Distinct microglial and macrophage distribution patterns in the concentric and lamellar lesions in Baló's disease and neuromyelitis optica spectrum disorders

Abstract

TMEM119 and purinergic receptor P2Y12 (P2RY12), which are not expressed by recruited peripheral blood macrophages, are proposed to discriminate microglia from macrophages. Therefore, we investigated the distribution patterns of microglia and macrophages in 10 concentric lesions from four autopsied Baló's disease cases and one neuromyelitis optica spectrum disorder (NMOSD) case, using quantitative immunohistochemistry for the markers TMEM119, P2RY12, CD68, CD163 and GLUT5. Three cases with Baló's disease had distal oligodendrogliopathy (DO) showing preferential loss of myelin‐associated glycoprotein and early active demyelination in the outermost demyelinating layer (termed DMY‐MO). In DMY‐MO with DO, TMEM119‐positive activated microglia expressing upregulated GLUT5 but markedly downregulated P2RY12 were significantly increased. These activated microglia expressed inducible nitric oxide synthase. Oligodendrocytes and their precursors showed apoptotic‐like nuclear condensation in DMY‐MO. TMEM119‐negative and CD68/CD163‐positive macrophages were distributed throughout the lesion center of DMY‐MO with DO and these cells demonstrated foamy morphology only in the inner portion but not in the outer portion. In concentric demyelinating lesions from another Baló's case and lamellar demyelinating lesions in an NMOSD case, which had late active demyelination without DO, the densities of TMEM119‐, GLUT5‐ and P2RY12‐positive microglia with ramified morphology were significantly increased in myelinated layers but not in demyelinating layers. In particular, in the NMOSD case, TMEM119‐positive microglia were confined to the outer portion of the myelinated layers. CD68‐positive macrophages with foamy morphology also expressing CD163 accumulated in myelinated as well as in demyelinated layers. These findings suggest that activated microglia expressing TMEM119 and GLUT5, but not P2RY12, are associated with apoptosis of oligodendrocytes in the leading edge of Baló's concentric lesions with DO, whereas TMEM119‐, GLUT5‐ and P2RY12‐positive microglia with ramified morphology are associated with myelin preservation in concentric lesions without DO in Baló's disease and NMOSD. These two types of microglia appear to play distinct roles in the formation of concentric lesions.

Hayashida et al. discriminated the distribution pattern of microglia and macrophages in concentric and lamellar lesions in Baló’s disease and neuromyelitis optica spectrum disorders. Activated microglia expressing TMEM119 and GLUT5, and lacking P2RY12, may play a critical role in the evolution of demyelinating layers in Baló’s disease. GLUT5 in microglia might be a potential therapeutic target for demyelinating diseases.

Introduction

Baló's disease is a rare demyelinating disease characterized by tumor‐like brain lesions with large concentric rings of alternating demyelinated and intact myelin layers (14, 17) and is considered a severe variant of multiple sclerosis (MS) (8, 35). Intriguingly, similar concentric lesions have been reported in neuromyelitis optica spectrum disorders (NMOSD), an autoimmune disorder caused by anti‐aquaporin 4 (AQP4) antibodies (13, 16, 18, 21, 23). We previously reported extensive loss of AQP4 and glial connexins (Cx) in both demyelinating and myelinated layers in Baló's disease (22, 24), which suggested perturbed interaction between oligodendrocytes and astrocytes. Although oligodendrocytic Cx32 and Cx47 are severely diminished in Baló's disease (22), it remains unknown how concentric rings develop.

Active demyelinating lesions in Baló's disease show MS pattern III lesions (termed distal oligodendrogliopathy; DO) characterized by apoptotic oligodendrocytes and early selective loss of myelin‐associated glycoprotein (MAG) and 2′,3′‐cyclic‐nucleotide 3′‐phosphodiesterase (CNPase) but relative preservation of other myelin proteins such as proteolipid protein and myelin oligodendrocyte glycoprotein (MOG) (19). Typically, active demyelination with DO features is restricted to the periphery of concentric lesions, which suggests radial expansion from an inactive lesion core (31). Thus, tissue preconditioning has been proposed to underlie concentric demyelination (31). Here, an initial insult induces oligodendrocyte apoptosis, while in the lesion edges, oligodendrocytes are preconditioned to produce stress proteins that protect against the subsequent insult. However, patients with Baló's disease frequently display a concentric pattern at the time of initial presentation on MRI. Successive formation of alternating layers of apoptotic and preconditioned oligodendrocytes cannot adequately account for the simultaneous emergence of these concentric rings (17).

The expression of inducible nitric oxide synthase (iNOS), a pro‐inflammatory factor, has been observed in macrophages in actively demyelinating plaques and microglia at the plaque edges in concentric lesions in Baló's disease (31). Therefore, macrophages and microglia are thought to play a key role in the formation of Baló's concentric rings. However, the distribution patterns and roles of microglia and macrophages remain unclear, in part because of a lack of specific markers that clearly discriminate these cells.

Recently, TMEM119, a transmembrane protein, was reported to be a highly specific marker for microglia that is not expressed by recruited peripheral blood macrophages or other immune or neural cell types (2). The purinergic receptor P2Y12 (P2RY12) was also proposed as a specific marker of healthy “homeostatic” rodent CNS microglial cells, discriminating these cells from resident macrophages and blood‐derived monocytes (5). Zrzavy et al reported that microglia highly expressing TMEM119 lost expression of P2RY12 in active MS lesions, whereas the expression of CD68, a phagocyte‐associated activation marker for microglia/macrophages and CD163 (scavenger receptor cysteine‐rich protein), a marker of perivascular macrophages, remained in infiltrated macrophages in both active and inactive MS lesions (37). In addition, glucose transporter 5 (GLUT5), a member of the facilitated‐diffusion glucose transporter family, is exclusively expressed in microglia in the human and rat brain (15, 28). However, the expression pattern of GLUT5 has not been investigated in demyelinating disease. In the present study, we investigate the distribution patterns of microglia and macrophages in the concentric and lamellar lesions in Baló's disease and NMOSD using these new specific markers that distinguish these cell types.

Materials and Methods

Autopsy samples

Immunohistological studies were performed on archival autopsied brain specimens containing concentric demyelinating lesions with lamellar configuration from three Filipino cases pathologically diagnosed with Baló's disease, one Filipino case with Baló‐like lesions and one Japanese NMOSD case with anti‐AQP4 antibody (16) (Table 1 and Figure S1). The NMOSD case showed optic nerve involvement and longitudinally extensive spinal cord lesions fulfilling Wingerchuk's criteria (36) and displayed pathologically typical lesions (16). Two cases of Baló's disease (Baló‐2 and ‐4) are the same as those in our previous reports (22, 24). Cases of myasthenia gravis (MG; n = 1), limbic encephalitis (n = 1), bacterial meningoencephalitis (n = 1) and cerebral infarction (n = 1) were used as controls.

Table 1.

Demographic features of autopsied Baló's disease cases

Autopsied cases	Age (years)	Sex	Disease duration (years)	Clinically estimated sites of lesions	Pathologically determined sites of lesions
Baló‐1	23	M	0.03	Cr	Cr (3)*
Baló‐2	23	M	0.5	Cr	Cr (1)
Baló‐3	39	M	0.03	Cr	Cr (1)
Baló‐4	40	F	0.6	Cr	Cr (3)
NMOSD † with lamellar lesions	65	F	26.0	O, S, Cl, Cr	Cr (2)

All autopsied Baló's disease cases presented a monophasic disease culminating in death. An NMOSD case had multiple relapses and finally died of exacerbation of infection.

Abbreviations: Cl = cerebellum; Cr = cerebrum; F = female; M = male; NMOSD = neuromyelitis optica spectrum disorder; O = optic nerve; S = spinal cord.

^*Number of lesions in parenthesis.

^†An NMOSD case seropositive for anti‐AQP4 antibodies.

Tissue preparation and immunohistochemistry

All autopsied cases were obtained from the Department of Neurology, Kyushu University, except for the NMOSD case with anti‐AQP4‐antibody, which was obtained from Iou Hospital. Because the cases with Baló's disease died prior to the introduction of the anti‐AQP4 antibody assay, the serostatus of these cases was not assessed. The age at autopsy ranged from 23 to 40 years for the Baló's disease cases (three male and one female), while that for the NMOSD case was 65 years. Disease duration was 0.03–0.6 years for the Baló's disease cases and 26.0 years for the NMOSD case (Table 1). Autopsy specimens were fixed in 10% buffered formalin and processed into paraffin sections. The sections were routinely treated with hematoxylin and eosin (H&E) and Klüver–Barrera (KB) stains. The primary antibodies used for immunohistochemistry are listed in Table 2. TMEM119 and P2RY12 were used as general and resting microglia‐specific markers, respectively, while GLUT5, Iba1, CD68 and CD163 were used as microglia/macrophage markers. All sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Endogenous peroxidase activity was blocked with 0.3% (v/v) H₂O₂/methanol. Sections were then incubated with primary antibody at 4°C overnight. After rinsing, sections were incubated with either a streptavidin‐biotin complex or enhanced indirect immunoperoxidase reagents using Envision (DakoCytomation, Glostrup, Denmark). Immunoreactivity was detected using 3,3′‐diaminobenzidine and sections were counterstained with hematoxylin.

Table 2.

Antibodies used for immunohistochemistry

Antigen	Type	Dilution	Antigen retrieval	Source
Astrocyte
AQP4	rabbit polyclonal	1:500	N.D.	Santa Cruz Biotechnology, Dallas, TX, USA
GFAP	rabbit polyclonal	1:1000	N.D.	DakoCytomation, Glostrup, Denmark
Oligodendrocyte/myelin
MAG	rabbit polyclonal	1:400	N.D.	Sigma Aldrich, St Louis, MO, USA
MOG	rabbit polyclonal	1:1000	N.D.	Sigma Aldrich, St Louis, MO, USA
MBP	rabbit polyclonal	1:5000	N.D.	IBL, Fujioka, Japan
CNPase	mouse monoclonal	1:1000	Autoclave/10 mM citrate buffer	Abcam, Cambridge, UK
NG2	rabbit polyclonal	1:200	N.D.	Sigma Aldrich, St Louis, MO, USA
Macrophage/monocyte/microglia
CD68	mouse monoclonal	1:200	Autoclave/10 mM citrate buffer	DakoCytomation, Glostrup, Denmark
TMEM119 (HPA051870)	rabbit polyclonal	1:100	Autoclave/10 mM citrate buffer	Sigma Aldrich, St Louis, MO, USA
GLUT5 (HPA005449)	rabbit polyclonal	1:400	Autoclave/10 mM citrate buffer	Sigma Aldrich, St Louis, MO, USA
CD163	mouse monoclonal	1:1000	Autoclave/10 mM citrate buffer	Leica Biosystems, Newcastle, UK
P2RY12	rabbit polyclonal	1:200	Autoclave/10 mM citrate buffer	Sigma Aldrich, St Louis, MO, USA
Others
C3d	rabbit polyclonal	1:1000	Autoclave/10 mM citrate buffer	DakoCytomation, Glostrup, Denmark
iNOS	mouse monoclonal	1:100	Autoclave/10 mM citrate buffer	Santa Cruz Biotechnology, Dallas, TX, USA
iNOS	rabbit polyclonal	1:200	Autoclave/10 mM citrate buffer	Thermo Fisher Scientific, Waltham, MA, USA
Arginase‐1	rabbit polyclonal	1:200	Autoclave/10 mM citrate buffer	Sigma Aldrich, St Louis, MO, USA

Abbreviations: AQP4 = aquaporin‐4; CNPase = 2′,3′‐Cyclic‐nucleotide 3′‐phosphodiesterase; GFAP = glial fibrillary acidic protein; GLUT5 = facilitated glucose transporter 5; iNOS = inducible nitric oxide synthase; MAG = myelin‐associated glycoprotein; MBP = myelin basic protein; MOG = myelin oligodendrocyte glycoprotein; N.D. = not done; P2RY12 = purinergic receptor P2Y12; TMEM119 = transmembrane protein 119.

Indirect immunofluorescence and confocal laser microscopy

Using the same set of paraffin sections described above, double immunofluorescence staining was performed with the following combinations of antibodies: mouse monoclonal anti‐CD68 and rabbit polyclonal anti‐GLUT5; mouse monoclonal anti‐iNOS and rabbit polyclonal anti‐TMEM119. All sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Sections were then incubated with primary antibodies at 4 °C overnight. After rinsing, sections were incubated with Alexa 488‐conjugated goat anti‐mouse IgG and Alexa 546‐conjugated goat anti‐rabbit IgG (Invitrogen), and then, counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI). Images were captured using a confocal laser microscope system (Nikon A1, Nikon, Tokyo, Japan). We used the sequential multiple fluorescence scanning mode to avoid nonspecific overlap of colors and captured all images under the same conditions of magnification, laser intensity, gain and offset values and pinhole setting.

Evaluation of demyelinating activity in concentric lesions

Demyelinating activity within the lesions was determined by the presence of macrophages or microglia containing myelin degradation products, according to Brück and colleagues (3). An early active lesion was defined by the presence of macrophages or microglia with minor myelin degradation products that were reactive for MAG or MOG. A late active lesion was defined by the presence of macrophages or microglia containing degradation products that were reactive for major myelin proteins, such as myelin basic protein (MBP). A macrophage‐rich inactive lesion was defined by the presence of macrophages containing neutral lipid degradation products without reactivity for myelin proteins (31).

Quantitative evaluation

To facilitate the analysis, we identified and named each alternating concentric demyelinating and myelinated layer (ring) in our autopsied cases with Baló's disease (Figure S1). The outermost demyelinating layer was named DMY‐MO and the outermost myelinated layer was named MY‐MO. All adjacent layers were then named numerically according to position (i.e., DMY‐1, MY‐1, DMY‐2 and MY‐2). For the quantitative evaluation, immunohistochemically stained sections were overlaid with a morphometric grid (0.4 mm²) using the 10 × objective (Olympus) and four fields per region were quantified. For each case, the average counts per square millimeter were calculated for each region of interest and compared using statistical analysis.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 7. Because of the uneven distribution of our data, the statistical analysis was performed using nonparametric tests. Differences between two groups were assessed using the Kruskal–Wallis test. For multiple testing, significant values were corrected using the Bonferroni–Dunn procedure. A P‐value <0.05 was considered statistically significant.

Ethics statement

The study was approved by The Kyushu University Institutional Review Board for Clinical Research.

Results

Expression pattern of microglia in histologically normal brain

In the normal cerebral white matter from an MG case (for control), TMEM119, P2RY12 and GLUT5 were expressed in the cell bodies and fine processes of the resting microglia (so‐called ramified microglia; Figure S2A,C,E). In the cerebral cortex, the expression pattern of TMEM119, P2RY12 and GLUT5 were similar to those in normal white matter and similar to each other (Figure S2B,D,F). Cell bodies of resting microglia were positive for CD68, although immunoreactivity was weak in their fine processes in both cerebral white and gray matter (Figure S2G,H). Perivascular macrophages were positive for CD163 (Figure S2I,J, arrows), while parenchymal microglia were negative.

TMEM119‐positive microglia preferentially accumulate in the outermost demyelinating layers showing DO in Baló's disease

CD68 and CD163 were most abundantly expressed in DMY‐MO, but were also present throughout the lesion center in the Baló‐2 case (Figure 1A–C). In contrast, immunoreactivities for GLUT5 and TMEM119 were markedly upregulated in DMY‐MO even compared with peri‐plaque white matter (PPWM) and myelinated layers where TMEM119‐ and P2RY12‐positive microglia were also seen (Figure 1D,E). In this case, several macrophages or microglia contained myelin debris that was reactive for MAG in DMY‐MO, which indicated early active demyelinating lesions (Figure 1F). DMY‐MO showed DO features, which were characterized by preferential loss of MAG compared with MOG (Figure 1G–I). In the inner portion of the DMY‐MO with DO, KB staining revealed sharply demarcated demyelination associated with CD68‐positive foamy macrophages and TMEM119‐positive activated microglia, which were characterized by swollen ramified cells with a larger cell body and shorter, thick processes (Figure 1J,K,M,N,P,Q). In contrast, in the outer portion of DMY‐MO with DO, CD68‐positive macrophages did not display a foamy morphology, although TMEM119‐positive microglia were highly activated (Figure 1L,O,R). These findings suggest that microglia detected by TMEM119 or GLUT5 are preferentially activated in DMY‐MO with DO features before CD68‐positive macrophages transition to a foamy morphology.

Figure 1.

Distinct distribution patterns of microglia and macrophages in a Baló's lesion. Case Baló‐2 (A–Q). Macroscopic view of a concentric demyelinating lesion (A–E). KB staining shows a concentric demyelinating lesion in the cerebral white matter (A). The myelin is relatively preserved in the outer‐most demyelinating layer (DMY‐MO) (A, arrowheads). The highest numbers of CD68‐ and CD163‐positive cells are found in layer DMY‐MO (B, C, arrowheads), but are also seen throughout the lesion center (B, C). In contrast, GLUT5 and TMEM119 are confined to cells in DMY‐MO (D, E, arrowheads). Immunostaining for MAG shows numerous foamy macrophages or microglia containing myelin debris in DMY‐MO (F). Higher magnification view of the outer layers of the concentric rings indicated by the square in panel A (G–I). In DMY‐MO, indicated by asterisks in panel G–I, expression of MAG is completely absent, whereas KB and MOG are relatively preserved (distal oligodendrogliopathy; DO) (G–I). Higher magnification view of DMY‐MO, indicated by a square, in panel G (K, N, Q). Panels J, M and P show the inner portion of the DMY‐MO, indicated by the dotted square in panels K, N and Q. The dotted lines show the boundary of DMY‐MO and the outermost myelinated layer (MY‐MO). KB staining shows a reduction in the density of myelin (J). CD68‐positive foamy macrophages and TMEM119‐positive activated microglia are abundant in the inner portion of layer DMY‐MO (M, P). Panels L, O and R show the outer portion of layer DMY‐MO, indicated by the square in panels K, N and Q; arc lines indicate the boundary of the DMY‐MO and the peri‐plaque white matter (PPWM). KB staining shows slightly decreased myelin density in the outer portion of layer DMY‐MO (L), while some CD68‐positive macrophages have infiltrated the lesion, but do not display a foamy morphology (O). TMEM119‐positive microglia are markedly activated in the outer portion of DMY‐MO (R). Scale bars: 1 mm (A–E, G–I), 10 µm (E), 50 µm (J, L, M, O, P and R), 200 µm (K, N and Q).

Marked upregulation of GLUT5 in TMEM119‐positive microglia in the outermost demyelinating layers showing DO in Baló's disease

DMY‐MO in the Baló‐1 case also displayed signs of early active demyelination with DO (Figure 2A–D). CD68‐ and CD163‐positive macrophages had abundantly infiltrated into DMY‐MO as well as DMY‐1 (Figure 2E,F). In contrast, TMEM119‐ and GLUT5‐positive microglia had predominantly accumulated in DMY‐MO (Figure 2G,H). P2RY12‐positive microglia were also observed at the outer and inner portions of DMY‐MO, but with far less infiltration than TMEM119‐ or GLUT5‐positive microglia (Figure 2I). Notably, immunoreactivities for CD68, CD163, TMEM119, GLUT5 and P2RY12 were sparse in MY‐MO (Figure 2E–I). In addition, double immunofluorescence revealed robust GLUT5‐positive microglial activation at the outer portion of DMY‐MO, while CD68‐positive macrophages were sparse (Figure 2J–M). Many GLUT5‐positive microglia were observed in the center of DMY‐MO and these cells were only weakly stained for CD68 (Figure 2N–Q). In contrast, foamy macrophages were strongly labeled for CD68 and almost negative for GLUT5 in DMY‐1 (Figure 2R–U). Double immunofluorescence revealed that TMEM119‐positive activated microglia produced iNOS in the outer portion as well as the center of layer DMY‐MO (Figure S3A–H), while immunoreactivity for arginase‐1 was not detected in these cells (Figure S3I–L).

Figure 2.

Accumulation of GLUT5‐positive microglia in a DO lesion in Baló's disease. Case Baló‐1 (A–U). Macroscopic view showing a large whiter matter concentric lesion with KB staining (A). Panel B shows KB staining with a higher magnification view of the area in the square in panel A (B). The outermost demyelinating layer (DMY‐MO) has relative preservation of myelin, indicated by an asterisk (B). Expression of MAG is almost completely absent, while the expression of MOG is preserved in DMY‐MO (C, D). CD68‐ and CD163‐positive macrophages have broadly infiltrated the demyelinating layers (E, F). In contrast, TMEM119‐ and GLUT5‐positive microglia are preferentially distributed in layer DMY‐MO (G, H). Immunoreactivity for P2RY12 is weak at the inner and outer portions of DMY‐MO (I). Higher magnification view of the outer portion of DMY‐MO, indicated by a square in panel H (J–M). Double immunofluorescence labeling for GLUT5 and CD68 showing that GLUT5‐positive microglia are markedly accumulated at the outer portion of DMY‐MO, whereas CD68‐positive macrophages are scarce (J–M). Numerous GLUT5‐positive microglia weakly co‐express CD68 at the lesion center of DMY‐MO (N–Q). In contrast, CD68 expression is upregulated on foamy macrophages, while GLUT5 expression is almost negative in DMY‐1 (R–U). Scale bars: 1 mm (A–I), 100 µm (J–M) and 50 µm (N–U).

Apoptotic oligodendrocytes and oligodendrocyte precursor cells in the demyelinating layer with accumulation of TMEM119‐ and GLUT5‐positive microglia

The Baló‐1 case had another large concentric demyelinating lesion in the cerebral white matter that showed signs of DO in the DMY‐MO layer (Figure S4A–E). TMEM119‐ and GLUT5‐positive microglia had predominantly accumulated in DMY‐MO (Figure S4F,G). In contrast, immunoreactivity for CD68 was observed in DMY‐MO as well as the other layers (Figure S4H). Strong immunoreactivity for iNOS was detected in DMY‐MO (Figure S4I). Numerous mature oligodendrocytes and NG2‐positive oligodendrocyte precursor cells (OPCs) were found in DMY‐MO; however, most of these cells had apoptotic‐like nuclear condensation (Figure S4J–M). Both mature oligodendrocytes and OPCs were almost undetectable in the other demyelinating layers, including DMY‐1 and −2, without TMEM119‐ and GLUT5‐positive microglia (Figure S4J,L). These findings suggest that apoptotic oligodendrocytes and OPCs with abnormal morphology are present in layer DMY‐MO, along with an accumulation of TMEM119‐ and GLUT5‐positive microglia.

Expression pattern of microglia and macrophage markers in lesions without DO features in Baló's disease

The Baló‐4 case had a cerebral concentric lesion showing a graded decline in myelin density toward the lesion center in the preserved myelin layers (Figure 3A). Immunostaining for MAG and MOG showed a similar demyelination pattern, while MBP‐positive macrophages or microglia were seen in all demyelinating layers, which indicated late active demyelination (Figure 3B–D). CD68‐positive cells had highly infiltrated in the PPWM, demyelinating and myelinated layers (Figure 3E), while TMEM119‐ and P2RY12‐positive microglia were mainly seen in the PPWM and myelinated layers (Figure 3F,G). There were numerous TMEM119‐, GLUT5‐ and P2RY12‐positive activated microglia in the PPWM (Figure 3H–J). While CD68 expression was also seen in activated microglia, CD163 was restricted to perivascular macrophages in the PPWM (Figure 3K,L). TMEM119‐, GLUT5‐ and P2RY12‐positive microglial density showed a relative decline in layer DMY‐MO, whereas CD68‐ and CD163‐positive macrophages were more abundant (Figure 3M–O). In layer MY‐MO, TMEM119‐, GLUT5‐ and P2RY12‐positive microglia were abundant, whereas CD68‐ and CD163‐positive macrophages were relatively reduced (Figure 3R–V). In layer MY‐2, TMEM119‐positive microglia were rare, while GLUT5‐positive microglia and CD68‐ and CD163‐positive macrophages with a foamy morphology were abundant (Figure 3W–Aa).

Figure 3.

Microglia and macrophages in lesions without DO in Baló's disease. Case Baló‐4 (A–Aa). KB staining showing a large concentric lesion in the cerebral white matter (A). MAG and MOG expression levels are diminished to the same extent in each demyelinating layer (B, C). MBP‐positive macrophages or microglia are seen in all demyelinating layers (D). CD68‐positive cells are seen in both demyelinating and myelinated layers, as well as in peri‐plaque white matter (PPWM) (E). TMEM119‐ and P2RY12‐positive microglia are observed in the myelinated layers (F, G). In the PPWM, activated microglia are positive for TMEM119, GLUT5, P2RY12 and CD68, whereas CD163 expression is restricted to perivascular macrophages (H–L). In the outermost demyelinating layer (DMY‐MO), immunoreactivities for TMEM119, GLUT5 and P2RY12 are diminished, while CD68 and CD163 are robustly detected in both parenchymal and perivascular (indicated by a star) macrophages (M–Q). In the outermost myelinated layer (MY‐MO), TMEM119, GLUT5 and P2RY12 are expressed by microglia, and some infiltrated macrophages are positive for CD68 and CD163 (R–V). In MY‐2, TMEM119‐positive microglia are rare (W), while GLUT5 and P2RY12 are expressed by microglia/macrophages with a foamy morphology (X, Y). CD68 and CD163 are expressed by foamy macrophages (Z, Aa). Scale bars: 1 mm (A–C, E–G), 10 µm (D) and 50 µm (H–Aa).

Focal accumulation of TMEM119‐positive microglia in the myelinated layers of lamellar lesions in the NMOSD case

The NMOSD case had a large lesion showing lamellar demyelinating structure without DO in the cerebral white matter (Figure S5A–C). All demyelinating layers showed late active demyelination, which had MBP‐positive macrophages or microglia (Figure S5D). Marked degeneration of GFAP‐positive astrocytes was detected in both the demyelinating and myelinated layers; however, immunoreactivity for AQP4 was diminished not only in the demyelinating layers, but also partly in the myelinated layers (Figure S5E–H). The expression of MBP was preserved in the myelinated layers (Figure S5I). A perivascular lesion distant from this lamellar lesion showed typical astrocytopathy, with loss of AQP4 on degenerating astrocytes without demyelination (Figure S5J–L). Complement deposition was not detected in this lesion (data not shown). TMEM119‐ and P2RY12‐positive microglia were accumulated in this lesion (Figure S5M–O). In the lamellar lesion, CD68‐positive macrophages were detected in both demyelinating and myelinated layers (Figure 4A,B). However, immunoreactivity for TMEM119 was predominantly seen in the outer portion of each myelinated layer (Figure 4C). P2RY12‐positive microglia were also mostly seen in myelinated layers (Figure 4D). Numerous CD68‐positive cells had infiltrated layer DMY‐MO (Figure 4E,F). In contrast, TMEM119‐positive cells had accumulated in layer MY‐MO with a linear pattern (Figure 4G) and there was diffuse infiltration of P2RY12‐positive cells in MY‐MO (Figure 4H). Higher magnification of layer DMY‐MO showed that CD68‐positive foamy microglia/macrophages were abundant, while TMEM119‐ and P2RY12‐positive microglia were scarce in this layer (Figure 4I–K). TMEM119‐ and P2RY12‐positive microglia without foamy morphology were localized in layer MY‐MO (Figure 4L–N). Immunoreactivity for iNOS was negative in microglia of myelinated layers (data not shown).

Figure 4.

Focal accumulation of TMEM119‐positive microglia in preserved myelin layers of an NMOSD case with lamellar lesions. Lamellar demyelinating lesion from the same case of NMOSD described in Figure S5. CD68‐positive cells have infiltrated both demyelinating and myelinated layers (A, B). TMEM119‐positive cells are confined to the outer portion of each myelinated layer in this lesion (C, arrows). P2RY12‐positive microglia are also abundantly present in myelinated layers and have accumulated to some extent in the outer portions of the myelinated layers (D, arrows). KB staining showing the outermost demyelinating layer (DMY‐MO) and the outermost myelinated layer (MY‐MO) in this lesion (E). CD68‐positive cells have predominantly infiltrated DMY‐MO compared with MY‐MO (E, F). In contrast, TMEM119‐and P2RY12‐positive cells have preferentially accumulated in the MY‐MO (G, H). Higher magnification views showing DMY‐MO (I–K) and MY‐MO (L–N). CD68 is expressed by macrophages with a foamy morphology in DMY‐MO (I), and a few TMEM119‐ and P2RY12‐positive microglia are detected (J, K). CD68‐positive cells are diffusely distributed throughout layer MY‐MO (L), whereas TMEM119‐positive microglia are focally localized in the outer portion of MY‐MO (M). P2RY12‐positive microglia are diffusely seen in MY‐MO (N). Scale bars: 1 mm (A–D), 200 µm (E–H), 100 µm (I–K) and 50 µm (L–N).

Quantitative immunohistochemical profile of microglia and macrophages in concentric lesions with or without DO

Based on the immunohistochemical patterns in each case (described above), we analyzed microglia/macrophage molecules by quantitative immunohistochemistry in relation to the demyelinating activity in the Baló's disease and NMOSD cases, with or without DO (Figure 5).

Figure 5.

Quantitative analysis of microglia and macrophages in the concentric demyelinating lesions. (A–F) Concentric demyelinating lesions with distal oligodendrogliopathy (DO). TMEM119‐ and GLUT5‐positive cells are significantly increased in early active (EA) lesions of the outermost demyelinating layer (DMY‐MO) compared with the normal‐appearing white matter (NAWM) (A, B). Conversely, these cells are significantly decreased compared with NAWM in late active (LA) lesions (A, B). Both demyelinating and myelinated layers show a significant reduction in P2RY12‐positive cells compared with NAWM (C). Numerous CD68‐ and CD163‐positive macrophages are still present in LA lesions (E, F). CD163‐positive cells are also significantly increased compared with NAWM in all myelinated layers with macrophage‐rich inactive (IA) lesions (E). (F) A schematic drawing of microglia/macrophage distribution in concentric lesions with DO. TMEM119‐positive, GLUT5‐positive and P2RY12‐negative activated microglia predominantly accumulate in the outer portion of DMY‐MO. These activated microglia in DMY‐MO with DO features express iNOS. Although CD68/CD163‐positive foamy macrophages are distributed in all demyelinating layers, these cells demonstrated foamy morphology in the inner portion but not in the outer portion. (G–L) Concentric demyelinating lesions without DO. All demyelinating layers, including DMY‐MO, show a significant reduction of TMEM119‐, GLUT5‐ and P2RY12‐positive cells compared with NAWM (G–I). In contrast, TMEM119‐ and GLUT5‐positive cells are significantly increased in MY‐MO compared with NAWM (G, H). CD68‐ and CD163‐positive macrophages are significantly increased throughout the demyelinating and myelinated layers (J, K). (L) In concentric lesions without DO, TMEM119‐positive, GLUT5‐positive and P2RY12‐positive activated microglia with ramified morphology are accumulated in the outer portion of the myelinated layers, while CD68/CD163‐positive macrophages with foamy morphology are distributed among all demyelinating layers. The values for TMEM119, GLUT5, P2RY12, CD68 and CD163 are cells per mm²; thus, the numbers in the y‐axes are directly comparable. *P < 0.05; **P < 0.01; ***P < 0.001.

On average, in concentric lesions with DO, DMY‐MO with early active demyelination contained four times more TMEM119‐ and GLUT5‐positive microglia compared with NAWM (P < 0.001). In contrast, in DMY‐1 and DMY‐2, there were one‐third or less of these microglia compared with NAWM (P < 0.001; Figure 5A,B). P2RY12‐positive microglia were significantly reduced in DMY‐MO and MY‐2 (P < 0.05), as well as in the other demyelinating and myelinated layers (P < 0.001; Figure 5C). Furthermore, CD68‐ and CD163‐positive macrophages were significantly increased in all demyelinating layers (P < 0.001; Figure 5D,E). In contrast, in the cases without DO, the numbers of TMEM119‐, GLUT5‐ and P2RY12‐positive microglia were significantly increased in MY‐MO compared with NAWM (P < 0.05), but were reduced in all demyelinating layers with late active demyelination (P < 0.01 for TMEM119 and GLUT5; P < 0.05 for P2RY12; Figure 5G–I). In addition, CD68‐ and CD163‐positive macrophages were significantly increased in all demyelinating and myelinated layers (P < 0.05 in MY‐MO and MY‐2, P < 0.01 in DMY‐MO, DMY‐1, MY‐1 and DMY‐2 for CD68; P < 0.001 for CD163; Figure 5J,K).

Distribution pattern of microglia and macrophages in other neurological diseases

In the case of bacterial meningoencephalitis, CD68 and CD163 were markedly upregulated in activated macrophages in the meninges, while TMEM119, GLUT5 and P2RY12 were nearly undetectable (Figure S6A–F). In contrast, TMEM119, GLUT5 and P2RY12 were upregulated in activated microglia at the cortical surface (Figure S6D–F). Double immunofluorescence labeling showed that macrophages and microglia displayed distinct CD68 and GLUT5 expression patterns (Figure S6G–J). Some macrophages robustly expressed both CD68 and GLUT5 (Figure S6G–J, arrows). In the case with cerebral infarction, necrotic changes, with infiltration of numerous CD68‐ and CD163‐positive macrophages, were observed in the entire subacute ischemic lesion area (Figure S6K–M). TMEM119‐and P2RY12‐positive microglia were nearly absent in the lesion center, while many activated microglia expressed TMEM119 at the lesion edge (Figure S6N,P). GLUT5‐positive activated microglia were detected around the lesion and some macrophages were weakly positive for this marker, even in the lesion center (Figure S6O).

Discussion

In the present study, we analyzed the expression and distribution patterns of microglia‐ and macrophage‐specific markers to elucidate the pathogenetic mechanisms involved in the formation of concentric lesions in Baló's disease and NMOSD. Our main findings were as follows: (i) TMEM119‐ and GLUT5‐positive microglia are abundant in layer DMY‐MO with DO, whereas CD68‐ and CD163‐positive macrophages were seen throughout the lesion; (ii) In layer DMY‐MO with DO, activated TMEM119‐ and GLUT5‐positive microglia accumulated in the outer portion, while CD68‐positive macrophages with a foamy morphology were abundant in the inner portion; (iii) P2RY12 was downregulated on microglia in layer DMY‐MO with DO features; (iv) TMEM119‐positive activated microglia expressed iNOS; (v) Apoptotic oligodendrocytes and OPCs exhibiting an abnormal morphology with nuclear condensation were seen in DMY‐MO with DO and (vi) TMEM119‐ and P2RY12‐positive microglia had accumulated in the myelinated layers in Baló's disease and NMOSD cases without DO features.

TMEM119, also known as osteoblast induction factor (Obif), is expressed by differentiating osteoblasts and has an important role in bone formation and normal bone mineralization (4, 33). In the CNS, TMEM119 is specifically expressed by microglia, but not peripheral blood‐derived macrophages (2, 5). Satoh et al observed TMEM119‐positive microglia in neurodegenerative diseases, but not in demyelinating lesions in MS (30). In contrast, Zrzavy et al reported that a large proportion of phagocytes present in active MS lesions are derived from TMEM119‐positive microglia (37). In addition, the proportion of TMEM119‐negative macrophages increased as the demyelinating lesions matured, which suggests that macrophages in chronic MS lesions originate from the systemic circulation (37). In this study, we showed that TMEM119‐positive microglia are abundant in DMY‐MO with DO features, which are a pathological hallmark of early active lesions in Baló's disease (31). Intriguingly, TMEM119‐positive activated microglia display a ramified morphology in DMY‐MO with DO features, but not a foamy morphology typical of CD68‐positive macrophages. This suggests that these microglia are likely to play an important role, apart from phagocytosis, in the evolution of lamellar demyelination and DO in Baló's disease. Activated microglia produce various pro‐inflammatory mediators, including glutamate, matrix metalloproteinases, reactive oxygen and nitrogen species, chemokines and cytokines, which can induce oligodendrocyte death (29). Several pro‐inflammatory cytokines and nitrogen species released by microglia have been detected in lesions in MS and Baló's disease (1, 7, 31), which suggests a correlation between microglial activity and oligodendrocyte damage. Indeed, we previously showed that TMEM119‐positive activated microglia produce iNOS in DMY‐MO with DO in Baló's disease, along with the presence of apoptotic oligodendrocytes (22, 31). Here, we found that OPCs exhibited an abnormal morphology with nuclear condensation in DMY‐MO with DO features and were comparatively scarce in the other demyelinating layers, which suggests that activation of TMEM119‐positive microglia may cause OPC damage. Indeed, in vitro, microglia activated with lipopolysaccharide (LPS) arrest OPC proliferation and induce OPC death (27). Furthermore, we observed downregulation of P2RY12, a marker of homeostatic microglia, in DMY‐MO with DO features, which is consistent with findings on active MS lesions in previous studies (25, 37). Hence, early activation of TMEM119‐positive microglia may contribute to the pathogenesis of DO. Intriguingly, we found that TMEM119‐ and P2RY12‐positive microglia were highly abundant in myelinated layers of cases without DO features. In contrast, CD68‐positive macrophages with a foamy morphology were abundant in the demyelinating layers. These findings suggest that TMEM119‐ and P2RY12‐positive microglia may be protective against demyelination. Microglia/macrophages exhibiting an anti‐inflammatory phenotype produce a variety of antioxidants, promote neurogenesis, clear cell and tissue debris and suppress inflammation (9). Moreover, as anti‐inflammatory microglia can stimulate remyelination (6, 26), TMEM119‐ and P2RY12‐positive microglia may maintain the myelinated layer and promote remyelination in Baló's disease. Alternatively, TMEM119‐positive microglia may infiltrate the myelinated layers and initiate demyelination. However, this is unlikely because these cells express P2RY12 and display a ramified morphology, similar to healthy homeostatic microglia.

GLUT5, encoded by SLC2A5, is a high‐affinity fructose transporter expressed in mammalian cells, with very low affinity for other carbohydrates such as glucose and galactose (4, 34). GLUT5 is a marker of human microglia (15, 28) and cultured macrophages (11, 20). However, the expression pattern of GLUT5 in human demyelinating diseases remained unknown. Here, we found highly activated GLUT5‐expressing microglia in layer DMY‐MO with DO features, similar to the distribution of TMEM119‐positive microglia. GLUT5 expression in activated microglia gradually diminished toward the lesion center, suggestive of a role of GLUT5‐positive microglia in active demyelination. Thus, GLUT5 may be a potential therapeutic target for the treatment of active demyelinating lesions. In fact, GLUT5 is considered a potential therapeutic target for diabetes and cancer (12) because it is upregulated in these diseases (10, 32). Interestingly, we found a difference in distribution patterns between GLUT5‐ and TMEM119‐positive microglia. In the MY‐2 of the Baló‐4 patient, GLUT5‐positive microglia were abundant, while TMEM119‐positive microglia were scarce. Moreover, in the case of cerebral infarction, GLUT5‐positive activated microglia were detected around the lesion, including the lesion center, where TMEM119‐positive microglia were nearly absent. These findings suggest that GLUT5 and TMEM119 are differentially expressed by microglia and that the GLUT5 expression may persist longer than the TMEM119 expression in hypoxic and inflammatory states. Functional studies are required to more fully clarify the role of GLUT5‐positive microglia in demyelinating diseases.

The present study has limitations. It was extremely difficult to obtain autopsied tissue from cases with Baló's disease, because of the rarity of this disorder. Therefore, we were able to perform only a limited number of histochemical and immunofluorescence labeling assays to assess coexpression profiles of the microglia/macrophage markers. Furthermore, because we divided the cases into two distinct groups according to DO features, the numbers in each group became even smaller. Hence, future research with a larger number of cases is needed.

In conclusion, we discriminated the distribution pattern of microglia and macrophages in concentric and lamellar lesions in Baló's disease and NMOSD. Activated microglia expressing TMEM119 and GLUT5 and lacking P2RY12, may play a critical role in the evolution of demyelinating layers in Baló's disease. GLUT5 in microglia might, therefore, be a potential therapeutic target for demyelinating diseases.

Conflict of Interest

Dr. Kira received consultant fees, speaking fees and/or honoraria from Novartis Pharma, Mitsubishi Tanabe Pharma, Boehringer Ingelheim, Teijin Pharma, Takeda Pharmaceutical Company, Otsuka Pharmaceutical, Astellas Pharma, Pfizer Japan, Sumitomo Dainippon Pharma and Eisai. Other authors declare that they have no conflict of interest.

Supporting information

Fig S1

Figure S1. Overview of demyelinating and myelinated layers in Baló's disease and NMOSD. Panel A shows a schematic of the concentric rings. We termed the outermost demyelinating layer DMY‐MO and the outermost myelinated layer MY‐MO. All demyelinating and myelinated layers were then named according to numerical order (ie, DMY‐1, MY‐1, DMY‐2 and MY‐2). Panel B–F shows representative images of the positional relationship of each layer analyzed in the cases of Baló's disease and NMOSD. Peri‐plaque white matter (PPWM) is the preserved myelin region adjacent to DMY‐MO. Scale bars: 1 mm.

Fig S2

Figure S2. Normal expression pattern of microglia and macrophages in a control brain. Corpus callosum (A, C, E, G, I) and cerebral cortex (B, D, F, H, J) from a case of myasthenia gravis. TMEM119 is expressed on microglial cell bodies and processes in both the corpus callosum and cortex (A, B). P2RY12 (C, D) and GLUT5 (E, F) are also preferentially expressed on cell bodies and processes of resting microglial cells. CD68 is mainly expressed on the microglial soma and faintly expressed on ramified processes in both cerebral white and gray matter (G, H). CD163 is specifically expressed in perivascular macrophages (I, J, arrows) but not parenchymal microglia. Scale bars: 50 µm.

Fig S3

Figure S3. TMEM119‐positive microglia in DMY‐MO produce iNOS. Case Baló‐1 (A–H). Panels A–D show higher magnification view of the outer portion of the outermost demyelinating layer (DMY‐MO), indicated by a square in Figure 2H. Double immunofluorescence showing that accumulated TMEM119‐positive microglia produce iNOS (A–D). Also in the center of DMY‐MO, TMEM119‐positive activated microglia are positive for iNOS (E–H). Arginase‐1 is not expressed in TMEM119‐positive activated microglia in layer DMY‐MO (I–L). Scale bars: 100 µm (A–D) and 50 µm (E–L).

Fig S4

Figure S4. Damaged oligodendrocytes and oligodendrocyte precursor cells in the outermost demyelinating layer are observed together with abundant TMEM119‐ and GLUT5‐positive microglia. Case Baló‐1 (A–M). Panel A shows macroscopic view of the concentric lesions by KB staining (A). Panel B is a higher magnification view of the area in the square in panel A (B). Immunolabeling for MAG (C), MOG (D) and CNPase (E) shows preferential loss of MAG and CNPase with myelin debris‐containing macrophages or microglia (C, inset), but not MOG, in the outermost demyelinating layer (DMY‐MO, asterisk in panel C). TMEM119 (F) and GLUT5 (G) are upregulated in the DMY‐MO, but not in the other layers (arrows). CD68 is abundantly expressed in layer DMY‐MO, as well as the other layers (H). iNOS immunoreactivity is robust in DMY‐MO (I, arrows). TMEM119 and iNOS are strongly expressed by activated microglia (F, I, insets). Panel J is a higher magnification view of the area in the square in panel E. Numerous mature oligodendrocytes are localized in DMY‐MO (J, asterisk); however, most of these oligodendrocytes have an apoptotic‐like morphology with nuclear condensation (K, arrows). Several NG2‐positive oligodendrocyte precursor cells (OPCs) are also present in DMY‐MO (L, asterisk), but these OPCs display an abnormal morphology with nuclear condensation (M, arrows). Scale bars: 1 mm (A–I), 500 µm (J, L) and 20 µm (K, M).

Fig S5

Figure S5. A case of NMOSD with anti‐aquaporin 4 antibody showing lamellar demyelinating lesions. Macroscopic view of a lamellar demyelinating lesion in the cerebral white matter from a case of neuromyelitis optica spectrum disorders (NMOSD) (A–F). MAG and MOG expression levels are decreased to the same extent in each demyelinating layer (B, C). MBP‐positive macrophages or microglia are seen in all demyelinating layers (D, inset). Immunoreactivity for GFAP is more abundant in myelinated layers than demyelinating layers (E). In contrast, AQP4 expression is decreased in both myelinated and demyelinating layers (F). Higher magnification view of the lesion indicated in the square in panel D (G–I). AQP4 is downregulated in GFAP‐positive degenerating astrocytes in both demyelinating and myelinated layers (G, H). Immunoreactivity for MBP is completely preserved in myelinated layers (I). Astrocytopathy showing loss of AQP4 in degenerating astrocytes is observed in a perivascular region, while MBP expression is preserved (J–L). Activated microglia bearing TMEM119, GLUT5 and P2RY12 are accumulated in perivascular astrocytopathic lesions (M–O). Scale bars: 1 mm (A–F), 500 µm (G–I) and 200 µm (J–O).

Fig S6

Figure S6. Distribution pattern of microglia and macrophages in meningoencephalitis and cerebral infarction. A case of bacterial meningoencephalitis (A–J). Panel A shows massive accumulation of CD68‐positive macrophages in the meninges (A). Panels B–F show higher magnification views of the lesion indicated by the square in panel A (B–F). Foamy macrophages in the meninges are strongly positive for CD68 and CD163 (B, C), while TMEM119, GLUT5 and P2RY12 are almost undetectable in these macrophages (D–F). In contrast, TMEM119, GLUT5 and P2RY12 are detected on microglia in the peri‐lesional cortical gray matter (D–F). Double immunofluorescence reveals that CD68 and GLUT5 have different expression patterns on macrophages and microglia at the boundary of the meninges and cortex (G–J). CD68 is predominantly expressed on foamy macrophages (I), while GLUT5 is expressed on microglia and a few foamy macrophages (H, J, arrows). A case with subacute cerebral infarction (K–P). H&E staining shows a focal necrotic lesion in the cerebral white matter (K). CD68‐ and CD163‐positive macrophages are abundant throughout the lesion (L, M). TMEM119 is upregulated at the edge of the lesion (N). GLUT5 is also upregulated at the edge of the lesion and expressed by some macrophages within the lesion center (O). P2RY12 expression is not seen in the center or edge of the lesion (P). Scale bars: 1 mm (A), 100 µm (B–E), 50 µm (G–J) and 500 µm (K–P).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.