Strain-Dependent Activation of Monocytes and Inflammatory Macrophages by Lipopolysaccharide of Porphyromonas gingivalis

Porphyromonas gingivalis is one of the pathogens associated with periodontal diseases, and its lipopolysaccharide (LPS) has been suggested as a possible virulence factor, acting by stimulation of host cells to secrete proinflammatory mediators. However, recent studies have shown that P. gingivalis LPS inhibited some components of the inflammatory response. The present study was designed to test the hypothesis that there are strain-dependent variations in the ability of P. gingivalis LPS to elicit the host inflammatory response. By using LPS preparations from two strains of P. gingivalis, W50 and A7346, the responses of mouse macrophages and human monocytes were evaluated by measuring the secretion of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α). Both direct and indirect (priming) effects were investigated. LPS from Salmonella typhosa was used as a reference LPS. P. gingivalis A7436 LPS induced lower secreted levels of NO from the tested cells than S. typhosa LPS but induced similar levels of TNF-α. In contrast, LPS from P. gingivalis W50 did not induce NO or TNF-α secretion. Preincubation of macrophages with LPS from S. typhosa or P. gingivalis A7436 prior to stimulation with S. typhosa LPS upregulated NO secretion and downregulated TNF-α secretion, while preincubation with P. gingivalis W50 LPS enhanced both TNF-α and NO secretory responses. These results demonstrate that LPSs derived from different strains of P. gingivalis vary in their biological activities in vitro. The findings may have an impact on our understanding of the range of P. gingivalis virulence in vivo.     

Serine Lipids of Porphyromonas gingivalis Are Human and Mouse Toll-Like Receptor 2 Ligands

The total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. In vitro, both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2^−/−) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2^−/− mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced by P. gingivalis that likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate.     

Impaired Immune Tolerance to Porphyromonas gingivalis Lipopolysaccharide Promotes Neutrophil Migration and Decreased Apoptosis

Periodontitis, a chronic inflammatory disease of the tissues supporting the teeth, is characterized by an exaggerated host immune and inflammatory response to periopathogenic bacteria. Toll-like receptor activation, cytokine network induction, and accumulation of neutrophils at the site of inflammation are important in the host defense against infection. At the same time, induction of immune tolerance and the clearance of neutrophils from the site of infection are essential in the control of the immune response, resolution of inflammation, and prevention of tissue destruction. Using a human monocytic cell line, we demonstrate that Porphyromonas gingivalis lipopolysaccharide (LPS), which is a major etiological factor in periodontal disease, induces only partial immune tolerance, with continued high production of interleukin-8 (IL-8) but diminished secretion of tumor necrosis factor alpha (TNF-α) after repeated challenge. This cytokine response has functional consequences for other immune cells involved in the response to infection. Primary human neutrophils incubated with P. gingivalis LPS-treated naïve monocyte supernatant displayed a high migration index and increased apoptosis. In contrast, neutrophils treated with P. gingivalis LPS-tolerized monocyte supernatant showed a high migration index but significantly decreased apoptosis. Overall, these findings suggest that induction of an imbalanced immune tolerance in monocytes by P. gingivalis LPS, which favors continued secretion of IL-8 but decreased TNF-α production, may be associated with enhanced migration of neutrophils to the site of infection but also with decreased apoptosis and may play a role in the chronic inflammatory state seen in periodontal disease.Adult periodontitis is a chronic inflammatory disease of the tissues supporting the teeth, which is characterized by a constant interaction of the microorganisms in subgingival plaque and host defense mechanisms. Most of the tissue destruction in periodontitis is caused not directly by bacteria but by indirect mechanisms (35), such as an exacerbated host response manifesting itself as excessive inflammation, ultimately leading to tissue destruction (4). The inflammation caused by the activation of Toll-like receptors (TLRs), a group of pathogen recognition receptors (PRRs), is very important for detection and removal of the invading pathogen and should benefit the host in the short term by the recruitment and activation of cells and factors that aid in pathogen clearance. However, a prolonged or excessive immune response which leads to chronic inflammation can be detrimental for the host (12).The resolution of an inflammatory response is a complex process and includes induction of immune tolerance and apoptosis of effectors cells (5). Immunologic tolerance is defined as transient unresponsiveness to an antigen that is induced by previous exposure to the same or a different antigen. Induction of immune tolerance leads to a reduced immune response to repeated challenge by microbial antigens (27). The innate oral mucosal immune response becomes tolerized during sustained exposure to bacterial structures, and this may be one mechanism for the regulation of the local immune response (29). Apoptosis is a form of regulated, physiologic cell death in which the nucleus undergoes condensation and fragmentation, mitochondria swell and lose their transmembrane potential, and the apoptotic cells are rapidly phagocytosed without their contents being released (36). This process contrasts with necrosis, a type of cell death in which the nuclear and plasma membranes break down and cellular contents often spill out, inducing a local inflammatory reaction (18). Many inflammatory conditions, such as rheumatoid arthritis and systemic lupus erythematosus, are considered to result from defective apoptosis of immune cells (11).Porphyromonas gingivalis is a Gram-negative anaerobic bacterium implicated as a major periodontal pathogen. Surface components of P. gingivalis, such as lipopolysaccharide (LPS), interact with host-expressed TLRs, which are key control elements in the innate immune response (34). TLR activation triggers release of chemokines and cytokines that are responsible for accumulation of inflammatory cells and clearance of infection. It has been well established that as a consequence of stimulation with bacterial products, monocytes and macrophages synthesize a broad spectrum of cytokines that have important proinflammatory effects and are related to periodontal tissue destruction (17).Elevated secretion of various cytokines, including interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor alpha (TNF-α), by host cells has been observed following stimulation with P. gingivalis LPS (30). IL-8 is one of the principal mediators of the inflammatory response and a potent chemoattractant of neutrophils. IL-8 attracts leukocytes to the site of infection, leading to neutrophil infiltration which, if not controlled, may culminate in host tissue damage. Therefore, downregulation of IL-8 production is vital in the prevention of chronic inflammation and tissue destruction caused by an influx of neutrophils (31). During the course of inflammatory processes, monocytes, lymphocytes, and mast cells produce TNF-α, a powerful priming agonist for neutrophils (22). It increases neutrophil adhesion to endothelium and promotes release of reactive oxygen species and phagocytosis. However, TNF-α has also been recognized as a critical player in the control of neutrophil life span and induction of apoptosis (24).In the gingival crevice, neutrophils are the predominant line of defense against invading pathogens (28). They ingest microbes and destroy them by the combination of reactive oxygen species and cytotoxic components of their granules. These cytotoxic molecules cause significant damage to the tissues if the life span and clearance of neutrophils are prolonged (20). The localized accumulation of functional neutrophils at the site of inflammation is important in the host defense against infection, but the proper elimination of neutrophils is equally important in the resolution of the inflammatory response (16).The aim of this study was to examine the induction of immune tolerance by P. gingivalis LPS and the effect of cytokines produced by tolerized human monocytes on neutrophil chemotaxis and apoptosis. Escherichia coli LPS was used as a positive control LPS, as it is a well-described inducer of immune tolerance (14). In this study, we show that P. gingivalis LPS is able to induce only partial immune tolerance in human monocytes compared to E. coli LPS. Production of TNF-α was significantly decreased in tolerized cells after stimulation with P. gingivalis LPS, but IL-8 production remained as high as in naïve cells. As a consequence, we postulated that P. gingivalis may contribute to tissue destruction by promoting influx of neutrophils and their survival at the site of infection.     

Disequilibrium of M1 and M2 Macrophages Correlates with the Development of Experimental Inflammatory Bowel Diseases

Ulcerative colitis, a major inflammatory bowel disease, is an idiopathic inflammatory disorder of the colonic mucosa, accompanied by an aberrant immune reaction to intestinal microflora. Macrophages are central mediators of intestinal immune homeostasis and inflammation. The relationship between macrophages and the pathogenesis of colitis is poorly understood. We aimed to characterize the changing populations and roles of M1/M2 macrophages in colitis. We demonstrated that M1 macrophages increased and M2 macrophages decreased in colitis, accompanied by Interleukin (IL)-23 and Tumor necrosis factor-α induction and IL-10 suppression. Transfer of M2 macrophages reduced dextran sodium sulfate-induced colitis by inducing IL-10 production and promoting regulatory T-cell generation. In vivo neutralization of IL-10 partially reduced the effects of M2 transfer. These findings suggest that macrophages play a critical role in colitis; specifically, disequilibrium of macrophage subsets promotes colitis development. A shift from the M1 to M2 phenotype reduces colitis by inducing IL-10; thus, mobilization of M2 macrophages could be a novel approach to colitis therapy.     

Mycobacterium massiliense Induces Inflammatory Responses in Macrophages Through Toll-Like Receptor 2 and c-Jun N-Terminal Kinase

Mycobacterium massiliense (Mmass) is an emerging, rapidly growing mycobacterium (RGM) that belongs to the M. abscessus (Mabc) group, albeit clearly differentiated from Mabc. Compared with M. tuberculosis, a well-characterized human pathogen, the host innate immune response against Mmass infection is largely unknown. In this study, we show that Mmass robustly activates mRNA and protein expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in murine bone marrow-derived macrophages (BMDMs). Toll-like receptor (TLR)-2 and myeloid differentiation primary response gene 88 (MyD88), but neither TLR4 nor Dectin-1, are involved in Mmass-induced TNF-α or IL-6 production in BMDMs. Mmass infection also activates the mitogen-activated protein kinase (MAPKs; c-Jun N-terminal kinase (JNK), ERK1/2 and p38 MAPK) pathway. Mmass-induced TNF-α and IL-6 production was dependent on JNK activation, while they were unaffected by either the ERK1/2 or p38 pathway in BMDMs. Additionally, intracellular reactive oxygen species (ROS), NADPH oxidase-2, and nuclear factor-κB are required for Mmass-induced proinflammatory cytokine generation in macrophages. Furthermore, the S morphotype of Mmass showed lower overall induction of pro-inflammatory (TNF-α, IL-6, and IL-1β) and anti-inflammatory (IL-10) cytokines than the R morphotype, suggesting fewer immunogenic characteristics for this clinical strain. Together, these results suggest that Mmass-induced activation of host proinflammatory cytokines is mediated through TLR2-dependent JNK and ROS signaling pathways.     

Lipopolysaccharide preparation extracted from Porphyromonas gingivalis lipoprotein-deficient mutant shows a marked decrease in toll-like receptor 2-mediated signaling

We recently demonstrated that a new PG1828-encoded lipoprotein (PG1828LP) was able to be separated from a Porphyromonas gingivalis lipopolysaccharide (LPS) preparation, and we found that it exhibited strong cell activation, similar to that of Escherichia coli LPS, through a Toll-like receptor 2 (TLR2)-dependent pathway. In order to determine the virulence of PG1828LP toward cell activation, we generated a PG1828-deficient mutant of P. gingivalis strain 381 by allelic exchange mutagenesis using an ermF-ermAM antibiotic resistance cassette. A highly purified preparation of LPS from a PG1828-deficient mutant (DeltaPG1828-LPS) showed nearly the same ladder-like patterns in silver-stained gels as a preparation of LPS from a wild-type strain (WT-LPS), as well as Limulus amoebocyte lysate activities that were similar to those of the WT-LPS preparation. However, the ability of the DeltaPG1828-LPS preparation to activate NF-kappaB in TLR2-expressing cells was markedly attenuated. Cytokine production by human gingival fibroblasts was also decreased in response to the DeltaPG1828-LPS preparation in comparison with the WT-LPS preparation, and the activity was comparable to the stimulation of highly purified lipid A of P. gingivalis by TLR4. Further, lethal toxicity was rarely observed following intraperitoneal injection of the PG1828-deficient mutant into mice compared to that with the wild-type strain, while the DeltaPG1828-LPS preparation showed no lethal toxicity. Taken together, these results clearly indicate that PG1828LP plays an essential role in inflammatory responses and may be a major virulence factor of P. gingivalis.     

Lipopolysaccharide Induces CD25-Positive, IL-10-Producing Lymphocytes Without Secretion of Proinflammatory Cytokines in the Human Colon: Low MD-2 mRNA Expression in Colonic Macrophages

Despite the huge number of colonized Gram-negative bacteria in the colon, the normal colon maintains its homeostasis without any excessive immune response. To investigate the potential mechanisms involved, human colonic lamina propria mononuclear cells (LPMCs) obtained from uninflamed mucosa were cultured with lipopolysaccharide (LPS) prepared from Bacteroides vulgatus (BV-LPS) or Bacteroides fragilis (BF-LPS), as representatives of indigenous flora, or pathogenic Salmonella minnesota (SM-LPS). Colonic LPMCs failed to produce inflammatory cytokines in response to any type of LPS. Colonic macrophages barely expressed mRNA for MD-2, an essential association molecule for LPS signaling via Toll-like receptor 4. Further, BV-LPS induced CD25 and Foxp3 expression in lymphocytes and CD4(+)CD25(+) cells expressed IL-10 mRNA. Thus, the low expression of functioning LPS receptor molecules and induction of IL-10-producing CD4(+)CD25(+) lymphocytes by indigenous LPS may play a central role in the maintenance of colonic immunological homeostasis.     

Effect of Lipopolysaccharide and Inflammatory Cytokines on Interleukin-6 Production by Healthy Human Gingival Fibroblasts

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) and inflammatory cytokines on regulation of interleukin-6 (IL-6) production by human gingival fibroblasts (HGF). The HGF cell lines used in this study, H-CL and F-CL, were established by the explant technique from healthy gingival tissue. Cultured cells were grown to confluency and incubated with various concentrations of LPS from Escherichia coli or Porphyromonas gingivalis or with the recombinant human cytokine tumor necrosis factor alpha (TNF-α), IL-1α, or IL-1β. Culture supernatants were collected at various times and assessed for IL-6 production by enzyme-linked immunosorbent assay. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 mRNA by the RNase protection assay. Both LPS preparations induced IL-6 production (1 to 4 ng of IL-6 per ml) by both HGF cell lines. Although TNF-α stimulated IL-6 production by HGF, >10-fold-larger amounts were induced with IL-1α and IL-1β. Furthermore, the addition of both IL-1α and TNF-α to cultured cells resulted in approximately 600- to 800-fold-higher levels of IL-6 than seen in control cultures, suggesting that these cytokines synergistically induced IL-6 production by HGF. IL-6 message in cultured cells was upregulated 20-fold by TNF-α, 1,000-fold by IL-1α and IL-1β, and 1,400-fold by IL-1α plus TNF-α. IL-1α and TNF-α alone upregulate IL-6 production in a dose- and time-dependent fashion. The addition of IL-1α and TNF-α to cultured HGF cells resulted in a synergistic induction of IL-6 after 8 h of incubation and when greater than 10 pg of this combination per ml was used. Our studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.     

Induction of Lethal Shock and Tolerance by Porphyromonas gingivalis Lipopolysaccharide in d-Galactosamine-Sensitized C3H/HeJ Mice

Lipopolysaccharide (LPS) obtained from Porphyromonas gingivalis was found to exhibit marked lethal toxicity in galactosamine-sensitized C3H/HeJ mice. Although no lethality was observed in mice intraperitoneally challenged with 1 mg of P. gingivalis LPS without galactosamine, when they were sensitized with 30 mg of galactosamine, challenge with 1 and 10 micrograms of LPS resulted in 67 and 100% lethality, respectively. The lethal dose of LPS was almost the same in LPS-responsive C57BL/6 mice and non-LPS-responsive C3H/HeJ mice. Furthermore, when 1 microgram of P. gingivalis LPS was administered to each mouse 90 min before the challenge with the same LPS with galactosamine, tolerance to the lethal action of LPS was induced, and the mice were completely protected from death, even at a dose 100-fold greater than the lethal dose of LPS. Neither a lethal effect nor induction of tolerance to the lethality of P. gingivalis LPS was exhibited by Salmonella LPS in galactosamine-sensitized C3H/HeJ mice. A protein-LPS complex derived from Pseudomonas aeruginosa, which exhibited strong lethality and induced tolerance to a subsequent challenge with a lethal dose of LPS in galactosamine-sensitized LPS-responsive mice, did not exhibit lethal toxicity in galactosamine-sensitized C3H/HeJ mice and failed to induce tolerance in these mice to the lethality of P. gingivalis LPS. These results indicate that P. gingivalis LPS plays the central role in the activation of non-LPS-responsive C3H/HeJ mice.     

Prenatal Exposure to Lipopolysaccharide Induces PTX3 Expression and Results in Obesity in Mouse Offspring

This study tested the hypothesis whether inflammation will directly lead to obesity. This study was designed to investigate the relationship between inflammation and obesity by intraperitoneally injecting pregnant mice with lipopolysaccharide (LPS) (75 μg kg^?1). The results showed that inflammation during pregnancy could lead to a significant increase in the levels of the inflammatory factor PTX3. The offspring of the LPS-treated mice displayed abnormal levels of fat development, blood lipids, and glucose metabolism, and fat differentiation markers were significantly increased. Our study also confirmed that PTX3 can increase the susceptibility to obesity by regulating the expression of adipogenic markers; this regulatory role of PTX3 is most likely caused by MAPK pathway hyperactivation. Our study is the first to find strong evidence of inflammation as a cause of obesity. We determined that PTX3 was an important moderator of obesity, and we elucidated its mechanism, thus providing new targets and theories for obesity therapy. Moreover, our study provides new ideas and directions for the early intervention of anti-inflammation in pregnancy.     

Determination of Active Phagocytosis of Unopsonized Porphyromonas gingivalis by Macrophages and Neutrophils Using the pH-Sensitive Fluorescent Dye pHrodo

Phagocytosis of pathogens is an important component of the innate immune system that is responsible for the removal and degradation of bacteria as well as their presentation via the major histocompatibility complexes to the adaptive immune system. The periodontal pathogen Porphyromonas gingivalis exhibits strain heterogeneity, which may affect a phagocyte''s ability to recognize and phagocytose the bacterium. In addition, P. gingivalis is reported to avoid phagocytosis by antibody and complement degradation and by invading phagocytic cells. Previous studies examining phagocytosis have been confounded by both the techniques employed and the potential of the bacteria to invade the cells. In this study, we used a novel, pH-sensitive dye, pHrodo, to label live P. gingivalis strains and examine unopsonized phagocytosis by murine macrophages and neutrophils and human monocytic cells. All host cells examined were able to recognize and phagocytose unopsonized P. gingivalis strains. Macrophages had a preference to phagocytose P. gingivalis strain ATCC 33277 over other strains and clinical isolates in the study, whereas neutrophils favored P. gingivalis W50, ATCC 33277, and one clinical isolate over the other strains. This study revealed that all P. gingivalis strains were capable of being phagocytosed without prior opsonization with antibody or complement.