Porphyromonas gingivalis Evades Immune Clearance by Regulating Lysosome Efflux

Abstract

Porphyromonas gingivalis, a major periodontal pathogen, invades autophagosomes of cells, including gingival epithelial cells, endothelial cells, gingival fibroblasts, macrophages, and dendritic cells, to escape antimicrobial autophagy and lysosome fusion. However, it is not known how P. gingivalis resists autophagic immunity, survives within cells, and induces inflammation. Thus, we investigated whether P. gingivalis could escape antimicrobial autophagy by promoting lysosome efflux to block autophagic maturation, leading to intracellular survival, and whether the growth of P. gingivalis within cells results in cellular oxidative stress, causing mitochondrial damage and inflammatory responses. P. gingivalis invaded human immortalized oral epithelial cells in vitro and mouse oral epithelial cells of gingival tissues in vivo. The production of reactive oxygen species (ROS) increased upon bacterial invasion, as well as mitochondrial dysfunction-related parameters with downregulated mitochondrial membrane potential and intracellular adenosine triphosphate (ATP), upregulated mitochondrial membrane permeability, intracellular Ca²⁺ influx, mitochondrial DNA expression, and extracellular ATP. Lysosome excretion was elevated, the number of intracellular lysosomes was diminished, and lysosomal-associated membrane protein 2 was downregulated. Expression of autophagy-related proteins, microtubule-associated protein light chain 3, sequestosome-1, the NLRP3 inflammasome, and interleukin-1β increased with P. gingivalis infection. P. gingivalis may survive in vivo by promoting lysosome efflux, blocking autophagosome-lysosome fusion, and destroying autophagic flux. As a result, ROS and damaged mitochondria accumulated and activated the NLRP3 inflammasome, which recruited the adaptor protein ASC and caspase 1, leading to the production of proinflammatory factor interleukin-1β and inflammation.

Keywords

bacteria inflammation cell biology keratinocytes microbiology periodontitis

Introduction

Periodontopathogens such as Porphyromonas gingivalis evade the immune system by invading nonphagocytic host cells.P. gingivalis induces periodontitis and other diseases, such as Alzheimer’s disease (Dominy et al. 2019), diabetes (Śmiga et al. 2021), cardiovascular disease (Kozarov et al. 2005), rheumatoid arthritis (Potempa et al. 2017), and even inflammation of the central nervous system (Shapira et al. 2002). P. gingivalis quickly adheres to the surface of host cells and incorporates itself into early phagosomes. Afterward, it replicates within these phagosomes, activating autophagy while inhibiting apoptosis (Andrian et al. 2006; Zheng et al. 2021). Autophagy is a powerful mechanism that eliminates invading pathogenic microorganisms (Levine and Kroemer 2019; Greabu et al. 2020). In autophagy, intracellular organelles, intracellular bacteria, and other components that require degradation are isolated into multimembrane vacuoles to form autophagosomes (Huang et al. 2009; Lee et al. 2018). Under normal physiological conditions, the autophagy process entails autophagosomes maturing into autolysosomes to degrade isolated cytoplasmic components and maintain homeostasis (Li et al. 2008; Ravikumar et al. 2008; Xiong et al. 2013). Bacteria commonly interact with the autophagic pathway to increase the free amino acid concentrations needed for bacterial biochemical pathways and inhibit host cell protein synthesis to reduce the cellular response to pathogens (Baxt et al. 2013; Huang and Brumell 2014). Particularly, P. gingivalis can invade different cell types, including gingival epithelial cells, endothelial cells, macrophages, and dendritic cells, through adhesins on its surface such as FimA and HagB (El-Awady et al. 2015; El-Awady et al. 2019). Afterward, P. gingivalis rapidly traffics to the autophagic pathway to avoid being killed by autophagy evasion and lysosome fusion (Bélanger et al. 2006; Zaitsu et al. 2016; Molero-Abraham et al. 2019), but the exact mechanism remains unclear. Gingival epithelial cells are the primary host interface for microbial colonization in the subgingival microbial community (Novak et al. 2008; Swamy et al. 2021). The interaction between gingival epithelial cells and periodontal bacteria determines the success or failure of colonization, as well as the maintenance of health or occurrence of disease in the host. In optimal conditions, bacteria and host cells coexist peacefully, responding to each other in order to maintain health. When this relationship is disrupted, such as when the bacterial burden increases or the immune system responds inappropriately, the disease process can be triggered, and the bacterial burden increases; thus, it is key to the pathogenesis of P. gingivalis (Tribble and Lamont 2010; Bostanci and Belibasakis 2012).

Furthermore, P. gingivalis possesses a variety of virulence factors, such as lipopolysaccharides (LPS), fimbriae, and gingipain, which make it a potent immune stimulator, triggering the release of proinflammatory cytokines and activating the inflammation-related signaling pathway (Sheets et al. 2008). Interleukin (IL)-1β is a proinflammatory cytokine that regulates the inflammatory response and is essential during the host’s defense against many pathogens (Zhou et al. 2011; Lee et al. 2020). The secretion of IL-1β occurs in 2 steps: first, LPS activates nuclear factor-κB (NF-κB) by way of Toll-like receptors, leading to the transcription of pro-IL-1β. Second, pro-IL-1β converts to IL-1β via the NLR family pyrin domain-containing 3 (NLRP3) inflammasome (Liu et al. 2018). The critical components of the NLRP3 inflammasome include NLRP3, the adaptor protein ASC, and caspase 1. Several studies have shown that P. gingivalis activates the NLRP3 inflammasome with the addition of extracellular adenosine triphosphate (ATP) at a multiplicity of infection (MOI = 100) in human and murine macrophages, gingival epithelial cells, and mouse models (Yilmaz et al. 2010; Morandini et al. 2014; Park et al. 2014; Johnson et al. 2015; Almeida-da-Silva et al. 2019; Ding et al. 2020; De Andrade et al. 2021). With an increased MOI (>100), P. gingivalis can function as a source of extracellular ATP (Jun et al. 2017; Paik et al. 2021).

We hypothesized that P. gingivalis could escape antimicrobial autophagy induced by promoting lysosome efflux to block autophagic maturation, resulting in intracellular survival, and that an increase in the number of intracellular P. gingivalis could cause cellular oxidative stress, resulting in mitochondrial damage and inflammatory responses. We observed invasion of P. gingivalis in human immortalized oral epithelial cells (HIOECs) and gingival tissues of mice. We measured the effects of P. gingivalis invasion on mitochondrial function, autophagy/mitophagy, lysosomes, and NLRP3 inflammasomes of HIOECs.

Materials and Methods

Animal Experiment

The ethical committee approved the mice experiments at the Stomatological Hospital of Shandong University (p1005): Reporting of the In Vivo Experiments (ARRIVE) guidelines. The animal experiment was performed as previously described (Liu and Choi 2022). Briefly, 6-wk-old C57BL/6 mice, randomly divided into 3 groups (n = 5/group), were group-housed and kept under specific pathogen-free conditions in the Laboratory Animal Facility at the School of Stomatology, Shandong University. After a 7-d resting period, all mice received Streptococcus danieliae (2 × 10⁹ colony-forming units [CFU]) to simulate the coaggregation of bacteria during the development of periodontitis. Three days later, the mice were anesthetized with isoflurane, and then 100 µL 2% carboxymethylcellulose containing 2 × 10⁹ P. gingivalis, Fusobacterium nucleatum (F. nucleatum), or vehicle alone (S. danieliae) was applied at the surface of the mandibular and maxillary molars 6 times at 2-d intervals with a micropipette and sterile tip. The mice were euthanized at 6 wk after the first infection.

In Vitro Experiments

The in vitro experiments included cell and bacteria culture, bacterial invasion to HIOECs, cell proliferation and apoptosis assay, lysosomal and mitochondrial morphology and quantity, confocal microscopy and fluorescence microscopy observation, transmission electron microscopy observation, measurement of intracellular reactive oxygen species (ROS), mitochondrial membrane permeability (MPTP) assay, mitochondrial membrane potential (ΔΨm) detection, intracellular Ca²⁺ measurement, intracellular and extracellular ATP production, immunoblot analysis, RNA and mitochondrial DNA (mtDNA), extraction and quantitative reverse transcription polymerase chain reaction (RT-PCR), and RNA sequencing (RNA-seq) analysis. All in vitro experiments were conducted in triplicate. Details of those experiments can be found in the Appendix Materials and Methods.

Statistical Analysis

Differences between groups were determined by a 1-way analysis of variance test followed by Tukey’s post hoc test because variables were normally distributed. All statistical procedures were performed with SPSS Statistics 23 software (SPSS, Inc.). All data are presented as mean ± SD. Significance was set at P < 0.05.

Results

Bacterial Invasion into Oral Epithelial Cells

Bacterial invasion into oral epithelial cells is an important mechanism in gingival tissue infection (Liu and Choi 2022). To confirm the invasion of P. gingivalis, F. nucleatum was used as a positive control, which has a strong invasive and adhesive ability (Ji et al. 2010; Irshad et al. 2012; Baek et al. 2015), and S. danieliae as a negative control, which has no invasive capability as commensal bacteria within the mouse oral cavity.P. gingivalis and F. nucleatum invaded HIOECs (Fig. 1A–D [Fig. 1A–D has been removed for data ownership reasons], Appendix Fig. 1), which differed from S. danieliae (Fig. 1E, F [Fig. 1E, F has been removed for data ownership reasons]). Next, we examined the invasion of bacteria in the gingival tissues of mice (Fig. 1G–I [Fig. 1G–I has been removed for data ownership reasons]). P. gingivalis and F. nucleatum invaded the epithelial cells of the gingival tissues (Fig. 1J–M [Fig. 1J–M has been removed for data ownership reasons]), whereas S. danieliae did not invade cells (Fig. 1N, O [Fig. 1N, O has been removed for data ownership reasons]). Surprisingly, the invasion of P. gingivalis induced the efflux of many exosomes that were apparently visible and located outside the cells (Fig. 1A [Fig. 1A has been removed for data ownership reasons], Appendix Fig. 1). The following series of experiments were performed to explain this phenomenon and its mechanism.

Effects of P. gingivalis on Cellular Behavior and ROS

We measured the viability, proliferation, and apoptosis of HIOECs after they were cocultured with P. gingivalis or its supernatant. The supernatant of P. gingivalis contains the toxic factors LPS, gingipain, fimbriae, and outer membrane vesicles (Ramenzoni et al. 2019), which are key virulence factors that stimulate proinflammatory cytokine production. CCK8 analysis of cell viability showed that P. gingivalis did not affect HIOECs’ viability, even at an MOI of 2,000 (Appendix Fig. 2A). In contrast, its supernatant slightly impaired cell viability at a dilution of 1:10 (Appendix Fig. 2B). According to EdU assay, cell proliferation was not affected after coculture withP. gingivalis (MOI = 1,000) or supernatant (1:10) for 24 h (Appendix Fig. 2C). Flow cytometry showed that neitherP. gingivalis nor its supernatant increased early- or late-stage apoptosis compared to untreated cells (Appendix Fig. 2D, E). Consequently, P. gingivalis (MOI = 1,000) and its supernatant (1:10) were chosen for the following study.

P. gingivalis induced mitochondrial dysfunction and oxidative stress in peripheral blood mononuclear cells (Yamaguchi et al. 2017). We measured ROS production to determine whether P. gingivalis invasion caused oxidative stress. Six hours after P. gingivalis infection, the production of ROS increased compared with ROS production after 12 h (Appendix Fig. 2F–H). However, the supernatant did not induce ROS. Collectively, these findings showed that ROS production was derived primarily from the effect of intact P. gingivalis instead of from the metabolites of P. gingivalis.

Mitochondrial Dysfunction Induced by P. gingivalis

Mitochondria are the principal source of ROS in cells. However, ROS accumulation may induce mitochondrial dysfunction. Therefore, to evaluate whether P. gingivalis caused mitochondrial dysfunction, we analyzed the ΔΨm, MPTP, Ca²⁺ influx, intracellular and extracellular ATP, and expression of mtDNA. Flow cytometry analysis and immunofluorescence staining demonstrated an insignificant decrease in cellular ΔΨm in the treated cells compared to untreated cells (Appendix Fig. 3A–C). In contrast, both P. gingivalis and its supernatant upregulated MPTP significantly (Appendix Fig. 3D–F).

We then used flow cytometry to measure cell Ca²⁺ influx. Intracellular Ca²⁺ increased substantially in P. gingivalis-infected cells (Appendix Fig. 3G, H). We measured ATP and mtDNA expression to further define the effects of P. gingivalis on mitochondrial function. P. gingivalis inhibited intracellular ATP level (Appendix Fig. 2I). However, P. gingivalis enhanced extracellular ATP levels (Appendix Fig. 2J). Furthermore, the expression of mtDNA increased significantly with P. gingivalis infection (Appendix Fig. 2K). The data for the supernatant showed that the metabolites of P. gingivalis had no significant influence on the mitochondria. Overall, these results indicated that P. gingivalis induced cellular mitochondrial dysfunction and increased extracellular ATP.

P. gingivalis Activated Mitophagy

Mitochondrial dysfunction and damage are generally accompanied by autophagy/mitophagy, a cellular protective mechanism that eliminates invading microorganisms and dysfunctional organelles (Carneiro and Travassos 2013; Liu et al. 2018). We detected significantly higher levels of mitophagy-related proteins Parkin (Fig. 2A, C, E, F) and PTEN-induced putative kinase protein (Pink) 1 (Fig. 2B, D, E, G). Thus,P. gingivalis enhanced the expression of Parkin and Pink1 in epithelial cells, indicating dysfunctional mitochondria and the occurrence of cellular mitophagy. Interestingly, confocal microscopy showed minimal colocalization of Pink1 and lysosomes in the P. gingivalis-treated group (Fig. 2B), suggesting that the degradation of dysfunctional mitochondria might be abnormal because lysosomes are known to degrade damaged organelles.

P. gingivalis Infection Triggers Autophagy/Mitophagy

To further delineate the development of autophagy/mitophagy, we analyzed the expression of microtubule-associated protein light chain 3 (LC3) and sequestosome 1 (p62/SQSTM1) upon P. gingivalis infection. LC3 is an autophagosome marker, and the transformation of cytoplasmic LC3I to membrane-bounded LC3II indicates an increase in autophagosome formation. In contrast, p62 is degraded in the middle and late stages of autophagy (Greabu et al. 2020), and the expression of p62 is negatively correlated to autophagic activity. Moreover, the upregulated expression of LC3II and p62 indicates impaired autophagic flux (Vardar Acar et al. 2022), defined as a measure of autophagic degradation activity (Du Toit et al. 2018). After P. gingivalis infection, we found that the mitochondria were fragmented and surrounded by LC3 with a disrupted grid structure (Fig. 3A). The expression of LC3II was significantly upregulated in P. gingivalis and the supernatant groups as observed by confocal microscopy and immunoblot analyses (Fig. 3A, C, D, F, G). Interestingly, p62 expression was significantly increased in the P. gingivalis-treated group (Fig. 3B, E, F, H). Surprisingly, many autophagosomes but few autolysosomes were present after P. gingivalis treatment (Fig. 3I–Q). Swollen and grid-structure disrupted mitochondria were also clearly visible in the P. gingivalis-treated group (Fig. 3N). In summary, P. gingivalis infection damaged the structure of mitochondria and triggered the autophagy/mitophagy process. The paradoxically high expression of p62 indicated that the autophagic flux might be obstructed, whereas the supernatant did not block autophagy.

Autophagy Was Blocked by the Absence of Intracellular Lysosomes after P. gingivalis Infection

To determine whether decreased intracellular lysosomes caused the interruption of the autophagy/mitophagy process, we used an immunofluorescence probe to analyze the colocalization and quantities of mitochondria and lysosomes. After infection with P. gingivalis for 6 h, the fluorescence intensity and flow cytometry data of mitochondria did not show a significant decrease (Fig. 4A–C). Mitochondria with a broken grid structure and lysosomes were visible using confocal microscopy, accompanied by a significantly reduced fluorescence intensity. The colocalization of the dysfunctional mitochondria and lysosomes was nearly invisible (Fig. 4A). The flow cytometry value and intracellular lysosome number relative value were significantly reduced after P. gingivalis stimulation (Fig. 4D, E). In contrast, the extracellular lysosome number increased (Fig. 4F, G), indicating that P. gingivalis promoted lysosome efflux. Furthermore, lysosomal-associated membrane protein 2 (LAMP2) expression was decreased inP. gingivalis-infected groups (Fig. 4H, I). However,P. gingivalis supernatant neither decreased lysosome number nor blocked autophagy. The results indicated that the blockage of autophagy might result from the absence of intracellular lysosomes.

P. gingivalis Activates NLRP3 Inflammasome

P. gingivalis can stimulate the secretion of proinflammatory cytokines and activate proinflammatory signaling pathways (Xu et al. 2021). To examine the effect of P. gingivalis on the production of proinflammatory cytokine IL-1β, first we measured expression levels of NLRP3 and IL-1β. P. gingivalis upregulated messenger RNA (mRNA) and protein expression of NLRP3 (Fig. 5A–D). Furthermore, P. gingivalis and its supernatant significantly promoted the gene and protein expression of NLRP3 and the adaptor protein ASC (Fig. 5E, H). Concurrently, P. gingivalis and its supernatant stimulated caspase 1 activation (Fig. 5F, H, I). Pro-IL-1β was hydrolyzed by activated caspase 1 to form mature IL-1β (Fig. 5G, H, J). Second, we assessed the activity of innate signaling by the NF-κB pathway in activating mature IL-1β. The expression of NF-κB protein p65 was also measured. The data showed that P. gingivalis and its metabolites downregulated mRNA and protein expression of phosphorylated p65 after a 6-h stimulation (Fig. 5K–M). To further confirm our results, we performed RNA-seq analysis (Appendix Figs. 4–10). Meanwhile, together with the dysfunction of mitochondria and ROS level, as well as colocalization of mitochondria and NLRP3, we concluded that the activation of IL-1β and NLRP3 may be related to ROS and mitochondrial dysfunction caused by P. gingivalis. NLRP3 and IL-1β in the supernatant may be activated by other mechanisms (Haque et al. 2008), so further investigation is needed.

Discussion

We report a possible mechanism by which P. gingivalis coexists with epithelial cells and escapes antimicrobial autophagy. In oral epithelial cells, the invasion of P. gingivalis inhibited autolysosome maturation by promoting lysosome efflux and suppressing autophagosome-lysosome fusion, thus blocking autophagic flux, resulting in intracellular survival. Consequently, an increase in the number of intracellular P. gingivalis causes cellular oxidative stress, resulting in mitochondrial damage and inflammatory responses.

During P. gingivalis invasion of oral epithelium, the host activates an immune-inflammatory response to clear the source of infection and initiate tissue repair (Hajishengallis and Diaz 2020). The entire process involves activating a series of inflammation and bone metabolism-related signal transduction pathways. Epithelial cell apoptosis enables the host to clear infected cells, thereby maintaining cellular homeostasis. Numerous investigators have reported that P. gingivalis does not induce apoptosis and can be internalized into cells and escape autophagy (Madianos et al. 1996; Dorn et al. 2001; Nakhjiri et al. 2001; Geng et al. 2017; Meghil et al. 2019; Zhang et al. 2021). Coinciding with previous studies, our results showed that neither P. gingivalis nor its supernatant induced cellular apoptosis. Furthermore, a possible explanation for the slight inhibition of cell proliferation caused by the supernatant (1:10) was that it was collected from bacteria culture stock solutions containing higher levels of virulence factors.

Bullon et al. (2011) found that P. gingivalis LPS affected mitochondrial function and might link cardiovascular disease with periodontitis. In our study, P. gingivalis, not its supernatant, induced ROS generation and mitochondrial dysfunction. Excessive ROS regeneration may in turn lead to oxidative modification and inactivation of mitochondrial macromolecules, which exacerbates mitochondrial dysfunction. We found that P. gingivalis infection resulted in mitochondrial dysfunction, which was manifested by decreasing ΔΨm and intracellular ATP levels, increasing MPTP, intracellular calcium, and mtDNA expression. Interestingly, the supernatant data revealed that P. gingivalis metabolites slightly affected the production of ROS and mitochondrial function. To avoid cellular damage, ROS-producing mitochondria will be continuously cleared by mitochondrial autophagy, a special autophagic process. Mitophagy is the targeted phagocytosis and destruction of mitochondria by the autophagic device to remove damaged mitochondria (Sena and Chandel 2012), which is generally regarded as the primary mechanism of mitochondrial quality control. Parkin possesses E3 ubiquitin-protein ligase activity and modifies substrates to regulate protein degradation and signal transduction. Pink1 acts upstream of Parkin and coordinates their respective expression levels (Haque et al. 2008). The interaction between Parkin and Pink1 promotes mitophagy and facilitates the degradation of damaged mitochondria in the form of intact organelles by the autolysosome pathway (Liu et al. 2018). We confirmed thatP. gingivalis initiated autophagy/mitophagy and survived in autophagosomes, which is in agreement with a report by Bélanger et al. (2006). However, the increased expression of p62 demonstrated that P. gingivalis blocked autophagic flux. We also observed that P. gingivalis induced the abnormal excretion of lysosomes by excessive efflux. As the final degradation sites of the autophagy-lysosome pathway, lysosomes are enclosed by the cellular plasma membrane and contain the hydrolase cathepsin that degrades macromolecules and cellular components (Ballabio and Bonifacino 2020). Lysosome accumulation outside cells was observed, and this phenomenon was also detected by a 3-dimensional time lapse in our previous study (Liu and Choi 2022). Collectively, these data suggest that blocking the fusion of autophagosome and lysosome may be the key mechanism by which P. gingivalis survives within the cells.

Our study established that the active invasion of P. gingivalis into HIOECs promoted lysosome efflux. As a result of lysosome efflux, P. gingivalis interfered with the fusion of autophagosomes and lysosomes to form autolysosomes; thereby, autophagy was halted at the primary stage, as evidenced by the upregulation of LC3II in the primary stage and p62 in the middle and late stages. In addition, P. gingivalis replicated within autophagosomes, resulting in elevated ROS levels and mitochondrial damage. When damaged mitochondria accumulated, they activated NLRP3, which recruited adaptor protein ASC and caspase 1, resulting in the release of pro-IL-1β to induce inflammation (Fig. 5N).

However, there were 2 limitations in our study. We did not clarify the specific effect of P. gingivalis infection on lysosomes, and we analyzed only 1 P. gingivalis strain and 1 cell type. We will address these limitations in future research.

This study expanded our understanding of the pathogenesis of P. gingivalis and determined how the bacterium invaded epithelial cells, triggered autophagy/mitophagy, and induced inflammation. The invasion of P. gingivalis resulted in increased lysosome efflux and suppressed the fusion of autophagosomes with lysosomes, blocking autophagy/mitophagy. Ultimately, the ROS and damaged mitochondria accumulated and activated the NLRP3 inflammasome, which recruited the adaptor protein ASC and caspase 1, leading to the release of pro-IL-1β and inducing inflammation. Through the reversal of the storage of intracellular lysosomes, it is possible to facilitate the clearance of P. gingivalis within cells, providing a smart way to eliminate pathogens during periodontal therapy.

Author Contributions

M. Liu, contributed to conception and design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; J. Shao, contributed to acquisition and interpretation, critically revised the manuscript; Y. Zhao, contributed to acquisition and data interpretation, critically revised the manuscript; B. Ma, contributed to conception and design, data interpretation, critically revised the manuscript; S. Ge, contributed to conception and design, data acquisition, critically revised the manuscript. All authors approved the submitted manuscript and agreed to be accountable for all aspects of the work.

Supplemental Material

sj-docx-1-jdr-10.1177_00220345221146097 – Supplemental material for Porphyromonas gingivalis Evades Immune Clearance by Regulating Lysosome Efflux

Supplemental material, sj-docx-1-jdr-10.1177_00220345221146097 for Porphyromonas gingivalis Evades Immune Clearance by Regulating Lysosome Efflux by M. Liu, J. Shao, Y. Zhao, B. Ma and S. Ge in Journal of Dental Research

Footnotes

Acknowledgements

The authors acknowledge Professor Wang Liyan at Cheeloo College of Medicine for providing the transmission electron microscope.

Correction (May 2024):

Article updated on p. 558 to remove Figure 1. Please see for more details.

A supplemental appendix to this article is available online.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (Nos. 82170964, 81901009, and 82100974), Construction Engineering Special Fund of “Taishan Scholars” of Shandong Province (Nos. ts20190975 and tsqn201909180), Shandong Province Key Research and Development Program (No. 2021ZDSYS18), Shandong Province Major Scientific and Technical Innovation Project (No. 2021SFGC0502), Shandong Natural Science Foundation (No. ZR2022QH280), and Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong. The authors thank AiMi Academic Services () for the English-language editing and review services.

ORCID iDs

M. Liu

S. Ge

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