCC” ( lines 92-94). I suggest the authors rewrite this paragraph because anti-inflammatory cytokines are responsible for hepatic fibrosis by modulate HSC. I suggest to read the manuscript and reference it (10.1371/journal.pone.0148868).
Author Response
Dear Reviewer 3,
Thank you very much for the careful review.
Changes in the revised manuscript are underlined.
Our responses are shown below.
Thank you very much for your kind comments. According to your suggestions, we revised our manuscript.
1) The author should describe the loop of immunomodulation between KCs and HSCs.
Ans. According to the comment, we added sentences in the main text (p3, “6. KCs-HSCs axis”).
2) In liver, …..I suggest the authors rewrite this paragraph because anti-inflammatory cytokines are responsible for hepatic fibrosis by modulate HSC. I suggest to read the manuscript (10.1371/joural.pone.0148868).
Ans. Thank you very much for your advice. We rewrite the paragraph according to your comment (p4, lines 12-20, and Fig. 1).
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors provided satisfactory answers for all my queries. The paper can now be accepted for publication.
