Soluble CD163 as a Marker of Macrophage Activity in Newly Diagnosed Patients with Multiple Sclerosis

<div><p>Background</p><p>Soluble CD163 (sCD163) is a macrophage specific protein known to be up-regulated in serum from patients with multiple sclerosis (MS).</p><p>Objective</p><p>To investigate sCD163 in serum and CSF (cerebrospinal fluid) from patients undergoing MS diagnostic work-up and analyse its potential as a diagnostic biomarker.</p><p>Methods</p><p>After a full MS diagnostic work-up, including collection of paired samples of CSF and serum, 183 patients were evaluated for inclusion in this study. Patients were divided into groups based on their diagnosis. Patients with normal clinical and paraclinical findings were grouped as symptomatic controls. Serum and CSF levels of sCD163 were determined by enzyme-linked immunosorbent assay (ELISA).</p><p>Results</p><p>sCD163 could be measured in all serum and CSF samples. A high sCD163 CSF/serum ratio in relation to molecular weight was found, strongly indicating local production in the CNS. Median levels of sCD163 were significantly decreased in serum and significantly elevated in CSF in patients with relapsing-remitting, and primary- progressive MS. There were, however, some overlaps of the measures between groups. In a receiver operating characteristic (ROC) analysis sCD163 CSF/serum ratio had an area under the curve of 0.72.</p><p>Conclusion</p><p>The sCD163 CSF/serum ratio was significantly increased in patients with MS and may reflect macrophage activation in MS lesions. These results suggest that primary progressive MS also is driven by inflammation in which the innate immune system plays a pivotal role.</p></div

ROC curves for the sCD163 ratio, the CXCL13 ratio, the NEO ratio, the NfL CSF, the OPN serum, and the IgG index.

<p>Fig 4 shows the ROC curves generated for the sCD163 ratio (4a), the CXCL13 ratio (4b), the NEO ratio (4c), the NfL CSF (4d), the OPN serum (4e), and the IgG index (4f). The data on sCD163 have previously been presented in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119681#pone.0119681.ref018" target="_blank">18</a>]. AUC, with 95% CI, is given for each parameter. The parameter results for patients with RRMS, PPMS, and CIS are combined as true positives and plotted against SC as true negatives. The diagonal dividing the ROC space represents the random event. Abbreviations: ROC (receiver operating characteristic), AUC (area under the curve), RRMS (relapsing-remitting MS), PPMS (primary-progressive MS), CIS (clinically isolated syndrome), SC (symptomatic controls), CSF (cerebrospinal fluid), CI (confidence interval).</p

Fig 3 shows the biomarker profile for each group of patients.

<p>The biomarker profile was constructed as an overview representing the major characteristics of each group in the cohort. The profile was determined by categorizing the GM levels of the sCD163 ratio, the CXCL13 ratio, the NEO ratio, the NfL CSF, the OPN serum, and the IgG index for each patient group in the following manner: Very high (significantly increased GM, p <0.001); High (significantly increased GM, p <0.01); Moderate (significantly increased GM, p <0.05); Low (zero is included in the CI). Abbreviations: NEO (neopterin), NfL (neurofilament light polypeptide), OPN (osteopontin), SC (symptomatic controls), RRMS (relapsing-remitting MS), PPMS (primary-progressive MS), CIS (clinically isolated syndrome), CI (confidence interval), GM (geometric means).</p

Biomarkers of Inflammation and Axonal Degeneration/Damage in Patients with Newly Diagnosed Multiple Sclerosis: Contributions of the Soluble CD163 CSF/Serum Ratio to a Biomarker Panel

<div><p>Background</p><p>Expression of soluble CD163 (sCD163), a macrophage/microglia biomarker, is increased in inflammatory conditions, and sCD163 levels in the cerebrospinal fluid (CSF) have recently been shown to be elevated in patients with multiple sclerosis (MS): the sCD163 CSF/serum ratio was elevated in patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and clinically isolated syndrome (CIS) compared with symptomatic controls.</p><p>Objective</p><p>To investigate the contributions of the sCD163 CSF/serum ratio to a biomarker panel focusing on inflammation and axonal degeneration in newly diagnosed MS; thus optimising a diagnostic biomarker panel for MS.</p><p>Methods</p><p>After a full MS diagnostic work-up, including collection of paired samples of CSF and serum, 125 patients were included in this study. Patients were divided into groups based on their diagnosis, and patients with normal clinical and paraclinical findings were defined as symptomatic controls. Serum and CSF levels, ratios, and indices of sCD163, CXCL13, osteopontin, neopterin, and CSF levels of neurofilament light polypeptide were determined by enzyme-linked immunosorbent assays (ELISAs). For sCD163 the results constitute a post-hoc analysis of already published data.</p><p>Results</p><p>All tested biomarkers, notably the sCD163 ratio, the CXCL13 ratio, the NEO ratio, the CSF level of NfL, the IgG index, and the serum level of OPN, were significantly correlated to RRMS, PPMS, and/or CIS. The individual biomarkers in single tests had a lower performance than the IgG index, however, their combined receiver operating characteristic (ROC) curve demonstrated excellent diagnostic discriminatory power.</p><p>Conclusion</p><p>The biomarker panel showed distinct profiles for each patient group and could be a valuable tool for clinical differentiation of MS subgroups. The combined ROC analysis showed that sCD163 contributes positively as a diagnostic marker to a panel of established MS biomarkers. Patients with PPMS were demonstrated to have significantly elevated levels of both inflammatory and degenerative markers.</p></div

ROC curves for the sCD163 ratio, the CXCL13 ratio, the NEO ratio, the NfL index, and the OPN serum combined with the IgG index individually as well as a combined ROC curve.

<p>Fig 5 shows the combined ROC curves generated for the IgG index and the sCD163 ratio (5a), the CXCL13 ratio (5b), the NEO ratio (5c), the NfL CSF (5d), the OPN serum (5e), and All biomarkers (5f). AUC, with 95% CI, is given for each parameter. The parameter results for patients with RRMS, PPMS, and CIS are combined as true positives and plotted against SC as true negatives. The diagonal dividing the ROC space represents the random event. Abbreviations: ROC (receiver operating characteristic), AUC (area under the curve), RRMS (relapsing-remitting MS), PPMS (primary-progressive MS), CIS (clinically isolated syndrome), SC (symptomatic controls), CI (confidence interval).</p

Median levels of sCD163 in serum and CSF samples as determined by ELISA.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098588#pone-0098588-t002" target="_blank">Table 2</a> shows the median levels of sCD163 in serum, CSF and, sCD163 CSF/serum ratio, and sCD163 index. Abbreviations: RRMS (relapsing-remitting MS), PPMS (primary-progressive MS), SPMS (secondary-progressive MS), CIS (clinically isolated syndrome), SC (symptomatic controls), n (number of persons), CSF (cerebrospinal fluid).</p

Pearson pairwise correlation analysis without and with the Bonferroni correction of biomarkers: sCD163, CXCL13, NEO, NfL, and OPN.

<p>Table 5 shows the results from the Pearson pairwise correlation analyses without and with the Bonferroni correction of biomarkers and their intercorrelation. The correlation coefficient, r, is displayed with the significance levels without / and with the Bonferroni corrections marked by * for p<0.05, and ** for p<0.001. All p-values are presented in tables Z and AA in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119681#pone.0119681.s001" target="_blank">S1 Dataset</a>. Abbreviations: CSF (cerebrospinal fluid), NfL (neurofilament light polypeptide), NEO (neopterin), OPN (osteopontin).</p><p>Pearson pairwise correlation analysis without and with the Bonferroni correction of biomarkers: sCD163, CXCL13, NEO, NfL, and OPN.</p

Results from linear regression analyses of differences in levels of biomarkers between patients with MS/CIS and SC.

<p>Table 6 shows the results of linear regression analyses on parameters identified in the previous correlation analysis. The regression analyses were performed on log transformed data and if the parameter was correlated to age and/or gender in the correlation analysis these were included in the linear regression. Fold differences in geometric means (GM) and CIs are shown for RRMS, PPMS, and CIS compared with the SC group. The level of significance is shown by the p-value and * marks a significant difference between groups. Abbreviations: RRMS (relapsing-remitting MS), PPMS (primary-progressive MS), CIS (clinically isolated syndrome), SC (symptomatic controls), n (number of persons), CSF (cerebrospinal fluid), CI (confidence interval) NfL (neurofilament light polypeptide), NEO (neopterin), OPN (osteopontin). The data on sCD163 have previously been presented in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119681#pone.0119681.ref018" target="_blank">18</a>].</p><p>Results from linear regression analyses of differences in levels of biomarkers between patients with MS/CIS and SC.</p

ROC curves generated for serum sCD163, CSF sCD163, sCD163 CSF/serum ratio, sCD163 Index, and IgG Index.

<p>AUC, with 95% CI, is given for each parameter. The parameter results for patients with MS/CIS are combined as true positives and plotted against SC as true negatives. The diagonal dividing the ROC space represents the random event. Abbreviations: ROC (receiver operating characteristic), AUC (area under the curve), CIS (clinically isolated syndrome), SC (symptomatic controls), CSF (cerebrospinal fluid), CI (confidence interval).</p