interstitial lung disease in systemic sclerosis is...

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


100 101 102 103 104

FL1-H


100 101 102 103 104

FL1-H


100 101 102 103 104

FL1-H


100 101 102 103 104

FL1-H


100 101 102 103 104

FL1-H


100 101 102 103 104

FL1-H


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

interstitial lung disease in systemic sclerosis is...

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