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

Abstract

Background: multiple sclerosis (MS) presentation varies depending on the location and severity of the lesions affecting different areas of the spinal cord and brain. Extensive research has focused on specific systems to detect the disease in its various stages. The objective of this study was to investigate the concentration of the soluble scavenger receptor for haptoglobin–haemoglobin complex (Hb-Hp), sCD163, which is mostly expressed by monocytes and protects tissues from oxidative damage, in patients with MS. Methods: enzyme-Linked Immunosorbent Assay (ELISA) analysis was conducted in plasma samples collected from twenty-three relapsing–remitting MS (RRMS) subjects treated with teriflunomide and ten healthy control subjects (HCs). Results: the study results showed no differences between RRMS subjects and HCs in the concentration of CD163. A significantly higher concentration of sCD163 in RRMS was found in men in comparison to women (p = 0.038, Cohen d = 0.97). Conclusions: a significant correlation between disease activity, estimated using plasma-soluble CD163 (sCD163) and clinical assessment of the Expanded Disability Status Scale (EDSS) (p = 0.021), was detected in female patients with RRMS.

1. Introduction

Multiple sclerosis (MS) is the most common inflammatory neurological disease in young adults, and its prevalence is increasing [1]. Currently, its prevalence ranges from 50 to 300 cases per 100,000 individuals. There are approximately 2.3 million people with MS worldwide today [2]. In the relapsing–remitting form of MS (RRMS), women are affected nearly three times more often than men, and the average age of onset is around 30 years [3,4,5], with notable differences in the number of relapses and disability accumulation [6]. It is predominantly a Th1-mediated disease [7], which increases in later stages [8]. Although many studies have shown differences in the concentrations of biomarkers between healthy controls and people with RRMS, 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 [9].

Approximately one in eight patients has a family history of MS [10]. The risk for a first-generation relative is 3–4%, 7–40 times higher than the general population [11,12]. It is known that there is a latitudinal gradient of MS. This is more widespread the farther the population is from the equator [13]. Individuals with Epstein–Barr virus (EBV) exhibit an elevated risk of developing adult-onset MS, where infectious mononucleosis is likely a marker for an abnormal immune response, which itself increases the risk for MS, not infection [14]. The first and key event in pathogenesis involves the activation of potentially autoreactive T lymphocytes that are positive for CD4 receptors (CD4+) in the periphery [15]. Activated peripheral lymphocytes then migrate through the blood–brain barrier (BBB) via a transmigration process involving the interaction between adhesion molecules on T lymphocytes and adhesion molecules present on capillary endothelial cells, with the simultaneous release of metalloproteinases (MPPs).

An accurate characterisation of the clinical phenotype of MS is important for communication, prognosis, and treatment decisions. The clinical phenotype can be classified into three groups: RRMS, secondary progressive (SPMS), and primary progressive (PPMS). The clinical signs and symptoms of MS vary depending on the location and severity of the lesions affecting different areas of the spinal cord and brain [16,17]. The onset of the disease is usually monosymptomatic. Sensory symptomatology is most often present, followed by motor and visual symptoms [18]. Extensive research efforts have focused on specific components of the immune system in an attempt to detect and diagnose the disease in its earliest stages; however, they currently lack the precision to serve as reliable biomarkers [19]. Further, the treatment of MS can be divided into three main categories that include the treatment of acute attacks, disease-modifying treatment, and the treatment of symptoms [20]. Remarkable progress in the treatment of RRMS has been achieved with drugs that modify the course of the disease. Among these is teriflunomide, which was prescribed for RRMS patients enrolled in this study. Teriflunomide, which is the active metabolite of leflunomide, has been used in the treatment of rheumatoid arthritis since 1998. It selectively inhibits dihydroorotate dehydrogenase, one of the key enzymes for pyrimidine synthesis, which reduces the proliferation of activated B and T cells [21].

CD163, a 130-kDa transmembrane protein with a short cytoplasmic tail, belongs to the scavenger receptor family characterised by class B cysteine-rich domains. It was first identified in 1987 and assigned its CD designation in 1996 [22,23]. CD163 expression is restricted to cells of the monocyte/macrophage lineage and is predominantly found on tissue macrophages, including red pulp macrophages in the spleen, Kupffer cells in the liver, interstitial and alveolar macrophages in the lungs, perivascular macrophages (PVMs) and microglia in the central nervous system (CNS) [24], as well as M2c macrophages that infiltrate tissues during the healing phase of inflammation [25]. Expression is significantly higher in macrophages than in circulating blood monocytes, suggesting that CD163 serves as a marker of macrophage lineage differentiation, with its expression increasing as cells progress along the differentiation pathway [26]. Membrane CD163 (mCD163) binds the haemoglobin–haptoglobin complex (Hb-Hp) with high affinity, which leads to endocytosis and cleaning of this complex, thus protecting tissues from oxidative damage mediated by free haemoglobin (Hb). The expression of both CD163 and Hb is highly upregulated by the major acute phase reactants, IL-6, and glucocorticoids [27]. The soluble form, sCD163, is released from the cell membrane by the cleavage of mCD163 by metalloproteinases [28]. It can be found in plasma and in other body fluids such as cerebrospinal fluid (CSF) [29]. Patients with demyelinating diseases have significantly elevated CSF levels that are consistent with neuropathological findings in active lesions in RRMS patients [30]. In the CSF, sCD163 is probably released by macrophages and microglia [31]. Studies have indicated that mCD163 could be a valuable biomarker for monitoring disease activity and therapeutic response. Stilund et al. [32] showed that in newly diagnosed patients, sCD163 levels in CSF and serum did not show significant correlations with the Expanded Disability Status Scale (EDSS), disease duration, time since the last attack, or number of attacks. However, the CSF/serum ratio was significantly elevated in patients with MS or clinically isolated syndrome compared to healthy controls.

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.

2. Materials and Methods

2.1. Subjects

Forty-six patients with RRMS treated with teriflunomide (Aubagio; Sanofi, Tours, France) for a minimum of 12 months were included in the study. The study sample consisted of individuals who met the inclusion criteria and agreed to participate. Inclusion criteria for the study required participants to be at least 18 years old and to have received a diagnosis of RRMS at least one year prior to study enrolment (initial recruitment occurred in 2022). Participants had no signs of clinical or neuroradiological disease activity for a minimum of three months before study inclusion. 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.

2.2. The Data Collection Procedures

Following neurological examination, peripheral blood (PB) was collected. Neurological examinations and peripheral blood (PB) collection were conducted at the Department of Neurology, University Hospital of Split, Croatia. PB analyses were performed at the Department of Medical Chemistry and Biochemistry, School of Medicine, University of Split, Croatia.

2.3. ELISA Analyses

Quantitative levels of sCD163 were analysed using the Human CD163 ELISA Kit (M130) (ab155428) based on the “sandwich” principle. All standards, controls, and samples were tested in duplicate. In brief, antibodies specific to Human sCD163 was coated into microtitre wells, and plasma samples and standards were added. sCD163 antibodies that were present in the samples and standards were bound to the wells by the immobilised antibody. After washing the wells, biotinylated anti-human sCD163 antibodies were added. Following another wash to remove unbound antibodies, HRP-conjugated streptavidin was introduced. TMB substrate solution was then pipetted into the wells, and the colour developed in proportion to the amount of bound sCD163. Finally, the stop solution was added, and the colour intensity was measured at 450 nm.

2.4. Transcranial Magnetic Stimulation (TMS) Procedure in the 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 the muscles of the upper and lower extremities while stimulating the primary motor cortex via navigated transcranial magnetic stimulation (TMS) [33]. The navigated transcranial magnetic stimulator (TMS) (Nexstim NBS System 4 of the manufacturer Nexstim Plc., Helsinki, Finland) is used for mapping the 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; Nexstim Plc, Helsinki, Finland) [33]. The MEP findings obtained from our previously published work [33] 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 the non-ASNF group.

2.5. 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.

3. Results

The final study included 23 RRMS patients with a mean age of 41.65 ± 8.89 years, and 10 healthy controls (HCs) whose mean age was 37 ± 13.9. The HCs included were (60%) women. The majority of MS patients were female (60.87%) with a high school education (73.9%). There were nine men (39.13%); the ratio of women to men was 1.56:1. The mean disease duration was 9.39 ± 5.73 years, and the median (Q1–Q3) EDSS (general score) was 2.5 (0–3.5). The mean duration of disease duration for women was 8.81 ± 1.6 years; for men, this was 10.22 ± 1.84 years. Among the 23 individuals with MS, 57.8% had received corticosteroid treatment three or more times over the course of their medical care but not three months before inclusion in the study. Patients received teriflunomide once a day at a dose of 14 mg. Subjects had no relapse occurrence three months before inclusion in the study.

Table 1. Demographic and clinical characteristics of the study group.

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

Abbreviations: ASNF—subjects with altered subclinical neurophysiological findings; non-ASNF—subjects without alterations in subclinical neurophysiological findings; HC—healthy control. Data for variables are presented as M—mean value, SD—standard deviation, min—minimum, max—maximum, and IQR—interquartile range.

presents the descriptive parameters of disease indicators included in this study. Out of the 23 RRMS subjects, 15 RRMS subjects (65.22%) had altered subclinical neurophysiological (ASNF) findings, with an EDSS score of 2.57 ± 1.47, while 8 patients (34.78%) had no alterations in their subclinical neurophysiological findings (non-ASNF), with an EDSS score of 1.06 ± 1.02 (Table 1).

No statistically significant difference was found in serum sCD163 protein concentrations between ASNF and non-ASNF MS subject subgroups, nor was there any difference between all MS subjects and HCs (p > 0.05) (Table 1).

To verify the association between disease activity (sCD163) and EDSS, Pearson’s correlation coefficient was calculated between the observed variables for RRSM subjects. No statistically significant correlation (r = 0.112, p = 0.609) was found between serum sCD163 concentration and EDSS. To assess the difference in sCD163 protein concentration concerning the sex of participants with RRMS, a t-test for independent samples was performed (

Table 2. The difference in sCD163 protein concentration in male and female RRMS subjects (N = 23) and correlation with EDSS.

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

Abbreviations: Data for variables are presented as M-mean values (m—men, f—women), SD—standard deviation.

). Men had a higher concentration of sCD163 than women (p = 0.038) and a longer disease duration. The correlation between sCD163 in female patients with RRMS, and disease activity assessed by EDSS, was verified with Pearson’s correlation coefficient (r = 0.94, p = 0.021) (Table 2). Since only 23 subjects participated, and given the comparison with existing studies in which the number of subjects varied from 20 to 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).

4. Discussion

Due to the autoimmune nature of MS, several studies have focused on specific components of the immune system, such as Myelin oligodendrocyte glycoprotein, Myelin basic protein, and Myelin-associated glycoprotein, to diagnose patients at the earliest possible stage of the disease. All the molecules examined have been shown to correlate with MS, but they currently lack sufficient accuracy for use as diagnostic biomarkers [34].

One of the investigated markers is sCD163, which can be measured in serum using an ELISA test. It is already a valuable biomarker for macrophage activation in various inflammatory conditions, including sepsis, macrophage activation syndrome, and liver disease [32]. Moreover, it serves as a general marker for the risk of comorbidity and mortality in several chronic inflammatory diseases, including rheumatoid arthritis, asthma, celiac disease, spondyloarthropathies, scleroderma, and Crohn’s disease [32]. Patients with demyelinating diseases have significantly elevated CSF levels consistent with neuropathological findings in active lesions in RRMS patients [32]. Macrophages are densely distributed in these active lesions and can also be found in chronic, active lesions, but in smaller numbers. In chronic lesions, macrophages are likely activated through mechanisms that involve both pro-inflammatory and anti-inflammatory stimuli [35]. In the CSF, sCD163 is probably released by macrophages and microglia. Stilund et al. [29] showed that patients with the progressive form exhibited elevated sCD163 levels in CSF that were consistent with the pathologic findings showing the presence of ongoing inflammation-mediated deterioration. In the CSF, sCD163 is released by microglia in all white matter lesions. Normal-appearing white matter (NAWM) in the brains of individuals with MS contains a high level of CD163-positive microglia in comparison to normal-appearing cortical grey matter (NAGM) [35]. Due to the involvement of CD163 in iron homeostasis, as a soluble scavenger receptor for the haptoglobin–haemoglobin complex, Hofmann et al. performed iron-sensitive magnetic resonance imaging of MS brains, thereby detecting a correlation between sCD163 and CSF via brain paramagnetic rim lesion counts [36]. Recently, Maliozzi et al. described a correlation between choroid plexus inflammation and frequency of CD163⁺ innate immune cells [37]. Treatment-naive multiple sclerosis patients show elevated sCD163 levels in CSF due to changes in T and B cell signaling [38]. Comparisons of CD163⁺ microglia expression in MS brain lesions and MS remyelinating donor brains (at autopsy) have not revealed any differences between them, in spite of the expected increase in regenerative (CD163⁺) microglia in the MS remyelinating group without lesions [39].

Like most autoimmune diseases, MS is more prevalent in women. In RRMS, women are affected three times more often than men. In our study, women outnumbered men 1.56-fold, which does not coincide with the study by Confavreux et al. [3]. This low ratio may be due to the small group of observed patients, or it may be attributed to the greater cooperation of men than women. Males have a typically later onset of MS than females. The onset in males, ages 30–40, typically coincides with the onset of testosterone decline [40].

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

The present study found no statistically significant differences in plasma sCD163 protein concentration between MS subjects regarding the severity of disease (determined by neurophysiological findings) [33], nor was there any difference between participants with RRMS and HCs. The results of sCD163 in patients with alterations in neurophysiologic findings were 834.57 ± 335.29 ng/mL, and 720.31 ± 156.98 ng/mL in patients with no alterations in subclinical neurophysiologic findings; in HCs, the results were 591.75 ± 225.10 ng/mL. These results align with the findings of De Fino et al. [41] and Stilund et al. [32], who observed no statistically significant differences between the tested groups of patients with MS and the HCs regarding serum levels of sCD163. De Fino et al. [41] reported a mean sCD163serum level of 470 ng/mL ± 464.2 in RRMS patients, while Stilund et al. [32] reported a mean sCD163 serum level of 1490 ng/mL the RRMS group, with a range of 390 to 4530 ng/mL. While we have not detected significant levels of sCD163 elevation, Farrokhi et al. [42] and Mona et al. [43] found significantly higher sCD163 serum levels in RRMS subjects compared to HCs. In this study, the sensitivity of the ELISA kit (0.03 ng/mL) was higher than that in the studies by Farrokhi et al. (1.56 ng/mL) and Mona et al. (0.94 ng/mL); however, we did not have age- and sex-matched patients. In a study conducted by Farrokhi et al. [42], 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 Farrokhi et al.’s study [42], the average sCD163 serum levels ] were 2160 ng/mL ± 1140 SD in RRMS subjects, while in the HCs, the average serum level was 1450 ng/mL ± 730. The ratio of RRMS/HCs was 1.5; the ratio to HCs in our investigation for patients with altered subclinical neurophysiological findings was 1.4, and the ratio for those without alterations was 1.2.

Furthermore, the present study found no association between serum sCD163 concentration and EDSS. This is in alignment with the findings of Mona et al. [43], Gjelstrup et al. [44], and Stilund et al. [32] who also found no statistically significant association between sCD163 and EDSS. Farrokhi et al. [42] obtained a significantly positive correlation between sCD163 and EDSS. This finding could be due to their exclusion criterion of patients who underwent immunomodulating treatment within a month before sampling. In the present study, men had a higher concentration of sCD163 than women, and they also had a longer duration of illness, but not statistically; however, other studies did not establish higher concentrations of sCD163 for men or longer disease duration [32]. Biological variability could affect how sCD163 reflects disease activity or progression in each sex. Sex differences in inflammatory disease activity lessen after age 50. This suggests that sex hormones strongly affect the condition, especially before menopause. In contrast, disparities in the neurodegenerative aspects of MS began to emerge after age 45 and became more pronounced with advancing age [45].

Additionally, in previous studies, men tended to reach EDSS milestones more quickly than women, had a higher mortality than women [46]; they 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 [47]. One possible reason for the lower levels of sCD163 in females in our study is that 17β-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 [48]; this could also be attributed to the small number of respondents. The present study showed an association between the concentration of (sCD163) in women patients with RRMS, and the EDSS score.

The potential usage of sCD163 as a diagnostic marker in MS is currently unclear. The elevated sCD163 CSF/serum ratio is indicative of macrophage activation in MS lesions [32]. sCD163 is unlikely to be used as a sole diagnostic marker in MS; however, according to our results, it may have potential as a correlative marker of effective teriflunomide treatment in female RRMS patients.

The limitations of this study are the cross-sectional form of the research conducted in a single-centred institution, the small sample size, and the single MS type. It is necessary to emphasise that due to the small sample size, the observed effect should be examined in future studies which would benefit from a larger sample size (especially females) and monitoring subjects for a longer period of time. It can be argued that the observed difference in our study might have practical value; however, further examinations are needed.

5. Conclusions

The present study findings reveal no statistically significant differences between all participants with RRMS and the HCs regarding the concentration of sCD163. However, in our study, there is a correlation between sCD163 concentration in female patients with RRMS who were treated with teriflunomide and disease activity as assessed by EDSS scores. The recommendations for further studies are a larger number of samples (especially females) and monitoring subjects for a longer period of time.