Streptococcal-vimentin cross-reactive antibodies induce microvascular cardiac endothelial proinflammatory phenotype in rheumatic heart disease

Abstract

Rheumatic heart disease (RHD) is characterized by the presence of anti-streptococcal group A antibodies and anti-endothelial cell antibodies (AECA). Molecular mimicry between streptococcal antigens and self proteins is a hallmark of the pathogenesis of rheumatic fever. We aimed to identify, in RHD patients, autoantibodies specific to endothelial autoantigens cross-reactive with streptococcal proteins and to evaluate their role in inducing endothelial damage. We used an immunoproteomic approach with endothelial cell-surface membrane proteins in order to identify autoantigens recognized by AECA of 140 RHD patients. Cross-reactivity of purified antibodies with streptococcal proteins was analysed. Homologous peptides recognized by serum cross-reactive antibodies were found through comparing the amino acid sequence of streptococcal antigens with human antigens. To investigate interleukin (IL)-1R-associated kinase (IRAK1) and nuclear factor-κB (NF-κB) activation, we performed a Western blot analysis of whole extracts proteins from unstimulated or stimulated human microvascular cardiac endothelial cells (HMVEC-C). Adhesion molecule expression and release of proinflammatory cytokines and growth factors were studied by multiplex bead based immunoassay kits. We observed anti-vimentin antibodies in sera from 49% RHD AECA-positive patients. Cross-reactivity of purified anti-vimentin antibodies with heat shock protein (HSP)70 and streptopain streptococcal proteins was shown. Comparing the amino acid sequence of streptococcal HSP70 and streptopain with human vimentin, we found two homologous peptides recognized by serum cross-reactive antibodies. These antibodies were able to stimulate HMVEC-C inducing IRAK and NF-κB activation, adhesion molecule expression and release of proinflammatory cytokines and growth factors. In conclusion, streptococcal-vimentin cross-reactive antibodies were able to activate microvascular cardiac endothelium by amplifying the inflammatory response in RHD.

Keywords: anti-endothelial cell autoantibodies; rheumatic heart disease; vimentin.

Figure 1

Identification of vimentin as an endothelial autoantigen of rheumatic heart disease (RHD). (a) Endothelial cell-surface membrane proteins were separated by two-dimensional electrophoresis (2DE). (b) After transfer onto nitrocellulose membrane, immunoblotting was performed with sera [diluted 1:50 in phosphate-buffered saline (PBS)-T] from two patients with RHD that resulted anti-endothelial cell antibodies (AECA)-positive. Three spots with molecular weights of approximately 55 kDa, strongly reactive with serum immunoglobulin (Ig)G, were identified (circle). The three spots identified were excised from 2DE gel, digested with trypsin, and then analysed by matrix-assisted laser desorption ionization (MALDI time-of-flight mass spectrometry (MS). MALDI-MS spectra of the tryptic peptides mixtures obtained from the three spots are shown (c) and all of them match vimentin (NP_003371·2) with scores of 171, 130 and 104, respectively. (d) The vimentin sequence is reported three times, but each one shows, in bold, the matched peptides for the relative spectrum reported on the left.

Figure 2

Anti-vimentin antibodies in patients and healthy donors. (a) Box-and-whisker plots of sera [diluted 1:100 in phosphate-buffered saline (PBS)-T] from anti-endothelial cell antibodies (AECA)-positive rheumatic heart disease (RHD) patients, AECA-negative RHD patients and healthy donors analysed by enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin (Ig)G anti-vimentin antibodies. The occurrence of specific antibodies was significantly higher in AECA-positive RHD patients compared with those in AECA-negative and in healthy donors (P < 0·001). (b) To identify the immunoreactive epitopes of vimentin, the C-and N-terminal subunits were cloned and expressed, and analysed using ELISA with AECA and vimentin-positive sera (diluted 1:100 in PBS-T). Immunoreactive epitopes were present in both the subunits. The broken lines represent the cut-off (mean ± 2 standard deviations for healthy donors). Results are expressed as absorbance at 490 nm. The immunoreactivity to the C-ter subunit appeared significantly higher than that to the N-ter subunit of vimentin (P < 0·001 by Mann–Whitney U-test). (c) Inhibition of anti-vimentin ELISA reactivity by pre-absorption of purified human anti-vimentin antibodies (1 μg), with streptococcal protein extract. The y-axis represents percentage of inhibition and the x-axis indicates inhibitor concentrations.

Figure 3

Identification of streptococcal proteins cross-reactive with human vimentin. Streptococcal extracts run in two-dimensional electrophoresis (2DE) (a) were immunoblotted and analysed by 20 μg/ml of purified anti C-ter vimentin antibodies (spot 1, b) and anti N-ter vimentin antibodies (spot 2, c). Spot 1 of 70 kDa and spot 2 of 45 kDa were excised by gel and analysed by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (d,e). The analysis of the spectra allowed the identification, respectively, of the streptococcal heat shock protein 70 (HSP70, NP_665335) with score 102, and the streptococcal streptopain (STRP1, P0C0J1) with score 71: the matching peptides are shown in bold in the respective sequences (f,g).

Figure 4

Identification and immunological characterization of the streptococcal peptides homologous to human vimentin. (a) Sequence homology between streptococcal heat shock protein (HSP)70 and C-ter human vimentin and between streptococcal STRP1 and N-ter human vimentin revealed two peptides named, respectively, peptides A and B (*identical amino acid). (b) Enzyme-linked immunosorbent assay (ELISA) using peptides as antigens with sera diluted 1:100 revealed that all nine sera from rheumatic heart disease (RHD) patients, positive to the C-ter vimentin, reacted with peptide A and four of nine sera from RHD patients reacted positive to the N-ter vimentin resulted positive to peptide B. The broken line represents the cut-off (mean ± 2 standard deviations for healthy donors). Results are expressed as absorbance at 490 nm.

Figure 5

Anti-streptococcal peptides antibodies activate interleukin (IL)-1R-associated kinase (IRAK)1 and nuclear factor (NF)-κB in endothelial cells. Human microvascular cardiac endothelial cells (HMVEC)-C cells either unstimulated or stimulated with purified anti-peptides A and B (200 μg/ml), anti-bovine serum albumin (BSA) antibodies (100 μg/ml) or lipopolysaccharide (LPS) (100 ng/ml) were analysed by Western blot for IRAK1 phosphorylation and NF-κB activation. (a) Phosphorylated levels of IRAK1 (p-IRAK1) were analysed in whole cell extracts by Western blot with anti-phospho-IRAK1 antibodies; for control, the blotted membranes were reprobed with anti-actin antibodies. Bound antibodies were visualized with horseradish peroxidase (HRP)-conjugated immunoglobulin (Ig)G and immunoreactivity was assessed by enhanced chemiluminescence (ECL). Inhibition of IRAK1 phosphorylation by treating HMVEC-C cells with purified anti-peptides A and B (200 μg/ml) pre-absorbed with human vimentin (20 μg/ml) is also shown; (b) NF-κB activation was analysed in whole cell extracts by Western blot with anti-phospho-NF-κB p65 Ser antibodies; as control, the blotted membranes were reprobed with anti-actin antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. Inhibition of NF-κB activation by treating HMVEC-C cells with purified anti-peptide A and anti-peptide B (200 μg/ml) pre-absorbed with human vimentin (20 μg/ml) is also shown.

Figure 6

Anti-streptococcal peptide antibodies induce cytokines, chemokines, growth factors, adhesion molecules and tissue factor (TF) production in endothelial cells. In order to determine concentration of a panel of cytokines, chemokines, growth factor adhesion molecules and TF, enzyme-linked immunosorbent assay (ELISA) or multiplex analysis of culture supernatants from culture supernatants of human microvascular cardiac endothelial cells (HMVEC)-C cells either unstimulated or stimulated with purified anti-peptides A and B (200 μg/ml), anti-bovine serum albumin (BSA) antibodies (100 μg/ml) or lipopolysaccharide (LPS) (100 ng/ml) was performed. The supernatants were harvested and analysed after 1, 6, 12 and 24 h. The comparison with the corresponding values (optical density or fluorescence intensity) obtained after anti-BSA stimulus revealed a significant increase of interleukin (IL)-6, IL-8, platelet-derived growth factor (PDGF), monocyte chemoattractant protein (MCP)-1, vascular cell adhesion molecule (VCAM)-1 and TF in response to anti-peptide A antibody stimulus and a significant increase of IL-8, VCAM-1 and TF in response to anti-peptide B antibody. *P < 0·05.