ADULT – Original Submission
Immunohistochemical Detection of Bacteria in the Resected Valves was Associated with Stromal Immune Checkpoint Protein Expression that may Contribute to Calcific Aortic Stenosis

https://doi.org/10.1053/j.semtcvs.2021.10.007Get rights and content

Aortic stenosis (AS) is a disease characterized by narrowing of the aortic valve (AV) orifice. The purpose of this study was to clarify the significance of bacterial detection and clinicopathological factors, including valve-infiltrating immune cells and disease severity, in relation to AS. After obtaining the written informed consent form from 50 patients with AS, we performed immunohistochemical analysis of lipopolysaccharide (LPS) for gram-negative bacteria and lipoteichoic acid (LTA) for gram-positive bacteria on surgically resected-AVs. Moreover, we evaluated the relationships among the presence of bacteria, immune checkpoint protein PD-L1 expression, and immune cell infiltrations such as CD8-positive T lymphocytes, CD163-positive macrophages, and FOXP3-positive regulatory T cell (Treg) in resected-aortic valves. LPS detection in the resected-aortic valve tissues was significantly associated with stromal PD-L1 expression, valve calcification, and LTA existence in resected samples. We showed that the presence of LPS was significantly related to high PD-L1 expression only in calcified-AVs, not in non-calcified-AVs. Moreover, the high expression of PD-L1 in AS samples without LPS was significantly associated with positive infiltration of CD163-positive macrophages and FOXP3-positive Tregs. Immunohistochemical bacterial detection in resected-aortic valves was associated with PD-L1 accumulation and valve calcification. PD-L1 significantly accumulated only in calcified valves with LPS existence.

Graphical abstract

LPS-activated VICs may be involved in calcification and immune checkpoint protein accumulation.

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Section snippets

INTRODUCTION

Aortic valve stenosis (AS) is a highly prevalent acquired aortic valve (AV) disease, and the 2- and 5-year survival rates of patients after the symptom onset have been reported as 50% and 25%, respectively.1, 2 Disease progression could be prevented if the pathogenic mechanisms underlying AS are elucidated. Over the years, efforts have been made for the detection of immune infiltration and bacterial detection along with destructive changes in AV.3 Recently, we reported that high expression of

Study design and patient samples

A total of 50 consecutive patients diagnosed with AS were included in this retrospective study, each of whom underwent surgical resection and valve implantation at Gunma University Hospital between 2010 and 2017. Informed consent was obtained using the opt-out method. Patients with traumatic aortic injury, infectious aortic disease, or other connective tissue disorders were excluded (Supplemental Fig. 1). All patients included in this study were confirmed to have no obvious signs of infection

Patients’ background characteristics

In the present study, 24 men and 26 women were included, and the median age of patients was 73 (56–87) years. The majority of patients were diagnosed with senile AS (n = 23; 46.0%) and tricuspid AV morphology (n = 49, 98.0%). Most patients were older adults (≥65 years; n = 45, 90.0%) and non-smokers (n = 34, 64.0%). For most patients, the left ventricular ejection fraction was ≥50% (39, 78.0%) and the same number of patients (78.0%) were classified as severe AS (AVA <0.75 cm2). The patient

DISCUSSION

In this study, we demonstrated that the relationship of LPS in VICs was significantly associated with calcified-aortic valves and not with non-calcified-aortic valves (Fig. 4). Our findings suggested that LPS existence in AV may cause progression of calcification and inflammation of AS. Remarkably, inflammation and LPS/TLR/NF-κB pathway evidently contributes to calcification and destructive changes in AV.15 Among the toll-like receptor ligands, we focused on gram-negative bacteria-derived LPS.

CONCLUSION

In conclusion, our data revealed that immunohistochemical bacterial detection in resected-aortic valves was associated with PD-L1 accumulation and valve calcification, and that PD-L1 expression was significantly high in calcified-aortic valves with LPS existence, not in non-calcified-aortic valves. Moreover, high PD-L1 in AS samples without LPS was significantly associated with immune cell infiltration. These results suggest that LPS-induced inflammation in AS might cause valve calcification

Acknowledgments

We thank Ms. Suto Yukiko, Ms. Mariko Nakamura, Ms. Sawa Nagayama, and Ms. Fumie Takada for their excellent assistance.

Authors' Contributions

Conceived and designed the study: BE, WT, and TY. Performed the experiments: BE. Analyzed the clinical data: BE, WT, NG, and TA. Wrote the paper: BE, TY, WT, HS, and TA. Funding: WT and TA. Final confirmation of the manuscript: KS and TA.

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    • Ethics approval and consent to participate: This study conformed to the Declaration of Helsinki and was approved by the Institutional Review Board of the Clinical Research at Gunma University Hospital (approval number: HS2020-014, May 19, 2020). Informed consent for this retrospective study was obtained by the opt-out method.

    Funding: This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS); grant numbers 19K18171.

    Conflicts of Interest: The authors have no conflicts of interest to disclose.

    1

    These authors contributed equally to this work.

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