Establishment of SV40 Large T-Antigen-Immortalized Yak Rumen Fibroblast Cell Line and the Fibroblast Responses to Lipopolysaccharide
, Ziqi Yue †, Li Che, Hui Li, Rui Hu, Liyuan Shi, Xiaohong Zhang, Huawei Zou, Quanhui Peng, Yahui Jiang and Zhisheng Wang *Round 1
Reviewer 1 Report
The manuscript reports the establishment of immortalized (SV40 large T antigen-transformed) yak rumen fibroblast cell line, and characterizes its response to lipopolysaccharide (LPS). The authors conduct experiments to confirm the cell line identity and its ability to proliferate and maintain in culture. In addition, the mRNA expression changes of some genes related to extracellular matrix production and degradation as well as inflammatory response are analyzed.
While the part of the study related to cell line establishment and basal characterization is scientifically sound and clearly presented, the part regarding the response to LPS requires revision, since the range of LPS concentrations used is non-physiological, and no clear concentration-dependencies are observed.
Language editing is necessary (particularly, parts highlighted in yellow in the manuscript pdf attached).
Specific points:
Page 2, Lines 53-55. The sentence: “At present, there have been some research has been carried out in different parts of organs and tissues of different species” is unclear and should be rephrased.
Page 2, Lines 73-75. The part of the sentence: “The morphologically fibroblastic cells with spindle-like, bipolar, or multipolar and elongated morphology and adhesive growth, which was typical of fibroblasts in the culture plates” should contain a verb.
Page 2, line 86: what is “dark blue” referring to when describing no evidence of senescence? This should be described in the Results text so that the method of measuring senescence (apparently SA-b-Gal staining) is clear.
Figure 2c – no error bars are shown (although the legend indicates data are presented as Means ± SEM).
Figure 4b legend – units of LPS concentration are probably not correct (µg/ml in Methods).
Figure 4b, 5, 6 – the range of LPS concentrations is too high and does not reflect biologically relevant concentrations (no more than 100 ng/ml).
Figures 5, 6 – it is not clear which values were compared when analyzing statistical significance (indicated by different letters).
Comments for author File: 
Comments.pdf
Language editing is necessary (particularly, parts highlighted in yellow in the manuscript pdf attached).
Specific points:
Page 2, Lines 53-55. The sentence: “At present, there have been some research has been carried out in different parts of organs and tissues of different species” is unclear and should be rephrased.
Page 2, Lines 73-75. The part of the sentence: “The morphologically fibroblastic cells with spindle-like, bipolar, or multipolar and elongated morphology and adhesive growth, which was typical of fibroblasts in the culture plates” should contain a verb.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
The manuscript titled " Establishment of SV40 large T antigen-immortalized Yak Rumen Fibroblast Cell Line and Extracellular matrix Responses to Lipopolysaccharide” describes in a reliable and detailed manner the immortalized yak rumen fibroblast cell line. I believe that this type of methodical publication is needed because well-characterized cell lines can be properly used in research. Also, the aspect of changes within the MMPs/TIMPs, the uPA system, and its participation in fibrosis is the important value of this work. However, there are some comments that need to be addressed:
Introduction
· This part needs to be complemented with very basic information about fibrosis and the importance of this pathological process in the yak rumen. My suggestion is to put this information after the sentence in lines 40-42 (”It is known that one of the main functions of fibroblasts is the synthesis of collagen and other extracellular matrices (ECM), which plays an important role in the process of tissue and organ fibrosis [14].”)
· In my opinion, in the introduction section, sentences such as:
”…At present, there have been some research has been carried out in different parts of organs and tissues of different species….”
”…There are few reports have been reported on the analysis of yak organism-related organ functions at the level of yak rumen fibroblasts…”
does not provide the necessary information, the more so that it is not supported by references, and also does not explain the purpose of this work.
· In my opinion, the introduction lacked a solid explanation of the taken up of the LPS topic, which was placed in the discussion section in the sentences from lines: 236-242. Therefore, I think this content should be incorporated into the introduction section to lead the reader straight to the goal of the work.
Discussion
· Line 249 – in the sentence ”…In physiological processes such as inflammation…” should be: In pathological processes…
· The following two sentences should be combined into one sentence:
(Lines 273-276) ”…However, to our best knowledge, there are no studies between MMPs/TIMPs and uPA system in yak rumen fibrosis. Currently, our work is the first systematic study of MMPs/TIMPs and uPA systems in yak rumen fibrosis…”
Author Response
# reviewer 2
The manuscript titled " Establishment of SV40 large T antigen-immortalized Yak Rumen Fibroblast Cell Line and Extracellular matrix Responses to Lipopolysaccharide” describes in a reliable and detailed manner the immortalized yak rumen fibroblast cell line. I believe that this type of methodical publication is needed because well-characterized cell lines can be properly used in research. Also, the aspect of changes within the MMPs/TIMPs, the uPA system, and its participation in fibrosis is the important value of this work. However, there are some comments that need to be addressed:
Introduction
- This part needs to be complemented with very basic information about fibrosis and the importance of this pathological process in the yak rumen. My suggestion is to put this information after the sentence in lines 40-42 (”It is known that one of the main functions of fibroblasts is the synthesis of collagen and other extracellular matrices (ECM), which plays an important role in the process of tissue and organ fibrosis [14].”)
Response: Thank you very much for your guidance and suggestions on writing the introduction in the manuscript. Based on your suggestion, I have added basic information about organ fibrosis “Organ fibrosis is a disease characterized by excessive deposition of ECM and the accumulation of myofibroblasts, which is closely related to chronic inflammatory diseases. The fibrotic signaling cascade that occurs during chronic inflammation, which is initiated by epithelial injury and results in irreversible organ damage, is regulated by various inflammatory mediators.” after the sentence in lines 40-42 (”It is known that one of the main functions of fibroblasts is the synthesis of collagen and other extracellular matrices (ECM), which plays an important role in the process of tissue and organ fibrosis [14].”) and linked its inflammatory response to draw attention to the occurrence of fibrotic diseases in patients with gastritis. See pages 1-2, lines 44 – 49; See page 2, lines 57 – 63.
(Miao Z, Ding Y, Bi Y, et al. Staphylococcus aureus on the effect of expression of MMPs/TIMPs and uPA system in bovine mammary fibroblasts[J]. Journal of Microbiology, Immunology and Infection, 2021, 54(3): 411-419.).
(Ueha S, Shand F H W, Matsushima K. Cellular and molecular mechanisms of chronic inflammation-associated organ fibrosis[J]. Frontiers in immunology, 2012, 3: 71.).
- In my opinion, in the introduction section, sentences such as:
”…At present, there have been some research has been carried out in different parts of organs and tissues of different species….”
”…There are few reports have been reported on the analysis of yak organism-related organ functions at the level of yak rumen fibroblasts…”
does not provide the necessary information, the more so that it is not supported by references, and also does not explain the purpose of this work.
Response: We have deleted these sentences based on your and another reviewer's suggestions.
In my opinion, the introduction lacked a solid explanation of the taken up of the LPS topic, which was placed in the discussion section in the sentences from lines: 236-242. Therefore, I think this content should be incorporated into the introduction section to lead the reader straight to the goal of the work.
Response: Thank you very much for your guidance. I have made corrections and placed the discussion section on the impact of LPS on fibroblasts after line 54 of the introduction.
In the discussion section, in lines 257-258, I added "LPS is a highly proinflammatory endotoxin in the circulatory system, past studies have raised concerns regarding ruminal LPS contribution to the pathogenesis of ruminal acidosis [21]. This sentence enriches the discussion section. See pages 9, lines 270 – 272.
Monteiro H F, Faciola A P. Ruminal acidosis, bacterial changes, and lipopolysaccharides[J]. Journal of Animal Science, 2020, 98(8): skaa248.
Discussion
- Line 249 – in the sentence ”…In physiological processes such as inflammation…” should be: In pathological processes…
Response: Thank you for your correction. I have made corrections to the sentence. See page 9, lines 286.
- The following two sentences should be combined into one sentence:
(Lines 273-276)”…However, to our best knowledge, there are no studies between MMPs/TIMPs and uPA system in yak rumen fibrosis. Currently, our work is the first systematic study of MMPs/TIMPs and uPA systems in yak rumen fibrosis…”
Response: Thank you for your correction. To the best of our knowledge, our study represents the first comprehensive investigation of the association between MMPs/TIMPs and the uPA system in yak rumen fibrosis at the gene level in vitro cell experiments. See page 10, lines 320-323.
Author Response File: Author Response.pdf
Reviewer 3 Report
Authors present establishment of a Yak rumen fibroblast cell line and its response to LPS treatment. This work is well presented although a few typos/grammatical errors needs to be fixed throughout the manuscript. To be accepted the following concerns need to be addressed.
1. In the title "ECM responses to LPS" doesn't seem relevant. you are measuring the fibroblast response to LPS.
2. It is hard to visualize if all the cells express the SV40T vector and if not what percentage of cells are transfected? Are the same cells stained for vimentin? It would be nice to see all the staining on the same cells.
3. please indicate statistical values on the figures for easy comprehension.
4. in Figure 5, mRNA level of ColA is reduced less than half. How does this translate to the protein level and ultimately the functionality of the cells?
5. FN level goes down at 4 ug/ml concentration but goes back up after. Please explain the reason and how this is related to the functionality of the cells.
6. All the mRNA data shows either 2-fold increase here and there at different dosages of LPS but not a dose-dependent increase or decrease. How is this explained and how are these changes related to the protein level and function?
7. IL6 an dIL10 are increased in a dose-dependent manner but not the others. Same a s before, how does this affect the protein level and function.
8. Fig1c baht does the black arrow pointing at?
9. Fig3a and b larger images would be more informative.
10. are the TIMPs significantly reduced? Please indicate significance values clearly.
There are typos or grammatical errors throughout the manuscript. Please re-read.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors addressed the concerns raised in the original review. The manuscript can be published in the present form.
Author Response
Dear reviewer:
Thank you for reviewing my manuscript and giving comments in your precious time. Thank you very much for your feedback on the revised manuscript. We are very pleased with your approval of the manuscript.
Reviewer 3 Report
I pretty much accepted the revised version and I think it could be published as it is.
Author Response
Dear reviewer:
Thank you for reviewing my manuscript and giving comments in your precious time. Thank you very much for your feedback on the revised manuscript. We are very pleased with your approval of the manuscript.
