interstitial lung disease in systemic sclerosis is...
TRANSCRIPT
Ana Paula Pereira Velosa1, Lais Brito1, Zelita Aparecida de Jesus Queiroz1, Solange Carrasco1,
Jurandir Tomaz de Miranda1, Cláudia Goldenstain-Shainberg1, Edwin Parra2, Vera Luiza Capelozzi2,
Walcy Rosolia Teodoro1
Interstitial lung disease in systemic
sclerosis is associated with autoimmunity
to α1(V) chain of type V collagen
Financial support: FAPESP (2016/05617-4; 2017/11865-3), LIM
1Rheumatology Division and 2Department of Pathology of the
Hospital das Clinicas HCFMUSP, Faculdade de Medicina,
Universidade de São Paulo, Brazil
Introduction
➢ Interstitial lung diseases (ILD) are among the most serious complications associated with SSc.
➢ Systemic sclerosis (SSc) is a chronic autoimmune disease, characterized by vascular damage, inflammation
and fibrosis of the skin and internal organs.
kidney crisis
Digital ulcers
Gastroesophageal reflux
disease
PAH
Cardiac involvement
ILD
Histological pattern of NSIP
Morales-Cárdenas et al., Autoimmun Rev 15(11):1094-1108 2016 Peripheral distribution
Non-Specific Interstitial Pneumonia (NSIP)
➢ The identification of biomarkers could predict the course of the disease
➢ Our group have shown the pathogenic role of ColV in clinical and
pre-clinical models of SSc.
Introduction
➢ ColV has been associated to autoimmunity in pulmonary fibrosis
Type V collagen (Col V)
Autoimmunity to COL V
Exposition of COL V immunogenic fragments
➢ Col V is a fibrillary protein
➢ Col V isoform [α1(V)2, α2(V)] is located embedded within collagen I, III and V heterotypic fibrils
Collagen I, III and V heterotypic fibrils
➢ Col V can be considered a sequestered antigen
➢ Potential to become an autoantigen when exposed to the immune system
Objective
Our aim was to evaluate the autoimmunity to α1(V) and α2(V) chains of the Col V in SSc-ILD
Methods
Early SSc patient
sera samples
positive to anti-
Col V (n=7)
Isolate IgG
Early-SSc
anti-ColV_IgG+
Biotinylated
Fluorescent
StreptavidinImmunofluorescence
Pulmonary tissue of SSc-ILD
Biotinylated ColV-IgG+
Sera samples
➢ Health individuals (n=6)
➢ Patients with Early-SSc (n=19)
Lung biopsies
➢ SSc-ILD patients (n=4)
Histologic pattern of NSIP
➢ Control individuals (n=6)
Patients-EULAR Preliminary Criteria
Early-SSc
Biotinylated
Adsorption
α1(V) chain
α2(V) chain
Immunofluorescence
Biotinylated Early-SSc anti-ColV_IgG+/ads_α1(V)
Biotinylated Early-SSc anti-ColV_IgG+/ads_α2(V)Anti-ColV_IgG+
Evaluation of the α1(V) and α2(V) chains immunogenicity in SSc
Positive anti-ColV
(n=7)
Methods
Immunogenic Col V peptides
Peptides Amino acids sequence
Col5A1(599) PPGPAGKPGRRG
Col5A1(779) GIRGLKGTKGEKGED
Col5A1(909) RGQRGPTGPRGERGPRG
Col5A1(1.049) KDGPPGLRGFPGDRG
Col5A1(1.439) LRGIPGPVGEQGLPG
Col5A2(275) PGEVGFAGSPFARGF
Col5A2(419) PGAIGTDGTPGAKGP
Col5A2(1.078) NIRFRYIVLQDTCSK
Keller MR et al. PLoS One 12;8(11):e79601, 2013; Tiriveedhi
V et al. Clin Exp Immunol 167(1):158-68, 2012; Park AC et al.
J Biol Chem 291(7):3359-70, 2016; Sullivan JA et al. Am JTransplant. 17(4): 944–956.
Flow cytometry assay
Col V peptides biotin
Streptoavidinmicrospheres
➢ Early-SSc sera samples with anti-ColV antibodies (n=7)
-Reaction was reveled with a fluorescent conjugate and analyzed in Flow Cytometer
Evaluation of immunogenic peptides of the Collagen V in Early-SSc sera samples
Control pulmonary tissue SSc-ILD patients pulmonary tissue
Lung Histologic Characterization
NSIP histological pattern
Results
Control pulmonary tissue SSc-ILD pulmonary tissue
50µm 50µm50µm
Early-S
Sc
anti-C
olV
_Ig
G+
Immunofluorescence with Early-SSc anti-ColV-IgG+ in pulmonary tissue
↑ immunostaining in vascular layers
↑ immunostaining along of the basal and
adventitial layers in the bronchioles
Weak green fluorescence in the vascular wall ↑ immunostaining along the thickened alveolar
septa
Results
Col V / Red
V VBr
V VBr
Control pulmonary tissue SSc-ILD pulmonary tissue
Early-SSc
anti-ColV_IgG+ / green
Evaluation of co-location of the Early-SSc anti-ColV-IgG+ and Col V in pulmonary tissue
Merge
V VBr Co-location of the Col V and anti-ColV-IgG+
-Vessels and bronchioles basal layers and along the
alveolar septa in SSc-ILD
Rabbit Anti-human Col V antibody
50µm50µm 50µm
50µm50µm 50µm
Early-S
Sc
anti-
ColV
_Ig
G+
/ads_
α1(V
)
Ea
rly-S
Sc
anti-
ColV
_Ig
G+
/ads_
α2(V
)Control pulmonary tissue SSc-ILD pulmonary tissue
After adsorption of Early-SSc anti-ColV-IgG+ with α(V) chains
ColV-IgG+/ads-α2(V) – immunostaining in vessel layers and along the thickened alveolar septa
SSc
lung:
Col
V-IgG+
CT
lung: C
olV-Ig
G+
1(V)
SSc
lung:
Col
V-IgG+/
ads-
1(V)
CT
lung: C
olV-Ig
G+/
ads-
2(V)
SSc
lung:
Col
V-IgG+/
ads-
2(V)
CT
lung: C
olV-Ig
G+/
ads-
0
10
20
30
40
50 * *
Imm
un
ofl
uo
res
ce
nc
e i
nte
ns
ity
(%
)SSc
Lung
CT
Lung
SSc
LungCT
Lung
SSc
Lung
CT
Lung
Anti-ColV-IgG+ Anti-ColV-IgG+/
ads-α1(V)
Anti-ColV-IgG+/ads-α2(V)
Anti-ColV_IgG+/ads-α1(V) - ↓ immunofluorescence intensity
Results
100 101 102 103 104
FL1-H
05-02-19 exp pepti COLV.003
100 101 102 103 104
FL1-H
05-02-19 exp pepti COLV.004
100 101 102 103 104
FL1-H
05-02-19 exp pepti COLV.005
100 101 102 103 104
FL1-H
05-02-19 exp pepti COLV.002
100 101 102 103 104
FL1-H
05-02-19 exp pepti COLV.003
100 101 102 103 104
FL1-H
05-02-19 exp pepti COLV.004
100 101 102 103 104
FL1-H
05-02-19 exp pepti COLV.005
100 101 102 103 104
FL1-H
05-02-19 exp pepti COLV.002
Pept Col5A1 (1.049) / Normal serum Pept Col5A1 (1.049) / Early-SSc patient sera
18,23% 64,34%
Results
Flow cytometry
Positive reactivity to immunogenic Col V peptide in control and Early-SSc patient sera
Antibodies detection to Col V peptides in early-SSc patients sera
SSc NS SSc NS SSc NS SSc NS SSc NS SSc NS SSc NS SSc SN0
20
40
60SSc: early-SSc patient sera
NS: Normal sera
Pep
t C
ol5
A1(5
99)
Pep
t C
ol5
A1(7
79)
Pep
t C
ol5
A1(9
09)
Pep
t C
ol5
A1(1
.049)
Pep
t C
ol5
A1(1
.439)
Pep
t C
ol5
A2(2
75)
Pep
t C
ol5
A2(4
19)
Pep
t C
ol5
A2(1
.078)
****
An
tib
od
ies d
ete
cti
on
(%
)
Results
-Early-SSc Anti-Col V sera samples - immunoreactivity for Col5A1(599) and Col5A1(1.049) peptides
Flow cytometry
➢ We found that the autoimmunity to Col V in Early-SSc is directed to α1(V) chain, emerging as
promisor biomarker of prognosis and target therapy in SSc-ILD patients
Conclusion
➢ The two immunogenic α1(V) chain peptides [Col5A1(599) and Col5A1(1.439)] probably are
related to autoimmunity to α1(V) chain in SSc
Vera Luiza Capelozzi
Edwin Roger Parra
Walcy Rosolia Teodoro
Ana Paula Pereira Velosa
Antonio dos Santos Filho
Cláudia G. Schainberg
Lais Brito
Researchers group
Lizandre Keren Ramos de Oliveira
Jurandir Tomaz de Miranda
Solange Carrasco
Vitória Elias
Zelita Aparecida de Jesus Queiroz
Division of Rheumatology
Department of Pathology
Acknowledgment