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Periodontitis (PD) is a widespread chronic inflammatory disease in the human population. Porphyromonas gingivalis is associated with PD and can citrullinate host proteins via P. gingivalis peptidyl arginine deiminase (PPAD). Here, we hypothesized that infection of human dental follicle stem cells (hDFSCs) with P. gingivalis and subsequent interaction with neutrophils will alter the neutrophil phenotype. To test this hypothesis, we established and analyzed a triple-culture system of neutrophils and hDFSCs primed with P. gingivalis. Mitogen-activated pathway blocking reagents were applied to gain insight into stem cell signaling after infection. Naïve hDFSCs do not influence the neutrophil phenotype. However, infection of hDFSCs with P. gingivalis prolongs the survival of neutrophils and increases their migration. These phenotypic changes depend on direct cellular contacts and PPAD expression by P. gingivalis. Active JNK and ERK pathways in primed hDFSCs are essential for the phenotypic changes in neutrophils. Collectively, our results confirm that P. gingivalis modifies hDFSCs, thereby causing an immune imbalance.

Pattern recognition receptors are critical for the detection of invading microorganisms. They activate multiple pathways that lead to the induction of proinflammatory responses and pathogen clearance. The intensity and duration of this immune reaction must be tightly controlled spatially and temporally in every tissue by different negative regulators. We hypothesized that monocyte chemoattractant protein-1-induced protein-1 (MCPIP-1) might play a role in maintaining immune homeostasis in the epithelium both under physiological conditions and upon bacterial infection. To this end, we examined the distribution of the MCPIP-1 transcript and protein in various tissues. The MCPIP-1 protein level was higher in epithelial cells than in myeloid cells. MCPIP-1 exerted RNase activity towards the interleukin (IL)-8 transcript and the lifespan of IL-8 was determined by the presence of the stem-loops/hairpin structures at the 3′UTR region of IL-8 mRNA. Moreover, using fully active, purified recombinant MCPIP-1 protein, we elucidated the mechanism by which MCPIP-1 controls the IL-8 mRNA level. In conclusion, we uncovered a novel IL-8-dependent mechanism via which MCPIP-1 maintains epithelial homeostasis. This study reveals for the first time that MCPIP-1 plays a crucial anti-inflammatory role not only in myeloid cells but also in epithelial cells.

Aspiration pneumonia is a life-threatening infectious disease often caused by oral anaerobic and periodontal pathogens such as Porphyromonas gingivalis. This organism produces proteolytic enzymes, known as gingipains, which manipulate innate immune responses and promote chronic inflammation. Here, we challenged mice with P. gingivalis W83 and examined the role of gingipains in bronchopneumonia, lung abscess formation, and inflammatory responses. Although gingipains were not required for P. gingivalis colonization and survival in the lungs, they were essential for manifestation of clinical symptoms and infection-related mortality. Pathologies caused by wild-type (WT) P. gingivalis W83, including hemorrhage, necrosis, and neutrophil infiltration, were absent from lungs infected with gingipain-null isogenic strains or WT bacteria preincubated with gingipain-specific inhibitors. Damage to lung tissue correlated with systemic inflammatory responses, as manifested by elevated levels of TNF, IL-6, IL-17, and C-reactive protein. These effects were unequivocally dependent on gingipain activity. Gingipain activity was also implicated in the observed increase in IL-17 in lung tissues. Furthermore, gingipains increased platelet counts in the blood and activated platelets in the lungs. Arginine-specific gingipains made a greater contribution to P. gingivalis-related morbidity and mortality than lysine-specific gingipains. Thus, inhibition of gingipain may be a useful adjunct treatment for P. gingivalis-mediated aspiration pneumonia.

After phagocytosis by macrophages, Staphylococcus aureus evades killing in an a-toxin-dependent manner, and then prevents apoptosis of infected cells by upregulating expression of antiapoptotic genes like MCL-1 (myeloid cell leukemia-1). Here, using purified a-toxin and a set of hla-deficient strains, we show that a-toxin is critical for the induction of MCL-1 expression and the cytoprotection of infected macrophages. Extracellular or intracellular treatment of macrophages with a-toxin alone did not induce cytoprotection conferred by increased Mcl-1, suggesting that the process is dependent on the production of a-toxin by intracellular bacteria. The increased expression of MCL-1 in infected cells was associated with enhanced NFκB activation, and subsequent IL-6 secretion. This effect was only partially inhibited by blocking TLR2, which suggests the participation of intracellular receptors in the specific recognition of S. aureus strains secreting a-toxin. Thus, S. aureus recognition by intracellular receptors and/or activation of downstream pathways leading to Mcl-1 expression is facilitated by a-toxin released by intracellular bacteria which permeabilize phagosomes, ensuring pathogen access to the cytoplasmatic compartment. Given that the intracellular survival of S. aureus depends on a-toxin, we propose a novel role for this agent in the protection of the intracellular niche, and further dissemination of staphylococci by infected macrophages.

Cysteine proteases (gingipains) from Porphyromonas gingivalis are key virulence factors in chronic periodontitis. Innate immune receptors CD14, Toll-like receptor (TLR) 2 and TLR4 are important in P. gingivalis recognition. We examined the ability of gingipains to cleave CD14, TLR2 and TLR4, and the consequences for the cellular response to bacterial challenge. Macrophages were exposed to Arg (RgpA and RgpB)- and Lys (Kgp)-gingipains, and residual expression of TLR2, TLR4 and CD14 was determined by flow cytometry. The cellular response to live bacteria following exposure to purified gingipains was evaluated by TNFα production and bacterial phagocytosis. RgpA and Kgp decreased CD14 detection in a concentration (p = 0.0000002)- and time (p = 0.03)-dependent manner, whereas RgpB had no significant effect. TLR2 and TLR4 expression were unaffected. Reduction in CD14 expression was more efficient with Lys-gingipain than with Arg-gingipain. A reduced CD14 surface level correlated with decreased TNFα secretion and bacterial phagocytosis following challenge with live P. gingivalis, but the response to heat-killed bacteria was unaffected. Therefore, gingipains reduce CD14 expression without affecting expression of the bacterial-sensing TLRs. Reduced CD14 expression depends on the gingipain hemagglutinin/adhesion site and results in macrophage hyporesponsiveness to bacterial challenge. Further studies are needed to determine if reduced CD14 expression is linked to periodontitis induced by P. gingivalis.

Porphyromonas gingivalis is a major contributor to the pathogenesis of periodontitis, an infection-driven inflammatory disease that leads to bone destruction. This pathogen stimulates pro-interleukin (IL)-1β synthesis but not mature IL-1β secretion, unless the P2X7 receptor is activated by extracellular ATP (eATP). Here, we investigated the role of P. gingivalis fimbriae in eATP-induced IL-1β release. Bone marrow-derived macrophages (BMDMs) from wild-type (WT) or P2X7-deficient mice were infected with P. gingivalis (381) or isogenic fimbria-deficient (DPG3) strain with or without subsequent eATP stimulation. DPG3 induced higher IL-1β secretion after eATP stimulation compared to 381 in WT BMDMs, but not in P2X7-deficient cells. This mechanism was dependent on K+ efflux and Ca2+-independent phospholipase A2 activity. Accordingly, non-fimbriated P. gingivalis failed to inhibit apoptosis via the eATP/P2X7 pathway. Furthermore, P. gingivalis-driven stimulation of IL-1β was Toll-like receptor 2 and MyD88 dependent, and not associated with fimbria expression. Fimbria-dependent down-modulation of IL-1β was selective, as levels of other cytokines remained unaffected by P2X7 deficiency. Confocal microscopy demonstrated the presence of discrete P2X7 expression in the absence of P. gingivalis stimulation, which was enhanced by 381-stimulated cells. Notably, DPG3-infected macrophages revealed a distinct pattern of P2X7 receptor expression with a marked focus formation. Collectively, these data demonstrate that eATP-induced IL-1β secretion is impaired by P. gingivalis fimbriae in a P2X7-dependent manner.

The pulmonary surfactant is a complex mixture of lipids and proteins that is important for respiratory lung functions, which also provides the first line of innate immune defense. Pulmonary surfactant protein-A (SP-A) is a major surfactant component with immune functions with importance during Staphylococcus aureus infections that has been demonstrated in numerous studies. The current study showed that S. aureus can efficiently cleave the SP-A protein using its arsenal of proteolytic enzymes. This degradation appears to be mediated by cysteine proteases, in particular staphopain A (ScpA). The staphopain-mediated proteolysis of SP-A resulted in a decrease or complete abolishment of SP-A biological activity, including the promotion of S. aureus phagocytosis by neutrophils, aggregation of Gram-negative bacteria and bacterial cell adherence to epithelium. Significantly, ScpA has also efficiently degraded SP-A in complete bronchi-alveolar lavage fluid from human lungs. This indicates that staphopain activity in the lungs is resistant to protease inhibitors, thus suggesting that SP-A can be cleaved in vivo. Collectively, this study showed that the S. aureus protease ScpA is an important virulence factor that may impair innate immunity of the lungs.

Toll-like receptors (TLRs) are key molecules in innate immunity that recognize a variety of pathogen-associated molecular patterns. Activation of TLRs by their agonists initiates several signaling cascades, which eventually result in the expression of immune modifiers. Despite the fact that MCPIP1 is reported as an important immune regulator involved in macrophage activation, modulation of its expression by all known TLR agonists has never been documented. In this study, we present for the first time that in human monocyte-derived macrophages all TLR agonists, except CpG, markedly induced the expression of MCPIP1. The level of the induced transcript, as well as the protein and time of their appearance varied depending on the agonist. Furthermore, we confirmed the strong and differential upregulation of MCPIP1 during bacteria, virus and fungus infection. MCPIP1 belongs to a group of early-response genes; however, in the present study, we show for the first time the sustained high level of MCPIP1 expression during long-term Staphylococcus aureus infection. Taken together, our results implicate MCPIP1 as a potent regulator of innate immunity, which can be strongly engaged in the pathogenesis of acute and chronic infective diseases.

Tannerella forsythia is a gram-negative bacterium strongly associated with the development and/or progression of periodontal disease. Here, we have shown that a newly characterized matrix metalloprotease-like enzyme, referred to as karilysin, efficiently cleaved the antimicrobial peptide LL-37, significantly reducing its bactericidal activity. This may contribute to the resistance of T. forsythia to the antibacterial activity of LL-37, since their vitality was found not to be affected by LL-37 at concentrations up to 2.2 µM. Furthermore, proteolysis of LL-37 by karilysin not only abolished its ability to bind lipopolysaccharide (LPS) to quench endotoxin-induced proinflammatory activity, but LL-37 cleavage also caused the release of active endotoxin from the LPS/LL-37 complex. Proteolytic inactivation of LL-37 bactericidal activity by karilysin may protect LL-37-sensitive species in the subgingival plaque and maintain the local inflammatory reaction driven by LPS from gram-negative bacteria. Consequently, the karilysin protease may directly contribute to periodontal tissue damage and the development and/or progression of chronic periodontitis.

Periodontal disease is an oral inflammatory disease affecting the supporting structures of teeth. Porphyromonas gingivalis, a major pathogenic agent for the disease, expresses a number of virulence factors, including cysteine proteases called the gingipains. The arginine- and lysine-specific gingipains, HRgpA and Kgp, respectively, are expressed as high molecular weight forms containing both catalytic and adhesin subunits. We examined the expression pattern of cytokines and their receptors in differentiated macrophages following exposure to active and inactive forms of the gingipains, using a cDNA array, quantitative PCR and ELISA analysis. Amongst other pro-inflammatory cytokines, results from the cDNA array suggested that interleukin-1β, granulocyte-macrophage colony stimulatory factor and interferon-γ were upregulated after exposure of the macrophages to the gingipains. Quantitative PCR analysis substantiated these observations and indicated that active or inactive forms of the high molecular weight gingipains were able to upregulate expression of transcripts for these cytokines. The strongly enhanced production of interleukin-1β and granulocyte-macrophage colony stimulatory factor by differentiated macrophages in response to active or inactive forms of the high molecular weight gingipains was confirmed at the protein level by ELISA analysis. The results indicate that the adhesin subunits of the gingipains mediate strong upregulation of the expression of pro-inflammatory cytokines in macrophages.