Serum CCL18 May Reflect Multiorgan Involvement with Poor Outcome in Systemic Sclerosis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In their study, the authors successfully resolved a specific scientific problem: demonstrating that the level of serum chemokine CCL18 reflects the degree of multiple organ changes in systemic sclerosis (SSc). This work may be of interest to readers of the Journal Biomolecules. I have a few minor comments to make.

(1) Statistical analysis. Results. Typically, the minimum significant correlation coefficient is considered to be 0.4, not 0.2.  Apparently, the data distribution was abnormal, since the authors used the Mann-Whitney U-test and the Kruskal–Wallis criterion.The rationale behind the authors' decision to present the results in the article's text differently, specifically as the mean (M) and median (Me), remains unclear. In the note to each table and figure, please indicate the form of presentation of the stated results.

(2) Discussion. It should be noted that the involvement of CCL18 in the pathogenesis of SSc is ambiguous, since CCL18 is associated with type 2 immune response (Th2, M2, Treg). Therefore, CCL18 promotes fibrosis on the one hand (through activation of M2, etc.), but on the other hand limits the immune mechanisms of autoimmune inflammation (Th1, Th17, M1).

(3) References: The authors did not fully consider the MDPI style requirements. Please find below some examples of using the MDPI style:

Umadevi, S.; Boopathi, R.S. A paradigm shift in bioavailability enhancement using solid self emulisifying drug delivery system. J. Appl. Pharm. Res. 2025, 13, 14–24.

Mostafa, N.; Taha, I.E.; Abourobe, N.M.; Ashour, E.A. Development and Characterization of Cannabidiol Self-Emulsifying Drug Delivery System: In Vitro and In Vivo Evaluation. Biomolecules 2026, 16, 21.

Author Response

Reviewer’s comment: 

(1) Statistical analysis. Results. Typically, the minimum significant correlation coefficient is considered to be 0.4, not 0.2.  Apparently, the data distribution was abnormal, since the authors used the Mann-Whitney U-test and the Kruskal–Wallis criterion. The rationale behind the authors' decision to present the results in the article's text differently, specifically as the mean (M) and median (Me), remains unclear. In the note to each table and figure, please indicate the form of presentation of the stated results.

Authors’ comment:

We thank the reviewer for this comment. The minimum significant correlation coefficient has been corrected to 0.4 in the Statistical Analysis subsection of the Methods. We have now revised the Methods section to explicitly state that the distribution of each continuous variable was assessed using exploratory normality testing. Based on these assessments, descriptive data are presented either as mean ± standard deviation or as median (interquartile range), depending on the distribution of the given parameter. Importantly, due to heterogeneous distributions and the limited size of several subgroups, non-parametric statistical tests were applied consistently for all inferential analyses to ensure robustness. To improve transparency, we have clarified in the legends of all tables and figures the form in which data are presented (mean ± SD or median (IQR)). In addition, instances where mean values are reported have been clearly indicated. (lines 172-182)

 

Reviewer’s comment:

(2) Discussion. It should be noted that the involvement of CCL18 in the pathogenesis of SSc is ambiguous, since CCL18 is associated with type 2 immune response (Th2, M2, Treg). Therefore, CCL18 promotes fibrosis on the one hand (through activation of M2, etc.), but on the other hand limits the immune mechanisms of autoimmune inflammation (Th1, Th17, M1).

Authors’ comment:

Thank you for this comment. In the revised Discussion, we have added the following sentence to address this comment: “In SSc, increased seCCL18 may reflect a dual immunological role, with predominance of Th2 cells, M2 macrophages, and regulatory T cells promoting fibrotic processes, while reducing Th1-, Th17-, and M1-mediated inflammatory pathways, highlighting its ambiguous role.” (lines 379-382)

 

Reviewer’s comment:

(3) References: The authors did not fully consider the MDPI style requirements. Please find below some examples of using the MDPI style:

Umadevi, S.; Boopathi, R.S. A paradigm shift in bioavailability enhancement using solid self emulisifying drug delivery system. J. Appl. Pharm. Res. 2025, 13, 14–24.

Mostafa, N.; Taha, I.E.; Abourobe, N.M.; Ashour, E.A. Development and Characterization of Cannabidiol Self-Emulsifying Drug Delivery System: In Vitro and In Vivo Evaluation. Biomolecules 2026, 16, 21.

Authors’ comment:

We thank the reviewer for pointing this out. The reference list has been carefully revised and updated to fully comply with the MDPI formatting requirements, in accordance with the provided examples.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study analyse CCL 18 levels in a single-center cohort of patients with systemic sclerosis. This cross-sectional controle-base study is based on a single assessment of the study marker. The topic is quite new. The statistical analysis should be performed again.

Major

It is not clear whether increased CCL18 was calculated based on assessments in the study or controle group.

The presentation of statistical analysis results is very complicated. As the authors defined increased CCL 18, a natural consequence is to show this subgroup. This will simplify the analysis. The current presentation of the results is not acceptable, very difficult to follow, and highly descriptive. There is no need to present data in tables and than in the text. Please focus on the topic!

As the results section is expected to be changed and much shorten, more specific comments will be provided later.

lines 51-53 The sentence ' Despite recent advances, disease course and prognosis remain highly variable, and widely accepted and routinely usable biomarkers for organ involvement and disease progression are lacking.' is  not clear - what is widely accepted?

line 180 - please reformulate the sentence 'Female sex was more common in the lcSSc subgroup'

Table 1 - the common term is arterial hypertension.

Author Response

Reviewer’s comment:

It is not clear whether increased CCL18 was calculated based on assessments in the study or control group.

Authors’ comment:

We thank the reviewer for pointing this out. Elevated serum CCL18 was defined exclusively based on the healthy control group, using the mean + 2 standard deviations of serum CCL18 concentrations measured in controls (>130 ng/mL). This cut-off was then applied uniformly to the systemic sclerosis cohort to classify patients as having elevated or normal seCCL18 levels, as previously described using a control-based mean + 2SD approach (Hoffmann-Vold et al., Chest, 2016). To avoid any ambiguity, we have now clarified this definition explicitly in the Methods section and mentioned it at the beginning of the Results section as well where subgroup analyses based on elevated seCCL18 are introduced. (lines 153-155 and lines 209-210).

 

Reviewer’s comment:

The presentation of statistical analysis results is very complicated. As the authors defined increased CCL 18, a natural consequence is to show this subgroup. This will simplify the analysis. The current presentation of the results is not acceptable, very difficult to follow, and highly descriptive. There is no need to present data in tables and then in the text. Please focus on the topic!

Authors’ comment:

We thank the reviewer for this comment. In response, the Results section was substantially revised and shortened. Analyses were refocused on patients with elevated seCCL18, defined using a healthy control–based cut-off, and duplicated numerical descriptions were removed from the main text.

Because no clinically validated diagnostic cut-off currently exists for seCCL18 and absolute concentrations are assay-dependent, analyses using continuous seCCL18 values were retained in a clearly subordinate role to explore biological gradients across different disease manifestations. In addition, subtype-specific analyses (diffuse and limited cutaneous systemic sclerosis) were preserved where clinically relevant, as these represent distinct disease entities with markedly different clinical courses and prognostic profiles.

 

Reviewer’s comment:

lines 51-53 The sentence ' Despite recent advances, disease course and prognosis remain highly variable, and widely accepted and routinely usable biomarkers for organ involvement and disease progression are lacking.' is not clear - what is widely accepted?

Authors’ comment:

Thank you for this comment. The sentence has been revised for clarity and replaced with the following formulation in the manuscript: “Despite recent advances in the understanding of disease pathogenesis, reliable biomarkers for routine disease monitoring and clinical management remain limited.” (lines 52-54)

 

Reviewer’s comment:

line 180 - please reformulate the sentence 'Female sex was more common in the lcSSc subgroup'

Authors’ comment:

We thank the reviewer for this suggestion. The sentence has been revised accordingly and now reads: “Female patients were more prevalent in the lcSSc subgroup... (lines 188-189)

 

Reviewer’s comment:

Table 1 - the common term is arterial hypertension.

Authors’ comment:

Thank you for the comment. The term has been changed to arterial hypertension.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The paper certainly looks better. Still, some improvement is possible.  

The most important thing is to edit the conclusion. Tone down.  These findings support the use of seCCL18 ??- perhaps these findings show some clinical usefulness..... However, the lack of an established cut-off point requires further studies validating its usefulness. Please think about it.

In addition, please discuss the potential benefits of CCL18 assessment in clinical practice. 

Author Response

Reviewer’s comment:

The paper certainly looks better. Still, some improvement is possible.  

The most important thing is to edit the conclusion. Tone down.  These findings support the use of seCCL18 ??- perhaps these findings show some clinical usefulness..... However, the lack of an established cut-off point requires further studies validating its usefulness. Please think about it.

Authors’ comment:

We thank the reviewer for this valuable suggestion. In response, we have revised the Conclusions (Abstract, Main text) to adopt a more cautious tone. Statements implying direct clinical use were rephrased to indicate some clinical utility rather than established applicability. In addition, we explicitly acknowledged the absence of a validated cut-off value and emphasised the need for prospective, multicenter studies to confirm the clinical usefulness of seCCL18. Corresponding modifications were made in both the Abstract, Discussion and the Conclusions.

(lines 38-42, 390-392, 460-465, 482-486)

Reviewer’s comment:

In addition, please discuss the potential benefits of CCL18 assessment in clinical practice. 

Authors’ comment:

We thank the reviewer for this important comment. In response, we have expanded the Discussion to explicitly address the potential benefits of seCCL18 assessment in clinical practice, emphasising its possible role as a complementary biomarker for disease burden, prognosis, and identification of patients who may benefit from closer cardiopulmonary monitoring. In addition, the Conclusions were revised to highlight its potential contribution to risk stratification and clinical assessment, while clearly acknowledging that further validation is required before routine clinical use.

(lines 468-471, 479-486)

Author Response File: Author Response.pdf