Transcript Profiles of Microglia/Macrophage Cells at the Borders of Chronic Active and Subpial Gray Matter Lesions in Multiple Sclerosis

Abstract

Objective: Microglia/macrophages line the border of demyelinated lesions in both cerebral white matter and the cortex in the brains of multiple sclerosis patients. Microglia/macrophages associated with chronic white matter lesions are thought to be responsible for slow lesion expansion and disability progression in progressive multiple sclerosis, whereas those lining gray matter lesions are less studied. Profiling these microglia/macrophages could help to focus therapies on genes or pathways specific to lesion expansion and disease progression.

Methods: We compared the morphology and transcript profiles of microglia/macrophages associated with borders of white matter (WM line) and subpial gray matter lesions (GM line) using laser capture microscopy. We performed RNA sequencing on isolated cells followed by immunocytochemistry to determine the distribution of translational products of transcripts increased in WM line microglia.

Results: Cells in the WM line appear activated, with shorter processes and larger cell bodies, whereas those in the GM line appear more homeostatic, with smaller cell bodies and multiple thin processes. Transcript profiling revealed 176 genes in WM lines and 111 genes in GM lines as differentially expressed. Transcripts associated with immune activation and iron homeostasis were increased in WM line microglia, whereas genes belonging to the canonical Wnt signaling pathway were increased in GM line microglia.

Interpretation: We propose that the mechanisms of demyelination and dynamics of lesion expansion are responsible for differential transcript expression in WM lines and GM lines, and posit that increased expression of the Fc epsilon receptor, spleen tyrosine kinase, and Bruton's tyrosine kinase, play a key role in regulating microglia/macrophage function at the border of chronic active white matter lesions. ANN NEUROL 2024;95:907-916.

Figure 1. Characterization of lesions and microglial lines.

Demyelinated lesions were identified using myelin-specific antibodies directed against proteolipid protein (PLP) (A, D, G). Microglia/immune cells were identified using antibodies directed against MHC Class II (CII) (B, E, H). Chronic active WM lesions (A) are surrounded by a dense line of MHC CII-positive cells at the lesion border (B). WM line microglia/macrophages have large cell bodies and short processes (C). Many subpial cortical (GM) lesions (D) have dense lines of microglia at their borders (E). These microglia have smaller cell bodies and more processes (F) than CII-positive cells at the border of chronic active WM lesions (C). WM lines at lesion borders were identified in frozen tissue sections using PLP (G) and CII (H) antibodies and then isolated by infrared laser capture microscopy. Panel I depicts microglia captured from chronic active WM at the lesion border. Scale bar = 500 µm

Figure 2. Transcript comparisons of microglia from WM lines versus GM lines.

Two hundred and eighty-seven transcripts were significantly increased: 176 in WM line microglia/macrophages and 111 in GM line microglia (A); P< 0.01 and |log2FC| >1.5 for clustered expression levels. (B) Network analysis shows interactions between DEGs and regulatory networks (C) (isolated nodes not shown). Significantly altered KEGG pathways (D) and the top enriched Gene Ontology (GO) Biological Processes Terms (E) for WM line (red) and GM line (blue) microglia.

Figure 3. Immune signaling transcripts are increased in WM line microglia.

Transcripts associated with FC epsilon signaling, NF-kappa B, and cytokine-mediated signaling were increased in WM lines compared to GM lines (A). Translation products were co-localized with MHC CII in WM and GM microglia lines (B-G). FCER1G, BTK, and SYK positive cells were increased in WM lines (H, J, L) compared to GM lines (I, K, M). MHC CII green; FCER1G, BTK, and SYK red. Scale bar = 100 µm. Quantification of BTK and SYK positive cells is shown in N

Figure 4. Iron homeostasis transcripts and iron levels are increased in WM line microglia.

Transcripts associated with iron ion homeostasis were significantly increased in WM lines compared to GM lines (A). WM microglial/macrophage lines (B) were often positive for iron (C). GM microglial lines (D) were negative for iron (E). Hepicidin (HAMP) was increased in WM microglial lines (F) compared to GM microglial lines (G). MHC CII green; HAMP red. Scale bar = 100 µm.