Download Viral myocarditis/pericarditis: pathogenesis.

Viral myocarditis/pericarditis: pathogenesis
Alida LP Caforio, MD, PhD
Dept Cardiological ,Thoracic and Vascular Sciences
University of Padova
E-mail: [email protected]
ESC congress 2010
Stockholm, 28 August-1 September 2010
Viral myocarditis/pericarditis:
pathogenesis
Disclosures:
None
Viral myocarditis
• Challenging diagnosis
• Polymorphic presentation
• It may heal or lead to dilated cardiomyopathy
(DCM)
• Pathophysiology of progression to DCM in man
incompletely understood
• Diagnosis (WHO) based on endomyocardial biopsy
(EMB), (histology, immunohistology and molecular
techniques)
• EMB infrequently used, viral etiology suspected,
not proven
Etiology of human myocarditis
INFECTIOUS
IMMUNE-MEDIATED
TOXIC
Bacterial
Allergens: e.g. penicillin
Drugs: e.g
catecholamine
cocaine
Spirochetal
Fungal
Alloantigens: e.g. hearttransplant rejection
Heavy
metals
Protozoal
Parasitic
Physical
agents
Rickettsial
Viral: coxsackievirus, cytomegalovirus, dengue
virus, echovirus, encephalomyocarditis, Epstein–
Barr virus, hepatitis A, hepatitis C virus, herpes
simplex virus, herpes zoster, HIV, influenza A and
B, Junin virus, lymphocytic choriomeningitis,
measles, mumps, parvovirus, poliovirus, rabies,
respiratory syncytial, rubella, rubeola, vaccinia,
varicella–zoster, variola, and yellow fever virus
Autoantigens: e.g. myosin Various
in giant-cell myocarditis and
Agents, e.g
in virus-negative myocarditis ,
myocarditis associated to
organ and non-organ-specific
autoimmune diseases
Caforio A and McKenna WJ, Drugs 1996
sting bites
Viral pericarditis
• Majority of patients recover spontaneously.
• A minority may develop idiopathic recurrent acute
pericarditis (IRAP), a likely autoimmune sequela with
benign prognosis.
• Myocarditis, e.g. small increase in myocytolitic enzymes,
may coexist.
• Viral etiology of acute pericarditis is often suspected but
not proven by current molecular diagnostic tools.
Myocarditis/pericarditis: Viral Epidemiology
• Temporal changes and population changes.
• Historically, group B coxsackieviruses (CVB1 to CVB6),
single-stranded RNA cytolytic viruses of the enterovirus
(EV) genus within the family of Picornaviridae most
frequently associated.
• Newly identified agents: adenovirus (ADV),
cytomegalovirus (CMV), PVB19 and herpesvirus 6
(HHV6).
Myocarditis/pericarditis: Viral Epidemiology
• Cardiotropic viruses are common environmental
pathogens.
• High prevalence of seropositivity in the absence of heart
disease.
• Only a minority of subjects develop
myocarditis/pericarditis during epidemics.
• Probable predisposing immunogenetic background.
Susceptibility to murine coxsackievirus
B3 (CVB3) myocarditis.
• Main determinants:
– virus genetic background and viral load
– genetic control of the immune response in the host,
e.g. Major histocompatibility complex (MHC) and non
MHC genes
– genetically determined and/or acquired dystrophin
deficiency?
• Additional factors:
– Male gender, age (young and old do worse),
pregnancy, exercise and malnutrition, selenium
deficiency
Woodroof JF, 1980; Martino TA, et al 1994; Schmidtke M et al, 2007;
Kadokami T, et al, 2000; Yajima T, et al, 2009; Maekawa Y, et al 2007;
Wolfgram et al 1986; Badorff C, et al 1999; Xiong D, et al 2002; Beck MA
et al. 1995; Gay RT, et al, 2006.
Time course of experimental viral
myocarditis in mice
Innate immunity
Adaptive immunity
Chronic Immunopathology
*Kawai C. Circulation 1999; Feldman AM &
McNamara D, N Engl J Med 2000
Mechanisms of viral entry into the cell
• CVB1 to CVB6 use the coxsackie and ADV receptor (CAR) for
attachment and entry into epithelial cells.
• Some CVB strains use decay accelerating factor (DAF, e.g. CD55)
and ADV use integrins ( 3 and  5) as coreceptors.
• The majority of CAR localizes at the intercalated disks in adult
cardiac myocytes, as well as at the cell-cell junctions of the atrioventricular node.
• CAR is colocalised with integrins  3 and  5 on the cardiac
myocyte sarcolemma and CAR expression is upregulated at the
intercalated disks, the sarcolemma and interstitial cells in DCM
hearts and in a myosin-induced autoimmune myocarditis rat model.
• In the absence of CAR expression in the cardiac myocyte the virus
does not infect the cardiac myocyte.
Yajima T & Knowlton KU, Circulation 2009; Shi Y, et al JACC 2009;
Noutsias M, et al Circulation 2001; Ito et al, Circ Res 2000
Positive
strand
Negative strand
Positive strand>Negative strand
=acute infection
Positive =Negative strand
=persistent infection,
restricted viral replication
Mechanisms of direct CVB-mediated
myocardial injury in experimental models (I)
Mechanism
Virus type
Reff
Picornavirus protease 2A cleavage of of
eukariotic initiation factor-4G, leading to
inhibition of host cell protein synthesis
CVB
Lamphear,1993
Enteroviral protease 2A cleavage of the hinge 3
region of dystrophin, leading to loss of
sarcolemmal integrity
CVB3
Badorff,1999
Inducible cardiac-restricted expression of CVB3
protease 2A alone, leading to cleavage of
dystrophin and DCM
None
Xiong, 2007
Viral propagation to adjacent cardiac myocytes
increase with dystrophin deficiency
CVB
Xiong, 2002
CVB3 proteases 2A and 3C induction of
apoptosis, via activation of the extrinsic
caspase-8 mediated pathway and intrinsic
mitochondria-mediated apoptosis pathway
CVB3
Chau, 2007
Pathogenesis in experimental CVB3 models:
lymphocyte-mediated myocardial injury
• After 6 to 7 days after experimental infection, the adaptive (antigenspecific) immune response begins, leading to infiltration of T
lymphocytes in the heart.
• Infiltration of T lymphocytes in the heart peaks at 7-14 days when
tissue damage is also highest and may lead to clearance of virusinfected myocytes (beneficial) by cytotoxic MHC class I restricted
lymphocytes, and/or to autoimmune cell-mediated myocardial injury
or necrosis of uninfected myocytes (detrimental)
• Infectious CVB3 undetectable by culture in the blood and in the
myocardium after day 15 and most animals recover.
• In susceptible mouse strains myocarditis persists weeks or months,
resulting in chronic pathological changes that resemble human
DCM, due to immunopathology (driven by persisting viral or virusinduced antigen) and/or virus-triggered autoimmunity (driven by host
determinants).
Kawai C. Circulation 1999; Feldman AM & McNamara D, N Engl J Med 2000
Yajima T & Knowlton KU, Circulation 2009
Features of persistent CVB infection in
murine models
• Decrease in the total amont of viral RNA.
• Decrease in the ratio of positive- to negative-strand
viral RNA
• Low levels of viral protein expression reflecting
restricted virus replication at the level of viral positivestrand genomic RNA synthesis
• Inability to isolate infectious virus from in vivo samples
during chronic disease
Klingel K et al 1992; Muir P, et al, 1994; Kyu 1992,
Wessely R, et al, 1998; Conaldi PG, et al, 1997
Gullberg M, et al 2010
Evidence for persistent Enteroviral
infection in DCM
• In vivo and in vitro evidence that some picornaviruses, e.g. poliovirus,
Theiler’s murine encephalomyocarditis virus, foot-and-mouth disease
virus, CVB3, CVB4 and CVB5 may establish persistent, noncytolytic
experimental infections.
• Persistence of Enteroviral RNA in human DCM myocardial tissue by PCR
• Cytopathic effects in myocyte cultures transfected with mutant CVB3
cDNA with restricted or defective replication without generation of
infectious virus progeny
• Transgenic expression of replication-restricted CVB3 mutant genome in
myocardial tissue induces DCM with excitation-contraction coupling
abnormalities in mice.
Klingel K et al 1992; Muir P, et al, 1994; Kyu 1992,
Wessely R, et al, 1998; Reetoo NK et al,2000; Conaldi PG, et al, 1997
Gullberg M, et al 2010

MURINE MODEL OF CVB3-INDUCED AUTOIMMUNE
MYOCARDITIS
Acute viral
Chronic lymphocitic
myocarditis myocarditis in the
(1-9 days)
absence
of virus _
__________ ____________
______________
Heart-specific*
antibodies
__________
A.BY/SnJ
Non resolving
Present
A.SW/SnJ
"
"
"
"
A.CA/SnJ
"
"
"
"
B10.S/SgSf
Resolving
Absent
Absent
B10.PL/SgSf
Resolving
Absent
Absent
C3H.NB/SnJ
Non resolving
Present
Present
Genetic strain
Present
* Autoantigen: cardiac myosin heavy chain
(Wolfgram LJ, J Immunol 1986)
MURINE MODEL OF MYOSIN -INDUCED AUTOIMMUNE MYOCARDITIS
Genetic strainMyocarditis Anti-myosin antibdies
___________ __________ __________________
A./J
Cardiac *
myosin
+
complete
Freund's
adjuvant
Severe
High titer
A.SW/SnJ
"
"
A.CA/SnJ
"
"
A.BY/SnJ Moderate
B10.A/SgSnJ "
Low titer
"
C57BL/6J Absent
Absent
C57BL/10J Absent
Absent
* Skeletal myosin did not induce autoimmune myocarditis
(Neu N, J Immunol 1987)
POSSIBLE MECHANISMS OF VIRUS-INDUCED/PRECIPITATED
AUTOIMMUNE HEART DISEASE
Virus
Molecular Breakdown in Breakthrough Tissue
suppressor of "rogue"
necrosis
mimicry
mechanisms autoreactive
T cell clones
Cross-reaction Disturbance in the host's
immune response
between
anti-viral
antibodies and
heart antigens
Insertion of
viral epitopes
into myocyte
membrane
Presentation
Induction of
of "sequestered" myocardial
myocardial
"neoantigen"
autoantigen
Breakdown of T cell tolerance to myocardial self antigens resulting
In autoimmune heart disease
1
2
3
4
5
6
7
8
Viral infection leading to cell
necrosis and viral Ag
presentation
CD8+ T-cell response to viral
Ag presentation leading to
clonal expansion of CD8+
cytotoxic T-cells
Molecular mimicry of viral
Ags
Release of cardiac Ags from
necrotic/apoptotic myocytes
Endocytosis and cardiac-Ag
presentation to CD4 + T-cells
CD4+ T-cells activation of B
cells
Secretion of cardiac-specific
AutoAbs
AutoAbs bind to
cardiomyocyte membrane
Receptors and perpetuate
cardiac necrosis/apoptosis
CBV3, group A Streptococcal M protein
and cardiac myosin: is molecular mimicry
involved in myocarditis/IDC?
• Polireactive autoantibody response against Streptococci , myosin
and other -helical proteins in acute rheumatic fever (ARF)
• Murine anti-Streptococcus/anti-myosin mAb are cytotoxic to
myocytes in culture
Adderson EE et al, J Immunol 1998
• Streptococcal M protein peptide (NT4) with similarity to myosin
induces autoimmune myocarditis in susceptible mice and induces
partial tolerance against CBV3 myocarditis
Huber SA et al, J Immunol 1996
Von Landenberg P, et al, 2007
Elliott, et al
PNAS 2003:13447
Genetics and Autoimmune Disease
• Complex poligenic control of susceptibility and
environmental factors are required to cause disease
• Major role of HLA class II and class I genes in determining
genetic risk of disease
• Involvement of genes related to immune regulatory
pathways
• Same genes may confer increased risk two more than one
autoimmune disease (e.g. Type 1 diabetes mellitus and
Graves’ disease
• Need of massive numbers of study samples and
collaboration of large consortia because of the small effect
of common alleles in genome-wide association studies
(GWAS)
N Engl J Med 2007; 357: 1-12
Nature 2007; 447: 661-678
Nature Genet 2007; 39:857-864
Major Criteria of Autoimmune Disease
Witebsky E, Rose NR
• Mononuclear cell infiltrate and abnormal HLA expression in
the target organ (organ-specific disease) or in various organs
(nonorgan-specific disease) in the absence of infectious
agents
• Circulating autoantibodies (Abs) and/or autoreactive
lymphocytes in patients (pts) and family members
• Abs and/or autoreactive lymphocytes within the affected
organ
• Identification and isolation of autoantigen(s) (Ags) involved
• Disease induction in animals after immunization with Ags
and/or passive transfer of serum, Abs and/or lymphocytes
• Efficacy of immunosuppression in pts
• Autoimmune disease= fullfillment of 2 or more major criteria
Methods
• Anti-heart autoantibody (AHA) screening
– Standard indirect immunofluorescence (IFL) on normal O
blood group human atrium and ventricle cryostat sections.
Skeletal muscle used to detect cross-reacting antibodies.
» Caforio et al. J Am Coll Cardiol 1990; 15 : 1527-34
» Caforio et al. Lancet 1994; 344: 773-77
O-s and AIDA positive patterns
Caforio et al. Heart 2010;96:779-84
Frequency of the cardiac antibodies detected by IFL in
heart disease
Caforio et al, Eur J Heart Fail, 2002; Caforio et al, Lupus 2005
Organ-specific
n (%)
Cross-reactive-1 Cross-reactive-2
n (%)
n (%)
Neg
n (%)
DCM (n=327)
83 (25)*^
35 (11)*^^
3 (1)
206 (63)
Myocarditis (n=161)
54 (33) *^
22 (14)**^
3 (6)
82 (51)
Relatives of DCM
(n=567)
176 (31)*^
7 (1)
12 (2)
372 (66)
Other heart disease
(OCD) (n=160)
1 (1)
7 (4)
5 (3)
147 (92)
Ischemic heart
failure (IHF) (n=141)
1 (1)
1 (1)
8 (6)
131 (92)
7 (2.5)
8 (3)
9 (3)
246 (91)
Normals (n=270)
* p=0.0001 vs OCD or IHF; **p=0.0002 vs OCD
^ p=0.0001 and ^^p =0.0003 vs normals
MHC as antigens in DCM-immunoblotting
Caforio et al, Circulation 1992
Frequency of Ab to myosin heavy chain (MHC) and
other contractile proteins in AM and DCM
Ag
Method
AM
%Ab pos
DCM
%Ab pos
OCD
%Ab pos
Norm
Ref
 and 
MHC
Western
blotting
NT
46 *’**
8
0
Caforio 1992
MLC 1v
Western
blotting
Western
blotting
NT
35
25
15
Caforio 1992
NT
46 *’**
17
0
Caforio 1992
Western
blotting
Western
blotting
NT
67**
42
NT
Latif 1993
NT
17**
0
NT
Latif 1993
Tropom
Western
blotting
NT
55**
21
NT
Latif 1993
Actin
Western
blotting
NT
71**
21
NT
Latif 1993
 MHC
ELISA
17 *’**
20 *’**
4
2
 MHC
ELISA
37 *’**
44 *’**
16
2.5
Goldman 1995
Caforio 1997
Lauer 1994
Non
myofibr
MHC
MLC-1
*P<0.05 vs normals; **P<0.05 vs other cardiac disease (OCD)
Frequency of Ab to non-muscle specific Ag in
AM and DCM
Ag
Method
AM
%Ab pos
DCM
%Ab pos
OCD
Normals Ref
%Ab pos
s.Na/KATPase
M7
ELISA+W NT
estern
blotting
ELISA
13*
26*
NT
2
Baba 2002
31*
10
0
Klein 1984
ANT
SPRIA
91*’**
57 *’**
0
0
Schulteiss1985
BCKD-E2 ELISA
100*’**
60*’**
4
0
Ansari 1994
Laminin ELISA
73
78
25-35
6
Wolff 1989
HSP-60
Western NT
blotting
85**
42
NT
Latif 1993
HSP60,70
Western NT
blotting
10-14**
1-2
3
Portig 1997
*P<0.05 vs normals; **P<0.05 vs other cardiac disease (OCD)
Frequency of Ab to non cardiac-specific receptors in AM
and DCM
Ab
Method
AM
%Ab
pos
NT
DCM
%Ab pos
OCD
Norm
%Ab pos
Ref
30-75 *’**
37
18
Limas 1989
Limas 1991
1 adrener. ELISA
inhibiting
NT
31 *’**
0
12
Magnusson
1990,1994
1 adrener. Bioassay
stimulat.
96 *’** 95 *’**
8
0
Wallukat
1991
1 adrener. ELISA
stimulat.
NT
38**
6
19
Chiale 1995
1 adrener. ELISA
stimulat.
NT
26**
10
1
Jahns 1999
M2 muscar. ELISA
NT
39*
NT
7.5
Fu 1993
1 adrener. LBI
inhibiting
*P<0.05 vs normals; **P<0.05 vs other cardiac disease (OCD)
Survival free from any progression (DCM, LVE, DFS) by cardiac
antibody status at diagnosis in relatives classified as normal at
baseline
Caforio et al, Circulation 2007;
115, January 2/9
Viral damage or ‘molecular mimicry’- placing
the blame in myocarditis
Rose NR,
Nature Med
2000:631-2
Warraich et al
JahnsR, et al
JCI 2004:1419-29
Jane-wit et al,
Li et al
Myocarditis/pe
ricarditis
pathogenesis:
One face or two faces?
Infection and
Autoimmunity
=
Two faces of
the same medal
M.C. Escher
Thank you for your attention!