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. 2021 Mar;31(2):283-296.
doi: 10.1111/bpa.12906. Epub 2020 Nov 18.

Overexpression of the ubiquitin-editing enzyme A20 in the brain lesions of Multiple Sclerosis patients: moving from systemic to central nervous system inflammation

Simona Perga  1   2   3 , Francesca Montarolo  1   2   4 , Serena Martire  1   2 , Brigitta Bonaldo  1   3 , Gabriele Bono  1   2 , Jessica Bertolo  1   2 , Roberta Magliozzi  5   6 , Antonio Bertolotto  1   2
Affiliations

Overexpression of the ubiquitin-editing enzyme A20 in the brain lesions of Multiple Sclerosis patients: moving from systemic to central nervous system inflammation

Simona Perga et al. Brain Pathol. 2021 Mar.

Abstract

Multiple Sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) in which inflammation plays a key pathological role. Recent evidences showed that systemic inflammation induces increasing cell infiltration within meninges and perivascular spaces in the brain parenchyma, triggering resident microglial and astrocytic activation. The anti-inflammatory enzyme A20, also named TNF associated protein 3 (TNFAIP3), is considered a central gatekeeper in inflammation and peripheral immune system regulation through the inhibition of NF-kB. The TNFAIP3 locus is genetically associated to MS and its transcripts is downregulated in blood cells in treatment-naïve MS patients. Recently, several evidences in mouse models have led to hypothesize a function of A20 also in the CNS. Thus, here we aimed to unveil a possible contribution of A20 to the CNS human MS pathology. By immunohistochemistry/immunofluorescence and biomolecular techniques on post-mortem brain tissue blocks obtained from control cases (CC) and progressive MS cases, we demonstrated that A20 is present in CC brain tissues in both white matter (WM) regions, mainly in few parenchymal astrocytes, and in grey matter (GM) areas, in some neuronal populations. Conversely, in MS brain tissues, we observed increased expression of A20 by perivascular infiltrating macrophages, resident-activated astrocytes, and microglia in all the active and chronic active WM lesions. A20 was highly expressed also in the majority of active cortical lesions compared to the neighboring areas of normal-appearing grey matter (NAGM) and control GM, particularly by activated astrocytes. We demonstrated increased A20 expression in the active MS plaques, particularly in macrophages and resident astrocytes, suggesting a key role of this molecule in chronic inflammation.

Keywords: A20/TNFAIP3; active lesions; central nervous system; inflammation; multiple sclerosis; neuropathology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Localization of A20 expression in human post‐mortem CC brain tissues by IHC and IF. WM (A‐D) and GM (E‐J) were identified by anti‐MOG immunostaining (A, E). The presence of microgliosis were identified by anti‐MHC II immunostaining (B, F). In WM, A20 was expressed by cells with astrocyte morphology (C‐C′) expressing GFAP astrocytic marker (D). In GM, A20 was expressed by cells with neuronal morphology with a perinuclear distribution (G‐G′) expressing NEFH neuronal marker (H) and by rare cells with astrocytic morphology (I‐I′) expressing GFAP astrocytic marker (J). The inserts in (C′, G′, and I′) show A20‐positive cells in WM and GM at high magnification. In D, H, and J, the arrows indicate the colocalization of the A20 and the specific cell marker signals, while the arrowheads show the lacking of colocalization. Original confocal microscopy magnification: 40x. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 2
Figure 2
Localization of A20 protein expression in WM of human post‐mortem MS brain tissues by IHC and IF. The presence of NAWM, pre‐active (pre‐AL), active (AL), and chronic active (CAL) lesions was evaluated with MOG and MHC II immunostaining (A, B, F, G, J, K). In NAWM, A20 was expressed by rare scattered ramified cells (C, C') expressing GFAP astrocytic (D, D′) and MHC II microglial markers (E, E′). In the area of active demyelination, including AL and CAL (H, H′, L, L′), A20 was expressed by a huge number of highly ramified cells expressing GFAP astrocytic (I, I′) and MHC II microglia markers (M, M′). The inserts in (C′, D′, E′, H′, I′, L′, and M′) show A20‐positive cells in NAWM and WML at high magnification. The arrows indicate the colocalization between A20 and the cell‐specific markers (MHC II or GFAP); the arrowheads indicate the lack of colocalization. Original confocal microscopy magnification: 40x. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3
Characterization of A20 expression in perivascular infiltrates in human post‐mortem MS brain tissue by IHC and IF. In the area of demyelination, A20 was found in perivascular infiltrates (C, C′), not expressing CD4 T helper (D‐G′), CD8+ T cytotoxic (H‐K′), or CD20 B lymphocytes markers (L‐O′). Conversely, A20 was expressed by a high number of CD68+ macrophages infiltrating the active demyelinated lesions in WM (P‐S′). The arrows indicate the colocalization between A20 and CD68 macrophage marker, while the arrowheads indicate the lack of A20 colocalization with cell‐specific markers (CD4, CD8 or CD20). Original confocal microscopy magnification: 40x. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 4
Figure 4
Localization of A20 protein in GM of human post‐mortem MS brain tissues by IHC and IF. The presence of active (aGML) and inactive (iGML) lesions in GM (GML) was evaluated with MOG and MHC II immunostaining (A B, E, F, I, J). In NAGM, A20 was expressed by some populations of cells with neuronal morphology, with somatic distribution (C, C′), expressing the NEFH marker (D). Furthermore, A20 was expressed by rare ramified cells resembling astrocytes (G, G′) expressing the GFAP astrocytic marker (H). Conversely, in the aGML A20 was expressed by a tick mantel of cells with astrocyte morphology (K, K′) expressing the GFAP astrocytic marker (L). The arrows indicate the colocalization between A20 and the neuronal nuclei in the NAGM (D) and the colocalization of A20 with GFAP+ astrocytes in the NAGM (H, L); the arrowheads indicate neurons that do not express A20 (D). Original confocal microscopy magnification: 40x. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 5
Figure 5
Densitometric analysis of A20 immunoreactivity measured on IHC images of human post‐mortem brain tissues of MS cases. The A20 OD was compared between NAWM and AL (A) or CAL (B) and between NAGM and aGML (D) or iGML (E) (paired Mann–Whitney U‐test, *P < 0.05, **P < 0.001, ***P < 0.0001). Normalized A20 OD signals, calculated by subtracting the OD value of the normal‐appearing area to that of the matched lesion area, were compared between AL and CAL (C) and between aGML and iGML (F) (Mann–Whitney U test, *P < 0.05, **P < 0.001, ***P < 0.0001). [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 6
Figure 6
A20 gene expression analysis in human post‐mortem brain tissues of MS and CC. The A20 expression was compared between WM of CC and NAWM and WML of MS cases (A), and between GM of CC and NAGM and aGML of MS cases (C) (Kruskal–Wallis with Dunn post hoc test, *P < 0.05, **P < 0.001, ***P < 0.0001). In addition, the A20 expression was compared between matched samples of normal‐appearing and lesioned areas of MS cases (B, D) (paired Mann–Whitney U test, *P < 0.05, **P < 0.001, ***P < 0.0001). Relative gene expression was calculated by the normalized comparative cycle threshold (Ct) method 2−ΔΔC. [Colour figure can be viewed at wileyonlinelibrary.com]
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