Myelin phagocytosis by astrocytes after myelin damage promotes lesion pathology - PubMed (original) (raw)

Myelin phagocytosis by astrocytes after myelin damage promotes lesion pathology

Gerald Ponath et al. Brain. 2017 Feb.

Abstract

Astrocytes are key players in the pathology of multiple sclerosis and can assume beneficial and detrimental roles during lesion development. The triggers and timing of the different astroglial responses in acute lesions remain unclear. Astrocytes in acute multiple sclerosis lesions have been shown previously to contain myelin debris, although its significance has not been examined. We hypothesized that myelin phagocytosis by astrocytes is an early event during lesion formation and leads to astroglial immune responses. We examined multiple sclerosis lesions and other central nervous system pathologies with prominent myelin injury, namely, progressive multifocal leukoencephalopathy, metachromatic leukodystrophy and subacute infarct. In all conditions, we found that myelin debris was present in most astrocytes at sites of acute myelin breakdown, indicating that astroglial myelin phagocytosis is an early and prominent feature. Functionally, myelin debris was taken up by astrocytes through receptor-mediated endocytosis and resulted in astroglial NF-κB activation and secretion of chemokines. These in vitro results in rats were validated in human disease where myelin-positive hypertrophic astrocytes showed increased nuclear localization of NF-κB and elevated chemokine expression compared to myelin-negative, reactive astrocytes. Thus, our data suggest that myelin uptake is an early response of astrocytes in diseases with prominent myelin injury that results in recruitment of immune cells. This first line response of astrocytes to myelin injury may exert beneficial or detrimental effects on the lesion pathology, depending on the inflammatory context. Modulating this response might be of therapeutic relevance in multiple sclerosis and other demyelinating conditions.

Keywords: T-lymphocytes; astrocyte; demyelination; multiple sclerosis and neuroinflammation; neuroimmunology.

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Figures

Figure 1

Hypertrophic astrocytes in active white matter multiple sclerosis lesions contain myelin debris. (A) Low magnification of an acute multiple sclerosis lesion (biopsy) filled with foamy macrophages (MBP, brown; CD68, purple). (B) Reactive astrocytes (GFAP, brown) within the acute lesion are hypertrophic with rarefied, thickened processes. (C) Higher magnification shows LFB-positive inclusions within astroglia (arrow). (D) Confocal microscopy confirms the presence of the myelin protein, PLP1 (red) in an astrocyte. (D’ and D”) Higher magnification shows PLP-positive puncta within ring-like structures labelling positive for the lysosomal marker LAMP1 (LAMP1, white; GFAP, cyan; nuclei, blue). (E) Low magnification of the edge of a chronic active lesion with macrophage infiltrates at the lesion rim and in the adjacent white matter (CD68, brown; MBP, purple). (F) GFAP staining shows reactive astrocytes in adjacent white matter, (I) reactive hypertrophic astrocytes at the lesion edge and (L) small, chronic-reactive astrocytes in the gliotic lesion centre. (G) As in acute lesions, hypertrophic astrocytes (GFAP, brown) at the lesion edge contain neutral lipid droplets, visualized with Oil Red O, and (H) LFB-positive inclusions, both indicative of myelin debris (arrows). Oil Red O-positive droplets are also seen in a macrophage next to the astrocyte (arrowhead). (J) Confocal microscopy of a ballooned astrocyte double-labelled for GFAP (cyan) and intracellular accumulation of MBP-positive myelin fragments (red; see inset). (K) PLP-positive myelin debris (red) within astrocytes (GFAP, cyan) is present in lysosomes (LAMP1, white; see inset) and (M) co-localizes with the scavenger receptor LRP1 (white; inset). Scale bars = 100 µm in A, B, E; 25 µm in C, D, F, G, H, I, L; 20 µm in J; and 10 µm in K and M.

Figure 2

Myelin debris-positive hypertrophic astroglia in progressive multifocal leukoencephalopathy lesions and metachromatic leukodystrophy. (A) Low magnification image of a subacute progressive multifocal leukoencephalopathy lesion with infiltrating microglia/macrophages at the lesion rim and the adjacent white matter (MBP, brown; CD68, purple). (B) Higher magnification shows foamy macrophages at the lesion edge. (C) Numerous hypertrophic astrocytes are present within the lesion and the lesion edge but not in adjacent white matter (GFAP, brown; myelin, LFB). (D and E) Astrocyte in myelinated white matter remote from the lesion without myelin inclusions. In contrast, a perivascular astrocyte at the lesion rim contains LFB-positive debris (arrow). (F and G) Confocal microscopy confirms the presence of MBP (magenta) in GFAP-labelled astrocytes (green) at the lesion rim (G; white arrows, inset) but not in adjacent white matter (F). (H) Low magnification of metachromatic leukodystrophy white matter shows areas of subcortical U-fibres with apparently intact myelin (H1; detail in I), of ongoing myelin damage (H2; detail in J) and of myelin-depleted white matter (H3; detail in K); double labelling for myelin and macrophages (MBP, purple; CD68, brown). (I–K) Numerous macrophages are scattered throughout the intact and demyelinated white matter (arrowheads). (L and O) Activated astrocytes in subcortical myelinated white matter are highly immunoreactive for GFAP (brown) but do not contain PLP-positive debris (GFAP, green; PLP, magenta) (arrows). (M and P) The zone with ongoing myelin damage is filled with hypertrophic astrocytes (GFAP, brown) that contain PLP-positive myelin fragments (arrows), visualized with confocal microscopy (GFAP, green; PLP, magenta). (N and Q) Gliotic fibres with occasional astroglial cell bodies predominate in myelin-damaged white matter (GFAP, brown). PLP-positive debris (magenta) is present in macrophages (arrowheads) that are interspersed among the GFAP-positive fibrous mesh (green). Scale bars = 1000 µm in A; 100 µm in B, C and H; 25 µm in D, E and I–N; and 20 µm in F, G, O–Q.

Figure 3

Myelin debris-positive hypertrophic astrocytes in the reactive zone of a subacute infarct. (A) Overview image of a subacute infarct double-labelled for MBP (purple) and CD68 (brown). Myelin is present at the infarct border (A1, detail in B). The reactive zone surrounding the infarct centre shows loss of myelin and contains infiltrating macrophages (A2, detail in C). The necrotic core is filled with foamy CD68+ macrophages (A3, detail in E). (D) Two astrocytes in the reactive zone contain hemosiderin (arrows; haematoxylin/eosin). (F) Reactive astrocytes in myelinated white matter at the lesion border. (G) Hypertrophic (gemistocytic) astrocytes in the reactive zone (arrows). (H) Astrocytes are absent from the infarct core. Arrowheads indicate hemosiderin-laden macrophages. (I) Confocal microscopy shows an activated astrocyte in intact white matter without myelin debris. (J) PLP1-positive myelin debris in lysosomes of a proliferating hypertrophic astrocyte within the reactive zone (arrows). (K) PLP1-positive debris is present in lysosomes of macrophages at the infarct core. (PLP1, red; LAMP1, white; GFAP, cyan). Scale bars = 100 µm in A; 25 µm in B–H; and 10 µm in I–K.

Figure 4

Kinetics and mechanism of myelin uptake in cultured astrocytes. (A) Primary rat cortical astrocytes (in vitro Day 14) containing fluorescent-labelled purified myelin after 24 h of myelin exposure (1 µg/ml) (S100B, magenta; myelin, green). (B) Intracellular localization of fluorescent myelin debris demonstrated with orthogonal views of confocal _z_-stack images of a representative astrocyte (S100B, magenta; myelin, green). (C) Accumulation of fluorescence in isolated cultured astrocytes and microglia cells after 30 min of exposure to various concentrations of fluorescent-labelled myelin (0.0625 μg/ml to 2 µg/ml). In co-cultures, astrocytes and microglia were separated with magnetic beads before quantification. Graphs show mean ± SD from three independent experiments of the quantification of Oregon Green® 488-labelled myelin. (D) Cultured astrocytes containing myelin fragments after 30 min of myelin exposure. Myelin particles partially co-localize with the cell surface receptor LRP1 (myelin, green; LRP1, magenta; H 33342, blue; ALDH1L1, grey in inset). (E) Partial co-localization of intracellular myelin debris with the early endosomal marker, EEA1, in astrocyte exposed to myelin for 60 min (EEA1, magenta; myelin, green). (F and G), Intracellular myelin debris co-localized with lysosomes after 120 min of myelin exposure demonstrated with the lysosomal markers LysoTracker™ and CellROX (both magenta). (G; inset) Rapid degradation of myelin debris (green) in CellROX-positive lysosomes shown with time-lapse recording. (H) Myelin uptake in cultured astrocytes is reduced by the competitive LRP1 ligand RAP (5 μg/ml), the dynamin-dependent endocytosis blocker Dynasore (0.1 μM; 30 min preincubation) and by LPS (10 ng/ml, 2 h preincubation). Data represent the means ± SD from three independent experiments. **Means were significantly heterogeneous [one-way ANOVA, F(3,9) = 70.55, P < 0.0001] and significantly different from the control condition (Tukey–Kramer test, P < 0.01). (I) Flow cytometry analysis of astrocytes after 45 min incubation with myelin (2 µg/ml + DMSO 1:500). Cells were gated for GLAST+ living cells. A representative graph from five independent experiments shows per cent of maximum fluorescence intensity of intracellular myelin. Pretreatment with Dynasore (0.1 μM in DMSO 1:500 for 30 min) resulted in 90% reduction of mean fluorescence intensity. DMSO (1:500) shows no effect of myelin uptake (data not shown). Scale bars = 30 µm in A; 15 µm in B and E–H.

Figure 5

Myelin uptake induces astroglial activation and proliferation. (A) Increased astroglial proliferation after myelin uptake (1 μg/ml for 72 h) compared to TGFβ-induced proliferation (10 ng/ml), measured by BrdU-incorporation. Myelin concentration 1 μg/ml. Data represent the means ± SD from four independent experiments. **Means were significantly heterogeneous [one-way ANOVA, F(2,21) = 21.41, P < 0.0001] and significantly different from the control condition (Tukey–Kramer test, P < 0.01). (B) GFAP expression increases for at least 9 days after myelin stimulation (western blot). (C) Myelin exposure of astrocytes for 24 h increases GFAP immunoreactivity. (D) Induction of intracellular reactive oxygen species in myelin-exposed astrocytes after 24 h (myelin, green; CellROX, magenta). Scale bars = 15 μm.

Figure 6

NF-κB and p38 MAP kinase activation in cultured astrocytes exposed to purified myelin. (A) Nuclear translocation (Hoechst dye 33342, cyan) of the NF-κB subunit p65 (a' and b', red) after 2 h of exposure with myelin debris (b and b') in GFAP-labelled astrocytes (a and b, grey). (B) astroglial p65 phosphorylation (flow cytometry); (C) time course (0–60 min) of IκBα degradation with maximum after 45 min of myelin exposure (flow cytometry). (C’) Blockade of IκBα degradation after 45 min of myelin exposure by pretreatment with Dynasore (0.1 µM in 1:500 DMSO, 30 min). (D) Increased phosphorylation of p38 MAPK after stimulation with myelin for 45 min and partial block after pretreatment with Dynasore (open black; 0.1 µM in 1:500 DMSO, 30 min; flow cytometry). DMSO (1:500) alone has no effect on IκBα degradation or p38 phosphorylation (data not shown). (B–D) Representative graphs and heat maps from three independent experiments are shown. (E) Transient increase in p38 MAPK phosphorylation after myelin exposure demonstrated by western blot. Scale bars = 15µm in A.

Figure 7

Induction of chemokines in astrocytes through myelin uptake. (A) Scatter plot profiling astroglial expression of 84 immune-related genes after incubation with myelin (4 μg/ml; 4 h exposure). Red dots indicate cytokines with a ≥3.5-fold increase in expression over baseline. (B) Representative ELISPOT protein arrays from three independent experiments showing induced and upregulated cytokine expression in astroglial supernatant (red squares: 1 = CCL5, 2 = CXCL9, 3 = CCL3, 4 = CCL20, 5 = CXCL2, 6 = CXCL10, 7 = CXCL3, 8 = CXCL5). (C) Directed migration of polarized CD4+ Th1 and microglia in conditioned medium from myelin-treated astrocytes (2 μg/ml for 24 h) in transwell-chambers. Data represent the means ± SD from three independent experiments [one-way ANOVA, F(3,8) = 26.08, P < 0.001; Tukey–Kramer test, *P < 0.05, **P < 0.01].

Figure 8

Quantification of chemokines and iNOS expression, and NF-κB nuclear localization in areas of myelin breakdown. Graphs display fluorescent immunoreactivity means ± SD of CXCL10, CCL20, CCL3, CCL5, iNOS and nuclear localization of p65 NF-κB in myelin-positive hypertrophic astrocytes, myelin-negative reactive astrocytes and myelin-laden macrophages, quantified by densitometric analysis. For statistical analysis one-way ANOVA with Tukey-Kramer test for individual comparisons from at least 20 cells per cell type per case was performed. *P < 0.05, **P < 0.01.

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Myelin phagocytosis by astrocytes after myelin damage promotes lesion pathology - PubMed (original) (raw)