Aggregatibacter actinomycetemcomitans targets NLRP3 and NLRP6 inflammasome expression in human mononuclear leukocytes

Periodontitis is an inflammatory condition that destroys the tooth-supporting tissues, as a result of local bacterial infection. Aggregatibacter actinomycetemcomitans is a Gram-negative facultative anaerobic species, highly associated with aggressive periodontitis. Periodontal inflammation is dominated by cytokines of the Interleukin (IL)-1 family. Prior to their secretion by mononuclear cells, IL-1 cytokines are processed by intracellular protein complexes, known as "inflammasomes", which can sense the bacterial challenge. The aim of this study was to investigate which inflammasomes are regulated in mononuclear cells in response to A. actinomycetemcomitans. The D7SS strain and its derivative leukotoxin and cytolethal distending toxin knock-out mutant strains were used to infect human mononuclear cells at a 1:10 cell: bacteria ratio, for 3 h. The expression of various inflammasome components in the cells was investigated by TaqMan quantitative real-time polymerase chain reaction (qPCR). The expressions of NOD-like receptor protein (NLRP)1, NLRP2 and Absent In Melanoma (AIM)2 inflammasome sensors, as well as their effector Caspase-1 were not affected. However, NLRP3 was up-regulated, while NLRP6 was down-regulated. This effect was not dependent on the leukotoxin or the cytolethal distending toxin, as demonstrated by the use of specific gene knock-out mutant strains. IL-1β and IL-18 expressions were also up-regulated by the bacterial challenge. In conclusion, A. actinomycetemcomitans enhances NLRP3 and reduces NLRP6 inflammasome expression, irrespective of its major virulence factors, confirming the high pathogenic profile of this species, and providing further insights to the mechanisms of periodontal inflammation.     

Quantification of Subgingival Bacterial Pathogens at Different Stages of Periodontal Diseases

Anaerobic gram-negative oral bacteria such as Treponema denticola, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Campylobacter rectus, and Fusobacterium nucleatum are closely associated with periodontal diseases. We measured the relative population (bacterial levels) of these oral pathogens in subgingival tissues of patients at different stages of Korean chronic periodontal diseases. We divided the individuals into those with chronic gingivitis (G), moderate periodontitis (P1), severe periodontitis (P2), and normal individuals (N) (n?=?20 for each group) and subgingival tissue samples were collected. We used real-time PCR with TaqMan probes to evaluate the change of periodontal pathogens among different stages of periodontitis. Bacterial levels of A. actinomycetemcomitans and C. rectus are significantly increased in individuals with chronic gingivitis and moderate periodontitis, but unchanged in severe periodontitis patients. These results suggest that analyzing certain bacterial levels among total oral pathogens may facilitate understanding of the role of periodontal bacteria in the early stages of periodontitis.     

Expression and characterization of Syrian golden hamster p16, a homologue of human tumor suppressor p16 INK4A

The p16(INK4A)/CDKN2A tumor suppressor gene is known to be inactivated in up to 98% of human pancreatic cancer specimens and represents a potential target for novel therapeutic intervention. Chemically induced pancreatic tumors in Syrian golden hamsters have been demonstrated to share many morphologic and biological similarities with human pancreatic tumors and this model may be appropriate for studying therapies targeting p16(INK4A)/CDKN2A. The purpose of this study was to investigate the fundamental biochemistry of hamster P16 protein. Using both in vivo and in vitro approaches, the CDK4 binding affinity, kinase inhibitory activity, and thermodynamic stability of hamster and human P16 proteins were evaluated. Furthermore, a structural model of hamster P16 protein was generated. These studies demonstrate that hamster P16 protein is biochemically indistinguishable from human P16 protein. From a biochemical perspective, these data strongly support the study of p16-related pancreatic oncogenesis and cancer therapies in the hamster model.     

Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils

Neutrophils are initially the predominant cells involved in the host defence of bacterial infections, including periodontal disease. Aggressive periodontitis is associated with Actinobacillus actinomycetemcomitans, a Gram-negative capnophilic microorganism. Infections caused by A. actinomycetemcomitans are not resolved by the host immune response despite the accumulation of neutrophils at the site of inflammation. To better understand the role of natural host defence mechanisms in A. actinomycetemcomitans infections, the interaction of phenotypically diverse strains of this pathogen with human neutrophils was assessed directly using techniques such as genetic labelling with the gene for green fluorescent protein, fluorescence-activated cell sorting and fluorescence imaging. The study included clinical isolates of A. actinomycetemcomitans represented by self-aggregating, biofilm-associated and isogenic planktonic variants. Data obtained showed that complement-mediated phagocytosis of A. actinomycetemcomitans was generally inefficient regardless of strain-specific serotype or leukotoxin production. Furthermore, the majority of ingested bacteria remained viable after exposure to neutrophils for 1 h. Interestingly, uptake of antibody-opsonized bacteria resulted in the rapid cell death of neutrophils. This was in contrast to ingestion of complement-opsonized bacteria, which did not affect neutrophil viability. The methods used in this study provided reliable and reproducible results with respect to adherence, phagocytosis and killing of A. actinomycetemcomitans when encountering human neutrophils.     

Localization of heat shock proteins in clinical Actinobacillus actinomycetemcomitans strains and their effects on epithelial cell proliferation

Actinobacillus actinomycetemcomitans is an important pathogen in periodontitis. In the present study we localized the GroEL- and DnaK-like heat shock proteins (Hsp) in subcellular fractions of 12 A. actinomycetemcomitans strains of various clinical origin and compared their effects on periodontal epithelial cell proliferation and viability. In all strains, GroEL-like protein was found in the membrane, cytoplasm, and periplasm, whereas DnaK-like protein was present in the cytoplasm and periplasm. No correlation was observed between the Hsp expression and the serotype or origin of A. actinomycetemcomitans strains. The bacterial membrane fractions that expressed the GroEL-like protein moderately or strongly induced epithelial cell proliferation more strongly than strains that expressed the protein weakly. The results suggest that GroEL-like Hsp may play a role in the virulence of A. actinomycetemcomitans by increasing epithelial proliferation.     

Periodontitis decreases the antiatherogenic potency of high density lipoprotein

Periodontitis, a consequence of persistent bacterial infection and chronic inflammation, has been suggested to predict coronary heart disease (CHD). The aim of this study was to investigate the impact of periodontitis on HDL structure and antiatherogenic function in cholesterol efflux in vitro. HDL was isolated from 30 patients (age 43.6 +/- 6.1 years, mean +/- SD) with periodontitis before and after (3.2 +/- 1.4 months) periodontal treatment. The capacity of HDL for cholesterol efflux from macrophages (RAW 264.7), HDL composition, and key proteins of HDL metabolism were determined. After periodontal treatment, phospholipid transfer protein (PLTP) activity was 6.2% (P<0.05) lower, and serum HDL cholesterol concentration, PLTP mass, and cholesteryl ester transfer protein activity were 10.7% (P<0.001), 7.1% (P=0.078), and 19.4% (P<0.001) higher, respectively. The mean HDL2/HDL3 ratio increased from 2.16 +/- 0.87 to 3.56 +/- 0.48 (P<0.05). HDL total phospholipid mass and sphingomyelin-phosphatidylcholine ratio were 7.4% (P<0.05) and 36.8% (P<0.001) higher, respectively. The HDL-mediated cholesterol efflux tended to be higher after periodontal treatment; interestingly, this increase was significant (P<0.05) among patients whose C-reactive protein decreased (53.7% reduction, P=0.015) and who were positive by PCR for Actinobacillus actinomycetemcomitans. These results suggest that periodontitis causes similar, but milder, changes in HDL metabolism than those that occur during the acute-phase response and that periodontitis may diminish the antiatherogenic potency of HDL, thus increasing the risk for CHD.     

Immune interactions with CD4+ T cells promote the development of functional osteoclasts from murine CD11c+ dendritic cells

Dendritic cells (DC) are innate immune effectors and are critically involved in regulating T cell immunity. Osteoclasts (OC) are bone-resorbing cells derived from the monocyte/macrophage lineage in response to receptor activator of NF-kappaB ligand (RANKL). DC and T cells form aggregates in the inflammatory infiltrates at active disease sites in human and in experimental rheumatoid arthritis and periodontitis. We investigated whether DC interactions with T cells in the bone environment can support the development of functional OC. In the present study, we demonstrate that upon proper activation by microbial or protein Ags (namely Actinobacillus actinomycetemcomitans, bovine insulin, and outer membrane protein-1) and during immune interactions with CD4+ T cells in vitro, murine BM-derived and splenic CD11c+ DC (CD11b- F4/80- Ly-6C- CD31-) develop into TRAP+ CT-R+ cathepsin-k+ functional OC in a RANKL/RANK-dependent manner. Rescue and blocking experiments using CD11c+ DC derived from Csf-1(-/-) op/op mice show that M-CSF is required "before" developing such osteoclastogenic potential upstream of RANKL/RANK signaling, suggesting that immature CD11c+ DC can indeed act like OC precursors. In addition, these CD11c+ DC-derived OC are capable of inducing bone loss after adoptive transfer in vivo. These data suggest a direct contribution of DC during immune interactions with CD4+ T cells to inflammation-induced osteoclastogenesis. Therefore, our findings not only provide further evidence for DC plasticity, but also extend the current paradigm of osteoimmunology.     

Lipids, apoptosis, and cross-presentation: links in the chain of host defense against Mycobacterium tuberculosis

Eicosanoids regulate whether human and murine macrophages infected with Mycobacterium tuberculosis die by apoptosis or necrosis. The death modality is important since apoptosis is associated with diminished pathogen viability and should be viewed as a form of innate immunity. Apoptotic vesicles derived from infected macrophages are also an important source of bacterial antigens that can be acquired by dendritic cells to prime antigen-specific T cells. This review integrates in vitro and in vivo data on how apoptosis of infected macrophages is linked to development of T cell immunity against M. tuberculosis.     

Actinobacillus (Haemophilus) actinomycetemcomitans in periodontal disease

Actinobacillus (Haemophilus) actinomycetemcomitans is a facultatively anaerobic, gram-negative coccobacillus which is a possible etiological agent in juvenile periodontitis (JP). In this study, bacterial flora, especially the occurrence of A. actinomycetemcomitans, in the periodontal pockets of one juvenile with gingivitis (G), one JP patients, five rapidly progressive periodontitis (RP) patients and one adult periodontitis(AP) patient, and one adult with healthy periodontium was investigated using a blood agar medium and a selective medium for A. actinomycetemcomitans. Eight hundred and sixty-five bacteria were isolated from the periodontal pockets, examined for their gram-stain, cell morphologies, relations to O2 and CO2 and catalase reaction, and divided into 21 groups on the basis of these characters. Among the isolates, 604 isolates were further characterized biochemically and identified. A. actinomycetemcomitans was found as 0.2% of the flora of a site in the JP patient, as 9% of the flora of a site in the G patient, and as 19% and 1%, respectively, of the flora of a site in the two RP patients. However, the organism was not detected in another lesion site of the JP patient. In our JP and RP patients, Fusobacterium, Wolinella, Streptococcus, and obligately anaerobic, gram-positive cocci were frequently found at high levels. The bacterial flora of the G and AP patients were more heterogeneous and included Bacteroides at relatively high proportions. These results indicate that A. actinomycetemcomitans is not always associated with JP but occurred in some patients with RP and G.     

Influence of CD4+CD25+ T cells on Plasmodium berghei NK65 infection in BALB/c mice

CD4(+) T cells co-expressing CD25 (CD4(+)CD25(+) T cells) have been identified as immunoregulatory suppressors modulating autoimmune response. Beside that, autoimmune response was supposed to be associated with malaria infection. Based on these data, we hypothesised that CD4(+)CD25(+) T cells may influence protective immunity to malaria parasites, while suppressing autoimmune response arising throughout the course of malarial infection. To test this possibility, we evaluated the kinetics of CD4(+)CD25(+) T cells during malaria infection and investigated the influence of CD25 depletion by anti-mouse CD25 monoclonal antibody (PC61) on the infection, using a mouse model of premunition to Plasmodium berghei NK65 malaria. The results showed that, during exacerbation of P. berghei NK65 infection, the proportion of CD4(+)CD25(+) T cells among CD4(+) T cells decreased, although that of CD4(+) T cells increased. CD25 depletion clearly delayed the growth of parasitaemia during parasite challenge, particularly in immunised mice. These findings demonstrated that CD4(+)CD25(+) T cells are able to influence protective immunity underlying premunition to P. berghei NK65 parasites.     

A novel RGD-toxin protein,Lj-RGD3, from the buccal gland secretion of Lampetra japonica impacts diverse biological activities

RGD (Arg-Gly-Asp) motif toxin proteins from snake venoms, saliva glands secretion of leech or tick have typical characteristics of inhibiting platelet aggregation, angiogenesis, and tumor growth. Here we report cloning and characterization of a novel RGD-toxin protein from the buccal gland of Lampetra japonica. In an attempt to study the activities of anticoagulant in the buccal gland secretion of L. japonica, we established buccal gland cDNA library and identified a gene encoding a predicted protein of 118 amino acids with 3 RGD motifs. The predicted protein was named Lj-RGD3. We generated the cDNA of Lj-RGD3 and obtained the recombinant protein rLj-RGD3. The polyclonal antibodies against rLj-RGD3 recognized the native Lj-RGD3 protein in buccal gland secretion in Western blot analyses. The biological function studies reveal that rLj-RGD3 inhibited human platelet aggregation in a dose-dependent manner with IC₅₀ value at 5.277 μM. In addition, rLj-RGD3 repressed bFGF-induced angiogenesis in the chick chorioallantoic membrane model. rLj-RGD3 also inhibited the adhesion of ECV304 cells to vitronectin. Furthermore, rLj-RGD3 induced apoptosis and significantly inhibited proliferation, migration, and invasion evoked by bFGF in ECV304 cells. Taken together, these results suggested that rLj-RGD3 is a novel RGD-toxin protein possessing typical functions of the RGD-toxin protein.     

Actinobacillus actinomycetemcomitans serotype-specific genotypes and periodontal status in Brazilian subjects

Periodontitis is associated with members of the oral microbiota, such as Actinobacillus actinomycetemcomitans. To our knowledge, this is the first study to evaluate, by PCR, the occurrence of the six known bacterium serotypes that included subjects with and without periodontitis. Our group comprised 49 Brazilian subjects. We studied 146 bacterial isolates from 23 patients with aggressive or chronic periodontitis and 26 subgingival specimens from subjects with or without periodontitis, all originating in our collection. Serotypes b and c were observed in similar frequencies, and no subject harboured d, e, or f serotype strains. Around 78% subjects had single-serotype infection. Mixed infection was seen only in aggressive periodontitis patients. An association between serotype b and healthy periodontium and between serotype c and chronic periodontitis was observed. Our results diverge from those previously reported, which may be explained by specific distribution patterns in distinct populations. The association of different serotypes with the same periodontal status or conversely of a serotype with different periodontal conditions indicates that organism serotyping should not be used as a sole reliable marker for predicting the outcome of the infection. Evaluation of factors involved in human oral cavity colonization by subsets of A. actinomycetemcomitans is essential for elucidating organism-host-environment relationships.     

UPR attenuates the proinflammatory effect of HPDLF on macrophage polarization

Human periodontal ligament fibroblast (HPDLF) is a major component of the resident cells in the periodontal microenvironment, and plays important roles in periodontitis through multiple mechanisms. Although lipopolysaccharide (LPS) has been shown to cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) in HPDLF, the mechanisms governing HPDLF function in periodontitis are unclear. In this study, we tested the ability of unfolded protein response (UPR) to regulate HPDLF in vitro and examined the underlying mechanisms. We found LPS-pretreated HPDLF induced macrophage polarization toward M1 phenotype. UPR activation reduced the inflammatory cytokine production and downregulated the expression of TLR4 in HPDLF. The phosphorylation of NF-κB p65 and I-κB was also inhibited by UPR activation. Our findings demonstrate that the connection of LPS, UPR, and HPDLF may represent a new concrete theory of innate immunity regulation in periodontal diseases, and suggest that targeting of UPR in HPDLF may be developed as a potent therapy for periodontitis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01234-0.     

Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a facultative anaerobic gram-negative bacterium associated with severe forms of periodontitis. A leukotoxin, which belongs to the repeats-in-toxin family, is believed to be one of its virulence factors and to have an important role in the bacterium''s pathogenicity. This toxin selectively kills human leukocytes by inducing apoptosis and lysis. Here, we report that leukotoxin-induced cell death of macrophages proceeded through a process that differs from the classical characteristics of apoptosis and necrosis. A. actinomycetemcomitans leukotoxin-induced several cellular and molecular mechanisms in human macrophages that led to a specific and excessive pro-inflammatory response with particular secretion of both interleukin (IL)-1β and IL-18. In addition, this pro-inflammatory cell death was inhibited by oxidized ATP, which indicates involvement of the purinergic receptor P2X₇ in this process. This novel virulence mechanism of the leukotoxin may have an important role in the pathogenic potential of this bacterium and can be a target for future therapeutic agents.     

Chemical composition and immunobiological activities of sodium dodecyl sulphate extracts from the cell envelopes of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Fusobacterium nucleatum

The chemical composition and immunobiological activities in vivo and in vitro of sodium dodecyl sulphate extracts (SDS-SE) derived from periodontopathic bacteria (three strains of Actinobacillus actinomycetemcomitans, two strains of Bacteroides gingivalis, and one strain of Fusobacterium nucleatum) were investigated. The main components of SDS-SE were protein and lipid, with negligible amounts of peptidoglycan and lipopolysaccharide. Immunopotentiating activity was detected in both delayed-type hypersensitivity and antibody formation against the elicitation of a protein antigen with the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and B. gingivalis 381 and 1021. On the other hand the SDS-SE of A. actinomycetemcomitans ATCC 29522 enhanced only the induction of a delayed-type hypersensitivity response. All the SDS-SE preparations had mitogenic activity to splenocytes from BALB/c nu/nu, C3H/HeN and C3H/HeJ mice. Migration-stimulating activity for human peripheral blood monocytes was detected especially in the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and Y4. All of the SDS-SE samples inhibited [3H]thymidine uptake in human gingival fibroblasts and caused degradation of the cells. The results suggest that the cell membrane components extractable with sodium dodecyl sulphate from periodontopathic bacteria are involved in the pathogenesis of periodontal disease.     

Effects of low-dose doxycycline on cytokine secretion in human monocytes stimulated with Aggregatibacter actinomycetemcomitans

Doxycycline is an antibiotic used in the treatment of a variety of inflammatory conditions, including periodontitis. Apart from its antimicrobial properties, this drug also has independent anti-inflammatory effects at sub-antimicrobial doses. The present study aimed to investigate the effects of low-doses of doxycycline (LDD) on cytokine production by human monocytic cells challenged with the periodontal pathogen Aggregatibacter actinomycetemcomitans, for up to 6 h. The simultaneous regulation of 12 cytokines were measured by a Human Cytokine Array Kit. To validate the array findings, selected cytokines were also measured by enzyme-linked immunosorbant assay (ELISA). A. actinomycetemcomitans stimulated the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, IL-6 and IL-8 by the cells after 6 h of challenge, and doxycycline significantly inhibited this effect. The kinetics of this regulation demonstrated an early (within 2 h) and significant (P<0.05) inhibition of pro-inflammatory cytokines, with a mild (0.5-fold) up-regulation of the anti-inflammatory cytokine IL-10. The results indicate that LDD acts as an anti-inflammatory agent in human monocytic cells stimulated with A. actinomycetemcomitans. This model provides clear evidence that some of the clinically proven benefits of LDD may be related to its ability to regulate inflammatory mediator release by monocytic cells. This property may contribute to the clinically proven benefits of this antibiotic as an adjunctive treatment for periodontitis.     

Inflammatory responses of a macrophage/epithelial cell co-culture model to mono and mixed infections with Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia

Accumulated evidence points to Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as three major etiologic agents of chronic periodontitis. Epithelial cells and macrophages play a major role in the host response to periodontopathogens, and the secretion of inflammatory mediators and matrix metalloproteinases (MMPs) by these host cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of a macrophage/epithelial cell co-culture model following mono or mixed infections with the above three periodontopathogens. An in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 monocytic cell line) was challenged with whole cells or lipopolysaccharides (LPS) of P. gingivalis, T. denticola, and T. forsythia, individually and in combination. Following stimulation, the production of interleukin-1 beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), regulated on activation normal T cell expressed and secreted (RANTES), prostaglandin E2 (PGE2), and MMP-9 were quantified by enzyme-linked immunoassays. We observed that mono or mixed infections of the co-culture model induced the secretion of IL-1beta, IL-6, IL-8, PGE2, and MMP-9. P. gingivalis and T. forsythia induced an increase in RANTES secretion, whereas T. denticola alone or in combination resulted in a significant decrease in RANTES levels. All LPS challenges induced an increase in chemokine, MMP-9, and PGE2 production. No synergistic effect on the production of cytokines, chemokines, PGE2, and MMP-9 was observed for any of the bacterial or LPS mixtures tested. This study supports the view that P. gingivalis, T. denticola, and T. forsythia may induce high levels of pro-inflammatory mediators and MMP-9 in periodontal lesions, thus contributing to the progression of periodontitis.     

Nonspecific adherence and fibril biogenesis by Actinobacillus actinomycetemcomitans: TadA protein is an ATPase

Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actinomycetemcomitans tadA mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A. actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration. TadA-T7 forms multimers that are stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defective in their ability to complement tadA mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of A. actinomycetemcomitans.