Abstract
OBJECTIVE
Our aim was to determine whether serum C-X-C motif chemokine 5 (CXCL5) may serve as a diagnostic biomarker for relapsing-remitting multiple sclerosis (RRMS) as well as a marker that can be used to predict treatment response.
METHODS
CXCL5 levels were measured by ELISA in sera of 20 RRMS patients under fingolimod treatment, 10 neuromyelitis optica spectrum disorder (NMOSD) patients, 15 RRMS patients presenting predominantly with spinal cord and optic nerve attacks (MS-SCON), and 14 healthy controls.
RESULTS
Fingolimod treatment significantly reduced CXCL5 levels. CXCL5 levels were comparable among NMOSD and MS-SCON patients.
CONCLUSION
Fingolimod might regulate the innate immune system. Serum CXCL5 measurement does not differentiate between RRMS and NMOSD.
Keywords: C-X-C motif chemokine 5, fingolimod, multiple sclerosis, neuromyelitis optica spectrum disorder
Highlight key points
Fingolimod treatment significantly reduced CXCL5 levels.
Regulation of the innate immune system could be one of the action mechanisms of fingolimod.
CXCL5 levels do not reflect the severity of NMOSD and MS and do not provide a prediction about prognosis after fingolimod treatment.
Multiple sclerosis (MS) is the most common autoimmune demyelinating disease of the central nervous system. Although myelin-reactive CD4+ T cells are the main drivers of disease pathogenesis, innate immune system cells recruited to MS lesions by chemokines contribute to disease pathogenesis. Neutrophils migrate to the inflammation site as the first defenders of the innate immune system and they are detected in the cerebrospinal fluid (CSF) of MS patients during relapses [1].
C-X-C motif chemokine 5 (CXCL5), also known as epithelial neutrophil-activating peptide 78, is a potent chemokine that signals through C-X-C motif chemokine receptor 2 and attracts neutrophils to the lesion site [2]. CXCL5 levels are elevated during MS attacks and correlate with disease severity [3]. Suppression of neutrophils attenuates disease severity in experimental autoimmune encephalomyelitis [4]. Fingolimod treatment was shown to decrease CXCL5 levels in a demyelination model using cerebellar slice cultures, suggesting that besides prevention of T lymphocyte migration, regulation of the innate immune system could contribute to the mechanism of action of fingolimod [5].
Neutrophil aggregation is a typical feature of neuromyelitis optica spectrum disorder (NMOSD). CXCL5 level was found to be correlated with disease severity in NMOSD but not in MS in contrast with previous study [3], raised the question whether CXCL5 might serve differentiation of NMOSD from relapsing-remitting MS (RRMS) presenting predominantly with spinal cord and optic nerve attacks (MS-SCON) [6].
In this study, we first aimed to investigate the possible utility of CXCL5 as a biomarker for the differentiation of NMOSD from MS presenting with clinical features reminiscent of NMOSD. Furthermore, we investigated the role of CXCL5 as a biomarker for the prediction of treatment response in MS patients.
MATERIALS AND METHODS
Subjects
Ten NMOSD (6 aquaporin-4 IgG seropositive) patients who fulfilled international diagnostic criteria for NMOSD [7], 15 MS-SCON patients, and 14 age/sex-matched healthy controls with no prior inflammatory or neurological disorder history were recruited (Table 1).
Table 1.
Clinical and demographical data of participants
| RRMS (n=20) |
NMOSDs (n=10) |
MS-SCON (n=15) |
Healthy control (n=14) |
|
|---|---|---|---|---|
| Gender (female/male) | 14/6 | 8/2 | 11/4 | 11/3 |
| Age, years (mean±SD) | 34.35±8.29 | 32.20±8.55 | 37.20±8.26 | 32.78±8.95 |
| Disease duration (mean±SD) | 6.40±2.96 | 12.60±10.19 | 10.80±6.18 | |
| EDSS (mean±SD) | 1.9±1.28 | 3.6±2.19 | 2.46±1.76 |
SD: Standard deviation; RRMS: Relapsing remitting multiple sclerosis; NMOSD: Neuromyelitis optica spectrum disorder; MS-SCON: MS presenting predominantly with spinal cord and optic nerve attacks.
All patients were in remission and not under steroid treatment. Both NMOSD and MS-SCON patients were under immunomodulating treatment (azathioprine, interferon-beta, and glatiramer acetate). All MS-SCON patients fulfilled the diagnostic criteria of MS and none of them had aquaporin-4 IgG, long extensive spinal cord lesions, or clinical features indicating brainstem, cerebellum, or hemisphere involvement. The study protocol was approved by the Koc University Clinical Research Ethical Committee with the protocol ID of 2016.123.IRB2.077 and written informed consent was obtained from all participants.
CXCL5 Assessment
We asserted that CXCL5 levels were reduced in patient groups due to immunomodulating medications. Thus, secondly, we tested whether CXCL5 levels could be used for the determination of treatment responsiveness in MS. For this purpose, CXCL5 levels were measured in sera of 20 RRMS patients before and 3 months after fingolimod (0.5 mg/day) treatment. All patients experienced a 3-month washout period before fingolimod treatment. Serum CXCL5 levels were measured with an ELISA kit (Elabscience, Houston, TX, USA) according to the manufacturer’s instructions, and results were expressed as pg/mL.
Statistical Analyses
Statistical analysis was performed using SPSS 20 software (SPSS Inc, Chicago, IL, USA) and the significance level was set at p<0.05. The variables were investigated using visual (histograms) and analytical methods (Kolmogorov–Smirnov/Shapiro–Wilk’s test) to determine whether they are normally distributed. Descriptive statistics were applied, and the characteristics of the groups were compared with paired or unpaired t-tests and ANOVA where appropriate.
RESULTS
CXCL5 levels were significantly higher in healthy controls compared to NMOSD (p=0.04) and MS-SCON (p=0.01) patients. However, CXCL5 levels of NMOSD and MS-SCON patients were comparable (Fig. 1). Moreover, CXCL5 levels did not correlate with EDSS score, number of attacks, and disease duration, as assessed by Pearson’s correlation test.
Figure 1.
Serum CXCL5 levels of NMOSD, MS-SCON, and HC groups. CXCL5: C-X-C motif chemokine 5, NMOSD: Neuromyelitis optica spectrum disorder, MS-SCON: MS presenting predominantly with spinal cord and optic nerve attacks, HC: Healthy controls. Statistical comparison was done with ANOVA (p=0.03) and Tukey’s post-hoc test (p=0.01, p=0.04). P<0.05 was considered for statistical significance. Bars indicate standard deviations and horizontal lines indicate mean values.
In keeping with our assertion, CXCL5 levels of RRMS patients in the washout period (921.37±343.33) were not significantly different from those of healthy controls (1073.77±257.14, p=0.24 with unpaired t-test) and a significant decrease was observed in CXCL5 levels after treatment (p=0.003 with paired t-test, Fig. 2). Post-treatment CXCL5 levels of RRMS patients (788.33±290.75) were significantly lower than those of controls (p=0.005, with unpaired t-test). There was no correlation between EDSS scores before and after treatment. Four patients who had more than one point increase in EDSS score and/or more than one attack during follow-up were considered treatment unresponsive. No significant difference was found between CXCL5 levels of treatment responsive and unresponsive patients before or after treatment (data not shown).
Figure 2.
Serum CXCL5 levels of RRMS patients before (0M) and 3 months (3M) after fingolimod treatment. Statistical comparison was done with Student’s t-test. P<0.05 was considered for statistical significance. Open circles represent drug unresponsive patient.
DISCUSSION
We may conclude that serum CXCL5 levels do not reflect the severity of NMOSD and MS and do not provide a prediction about prognosis after fingolimod treatment. Our results also argue against the possible utility of CXCL5 in the differential diagnosis of MS presenting with clinical features reminiscent of NMOSD. It should be noted that immunomodulating drugs might plausibly have suppressed CXCL5 levels, thereby masking differences between patient groups and correlations between clinical features. Therefore, our results do not rule out the possibility that CXCL5 measurements in treatment-naïve patients might be utilized for diagnostic purposes. Furthermore, serum CXCL5 levels probably do not accurately reflect cerebral levels of CXCL5, which is more representative of neuroinflammation in MS. Nonetheless, it has been shown that CSF CXCL5 levels did not show any differences between neuroinflammatory disease groups such as clinically isolated syndrome, RRMS, or secondary progressive MS patients [8].
Suppression of CXCL5 levels after fingolimod treatment supports the modulating role of this drug on innate immunity [9]. In chronic neuroinflammatory diseases, CXCL5 is released from both cerebral and blood-born innate immunity cells. Fingolimod has been shown to decrease CXCL5 expression in both astrocytes and microglia [10]. Moreover, MS patients under IVIg treatment also show reduced CXCL5 levels [11]. Our results also show that one of the actions of a wide array of immunomodulating treatment modalities (e.g. fingolimod, azathioprine, interferon-beta, glatiramer acetate, etc.) is suppression of CXCL5, thereby reducing recruitment of neutrophils into the brain parenchyma. This effect might be achieved through the inhibition of Th1 and Th17-type immune responses, which activate cells of innate immunity.
One of the limitations of our study is the low sample size, and the other is that NMO and MS-CON patients are under immunosuppressive therapy. However, NMO and MS-CON patients can generally be diagnosed after long-term follow-up makes it difficult to catch these patients during the untreated period. Although our study suggests that CXCL5 is not a good candidate biomarker for the differentiation of NMO and MS-CON, it provides a new clue about the mechanism of action of fingolimod treatment. Studies in this area with a larger cohort will shed light on fingolimod’s effect on the innate immune system.
Conclusion
In conclusion, our results indicate that CXCL5 levels may possibly not be used as a prognostic and diagnostic biomarker in chronic MS or NMOSD patients under long-term treatment. However, whether serum/CSF measurements of CXCL5 levels in treatment-naïve patients may exhibit a biomarker quality still tends to be assessed.
Footnotes
Cite this article as: Karaaslan Z, Yilmaz V, Yuceer H, Sanli E, Akcay HI, Kurtuncu M, et al. Serum CXCL5 as a biomarker in multiple sclerosis and neuromyelitis optica spectrum disorder. North Clin Istanb 2023;10(3):341–344.
Ethics Committee Approval
The Koc University Clinical Research Ethics Committee granted approval for this study (date: 30.05.2016, number: 2016.123.IRB2.077).
Conflict of Interest
No conflict of interest was declared by the authors.
Financial Disclosure
The authors declared that this study has received no financial support.
Authorship Contributions
Concept – ET, VY, RT; Design – VY; Supervision – ET, MK, RT; Fundings – ET, VY; Materials – RT, MK, HIA; Data collection and/or processing – ZK, HY, ES; Analysis and/or interpretation – ZK, VY; Literature review – ZK, VY; Writing – ZK, VY, ET; Critical review – MK, RT, ET.
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