Monitoring Early Detection of Experimental Systemic Sclerosis-

Interstitial Lung Disease through Collagen V in Human Liquid Biopsy

Background. Systemic sclerosis (SSc) is a rare disorder characterized by immunologic

abnormalities, organ fibrosis and vasculopathy. Interstitial lung disease (ILD), also
named pulmonary fibrosis, is a frequent manifestation of SSc. ILD in SSc is often
associated with a decline in lung function within the first several years of lung disease
onset. Therefore, effective screening to improve early diagnosis of patients with SSc
with associated ILD (SSc-ILD) is crucial. Although techniques such as HRCT (High-
Resolution Computed Tomography) and pulmonary function tests (PFTs) can be used to
detect early development or progression of SSc-ILD, it is essential to find
supplementary consistent and easily obtained markers of disease progression.
Biomarkers may have a role in this respect since they can be readily obtained, are
informative, and can be monitored over time. A focused assessment of peripheral blood
levels of Collagen V (Col V) exemplifies how biomarkers can link molecular mediators
to clinical outcomes.
Objective. Our aim was to evaluate Col V from human early-SSc sera employing lung
tissue of mice SSc-ILD, as antigen source.
Method. Frozen sera samples from 18 female patients (median, 29yrs) with early-SSc
were included in the study. Patients presenting puffy fingers, Raynaud’s phenomenon,
abnormal capillaroscopy with scleroderma pattern, antinuclear, anticentromere and
antitopoisomerase-I antibodies were categorized as early-SSc (EULAR Preliminary
Criteria). Female C57BL/6 mice (n=XXXX) were immunized subcutaneously with
human Col V (150 μg) in complete Freund´s adjuvant, followed by two intramuscular
boosters. The control group (n=XXXX) did not receive Col V. The animal´s groups
were submitted to euthanasia on 120 days after immunization. Lung sections were fixed
in 10% buffered formalin, embedded in paraffin, and sectioned at 4 µm. Hematoxylin-
eosin, western blot, immunofluorescence and histomorphometry were performed to
phenotype and quantify Col V expression.
Results. We found that sera samples from patients with early-SSc were reactive to Col
V α1(V) in six patients (33%). The early-SSc patients-anti-Col V positive sera (anti-
ColV) was used to evaluate the spectrum of reactivity in SSc-ILD mice lung by
immunofluorescence. The mice SSc-ILD lung tissue samples immunostained with anti-
ColV showed increased green fluorescence in the vascular basement membrane,
bronchiolar smooth muscle, and adventitial layer, contrasting with the tenue
immunostaining in control lungs. Histomorphometric analysis showed a significant
increased immunostaining in SSc-ILD lung issue mice incubated with early-SSc anti-
ColV IgG compared to control lungs (p=0.009).
Conclusion. These results show that Col V is a relevant diagnostic biomarker for SSc-
associated ILD and could be used to assess the severity of lung fibrosis through liquid
biopsy and can help to link therapeutic targets to treatable traits in SSc.