The anti-inflammatory enzyme A20 in the neuropathology of Multiple Sclerosis

More Than Neurons, 1-3 December, Turin The anti-inflammatory enzyme A20 in the neuropathology of Multiple Sclerosis Dr. Simona Perga, PhD Neuroscience Institute Cavalieri Ottolenghi (NICO) & Multiple Sclerosis Center (CReSM), San Luigi University Hospital, Orbassano (TO), Italy

Multiple Sclerosis Multiple sclerosis is a autoimmune neurological disease, characterized by chronic inflammation with immune cells infiltration within the CNS, demyelination and axonal damage Healthy brain Brain with damage (lesions or plaques) caused by MS

Multiple sclerosis risk factors MS Nat Rev Neurol 6, 429-437 (2010)

2013 2013

2013 2013 6q23.3 (OLIG3-TNFAIP3) TNFAIP3 gene A20 protein

Coelic disease Multiple sclerosis 2013 2013 Rheumatoid arthritis Type I diabetes Crohn s disease TNFAIP3/ A20 Lupus B-cell lymphoma 6q23.3 (OLIG3-TNFAIP3) TNFAIP3 gene A20 protein

A20 is a key negative regulator of inflammation by the inhibition of the NF-kB pathway TNF TNF-R IL-1R IL-1B TLR-4 LPS Ma and Malynn, Nature Reviews, 2012

A20 deficiency in mice suggest mechanisms of human autoimmune disease physiopathology Martin and Dixit, Nature Genetics, 10:822-823

A20 is down-regulated in treatment-naive MS patients peripheral blood cells 2010 2011

Log A20 relative expression A20 relative expression Log A20 relative expression Low A20 blood expression level correlate with a worse disease course in MS patients r= - 0.618 Treatment naïve MS patients Log DEDSS r= - 0.586 Gilli F. et al., ArchNeurol 2011 Log RR

A20 expression id down-regulated in the monocytes of MS treatment-naive patients 2014 CD14+ MONOCYTES CD4+ LYMPHOCITES

The role of ubiquintin-editing enzyme A20 in the CNS Trends in Molecular Medicine, October 2013. Vol. 19, N. 10

The role of ubiquintin-editing enzyme A20 in the CNS Trends in Molecular Medicine, October 2013. Vol. 19, N. 10 Moving from periphery to central nervous system : a possible role for A20?

Aim of the work Evaluate the presence and possible role of A20 protein within human post-mortem brain tissues in control and MS cases. Ministero della Salute - Giovani Ricercatori 2010; project title; The ubiquintin-editing enzyme A20 (TNFAIP3) as a peacekeeper in inflammation and immunity: a link between TNFAIP3 deregulation and Multiple Sclerosis (grant n. GR-2010-2315964). AISM/FISM 2014; project title: The deubiquitinase A20/TNFAIP3 in the immunopathology of Multiple Sclerosis (grant n. 2014/R/14).

Human SNAP-Frozen Post-Mortem Brain Tissues United Kingdom Multiple Sclerosis Society Tissue Bank - Centre for Neuroscience, Imperial College of London 13 MS cases 10 SPMS 3 PPMS 5 CCs Neuropathological analysis 10 µm-thick cryosections for IHC and IF 40 snap-frozen tissue blocks Biomolecular analysis Gene and protein expression

Technical difficulties linked to the use of human post-mortem brain (post-fixed) frozen material Subject conditions Presence of systemic illnesses Age of death Cause of death Post-mortem delay Brain removal steps Method of brain removal/storage Temperature Fixation

Tissues characterization Anti-MOG Myelin White matter (WM) Grey matter (GM) Anti-MHC class II Inflammatory activity Monocytes Macrophages Dendritic cells Lymphocytes B Microglia

Lesions characterization White Matter Lesions NAWM Pre-Active lesion pre-al Active lesion (AL) Chronic Active lesion (CAL) MOG + MHCII - MOG + MHCII + MOG - MHCII ++ MOG - Plaque edge MHCII + Time Grey Matter Lesions (cognitive disability) Shadow or Remyelinating Lesions (MOG+; MHC-) Inactive Lesions (MOG-; MHC -)

Tissue characterization: WM and GM in control cases MHCII MOG WM WM GM 50 µm 50 µm A20 GM 250 µm WM 50 µm GM 50 µm Control case - C025 P1D1 Gender: M - Age of death: 35 - Cause of death: Carcinoma of tongue

Plaques characterization: PRE-ACTIVE LESIONS A20 MOG MHCII A20 pre-al pre-al pre-al AL AL AL WM 250 µm WM 250 250 µm µm WM 250 µm pre-al WM 50 µm SPMS case - MS230 P3C2 Gender: F - Age of death: 42 - Cause of death: MS

A20 Plaques characterization: ACTIVE LESIONS MOG MHCII A20 WM WM WM AL AL AL 100 µm 100 µm 100 µm AL 25 µm SPMS case - MS154 P5B8 Gender: F - Age of death: 34 - Cause of death: Pneuomonia

Plaques characterization: CRONIC ACTIVE LESIONS A20 MOG MHCII A20 CAL CAL CAL CAL WM 250 µm 250 µm 250 250 µm µm WM WM 50 µm PPMS case - MS383 P4B1 Gender: M - Age of death: 42 - Cause of death: Aspiration pneumonia

Densitometric analysis of A20 immunoreactivity in the MS lesions 160 *** ALs: n= 8 CALs n= 11 1.4 1.3 A20 optic density (OD) 140 120 100 Lesion 100 µm AL A20 NAWM A20 fold change* 1.2 1.1 1.0 0.9 NAWM AL CAL A20 optic density (OD) 80 NAWM NAWM 200 *** 180 160 140 120 100 NAWM NAWM AL AL CAL CAL A20 is over-expressed both in ALs and CALs edge.

Correlation between A20 immunoreactivity in lesions and demographical and clinical characteristics of MS patients A20 expression (OD) in lesions? Gender Diagnosis Disease length Cause of death Age of death Plaque dimension

A20 optic density (OD) Correlation between A20 immunoreactivity in lesions and demographical and clinical characteristics of MS patients A20 expression (OD) in lesions R= -0.42 p = 0.07? Gender Diagnosis Disease length Cause of death Age of death Plaque dimension Age of death

Identification of cell populations expressing A20 in the CNS A20/DAPI NAWM 25 µm PPMS case - NAWM - MS473 A3C4 Gender: F - Age of death: 39 - Cause of death: Bronchopneuominia, MS

MHC+ cells express A20 in the active lesions A20 A20/MHCII/DAPI 100 µm MHCII 100 µm DAPI 100 µm 25 µm PPMS case - AL - MS473 A3C4 Gender: F - Age of death: 39 - Cause of death: Bronchopneuominia, MS

CD68+ macrhopages express A20 in the active lesions A20 A20/CD68/DAPI 100 µm CD68 100 µm DAPI 100 µm 25 µm SPMS case - CAL edge - MS230 P3C2 Gender: F - Age of death: 42 - Cause of death: MS

Identification of CNS resident cell populations expressing A20 A20 GFAP 50 µm 50 µm MHCII SPMS case Gender: M - Age of death: 53 - Cause of death: Advanced MS, urinary tract infection 50 µm

A20 is expressed by GFAP+ astrocytes A20 GFAP DAPI SPMS case - CAL edge - MS230 P3C2 Gender: F - Age of death: 42 Cause of death: MS 100 µm 100 µm 100 µm A20/GFAP/DAPI 100 µm

CAL plaque edge In AL and CAL, A20+ cells show the same pattern of distribution of GFAP+ astrocytes, resembling astrogliosis A20 GFAP WM WM 50 µm 50 µm Reactive astrogliosis!! PPMS case CAL edge - MS383 P4B1 Gender: M - Age of death: 42 - Cause of death: Aspiration pneumonia

Conclusions A20 protein is expressed in human control and MS brain tissues, both in WM, within highly ramified cells and in GM, within morphologically neuron-like cells. A20 protein is over-expressed in human MS WM lesions (pre-als, ALs, and CALs) in infiltrated inflammatory macrophages (lymphocytes, dendritic cells?) and astrocytes (microglia?). In the CAL lesions, it is possible to observe an intricate network of A20+ ramified cells (astrocytes) resembling astrogliosis

Future perspective Conclude the characterization of A20 in human MS brain tissues: - evaluating the A20 expression in other cell types (B and T lymphocytes, resting and activated microglia, oligodendrocytes and neurons); - analyzing A20 expression in GM lesions and shadow plaques; - confirming the A20 over-expression in lesions by WB and quantitative RT-PCR Define the signaling pathway in which A20 is involved in immune and CNS activated cells in human MS brain lesions (TNF-R1/R2? NF-kB pathway activation?) Dissect the role of A20 in the cell populations of interest in MS (astrocytes and macrophages) by inducing the EAE in transgenic mice with conditional A20 deletion Does the over-expression of A20 in lesions promotes the damage or counteracts it, playing a protective role?

Aknowdelgmets Neuroscience Institute Cavalieri Ottolenghi (NICO) & Regional Multiple Scerosis Centre (CReSM) University Hospital S. Luigi Gonzaga, IT Antonio Bertolotto Francesca Montarolo Brigitta Bonaldo Gabriele Bono Serena Martire Neurobiology s lab CReSM staff Department of Neurosciences, Biomedicine and Movement, University of Verona, Verona, IT Roberta Magliozzi This study was supported by FISM/AISM (Grant 2014) and Italian Ministry of Health Young Researcher 2010

A20 2 Plaques characterization: CHRONIC ACTIVE LESIONS MOG MHCII A20 I 250 µm 250 µm 250 µm WM WM WM I 50 µm PPMS case - MS383 P4B1 Gender: M - Age of death: 42 - Cause of death: Aspiration pneumonia

MS SIGNATURE 347 transcripts (among which A20) considered the most discriminant gene-set by comparing transcription profiles of MS patients vs. healthy controls before pregnancy During GESTATION the MS signature lose its discrimination power in agreement with the normalization of the MS phenotype. Eight transcripts were found to change most significantly during pregnancy. /A20 Gilli F. et al., PlosOne 2010

Human genetic studies have strongly linked polymorphisms and mutations in the gene encoding A20 to inflammatory, autoimmune and malignant diseases. Moreover, its dysregulation is observed in several human autoimmune disorders. Associated diseases SNPs Lymphoma mutations Ma and Malynn, Nature Reviews, 2012

Hypothetical view of immune responses in acute multiple sclerosis lesions. Independent of the causative event, two steps are required to induce an immune response in the central nervous system (CNS): a pro-inflammatory milieu in the CNS, leading to upregulation of major histocompatibility complex (MHC) molecules, co-stimulatory receptors and inflammatory cytokines and an antigen-driven acquired immune response. T- and B-cell responses are primed in the peripheral lymphoid tissue by antigens that are released from the CNS or by cross-reactive foreign antigens. Dendritic cells that present neural antigens are strong stimulators of T-cell responses. After clonal expansion, T and B cells infiltrate the CNS. Clonally expanded B cells reencounter their specific antigen, mature to plasma cells and release large amounts of immunoglobulin-gamma (IgG) antibodies. These antibodies bind soluble or membrane-bound antigen on expressing cells. Clonally expanded CD8+ T cells also invade the brain and could encounter their specific peptide ligand, presented by glial or neuronal cells on MHC class I molecules. The recognition of specific MHC peptide complexes on these cells prompts direct damage to expressing cells. CD4+ T cells migrate into the CNS and encounter antigens that are presented by microglial cells on MHC class II molecules. Reactivation of these cells leads to heightened production of inflammatory cytokines. These cytokines attract other immune cells, such as macrophages, which contribute to inflammation through the release of injurious immune mediators and direct phagocytic attack on the myelin sheath.