Development of a PHBV nanoparticle as a peptide vehicle for NOD1 activation.

NOD1 is an intracellular receptor that, when activated, induces gene expression of pro-inflammatory factors promoting macrophages and neutrophils recruitment at the infection site. However, iE-DAP, the dipeptide agonist that promotes this receptor's activation, cannot permeate cell membranes. To develop a nanocarrier capable of achieving a high and prolonged activation over time, iE-DAP was encapsulated in nanoparticles (NPs) made of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The physicochemical properties, colloidal stability, encapsulation efficiency, and cellular uptake of iE-DAP-loaded PHVB NPs were analyzed. Results evidenced that physicochemical properties of iE-DAP-loaded NPs remained stable over time, and NPs were efficiently internalized into cells, a process that depends on time and concentration. Moreover, our results showed that NPs elicited a controlled cargo release in vitro, and the encapsulated agonist response was higher than its free form, suggesting the possibility of activating intracellular receptors triggering an immune response through the release of NOD1 agonist.

Nucleotide-binding oligomerization domain 1 acts in concert with the cholecystokinin receptor agonist, cerulein, to induce IL-33-dependent chronic pancreatitis.

Nucleotide-binding oligomerization domain 1 (NOD1) fulfills important host-defense functions via its responses to a variety of gut pathogens. Recently, however, we showed that in acute pancreatitis caused by administration of cholecystokinin receptor (CCKR) agonist (cerulein) NOD1 also has a role in inflammation via its responses to gut commensal organisms. In the present study, we explored the long-term outcome of such NOD1 responsiveness in a new model of chronic pancreatitis induced by repeated administration of low doses of cerulein in combination with NOD1 ligand. We found that the development of chronic pancreatitis in this model requires intact NOD1 and type I IFN signaling and that such signaling mediates a macrophage-mediated inflammatory response that supports interleukin (IL)-33 production by acinar cells. The IL-33, in turn, has a necessary role in the induction of IL-13 and TGF-ß1, factors causing the fibrotic reaction characteristic of chronic pancreatitis. Interestingly, the Th2 effects of IL-33 were attenuated by the concomitant type I IFN response since the inflammation was marked by clear increases in IFN-γ and TNF-α production but only marginal increases in IL-4 production. These studies establish chronic pancreatitis as an IL-33-dependent inflammation resulting from synergistic interactions between the NOD1 and CCKR signaling pathways.

NOD-like receptors mediated activation of eosinophils interacting with bronchial epithelial cells: a link between innate immunity and allergic asthma.

Key intracytosolic pattern recognition receptors of innate immunity against bacterial infections are nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). We elucidated the NOD1 and NOD2-mediated activation of human eosinophils, the principal effector cells for allergic inflammation, upon interacting with human bronchial epithelial BEAS-2B cells in allergic asthma. Eosinophils constitutively expressed NOD1,2 but exhibited nonsignificant responses to release chemokines upon the stimulation by NOD1 ligand γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and NOD2 ligand muramyl dipeptide (MDP). However, iE-DAP and MDP could significantly upregulate cell surface expression of CD18 and intercellular adhesion molecule (ICAM)-1 on eosinophils and ICAM-1 on BEAS-2B cells, as well as induce chemokines CCL2 and CXCL8 release in the coculture system (all P<0.05). Both eosinophils and BEAS-2B cells were the main source for CXCL8 and CCL2 release in the coculture system upon iE-DAP or MDP stimulation. Direct interaction between eosinophils and BEAS-2B cells is responsible for CCL2 release, and soluble mediators are implicated in CXCL8 release. ERK and NF-κB play regulatory roles for the expression of adhesion molecules and chemokines in coculture. Treatment with NOD1,2 ligand could induce the subepithelial fibrosis and significantly enhance the serum concentration of total IgE, chemokine CCL5 for eosinophils and T helper type 2 (Th2) cells and asthma Th2 cytokine IL-13 in bronchoalveolar lavage fluid of ovalbumin-sensitized allergic asthmatic mice (all P<0.05). This study provides further evidence of bacterial infection-mediated activation of NOD1,2 in triggering allergic asthma via the activation of eosinophils interacting with bronchial epithelial cells at inflammatory airway.

Analgesic effects of chemically synthesized NOD1 and NOD2 agonists in mice.

Intracellular nucleotide-binding oligomerization domain (NOD)-like receptors, NOD1 and NOD2, recognize the diaminopimelic acid (DAP)-containing peptide moiety and muramyldipeptide (MDP) moiety of bacterial peptidoglycan, respectively. Muramyldipeptide has been reported to exert analgesic activity to decrease the frequency of acetic acid-induced writhing movements in mice. In this study, we demonstrated the analgesic activities of NOD1 as well as NOD2 agonists. Intravenous injection of NOD2-agonistic MDP, 6-O-stearoyl-MDP (L18-MDP), and MDP-Lys (L18) exhibited analgesic activity at 10, 50, and 2.0 µg/head, respectively, in BALB/c mice. NOD1-Agonistic FK156 (D-lactyl-L-Ala-D-Glu-meso-DAP-L-Gly) and FK565 (heptanoyl-D-Glu-meso-DAP-D-Ala) were also analgesic at 50 µg/head and 1.0 µg/head, respectively. The analgesic effect of FK565 appeared from 30 min, reached maximum activity at 8 h, and continued until 24 h. The FK565 exhibited activity by various administration routes; intravenous, intraperitoneal, intramuscular, sublingual (1.0 µg/head each), subcutaneous, intragastric (oral), intragingival (10 µg/ head each) and intracerebroventricular (0.01 µg/head). The analgesic activity of FK565 was observed even in tumor necrosis factor (TNF)-α knockout, interleukin (IL)-1α/ß double knockout, and their triple knockout mice. Naloxane, a non-selective antagonist for the opioid receptor, completely inhibited the analgesic effect of FK565. These findings suggest that NOD1 and NOD2 activation induces an analgesic effect via opioid receptors in a TNF-α and IL-1α/ß independent manner.

Antitumor effects of novel immunoactive peptides, FK-156 and its synthetic derivatives.

Effects produced by intratumor or systemic application of FK-156 and its synthetic derivatives on the syngeneic P388-DBA/2 mouse system were investigated. Among 21 compounds tested, FK-156, FK-565, FR-46758, FR-48217, FR-46091 and FR-47920 substantially suppressed tumor growth when directly injected into a tumor mass and further experiments showed that FK-156, FK-565 and FR-46758 were effective even when administered subcutaneously into site remote from tumor. The mechanisms of growth inhibition are strongly suggested to be host mediated, because these three compounds have remarkably low cytotoxicity against P388 cells in vitro. A single dose of FK-565, however, markedly decreased body weight in healthy DBA/2 mice, whereas FK-156 and FR-46758 did not. These results indicate the superiority of FK-156 and FR-46758 as immunotherapeutic agents over FK-565 with respect to their safety for treatment of cancer. Although significant life-span prolongation could not be seen in the two-injection regimen of six compounds in either system, systemic multiple injections of FK-156 and FR-46758 provided a statistically significant increase in the median survival time of P388 tumor bearing mice.