Autoimmunity and Chronic Inflammation-O.Boyer

Autoimmunity and chronic inflammation: the example of myositis
Olivier BOYER, MD, PhD

INSERM U1234
Pathophysiology, autoimmunity, neuromuscular diseases and regenerative therapies
Rouen, Normandy, France
Acknowledgements: Catalina Abad, Laurent Drouot, Audrey Aussy, Yves Allenbach (Paris), Alain Meyer (Strasbourg)
Objectives
• To show that:
one tissue (i.e., muscle) can be targeted by different autoimmune processes

autoantibodies can be biomarkers but also pathogenic effectors
developing mouse models is helpful to study pathogenesis
autoimmunity can be associated to cancer
For Jaipur IUIS IIS FIMSA course Oct 12-16, 2019 participants only – DO NOT DISTRIBUTE SLIDES
Myositis: first description
Wagner E. (1886-7). Ein fall von acuter polymyositis. Deutsches Archiv f klin Med Leipz xl.
First description of myositis by Wagner, 1886:

• Muscle weakness
• Myalgia
• Dermatological signs
• Evolution: dyspnea, death  pathological examination of muscle
Myositis
• Acquired (non monogenic) autoimmune diseases

• There is not a single myositis but several distinct entities with different treatments and prognosis
• autoantibody-mediated autoimmunity like myasthenia gravis (e.g., immune-mediated necrotizing myopathy)
• cell-mediated autoimmunity like multiple sclerosis(e.g., dermatomyositis)
• May affect adults as well as children (e.g., juvenile dermatomyositis)
• May be associated to cancer (i.e., dermatomyositis)
• Inflammatory (macrophages) and adaptive immunity (T) cells infiltrate muscle
• Immune attack causes necrosis of muscle fibers and release of muscle enzymes (creatine kinase)
• Necrosis elicits muscle fiber regeneration from muscle adult stem cells (i.e. satellite cells)
Myositides: classification
Griggs et al. ENMC- Hoogendijk et al. Troyanov et al.

(1995) (2004) (2005)
Inclusion-body Immune-mediated Overlap

Dermatomyositis Polymyositis
myositis necrotizing myopathy myositis
aAb ± clinical signs
H&E CD8

(1995) (2004) (2005)

- Red ragged fibers (COX negative)

- Rimmed vacuoles
- Protein inclusions
- Inflammatory infiltrate (CD8)
- HLA class I upregulation

(1995) (2004) (2005)

H&E

(1995) (2004) (2005)

- Adult and juvenile forms

- Targets skin and muscle
- Risk of cancer in adults
H&E - IFN-I signature
- Perifascicular atrophy
- Inflammatory infiltrate (B, CD4)

(1995) (2004) (2005)


(1995) (2004) (2005)

- Myofiber necrosis
- Regeneration
- Myofiber C5b-9 deposits
- Moderate (if any) inflammation
- Anti-SRP or HMGCR aAb

(1995) (2004) (2005)

aAb ± clinical signs
Pathogenesis of myositis:
 Humoral immunity
 Cellular immunity
Auto-antibodies: from biomarkers to pathogenic cues
IMNM
Simon et al., Front Immunol, 2016

Auto-antibodies: from biomarkers to pathogenic cues
Auto-antibodies in necrotizing autoimmune myopathies
 recently recognized group of acquired myopathies

 progressive muscle weakness
 severe, debilitating disease
 myofiber necrosis with high CK levels
Kipfer et al. Lancet 2015
 aAbs against ER molecules:

anti-SRP (signal recognition particle)
anti-HMGCR
Signal Recognition Particle (SRP)
Ribonucleoproteic complex
Translocation of nascent polypeptide
- ARN 7SL
to the endoplasmic reticulum
- 6 polypeptides
14kDa 9kDa
7SL ARN mRNA
68kDa 72kDa
54
54
19kDa 54kDa
ER membrane
SRP receptor
Translocon
HMG-CoA reductase (HMGCR)
• Target : HMG-coA reductase (HMGCR)
• Discovered in 2011 by Mammen et al. in NAM patients taking statins
N cytosol
endoplasmic reticulum
Cholesterol
Anti-SRP and anti-HMGCR aAb are pathogenic in IMNM
• aAb levels correlate with disease severity
aAb levels correlate with CK under therapy
Benveniste et al. Arthritis Rheum 2011
Momomura et al. Mod Rheum 2014

• aAb induce myotube atrophy in vitro
aAb induce myotube atrophy in vitro
Plasma
Ctrl anti-HMGCR
IgG
Ag-specific aAb
Arouche-Delaperche et al. Ann Neurol 2017


• aAb decrease myotube formation in vitro
aAb inhibit myotube formation in vitro
Ctrl anti-SRP Plasma
IgG
Ag-specific aAb
IL-4
in vitro : no complement, no ADCC
Arouche-Delaperche et al. Ann Neurol 2017


• passive transfer of aAb to mice induces a muscle deficit
What question should you ask yourself before doing the experiment?
What question should you ask yourself before doing the experiment?
human vs mouse identity

SRP54 : 100% HMGCR (catalytic domain): 95%
confirm recognition experimentally
Anti-SRP and anti-HMGCR aAb are pathogenic in mice
CYC Plasma or IgG injections
C57BL/6
D-1 D1 D2 D3 D4 D5 D6 D7 D8
Grip test, blood Grip test

Grip training Muscle strength
Blood & organs
*** *
*** 250 p=0.055
120 **
**
0
Muscle strength (g)
 Grip strength (g)
Muscle strength (g)

200
100
-20 -20
 Gripstrength(g)
150
80
-40 -40
100
60
-60 50 -60
Co ntrol IgG Control IgG Control IgG Control IgG
IgG-d ep letedplasma(P3) IgG-depleted plasma (P3) IgG-depleted plasma (P4) Anti-HMGCR+ IgG (P4)
Anti-SR +
P IgG(P3) Anti-SRP+ IgG (P3) +
Anti-HMGCR IgG (P4)
Bergua et al. Ann Rheum Dis 2018 Anti-SRP Anti-HMGCR
Anti-SRP and anti-HMGCR aAb are pathogenic in mice
H&E H&E 6
*
Necrotic fibres
4
Control Anti-SRP+
2
0
Control IgG Anti-HMGCR+ IgG
Anti-HMGCR+ Anti-HMGCR + Control IgG
Anti-HMGCR+ IgG (P5)
X400 X400
Quantification of necrotic muscle fibres in tibialis, triceps and
H&E F4/80+
gastrocnemius muscles
x400
MYH3
Bergua et al. Ann Rheum Dis 2018

Pathogenic effects are prolonged in immunodeficient mice
IgG injections
Rag2-/-
C57BL/6
D-1 D0 D8 D14 D21 D22
Grip test blood Grip test Grip test Grip test

Grip training Muscle strength
Blood & organs
50 ** 50
** **
** **

0 0
-50 -50
-100 -100
D8 D14 D21 D8 D14 D21 D8 D14 D21 D8 D14 D21
Control IgG Control IgG

+
Anti-SRP IgG Anti-HMGCR+ IgG
SRP > HMGCR
Bergua et al. Ann Rheum Dis in press


• immunization induces a muscle deficit
• complement (classical pathway) is involved
Immunisation with recombinant SRP provokes a muscle deficiency
20000
**
** 40 ***
Anti-SRP Ab (MFI) 15000 ** ***
SRP

20
Boosts
10000 0
5000 -20
-40
0
D0 D14 D21 D28 D38 D60 -60
D14 D28 D38 D60 D14 D28 D38 D60
ALBIA
1.5 ** 150 *
** OVA immunisation
** SRP immunisation
Anti-OVA Ab (DO)
Muscle strength (g)

*
OVA 1.0 100
0.5 50
0.0 0
D0 D14 D21 D28 D38
ELISA

Immunisation with recombinant HMGCR provokes a muscle deficiency
8000 2.0
**
6000 1.5
HMGCR Anti-HMGCRAb(MFI) **
Anti-OVAAb(DO)
4000 1.0
2000 0.5
0 0.0 160
D0 D7 D14 D0 D7 D14 **
Muscle strength (g)

140
ALBIA ELISA
120
100
80
2.0
8000
** 60
**
Anti-OVA Ab (DO)
1.5
6000
OVA OVA immunisation
Anti-HMGCRAb(MFI)
1.0 HMGCR immunisation
4000
0.5
2000
0.0
D0 D7 D14 0
D0 D7 D14
ELISA ALBIA


Involvement of complement
)
g
(
h
C3-/- CYC Plasma or IgG injections CYC IgG and complement injections
t
C3WT
C57BL/6
g
D-1 D1 D2 D3 D4 D5 D6 D7 D8 D-1 D1 D2 D3 D4 D5 D6 D7 D8
n
e
Grip test, blood Grip test Grip test, blood Grip test
r
Grip training Muscle strength Grip training Muscle strength

t
Blood & organs Blood & organs

s
e
C5b-9
l
** Anti-SRP+ IgG (P3)

**
c
6 0 120 150 ***
Muscle strength (g)

* Anti-SRP+ IgG (P3) + C'
s
100 Anti-HMGCR+ IgG (P4)

Muscle strength (g)
100
4 0
Anti-HMGCR+ IgG (P4) + C'
u
80
50
60
2 0
M
+ +
A n ti- S R P Ig G (P 3 ) Anti-HMGCR IgG(P4)
40 0
C 3 w t
C3 wt
C 3 - /- C3-/-
Bergua et al. Ann Rheum Dis 2018

• anti-SRP and anti-HMGCR are useful for diagnosis

• support the use of plasmapheresis, and of B cell or complement-targeting
therapies
Pathogenesis of myositis:
 Humoral immunity
 Cellular immunity
Icos-/- NOD mice
• NOD: non obese diabetic
• Auto-reactive response toward pancreas
Icos : inducible costimulation
• Icos-/- NOD: no diabetes
• Muscle weakness
Prévot N. et al, Eur J Immunol, 2010
May Icos-/- NOD mice represent (the first) model of spontaneous autoimmune myositis ?
Myopathy but no diabetes in ICOS/ICOSL deficient NOD mice
Incidence of
Survival (%) Δ Weight
diabetes (%)
100 1.50
100 * toto ***
80
80
1.25
* ***
NOD wt
60 Icos NOD
-/-
60
40 40 1.00
*
* **
**
Icosl-/- NOD
Icos-/-
20 20
0
0 10 20 30 40 50
0
0 10 20 30 40 50
0.75
11 16 21 26 31 36 41
Icosl-/-
Age (weeks) Age (weeks) Age (weeks)
Grip Myopathy
strength (g) clinical score
150 10 **
Icos-/- ** **
**
wt Icosl-/- ** **
8 **
Icos-/- **
**
*** * **
* **
j
*** NOD
100
*** 6
** **
** j
-/- NOD
-/- •Decrease in muscle strength
** ** ** Icoslj Icos
Icosl NOD
• Myopathy onset around 25 weeks
-/-
*** 4 **
50 **
wt **
** 2
0 0
07 19 31 07 19 31 07 19 31 25 29 33 37 41 25 29 33 37 41 25 29 33 37 41
Age (weeks) Age (weeks)
Myopathy affects locomotor ability in Icos-/- NOD mice
Cadence Regularity
(step/sec) index (%)
***
30 *** 100
Catwalk analysis 80 wt
20
60
Icos-/-
wt 40
10
20 Icos-/-
0 0
Mean Print
intensity (%) area (cm²)
100 0.5
** ***
80 0.4
60 0.3
35 weeks-old mice 40 0.2
20 0.1
0 0.0
Front paws Hind paws Front paws Hind paws
Predominance of CD4+ T cell infiltrates
MYH3 H&E CD4
Icos-/-
x100
wt
x100
• Myofiber necrosis/regeneration
• Endomysial mononuclear infiltrates
• CD4+ T cells
Is this sufficient evidence to conclude to the pathogenic role of T cells?
What would you do?
CD4+ T-cell requirement for myositis development
Percent myopathy
100
Sorted splenocytes
80
CD4+
60 CD4+ CD8+
40
20 CD8+
Icosl-/- NOD.scid 0 CD4- CD8-
20 30 40 50
Days after transfer
CD4+ from muscle infiltrates

IFNγ IL-4 IL-17
• CD4+ but not CD8+ T cells mediate myositis (Th1 bias) Briet C. et al, Front Immunol, 2017
Prévot N. et al, Eur J Immunol, 2010
MHC class II requirement (Icosl-/-) for myositis development
In c id e n c e o f m y o s it is ( % ) In c id e n c e o f m y o s it is ( % )
100 100
80 CIITAwt 80 β2m-/-
60 60
β2mwt
40 40
20
CIITA-/- 20
0 0
0 10 20 30 40 50 0 10 20 30 40 50
A g e (w e e k s ) A g e (w e e k s )
Briet C. et al, Front Immunol, 2017

Cancer / DM association : a long story
• First description in 1916
• Global rate of cancer in DM: 7% - 32%
• Occurrence within 3 years around DM diagnosis
• Different types of cancer (ovarian, lung, breast)
• Absence of cancer association in juvenile DM
CADM are strongly associated with specific aAbs
• Anti-TIF1g aAbs
• Anti-NXP2 aAbs
Aussy et al., Arthritis Rheum 2019
• ANA negative
Association of antinuclear antibody status with clinical features and malignancy risk in adult-onset
dermatomyositis
Hoesly PM et al. JAAD 2018
Description of anti-TIF1g+ DM
Anti-TIF1γ aAb+ DM
Œdema, severe cutaneous signs, dysphagia +++, mild muscle weakness
No calcinosis, no ILD
JDM (30 - 40 %) Adult DM

No cancer (15 – 30 %)
Rate of Series of anti-TIF1g+ aAbs adult DM
cancer
84% Hida A et al. Am Acad Neurology. 2016.
78% Aussy et al. submitted

Higher frequency of 75% Targoff I et al.. Arthritis Rheum. 2006.
associated cancer 73% Fujimoto M et al. Arthritis Rheum. 2012.
71% Kaji K et al. Rheumatology. 2007.
46% Best M et al. J Eur Acad Dermatol Venereol. 2017.
22% Fiorentino DF et al. J AM Acad Dermatol. 2015.
Risk of cancer Short delay between DM and

cancer
x5.5 vs general population
Within 1 year before or after
X 10-20 vs other myositides DM diagnosis
Anti-TIF1g IgG2 are associated with cancer
Positive predictive value of

occurrence of cancer = 100% if
anti-TIF1g-IgG2+ > 385 of MFI
No cancer occurred after 2 years
in absence of IgG2
analysis performed in patients in whom cancer was not known at time of diagnosis of DM
Aussy et al., Arthritis Rheum 2019

Conclusions
one tissue (i.e., muscle) can be targeted by different autoimmune processes

autoantibodies can be biomarkers but also pathogenic effectors
developing mouse models is helpful to study pathogenesis
autoimmunity can be associated to cancer

Autoimmunity and Chronic Inflammation-O.Boyer

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Autoimmunity and chronic inflammation: the example of myositis

Olivier BOYER, MD, PhD

Rouen, Normandy, France

one tissue (i.e., muscle) can be targeted by different autoimmune processes

First description of myositis by Wagner, 1886:

• Acquired (non monogenic) autoimmune diseases

Griggs et al. ENMC- Hoogendijk et al. Troyanov et al.

Inclusion-body Immune-mediated Overlap

aAb ± clinical signs

Griggs et al. ENMC- Hoogendijk et al. Troyanov et al.

Inclusion-body Immune-mediated Overlap

- Red ragged fibers (COX negative)

Griggs et al. ENMC- Hoogendijk et al. Troyanov et al.

Inclusion-body Immune-mediated Overlap

Griggs et al. ENMC- Hoogendijk et al. Troyanov et al.

Inclusion-body Immune-mediated Overlap

- Adult and juvenile forms

Griggs et al. ENMC- Hoogendijk et al. Troyanov et al.

Inclusion-body Immune-mediated Overlap

Griggs et al. ENMC- Hoogendijk et al. Troyanov et al.

Inclusion-body Immune-mediated Overlap

Griggs et al. ENMC- Hoogendijk et al. Troyanov et al.

Inclusion-body Immune-mediated Overlap

aAb ± clinical signs

Simon et al., Front Immunol, 2016

 recently recognized group of acquired myopathies

 aAbs against ER molecules:

• Target : HMG-coA reductase (HMGCR)

• Discovered in 2011 by Mammen et al. in NAM patients taking statins

• aAb levels correlate with disease severity

Benveniste et al. Arthritis Rheum 2011

Momomura et al. Mod Rheum 2014

• aAb levels correlate with disease severity

Arouche-Delaperche et al. Ann Neurol 2017

• aAb levels correlate with disease severity

Ctrl anti-SRP Plasma

in vitro : no complement, no ADCC

Arouche-Delaperche et al. Ann Neurol 2017

• aAb levels correlate with disease severity

• passive transfer of aAb to mice induces a muscle deficit

• passive transfer of aAb to mice induces a muscle deficit

human vs mouse identity

CYC Plasma or IgG injections

Grip test, blood Grip test

 Grip strength (g)

Muscle strength (g)

Bergua et al. Ann Rheum Dis 2018 Anti-SRP Anti-HMGCR

Bergua et al. Ann Rheum Dis 2018

Grip test blood Grip test Grip test Grip test

 Grip strength (g)

Control IgG Control IgG

SRP > HMGCR

Bergua et al. Ann Rheum Dis in press

• aAb levels correlate with disease severity

 Grip strength (g)

Muscle strength (g)

Bergua et al. Ann Rheum Dis in press

Muscle strength (g)

Bergua et al. Ann Rheum Dis in press

• aAb levels correlate with disease severity

Grip training Muscle strength Grip training Muscle strength

Blood & organs Blood & organs

** Anti-SRP+ IgG (P3)

6 0 120 150 ***