Viridans Streptococcal Biofilm Evades Immune Detection and Contributes to Inflammation and Rupture of Atherosclerotic Plaques

Abstract

Background: Bacterial DNA from the oral cavity, respiratory tract, gut, and skin has been detected in atherosclerotic plaques, suggesting a role in chronic inflammation linked to atherosclerosis. Chronic bacterial infections often form biofilms resistant to antibiotics and immune detection, giving rise to a new generation of virulent bacteria in suitable conditions. This study explores the role of the immune system in bacterial-induced inflammation of atherosclerotic plaques.

Methods: Coronary plaques from 121 sudden death victims and endarterectomy samples from 96 surgical patients were analyzed using bacterial real-time quantitative polymerase chain reaction, immunohistochemistry, and genome-wide expression analysis. TLR (toll-like receptor) signaling was examined in bacterial-activated TLR cell lines.

Results: Of the bacteria detected, oral viridans group streptococcal DNA was the most common, being found in 42.1% of coronary plaques and 42.9% of endarterectomies. Immunopositivity for viridans streptococci correlated with severe atherosclerosis (P<0.0001) in both series and death from coronary heart disease (P=0.021) or myocardial infarction (P=0.042). Viridans streptococci colonized the core of the atheroma as a biofilm unrecognized by macrophages of the innate immune system. In contrast, immunopositive streptococci that appeared to have originated from the biofilm infiltrated the ruptured fibrous cap of the atheroma in endarterectomy samples and coronary plaques and were detected by pattern-recognizing receptors and coexpressed with the adaptive immune response. Among the viridans streptococcal strains, TLR2 was the most activated bacterial-signaling pathway. Genome-wide expression analysis of endarterectomy samples showed upregulation of bacterial recognition pathways.

Conclusions: Latent chronic bacterial inflammation evades immune detection and may contribute to the pathogenesis of complicated atherosclerotic plaques and fatal myocardial infarction.

Keywords: bacteria; biofilm; coronary heart disease; immune system; myocardial infarction.

Figure 1. Occurrence of bacterial DNAs and association of streptococcal immunopositivity with atherosclerosis and death due to CHD.

A, A comparison of the frequencies (%) of different bacterial DNA shows similar profiles in the atherosclerotic plaques of the TSDS autopsy coronary samples (black bar) and the TVS clinical endarterectomy samples (gray bar). B, Positive viridans group streptococcal immunostaining (%) among the TSDS autopsy coronary samples (black bar) and TVS clinical endarterectomy specimens (gray bar) shows a highly significant association in both series with the severity of atherosclerosis, using the score defined by the American Heart Association. C, The viridans streptococcal immunopositivity score in the TSDS autopsy series associated with death due to CHD and MI along with age but did not associate with postmortem time, sex, or BMI. AHA indicates American Heart Association; BMI, body mass index; CHD, coronary heart disease; MI, myocardial infarction; RT‐qPCR, real‐time quantitative polymerase chain reaction; TSDS, Tampere Sudden Death Study; and TVS, Tampere Vascular Study.

Figure 2. Immunostaining (1:2000) with pooled viridans streptococci antibodies (brown) and ImageJ software–created color convoluted images (red*) of coronary arteries in the TSDS autopsy series and in the carotid endarterectomy and left internal thoracic control samples in the TVS clinical series.

A through C, Coronary artery with intimal thickening without streptococcal immunopositivity. D through F, Narrowed fibrocalcific plaque with biofilm‐type immunoreactivity inside the plaque and in the subintima over the plaque (arrows). G through I, Rupture and thrombosis (arrows) of a soft thin‐walled coronary atheroma (left) with fatal coronary thrombosis. The remnants of the ruptured fibrous cap are stained by a viridans streptococcal antibody cocktail (arrow). J through L, A control LITA artery sample taken during coronary bypass shows no immunopositivity. M through O, Carotid endarterectomy samples have immunopositive infiltrates (arrows) in the fibrous capsule. P through R, Streptococcal biofilm (arrows) inside the necrotic core of a carotid atheroma. S through T, A viridans streptococcal antibody–positive biofilm borders the lipid core of a coronary atheroma (arrows). U through V, Preimmune serum (1:500) originating from the same mouse before raising antibodies does not stain the biofilm. W through Z, Staining of a tight coronary stenosis site with a viridans streptococcal antibody and macrophage marker CD68 shows that macrophages do not detect bacterial biofilms (arrow). CD68 stains cholesterol‐containing areas (arrows) harboring macrophage‐derived foam cells. Ab indicates antibody; LAD, left anterior descending coronary artery; LITA, left internal thoracic artery; TSDS, Tampere Sudden Death Study; TVS, Tampere Vascular Study; and VStrep, viridans streptococcus.

Figure 3. Immunohistochemical studies on streptococcal biofilms and dispersed virulent streptococci, using conventional peroxidase staining (brown) and ImageJ software to separate the DAB channel with color convolution and to threshold it into red* (biofilm) or green* (virulent streptococci), depending on the intensity of staining (Data S1).

A and B, A coronary atheroma of a patient who experienced SCD showing a streptococcal biofilm (red*), along with several dispersed virulent streptococci that express intense staining of the capsule (green*). A neovascular channel (nvc) is seen near the biofilm. C and D, CD68‐positive macrophages do not detect bacterial biofilms but do recognize discrete virulent streptococci. E through G, An endarterectomy sample from a symptomatic carotid atherosclerotic plaque, showing a streptococcal biofilm (red*) inside the atheroma (dashed line between atheroma and fibrous cap), along with discrete strongly stained streptococci (green*) that have been dispersed from the biofilm and have infiltrated the fibrous cap of the atheroma. H through J, Ruptured coronary atheroma of another patient who experienced SCD with a myocardial infarction, showing infiltration of the rupture site by strongly stained virulent streptococci (green*) that seem to be largely engulfed by macrophages but also occur as free infiltrates. K and L, Ruptured coronary atheroma showing a bacterial biofilm inside the atheroma (red*) and strongly stained virulent streptococci (green*) infiltrating the fibrous cap. Ab indicates antibody; nvc, neovascular channel; SCD, sudden cardiac death; and VStrep, viridans streptococcus.

Figure 4. TLR preferences of different oral bacteria and Chlamydia pneumoniae with demonstration of active TLR2 dependent bacterial recognition pathway in ruptured coronary atheroma.

A, Stimulation of HEK‐TLR cell lines with reference bacteria. Reference bacteria were heat‐inactivated before being added into cultures and cultured as triplicates. Mean values of NF‐kB luciferase activities are presented as % medium values. B, Fatal rupture of an atheroma in the left anterior descending coronary artery in a 67‐year‐old man. The occluding thrombus is removed over the rupture site. C, HE‐stained histological cross‐section from the ruptured atheroma, with the contents of the atheroma (arrow) pouring out. D, A diagram of the tissue components of the ruptured atheroma. E through G, Positive immunostaining of the rupture site with a pooled streptococcal antibody cocktail (1:2000), showing biofilm‐like staining. H through J, Macrophage marker CD68; K through M, Toll‐like receptor 2; N through P, CD14; Q through S, NFkB. T through V, Negative staining with preimmune serum (1:1000). The middle column represents a detail of the ruptured fibrous cap from the left column (marked with a white square). The right column represents pseudo‐colored images created with ImageJ software to visualize DAB–stained (yellow*) and HE‐stained nuclei regions (blue) in the original photos. Ab indicates antibody; HE, hematoxylin and eosin; HEK, human embryonic kidney; NFkB, nuclear factor kappa B; TLR, toll‐like receptor; and VStrep, viridans streptococcus.

Figure 5. Entry of viridans group streptococci into coronary plaque may occur through neovessels.

A through C, A neovascular thin‐walled channel filled with erythrocytes and surrounded by a layer of viridans streptococcal immunopositivity is seen at the boundary between adventitia and media near a large fibrocalcific atheroma of a 61‐year‐old man, who died of recurrent myocardial infarction. There were also immunopositive infiltrates at the border of the atheroma. The coronary sections were stained with viridans streptococcal antibody cocktail. More contrast was achieved by using the color (red) convolution option of the ImageJ software. Ab indicates antibody; and VStrep, viridans streptococcus.

Figure 6. The wall of a ruptured coronary atheroma with occluding thrombosis (A) in the Tampere Sudden Death Study series showed strong staining (B) with the Viridans streptococcal antibody cocktail (1:1000).

Staining with Streptococcus pyogenes mAb (C) and Escherichia coli mAb (D) was negative. Viridans streptococcal immunopositivity was colocalized with CD68‐positive macrophages (E) and bacteria‐recognizing receptors of the TLR signaling pathway (F through J), as well as with lymphocytes expressing T cell markers CD3 and CD3z (CD 247), which together form a T cell receptor complex on the surface of T cells, indicating the activation of the adaptive immune system (K and L). DAB staining to detect necrotic cells was negative (M), whereas preimmune sera (1:1000) nonspecifically detected streptococci (N). Control staining using IgG1 isotype (1:500) was negative (O). Genome‐wide expression analysis (P) on the activity of the signaling pathways of germline‐encoded pattern recognition receptors for gram‐positive and gram‐negative bacteria in atherosclerotic plaque samples from the Tampere Vascular Study in comparison to healthy controls. Ab indicates antibody; FC, fold change; LITA, left internal thoracic artery; MyD88, myeloid differentiation primary response protein; NFkB, nuclear factor kappa B; TLR, toll‐like receptor; and VStrep, viridans streptococcus.