Complement receptor 3 blockade promotes IL-12-mediated clearance of Porphyromonas gingivalis and negates its virulence in vivo

The ability of certain pathogens to exploit innate immune function allows them to undermine immune clearance and thereby increase their persistence and capacity to cause disease. Porphyromonas gingivalis is a major pathogen in periodontal disease and is associated with increased risk of systemic conditions. We have previously shown that the fimbriae of P. gingivalis interact with complement receptor 3 (CR3; CD11b/CD18) in monocytes/macrophages, resulting in inhibition of IL-12p70 production in vitro. The in vivo biological implications of this observation were investigated in this study using a CR3 antagonist (XVA143). XVA143 was shown to block CR3 binding of P. gingivalis fimbriae and reverse IL-12p70 inhibition; specifically, CR3 blockade resulted in inhibition of ERK1/2 phosphorylation and up-regulation of IL-12 p35 and p40 mRNA expression. Importantly, mice pretreated with XVA143 elicited higher IL-12p70 and IFN-gamma levels in response to P. gingivalis i.p. infection and displayed enhanced pathogen clearance, compared with similarly infected controls. The notion that CR3 is associated with reduced IL-12p70 induction and impaired P. gingivalis clearance was confirmed using i.p. infected wild-type and CR3-deficient mice. Moreover, XVA143 dramatically attenuated the persistence and virulence of P. gingivalis in experimental mouse periodontitis, as evidenced by reduced induction of periodontal bone loss. Therefore, CR3 blockade may represent a promising immunomodulatory approach for controlling human periodontitis and possibly associated systemic diseases.     

The C5a receptor impairs IL-12-dependent clearance of Porphyromonas gingivalis and is required for induction of periodontal bone loss

The C5a anaphylatoxin receptor (C5aR; CD88) is activated as part of the complement cascade and exerts important inflammatory, antimicrobial, and regulatory functions, at least in part, via crosstalk with TLRs. However, the periodontal pathogen Porphyromonas gingivalis can control C5aR activation by generating C5a through its own C5 convertase-like enzymatic activity. In this paper, we show that P. gingivalis uses this mechanism to proactively and selectively inhibit TLR2-induced IL-12p70, whereas the same pathogen-instigated C5aR-TLR2 crosstalk upregulates other inflammatory and bone-resorptive cytokines (IL-1β, IL-6, and TNF-α). In vivo, the ability of P. gingivalis to manipulate TLR2 activation via the C5a-C5aR axis allowed it to escape IL-12p70-dependent immune clearance and to cause inflammatory bone loss in a murine model of experimental periodontitis. In the latter regard, C5aR-deficient or TLR2-deficient mice were both resistant to periodontal bone loss, in stark contrast with wild-type control mice, which is consistent with the interdependent interactions of C5aR and TLR2 in P. gingivalis immune evasion and induction of bone-resorptive cytokines. In conclusion, P. gingivalis targets C5aR to promote its adaptive fitness and cause periodontal disease. Given the current availability of safe and effective C5aR antagonists, pharmacological blockade of C5aR could act therapeutically in human periodontitis and reduce associated systemic risks.     

Bacterial fimbriae stimulate proinflammatory activation in the endothelium through distinct TLRs

The major and minor fimbriae proteins produced by the human pathogen Porphyromonas gingivalis are required for invasion of human aortic endothelial cells and for the stimulation of potent inflammatory responses. In this study, we report that native forms of both the major and minor fimbriae proteins bind to and signal through TLR2 for this response. Major and minor fimbriae bound to a human TLR2:Fc chimeric protein with an observed K(d) of 28.9 nM and 61.7 nM, respectively. Direct binding of the major and minor fimbriae to a human chimeric CD14-Fc protein also established specific binding of the major and minor fimbriae to CD14 with classic saturation kinetics. Using a P. gingivalis major and minor fimbriae mutant, we confirmed that TLR2 binding in whole cells is dependent on the expression of the major and minor fimbriae. Although we did not observe binding with the major or minor fimbriae to the TLR4-Fc chimeric protein, signaling through TLR4 for both proteins was demonstrated in human embryonic kidney 293 cells transfected with TLR4 and only in the presence MD-2. Transient transfection of dominant-negative forms of TLR2 or TLR4 reduced IL-8 production by human aortic endothelial cells following stimulation with major or minor fimbriae. The ability of two well-defined microbe-associated molecular patterns to select for innate immune recognition receptors based on accessory proteins may provide a novel way for a pathogen to sense and signal in appropriate host environments.     

Importance of TLR2 in early innate immune response to acute pulmonary infection with Porphyromonas gingivalis in mice

The periodontal pathogen Porphyromonas gingivalis is implicated in certain systemic diseases including atherosclerosis and aspiration pneumonia. This organism induces innate responses predominantly through TLR2, which also mediates its ability to induce experimental periodontitis and accelerate atherosclerosis. Using a validated mouse model of intratracheal challenge, we investigated the role of TLR2 in the control of P. gingivalis acute pulmonary infection. TLR2-deficient mice elicited reduced proinflammatory or antimicrobial responses (KC, MIP-1alpha, TNF-alpha, IL-6, IL-12p70, and NO) in the lung and exhibited impaired clearance of P. gingivalis compared with normal controls. However, the influx of polymorphonuclear leukocytes into the lung and the numbers of resident alveolar macrophages (AM) were comparable between the two groups. TLR2 signaling was important for in vitro killing of P. gingivalis by polymorphonuclear leukocytes or AM and, moreover, the AM bactericidal activity required NO production. Strikingly, AM were more potent than peritoneal or splenic macrophages in P. gingivalis killing, attributed to diminished AM expression of complement receptor-3 (CR3), which is exploited by P. gingivalis to promote its survival. The selective expression of CR3 by tissue macrophages and the requirement of TLR2 inside-out signaling for CR3 exploitation by P. gingivalis suggest that the role of TLR2 in host protection may be contextual. Thus, although TLR2 may mediate destructive effects, as seen in models of experimental periodontitis and atherosclerosis, we have now shown that the same receptor confers protection against P. gingivalis in acute lung infection.     

Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo.

The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood. In this study, we investigated the type of Ag-specific CD4(+) Th and CD8(+) T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens. We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling. Coinjections of E. coli LPS + OVA or P. gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4(+) and CD8(+) T cells, but strikingly different cytokine profiles. E. coli LPS induced a Th1-like response with abundant IFN-gamma, but little or no IL-4, IL-13, and IL-5. In contrast, P. gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-gamma. Consistent with these results, E. coli LPS induced IL-12(p70) in the CD8alpha(+) dendritic cell (DC) subset, while P. gingivalis LPS did not. Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-alpha. Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E. coli LPS, but not P. gingivalis LPS. Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo.     

Differential interactions of fimbriae and lipopolysaccharide from Porphyromonas gingivalis with the Toll-like receptor 2-centred pattern recognition apparatus

The lipopolysaccharide (LPS) and fimbriae of Porphyromonas gingivalis play important roles in periodontal inflammation and pathogenesis. We investigated fimbriae and LPS from several P. gingivalis strains in terms of relative dependence on Toll-like receptor (TLR) signalling partners or accessory pattern-recognition molecules mediating ligand transfer to TLRs, and determined induced assembly of receptor complexes in lipid rafts. Fimbriae could utilize TLR1 or TLR6 for cooperative TLR2-dependent activation of transfected cell lines, in contrast to LPS and a mutant version of fimbriae which displayed preference for TLR1. Whether used to activate human cell lines or mouse macrophages, fimbriae exhibited strong dependence on membrane-expressed CD14 (mCD14), which could not be substituted for by soluble CD14 (sCD14). In contrast, sCD14 efficiently substituted for mCD14 in LPS-induced cellular activation. LPS-binding protein was more important for LPS- than for fimbria-induced cell activation, whereas the converse was true for CD11b/CD18. Cell activation by LPS or fimbriae required lipid raft function and formation of heterotypic receptor complexes (TLR1-2/CD14/CD11b/CD18), although wild-type fimbriae additionally recruited TLR6. In summary, TLR2 activation by P. gingivalis LPS or fimbriae involves differential dependence on accessory signalling or ligand-binding receptors, which may differentially influence innate immune responses.     

Virulence of Porphyromonas gingivalis is altered by substitution of fimbria gene with different genotype

Porphyromonas gingivalis is a periodontal pathogen whose fimbriae are classified into six genotypes based on the diversity of the fimA genes encoding each fimbria subunit. It was suggested that P. gingivalis strains with type II fimbriae were more virulent than type I strains. For the present study, we generated the mutants in which fimA was substituted with different genotypes to study virulence of type II fimbriae. Using plasmid vectors, fimA of ATCC33277 (type I strain) was substituted with type II fimA, and that of OMZ314 (type II strain) with type I fimA. The substitution of type I fimA with type II enhanced bacterial adhesion/invasion to epithelial cells, whereas substitution with type I fimA resulted in diminished efficiency. Following bacterial invasion, type II clones swiftly degraded cellular paxillin and focal adhesion kinase, and inhibited cellular migration, whereas type I clones and DeltafimA mutants did not. BIAcore analysis demonstrated that type II fimbriae possess greater adhesive abilities for their receptor alpha5beta1-integrin than those of type I. In a mouse abscess model, the type II clones significantly induced serum IL-1beta and IL-6, as well as other infectious symptoms. These results suggest that type II fimbriae are a critical determinant of P. gingivalis virulence.     

TLR2 transmodulates monocyte adhesion and transmigration via Rac1- and PI3K-mediated inside-out signaling in response to Porphyromonas gingivalis fimbriae

We present evidence for a novel TLR2 function in transmodulating the adhesive activities of human monocytes in response to the fimbriae of Porphyromonas gingivalis, a pathogen implicated in chronic periodontitis and atherosclerosis. Monocyte recruitment into the subendothelium is a crucial step in atherosclerosis, and we investigated the role of P. gingivalis fimbriae in stimulating monocyte adhesion to endothelial cells and transendothelial migration. Fimbriae induced CD11b/CD18-dependent adhesion of human monocytes or mouse macrophages to endothelial receptor ICAM-1; these activities were inhibited by TLR2 blockade or deficiency or by pharmacological inhibitors of PI3K. Moreover, this inducible adhesive activity was sensitive to the action of Clostridium difficile toxin B, but was not affected by Clostridium botulinum C3 exoenzyme, pertussis toxin, or cholera toxin. Accordingly, we subsequently showed through the use of dominant negative signaling mutants of small GTPases, that Rac1 mediates the ability of fimbria-stimulated monocytes to bind ICAM-1. A dominant negative mutant of Rac1 also inhibited the lipid kinase activity of PI3K suggesting that Rac1 acts upstream of PI3K in this proadhesive pathway. Furthermore, fimbriae stimulated monocyte adhesion to HUVEC and transmigration across HUVEC monolayers; both activities required TLR2 and Rac1 signaling and were dependent upon ICAM-1 and the high-affinity state of CD11b/CD18. P. gingivalis-stimulated monocytes displayed enhanced transendothelial migration compared with monocytes stimulated with nonfimbriated isogenic mutants. Thus, P. gingivalis fimbriae activate a novel proadhesive pathway in human monocytes, involving TLR2, Rac1, PI3K, and CD11b/CD18, which may constitute a mechanistic basis linking P. gingivalis to inflammatory atherosclerotic processes.     

TLR2-dependent modulation of osteoclastogenesis by Porphyromonas gingivalis through differential induction of NFATc1 and NF-kappaB

Osteolytic diseases, including rheumatoid arthritis, osteomyelitis, and periodontitis, are usually associated with bacterial infections. However, the precise mechanisms by which bacteria induce bone loss still remain unclear. Evidence exists that Toll-like receptor (TLR) signaling regulates both inflammation and bone metabolism and that the receptor activator of NF-κB ligand (RANKL) and its receptor RANK are the key regulators for bone remodeling and for the activation of osteoclasts. Here, we investigate the direct effects of the periodontal pathogen Porphyromonas gingivalis on osteoclast differentiation and show that P. gingivalis differentially modulates RANKL-induced osteoclast formation contingent on the state of differentiation of osteoclast precursors. In addition, although an optimal induction of cytokines by P. gingivalis is dependent on TLR2 and TLR4, as well as myeloid differentiation factor 88 and Toll/IL-1R domain-containing adaptor-inducing IFN-β, P. gingivalis utilizes TLR2/ myeloid differentiation factor 88 in modulating osteoclast differentiation. P. gingivalis modulates RANKL-induced osteoclast formation by differential induction of NFATc1 and c-Fos. More importantly, RANKL-mediated lineage commitment also has an impact on P. gingivalis-induced cytokine production. RANKL inhibits P. gingivalis-induced cytokine production by down-regulation of TLR/NF-κB and up-regulation of NFATc1. Our findings reveal novel aspects of the interactions between TLR and RANK signaling and provide a new model for understanding the mechanism underlying the pathogenesis of bacteria-mediated bone loss.     

Peptide mapping of bacterial fimbrial epitopes interacting with pattern recognition receptors

The fimbriae of the oral pathogen Porphyromonas gingivalis induce Toll-like receptor 2 (TLR2)-dependent macrophage activation upon their recognition by CD14 and the beta(2) integrin CD11b/CD18. To map functional epitopes of fimbriae that interact with these pattern recognition receptors (PRRs), we examined 20 synthetic peptides covering the entire length of the 41-kDa fimbrillin subunit. Using direct or competitive inhibition assays for receptor binding or cell activation, the CD14 binding activity of fimbriae was localized to residues 69-90 and was essential for TLR2-dependent cytokine induction. The CD11b/CD18 binding activity of fimbriae was localized to two neighboring epitopes defined by residues 166-185 and 206-225. Unlike epitope 69-90 that constitutively bound CD14, the CD11b/CD18 binding activity of epitopes 166-185 and 206-225 was inducible by integrin activators. The CD11b/CD18 binding activity played a contributory role to TLR2-dependent induction of tumor necrosis factor-alpha by fimbriae but was involved in specific down-regulation of interleukin-12. Cell activation by a combination of fimbrillin peptides corresponding to the CD14 and CD11b/CD18 binding activities resulted in higher tumor necrosis factor-alpha responses than would be expected from a simply additive effect, attributable to CD14-dependent inside-out signaling leading to enhanced binding interactions with CD11b/CD18. These data suggest that P. gingivalis fimbriae display a modular structure that interacts through discrete epitopes and in a regulated mode with distinct PRRs, which in turn differentially modulate the state of cell activation. Elucidation of pathogen interactions with PRRs at the molecular level may glean insight into host defense mechanisms as well as into microbial strategies that subvert innate immunity.     

Maturation of monocyte-derived dendritic cells with Toll-like receptor 3 and 7/8 ligands combined with prostaglandin E2 results in high interleukin-12 production and cell migration

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that play a key role in regulating T cell-based immunity. In vivo, the capacity of DC to activate T cells depends on their ability to migrate to the T cell areas of lymph nodes as well as on their maturation state. Depending on their cytokine-secreting profile, DC are able to skew the immune response in a specific direction. In particular, IL-12p70 producing DC drive T cells towards a T helper 1 type response. A serious disadvantage of current clinical grade ex vivo generated monocyte-derived DC is the poor IL-12p70 production. We have investigated the effects of Toll-like receptor (TLR)-mediated maturation on ex vivo generated human monocyte-derived DC. We demonstrate that in contrast to cytokine-matured DC, DC matured with poly(I:C) (TLR3 ligand) and/or R848 (TLR7/8 ligand) are able to produce vast amounts of IL-12p70, but exhibit a reduced migratory capacity. The addition of prostaglandin E(2) (PGE(2)) improved the migratory capacity of TLR-ligand matured DC while maintaining their IL-12p70 production upon T cell encounter. We propose a novel clinical grade maturation protocol in which TLR ligands poly(I:C) and R848 are combined with PGE(2) to generate DC with both high migratory capacity and IL-12p70 production upon T cell encounter.     

Human Toll-like receptor 4 responses to P. gingivalis are regulated by lipid A 1- and 4'-phosphatase activities

Signal transduction following binding of lipopolysaccharide (LPS) to Toll-like receptor 4 (TLR4) is an essential aspect of host innate immune responses to infection by Gram-negative pathogens. Here, we describe a novel molecular mechanism used by a prevalent human bacterial pathogen to evade and subvert the human innate immune system. We show that the oral pathogen, Porphyromonas gingivalis , uses endogenous lipid A 1- and 4'-phosphatase activities to modify its LPS, creating immunologically silent, non-phosphorylated lipid A. This unique lipid A provides a highly effective mechanism employed by this bacterium to evade TLR4 sensing and to resist killing by cationic antimicrobial peptides. In addition, lipid A 1-phosphatase activity is suppressed by haemin, an important nutrient in the oral cavity. Specifically, P. gingivalis grown in the presence of high haemin produces lipid A that acts as a potent TLR4 antagonist. These results suggest that haemin-dependent regulation of lipid A 1-dephosphorylation can shift P. gingivalis lipid A activity from TLR4 evasive to TLR4 suppressive, potentially altering critical interactions between this bacterium, the local microbial community and the host innate immune system.     

Heterogeneous expression of Toll-like receptor 4 and downregulation of Toll-like receptor 4 expression on human gingival fibroblasts by Porphyromonas gingivalis lipopolysaccharide.

Porphyromonas gingivalis (P. gingivalis) is implicated in the initiation and progression of periodontitis. Human gingival fibroblasts (HGFs) are the major constituent of gingival connective tissue. P. gingivalis or its components such as lipopolysaccharide (LPS) upregulate the production of various inflammatory cytokines including interleukin (IL)-1 and IL-6 in HGFs. Recently, we demonstrated that the binding of P. gingivalis LPS to Toll-like receptor 4 (TLR4) on HGFs activates various second messenger systems (Biochem. Biophys. Res. Commun. 273, 1161-1167, 2000). In the present study, we examined the level of TLR4 expression on HGFs by flow cytometric analysis (FACS), and studied the levels of IL-1 and IL-6 in the culture medium upon LPS stimulation of HGFs by enzyme-linked immunosorbent assay (ELISA). Upon stimulation by P. gingivalis LPS for 24 h, HGFs that expressed a high level of TLR4 secreted significantly higher levels of IL-1 and IL-6 than HGFs that expressed a low level of TLR4. On the other hand, after stimulation with P. gingivalis LPS for 24 h, the level of TLR4 on the surface of HGFs decreased. These results suggest that the level of TLR4 expression on HGFs reflects the extent of inflammation in the gingival tissue, and that P. gingivalis LPS downregulates TLR4 expression on HGFs. These findings may be used to control inflammatory and immune responses in periodontal disease.     

IL-8 and IDO expression by human gingival fibroblasts via TLRs

Human gingival fibroblasts (HGFs), a predominant cell type in tooth-supporting structure, are presently recognized for their active role in the innate immune response. They produce a variety of inflammatory cytokines in response to microbial components such as LPS from the key periodontal pathogen, Porphyromonas gingivalis. In this study, we demonstrated that HGFs expressed mRNA of TLRs 1, 2, 3, 4, 5, 6, and 9, but not TLRs 7, 8, and 10. Stimulation of HGFs with highly purified TLR2 ligand (P. gingivalis LPS), TLR3 ligand (poly(I:C)), TLR4 ligand (Escherichia coli LPS), and TLR5 ligand (Salmonella typhimurium flagellin) led to expression of IL-8 and IDO. A potent TLR 9 ligand, CpG oligodeoxynucleotide 2006 had no effect, although HGFs showed a detectable TLR9 mRNA expression. No significant enhancement on IL-8 or IDO expression was observed when HGFs were stimulated with various combinations of TLR ligands. Surprisingly, the TLR9 ligand CpG oligodeoxynucleotide 2006 was able to specifically inhibit poly(I:C)-induced IL-8 and IDO expression. TNF-alpha enhanced TLR ligand-induced IL-8 production in HGFs, whereas IFN-gamma enhanced TLR ligand-induced IDO expression. HGF production of IDO in response to P. gingivalis LPS, IFN-gamma, or the two in combination inhibited T cell proliferation in MLRs. The observed T cell inhibition could be reversed by addition of either 1-methyl-dl-tryptophan or l-tryptophan. Our results suggest an important role of HGFs not only in orchestrating the innate immune response, but also in dampening potentially harmful hyperactive inflammation in periodontal tissue.     

The bacterial preparation OK432 induces IL-12p70 secretion in human dendritic cells in a TLR3 dependent manner

Dendritic cells (DC) used in therapeutic cancer immunotherapy have to be able to stimulate T cells resulting in an immune response that can efficiently target the cancer cells. One of the critical hurdles has been the lack of IL-12p70 production when maturating the DC, which is rectified by using the bacterial preparation OK432 (trade name Picibanil) to mature the cells. In order to identify the mechanism behind OK432 stimulation of DC, we investigated the contribution of different TLR to examine their involvement in IL-12p70 production. By combining different inhibitors of TLR signaling, we demonstrate here that TLR3 is responsible for the IL-12p70 production of DC induced by OK432. Moreover, our data suggest that the ligand triggering IL-12p70 secretion upon TLR3 stimulation is sensitive to proteinase and partly also RNAse treatment. The fact that a bacterial compound like OK432 can activate the TLR3 pathway in human DC is a novel finding. OK432 demonstrates a critical ability to induce IL-12p70 production, which is of great relevance in DC based cancer immunotherapy.     

Limited contribution of Toll-like receptor 2 and 4 to the host response to a fungal infectious pathogen, Cryptococcus neoformans

The present study was designed to elucidate the role of Toll-like receptor (TLR) 2 and TLR4 in the host response to Cryptococcus neoformans. Both TLR2 knockout (KO) and TLR4KO mice produced interleukin-1beta (IL-1beta), IL-6, IL-12p40 and tumor necrosis factor-alpha (TNF-alpha) in sera and cleared this fungal pathogen from infected lungs at a comparable level to control littermate (LM) mice. Synthesis of these cytokines was not significantly different in the lungs of these KO mice and LM mice, although IL-1beta, IL-6 and IL-12p40 tended to be lower in TLR2KO, but not TLR4KO, mice than in controls. In addition, there was no significant reduction detected in the synthesis of IL-12 and TNF-alpha by bone marrow-derived dendritic cells from TLR2KO and TLR4KO mice upon stimulation with live yeast cells. Finally, HEK293 cells expressing either TLR2/dectin-1 or TLR4/MD2/CD14 did not respond to C. neoformans in the activation of nuclear factor kappa B (NFkappaB) detected by a luciferase assay. Our results suggest that TLR2 and TLR4 do not or only marginally contribute to the host and cellular response to this pathogen.     

T cell-independent, TLR-induced IL-12p70 production in primary human monocytes

IL-12p70 is a key cytokine for the induction of Th1 immune responses. IL-12p70 production in myeloid cells is thought to be strictly controlled by T cell help. In this work we demonstrate that primary human monocytes can produce IL-12p70 in the absence of T cell help. We show that human monocytes express TLR4 and TLR8 but lack TLR3 and TLR7 even after preincubation with type I IFN. Simultaneous stimulation of TLR4 and TLR8 induced IL-12p70 in primary human monocytes. IL-12p70 production in peripheral blood myeloid dendritic cells required combined stimulation of TLR7/8 ligands together with TLR4 or with TLR3 ligands. In the presence of T cell-derived IL-4, but not IFN-gamma, stimulation with TLR7/8 ligands was sufficient to stimulate IL-12p70 production. In monocytes, type I IFN was required but not sufficient to costimulate IL-12p70 induction by TLR8 ligation. Furthermore, TLR8 ligation inhibited LPS-induced IL-10 in monocytes, and LPS alone gained the ability to stimulate IL-12p70 in monocytes when the IL-10 receptor was blocked. Together, these results demonstrate that monocytes are licensed to synthesize IL-12p70 through type I IFN provided via the Toll/IL-1R domain-containing adaptor inducing IFN-beta pathway and the inhibition of IL-10, both provided by combined stimulation with TLR4 and TLR8 ligands, triggering a potent Th1 response before T cell help is established.     

Proinflammatory gene expression in mouse ST2 cell line in response to infection by Porphyromonas gingivalis

Porphyromonas gingivalis is a predominant periodontal pathogen, whose infection causes inflammatory responses in periodontal tissue and alveolar bone resorption. Various virulence factors of this pathogen modulate host innate immune responses. It has been reported that gingipains degrade a wide variety of host cell proteins, and fimbriae are involved in bacterial adhesion to and invasion of host cells. In the present study, we profiled ST2 stromal cell gene expression following infection with the viable P. gingivalis strain ATCC33277 as well as with its gingipain- and fimbriae-deficient mutants, using microarray technology and quantitative real-time polymerase chain reaction. Using a mouse array of about 20,000 genes, we found that infection with the wild strain elicited a significant upregulation (greater than 2-fold) of expression of about 360 genes in ST2 cells, which included the chemokines CCL2, CCL5, and CXCL10, and other proinflammatory proteins such as interleukin-6 (IL-6) and matrix metalloproteinase-13 (MMP-13). Further, infection with the gingipain-deficient mutant elicited a reduced expression of the CXCL10, IL-6 and MMP-13 genes, suggesting that gingipains play an important role in inducing the expression of those genes following P. gingivalis infection. On the other hand, the pattern of global gene expression induced by the fimbriae-deficient mutant was similar to that by the wild strain. These results suggest that P. gingivalis infection induces gene expression of a wide variety of proinflammatory proteins in stromal cells/osteoblasts, and gingipains may be involved in inducing several of the proinflammatory factors.     

Neonatal plasma polarizes TLR4-mediated cytokine responses towards low IL-12p70 and high IL-10 production via distinct factors

Human neonates are highly susceptible to infection, which may be due in part to impaired innate immune function. Neonatal Toll-like receptor (TLR) responses are biased against the generation of pro-inflammatory/Th1-polarizing cytokines, yet the underlying mechanisms are incompletely defined. Here, we demonstrate that neonatal plasma polarizes TLR4-mediated cytokine production. When exposed to cord blood plasma, mononuclear cells (MCs) produced significantly lower TLR4-mediated IL-12p70 and higher IL-10 compared to MC exposed to adult plasma. Suppression by neonatal plasma of TLR4-mediated IL-12p70 production, but not induction of TLR4-mediated IL-10 production, was maintained up to the age of 1 month. Cord blood plasma conferred a similar pattern of MC cytokine responses to TLR3 and TLR8 agonists, demonstrating activity towards both MyD88-dependent and MyD88-independent agonists. The factor causing increased TLR4-mediated IL-10 production by cord blood plasma was heat-labile, lost after protein depletion and independent of lipoprotein binding protein (LBP) or soluble CD14 (sCD14). The factor causing inhibition of TLR4-mediated IL-12p70 production by cord blood plasma was resistant to heat inactivation or protein depletion and was independent of IL-10, vitamin D and prostaglandin E2. In conclusion, human neonatal plasma contains at least two distinct factors that suppress TLR4-mediated IL-12p70 production or induce IL-10 or production. Further identification of these factors will provide insight into the ontogeny of innate immune development and might identify novel targets for the prevention and treatment of neonatal infection.     

Frailty in Old Age Is Associated with Decreased Interleukin-12/23 Production in Response to Toll-Like Receptor Ligation

Aging is associated with progressive alterations of immune functions, leading to higher susceptibility to bacterial and viral infections and reduced vaccine responses. Data concerning cytokine production in response to Toll-like receptor (TLR) ligands are highly variable in old people, reflecting the heterogeneity of the geriatric population. The aim of our study was to define the relative contribution of age and clinical status on TLR-induced interleukin (IL)-12p70 and IL-23 production as these cytokines play an important role in the protection against intracellular and extracellular pathogens, respectively. For this purpose, we recruited 100 subjects (aged 23–96 years) in the general population or hospitalized for chronic diseases. We collected information on clinical status (medical history, ongoing comorbidities, treatments and geriatric scales), biological parameters (biochemical and hematological tests, telomere length determination, cytomegalovirus serology). Whole blood samples were stimulated with a combination of TLR4 and TLR7/8 ligands. We performed univariate and stepwise backward multivariate analyses regression to define which set of clinical variables could be predictive for IL-12p70 and IL-23 production in these conditions. Our results indicated that age was not correlated with TLR-mediated IL-12p70 and IL-23 production. In contrast, poor nutritional status and frailty in subjects >75 years were associated with decreased IL-12p70 and IL-23 production. By intracytoplasmic staining, we confirmed that production of IL-12/23p40 by conventional dendritic cells (DCs) upon TLR ligation was decreased in frail patients. However, proportion of DCs and monocytes subsets, phenotypic maturation and proximal signaling events were found to be comparable in frail and healthy old subjects. These results suggest the importance of age-associated clinical parameters and not age by itself in the alteration of innate immune responses in old individuals and emphasis the importance of innate immune responses in the susceptibility of frail geriatric patients to infections.