Arginine-specific gingipains (RgpA/RgpB) knockdown modulates neutrophil machinery

ABSTRACT

Background

Gingipains are important virulence factors present in Porphyromonas gingivalis. Arginine-specific gingipains (RgpA and RgpB) are critically associated with increased proteolytic activity and immune system dysfunction, including neutrophilic activity. In this study, we assessed the impact of gingipains (RgpA and RgpB) on neutrophil function.

Methods

Peripheral blood samples were obtained; neutrophils were isolated and incubated with P. gingivalis A7436, W50, and the double RgpA/RgpB double knockout mutant E8 at MOI 20 for 2 hours. Neutrophil viability was assessed by Sytox staining. Phagocytic capacity and apoptosis were measured by flow cytometry. Superoxide release was measured by superoxide dismutase and cytochrome c reduction assay. Gene expression of TLR2, p47-phox, p67-phox, and P2 × 7was measured by qPCR. Inflammatory cytokine and chemokine production was measured by IL-1β, IL-8, RANTES, and TNF-α in cell supernatants.

Results

Neutrophil TLR2 gene expression was reduced in the absence of RgpA/RgpB (p < 0.05), while superoxide production was not significantly impacted. RgpA/RgpB^−/− significantly impaired neutrophil phagocytic function (p < 0.05) and increased TNF-α production when compared with the wild-type control (p < 0.05). Neutrophil apoptosis was not altered when exposed to RgpA/RgpB^−/− E8 (p > 0.05).

Conclusion

These data suggest that arginine-specific gingipains (RgpA/RgpB) can modulate neutrophil responses against P. gingivalis infection.

Summary of key findings

P. gingivalis-derived arginine-specific gingipains impaired the phagocytic and apoptotic function in neutrophils.

KEYWORDS:

• Porphyromonas gingivalis
• gingipains
• neutrophils
• cytokines
• phagocytosis
• superoxides
• innate immunity

Introduction

Periodontitis is a chronic multifactorial inflammatory disease associated with a dysbiotic biofilm causing tooth loss [1]. Porphyromonas gingivalis is a Gram-negative, non-motile, proteolytic, obligatory anaerobic species that forms black-pigmented colonies on blood agar plates and requires iron under the form of hemin to grow [2–5]. P. gingivalis dysregulates innate immunity pathways in a susceptible host and facilitates an increase in overall community biomass [6]. P. gingivalis and host interaction is mediated by virulence factors that activate cell receptors, eliciting the immune response [7]. Gingipains are among the most important virulence factors of P. gingivalis [8,9]. The proteolytic activity of the gingipains is aimed at bacterial access nutrients [10], stimulation of host’s cytokines [11–13], and regulation of receptors [14,15] and components of the complement system [16]. The gingipain family comprises three related cysteine proteases that hydrolyze peptide bonds at the carbonyl groups of arginine (Arg-Xaa) and lysine residues (Lys-Xaa) [17]. The Lys-specific gingipain (Kgp), encoded by the Kgp gene [18], degrades host proteins and tissues, aids in immune evasion, enhances bacterial adherence to host cells and tissues, and facilitates iron acquisition and the maturation of other virulence factors [19–21]. RgpA and RgpB, encoded by the RgpA and RgpB genes, are Arg-Xaa gingipains, that participate in many of the pathological effects of gingipains associated with periodontal destruction, such as increased collagenase activity [22,23], activation of matrix metalloproteinase 2 [24], increased RANKL/OPG ratio [13], and alveolar bone loss [25,26] are activated by the Arg-Xaa group [27].

Neutrophils are the most prevalent cells in the gingival crevicular fluid in periodontitis, playing a pivotal role in periodontal health maintenance and the defense against microorganisms and virulence factors [28]. Neutrophil numbers in the periodontal tissues are maintained with a gradient of interleukin (IL)-8 produced by the gingival epithelium and other neutrophils. This equilibrium can be disrupted when IL-8 is digested by Rgps [11] and P. gingivalis serine phosphatase [29]. Rgps also down-regulate and degrade intercellular adhesion molecule (ICAM)-1, preventing a transient neutrophil migration into gingival tissues, allowing the development of mature pathogenic biofilm, and inciting inflammation [30]. Consequently, inflammation leads to heightened recruitment and activity of neutrophil functions such as superoxide production, phagocytosis, and cytokine release, contributing to periodontal destruction [31].

Although neutrophils initially follow the IL-8 gradient, in infected or inflamed sites, they move towards chemo-attractants derived from bacteria or local activation of the complement system [32]. Interestingly, RgpA regulates the cleavage of C3 into C3b and C5 into C5a, both of which are potent chemotactic factors favoring leukocytic infiltration [10,33]. Neutrophils are ‘tagged’ by Rgps to confuse the recognition signals of phagocytosis by macrophages, which in turn results not only in the enhancement of the dying cells clearance but also stimulation of the uptake of healthy and functional neutrophils by macrophages [34], indicating that P. gingivalis has developed a pathway for subverting the host defense dependent on neutrophils [34,35].

Since neutrophils and P. gingivalis play a key role in the pathogenesis of periodontal disease, mapping specific neutrophil functions affected by Arg-gingipains from P. gingivalis is crucial for understanding the disease. In this study, we tested the hypothesis that RgpA and RgpB regulate the oxidative stress response, viability, and phagocytic and apoptotic functions in neutrophils.

Materials and methods

Ethical compliance

Informed consent was obtained from all subjects, and ethical approval was granted by the Forsyth Institutional Review Board (protocol number 11–03) before collecting patient blood samples at the Forsyth Institute Center for Clinical and Translational Research.

P. gingivalis culture

The study design is shown in Figure 1(a). P. gingivalis wild-type strains A7436 and W50 were cultured in blood agar plates and individually stored in an anaerobic chamber (5% CO₂, 10% H₂, and 85% N₂ atmosphere) at 37°C. The colonies were re-plated every three days and 24 hours before each experiment; bacterial suspensions were prepared in BHI broth* from the primary culture at their log phase of growth. Concentrations were determined by an optical density of 600 nm^# corresponding to 1 × 10⁹ bacteria/mL. To test the role of Rgp’s, RgpA/RgpB double mutant E8 was obtained from Dr. J. Aduse-Opoku (Queen Mary’s School of Medicine and Dentistry, London, UK) and grown as above.

Figure 1.  (a) Human neutrophil isolation and experimental workflow with P. gingivalisstrains. (b) TLR2 gene expression. (c) Time course of the fMLP-induced O²- production by human neutrophils (0.5 × 10⁶) in response to P. gingivalis strains compared with fMLP stimulation alone. PBS was used as a negative control (n = 8). *: p < 0.05 statistical difference when compared with baseline PBS. (d) Gene expression of P2X7, p47-phox, and p67-phox in response to different P. gingivalis strains. *: p < 0.05 statistical difference when compared with the control group. #: p < 0.05: statistical difference when compared with W50 group. (e) Iron release optical density (565 nm). *: p < 0.05 statistical difference when compared with the control group. (f) Levels of IL-1β, IL-8, RANTES, and TNF-α (pg/mL) * p < 0.05 statistical difference from PMN. # p < 0.05 statistical difference from E8. Panel a created with BioRender. One-way ANOVA followed by Tukey’s posthoc test was performed for graphs on B, D, E and F. Two-way ANOVA followed by Tukey’s posthoc test was performed on C.

Neutrophil isolation

Donors were nonsmokers and did not present any systemic or periodontal disease. Their average age was 32 (±5.3) years, with a gender distribution of 3 females and 5 males. Approximately 50 mL of peripheral venous blood samples were collected. The samples were placed into vacutainer tubes containing 25 units/ml heparin. Polymorphonuclear neutrophils were isolated by gradient centrifugation as described [36].

Superoxide assay

Neutrophils (0.5 × 10⁵) were infected with P. gingivalis A7436, W50 or E8 at a multiplicity of infection (MOI) of 20. Superoxide production (O [2-] production was monitored in a spectrophotometer (VMax, SpectraMax 340PC, Molecular Devices, Sunnyvale, CA) at 37°C by reducing Cytochrome C at an absorbance of 550 nm. Superoxide dismutase was used as a negative control, and N-fMLP^‖ (N-formyl-methionine-leucine-phenylalanine) at 1 µM was used as a positive control. Superoxide production was evaluated linearly and expressed in O [2]/min/cell.

Viability, phagocytosis, and apoptosis

Porphyromonas gingivalis strains isolates were cultured overnight in Brain Heart Infusion (BHI) broth (NutriSelect® Plus, Millipore, Burlington, MA). Post-cultivation, the isolates were subjected to three successive washes with sterile phosphate-buffered saline (PBS) and resuspended to a final concentration of 1 × 10^9 cells/mL in PBS. This concentration was quantified using a spectrophotometer (BioPhotometer, Eppendorf, Hamburg, Germany) at an optical density of 660 nm. For opsonization, P. gingivalis were incubated with human serum (H4522, Millipore, Burlington, MA) at 37°C for 30 minutes under shaking conditions and labeled with fluorescein isothiocyanate (FITC; 100 mg/mL in 1× PBS) to allow detection upon phagocytosis by PMNs. Subsequently, the labeled P. gingivalis were incubated with neutrophils at a MOI of 20 in triplicate at 37°C. Apoptotic cell detection was performed using the CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (ThermoFisher Scientific, Waltham, MA) following the manufacturer’s protocol. Additionally, Sytox® (ThermoFisher Scientific, Waltham, MA) was utilized to identify damaged cells. After a 2-hour incubation period, samples were washed with PBS and resuspended in 500 μL of PBS for flow cytometric analysis. The analysis was conducted using a FACScan flow cytometer (BD Bioscience, San Jose, CA) with excitation at 525 nm and detection using a 530/30 nm bandpass filter.

Expression of TLR-2, p47-phox and P2X7

Following infection, polymorphonuclear leukocytes (PMNs) were lysed using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA (tRNA) was quantified, and 1 µg was used for reverse transcription (RT) with a high-capacity cDNA RT kit (Applied Biosystems, Foster City, CA, USA) in 30 µL reactions. The RT conditions were set to 25°C for 10 minutes, 37°C for 60 minutes, and 95°C for 5 minutes. Quantitative real-time PCR (qPCR) was performed using TaqMan fast advanced master mix (Applied Biosystems) with pre-designed probes targeting TLR-2, P2X7, p47-phox, p67-phox, and β-actin (TaqMan, Applied Biosystems, Thermo Fisher Scientific, Waltham, MA). The NCBI Reference Sequences utilized were TLR-2 (NM_001318787.2), P2×7(NM_002562.5), p47-phox (NR_110044.1), p67-phox (NM_000433.3), and β-actin (BNM_001101.3). For each qPCR reaction, 10 ng of cDNA (50 ng/µL) were used in a 20 µL volume, performed in triplicate using a standard 96-well format. The reactions were carried out in a StepOnePlus real-time PCR system (Applied Biosystems) with the following thermal cycling conditions: 50°C for 2 minutes, 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. The cycle threshold (CT) values were determined using StepOnePlus Software v2.3 (Applied Biosystems). Data were analyzed using the 2-ΔΔct method, and TLR-2, P2X7, p47-phox, and p67-phox mRNA levels were calculated by comparing with corresponding control samples running simultaneously, each normalized to β-actin mRNA as endogenous controls.

Cytokine generation

To measure cytokine release by neutrophils in response to gingipains, cells were infected with P. gingivalis A7436, W50, or E8, incubated for 2 hours at 37°C, and a relative humidity of about 95% before collecting the supernatants. All assays were performed on 100 mL of culture supernatant using four multiplex magnetic bead panels on a flexible laser analyzer platform (Luminex 200, Luminex, Austin, TX). IL-1β, IL-8, RANTES, and TNF-α were measured by a human cytokine/chemokine panel (Millipore, MILLIPLEX, Billerica, MA) with no dilution. All assays were performed following the manufacturer’s protocol [37] and analyzed (Bio-Plex Manager, Version 5.0, Bio-Rad, Hercules, CA).

Iron release

To measure the iron (Fe²⁺) released by neutrophils in response to gingipains, cells (1 × 10⁶) were infected with P. gingivalis A7436, W50, or E8 at MOI = 20. After 2 hours, supernatants were harvested and transferred to a 96-well plate. Iron was evaluated using QuantiChromTM Iron Assay Kit (DIFE-250) following the manufacturer’s protocol (BioAssay Systems, Hayward, CA).

Statistical analyses

All raw data were used in averages of at least three experiments repeated in triplicate. After statistical analysis, data were displayed in mean and standard error. All analyses were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. In the case of non-parametric data, the Kruskal-Wallis test was used, followed by the Dunn-Bonferroni post hoc test in case of statistical significance. A 95% confidence level was attributed before running the statistical analysis.

Results

PMN superoxide generation and TLR2 expression under P. gingivalis challenge

To evaluate the impact of gingipain RgpA/RgpB on human PMN function, freshly isolated neutrophils were exposed to the challenge by P. gingivalis W50 or E8 (MOI = 20). P. gingivalis A7436 was used as a control (Figure 1a). Neutrophil TLR2 gene expression was decreased in the absence of RgpA/RgpB when compared to the A7436 (p < 0.05) (Figure 1b). Superoxide production was depleted to negative control levels (PBS) in the RgpA/RgpB knockdown group. The presence of RgpA/RgpB gingipains resulted in peaks of superoxide production after 2 and 4 minutes of stimulation (p < 0.05) (Figure 1c). There was an upregulation of P2×7and p47-phox gene expressions in the RgpA/RgpB positive strains and, conversely, a non-significant expression in the knockdown group (Figure 1d). Regardless of the presence of RgpA/RgpB gingipain, no impact was found on p67-phox gene expression (p > 0.05) (Figure 1d), suggesting an arginine-specific mechanism of P2×7and p47-phox mediated regulation of superoxide formation in neutrophils in response to gingipains.

RgpA/RgpB knockdown upregulates neutrophil-derived iron release

To check the possibility that RgpA/RgpB gingipains interfere with iron release ability from neutrophils, iron concentration was assessed after neutrophils were incubated with different P. gingivalis strains for 2 hours (Figure 1e). Data revealed a significant increase in iron release by neutrophils in the absence of RgpA/RgpB compared to the PMN control group (p < 0.05), suggesting gingipain-induced negative feedback on the neutrophil iron release.

Lack of RgpA/RgpB upregulates TNF-α and downregulates RANTES in a strain-specific manner

The IL-1β and IL-8 release were not dependent on RgpA/RgpB gingipain expression while RANTES gene expression was significantly decreased in response to all P. gingivalis strains (p < 0.05) (Figure 1f). Interestingly, TNF-α levels in the RgpA/RgpB knockdown E8 were significantly higher when compared to its strain-specific wild-type control (W50), revealing a negative TNF-α feedback response related to RgpA/RgpB absence (p < 0.05) (Figure 1f).

Human neutrophil phagocytic capacity is impaired in the absence of gingipains

Following a two-hour incubation period, the phagocytic capacity of neutrophils in response to P. gingivalis was assessed. The lack of RgpA/RgpB notably impaired the neutrophil phagocytic potential when comparing the E8 to the A7436 (p < 0.05) (Figure 2b). Although no difference was found between W50 and E8, only the RgpA/RgpB knockdown significantly reduced the percentage of phagocytic events compared to the wild-type controls, suggesting the contribution of gingipains in governing neutrophil phagocytic function (Figure 2b).

Figure 2.  (a) Experimental workflow. (b) Representative flow cytometry density plots. (c) Influence of three different strains of P. gingivalis (A7436, W50, and E8) on PMN phagocytic capacity. *: p < 0.05 statistical difference when compared with the control group. #: p < 0.05: statistical difference when compared with W50 group. Panel A was created with BioRender. One-way ANOVA followed by Tukey’s posthoc test was performed.

The RgpA/RgpB profile does not regulate neutrophil apoptotic cell death

After 2 hours of incubation, apoptotic cell death was visualized in neutrophils exposed to P. gingivalis (Figure 3b). Data revealed no statistical significance, suggesting that apoptotic cell death was not dependent on RgpA/RgpB gingipain status (Figure 3b).

Figure 3.  (a) Experimental workflow. (b) Results from caspase-3/7 activity assayed by measuring the fluorescence of AMC; the activity of caspase-3 was expressed in % of relative fluorescence units (RFU), which is translated to PMN apoptosis. (c) Results from Sytox staining of % PMN necrosis. Data are the mean of six independent experiments, and bars are SE. *: p < 0.05 statistical difference. Panel A was created with BioRender. One-way ANOVA followed by Tukey’s posthoc test was performed.

Discussion

P. gingivalis is a keystone etiologic agent associated with dysbiosis and disrupts the host’s innate responses in the periodontium. When neutrophil functions such as migration, peptides/enzyme production, and superoxide release are defective, tissue breakdown may be catalyzed during periodontal disease [38]. Gingipains are one of the principal proteinases exerted by this bacterium. They are associated with immunoglobulin degradation, disruption of the coagulation cascade, iron caption, collagen destruction, cytokines lysing, and destruction of pro-adhesion molecules [39]. RgpA/RgpB gingipains are involved in several mechanisms that shape the neutrophilic response to P. gingivalis infection. Neutrophil extracellular traps (NETs), which are networks with antimicrobial properties, are directly affected by the presence of Rgps, where a proteolytic activation of the protease-activated receptor-2 (PAR-2) takes place, inducing the malformation of NETs lacking bactericidal properties [40]. We investigated whether the absence of gingipains RgpA/RgpB impacted neutrophil function and immune regulation. We found that arginine-specific gingipains (RgpA/RgpB) can modulate neutrophil functions and regulate the survival of P. gingivalis. The RgpA/RgpB absence led to an impaired phagocytic function, suggesting that gingipains play an important role in P. gingivalis survival. Our human data corroborates a murine periodontitis study demonstrating that eliminating RgpA from P. gingivalis increased neutrophil phagocytosis [26]. A plausible mechanistic explanation for these findings is that Arg-gingipains can degrade components of the complement system [16,41,42], leading to a decrease in bacterial opsonization and leukocyte phagocytosis.

Superoxide produced by neutrophils is essential for the elimination of phagocytosed microorganisms [43]. Our data revealed neutrophil superoxide production was not impacted by the absence of arginine-specific gingipains. Neutrophil apoptosis and its subsequent clearance by macrophages are important events during the resolution of inflammation [44]. The inability to effectively complete the efferocytic process fuels a prolonged inflammatory response that impairs resolution and subsequent events to achieve tissue homeostasis [45]. In this study, neutrophil apoptosis was not impacted regardless of the RgpA/RgpB presence, suggesting that although arginine-specific gingipains can increase the number of living neutrophils, they are functionally compromised. These findings corroborate with the previous literature, where a human study found fewer apoptotic neutrophils in gingival tissue sections from periodontitis patients compared to healthy subjects [46]. Further, some studies have demonstrated that P. gingivalis LPS can prolong the lifetime of neutrophils [47] and may lead to diminished secretion of proapoptotic signals, resulting in persistent neutrophil migration with a delay in their physiological clearance [48].

Our data demonstrated that P. gingivalis had no significant impact on PMN apoptosis independent of the presence of arginine-specific gingipains. An increased expression of TLR2 was found in both W50 and E8 groups, corroborating with studies that have demonstrated the TLR2-PI3K stimulus as a way in which P. gingivalis avoids clearance from immune cells, such as neutrophils [49].

Increased number of living neutrophils could result in greater iron concentration in the microenvironment, favoring its scavenging by P. gingivalis and boosting the growth and survival of this microorganism. Iron availability regulates P. gingivalis virulence and influences its growth and survival [50]. We quantified iron concentration to check whether P. gingivalis increased iron secretion in neutrophils. P. gingivalis induced neutrophil iron release regardless of arginine-specific gingipains RgpA/RgpB. Although the E8 group showed a statistically significant upregulation of iron release, the average values are comparable to those of the other strain groups. This suggests that the observed difference may not translate into a biologically relevant effect.

As P. gingivalis can degrade cytokines disrupting the immune system, we measured the IL-1β, IL-8, RANTES, and TNF-α levels. There was no significant impact on IL-1β levels, agreeing with previous research that demonstrated P. gingivalis did not affect IL-1β production by dendritic cells [51]. P. gingivalis can degrade IL-8 and RANTES, essential components of the innate and adaptive immune response, independently of arginine-specific gingipains RgpA/RgpB in line with P. gingivalis proteases degrading IL-8 [52]. P. gingivalis can disturb the cytokine network by eliminating a variety of cytokines [53]. Our results showed that RgpA/RgpB led to TNF-α degradation in line with the literature, showing that P. gingivalis cysteine proteinases can digest TNF-α [54].

Conclusion

Taken together, our data suggested that P. gingivalis can evade neutrophil phagocytosis through gingipains by multiple mechanisms, including the modulation of neutrophils function, apoptosis, and immune regulation, and thus compromise the host’s ability to eliminate infection and sustain chronic periodontal inflammation.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/20002297.2024.2376462.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This study was supported by grants from the National Institutes of Health [AG062496], IADR PRG Philips Young Investigator Award, FAPESP/Brazil 2012/22439-1 and the J. William Fulbright Scholarship sponsored by CAPES (finance code 001).