Porphyromonas gingivalis systemic infection is associated with calcific aortic valve stenosis through IL-1-dependent signaling pathways

Abstract

Abstract Background Calcific aortic valve stenosis (CAVS) is characterized by inflammation mediated valvular calcification. Porphyromonas gingivalis, a keystone pathogen for periodontitis, has been associated with various chronic inflammatory diseases. We assessed the impact of intravenous inoculation with P. gingivalis on the progression of CAVS. Methods Wide-type (WT) C57BL/6, ApoE deficient (ApoE-/-), and IL1B deficient (IL-1β-/-) mice were fed with normal chow or high-fat diet (HFD). A series of metagenomic, morphometric, and molecular signaling experiments, including 16S rRNA gene sequencing, RNA sequencing (RNA-Seq), quantitative real-time polymerase chain reaction (qPCR), immunostaining, and western blotting, were conducted. Valvular calcification and aortic stenosis were evaluated by Alizarin red staining, and mice echocardiography, respectively. Results By using 16S rRNA gene sequencing and qPCR, we found that P. gingivalis was the 3rd most significantly enriched bacterial species in aortic valves from CAVS patients compared with controls. Morphometric assays showed predominant P. gingivalis infiltration inside of human aortic valves. Intravenous inoculation of P. gingivalis in either WT or ApoE-/- mice resulted in increased bacterial infiltration in aortic valves, irrespective of chow- or HFD. Alizarin red staining and echocardiographic evaluation further revealed that P. gingivalis bacteremia markedly enhanced valvular calcification, leading to hemodynamically significant aortic stenosis in the murine models, which were ameliorated by intraperitoneal metronidazole prophylaxis. RNA-Seq and qPCR validation showed that the gene expression of IL1B, RUNX2, and BMP2 was upregulated in P. gingivalis-stimulated valvular interstitial cells (VIC). Similar changes on IL1B were observed in aortic valves form P. gingivalis-challenged WT and ApoE-/- mice, and in valvular specimens from CAVS patients. qPCR and Alizarin red staining showed that IL1B knockdown decreased P. gingivalis-induced RUNX2 and BMP2 expression, and attenuated calcification in VICs. Consistently, P. gingivalis-induced changes on bacterial infiltration, valvular calcification, and hemodynamically significant aortic stenosis were markedly blunted in IL-1β-/- mice. Furthermore, the level of serum IL-1β and SAA were increased in response to P. gingivalis challenge in ApoE-/- mice, but not in WT or IL-1β-/- mice. Conclusions This study demonstrated, for the first time, that P. gingivalis infiltration in aortic valves promoted IL-1β-mediated local inflammation and valvular calcification, thus resulting in the progression of CAVS.Central Illustration: Pg promotes CAVS