Identification of Autoimmunity to Peptides of Collagen V α1 Chain as Newly Biomarkers of Early Stage of Systemic Sclerosis

Abstract

Patients with Systemic sclerosis (SSc) presents immune dysregulation, vasculopathy, and fibrosis of the skin and various internal organs. Pulmonary fibrosis leads to SSc-associated interstitial lung disease (ILD), which is the main cause of morbidity and mortality in SSc. Recently autoimmunity to type V collagen (Col V) has been characterized in idiopathic pulmonary fibrosis and show promise to be related to the development in SSc. Our aim was to evaluate autoimmunity to Col V α1(V) and α2(V) chains and to the antigenic peptides of these Col V chains in early-SSc sera employing lung tissue of SSc-ILD, as antigen source. We found that sera samples from patients with early-SSc were reactive to Col V (41.18%) and presented immunoreactivity for Col5A1(1.049) and Col5A1(1.439) peptides. The IgG isolated from early-SSc patients-anti-Col V positive sera (anti-ColV IgG) was adsorbed with α1(V) chain (anti-ColV IgG/ads-α1(V)) and α2(V) chain (anti-ColV IgG/ads-α2(V)) and biotinylated to evaluate the spectrum of reactivity in SSc-ILD patients lung biopsies by immunofluorescence. The SSc-ILD lung tissue samples immunostained with anti-ColV IgG showed increased green fluorescence in the vascular basement membrane, bronchiolar smooth muscle, and adventitial layer, contrasting with the tenue immunostaining in control lungs. Col V protein expression in these pulmonary compartments immunostained with early-SSc anti-ColV IgG was confirmed by immune colocalization assays with commercial anti-human Col V antibodies. In addition, SSc-ILD lung tissues immunostained with anti-ColV IgG/ads-α1(V) (sample in which Col V α1 chain-specific antibodies were removed) showed decreased green fluorescence compared to anti-ColV IgG and anti-ColV IgG/ads-α2(V). Our data show that autoimmunity to Col V in early-SSc was related to peptides of the α1(V) chain, suggesting that these antibodies could be biomarkers of SSc stages and potential target of immunotherapy with Col V immunogenic peptides.

Keywords: autoimmunity; biomarker; early-SSc; type V collagen; α1(V) chain.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Velosa, Brito, de Jesus Queiroz, Carrasco, Tomaz de Miranda, Farhat, Goldenstein-Schainberg, Parra, de Andrade, Silva, Capelozzi and Teodoro.

Figures

Figure 1

Detection of systemic antibodies to α1(V) and α2(V) peptides. The early-SSc patient and normal sera were incubated with streptavidin beads coated with Col5A1(599), Col5A1(779), Col5A1(909), Col5A1(1049), Col5A1(1439), Col5A2(275), Col5A2(419) and Col5A2(1078) peptides. (A) Flow cytometry was used to detect IgG antibodies bound to Col V peptides. Data shown are derived from serum collected from an early-SSc patient and a normal individual and are representative of n=7 patients sera tested positive to anti-Col V and n=6 normal sera. (B) Graphic of the anti-Col V peptides detection by flow cytometry, expressed as percentage of the fluorescent bead-bonded anti-Col V peptides of the α1(V) chain and α2(V) chain in sera from early-SSc patients and normal individuals. The filled points and the empty points represent the individual values of the detection of anti-Col V peptide antibodies, respectively, in the serum of patients and controls. Among the eight tested peptides, the Pept Col5.1 (1049) and Pept Col5.1 (1439) were more expressed in early-SSc sera in relation to control. (Student t test; *P < 0.05).

Figure 2

Histological sections of lung samples from control subjects without associated pulmonary pathology (A) and from SSc-ILD patients with a histological pattern of Non-Specific Interstitial Pneumonia (NSIP) (B, C) (Hematoxylin & Eosin staining; original magnification: A–C, X400). Immunofluorescence in lung sections from control samples (D, G, J) and SSc-ILD patients (E, F, H, I, K, L) immunostained with early-SSc biotinylated anti-ColV IgG (D–F), anti-ColV IgG/ads-α1(V) (G–I) and anti-ColV IgG/ads-α2(V) (J–L). Note the green immunofluorescence along of periadventitial layer of the bronchovascular axis (E, K) and alveolar septa (F, L) layers (arrows). The reaction was reveled with ALEXA Fluor 488 streptavidin. Original magnification: 400X, (D–L).

Figure 3

Box plot graphs of the immunofluorescence staining quantification of biotinylated early-SSc anti-ColV IgG, anti-ColV IgG/ads-α1(V) and anti-ColV IgG/ads-α2(V) detection, expressed as percentage, in lung tissue from defined-SSc patients and normal lungs. Each box corresponds to individual immunofluorescence intensity and lung tissue in defined-SSc patients and normal lungs. The solid bar represents the values of anti-Col V α1 or α2 chains between the 25th and 75th percentiles, the white bar shows the median value, and the top and bottom brackets show the extreme values whereas black dotted lines indicate the median anti-Col V level. *P

Figure 4

Immunofluorescence in lung sections from control and SSc-ILD patients’ pulmonary tissues shows Col V immunostained in red (A, B, arrows) and the immunolabeling of biotinylated anti-ColV IgG from early-SSc immunostained in green (C, D, arrows). The merged images show the colocalization of Col V and early-SSc anti-ColV IgG in the vessels and bronchiole basal layers and along the alveolar septa in SSc-ILD pulmonary tissue as seen in a yellowish green immunostaining (F, arrows). The nuclei are stained in blue (E, F). Note, in the detail, the colocalization of Col V and early-SSc anti-ColV IgG in yellowish green fluorescence in the vessel layer from a SSc-ILD tissue sample (F). Original magnification: 400X, (A–H).