Unleashing the potential of NOD- and Toll-like agonists as vaccine adjuvants.

Innate immunity confers an immediate nonspecific mechanism of microbial recognition through germ line-encoded pattern recognition receptors (PRRs). Of these, Toll-like receptors (TLRs) and nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) have shaped our current understanding of innate regulation of adaptive immunity. It is now recognized that PRRs are paramount in instructing an appropriate adaptive immune response. Their ligands have been the focus of adjuvant research with the goal of generating modern vaccine combinations tailored to specific pathogens. In this review we will highlight the recent findings in the field of adjuvant research with a particular focus on the potential of TLR and NLR ligands as adjuvants and their influence on adaptive immune responses.

Aspartate alleviates liver injury and regulates mRNA expressions of TLR4 and NOD signaling-related genes in weaned pigs after lipopolysaccharide challenge.

Pro-inflammatory cytokines play a critical role in many models of liver injury. In addition, aspartate (Asp) plays an important role in many biological and physiological processes including liver physiology. We hypothesized that Asp could alleviate lipopolysaccharide (LPS)-induced liver injury. Forty-eight weanling pigs were assigned to four treatments including: (1) non-challenged control; (2) LPS challenged control; (3) LPS+0.5% Asp; (4) LPS+1.0% Asp. After 20-d feeding with control (0% Asp), 0.5% or 1.0% Asp supplemented diets, pigs were injected with saline or LPS. At 4 (early phase) and 24 h (late phase) post-injection, blood and liver samples were obtained. Asp attenuated liver injury indicated by reduced serum aspartate aminotransferase activity and increased ratio of serum alanine aminotransferase and aspartate aminotransferase at 24 h, and less severe histological liver damage induced by LPS challenge at 4 or 24 h. In addition, Asp supplementation to LPS challenged pigs decreased mRNA expressions of tumor necrosis factor (TNF)-α and cyclooxygenase-2 linearly and quadratically at 4 h, and increased mRNA expressions of these pro-inflammatory mediators linearly and quadratically at 24 h. Finally, Asp decreased mRNA expression of toll-like receptor 4 (TLR4) signaling related genes (TLR4, myeloid differentiation factor 88, IL-1 receptor-associated kinase 1, TNF-α receptor-associated factor (6), nucleotide-binding oligomerization domain protein (NOD) signaling related genes (NOD1, NOD2 and receptor-interacting serine/threonine-protein kinase 2) and nuclear factor-κB p65 linearly or quadratically at 4 h. However, Asp increased mRNA expressions of these signaling molecules linearly or quadratically at 24 h. These results indicate that, at early and late phases of LPS challenge, Asp exerts opposite regulatory effects on mRNA expression of hepatic pro-inflammatory cytokines and TLR4 and NOD signalling related genes, and improves liver integrity.

Human fetal membranes generate distinct cytokine profiles in response to bacterial Toll-like receptor and nod-like receptor agonists.

Bacterial infection-associated inflammation is thought to be a major cause of preterm premature rupture of membranes. Proinflammatory cytokines, such as interleukin 1B (IL1B), can weaken fetal membranes (FM) by upregulating matrix metalloproteinases and inducing apoptosis. The mechanism by which infection leads to inflammation at the maternal-fetal interface and subsequent preterm birth is thought to involve innate immune pattern recognition receptors (PRR), such as the Toll-like receptors (TLR) and Nod-like receptors (NLR), which recognize pathogen-associated molecular patterns (PAMPs). The objective of this study was to determine the cytokine profile generated by FMs in response to the bacterial TLR and NLR agonists peptidoglycan (PDG; TLR2), lipopolysaccharide (LPS; TLR4), flagellin (TLR5), CpG ODN (TLR9), iE-DAP (Nod1), and MDP (Nod2). PDG, LPS, flagellin, iE-DAP, and MDP triggered FMs to generate an inflammatory response, but the cytokine profiles were distinct for each TLR and NLR agonist, and only IL1B and RANTES were commonly upregulated in response to all five PAMPs. CpG ODN, in contrast, had a mild stimulatory effect only on MCP-1 and primarily downregulated basal FM cytokine production. IL1B secretion induced by PDG, LPS, flagellin, iE-DAP, and MDP was associated with its processing. Furthermore, FM IL1B secretion in response to TLR2, TLR4, and TLR5 activation was caspase 1-dependent, whereas Nod1 and Nod2 induced IL1B secretion independent of caspase 1. These findings demonstrate that FMs respond to different bacterial TLR and NLR PAMPs by generating distinct inflammatory cytokine profiles through distinct mechanisms that are specific to the innate immune PRR activated.

Innate immune receptors in human airway smooth muscle cells: activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 agonists.

BACKGROUND: Pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs), recognize microbial components and trigger a host defense response. Respiratory tract infections are common causes of asthma exacerbations, suggesting a role for PRRs in this process. The present study aimed to examine the expression and function of PRRs on human airway smooth muscle cells (HASMCs). METHODS: Expression of TLR, NLR and RLR mRNA and proteins was determined using real-time RT-PCR, flow cytometry and immunocytochemistry. The functional responses to ligand stimulation were investigated in terms of cytokine and chemokine release, cell surface marker expression, proliferation and proteins regulating the contractile state. RESULTS: HASMCs expressed functional TLR2, TLR3, TLR4, TLR7 and NOD1. Stimulation with the corresponding agonists Pam3CSK4, poly(I:C), LPS, R-837 and iE-DAP, respectively, induced IL-6, IL-8 and GM-CSF release and up-regulation of ICAM-1 and HLA-DR, while poly(I:C) also affected the release of eotaxin and RANTES. The proliferative response was slightly increased by LPS. Stimulation, most prominently with poly(I:C), down-regulated myosin light chain kinase and cysteinyl leukotriene 1 receptor expression and up-regulated ß2-adrenoceptor expression. No effects were seen for agonist to TLR2/6, TLR5, TLR8, TLR9, NOD2 or RIG-I/MDA-5. CONCLUSION: Activation of TLR2, TLR3, TLR4, TLR7 and NOD1 favors a synthetic phenotype, characterized by an increased ability to release inflammatory mediators, acquire immunomodulatory properties by recruiting and interacting with other cells, and reduce the contractile state. The PRRs might therefore be of therapeutic use in the management of asthma and infection-induced disease exacerbations.

Adjuvant effect of microencapsulated NOD ligands studied in a human phagocytic cell line.

BACKGROUND: Modern subunit vaccines, which are of high purity compared with traditional vaccines, are often incapable of inducing strong immune responses as necessary to build an immunological memory. The desired level of immune response can be achieved only by codelivering immune-modulating agents along with the antigenic epitopes present in these high-purity formulations. This study aimed to explore the adjuvant effect of nucleotide oligomerization domain (NOD) receptor agonists as immunomodulators encapsulated in polymeric microparticles as carriers. METHODS: Microparticles (MP) prepared from poly[(rac-lactide)-co-glycolide] (PLGA) (Mn = 5 kDa, PD = 3.2) by the water-in-oil-in-water (w/o/w) emulsion/solvent evaporation technique were characterized in terms of size, surface morphology, payload and endotoxin content. As NOD agonists, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP; NOD 2) and γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP; NOD 1) were encapsulated. The immunomodulatory potential of these ligand-loaded MP was evaluated with a human acute monocytic leukemia cell line (THP-1X Blue-CD14 cells). RESULTS: The MP prepared had a phagocytosable size (<10 µm) with a unimodal size distribution and low endotoxin content (<0.5 EU/mL). A dose-dependent cell activation could be established for MDP-loaded microparticles. CONCLUSIONS: MP with suitable characteristics for phagocytosis can be prepared and loaded with NOD agonists. The capability of these ligand-loaded microparticles to activate monocytes suggests their broader exploration as vaccine carriers.

Modulation of pattern recognition receptor-mediated inflammation and risk of chronic diseases by dietary fatty acids.

Chronic inflammation is known to promote the development of many chronic diseases. Pattern recognition receptors (PRRs), Toll-like receptors (TLRs), and nucleotide-binding oligomerization domain proteins (NODs) mediate both infection-induced inflammation and sterile inflammation by recognizing pathogen- associated molecular patterns and endogenous molecules, respectively. PRR-mediated inflammation is an important determinant in altering the risk of many chronic diseases. Saturated fatty acids (SFAs) can activate PRRs, leading to enhanced expression of pro-inflammatory target gene products. However, n-3 polyunsaturated fatty acids (PUFAs) inhibit agonist-induced activation of PRRs. These results suggest that SFAs and n-3 PUFAs can reciprocally modulate PRR-mediated inflammation, and that PRRs and their downstream signaling components are molecular targets for dietary strategies to reduce chronic inflammation and subsequent risk of chronic diseases. This advancement in knowledge provides a new paradigm for understanding the mechanism by which different dietary fatty acids modify risk of chronic diseases including insulin resistance, atherosclerosis, and cancer.

Gram-positive bacteria enhance HIV-1 susceptibility in Langerhans cells, but not in dendritic cells, via Toll-like receptor activation.

Although numerous studies have shown a higher risk of acquiring HIV infection in the presence of other sexually transmitted diseases, the biologic mechanisms responsible for enhanced HIV acquisition are unclear. Because Langerhans cells (LCs) are suspected to be the initial HIV targets after sexual exposure, we studied whether microbial components augment HIV infection in LCs by activating Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) pattern recognition receptors. We found that TLR1/2 and TLR2/6 agonists dramatically enhanced both HIV susceptibility and replication in immature monocyte-derived LCs, whereas TLR3-5, TLR7-9, and NOD1,2 agonists did not significantly affect HIV infection. The same infection-enhancing effects were observed when LCs were incubated with other related bacterial components as well as with whole Gram(+) bacteria. In resident LCs in human skin, TLR2 agonists also significantly increased HIV susceptibility. By contrast, TLR2 agonists and related bacterial components decreased HIV susceptibility in monocyte-derived dendritic cells (DCs). We found that TLR2 activation of LCs, but not DCs, resulted in a significant down-regulation of APOBEC3G, which is a cellular restriction factor for HIV. Given these data, we hypothesize that ligation of TLR2 by Gram(+) bacterial products may underlie enhanced sexual transmission of HIV that occurs with concomitant bacterial sexually transmitted disease infections.

Expression of functional Toll-like receptors and nucleotide-binding oligomerization domain proteins in murine cementoblasts and their upregulation during cell differentiation.

BACKGROUND AND OBJECTIVE: While the primary role of cementoblasts is to synthesize the components of cementum, we have reported that immortalized murine cementoblasts (OCCM-30) express functional Toll-like receptor (TLR)-2 and -4, and these receptors are involved in the alteration of gene expression associated with cementum formation and in the upregulation of osteoclastogenesis-associated molecules, such as receptor activator of nuclear factor-kappaB (NF-kappaB) ligand. We hypothesized that cementoblasts express a wide range of pattern recognition receptors in a manner comparable to osteoblasts, which are known to express various functional TLRs and nucleotide-binding oligomerization domain (NOD) proteins. MATERIAL AND METHODS: Murine cementoblasts and pre-osteoblasts were used. The gene and protein levels of TLRs/NODs were analyzed using real-time polymerase chain reaction and flow cytometry. Interleukin-6 (IL-6) and activated NF-kappaB were measured using enzyme-linked immunosorbent assay. RESULTS: The expressions of TLR-1, -2, -4, -6 and -9, CD14, NOD-1 and -2 were detected in cementoblasts and were upregulated upon differentiation induced by ascorbic acid. Similar patterns were observed in the mouse MC3T3-E1 osteoblast cell line. Synthetic ligands, Pam3CSK4 (TLR-1/2 agonist), Pam2CGDPKHPKSF (TLR-2/6 agonist), lipid A (TLR4 agonist), CpG DNA (TLR-9 agonist), FK565 (NOD1 agonist) and muramyldipeptide (NOD2 agonist), effectively induced NF-kappaB activation in cementoblasts and/or ascorbic acid-treated cementoblasts. Furthermore, these ligands induced IL-6 production in a NF-kappaB-dependent manner in cementoblasts and/or ascorbic acid-treated cementoblasts. CONCLUSION: These results indicate that cementoblasts possess functional TLR and NOD signaling systems and have a similar capacity to osteoblasts in responding to a wide variety of pathogens.