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Journal of Advanced Research logoLink to Journal of Advanced Research
. 2023 Apr 24;57:93–105. doi: 10.1016/j.jare.2023.04.007

The exoprotein Gbp of Fusobacterium nucleatum promotes THP-1 cell lipid deposition by binding to CypA and activating PI3K-AKT/MAPK/NF-κB pathways

Song Shen a, Tianyong Sun a, Xiangjiu Ding b, Xiufeng Gu a, Yushang Wang a, Xiaomei Ma a, Zixuan Li a, Haiting Gao a, Shaohua Ge a,⁎, Qiang Feng a,c,⁎
PMCID: PMC10918358  PMID: 37100345

Graphical abstract

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Keywords: Fusobacterium nucleatum, Lipid deposition, Gbp, CypA, RNA-seq, Drug repositioning

Highlights

  • •

    F. nucleatum was prevalent in diffuse intimal thickening as well as in plaque tissues;

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    F. nucleatum participated in the foaming process of THP-1 cells by activating MAPK, PI3K-AKT, and NF-κB signaling cascades;

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    The exoprotein of F. nucleatum, Gbp, promoted foam cell formation by interacting with CypA and CD147 and activating the PI3K-AKT, MAPK and NF-κB signaling pathways;

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    Drug candidates targeting PI3K-AKT, MAPK and NF-κB signaling pathways could dramatically attenuate F. nucleatum induced macrophage foaming.

Abstract

Introduction

Growing evidence has shown the correlation between periodontitis and atherosclerosis, while our knowledge on the pathogenesis of periodontitis-promoting atherosclerosis is far from sufficient.

Objectives

Illuminate the pathogenic effects of Fusobacterium nucleatum (F. nucleatum) on intracellular lipid deposition in THP-1-derived macrophages and elucidate the underlying pathogenic mechanism of how F. nucleatum promoting atherosclerosis.

Methods and results

F. nucleatum was frequently detected in different kinds of atherosclerotic plaques and its abundance was positively correlated with the proportion of macrophages. In vitro assays showed F. nucleatum could adhere to and invade THP-1 cells, and survive continuously in macrophages for 24 h. F. nucleatum stimulation alone could significantly promote cellular inflammation, lipid uptake and inhibit lipid outflow. The dynamic gene expression of THP-1 cells demonstrated that F. nucleatum could time-serially induce the over-expression of multiple inflammatory related genes and activate NF-κB, MAPK and PI3K-AKT signaling pathways. The exoprotein of F. nucleatum, D-galactose-binding protein (Gbp), acted as one of the main pathogenic proteins to interact with the Cyclophilin A (CypA) of THP-1 cells and induced the activation of the NF- κB, MAPK and PI3K-AKT signaling pathways. Furthermore, use of six candidate drugs targeting to the key proteins in NF- κB, MAPK and PI3K-AKT pathways could dramatically decrease F. nucleatum induced inflammation and lipid deposition in THP-1 cells.

Conclusions

This study suggests that the periodontal pathogen F. nucleatum can activate macrophage PI3K-AKT/MAPK/NF-κB signal pathways, promotes inflammation, enhances cholesterol uptake, reduces lipid excretion, and promotes lipid deposition, which may be one of its main strategies promoting the development of atherosclerosis.

Introduction

Periodontitis is a prevalent oral inflammatory disease caused by the colonization of Gram-negative pathogens around teeth [1]. The periodontal pathogens can invade the blood vessel, transplant and colonize in specific tissues and organs to affect the overall health of the human body [2]. At present, growing studies suggest that periodontitis is an important risk factor for atherosclerosis [3], [4], [5]. Four underlying mechanisms link periodontitis with atherosclerosis: (1) oral pathogens invade the blood vessel and induce low-level bacteremia; (2) bacterial toxins produced by oral pathogens initiate inflammation in the blood vessel; (3) periodontal inflammatory mediators are released into bloodstream, which leads to systemic inflammation; (4) the interplay between host and particular oral pathogen components activates the autoimmunity to host proteins [6].

Fusobacterium nucleatum (F. nucleatum), one of the keystone pathogens in periodontitis, is widely distributed in the mouth and intestine of the human body, and has attracted wide attention in recent years [7]. Studies have shown that it can migrate from the oral cavity to colonize multiple human tissues and organs and cause a series of systemic diseases, such as colon cancer, adverse pregnancy outcomes, and rheumatoid arthritis [8]. F. nucleatum has been proven to be closely relevant to atherosclerosis. This is because it could be frequently detected in atherosclerotic plaques and its abundance was positively correlated with the severity of this disease [9], [10]. F. nucleatum infection can reduce the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and induce the death of endothelial cells [11]. The heat-shock protein GroEL of F. nucleatum could induce the expression of chemokines, such as interleukin (IL)-8 and E-selectin in human microvascular endothelial cells [12].

Current studies of F. nucleatum on atherosclerosis are mainly focused on vascular endothelial damage, while few concentrate on its pathogenic effects on the main component of atherosclerotic plaques, namely foam cells [13], [14]. Mainly derived from macrophages, foam cells are triggered by uncontrolled lipid uptake and handling, as well as the impairment of cholesterol transport, which accumulates excess cholesterol from oxidized low-density lipoprotein (oxLDL) particles [15]. In this process, scavenger receptors on the surface of macrophages, such as cluster of differentiation 36 (CD36), are overexpressed and enhance oxLDL uptake [16]. Cholesterol transporters like adenosine triphosphate (ATP)-binding cassette transporter G1 and A1 (ABCG1 and ABCA1) are down-regulated and thereby reduce cholesterol outflow, which accelerates cholesterol deposition and macrophage foaming. A study showed that F. nucleatum speeded up macrophage foaming by the overexpression of miRNAs 155, 146a, and 23b, which lead to aberrant pro-inflammatory reactions and lipid metabolism [17]. As key sentinel cells, macrophages are an important target for abnormal lipid metabolism caused by exogenous bacterial stimuli [18] and plays a critical role in the elimination of microorganisms in the blood vessel [19].

Therefore, elucidating the pathogenesis of how F. nucleatum promotes macrophage foaming is essential to addressing why periodontitis contributes to the development of atherosclerosis. In this study, the effects of F. nucleatum on atherosclerosis were explored by analyzing the lipid metabolism and the genes/proteins expressing abnormalities in THP-1 cells. The time-series RNA-seq was applied to present the continuous alteration of gene expression in THP-1 cells with/without the stimulation of F. nucleatum. High performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis was performed on the toxic protein of F. nucleatum and its host receptors to reveal the main strategy of F. nucleatum promoting macrophage foaming. Drug repositioning analysis was applied to select candidate chemicals to validate the main pathways and proteins through which F. nucleatum induces macrophage foaming.

Materials and methods

Detailed methodology is available in Supplementary Material.

Results

Frequent detection of F. Nucleatum in different kinds of atherosclerotic plaques

The RNA-seq data of atherosclerotic lesion samples from the GEO database with accession No. GSE104140 were analyzed to determine the prevalence and abundance of F. nucleatum in atherosclerotic plaques. It was found that F. nucleatum was prevalent in diffuse intimal thickening, fibroatheroma with calcification, and non-calcified fibroatheroma samples (Fig. 1A). Genes co-expressed with F. nucleatum were screened, and GO and KEGG enrichment analysis were performed. It was discovered that the abundance of F. nucleatum was mainly associated with the immune-related biological progress and pathways (Fig. 1B). Then, F. nucleatum co-expressed genes overlapped with all immune-related genes. A total of 55 genes were obtained, which were mainly enriched into cytokine-cytokine receptor interaction, lipid and atherosclerosis, PI3K-AKT and MAPK signaling pathway, etc (Fig. 1C-D).

Fig. 1.

Fig. 1

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The occurrence of F. nucleatum in different kinds of atherosclerosis plaques and its correlation with immune genes and cells. (A) The abundance of F. nucleatum in atherosclerotic lesion samples in GSE104140 dataset. (B) The biological processes of the genes correlated with the abundance of F. nucleatum in GSE104140 dataset. (C) Venn diagram of F. nucleatum co-expressing genes and all immune-related genes in GSE104140 dataset. (D) Functional annotation of F. nucleatum correlated immune genes in GSE104140 dataset. (E) The abundance of F. nucleatum in atherosclerotic lesion samples in GSE120521 dataset. (F) The biological processes of the genes correlated with the abundance of F. nucleatum in GSE120521 dataset. (G) Venn diagram of F. nucleatum co-expressing genes and all immune-related genes in GSE120521 dataset. (H) Functional annotation of F. nucleatum correlated immune genes in GSE120521 dataset. (I) Correlation between the proportion of macrophages and the abundance of F. nucleatum in GSE104140 dataset. (J) Correlation between the proportion of macrophages and the abundance of F. nucleatum in GSE120521 dataset. (K) TEM images of F. nucleatum-invading THP-1 cells (scale bar 1 μm). (L) The number of internalized F. nucleatum incubating with THP-1 cells for 0, 1, 2, 4, 6, 12, and 24 h.

GSE120521 dataset was downloaded and analyzed by the same procedure to verify the above results. The results showed that F. nucleatum was prevalent in both stable and unstable plaques. Besides, F. nucleatum-related genes also mainly belonged to immune-response biological progress and pathways, such as a response to lipopolysaccharide and the MAPK signaling pathway (Fig. 1E-H). Prostaglandin E receptor-1 (PTGER-1) associated with the inflammatory responses to pathogen infection [20], exhibited a positive correlation with the abundance of F. nucleatum in both datasets (Figure S1A-B). CIBERSORTx was used to calculate the composition and proportion of immune cells in both datasets. It was found that macrophages occupied the highest proportion among all types of immune cells (Figure S1C-E), whose proportion was positively correlated with the abundance of F. nucleatum (Fig. 1I-J). The above results implied that F. nucleatum may interact with immune-related genes and macrophages to contribute to the development of atherosclerosis.

F. nucleatum was used to infect macrophages. It was found that long shuttle-shaped F. nucleatum could not only adhere to the surface of THP-1 cells but also be present in the interior of THP-1 cells (Fig. 1K). Gentamicin protection assays at 0, 1, 2, 4, 6, 12, and 24 h showed that F. nucleatum could survive continuously in macrophages for up to 24 h (Fig. 1L). This indicates that macrophages cannot timely eliminate this pathogenic bacterium, which might be an important prerequisite for its pathogenicity in the human body.

Promotion of intracellular lipid accumulation by F. Nucleatum through altering the expression of lipid metabolism genes

THP-1 cells were stimulated with different multiplicities of infection (MOIs) of F. nucleatum, and the number of apoptotic cells accumulated with the increase of MOI (Fig. 2A). THP-1 cells were more pronounced to apoptosis at MOI＞50 (P < 0.0001). Therefore, an MOI of 50 was chosen for the subsequent treatment in F. nucleatum stimulated group (F. nucleatum group), and F. nucleatum + oxLDL stimulated group (F. nucleatum + oxLDL group). In addition, we set up a negative control (NC) group and a positive control group, oxLDL stimulated group (oxLDL group), to compare the effects of F. nucleatum on macrophage foaming. More details can be found in Material and Methods.

Fig. 2.

Fig. 2

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The pathogenic effects of F. nucleatum on THP-1 cells. (A) Apoptosis of THP-1 cells induced by different MOIs of F. nucleatum stimulation. (B) Oil red O staining image of THP-1 cells. (C) Total cholesterol, free cholesterol, and cholesterol esters of THP-1 cells. (D) mRNA expression of IL1β, IL6, MMP9, CD36, LXR, ABCG1, ABCA1 and PPARG in different groups of THP-1 cells. **P < 0.01, ***P < 0.001 and ****P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The accumulation of intracellular lipids is a crucial index for the formation of foam cells. THP-1 cells were stimulated with F. nucleatum, oxLDL, or both for 24 h, and the lipid deposition among four groups was compared by Oil Red O staining. Compared with the NC group, the stimulation of macrophages with F. nucleatum alone (F. nucleatum group) resulted in a similar level of lipid deposition as oxLDL, while lipid deposition in F. nucleatum + oxLDL group far exceeded that of a single stimulus (Fig. 2B). Similarly, intracellular levels of total cholesterol (P < 0.0001), free cholesterol (P < 0.0001), and cholesteryl esters (P < 0.01) increased significantly after F. nucleatum stimulation alone (Fig. 2C). These indicators in the F. nucleatum + oxLDL group dramatically increased compared with any single stimulus. In additon, the morphology of THP-1 cells stimulated with F. nucleatum changed from the initial oval to an irregular shape with the extension of longer pseudopods (Fig. 2B).

RT-qPCR was utilized to examine the expression of genes associated with inflammation and lipid metabolism. As shown in Fig. 2D, compared to NC and oxLDL groups, F. nucleatum stimulation significantly promoted the expression of inflammation-related genes, such as IL1β (P < 0.0001), IL6 (P < 0.0001), and matrix metallopeptidase 9 (MMP9) (P < 0.0001). F. nucleatum stimulation up-regulated the expression of CD36 (P < 0.0001), which was bound up with cholesterol uptake and decreased the expression of cholesterol efflux-related genes, such as recombinant liver × receptor (LXR) (P < 0.0001), ABCG1 (P < 0.0001), ABCA1 (P < 0.0001), and peroxisome proliferator-activated receptor gamma (PPARG) (P < 0.0001). This gave rise to cholesterol accumulation in THP-1 cells. These results suggested that F. nucleatum, an independent factor, can promote lipid deposition and the formation of foam cells by altering the expression of inflammatory and lipid metabolism genes.

Time-series gene expression induced by F. Nucleatum in THP-1 cells

A time-series RNA-seq analysis was made of THP-1 cells at 2, 6, 12, 24, and 48 h to clarify the dynamics of gene expression during the stimulation of F. nucleatum or oxLDL. Firstly, maSigPro was used for independently identifying genes with significantly different expression profiles over time after stimulation with oxLDL, F. nucleatum or both based on quadratic regression models. As shown in Fig. 3A, three stimulation groups had a series of altered expressing genes compared with the NC group. Among them, the oxLDL group had 1083 differentially expressed genes (DEGs), while F. nucleatum and F. nucleatum + oxLDL groups had 4326 and 4790 DEGs, respectively, which differed from the NC group. Of note, DEGs in F. nucleatum and F. nucleatum + oxLDL groups were similar, with 4083 genes being the same (Table S1).

Fig. 3.

Fig. 3

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Dynamics of gene-activating process of NC, oxLDL, F. nucleatum and F. nucleatum + oxLDL groups of THP-1 cells. (A) Venn diagrams of overlapping DEGs among different groups. (B) Gene clusters with different pattern in response to F. nucleatum stimulation and the trends in other three groups. (C-E) GOs and KEGG pathways annotated by genes of cluster 2, 3 and 5.

The time-series expression pattern of these genes in the F. nucleatum group was analyzed, and the genes with similar expression patterns were classified into one cluster. The results indicated that the stimulation of F. nucleatum alone could induce nine different kinds of gene expression patterns (an orange curve). The patterns of those genes in NC, oxLDL and F. nucleatum + oxLDL groups were also presented with green, red and blue curves, respectively (Fig. 3B). Of the nine clusters, the abundance and pattern of F. nucleatum and F. nucleatum + oxLDL groups were consistent, while those of NC and oxLDL groups were similar.

Next, the genes enriched in each cluster were annotated to GO and KEGG databases and the main affected biological processes or pathways were obtained. The results showed that genes in the same cluster could be categorized into the same or similar functional units (Table S2). Lipid accumulation and inflammatory response induced by F. nucleatum stimulation were the focus of this study. Thus, it was found that cluster 2 consisting of 412 genes which were rapidly up-regulated after F. nucleatum stimulation but dramatically decreased 12 h later had a relationship with acute immune inflammatory responses, mainly activated MAPK, NF-κB signaling pathways (Fig. 3C). Cluster 3 showed a significant down-regulation within 6 h of F. nucleatum stimulation while recovering gradually with the duration of F. nucleatum stimulation. It was mainly involved in lipid transport and localization, and activated vascular endothelial growth factor (VEGF) and rhoptry-associated protein 1 (Rap1) signaling pathways (Fig. 3D). Cluster 5 dramatically increased and reached the plateau within 6 h of F. nucleatum stimulation, which was enriched in pathways related to inflammatory responses and biological processes, such as MAPK, PI3K-AKT, and atherosclerosis-related pathways (Fig. 3E). The functional annotation results of the other six gene clusters were displayed in Table S2.

As shown in Fig. 2D, inflammation and intracellular lipid accumulation-related genes experienced dramatic changes under the stimulation of F. nucleatum or F. nucleatum + oxLDL. Nevertheless, a group of “non-cononical” genes may still get involved in this process. Taking advantage of the RNA-seq data of multiple time points among four groups, we used the classical inflammatory and lipid accumulation-related genes as a “bait” to find out the genes with similar expressing tendencies, which may have the same or similar functions. Mfuzz was applied to identify gene clusters with time-series dynamic patterns in each of the four groups, respectively (Figure S2). As shown in Figure S3, genes with similar expression patterns of IL1β and IL6 among four groups were mainly annotated to inflammation-related pathways. However, genes with similar expression patterns of ABCG1 and PPARG were mainly annotated to lipid transport and metabolism pathways, which indicated that genes with similar time-series expression patterns may have similar functions. Venn diagram showed that the dual adapter for phosphotyrosine and 3-phosphoinositides 1 (DAPP1), nuclear receptor co-activator protein 7 (NCOA7), activating transcription factor 5 (ATF5), phosphodiesterase 4B (PDE4B), and serpin family B member 8 (SERPINB8) had the same expression pattern with IL1β among the four groups (Figure S3A). Of which, DAPP1 is a pivotal gene associated with abnormal endothelial function in early coronary atherosclerosis and is involved in the biological processes related to inflammation [21]. NCOA7 promotes the expression of inflammation related cytokines such as IL1β and IL6 [22]. Meanwhile, Niemann-Pick disease C1 like intracellular cholesterol transporter 1 (NPC1L1), related to cholesterol homeostasis and promoted atherogenesis [23], showed the same expression trend as ABCG1 (Figure S3B). Cleft lip and palate associated trans-membrane protein 1 (CLPTM1), associated with low-density lipoprotein cholesterol levels, shared a common pattern with PPARG (Figure S3C) [24]. It is worthwhile to deeply study the role and mechanism of these genes in the process of F. nucleatum induced macrophage foaming.

Expression of NF-κB, MAPK and PI3K-AKT signaling pathways activated by F. Nucleatum

The analysis of DEGs was performed on NC and the other three experimental groups at each time point, with a view to further investigate the genes and pathways mostly influenced by the stimulation of F. nucleatum. NC and F. nucleatum groups were compared to identify 2470, 3326, 3085, 2823, 3008 up-regulated genes and 2995, 3493, 3180, 2999, 3098 down-regulated genes at 2, 6, 12, 24, 48 h (Figure S4A). Venn plots revealed the consistent up-regulation of 412 genes and down-regulation of 287 genes in the whole process (Figure S4C). Compared with the NC group, the F. nucleatum + oxLDL one was found to have 291 up-regulated genes and 308 down-regulated genes after the intersection of DEGs at each time point (Figure S4B- D). In the process of F. nucleatum stimulation, a large number of genes was activated instantaneously and expressed continuously, which indicated that macrophages could timely sense and respond to the stimulation of pathogenic microorganisms.

To reveal the molecular bases of lipid deposition induced by F. nucleatum or F. nucleatum + oxLDL stimulation, DEGs in GO and KEGG databases were annotated, respectively. As presented in Figure S5, the genes up-regulated by F. nucleatum stimulation were mainly related to inflammatory response, such as NF-κB and MAPK signaling pathways, and atherosclerosis pathways (Figure S5A). In contrast, genes down-regulated by F. nucleatum stimulation primarily affected cell migration and metabolic processes (Figure S5B). In the F. nucleatum + oxLDL group, the up-regulated genes were also enriched in immune-related pathways, such as NF-κB, MAPK pathways and biological processes that affected lipids and atherosclerosis (Figure S5C), while the down-regulated genes were mainly related to cardiac and arterial developmental processes (Figure S5D). These results demonstrated that DEGs induced by F. nucleatum and F. nucleatum + oxLDL stimulation had similar functions.

To compare their similarity in the level of gene composition, a Veen diagram was drawn, and obtained DEGs at each time point of the two groups (Figure S4C-D). Recurrent DEGs were overlapped, and 240 up-regulated genes and 160 down-regulated genes at each time point of the two groups were obtained (Fig. 4A-B). Heat-map analysis revealed that overlapping DEGs had a consistent time-series expression pattern in two groups (Fig. 4C). GO and KEGG analyses showed that the up-regulated DEGs were abundant in NF-κB, MAPK and PI3K-AKT signaling pathways, and involved in the biological process of the immune response, iron death, autophagy and tumorigenesis, etc (Fig. 4D-E).

Fig. 4.

Fig. 4

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DEGs and their functions shared by F. nucleatum and F. nucleatum + oxLDL groups at all time points. (A, B) Venn diagram of up-regulated/down-regulated genes in F. nucleatum and F. nucleatum + oxLDL groups among all time points. (C) Time serial expression of the overlapping DEGs in NC, F. nucleatum and F. nucleatum + oxLDL groups. (D) Top 15 enriched GO biological processes of the overlapping DEGs. (E) Top 15 enriched KEGG pathways of the overlapping DEGs. (F) PPI network of the overlapping DEGs (high confidence 0.7). (G) The significant modules obtained from PPI network (MCODE:6.75, 5.0, 4,67). (H) KEGG pathways of potential targets. Up-regulation marked in red; Down-regulation marked in green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Moreover, these overlapping DEGs of two groups were put into the STRING database for protein–protein interaction (PPI) analysis and 293 edges and 172 nodes were identified. The up-regulated genes and important nodes were strongly associated with immunological processes (such as CXCL8, IL10 and SRC) (Fig. 4F). Cytoscape showed that the highest scoring sub-networks were in connection with immune inflammatory response, oxidative stress, autophagy and ubiquitination, respectively (Fig. 4G). The KEGG analysis of 172 nodes using ClueGO showed that NF-κB, MAPK and PI3K-AKT and other pathways related to immune inflammation, tumor formation, atherosclerosis and ferroptosis were significantly enriched in these overlapping DEGs (Fig. 4H). These results indicated that NF-κB, MAPK and PI3K-AKT are the major activating signal pathways of F. nucleatum stimulating macrophages.

The application of ssGSEA analysis in all RNA-seq data helped assess the expression characteristics of NF-κB, MAPK, and PI3K-AKT pathways at each time point of four groups. The results showed that these three pathways were expressed throughout the experimental period (48 h) (Fig. 5A), and activated by F. nucleatum stimulation more pronouncedly (Fig. 5B). Next, Pearson correlation analysis identified the genes co-expressed with NF-κB, MAPK, and PI3K-AKT pathways and annotated in GO and KEGG databases. The results showed that, other pathways related to lipid metabolisms and inflammation, such as PPAR, IL-17, and toll-like receptor signaling pathways, were also markedly activated during F. nucleatum stimulation in addition to these three pathways (Fig. 5C-E).

Fig. 5.

Fig. 5

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The key pathways dynamically activated in different groups. (A) Dynamics of MAPK, PI3K-AKT, NF-κB pathways in different groups. (B) Abundance of the MAPK, PI3K-AKT, NF-κB pathways on 2, 6, 12, 24, and 48 h in four groups. (C-E) GO and KEGG annotation of MAPK, PI3K-AKT and NF-κB pathway related genes.

Activation of NF-κB, MAPK, and PI3K-AKT pathways by the interaction between D-galactose-binding protein (Gbp) and Cyclophilin A (CypA)

Identifying the effector of F. nucleatum on THP-1 cells is an important step for further deciphering the pathogenesis of this pathogenic bacterium. A biotin pull-down assay and HLPC-MS analysis were performed to identify eight candidate proteins from F. nucleatum that may interact with host proteins (Table S3). Uniprot database showed that Gbp was primarily located in the periplasm of bacterial cells. Periplasmic space protein is the first communicating medium for pathogenic bacteria and host cells [25], which makes it easier to be recognized by macrophage. Hence, Gbp was selected as a candidate protein for subsequent experiments.

As shown in Figure S6a and S6b, the expression plasmid of Gbp was successfully constructed, and the Gbp protein in Escherichia coli (E. coli), which comprises 341 amino acid residues with a predicted molecular mass of 36.4 kDa, was purified. Next, the cells with different concentrations (0, 0.001, 0.01, 0.05, 0.1, 0.5, 1 mg/mL) of Gbp were stimulated. It was found that Gbp had no impact on cell viability at 0.1 mg/mL (Figure S6C), which was selected for subsequent studies. BODIPY staining was applied to present intracellular lipid droplets. It was noticed that Gbp could significantly increase the content of lipid droplets in THP-1 cells (P < 0.0001) (Fig. 6A-B). Meanwhile, Gbp dramatically promoted the expression of inflammation-related genes (IL1β, IL6, MMP9) (P < 0.0001), lipid uptake-related genes (CD36) (P < 0.0001) and inhibited the expression of lipid efflux-related genes (ABCG1, PPARG, LXR) (P < 0.0001) (Fig. 6C). These results suggested that Gbp is an important effector of F. nucleatum to promote lipid deposition of THP-1 cells.

Fig. 6.

Fig. 6

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The pathogenic effects of Gbp on THP-1 cells. (A, B) Fluorescence image and quantification of BODIPY staining (scale bar 50 μm). (C) The expression of IL-1β, IL-6, MMP9, CD36, PPARG, ABCG1 and LXR. (D) Co-IP analysis of Gbp and CypA. (E) mRNA expression of CypA. (F) ELISA test of CypA. (G) The abundance CypA and CD147. (H) mRNA level of CD147. (I) The phosphorylation of P65, AKT, ERK, and P38. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Next, the corresponding host proteins that could interact with Gbp were identified. His-tagged Gbp was used to perform a His pull-down assay with THP-1 proteins. HLPC-MS analysis identified eight candidate proteins that may interact with His-Gbp (Table S4). Among these candidates, CypA was successfully validated to interact with Gbp by a co-immunoprecipitation (Co-IP) assay (Fig. 6D). RT-qPCR showed that Gbp promoted the expression of CypA to a large extent (Fig. 6E). Previous studies showed that CypA could act as a receptor inside cells [26], or in special cases, secreted extracellularly to participate in the biological processes of the inflammatory response, vascular remodeling and apoptosis [27]. Whether Gbp stimulation could induce the overexpression of CypA within cells or promote its exocrine secretion was analyzed. An enzyme-linked immunosorbent assay (ELISA) and Western blotting showed that Gbp greatly increased the abundance of intracellular and extracellular CypA simultaneously (Fig. 6F-G). Extracellular CypA can bind to the membrane receptor CD147 on monocytes, to activate ERK and NF-κB signaling pathways, and participate in the transduction of multiple signal [28], [29], [30]. The results of this study showed that CD147 saw a dramatic increase on both mRNA and protein levels under the stimulation of Gbp (Fig. 6G-H). The expression of the genes in PI3K-AKT, MAPK, and NF-κB signaling pathways were examined as CD147 is the upstream protein of these pathways. It was noted that Gbp stimulation markedly activated these signaling pathways by promoting the phosphorylation of AKT (P < 0.0001), ERK (P < 0.0001), p38 (P < 0.0001) and p65 (P < 0.05) (Fig. 6I).

CypA was knocked down by small interfering RNA (siRNA) to further demonstrate that Gbp functioned in conjunction with CypA (Figure S6D-E). With the stimulation of Gbp, CypA knockdown dramatically reduced the formation of lipid droplets (P < 0.0001) (Fig. 7A), decreased the expression of inflammatory factors IL1β (P < 0.0001), IL6 (P < 0.0001), MMP9 (P < 0.0001), and increased the expression of lipid efflux-related genes PPARG (P < 0.0001), ABCG1 (P < 0.05) (Fig. 7B). Of PI3K-AKT, MAPK, and NF-κB pathways, CypA knockdown decreased the expression of AKT (P < 0.0001), JNK (P < 0.0001), ERK (P < 0.0001), p38 (P < 0.0001) and p65 (P < 0.0001) (Fig. 7C). These results showed that the pathogenic effect of Gbp on THP-1 cells was exerted by its interaction with CypA.

Fig. 7.

Fig. 7

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Effects of CypA and CD147 on the pathogenicity of Gbp. (A) Oil red O staining image of THP-1 cells. (B) mRNA level of IL1β, IL6, ABCG1, PPARG and MMP9 before (dark colour) and after (light colour) knockdown of CypA. (C) The phosphorylation of P65, AKT, JNK, ERK, and P38 before and after knockdown of CypA. (D) mRNA level of IL1β, IL6, ABCG1, PPARG and MMP9 before (dark colour) and after (light colour) knockdown of CD147. (I) The phosphorylation of P65, AKT, ERK, and P38 before and after knockdown of CD147. ***P < 0.001 and ****P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The necessity of CD147 in Gbp-induced signaling transduction and cytological behavior changes was verified by the use of siRNA knockdown. As shown in Fig. 7A, CD147 knockdown dramatically reduced the formation of lipid droplet (P < 0.0001), decreased the expression of IL1β (P < 0.0001), IL6 (P < 0.0001), MMP9 (P < 0.0001), and increased the expression of PPARG (P < 0.001), ABCG1 (P < 0.0001) (Fig. 7D). Meanwhile CD147 knockdown suppressed the activation of PI3K-AKT, MAPK, and NF-κB pathways by reducing p-AKT (P < 0.0001), p-ERK (P < 0.0001), p-P38 (P < 0.0001), p-P65 (P < 0.0001) (Fig. 7E). These results suggested that CD147 is an important mediator for Gbp to exert pathogenic effects on THP-1 cells.

Attenuation of F. nucleatum-induced lipid deposition by drugs targeting NF-κB, MAPK, and PI3K-AKT pathways

To verify the role of NF-κB, MAPK, and PI3K-AKT pathways in lipid deposition induced by F. nucleatum, a drug repositioning analysis was performed based on the gene-expression pattern regulated by F. nucleatum stimulation, and a series of candidate therapeutic agents were identified (Fig. 8A). Targets of these drugs were retrieved by the Drug-Gene Interaction database (DGIdb) (Figure S7A) and KEGG pathway enrichment analysis (Figure S7B). Six different candidate drugs were selected, including tretinoin, isotretinoin, liothyronine, betulin, trichostatin, and tanespimycin, as they could interfere with the PI3K-AKT, MAPK and NF-κB signaling pathways (Figure S7C-F).

Fig. 8.

Fig. 8

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Drug candidates identified to adverse F. nucleatum induced lipid deposition. (A) Computational drug repositioning analysis between NC and F. nucleatum + oxLDL group. (B) Candidate drugs attenuated the phosphorylation of AKT, JNK, ERK, and P38. (C) Candidate drugs attenuated the expression of IL-1β, IL-6, PPARG, and ABCG1. (D) BODIPY staining of THP-1 cells with F. nucleatum and different candidate drugs (scale bar 30 μm). ***P < 0.001 and ****P < 0.0001.

A CCK8 viability assay was applied to select the optimal concentration of each drug (Figure S8A). We chose drug concentrations that have no effect on cell viability for subsequent experiments, namely 10 μM of tretinoin, 10 μM of isotretinoin, 100 nM of liothyronine, 10 μM of betulin, 1 μM of trichostatin, and 100 nM of tanespimycin. Next, the effects of each candidate drug on MAPK and PI3K-AKT pathways under the stimulation of F. nucleatum were tested. The results showed tretinoin could impede the PI3K-AKT pathway by inhibiting the phosphorylation of AKT (P < 0.001), and inhibit the MAPK signaling pathway by decreasing the expression of p-JNK (P < 0.001), p-ERK (P < 0.01), and p-P38 (P < 0.001); isotretinoin and liothyronine could suppress MAPK pathway by reducing p-JNK (P < 0.005); trichostatin could attenuate the activation of MAPK via decreasing p-JNK (P < 0.001) and p-P38 (P < 0.001); betulin could restrain the expression of p-JNK (P < 0.001), p-ERK (P < 0.05), and p-P38 (P < 0.001) in MAPK pathway; while tanespimycin could repress p-ERK (P < 0.05), and p-P38 (P < 0.01) (Fig. 8B, S8B).

Inflammatory and lipid deposition related-gene analysis showed that all of these six drugs dramatically reduced the expression of inflammatory factors IL1β (P < 0.001) and IL6 (P < 0.001), and all drugs except trichostatin increased the expression of PPARG (P < 0.001). Besides, tretinoin and isotretinoin enhanced the expression of ABCG1 (P < 0.001) (Fig. 8C). BODIPY staining experiments intuitively demonstrated that all of these candidate drugs effectively down-regulated F. nucleatum-induced intracellular lipid deposition (P < 0.001) (Fig. 8D, S8C). These results further proved that NF-κB, MAPK, and PI3K-AKT pathways were the main mediators by which F. nucleatum exerted its pathogenic effects on THP-1 cells and also suggested that the great potential of these drugs to treat F. nucleatum induced atherosclerosis by targeting these pathways.

Discussion

Increasing evidence indicates that atherosclerosis is a chronic inflammatory disease [31]. DNA testing showed that pathogens in severe chronic periodontitis were identified in 50% of atherosclerotic plaque [32]. Similarly, it was found that F. nucleatum existed in all atherosclerosis lesions via the analysis of two public RNA-seq datasets. A study showed that F. nucleatum could invade host cells under aerobic conditions and survive for 48 h [33]. This study further proved that F. nucleatum is capable of adhering, invading and surviving in macrophages, which may be one of the main strategies for F. nucleatum to exert its pathogenicity.

As is known to all, macrophage-derived foam cells are associated with atherosclerosis [34]. The present study showed that F. nucleatum promoted macrophage foaming by increasing the expression of the lipid-uptake receptors, such as CD36 [35], and reducing the expression of cholesterol efflux-related transporter proteins ABCG1 and ABCA1 [36]. The key upstream transcription factors, PPARG and LXR [37], were also significantly down-regulated in response to F. nucleatum stimulation. Furthermore, it was demonstrated that F. nucleatum enhanced lipid deposition by activating NF-κB, MAPK, and PI3K-AKT pathways, which is the key event in the development of macrophage-derived foam cells [38], [39]. Herein, the result indicated that F. nucleatum induced macrophage foaming by activating series of classical signaling pathways.

Moreover, a protein called Gbp was discovered in F. nucleatum. Involved in the active transport of glucose, galactose and mediating bacterial chemotaxis [40], [41], [42], this protein may interact with the CypA of THP-1 cells to promote inflammation and lipid deposition. Ubiquitously distributed in intracellular and extracellular space [43], CypA was first identified as a receptor for Cyclosporine A and later proved to be secreted extracellularly as well [27], [44]. A previous study showed that CypA promoted the cellular uptake of LDL by enhancing the expression of scavenger receptors [45]. Likewise, this research firstly demonstrated that F. nucleatum exoprotein Gbp might facilitate lipid deposition through its interaction with CypA, which communicated information to CD147 to activate the PI3K-AKT, MAPK, and NF-κB signaling pathways.

Candidate drugs targeting NF-κB, MAPK, and PI3K-AKT pathways were used to verify whether the pathogenesis of F. nucleatum on THP-1 cells was mainly through these classical pathways. The results showed that these drugs significantly inhibited the activation of these signaling pathways, which thus reduced the level of lipid deposition. The result of this study demonstrated the main pathways by which F. nucleatum induced lipid deposition in macrophages, and also provided new drug candidates to treat F. nucleatum induced atherosclerosis.

To sum up, the present study suggests that F. nucleatum is involved in the process of atherosclerosis development through the activation of MAPK, PI3K-AKT, and NF-κB signaling pathways via the Gbp-CypA-CD147 complex. These results provide new knowledge on how periodontitis promotes atherosclerosis, while this may be one of the mechanisms of F. nucleatum promoting macrophage foaming, and there are still many potential ways to be clarified. Besides, further studies are needed to explore the therapeutic potential of targeting the Gbp-CypA-CD147 complex and its downstream signaling pathways for the prevention or treatment periodontitis associated atherosclerosis.

Compliance with ethics requirements

This study does not involve the use of human and animal subjects.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We sincerely thank all colleagues in our laboratory for all of the kindly advices and support. We sincerely thank the foundation support of the National Natural Science Foundation of China (No. 82071122, 82270980), the National Young Scientist Support Foundation (2019), Excellent Young Scientist Foundation of Shandong Province (No. ZR202102230369), Taishan Young Scientist Project of Shandong Province, Periodontitis innovation team of Jinan City (2021GXRC021), Major Innovation Projects in Shandong Province (No. 2021SFGC0502), Oral Microbiome Innovation Team of Shandong Province (No. 2020KJK001), Shandong Province Key Research and Development Program (No. 2021ZDSYS18), Shandong Province Major Scientific and Technical Innovation Project (No. 2021SFGC0502), the Construction Engineering Special Fund of “Taishan Scholars” of Shandong Province (No. ts20190975).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jare.2023.04.007.

Contributor Information

Shaohua Ge, Email: shaohuage@sdu.edu.cn.

Qiang Feng, Email: fengqiang@sdu.edu.cn.

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (31.3KB, docx)
Supplementary data 2
mmc2.docx (3.9MB, docx)
Supplementary data 3
mmc3.xlsx (81.4KB, xlsx)
Supplementary data 4
mmc4.xlsx (503.1KB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary data 1
mmc1.docx (31.3KB, docx)
Supplementary data 2
mmc2.docx (3.9MB, docx)
Supplementary data 3
mmc3.xlsx (81.4KB, xlsx)
Supplementary data 4
mmc4.xlsx (503.1KB, xlsx)

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