Neutrophils and Lymphocytes: Yin and Yang of Lung Fibrosis and Patient Outcome in Diffuse Interstitial Lung Diseases

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a well-written original research article dealing with the roles of neutrophils and lymphocytes in idiopathic pulmonary fibrosis (IPF) and other fibrotic interstitial lung diseases (f-ILD). The different alterations of the populations of these cells in the bronchoalveolar lavage in IPF and f-ILD and their correlations with disease severity and outcomes are well-described and indicate towards their potential use as biomarkers in clinical practice. The following minor comments could further improve the manuscript:

1. In the last two paragraphs, the authors mention, among other things, different limitations of the study. It would be more clear if the limitations were synopsised in a single paragraph along with the strengths of the study as well.

2. A separate last paragraph synopsising the findings of the study and their importance in the context of personalised medicine along with the authors' conclusions would be useful to be included.

3. Minor linguistic corrections are needed, e.g. "de" appears twice in the text instead of "the", abbreviations are not always explained the first time they appear in the text, etc.

Author Response

Comments #1 and #2 - We have tried to merge the limitations of the study in a single paragraph, to leave in a final paragraph the main conclusions and usefulness of the study. I hope I have caught the idea of your comments. Conclusion has been re-rewritten as suggested by the other reviewers.

Comment #3 - Sorry, sometimes mind and fingers are not synchronized. Thanks for finding out!

Reviewer 2 Report

Comments and Suggestions for Authors

In this retrospective study conducted on BAL cells from patients with ILD, the authors investigated the frequency of different cell populations and their role in the development of fibrosis in association with patient survival. The work is interesting and supports the application of early treatment to ensure the preservation of lung function and increase outcome.

However, I feel that the frequency of macrophage, lymphocyte and neutrophil populations alone is insufficient data to support the discussion. I am aware that as this is a retrospective study, however, I think it is important to also add some data from a smaller population, such as indicating the levels of cytokines in BAL fluid or BAL microbilogy data. In addition, to support the role of neutrophils it would be important to quantify the NETs in the BAL fluid, quantifying for example citrullinated histone H3. 

Furthermore, I think the methods should be implemented with a better description of the study population, in terms of the method of determining the presence of fibrosis and treatments.

 

Author Response

Comment #1 - I fully agree that having all that information would be very useful to draw firm conclusions and identify potential therapeutic targets that could be acted upon to improve the chances of these patients. Unfortunately, the parameters you request are not standard practice in our hospital and therefore they are not available. Neither are the physical samples that are discarded after flow cytometry analysis, especially considering that the evolutionary perspective has been achieved by having patients analyzed more than 20 years ago.

The retrospective nature of the study is clearly stated in the manuscript. We would like to inform you that this study was initially designed to see how flow cytometry was contributing to the accurate diagnosis of these patients, since clinicians throughout our region insistently requested this determination, apart from the cytopathological study. We will publish another manuscript very soon in which using logistic regression analysis we have been able to demonstrate that flow cytometry clearly helps in the differential diagnosis of many pathologies.

However, I fully agree that PROSPECTIVE studies investigating the molecular mechanisms involved in the deleterious effect of neutrophils, and more importantly, the beneficial role of lymphocytes are necessary.

In relation to the microbiological findings, inclusion and exclusion criteria have been added to the methods. Among exclusion criteria were pulmonary infections, these patients were categorized separately to ILDs. It is common for many BALs to be performed in patients with ILD in the setting of pulmonary infections that complicate the patient's clinical condition. These samples usually contain large amounts of neutrophils, so taking into account the objective of this work, these patients were excluded.

Regarding  smaller population, our flow cytometry analysis it is able to evaluate the levels of total lymphocytes, macrophages, neutrophils, CD117++ mastocytes and CD1a+ dendritic cells, in addition to CD4 and CD8 T lymphocytes, CD19+CD20+ B lymphocytes and CD56+CD3- NK cells. However, no clear association with fibrosis could be established for any of these subpopulations.

As you may know, the use of flow cytometry in the clinical management of ILD is limited in most cases to the analysis of the CD4/CD8 lymphocyte ratio in ILD with predominance of lymphocytes, and very few studies (with very limited series of patients) have extended the use of this methodology (highly accurate for quantifying leukocyte subpopulations) to the rest of the leukocyte subpopulations. The accuracy provided by flow cytometry in the quantification of these cells has shown that this tool should be introduced in the diagnosis of ILD patients as it can help in the clinical management of patients, who might benefit from a more personalized medicine. And that, you will agree with me, is a clear contribution of our work.

Comment #2 - We have improved the description of the patient populations and the description to determine the presence of fibrosis. Besides, we have detailed a bit better treatment description, although as described it was administered according to standard practice [34]

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript explores the relationship between immune cell counts (neutrophils and lymphocytes) and patient outcomes in IPF and non-IPF ILDs, offering valuable insights into disease progression and survival. Notably, the findings on corticosteroid treatment and its potential impact on fibrotic ILDs are significant. However, several key issues need to be addressed before publication.

Major Points:

1. The analysis of fibrotic patterns in ILDs was qualitative, lacking semi-quantitative assessment, which limits the robustness of the results. Additionally, Table 1 shows that most non-IPF ILDs did not exhibit fibrosis, raising questions about the definition of F-ILD. The statement "Pulmonary fibrosis was computed with the presence of radiological patterns associated with fibrosis (fibrosis, reticular and honeycomb)" is incorrect. Fibrosis should be described using reticular changes, traction bronchiectasis, and honeycombing.

2. The description of the control group in the Methods section needs clarification. Specify the criteria used to confirm the absence of pulmonary disease in the 112 BAL samples and the rationale for selecting MGUS patients as a survival control, including the exclusion of adverse prognostic factors.

3. The inclusion and exclusion criteria for study participants are missing in the Methods section. Clear criteria are essential for ensuring accurate participant selection and study consistency.

4. In Figure 3, the ROC analysis shows a low AUC for lymphocytes (0.423), indicating a weak association with overall survival. The conclusion that lymphocytes counteract the effects of neutrophils is overstated and should be more cautiously interpreted, considering the moderate predictive value.

5. The explanation of the combined effects of neutrophils and lymphocytes on F-ILD and IPF incidence is too lengthy and complex. A more concise presentation is needed. Additionally, the claim that lymphocytes counteract neutrophils' harmful effects in promoting IPF should be more cautiously phrased, considering the complexity of immune interactions.

6. BAL is not routinely recommended in the clinical management of IPF unless required to differentiate it from other conditions, such as chronic hypersensitivity pneumonitis, due to the risk of inducing acute exacerbation (AE). The study's application of BAL cell typing for antifibrotic therapy candidacy is limited in clinical practice, and this should be fully addressed.

7. In the Discussion, conclusions about the impact of neutrophils and lymphocytes on prognosis are overly definitive, especially regarding the antifibrotic role of lymphocytes. Cautious phrasing should be used, clearly indicating that further research is needed. Terms like "possibly" or "tends to" should be used when discussing BAL lymphocyte counts and survival in non-IPF fibrotic patients to avoid overstatement.

Minor Points:

1. The Methods section should include a detailed description of the BAL procedure for ILD patients and explain how OS was determined, including the follow-up period.

2. Clarify how information on corticosteroid and other immunosuppressant treatments was obtained for ILD patients in the Methods section.

3. The statement that neutrophil and lymphocyte counts "impact" patient overall survival in non-IPF ILD is overstated. It would be more accurate to describe them as "associated with" survival, reflecting a correlation rather than causation.

Author Response

Comment #1 - The study was focused on the lung cellular microenvironment regardless of the degree of fibrosis involvement. Moreover, semiquantitative information at the time of our study was very limited. Nevertheless, it is a very interesting question to be addressed in future studies once fibrosis quantification methods are extended to all centers.

We included all BAL samples received in our laboratory between 2000 and 2018 regardless of the type of pathology, phase or stage of the disease or the clinical condition of the patient. Samples were included according to the inclusion and exclusion criteria described in the new version of the manuscript. The fibrosis rate in the real-life patients in our series was as described in Table-1 and they are in the ranges described in the literature for these pathologies https://doi.org/10.1016/j.resinv.2024.05.005

We have corrected the definition of fibrosis, as suggested. Thanks.

Comment #2 - In the manuscript, we now describe “As control-BAL group, 112 BAL samples were included from patients with suspected lung disease (mainly due to the presence of micronodules on radiological images) that after years of follow-up did not show clinicopathological evidence of lung disease in the electronic medical record”. These data have been of GREAT utility, since there are almost NO reference values of leukocyte BAL subsets analyzed by flow cytometry.

Rationale of using MGUS patients was to demonstrate that ILDs with good prognosis (high lymphocytes and low neutrophils) have survival curves comparable to the general population, and therefore, they are patients in whom clinicians could consider more conservative treatments. The reason for using MGUS patients it is that it was the only series for what our group had long-term evolutionary data to compare patients with ILD. From the work published in [33] we were able to conclude that MGUS patients without cytogenetic alterations or circulating tumor plasma cells in peripheral blood, had a null risk of progressing to myeloma and therefore had survivals comparable to the general population. They were therefore useful to compare our ILD cases that also had a long follow-up.

We have modified the text to make these points clearer.

Comment #3 - We have added inclusion and exclusion criteria.

Comment #4 - Actually, the AUC value is 0.577, it was my mistake not realizing that being a protective factor the curve goes in the opposite direction and I took the “area over the curve” instead of the area under the curve or AUC. We have corrected this error in the figure.

However, neither AUC, for lymphocytes or neutrophils, is too high. And this is precisely because of the interaction that exists between these two cells, with one protecting and the other negatively affecting the patient's outcome. The low AUC is due to the fact that both cells are mixed in different proportions in bronchoalveolar lavages and it is difficult to analyze them separately, since a high number of lymphocytes can be associated with a low or high number of neutrophils and vice versa

Nevertheless, the dose dependent protective role of lymphocytes is clearly demonstrated in Figures 3B, 4 and 5. Even so, we have tried to moderate this statement, replacing “impact” by “association with”, etc. This is connected with the 3^rd minor points.

Comment #5 - An attempt has been made to address these issues by making our statements less categorical and being as concise as possible. I hope they are in line with what has been suggested.

Comment #6 - Although in our experience IPF exacerbations after BAL are extremely infrequent, it is true that they can occur in patients with IPF in whom antifibrotic treatment should be imperative from diagnosis. However, this analysis may have great relevance in F-ILD in which the initiation of antifibrotic treatment must await progression, and the N/L ratio could anticipate treatments in higher-risk patients. Nevertheless, we have rewritten the discussion and conclusion to moderate these statements.

Comment #7 - We have re-written the discussion and conclusion as suggested.

Minor point #1 - We have included in the method section that “BAL sampling procedure was performed following the official American thoracic society clinical practice guidelines [32]”.

The OS statement was already explained in the Statistic section “Overall survival (OS) was defined as the time from first BAL analysis to death, with living patients censored on the date of last follow-up.”

Minor point #2 - That information has been included in the methods as suggested.

Minor point #3 - Corrected as suggested.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I think the manuscript is acceptable for publication in the present form

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed all major and minor concerns, greatly improving the quality of the manuscript. The study is now well-prepared for publication and offers important insights into the relationship between immune cell counts and patient outcomes in ILDs. Therefore, I recommend the manuscript for acceptance.