Soluble CD163 Levels Correlate with EDSS in Female Patients with Relapsing–Remitting Multiple Sclerosis Undergoing Teriflunomide Treatment

Response to Reviewer

Reviewer #1 Comments

Thank you very much for taking the time to review this manuscript and for valuable comments. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in yellow in the re-submitted files.

Please note that we addressed all your suggestions and comments in the updated manuscript.

Response to Reviewer #1 Comments

Thank you very much for taking the time to review this manuscript and for valuable comments. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in yellow in the re-submitted files.

Please note that we addressed all your suggestions and comments in the updated manuscript.

Comments 1: The introduction is quite lengthy. Please focus on the study context and omit information such as side effects of Teriflunomide.

Response 1: Thank you for the comment. We have removed this sentence: “Areas of low incidence are found around the equator, and the prevalence in Asia is less than 30 cases per 100,000 population. Genetic and environmental factors play a role in the development of MS. Other symptoms are less common such as personality changes, deafness blindness, and dementia. The most common side effects are: headache, diarrhea, nausea, and elevated Alanine aminotransferase values [20].”

Comments 2: The last two paragraph of the introduction describe the same thing in different words. Please merge them in one paragraph, eliminating redundancy.

Response 2: Thank you for the comment. We merge two paragraphs into one as follows:

“The present study objective is to investigate sCD163 protein plasma concentration in teriflunomide-treated RRMS patients. Additionally, this Research seeks to explore the potential correlation between sCD163 levels and the EDSS score and evaluate its utility as a biomarker for monitoring disease activity and response to teriflunomide therapy.”

Comments 3: The authors should describe the full inclusion and exclusion criteria.

Response 3: Thank you for this valuable comment. “Inclusion criteria for the study required participants to be at least 18 years old and to have received a diagnosis of relapsing–remitting multiple sclerosis at least one year prior to study enrollment (initial recruitment occurred in 2022). Participants were also required to have shown no signs of clinical or neuroradiological disease activity for a minimum of three months before assessment. In addition, individuals must have been on a consistent regimen of the neuromodulatory medication teriflunomide for at least 12 months and had not undergone any rehabilitation in the three months preceding the start of the study. Exclusion criteria included any comorbid condition that impaired walking ability, a history of neurological disorders other than RRMS, psychiatric illness, substance or alcohol abuse, recent traumatic brain injury or brain surgery, or previous stroke.”

Comments 4: It is not clear if all patients received teriflunomide for exactly 12 months prior to the study or at least 12 months. If the latter is the case, please provide the details of the treatment duration.

Response 4: Thank you for this comment. We add in Materials and Methods the following:

“Individuals must have been on a consistent regimen of the neuromodulatory medication teriflunomide for at least 12 months and must not have undergone any rehabilitation in the three months preceding the start of the study.”

Comments 5: “Table 1 presents the basic characteristics of subjects with RRMS and healthy controls (HC) included in this study.” This is completely incorrect. Table 1 shows only EDSS for MS cases. Also, are data shown in Table 1 from the previous study? How are they related to the current study in relation to teriflunomide treatment?

Response 5: Thank you for pointing this out. We add: “Table 1 presents Descriptive parameters of disease indicators included in this study.“ Table 1 is not from a previous study, however, subclinical neurophysiological findings, refer to an assessment of the functional integrity of the corticospinal tract by recording motor evoked potentials (MEPs), are from an earlier study.

Comments 6: There is no information on HCs.

Response 6: Thank you for the comment. At the beginning of the section Results, we add:

The final study included twenty-three RRMS subjects with a mean age of 41.65 “± 8.89” and ten HCs with a mean age of 37 ± “13.9. HCs included were (60%) women”. Most people with MS were women (60.87%), and had a high school education (73.9%). There were 9 men (39.13%), therefore, the ratio of women to men was 1.56:1.

Comments 7: Line 117: “the median EDSS score was 2.5 (3.0).” What does this mean? What is the median value?

Response 7: Thank you for the comment. The result is updated as follows:

“The mean disease duration was 9.39 ± 5.73 years, which had a mild EDSS score. The median (Q1– Q3) EDSS (general score) was 2.5 (0-3.5).”

Comments 8: There is no information on statistical methodology

Response 8: Thank you for the comment. The result is updated as follows: Statistical Analysis: „Jamovi 2.3 software was used for data analysis. One-way analysis of variance and independent group t-test were used to test for differences. Pearson's correlation coefficient was used to calculate correlations. Significance levels were set at P<0.05 and P<0.01, respectively.“

In addition, we expanded the main content of the manuscript to more than 3500+ words by introducing updated references in Discussion:

 “In the CSF, sCD163 is released by microglia in all white matter lesion. Normal-appearing white matter (NAWM) of MS brain contains high content of CD163 positive microglia in comparison to normal-appearing cortical grey matter (NAGM). (Elkjaer, M.L.) Due to CD163 involvement in iron homeostasis, as soluble scavenger receptor for haptoglobin–hemoglobin complexe, Hofmann et al. performed method of iron-sensitive magnetic resonance imaging of MS brains thereby detecting correlation of sCD163 in CSF with brain paramagnetic rim lesion counts (Hofmann, A.). Recently, Maliozzi et al. described correlation of choroid plexus inflammation and frequency of CD163⁺ innate immune cells. (Magliozzi, R.; 2024)

Treatment naive multiple sclerosis patients show elevated sCD163 in CSF due to changes of T and B cell signaling (Magliozzi, R.; 2021),  Comparison of CD163⁺ microglia expression in MS brain lesions and MS remyelinating donor brains (at autopsy) has not found difference between them inspite expected increased regenerative (CD163⁺) microglia in MS remyelinating group without lesions (Chen, J.Q.A.).

Response to Reviewer #2 Comments

Thank you very much for taking the time to review this manuscript and for valuable comments. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in yellow in the re-submitted files.

Please note that we addressed all your suggestions and comments in the updated manuscript.

Comment 1: The rationale and novelty of the study are not clearly emphasized in the introduction. A brief paragraph highlighting the gap in current evidence on sex differences in immune biomarker profiles in RRMS would strengthen the background.

Why did the authors decide to study the correlations between sCD163 and EDSS? Any clinical evidence and any clinical application based on the findings

Response 1: Thank you for this valuable comment. The introduction is updated as follows:

“Multiple sclerosis is an autoimmune disease, which develops in genetically susceptible individuals exposed to environmental factors (Ramagopalan, S.V.); Women are more affected than men, with notable differences in number of relapses and disability accumulation among them (Ribbons KA). It is predominantely Th1 mediated disease (O’connor KC), and in later stages more Th17 mediated (Moser T). Although many studies showed differences in concentrations of biomarkers between healthy controls and people with RRMS, or relapsed and remission, there is scarce information regarding differences among males and females.

Sex hormones and sex-linked genetic inheritances may contribute to the increased susceptibility to certain autoimmune diseases. This is supported by observations that fluctuations in sex hormone levels, such as those occurring during pregnancy or menopause, or through the use of exogenous hormones like Hormone replacement therapy

 are often associated with changes in disease symptoms.” (Houtchens M. K.)

The discussion is updated as follows:

“Males have typically later onset than females in MS. The onset in males, ages 30-40, typically coincides with the onset of testosterone decline. (Gray, A.)

This could suggest a protective effect of physiologically high testosterone levels.

Receptors for sex hormones are expressed on various immune cells, including CD4⁺ and CD8⁺ T cells, monocytes, and macrophages. When sex hormones are added to in-vitro immune cells, they alter production of cytokines. Th1 lymphocyte responses are characterised by production of IFN-Y while, Th2 type by the production of IL-13, IL-5, IL-4, and IL-10.” (Voskuhl R. R.)

“Nguyen et al. showed a higher percentage of CD3+ T-cells producing TNF-α in men compared to women. The percentage of cells producing IFN- γ and IL-2 showed no differences.

There are no differences between genders in intracellular levels of IL-10. Furthermore, they showed a correlation between EDSS and IFN-γ in females but not in males and between EDSS and TNF-α also in females but not in males.” (Nguyen, L. T.)

“Female predominance may stem from distinct immune responses and hormone differences.” (Eikelenboom, M. J.)

We decided to study the correlations between soluble CD163 and the EDSS because sCD163 is released from macrophage/ microglia that play a crucial role in regulating inflammation, adapting their behavior in response to signals from their surroundings. Zhang et al. shown that in active multiple sclerosis lesions, both pro-inflammatory and anti-inflammatory or tissue-repairing macrophages are present, indicating their involvement in different phases of the disease process. (Zhang Z, et al. Parenchymal accumulation of CD163+ macrophages/microglia in multiple sclerosis brains. J Neuroimmunol 237: 73–79.)

Clinical evidence: Stilund et al. found correlations between baseline CSF sCD163 levels and disability progression measured by EDSS, with negative correlations indicating that higher sCD163 levels might be associated with less disability progression (r = −0.33, p = 0.01). (Stilund M, Gjelstrup MC, Christensen T, Møller HJ, Petersen T. A multi-biomarker follow-up study of patients with multiple sclerosis. Brain Behav. 2016 Jul 11;6(9):e00509. doi: 10.1002/brb3.509. PMID: 27688939; PMCID: PMC5036432.).

In other studies, the cerebrospinal fluid (CSF) to serum ratio of soluble CD163 (sCD163) was notably higher in individuals with multiple sclerosis, particularly in those with primary progressive MS (PPMS). This elevation points to localized activation of macrophages within the central nervous system.

Clinical application: The sCD163 CSF/serum ratio might serve as a biomarker reflecting macrophage activation in MS lesions, particularly in PPMS, where inflammation is less overt but still present. The combination of sCD163 with other established biomarkers may improve diagnostic accuracy and disease activity monitoring, but further research is needed to validate its role as a longitudinal marker for treatment efficacy or disability progression. Correlations with clinical disability and EDSS are inconsistent, CD163 might help predict time to disease activity, assisting in making early, proactive decisions. (Stilund M, Gjelstrup MC, Christensen T, Møller HJ, Petersen T. A multi-biomarker follow-up study of patients with multiple sclerosis. Brain Behav. 2016 Jul 11;6(9):e00509. doi: 10.1002/brb3.509. PMID: 27688939; PMCID: PMC5036432.)

Comment 2: The small sample size (n=23 RRMS patients) limits the generalizability of the findings, particularly the statistically significant correlation observed only in female patients. This limitation is acknowledged but would benefit from a more nuanced discussion regarding its impact on subgroup analysis reliability and potential for type I error.

Response 2: Thank you for the comment. Although effect size is large according to Cohen’s benchmarks (1988), it is needed to emphasize that due to a small sample size, the observed effect should be examined in futures studies that would benefit from larger sample size. It can be argued that the observed difference in our study might have a practical value, but further examinations are needed. Since only 23 subjects were able to participate and comparison with existing studies in which the number of subjects varied from 20-487, post hoc test power was calculated. The analysis showed a test power of 48-76%, with a cut-off value of 5% and an effect size (Cohen d) of 0.5

Comment 3: The observed correlation between sCD163 and EDSS in women is intriguing but requires more cautious interpretation. Given the cross-sectional nature of the study, it is not possible to infer a temporal or causal relationship. Please discuss this limitation more clearly and suggest prospective or longitudinal follow-up as a necessary next step. Moreover, what can be the reason that the results differ between males and females? It needs to be clinically and basically discussed.

Response 3: Thank you for pointing this out. Last sentences in our manuscript: “The recommendations for further studies are larger number of samples (especially females) and monitoring of subjects for a longer period.”

The Discussion is updated as follows:

“Biological variability could affect how sCD163 reflects disease activity or progression in each sex. The observation that gender-related differences in inflammatory disease activity diminished after individuals reached 50 years of age suggests that sex hormones significantly influence the progression of the condition, especially before menopause (Magyari M et al.). In contrast, disparities in the neurodegenerative aspects of MS began to emerge after age 45 and became more pronounced with advancing age.”

“Additionally, in their previous study men tended to reach EDSS milestones more quickly than women, had a higher mortality than the women (Koch-Henriksen N,) had fewer lesions than women, but a tendency to have a higher proportion of lesions that evolved into black holes, and exhibited greater grey matter loss compared to their female counterparts (Pozzilli C). The possible reason for lower female sCD163 in our study is that 17b-estradiol endogenous estrogens produced in females, exerts mainly anti-inflammatory effects by inhibiting pro-inflammatory cytokines, such as IL-6, IL-1, and TNF-α.” (Straub RH.)

Comment 4: The study claims no difference in sCD163 levels between MS and healthy controls, which contrasts with findings from some previous studies. The discussion would be enhanced by critically evaluating the methodological differences—such as ELISA kit sensitivity, disease duration, or exclusion criteria—that may explain these discrepancies.

Response 4: Thank you for the comment. The Discussion is updated as follows:

While we have not detected significant sCD163 elevation, Farrokhi et al. [37] and Mona et al. [38] found significantly higher sCD163 serum levels in RRMS subjects compared to HCs. “In this study, the sensitivity of the ELISA kit (30 pg/ml) was higher than that in Farrokhi et al. (1.56 ng/Ml) and Mona et al. (0.94 ng/mL), however, we didn’t have age and sex-matched patients. In a study conducted by Farrokhi et al. [37], there were twice as many men as women compared to our study, where the ratio of women to men was 1.56:1, the average duration of the disease was about 5 years shorter, and patients were excluded if they relapsed”. In a study conducted by Mona et al. ratio of women to men was 2.5:1, with an average age about 13 years shorter than ours; that biological variability could affect higher sCD163.

Comment 5: The influence of prior corticosteroid treatments on sCD163 levels is not thoroughly examined. Considering that over half of the MS participants received corticosteroids multiple times, it is important to comment on how this may have affected the inflammatory profile and interpretation of sCD163 as a biomarker

Response 5:  Thank you for the comment. Corticosteroids were associated with a significant increase in leukocytes (primarily classical monocytes), defined by increased counts of granulocytes and monocytes after the first corticosteroid infusion. However, concerning the impact of topic of corticosteroid impact on sCD163 levels in CSF or blood in multiple sclerosis, available immunophenotyping data suggest that immune cell profiles return to baseline within weeks after corticosteroid treatment. (Höpner L, Proschmann U, Inojosa H, Ziemssen T, Akgün K. Corticosteroid-depending effects on peripheral immune cell subsets vary according to disease modifying strategies in multiple sclerosis. Front Immunol. 2024 Jun 13;15:1404316. doi: 10.3389/fimmu.2024.1404316. PMID: 38938576; PMCID: PMC11208457.)

Our patients took teriflunomide for a minimum of 12 months and had not undergone any rehabilitation in the three months preceding the start of the study.

Comment 6: The statistical reporting lacks detailed effect size metrics (e.g., Cohen’s d or confidence intervals for correlation coefficients), which are essential for understanding the practical significance of the findings. Adding these values would improve the rigor and transparency of the results section.

Response 6: Thank you for the comment. The Results is updated as follows: “Since only 23 subjects were able to participate and comparison with existing studies in which the number of subjects varied from 20-487, post hoc test power was calculated. The analysis showed a test power of 48-76%, with a cut-off value of 5% and an effect size (Cohen d) of 0.5”, d=0.97 CI95% (0.087, 1.854)

Comments 7: The methods section refers to subclinical neurophysiological findings based on transcranial magnetic stimulation, but this variable is only briefly mentioned in the results. Consider either expanding the explanation of how these findings relate to sCD163 or streamlining this component if it is not central to the main outcome.

Response 7: Thank you for the comment. The Results is updated as follows:

“2.4. Transcranial magnetic stimulation (TMS) procedure in assessment of subclinical motor status

Subclinical neurophysiologic assessment refers to the evaluation of the functional integrity of the motor pathway (corticospinal tract) by recording motor evoked potentials (MEPs) from muscles of the upper and lower extremities while stimulating primary motor cortex by navigated transcranial magnetic stimulation (TMS) [35].  Navigated transcranial magnetic stimulator (TMS) (Nexstim NBS System 4 of the manufacturer Nexstim Plc., Helsinki, Finland) is used for mapping primary motor cortex for the representation of upper and lower extremity muscles using an individual subject's head MRI (3D optical tracking unit, Polaris®Vicra) [35].  The MEP findings obtained from our previously published work [35] were used to classify RRMS subjects as ASNF and non-ASNF in this work. RRMS subjects with ASNF had prolongation in MEP latency or an absent MEP response, while no alterations were detected in eliciting MEP response or in MEP latency findings in target extremity muscles in non-ASNF group.”

In addition, we expanded the main content of the manuscript to more than 3500+ words by introducing updated references in Discussion:

“In the CSF, sCD163 is released by microglia in all white matter lesion. Normal-appearing white matter (NAWM) of MS brain contains high content of CD163 positive microglia in comparison to normal-appearing cortical grey matter (NAGM). (Elkjaer, M.L.) Due to CD163 involvement in iron homeostasis, as soluble scavenger receptor for haptoglobin–hemoglobin complexe, Hofmann et al. performed method of iron-sensitive magnetic resonance imaging of MS brains thereby detecting correlation of sCD163 in CSF with brain paramagnetic rim lesion counts (Hofmann, A.). Recently, Maliozzi et al. described correlation of choroid plexus inflammation and frequency of CD163⁺ innate immune cells. (Magliozzi, R.; 2024)

Treatment naive multiple sclerosis patients show elevated sCD163 in CSF due to changes of T and B cell signaling (Magliozzi, R.; 2021),  Comparison of CD163⁺ microglia expression in MS brain lesions and MS remyelinating donor brains (at autopsy) has not found difference between them inspite expected increased regenerative (CD163⁺) microglia in MS remyelinating group without lesions (Chen, J.Q.A.).

Variable

Group

M

SD

Median

Min

Max

IQR

Range

F

df

P

EDSS

ASNF

2,57

1,47

3,50

0

4,00

1,50

4,00

non-ASNF

1,06

1,02

1,00

0

2,50

2,00

2,50

sCD163

ASNF

834,57

335,29

789,00

298

1322

510,00

1024

(ng/ml)

non-ASNF

720,31

156,98

699,75

486

978

137,50

492

2,08

2/28

0,143

HC

591,75

225,10

526,50

297

902,5

392,13

605,5

Variable

sCD163 (ng/ml)

SD

t

df

P

Mm

947

227

2.22*

21

0.038

Mf

697

285

Variable

EDSS

correlation

Median

(Q1–Q3)

r

p

Mm

2.5

(0.5-3.5)

0.33

0.382

Mf

2.25

(0-3.5)

0.94

0.021

Variable

Group

M

SD

Median

Min

Max

IQR

Range

F

df

P

EDSS

ASNF

2,57

1,47

3,50

0

4,00

1,50

4,00

non-ASNF

1,06

1,02

1,00

0

2,50

2,00

2,50

sCD163

ASNF

834,57

335,29

789,00

298

1322

510,00

1024

(ng/ml)

non-ASNF

720,31

156,98

699,75

486

978

137,50

492

2,08

0,143

HC

591,75

225,10

526,50

297

902,5

392,13

605,5

Variable

sCD163 (ng/ml)

SD

t

df

P

Mm

947

227

2.22*

21

0.038

Mf

697

285

Variable

EDSS

correlation

Median

(Q1–Q3)

r

p

Mm

2.5

(0.5-3.5)

0.33

0.382

Mf

2.25

(0-3.5)

0.94

0.021