main references: -Epidemiology and Pathophysiology of Multiple Sclerosis By Melanie Ward, MD; Myla D. Goldman, MD, MSc, FAAN -Multiple sclerosis, Nature disease primer Massimo Filippi1,2*, Amit Bar- Or3, Fredrik Piehl4,5,6, Paolo Preziosa1,2, Alessandra Solari7, Sandra Vukusic8 and Maria A. Rocca1,2

1. MULTIPLE
SCLEROSIS
MECHANISMS AND PATHOPHYSIOLOGY
Lobna Ahmed​

2. AGENDA
Introduction​.
Immune pathophysiology.
Inflammation in different types of MS.
Lesions pathology.
Risk factors.

3. INTRODUCTION
• Multiple sclerosis (MS) is a chronic, inflammatory,
demyelinating and neurodegenerative disease of the central
nervous system (CNS).
• MS is a heterogeneous, multifactorial, immune-​mediated
disease that is caused by complex gene–environment
interactions.
• The pathological hallmark of MS is the accumulation of
demyelinating lesions that occur in the white matter and the
grey matter of the brain and spinal cord.
3

4. IMMUNE PATHOPHYSIOLOGY
• Normally, CNS autoreactive immune cells are deleted
during development through central tolerance in the
thymus (T cells) or bone marrow (B cells).
• Although some may escape this mechanism and be
released into the circulation, peripheral tolerance
mechanisms typically prevent them from causing
disease.
4

5. Interactions
between several
immune cell
types
T-cells
Microgli
a
Other
resident
cells of
the CNS
B-cells
5
MS is primarily T cell-
​mediated disease.
Traditional
view
Shifted to

6. T-CELL INVOLVEMENT
• The historical view of MS, on the basis of studies of patients and studies using the most
commonly used animal model of MS (that is, experimental autoimmune encephalomyelitis
(EAE)), is that relapses are principally mediated by aberrantly activated and/or
insufficiently regulated pro-​inflammatory CNS-​specific effector T cells, including CD4+ T
cells and CD8+ T cells, that traffic to the CNS parenchyma and cause perivascular
demyelination, glial cell activation and neuro-​axonal injury.
6

7. Primary T-cell subsets implicated in MS include
7
Il-17-expressing CD4+ T
cells (T-helper 17)
IFN-gamma secreting CD4+
T cells (T-helper 1)
CD8+ T cells
Granulocyte– macrophage colony
stimulating factor (GM-CSF)
expressing CD4+ and CD8+ T cells

8. • The aberrant T cell activation in MS requires antigen presentation to T cells by antigen-.presenting cells
(APCs) such as B cells and myeloid cells (macrophages, dendritic cells and microglia) in the periphery
and the CNS.
• Myelin-​related antigens are suspected to be involved, although there is no consensus, and some studies
have suggested antigens on the neuronal or glial cell surface.
• Pro-​inflammatory APCs such as B cells and myeloid cells can drive TH1 cell and TH17 cell responses,
which might have a role in immune cell interactions and the trafficking that underlies relapses in MS.
8
Efficacy of some MS DMTs may at least partially relate to
shifting T-cell differentiation from TH1 and TH17 to TH2
phenotypes, which have a less inflammatory profile

9. B-CELL INVOLVEMENT
• The role of B cells in MS pathophysiology has been increasingly recognized and
characterized in recent years on the basis of impressive results of selective B cell-
​targeting therapies (such as anti-​CD20 antibodies) in MS.
• Healthy individuals typically have low levels of antibodies in the CNS (the normal ratio is
~1:300 of CNS to periphery); patients with MS have an abnormally increased production
of antibodies within the CNS, which can be detected, for example, as increased
immunoglobulin synthesis rates and the presence of cerebrospinal fluid-​restricted
oligoclonal bands (OCBs).
9

10. • This finding was the basis for anti-​B cell therapies in MS, although interestingly, the reduction in relapse
rate with anti-​CD20 therapy was associated with little or no change to the cerebrospinal fluid
immunoglobulin profile in patients, suggesting an antibody independent role of B cells in MS relapses.
• These antibody-​independent functions are likely to be the contribution of B cells to cascades of cellular
immune interaction in the periphery and/or their ability to attract and activate T cells and myeloid cells
in the CNS.
10

11. Pro-
inflammatory
cytokines
Anti-
inflammatory
cytokines
11
B-cells can produce both
Lymphotoxin-a
IL-6
TNF-a
GM-CSF
IL-10
IL-35
Transforming
growth factor ß
Their
production
may be
impaired in
MS patient

12. 12
Krumbholz, M., Derfuss, T., Hohlfeld, R. et al. B cells and
antibodies in multiple sclerosis pathogenesis and therapy.
Nat Rev Neurol 8, 613–623 (2012).
https://doi.org/10.1038/nrneurol.2012.203

13. 13
Within the CNS,
inflammatory B-cell
infiltrates can be found in
the meninges
of patients with MS, and a
higher burden of these
infiltrates correlates with
the
degree of cortical lesions
and neurodegeneration as
well as clinical disability.
Antigen presentation to T-
cells and secretion of
molecules that may be
directly toxic to
oligodendrocytes.
Act as a reservoir for
Epstein-Barr virus (EBV).
Other pathologic mechanisms of
B-cells in MS

14. 14
Krumbholz, M., Derfuss, T., Hohlfeld, R. et al. B cells and antibodies in multiple sclerosis pathogenesis and therapy. Nat Rev Neurol 8, 613–623 (2012).
https://doi.org/10.1038/nrneurol.2012.203

15. MICROGLIA INVOLVEMENT
• Microglia are immune cells residing in the CNS and can shift between both pro- and anti-
inflammatory phenotypes.
• Microglia have previously been implicated in genetic leukoencephalopathies and
neurodegenerative diseases and are increasingly recognized in MS pathophysiology.
• Microglia contribute to both acute and chronic lesion formation in MS and, on the opposite
spectrum, can also facilitate remyelination and neuronal repair
15

16. In early active MS
lesions
• 40% of phagocytic
cells are pro-
inflammatory
microglia.
In mixed
active/inactive
lesions
• (Also called
chronic, slowly
expanding or
smoldering
lesions), activated
microglia can be
found in the
periphery of these
lesions.
In progressive MS
• Activated microglia
and macrophages
may mediate
neurodegeneration
by several
mechanisms.
16
 Consequently, interest has increased in therapeutic agents potentially targeting microglia.
 Bruton tyrosine kinase is expressed in B cells and in CNS microglia, and several therapeutic
agents targeting inhibition of Bruton tyrosine kinase are currently in clinical trials for both
relapsing and progressive MS.

17. • Despite the fact that the CNS was considered immune privileged, with
the BBB thought to restrict entry of cells and macromolecules from the
circulation, BBB breakdown has been observed in patients with MS,
which is speculated to facilitate the migration of pro-​inflammatory cells
into the CNS parenchyma.
• In addition, a lymphatic drainage system has been demonstrated in the
CNS.
• The immune system can interact continuously with the CNS as part of
normal immune surveillance and, in MS, bidirectional trafficking likely
takes place during the course of disease.
• After activation in the periphery, immune cells upregulate cell surface
molecules such as chemokine receptors and adhesion molecules,
which enables efficient tissue infiltration, including to the CNS.
17
Immune cells
entry to the CNS
 BBB break down.
 Subarachnoid space.
 Blood-CSF barrier.

18. 18
Duffy, S. S., Lees, J. G., & Moalem-Taylor, G. (2014). The contribution of
immune and glial cell types in experimental autoimmune encephalomyelitis
and multiple sclerosis. Multiple sclerosis international, 2014.

19. 19
Mansilla, M.J., Presas-Rodríguez, S., Teniente-Serra, A. et al. Paving the way
towards an effective treatment for multiple sclerosis: advances in cell therapy.
Cell Mol Immunol 18, 1353–1374 (2021). https://doi.org/10.1038/s41423-020-
00618-z
Proposed mechanisms of action of
approved treatments for multiple
sclerosis and cell-based therapies.
Representation of the mechanisms
of action of current treatments
(black boxes) and cell-based
therapies (gray boxes).

20. INFLAMMATION IN
DIFFERENT TYPES
OF MS
20

21. 21
Relapsing
MS
Progressive
MS
Characterized by acute inflammatory
activity associated with disruption of BBB,
as evidenced by gadolinium-enhancing
lesions on MRI.
Classic acute lesions begin with
infiltrates of inflammatory B, T, and
plasma cells and macrophages
surrounding
a central vein.
Peripheral
immune
responses
targeting
the CNS
Intrinsic immune
processes
within the CNS
behind an
apparently
intact BBB
Characterized by a lower frequency of
inflammatory relapses (waves of
infiltration of activated immune cells into
the CNS in a perivascular distribution).
+
Evident CNS compartmentalized
inflammation.

22. 22
Mansilla, M.J., Presas-Rodríguez, S., Teniente-Serra, A. et al. Paving the way
towards an effective treatment for multiple sclerosis: advances in cell therapy.
Cell Mol Immunol 18, 1353–1374 (2021). https://doi.org/10.1038/s41423-020-
00618-z

23. 23
Other mechanisms
of
neurodegeneration
in progressive MS
Chronic microglia
activation
Meningeal
inflammation
Mitochondrial injury
Impaired ion
homeostasis
In SPMS lymphoid
follicles consisting of
B and T-lymphocytes,
macrophages, and
plasma cells are
frequently found in
the meninges and
perivascular spaces.
In PPMS, it’s more
diffuse without the
presence of follicles.

24. 24
a | Immune infiltrates accumulate in
meningeal lymphoid-like structures (top) and
perivascular regions (bottom).
b | Immunostaining of a post mortem brain
section from an individual with multiple
sclerosis shows that CD20+ B cells and
CD3+ T cells accumulate in meningeal
lymphoid structures.
c | Immunostaining for myelin MOG,
suggests that infiltration of CD20+ B cells
and CD3+ T cells into the meninges
(asterisk) is associated with subpial cortical
grey matter lesions.
d | Immunofluorescent staining shows that
CD20+ B cells and CD3+ T cells accumulate
in perivascular inflammatory infiltrates.
e | Immunostaining for MOG suggests that
infiltration of CD20+ B cells and CD3+ T cells
into the perivascular spaces (asterisk) is
associated with white matter demyelination.
Cencioni, M.T., Mattoscio, M., Magliozzi, R. et al. B cells in multiple sclerosis —
from targeted depletion to immune reconstitution therapies. Nat Rev Neurol 17,
399–414 (2021). https://doi.org/10.1038/s41582-021-00498-5

25. PATHOLOGY
• White matter lesions.
• Normal appearing white matter.
• Grey matter lesions.
25

26. WHITE MATTER LESIONS
Active
demyelinating
Inactive
Chronic
active
Slowly
expanding
26
The earliest phases of MS (CIS and RRMS) are typically characterized by active
demyelinating lesions. These lesions have heavy lymphocyte infiltration, activated
microglia (particularly at the lesion edge), macrophages and large, reactive astrocytes.
PPMS and SPMS are mainly characterized by inactive lesions, which are sharply
circumscribed, hypocellular and have well-​defined demyelination, reduced axonal density,
reactive astrocyte gliosis, variable microglial activation only in the peri-plaque
white matter (without macrophages) and a lower density of lymphocytes than active
lesions.
More frequent in patients with MS with a longer disease duration and in SPMS and
are characterized by macrophages at the edge of the lesion, with fewer macrophages
in the lesion center.
Typically found in patients with SPMS, are characterized by an inactive center with
demyelination, activated microglia at the lesion edge and few macrophages containing
myelin debris, but transected axons are also observed, suggesting a very slow rate of
ongoing demyelination and axonal damage.

27. NORMAL- APPEARING WHITE MATTER
• Macroscopically normal white matter (that is, normal appearing white matter (NAWM)) often shows
signs of diffuse inflammation and neuro-​axonal damage.
• Abnormalities of NAWM have been observed in patients with RRMS but are more severe in those
with progressive disease and include decreased fiber density owing to axonal degeneration and
demyelination, small round cell infiltration (mainly lymphocytes), macrophage infiltration, widespread
microglia activation and gliosis.
• These diffuse changes poorly correlate with the number, size, location and destructiveness of focal
white matter lesions in the brain and spinal cord, suggesting that they might occur independently.
27

28. GREY MATTER LESIONS
• Extensive cortical demyelination is observed in the forebrain and cerebellum in patients with MS, occurs
from the earliest phases of the disease (that is, also in patients with radiologically isolated syndrome
and is more widespread in patients with PPMS and SPMS.
• Lesions can also occur within deep grey matter nuclei and in the grey matter of the spinal cord, in which
grey matter demyelination is more extensive and widespread than in the white matter.
• Cortical lesions are predominantly found in cortical sulci and in deep invaginations of the brain surface
and are often topographically related to inflammatory infiltrates in the meninges.
• Compared with white matter lesions, cortical lesions typically display less BBB breakdown, less
oedema, a lower degree of inflammation and more efficient myelin repair occurring after demyelination,
suggesting that different mechanisms determine lesion formation in the white matter and the grey
matter.
28

29. 29
Types of cortical
lesions in MS
Type 1
Type 2
Type 3
Type 4
Located at the cortico-​subcortical border and
affect both the grey matter and the white matter.
Small perivenous intracortical lesions that do not
affect white matter or the pial surface of the brain
Extend inward from the subpial layers of the
cortex
(subpial lesions).
Extend through the whole width of the cortex but
without passing the border between the cortex
and the white matter.
Type 3 cortical lesions are the most frequent in patients with MS and are characterized by
subpial areas of demyelination, which involve the cortical ribbon of several gyri and are often
related to meningeal inflammatory infiltrates usually not extending beyond layers 3 and 4 of the
cortex.

30. 30
The four types of cortical lesion identified by DIR
imaging.
a | Mixed white matter and gray matter lesion (type I).
b | Intracortical lesions (type II).
c | Subpial lesion (type III).
d | 'Worm-like' lesion (type IV).
Calabrese, M., Filippi, M. & Gallo, P. Cortical lesions in multiple sclerosis.
Nat Rev Neurol 6, 438–444 (2010). https://doi.org/10.1038/nrneurol.2010.93

31. REMYELINATION AND DEGENERATION
• Remyelination gives rise to the so-​called shadow plaques that are characterized by global or patchy
remyelination, a sharp demarcation from the surrounding NAWM and axons with thin myelin sheaths
and shortened internodes.
• The extent of remyelination is very heterogeneous, although it is generally limited and restricted to
the lesion border or is patchy, and has been demonstrated in ~40–50% of white matter lesions and in up
to 90% of grey matter lesions, although different values have been reported in some studies.
• The variability in remyelination depends on several factors, including patients’ age, disease
duration, lesion location, the presence of oligodendrocyte progenitor cells and axonal integrity.
• Substantial remyelination is frequently observed during the earlier phases of MS and in younger
individuals, whereas it is more sparse or absent in PPMS and SPMS.
• Neuro-​axonal loss is of particular interest in MS, as it corresponds to neurodegeneration which
occurs from the earliest phases of disease and might contribute to irreversible clinical disability.
31

32. RISK FACTORS
32
Genetic factors
Environmental
factors
Life style
factors

33. GENETIC FACTORS
• The prevalence of familial MS is ~13% for all MS phenotypes.
• The heritability of MS is polygenic and involves polymorphisms in several genes, each of
which is associated with a small increase in disease risk. Among these, polymorphisms in
HLA class I and HLA class II genes convey the highest risk of MS.
• Genome-​wide association studies have identified >200 genetic risk variants for MS; each
variant has a small effect on risk of disease, and different combinations of these variants likely
contribute to genetic susceptibility in different patients.
33

34. 34
• HLA complex contains multiple genes related to immune system functioning, and HLA-DRB1*15:01 is
associated with increased risk of MS and is present in up to 30% of the population in the United
States and northern Europe, while HLA-A*02 is associated with decreased MS risk.
• Combinations of genetic factors also likely contribute to disease risk; for example, the presence of
HLA-DRB1*15:01 and the lack of HLA-A*02 are associated with higher risk of MS than the presence
of HLA-DRB1*15:01 alone.
• Gene–environmental interactions that may contribute to MS pathogenesis include vitamin D levels,
obesity in childhood, EBV infection, and smoking.
• In clinical practice, testing for genetic variants associated with MS risk is not currently
recommended even in patients with a family history of MS.

35. ENVIRONMENTAL AND LIFE STYLE FACTORS
• Substantial evidence supports a period of susceptibility to environmental risk factors for MS
during adolescence although exposure to some factors might be relevant during other phases
of life (such as low vitamin D level during pregnancy).
• The most well established risk factors are Epstein–Barr virus (EBV) infection in
adolescence and early adulthood, tobacco exposure through active or passive smoking, a lack
of sun exposure, low vitamin D levels and obesity during adolescence.
35

36. • EBV is a herpes virus frequently acquired in childhood and is often asymptomatic. Later-onset
infection in adolescence and adulthood is more commonly associated with clinical illness
manifesting as infectious mononucleosis. Following infection, latent EBV remains in host B
lymphocytes.
• Across multiple meta-analyses, EBV antibody seropositivity and infectious mononucleosis have
consistently been associated with MS risk.
• EBV seropositivity is high not only in patients with MS but also in the general population; thus,
the role of EBV in MS pathogenesis is likely multifactorial and may include interactions between
EBV, predisposing genetic factors, and environmental risk factors.
36
Epstein-Barr Virus

37. 37
EBV nuclear
antigen 1
(EBNA1)
 It’s expressed in EBV-infected B cells and contributes to transcription regulation and viral genome
maintenance.
 Proposed mechanisms by which EBV may contribute to MS pathogenesis include molecular mimicry
between EBNA1 and glial cell adhesion molecules expressed by CNS oligodendrocytes and
astrocytes resulting in cross-reactive antibodies.
 In human tissue studies, a significantly higher number of EBV-related proteins were present in chronic
MS lesions compared with non-MS controls, and 85% of patients with MS had EBV-encoded RNA-
positive B cells in their brain compared with rare occurrence in control brains.

38. • Smoking has been associated with increased risk of MS, as well as conversion
from CIS to clinically definite MS and conversion of relapsing to secondary
progressive MS.
• Smoking may also reduce efficacy of at least some DMTs including interferon and
natalizumab.
• Passive smoke exposure has also been associated with increased MS risk.
Interestingly, use of oral tobacco has been associated with decreased risk of MS,
suggesting that lung inflammation from smoking may be the driver of increased MS
risk in smokers.
38
Smoking

39. • Obesity in adolescence and childhood has been associated with increased risk of both
pediatric and adult-onset MS, with a seemingly stronger association in females compared
with males.
• In pediatric patients, obesity may precede MS diagnosis by several years.
• Abdominal obesity has also been associated with more severe disability in patients with
known MS.
• Proposed mechanisms by which obesity may be involved in MS pathogenesis include
promotion of a proinflammatory milieu, decreased vitamin D bioavailability, and interactions with
predisposing genetic MS risk factors and EBV infection.
39
Obesity

40. • Gut microbiome may influence systemic inflammation.
• Gut colonization in early life is influenced by a variety of factors including method of delivery,
breastfeeding, antibiotic exposure, and genetic, environmental, and maternal factors.
• Diet significantly influences microbiome composition throughout the remainder of an individual’s
life.
• Species of Bacteroides and Firmicutes within the gut use indigestible fiber and complex
carbohydrates to produce short chain fatty acids that can ultimately increase production of
regulatory T cells important in suppressing autoreactive immune cells in MS.
• Bacteria can also produce cytokines that can influence both pro- and anti-inflammatory immune
cells.
• A unifying “MS microbiome” has not been demonstrated, but a higher quality diet may assist in
improving common symptomatic issues.
40
Microbiome and diet

41. THANK YOU
41