Gene Expression Profiling of Multiple Sclerosis Pathology Identifies Early Patterns of Demyelination Surrounding Chronic Active Lesions

Abstract

In multiple sclerosis (MS), activated microglia and infiltrating macrophages phagocytose myelin focally in (chronic) active lesions. These demyelinating sites expand in time, but at some point turn inactive into a sclerotic scar. To identify molecular mechanisms underlying lesion activity and halt, we analyzed genome-wide gene expression in rim and peri-lesional regions of chronic active and inactive MS lesions, as well as in control tissue. Gene clustering revealed patterns of gene expression specifically associated with MS and with the presumed, subsequent stages of lesion development. Next to genes involved in immune functions, we found regulation of novel genes in and around the rim of chronic active lesions, such as NPY, KANK4, NCAN, TKTL1, and ANO4. Of note, the presence of many foamy macrophages in active rims was accompanied by a congruent upregulation of genes related to lipid binding, such as MSR1, CD68, CXCL16, and OLR1, and lipid uptake, such as CHIT1, GPNMB, and CCL18. Except CCL18, these genes were already upregulated in regions around active MS lesions, showing that such lesions are indeed expanding. In vitro downregulation of the scavenger receptors MSR1 and CXCL16 reduced myelin uptake. In conclusion, this study provides the gene expression profile of different aspects of MS pathology and indicates that early demyelination, mediated by scavenger receptors, is already present in regions around active MS lesions. Genes involved in early demyelination events in regions surrounding chronic active MS lesions might be promising therapeutic targets to stop lesion expansion.

Keywords: active lesions; demyelination; lipid uptake; microarray; microglia; multiple sclerosis; scavenger receptor.

Figure 1

Schematic overview of the different microarray analyses done. Roman numbers indicate direct comparisons. (I) chronic active rim vs. inactive rim, (II) chronic active peri-lesional (PL)-normal appearing white matter (NAWM) vs. inactive PL-NAWM, and (III) control vs. chronic active PL-NAWM. Arabic numbers indicate the sequence used for cluster analysis. (1) Control white matter (WM), (2) chronic active PL-NAWM, (3) chronic active rim, (4) inactive rim, (5) inactive PL-NAWM.

Figure 2

Top 50 significantly upregulated and downregulated genes in multiple sclerosis lesion subregions. For a description of the comparisons, see Figure 1 and text. Upregulated genes are indicated in red and downregulated genes are indicated in blue. Genes colored and marked in bold have been selected for further investigation. Genes expressed in cluster 3 are marked by an asterisk. Further details on the genes and p values are provided in Tables S3A–F in Supplementary Material. FC, fold change.

Figure 3

Cluster analysis of gene expression in and around multiple sclerosis (MS) lesions. Analysis was done with the sequence: (1) control white matter (WM), (2) chronic active peri-lesional (PL)-normal appearing white matter (NAWM), (3) chronic active rim, (4) inactive rim, (5) inactive PL-NAWM (also shown in Figure 1), which resulted in six different expression patterns, representing overall differences between control and MS (clusters 1 and 2), specific upregulation around expanding chronic active lesions (cluster 3), specific upregulation in active rims (cluster 4), upregulation in active rims and (peri)-rims of inactive lesions (cluster 5), or upregulation in and around inactive lesions (cluster 6). N indicates the number of significantly regulated genes within a cluster.

Figure 4

Expression of CHIT1, GPNMB, and OLR1 in and around multiple sclerosis (MS) lesions. Protein expression of CHIT1, GPNMB, and OLR1 in control tissue and in the center, rim, and peri-lesional (PL)-normal appearing white matter (NAWM) of chronic active and inactive MS lesions determined by immunohistochemistry. Scale bar = 100 µm.

Figure 5

Upregulation of CHIT1 after uptake of multiple sclerosis (MS) myelin in vitro. At the mRNA level, both CHIT1 (p = 0.02) and GPNMB (p = 0.007) are upregulated in THP-1 macrophages after incubation with myelin from MS donors for 5 days, followed by 3 days incubation in normal medium. Fold change from macrophages cultured without myelin, n = 3, *p < 0.05.

Figure 6

Functional role of scavenger receptors in myelin phagocytosis in vitro. MSR1, CXCL16, OLR1, and CD68 were downregulated with antisense oligonucleotides in the human macrophage cell line THP-1. Silencing efficiency was determined on mRNA level with quantitative polymerase chain reaction (A) and on protein level with immunocytochemistry [(B); only shown for MSR1]. Uptake of pHrodo-labeled myelin was validated by flow cytometry (C) and compared for myelin obtained from control and multiple sclerosis (MS) brain tissue (D). The number of cells that had phagocytosed myelin, and the total amount of myelin phagocytosed (geomean pHrodo) were calculated. The number of independent experiments (n) was 6 (MSR1), 4 (CXCL16), 3 (OLR1), and 3 (CD68). (E) Fucoidan was used to inhibit a broad spectrum of scavenger receptors in THP-1 cells. Provided is the number of cells that had phagocytosed myelin, the total amount of myelin phagocytosed (geomean pHrodo), and the viability of cells at the time point of analysis (n = 3). Scale bar in panel (B) = 200 µm *p < 0.05, **p < 0.01, ***p < 0.005.