A synthetic Nod2 agonist, muramyldipeptide (MDP), and two Nod1 agonists, FK565 and FK156, mimic the bacterial peptidoglycan moiety and are powerful adjuvants that induce cell-mediated immunity, especially delayed-type hypersensitivity. In this study, we used human dendritic cell ( Freund's complete adjuvant (10), which contains killed mycobacterial cells, has been widely used as a powerful adjuvant to induce cell-mediated immunity, represented by delayed-type hypersensitivity, as well as to enhance humoral immunity against test antigens in laboratory animals. A series of studies on the mycobacterial component responsible for the unique adjuvant activity of Freund's complete adjuvant revealed that Wax D is the active entity, being composed of peptidoglycan (PGN), arabinogalactan, and mycolic acid, and thereafter the PGN moieties of various bacteria were revealed to also be active in this respect (37). In the mid-1970s, the minimal essential structure of PGN for adjuvant activity was demonstrated to be muramyldipeptide (MDP; N-acetylmuramyl-Lalanyl-D-isoglutamine) by use of a chemically synthesized compound (8,24). MDP reproduced various bioactivities of PGN (39,40), although the activities of MDP were generally weaker than those of PGN and MDP was scarcely active in some experiments. PGN activates macrophages via Toll-like receptor 2 (TLR2) (35,42,51), whereas MDP lacks TLR2-agonistic activity (41,46,49,51).Fleck et al. (9) reported that desmuramylpeptide (DMP)containing meso-diaminopimelic acid (meso-DAP) was also active as an adjuvant to induce cell-mediated immunity. meso-DAP-type PGN is found in most gram-negative bacteria and in some gram-positive bacteria, including mycobacteria, while most gram-positive bacteria such as Staphylococcus and Streptococcus strains possess L-lysine (Lys)-type PGN (34). In their report, Fleck et al. (9) suggested by mistake that Lys-type DMPs were similarly active to meso-DAP-type DMPs in this respect. Thereafter, French and Japanese investigators chemically synthesized adjuvant-active meso-DAP-type DMPs (1).In the course of the study, the Fujisawa Pharmaceutical Company generated a DMP, D-lactyl-L-alanyl-␥-D-glutamyl-meso-DAP-glycine, by chemically mimicking a counterpart purified from fermentation broths of Streptomyces strains, and this DMP was designated FK156; the company then synthesized various derivatives of FK156, among which the leading compound was FK565, or heptanoyl-D-glutamyl-meso-DAP-␥-Dalanine (13). Recently, an intracellular molecule carrying nucleotidebinding oligomerization domain 2 (Nod2) was revealed to be a receptor for MDP (12,17). Thereafter, another Nod family molecule, Nod1, was demonstrated to recognize a PGN motif containing meso-DAP (7, 11). We found that FK156 and FK565 were Nod1 agonists similar to ␥-D-glutamyl-meso-DAP (45), which is the minimal active structure of a Nod1 agonist (7,22).

The cytokine‐inducing activities of fungal polysaccharides were examined in human monocytes in culture, with special reference to CD14 and Toll‐like receptors (TLRs). Tumor necrosis factor alpha (TNF‐α) production by monocytes was markedly induced in a dose‐dependent manner upon stimulation with cell walls from Candida albicans and mannan from Saccharomyces cerevisiae and C. albicans, although relatively high concentrations (10 to 100 μg/ml) of stimulants were required for activation as compared with the reference lipopolysaccharide (LPS) (1 to 10 ng/ml). The yeast form C. albicans and its mannan and cell wall fractions exhibited higher TNF‐α production than respective preparations from the hyphal form. Only slight TNF‐α production was induced by the S. cerevisiae glucan. The TNF‐α production triggered by reference LPS and purified fungal mannans required the presence of LPS‐binding protein (LBP), and these responses were inhibited by anti‐CD14 and anti‐TLR4 antibodies, but not by anti‐TLR2 antibody. In contrast to the activity of LPS, the activity of purified S. cerevisiae mannan was not inhibited by polymyxin B. These findings suggested that the mannan‐LBP complex is recognized by CD14 on monocytes and that signaling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by LPS.

The function of interleukin 15 (IL-15) in unmethylated CpG oligodeoxynucleotide (CpG)-induced immune responses remains unknown. Here, in response to CpG, both wild-type and natural killer cell-depleted mice produced IL-12 and became resistant to a lethal dose of Listeria monocytogenes. In contrast, CpG-treated IL-15-deficient mice produced little IL-12 and succumbed to L. monocytogenes. CpG-stimulated conventional dendritic cells (cDCs) were the main producers of both IL-15 and IL-12, but cDCs did not produce IL-12 in the absence of plasmacytoid DCs (pDCs). The cDC-derived IL-15 induced CD40 expression by cDCs. Interaction between CD40 on cDCs and CD40 ligand on pDCs led to IL-12 production by cDCs. Thus, IL-15-dependent crosstalk between cDCs and pDCs is essential for CpG-induced immune activation.

Periodontitis is a localized infectious disease caused by periodontopathic bacteria, such as Porphyromonas gingivalis. Recently, it has been suggested that bacterial infections may contribute to the onset and the progression of Alzheimer’s disease (AD). However, we do not have any evidence about a causative relationship between periodontitis and AD. In this study, we investigated by using a transgenic mouse model of AD whether periodontitis evoked by P. gingivalis modulates the pathological features of AD. Cognitive function was significantly impaired in periodontitis-induced APP-Tg mice, compared to that in control APP-Tg mice. Levels of Amiloid β (Aβ) deposition, Aβ40, and Aβ42 in both the hippocampus and cortex were higher in inoculated APP-Tg mice than in control APP-Tg mice. Furthermore, levels of IL-1β and TNF-α in the brain were higher in inoculated mice than in control mice. The levels of LPS were increased in the serum and brain of P. gingivalis-inoculated mice. P. gingivalis LPS-induced production of Aβ40 and Aβ42 in neural cell cultures and strongly enhanced TNF-α and IL-1β production in a culture of microglial cells primed with Aβ. Periodontitis evoked by P. gingivalis may exacerbate brain Aβ deposition, leading to enhanced cognitive impairments, by a mechanism that involves triggering brain inflammation.

Cysteine proteinases (gingipains) elaborated from Porphyromonas gingivalis exhibit enzymatic activities against a broad range of host proteins and are considered key virulence factors in the onset and development of adult periodontitis and host defense evasion. In this study, we examined the ability of arginine-specific gingipains (high molecular mass Arg-specific gingipain (HRGP) and Arg-specific gingipain 2) and lysine-specific gingipain (KGP) to cleave monocyte CD14, the main receptor for bacterial cell surface components such as LPS. Binding of anti-CD14 mAb MY4 to human monocytes was almost completely abolished by 0.3 M HRGP and KGP treatments for 15 min, and 1 M RGP2 for 30 min. In contrast, the expressions of Toll-like receptor 4, and CD18, CD54, CD59, and HLA-A, -B, -C on monocytes were slightly increased and decreased, respectively, by 0.3 M HRGP and KGP. This down-regulation resulted from direct proteolysis, because 1) gingipains eliminated MY4 binding even to fixed monocytes, and 2) CD14 fragments were detected in the extracellular medium by immunoblot analysis. Human rCD14 was degraded by all three gingipains, which confirmed that CD14 was a substrate for gingipains. TNF-␣ production by monocytes after HRGP and KGP treatments was decreased at 1 ng/ml, but not at 20 g/ml LPS, indicating that gingipains inhibited a CD14-dependent cell activation. These results suggest that gingipains preferentially cleave monocyte CD14, resulting in attenuation of the cellular recognition of bacteria, and as a consequence sustain chronic inflammation. The Journal of Immunology, 2000, 165: 411-418.A n oral chronic inflammation, i.e., periodontal disease, is one of the major diseases afflicting mankind and caused by a bacterial infection leading to gingival inflammation, destruction of periodontal tissues, loss of alveolar bone, and culminating in tooth loss (1, 2). Porphyromonas gingivalis has been implicated as a principal bacterium not only in adult periodontitis, but also in rapidly progressive periodontitis (1, 2). P. gingivalis possesses a number of putative virulence factors such as LPS, fimbriae, toxic products of metabolism, and proteinases, all of which enable this anaerobe to cause the disease either directly or indirectly by activation of host cells to release inflammatory mediators (3).It is now clear that all of the trypsin-like proteinase activity of P. gingivalis is due to two cysteine proteinases (4). Two types of cysteine proteinases specific for Arg-X (50 and 95 kDa) (5) and Lys-X (105 kDa) bonds have been purified and characterized and are referred to as arginine-specific gingipain (RGP) 3 (3) and lysine-specific gingipain (KGP), respectively (6). The 95-kDa high molecular mass RGP (HRGP or HRgpA) differs from the 50-kDa RGP (RGP2 or RgpB) in that the protein noncovalently complexes with the hemagglutinin/adhesin domain in the same manner as KGP. It has been shown that gingipains play a critical role in the onset of inflammation through enhancement of vascular permeability by activation of the kallikr...