COVID-19: Immunohistochemical Analysis of TGF-β Signaling Pathways in Pulmonary Fibrosis

Round 1

Reviewer 1 Report

The retrospective study by Paula et al examined the histopathological features and immunohistochemical biomarkers involved in TGF-β 1 signaling pathway and pulmonary fibrosis. They examined tissue specimens from control and covid 19 patients. They reported that alveolar damage was significantly increased in Covid group. Further they observed that a significant increase in the immunoexpression of tissue biomarkers in Covid group. Moreover, Caveolin was significantly decreased in Covid group. The authors conclude that TGF-β1 pathway is involved in the development of pulmonary fibrosis. They further suggest to use TGF-β inhibitors in the patients who recovered from Covid to mitigate the development of pulmonary fibrosis and its effects post recovery.

The manuscript is well written and easy to follow. The schematics are drawn well which facilitates easy reading. There are a few typos which needs to be corrected, page 4, line 104 etc

The authors should clarify why MMP9 was chosen for histology. Although it is well known that other MMPs like 1, 7 are also involved in lung fibrosis.

Do authors have histology of collagen markers to confirm the presence of collagen deposition?

Line 157 page 7. Collagen I was decreased in Covid group compared to control. But text states it the opposite. The authors must correct it. 

Table 2 indicates that Collagen I was decreased and Collagen III was increased in Covid patients compared to control. Why tow collagens exhibited different effects? These results needs to be discussed.

Line 226 4B? Please correct labelling

Current study does not provide data to show that corticosteroids had any impact on lung fibrosis. 

Since sample size is less, the data should be interpreted cautiously for role of TGF-β signaling in Covid 19 patients. Only one marker was used to show the presence of fibrosis, which is very preliminary.

 

Author Response

 

We thank the Reviewers and the Editors for the valuable suggestions and the opportunity to improve our manuscript (Title: COVID-19: TGF-β signaling pathways in pulmonary fibrosis).

 

Question 1

The manuscript is well written and easy to follow. The schematics are drawn well which facilitates easy reading. There are a few typos which needs to be corrected, page 4, line 104 etc.

Answer: We appreciate the positive feedback regarding the writing and schematics performed. Suggested corrections were made (line 112):

“The tissue immunoexpression of ACE-2, AKT-1, Cav-1, CD44v6, COX-2, IL-4, MMP-9, SMA, Sphingosine-1 and TGF-β1 in the COVID-19, H1N1 and CONTROL groups are shown in Figure 1, 2 and 3.”

 

Question 2

The authors should clarify why MMP9 was chosen for histology. Although it is well known that other MMPs like 1, 7 are also involved in lung fibrosis.

Answer: Thanks. We agree with the reviewer that MMPs 1 and 7 are also involved in the process of pulmonary fibrosis. The choice of MMP-9 was due to our laboratory expertise with this protein in previous works related to this theme. Below, some links to previously published scientific articles demonstrating the role of MMP-9 in the tissue remodeling process.

• DOI: 10.3389/fimmu.2021.748417
• DOI: 10.1111/jop.12213

 

Question 3

 Do authors have histology of collagen markers to confirm the presence of collagen deposition?

Answer: The presence of type I (mature) and III (immature) Collagen in the current study was demonstrated by Sirius Red histological/histochemical staining. This trichrome method allows quantifying and qualifying type I (mature Collagen in red) and type III (immature Collagen in green) collagens, employing polarized light. Due to the difference in color, intensity and birefringence of stained tissues. Our laboratory has expertise with this proven technique, as observed in some previously published works:

• DOI: 3389/fimmu.2021.685919
• DOI: 10.1590/S0102-865020160050000005

The authors also clarified this point in the methods section as follow (line 331-338):

“Histological sections were stained with Sirius Red (Direct Red: Aldrich Chemical Company Inc, Milwaukee, WI) to evaluate Collagen I (mature) and III (immature) depositions. The slides were photographed at a magnification of 400x (high power field or HPF) under polarized light resulting in 20 images for every case. The Collagen I to III evaluation was performed using Image-Pro Plus 4 version 4.5 (Media Cybernetics, Rockville, MD), where polarized areas (in red for mature Collagen I or in green for immature Collagen III) were identified. The values of the Collagen I (mature) and III (immature) analysis were expressed percentage per HPF.”

Please see the attachment.

 

Question 4

Line 157 page 7. Collagen I was decreased in Covid group compared to control. But text states it the opposite. The authors must correct it.

Answer: Correct. The results were double-checked and corrected as shown below (underlined in yellow, line 154-171):

“The results observed in the COVID-19 patients indicate high levels of Collagen III (immature) compared to the CONTROL group (Table 2). This precursor found in the alveolar compartment is associated with an unfavorable condition and a higher risk of death [25]. Collagen I (mature) was significantly decreased in the COVID-19 group compared to the others (Table 2). Collagen I (mature) is the main structural protein in the pulmonary septal interstitium [26]. It is found in large quantities during chronic pulmonary fibrosis, promoting the destruction of septal architecture and consequent impairment of gas exchange. In addition, the presence of Collagen I (mature) seems to be directly related to the mechanical/biochemical signaling of the actin-myosin contractile system [27]. Type III Collagen is considered an immature supporting fiber that precedes a type I Collagen (mature) polymerization. In the tissue remodeling process, under conditions involving inflammatory events, the ECM turnover results in quantitative and qualitative changes of collagens. Therefore, damaged mature molecules (type I mature Collagen) are degraded and replaced by new ones (type III immature Collagen) to re-establish the homeostasis process [28]. However, considering an increased percentage of type III Collagen (immature) was found in the COVID-19 group, its complementary percentage of type I Collagen (mature) would be reduced.”

The authors also added “mature” and “immature” terms following the types of the Collagens (I and III respectively) to clarify the results, discussion and methods sections.

 

Question 5

Table 2 indicates that Collagen I was decreased, and Collagen III was increased in Covid patients compared to control. Why two collagens exhibited different effects? These results need to be discussed.

Answer: Thanks. We tweaked the thread questioning as requested below highlighted yellow (Lines 146-153; 154-171).

“SARS-CoV-2 migrates to the lower respiratory tract and mainly affects type II pneumocytes [6], followed by the secretion of cytokines [20]. The inflammation process is followed by the alveolar edema and hyaline membranes over the damaged alveolar septa [21]. At the end of this process, septal terminal fibrosis appears, characterized by exacerbated proliferation of fibroblasts and excessive deposition of ECM [21,22]. Collagens I (mature) and III (immature) are the primary components of ECM [23]. Collagen III (immature) seems to predominate at the beginning of the fibrotic process, followed by de Collagen I (mature) [24].

The results observed in the COVID-19 patients indicate high levels of Collagen III (immature) compared to the CONTROL group (Table 2). This precursor found in the alveolar compartment is associated with an unfavorable condition and a higher risk of death [25]. Collagen I (mature) was significantly decreased in the COVID-19 group compared to the others (Table 2). Collagen I (mature) is the main structural protein in the pulmonary septal interstitium [26]. It is found in large quantities during chronic pulmonary fibrosis, promoting the destruction of septal architecture and consequent impairment of gas exchange. In addition, the presence of Collagen I (mature) seems to be directly related to the mechanical/biochemical signaling of the actin-myosin contractile system [27].

Type III Collagen is considered an immature supporting fiber that precedes a type I Collagen (mature) polymerization. In the tissue remodeling process, under conditions involving inflammatory events, the ECM turnover results in quantitative and qualitative changes of collagens. Therefore, damaged mature molecules (type I mature Collagen) are degraded and replaced by new ones (type III immature Collagen) to re-establish the homeostasis process [28]. However, considering an increased percentage of type III Collagen (immature) was found in the COVID-19 group, its complementary percentage of type I Collagen (mature) would be reduced.”

 

Question 6

Line 226 4B? Please correct labelling.

Answer: Thank you for the note. The corrected subtitle follows:

"TGF-β Signaling in Pulmonary Fibrosis" (Line 229).

 

Question 7

 The current study does not provide data to show that corticosteroids had any impact on lung fibrosis.

Answer: As indicated, the data are now presented in Table 3 (Line 107-110).

"Table 3. Relation of Corticosteroid use and PF development".

 

Question 7

Since sample size is less, the data should be interpreted cautiously for role of TGF-β signaling in Covid 19 patients. Only one marker was used to show the presence of fibrosis, which is very preliminary.

Answer: As these were post-mortem tissue samples from positive for COVID-19 patients hospitalized in the Intensive Care Unit, biopsies were performed only with family consent and in particular conditions, which mainly included the safety of the health professionals involved in this process. Thus, the number of samples was limited. However, we add this item to the “Limitations of Study” (Line 300-301). Below is the text of the article referring to the sampling:

“Once the number of samples is small, the results inferred in this study are considered preliminary, suggesting that future studies with larger samples be carried out.”

Regarding the TGF-β marker as the only marker to verify the presence of fibrosis, we emphasize that the other tissue markers present in the study support the presence of fibrosis in the alveolar environment. Furthermore, we performed a histopathological analysis, demonstrating lesions caused by COVID-19, which precede pulmonary fibrosis, as well as Sirius Red staining, qualifying and quantifying the presence of immature Collagen III and mature Collagen I.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript by Caroline Busatta Vaz de Paula et al is well written. However, there are some critical points which must be improved berore the acceptance. 

1. The time of biopsy after death should be specified in methods.  
2. Since authors performed just immunohistochemical studies, it should be reflected in the title. The title of the manuscript sounds more like a review type of paper. 
3. The lack of other molecular studies is a missing opportunity. Western blot analysis or PCR of biopsy tissue samples could be done. Histological densitometric data is not so accurare.
4. The graphic panels are too small and should be increased. 

Author Response

We thank the Reviewers and the Editors for the valuable suggestions and the opportunity to improve our manuscript (Title: COVID-19: TGF-β signaling pathways in pulmonary fibrosis).

Question 1

The time of biopsy after death should be specified in methods.

Answer: The acquisition time of samples as specified in the Methods section, Line 313:

“The time of acquisition after death is less than 2 hours.”

Question 2

 Since authors performed just immunohistochemical studies, it should be reflected in the title. The title of the manuscript sounds more like a review type of paper.

Answer: Thanks. As suggested, we emphasize in the title that the study was performed with immunohistochemistry (Line 2-3) as follow:

“COVID-19: immunohistochemical analysis of TGF-β signaling pathways in pulmonary fibrosis”

 

Question 3

The lack of other molecular studies is a missing opportunity. Western blot analysis or PCR of biopsy tissue samples could be done. Histological densitometric data is not so accurare.

Answer: We agree that both techniques can be evaluated in tissue samples. However, as presented in the study, the tests were performed in formalin-fixed paraffin-embedded samples (FFPE). FFPE samples are still a significant challenge in studies involving techniques that require RNA extraction or protein assays, such as gene expression and WB studies, due to the degradation of RNA and proteins during this processing. For this reason, we chose the immunohistochemistry technique that is most suitable for FFPE samples.

DOI: 10.1038/s41598-020-75659-5

DOI: 10.1161/ATVBAHA.120.314860

 

Question 3

The graphic panels are too small and should be increased.

Answer: Thanks. Figures 1, 2 and 3 were resized (Lines 114-122).

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors tried to address all the concerns. I have only minor comment.

Line 300: Once should be replaced with Since.

Reviewer 2 Report

The manuscript was significantly improved and could be accepted in present form