Current Pharmaceutical Design - Volume 12, Issue 32, 2006

In higher vertebrates and in mammals Toll- like receptors (TLRs) represent a bridge between innate and adaptive immunity. In this respect, a number of recent studies have led to the discovery of various TLRs in mice and in men, as well as to the characterization of structure and function of these receptors. In virtue of TLR ability to trigger a cascade of inflammatory and regulatory events, they are currently investigated as specific targets for novel drugs potentially useful in the treatment of various infectious and autoimmune diseases. On these grounds, the present issue of Current Pharmaceutical Design, entitled: "Toll-like receptors and innate immunity: Potential drug targets for treatment of infectious, inflammatory, and autoimmune diseases", will point out the main aspects of biology, function and potential drug targeting of TLRs. The in silico investigation of the phylogenesis of immune receptors will be described by Panaro and associates [1], demonstrating how some receptors are relatively conserved from insects to mammals. Hoebe and associates [2] and Ishi and associates [3] will provide basic information on the structure-function of the broad spectrum of TLRs, also emphasizing novel avenues for future immunotherapy. Butchar and associates [4] will review TLR4 activation by endotoxin with special reference to the regulatory mechanisms of TLR4 signaling. Recognition of beta-glucan, a major component of the fungal cell wall, will be described by Muta [5], as an example of the phylogenesis of innate immunity. Takada and Uehara [6] will consider another bacterial component, peptidoglycan, for its capacity to enhance TLRmediated responses via the NOD pathway. In two companion papers de la Barrera and associates [7] and Lorenz [8] will describe the involvement of TLRs in infectious diseases, even including bacterial infections with special emphasis on novel therapeutic strategies. Netea and associates [9] will review the mechanisms of TLR activation by fungi and potential therapeutic approaches. Nochi and Kiyono [10] and Ishihara and associates [11] will provide information on the innate mucosal immune system and on TLR-drug targeting in gastrointestinal inflammatory diseases. Stoll and associates [12] will review the involvement of endotoxin and TLR4 in vascular inflammation and potential therapeutic targets. In three consecutive papers Amati and associates [13], Pepe and associates [14] and Pepe and associates [15] will describe the TLR-mediated activation of dendritic cells, their involvement in Leishmanmia infantum and Candida albicans infections and novel protocols of TLR drug targeting. Finally, Kumazawa and associates [16] will illustrate the effects of flavonoids as therapeutic agents able to block TLR-mediated pathways leading to production of proinflammatory cytokines. References [1] Panaro, MA, Acquafredda A, Sisto M, Lisi S, Saccia M, Mitolo V. Evolution of a "conserved" amino acid sequence: a model study of an in silico investigation of the phylogenesis of some immune receptors. Curr Pharm Design 2006; 12(32): 4091-4121. [2] Hoebe K, Jiang Z, Georgel P, Tabeta K, Janssen E, Du X, Beutler B. TLR signaling pathways: opportunities for activation and blockade in pursuit of therapy. Curr Pharm Design 2006; 12(32): 4123-4134. [3] Ishi KJ, Uematsu S, Akira S. "Toll" gates for future immunotherapy. Curr Pharm Design 2006; 12(32): 4135- 4142. [4] Butchar JP, Parsa KVL, Marsh CB, Tridandapani, S. Negative regulators of Toll-like receptor 4-mediated macrophage inflammatory response. Curr Pharm Design 2006; 12(32): 4143-4153. [5] Muta T. Molecular basis for invertebrate innate immune recognition of (1-3)-b-D-glucan as a pathogen - associated molecular pattern. Curr Pharm Design 2006; 12(32): 4155-4161. [6] Takada H, Uehara A. Enhancement of TLR-mediated innate immune responses by peptidoglycans through NOD signaling. Curr Pharm Design 2006; 12(32): 4163-4172. [7] de la Barrera S, Aleman M, Sasiain M del C. Toll-like receptors in human infectious diseases. Curr Pharm Design 2006; 12(32): 4173-4184. [8] Lorenz E. TLR2 and TLR4 expression during bacterial infections. Curr Pharm Design 2006; 12(32): 4185- 4193. [9] Netea MG, Ferwerda G, van der Graaf CAA, Van der Meer JWM, Kullberg BJ. Recognition of fungal pathogens by Toll -like receptors. Curr Pharm Design 2006; 12(32): 4195-4201. [10] Nochi T, Kiyono H. Innate immunity in the mucosal immune system. Curr Pharm Design 2006; 12(32): 4203-4213. [11] Ishihara S, Rumi MAK, Ortega-Cava C-F, Kazumori H, Kadowaki Y, Ishimura N, Kinoshita Y. Therapeutic targeting of Toll-llike receptors in gastrointestinal inflammation. Curr Pharm Design 2006; 12(32): 4215- 4228. [12] Stoll LL, Denning GM, Weintraub NL. Endotoxin, TLR4 signaling, and vascular inflammation: potential therapeutic targets in cardiovascular disease. Curr Pharm Design 2006; 12(32): 4229-4245....

In this paper we analyze a 55-amino acid (aa) sequence which is relatively well conserved in several seventransmembrane receptor families (from Insects to Mammals) and in some Viruses. This sequence, which covers the second transmembrane domain, the first extracellular loop and the third transmembrane domain, appears in its complete configuration in most of the seven-transmembrane receptor families, as well as in the protein products of some viruses. Other seven-transmembrane receptors and viruses exhibit reduced configurations of the conserved sequence, lacking either aa 31 or aa 30-31. 53-aa configurations are typically found in most chemokine receptor (CKR) subfamilies, as well as in some viral protein products. However, the CCR1, CCR3, and CCR6 subfamilies comprise a 54-aa configuration and the CKRrelated protein products, ChemR23 and RDC1, include the complete 55-aa sequence. For each CKR subfamily the ”modal sequence” of the conserved segment was constructed by selecting the most frequently occurring aa at each position. Then, pairwise alignments were made between: (i) the modal CKR sequences, and (ii) the sequence (53-aa) of the Yaba-like disease virus - 7L protein. From the alignments two consensus matrices were derived: (i) the consensus 1 matrix with reference to the whole conserved segment, and (ii) the consensus 2 matrix with reference to aa 22-29, which appear to be the most variable segment of the sequence. Based on the obtained consensus values and with reference to this specific conserved segment, the following conclusions are proposed: (1) ChemR23 and RDC1 are probably the more primitive CKR forms; (2) CCR1 and CCR3 may be grouped in a single cluster; (3) CCRs 2, 4, and 5 are closely related to each other and may be grouped in a cluster; CCR7 is likely to be evolutionarily related to this cluster; (4) CXCRs 2, 3, and 4 and CCX CKR appear to be evolutionarily related to each other and very likely derived from an CCR6-like gene; (5) CCR2/4/5 and CCR7 may have derived either from CCR1/3-like or CCR6-like genes; (6). The Yaba-like disease virus - 7L protein most likely derived, through “molecular piracy”, from a CCR8-like gene. We also discuss possible, more remote, evolutionary links between CKRs, formylpeptide receptors, and possibly the highly conserved 18S rRNA genes.

The identification of the TLRs as key sensors of microbial infection has presented a series of new targets for drug development. The TLRs are linked to the most powerful inflammatory pathways in mammals. The question arises from the start: do we wish to stimulate TLR signaling in order to eradicate specific infections and/or neoplastic diseases? Or do we wish to block TLR signaling to treat inflammatory diseases? If we accept that it would be useful to modulate TLR signaling, the next step is to identify the correct molecular target(s) for the task. Perhaps it might even be possible to exercise selectivity, modulating some aspects of TLR signaling and not others. Classical and reverse genetic analyses offer insight into the possibilities that exist, and point to specific checkpoints within signaling pathways at which modulation might normally be imposed.

Toll-like receptors (TLRs) are evolutionary conserved transmembrane proteins that recognize a unique pattern of molecules derived from pathogens or damaged cells, triggering robust but defined innate immune responses. TLRmediated innate and/or adaptive immune responses play an important role in a variety of diseases including infectious diseases, sepsis, autoimmune diseases, allergy, and atherosclerosis. Each TLR displays a differential expression pattern, intracellular localization and signaling pathway, resulting in distinct immune responses. A variety of new TLR ligands including agonists (e.g. urinary Tamm-Horsfall glycoprotein as a TLR4 ligand, siRNA as TLR3 or 7 ligand, Plasmodium falciparum Hemozoin as a TLR9 ligand, Profilin-like protein in Toxoplasma gondii as a TLR11 ligand) and antagonists (G-rich oligodeoxynucleotides as antagonist for TLR9) have been identified, and some of other TLR ligands are already under clinical trials. The manipulation or intervention of TLR-mediated immune responses is a possible multiple ‘Toll’ gate for future developments of immunotherapies.

Activation of macrophages through TLR4, the receptor for the bacterial endotoxin LPS, results in a potent inflammatory response aimed at eliminating the invading pathogen. Excessive production of inflammatory mediators is harmful to host tissue and in extreme cases can result in fatal outcomes. This inflammatory response is, therefore, tightly regulated by negative regulatory mechanisms that act to maintain homeostasis. This review will summarize recent advances in our current understanding of molecular mechanisms that regulate macrophage TLR4 signaling.

Innate immunity responds to various pathogen-associated molecular patterns (PAMPs) to evaluate the biological nature of foreign materials by using limited numbers of receptors. Analyses of interactions between PAMPs and its receptors are essential to understand the molecular basis regarding how we discriminate self and non-self materials. Upon infection of horseshoe crabs, an arthropod species, rapid hemolymph coagulation is induced to engulf invading microorganisms by a cascade-type reaction. The reaction is very sensitive to lipopolysaccharide and (1→3)-β-D-glucans on Gramnegative bacteria and fungi, respectively, and hence is utilized as assay reagents that detect and quantitate these PAMPs with a name of “limulus test.” In this mini-review, recognition of (1→3)-β-D-glucans by a unique serine protease zymogen factor G of horseshoe crab is described. Molecular dissection and detailed kinetic analyses have revealed that multivalent binding to polymers of a simple target structure is one of the principles that allows stable and specific recognition of PAMPs by pattern recognition receptors in innate immunity.

Toll-like receptors (TLRs) recognize common motifs, pathogen-associated molecular patterns (PAMPs), in microorganisms. Bacterial PAMPs are mainly distributed on cell-surfaces. Peptidoglycans (PGNs) are ubiquitous constituents of bacterial cell walls. Muramyldipeptide (MDP; N-acetylmuramyl-L-alanyl-D-isoglutamine) is a common and key structure of PGNs and exhibits most the of bioactivities of PGNs. Recently, the intracellular receptor for MDP was revealed to be NOD2. Another bioactive moiety of PGNs, diaminopimelic acid (DAP) containing desmuramylpeptides (DMPs), senses another intracellular receptor, NOD1. MDP-primed mice exhibited hyper-responses to endotoxin and other bacterial components, which sense Toll-like receptors (TLRs), although MDP itself does not exhibit apparent activity in mice. On the other hand, DMPs exhibited definite activity in mice, and the most powerful DMP, FK565, exhibited stronger priming activity than MDP. In human monocytic cells, both MDP and DMPs exhibited definite activities; marked synergistic interleukin (IL)-8 secretion was induced by DMPs and MDP in combination with synthetic TLR agonists, and suppression of the mRNA expressions of NOD1 and NOD2, respectively, by RNA interference specifically inhibited synergistic IL-8 secretion. In human dendritic cells (DCs), synergistic T helper type 1 responses are induced by combined stimulations of synthetic NOD and TLR agonists. Considering these findings altogether, in host-bacteria interactions, host cells should recognize bacteria via both TLRs and NODs, which might induce synergistic innate and adaptive immune responses.

Toll-like receptors (TLRs) have emerged as critical players in immunity. They are capable of sensing organisms ranging from protozoa to bacteria, fungi or viruses upon detection of the pathogen as well as recognizing endogenous ligands, and triggering transduction pathways. Following activation of the innate immune system, strong inflammatory signals are generated inducing inflammation and activation of the adaptive immune response. However, the deregulation of TLRs signaling pathways may be conducive to the pathogenesis of many infectious diseases. Therefore, innate and adaptive immunity are not simply sequential and complementary mechanisms of resistance to pathogen, they regulate each other through cellular contacts and the secretion of soluble mediators. Herein, we summarize recent findings on TLRs signaling in infectious diseases and how pathogens have developed strategies to evade these pathways. In this context, a potential modulation of the innate immune response could have therapeutic benefit through the development of new drugs as well as vaccination strategies to be employed in infectious diseases.

The family of the toll-like receptors comprises a minimum of 10 members identified in humans so far. These transmembrane receptors act as important signaling intermediates between the host and the invading pathogens. The following review describes the complexities encountered by researchers studying toll-like receptor (TLR) expression changes during bacterial infections. Mutations in some of the TLRs, most prominently TLR4 and TLR2, have been associated with increased susceptibility to infectious diseases. While it is tempting to correct the phenotypic effect of such mutations, in vitro and in vivo research has shown that TLR activity and function comprises a complex regulatory network. Heterodimer formation, synergy, and cross-tolerance have previously been described. More recently, interdependence of TLR2 and TLR4 expression has been identified. In addition, TLR expression follows a specific timeline that may be dependent on the invading pathogen. Lastly, mutations in invading pathogens have been shown to alter the expression profile of TLR2 and TLR4, indicating that therapies against bacterial pathogens will have to target multiple TLRs. Despite the complexities involved in TLR function, the significant progress made in our understanding of the role these proteins play in human diseases also indicates their potential value as therapeutic agents.

Toll-like receptors (TLRs) have been identified as a major class of pattern-recognition receptors. Recognition of pathogen-associated molecular patterns (PAMPs) by TLRs, either alone or in heterodimerization with other TLR or non-TLR receptors, induces signals responsible for the activation of innate immune response. Recent studies have demonstrated a crucial involvement of TLRs in the recognition of fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. By studying fungal infection in knock-out mice deficient in either TLRs or TLRassociated adaptor molecules, it appeared that specific TLRs such as TLR2 and TLR4 play differential roles in the activation of the various arms of the innate immune response. Recent data also suggest that TLRs offer escape mechanisms to certain pathogenic microorganisms, especially through TLR2-driven induction of antiinflamatory cytokines. These recent developments provide crucial information for understanding the mechanisms of fungal recognition by cells of the immune system, and provide hope for designing new therapeutical approaches to fungal infections.

The mucosal immune system is equipped with unique innate and acquired defense mechanisms which provide a first line of protection against ingested and inhaled infectious agents. Peyer's patches (PPs) and nasopharynx-associated lymphoid tissue (NALT) have been shown to be important inductive sites for the initiation of the acquired phase of antigen- specific immune responses. In addition, the mucosal innate immune system acts as both a physical and an immunological boundary, playing a key role in the sensing and eliminating of pathogens and in the creating of symbiosis. The mucus layer covering the mucosal epithelium acts as a first physical and biochemical barrier. An additional layer of physical protection against microorganisms is provided by a tightly interlaced cell-to-cell network of epithelial cells and intraepithelial lymphocytes. Various antimicrobial peptides produced by the epithelium and secreted into the mucosal lumen can directly kill the invading pathogenic bacteria. Finally, Toll-like receptors (TLRs) associated with the mucosal compartment have been shown to recognize the pathogen-associated molecular patterns (PAMPs) of a variety of pathogenic and commensal microorganisms. Therefore, a greater understanding of the immunological progression from mucosal innate to acquired immune systems should facilitate the development of new generation of mucosal vaccines to prevent and control infectious diseases.

Toll-like receptors (TLRs) are sensors of microbial products that initiate host defense responses in multicellular organisms. They are mainly linked to innate immunity and bridging to adaptive immunity, signaling through different TLRs responsible for a wide range of biological responses. The intracellular signaling pathways through Toll/interleukin- 1 receptor (IL-1R) domains result in recruitment of the cytoplasmic adaptor molecules, with subsequent activation of a signaling cascade leading to nuclear factor-κ B (NF-κB). TLR-signaling induces host inflammatory response and the inflammation becomes more severe in the absence of several extra and intra cellular negative regulators of TLR-signaling. In the intestine, TLR-dependent activation of NF-κB plays a vital role in maintaining epithelial homeostasis as well as regulating infections and inflammation, while dysregulation of TLR-signaling is associated with the pathogenesis of inflammatory bowel diseases (IBD). Recent findings regarding innate immunity-mediated regulation of intestinal pathophysiology prove that development of new drugs targeting TLRs including antagonists of TLR-signaling and agonists of their negative regulators has a potential impact on therapeutic strategies for intestinal inflammatory diseases.

Cardiovascular disease ranks among the leading causes of morbidity and mortality in adult populations in the Western world. Significant progress in understanding the etiology of cardiovascular disease has come from recent recognition that chronic inflammation plays a key role in its development. The principal mediators of this inflammatory response, and the mechanisms by which they work, however, are incompletely understood. Moreover, the complex nature of the inflammatory response poses significant challenges to the development of effective and targeted treatments. Potentially promising targets to reduce inflammation in atherosclerosis include Toll-like receptor (TLR) pathways and anti-inflammatory factors that modulate TLR signaling. In this review, we outline studies that provide insight into the links between cardiovascular disease and inflammation, focusing on innate immunity and endotoxin/TLR4 signaling. We also discuss the contribution of specific host immune/inflammatory responses to atherogenesis, and describe cellular signaling pathways (lipopolysaccharide-binding protein [LBP], CD14, MD-2, TLR4, MyD88, and NF-κB, among others) that play key roles in innate immune signaling. Finally, we discuss the therapeutic potential of modulating these cellular signaling pathways as future strategies for the prevention and treatment of cardiovascular disease, including such approaches as specific targeting of the TLR4 signaling pathway, antibiotic therapy, drug classes with broad anti-inflammatory activity (statins, thiazolidinediones), and the potential of vaccine development. Because of the complexity of the links between low-level chronic infections, inflammation, and atherosclerosis, treatment and prevention of cardiovascular disease will likely require an integrated approach that utilizes a combination of these strategies to target the underlying inflammatory processes.

Dendritic cells (DCs) represent a bridge between innate and adaptive immunity, being the maturation process dependent on the binding of pathogen-associated molecular patterns (PAMPs) to Toll-Like Receptors (TLRs) expressed on their surface. TLRs associated to adaptor proteins, following binding to PAMPs, are able to skew specific immune responses towards the T helper (h)1- or the Th2-type according to the antigenic stimulation involved. Of note, other receptors different from TLRs are expressed on DCs which are also able to recognize PAMPs. Among them, one should mention the DC-specific ICAM-3-grabbing nonintegrin, the mannose receptor, Dectin-1 (the major β-glucan receptor) and NOD2. Finally, the possibility to interfere therapeutically with the TLR-dependent and -independent signaling pathways in DCs is reviewed. According to current literature, DC activation, their antigen uptake capacity and migration can be enhanced with different experimental procedures whose use in humans is still under evaluation. However, just recently a probiotic cocktail VSL3, successfully used in patients with pouchitis, seems to act on DCs, promoting abundant release of Interleukin- 10 in the gut. These novel therapeutic strategies based on the modulation of the signaling pathways in DCs seem to be encouraging for the treatment of inflammatory and autoimmune diseases.

Dendritic cells (DCs), instructed by the priming signals from microbial factors, can produce interleukin (IL)- 12p70 and promote T helper (Th)1 proliferation and interferon (IFN)-γ production. This event seems to be critical for the containment of infections caused by intracellular pathogens, even including Leishmania infection. In the present in vitro study we have investigated: 1) phagocytic capacities and IL-12 production by human monocyte-derived DCs and macrophages (Ms), infected with Leishmania infantum promastigotes; 2) IFN-γ production by human CD4+ T cells coincubated with DCs or macrophages pulsed with live promastigotes. Monocyte-derived myeloid DCs and Ms from healthy donors were infected with live metacyclic Leishmania infantum (MON-1) promastigotes, previously opsonized with 5% autologous serum, at 1:4 cell/parasite ratio. Percentage and index of phagocytosis were calculated after 2, 24 and 48 h of incubation. IL-12 production was evaluated by an ELISA in supernatants from 48 h Leishmania-infected or lipopolysaccharides (LPS)-stimulated DCs and Ms, also in the presence of phytohemagglutinin-activated or inactivated CD4+ T cells. For IFN-γ production, CD4+ T cells were repeatedly stimulated with DCs or Ms, pulsed with live Leishmania promastigotes or activated with LPS. The number of IFN-γ-secreting cells was evaluated by an ELISpot assay. Results showed that Ms have a higher phagocytic capacity towards L. infantum promastigotes than DCs. Moreover, unlike Ms, Leishmania-infected DCs were able to release IL-12p70; this production significantly increased in the presence of activated CD4+ T cells. Finally, DCs pulsed with live parasites and added to autologous CD4+ T cells induced a higher number of IFN- γ-secreting cells than Ms, thus indicating their ability to polarize Th cells toward the Th1 subset. These data indicate that DCs are able to promote protective Th1 immune responses in our experimental model of Leishmania infantum infection, thus representing the grounds for initiating immunoterapeutic and vaccinal strategies.

Nowadays, infections with Candida albicans (C.a.) are very frequent, mostly in the so-called immunocompromised host. Therefore, research has been focused on the types of immune response elicited by C.a., with the aim to develop novel therapeutic strategies. Neutrophils and macrophages (M) are deeply involved in the host defense against C.a., and also dendritic cells (DCs) seem to be very active in the host protection. In particular, DCs display an array of surface receptors able to interact with fungal components, even including Toll-like receptors. Here, we will illustrate the in vitro immune response of human monocyte-derived DCs infected with C.a. . In this test system, DCs exert phagocytic and killing activities with a magnitude similar to that of M. Moreover, in the presence of autologous CD4+ cells, DCs produce T-helper (h) 1 type cytokines. This Th1 polarizing activity is mediated by interleukin- 12 released by infected CDs in the presence of CD4+ cells. Taken together, these data suggest a protective role played by DCs in the course of C.a. infection and the possibility to develop new strategies of immune intervention.

Flavonoids have beneficial activities which modulate oxidative stress, allergy, tumor growth and viral infection, and which stimulate apoptosis of tumor cells. In addition to these activities, dietary flavonoids are able to regulate acute and chronic inflammatory responses. Here we describe new aspects of regulatory mechanisms by which flavonoids suppress production of tumor necrosis factor-α (TNF-α) by macrophages, microglial cells and mast cells stimulated with lipopolysaccharide (LPS) and others via toll-like receptors (TLRs), and TNF-α-mediated acute and chronic inflammatory responses. Treatment with flavonoids such as luteolin, apigenin, quercetin, genistein, (-)-epigallocatechin gallate, and anthocyanidin resulted in significant downregulation of LPS-elicited TNF-α and nitric oxide (NO) production and diminished lethal shock. In chronic diseases, pathogenesis of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis which is triggered by TNF-α, was improved by the oral administration of flavonoids after the onset of CIA. Here, we discuss that inhibitory effects of flavonoids on acute and chronic inflammation are due to regulation of signaling pathways, including the nuclear factor κB (NF-κB) activation and mitogen-activated protein (MAP) kinase family phosphorylation. FcεRI expression by NF-κB activation was also reduced by flavonoids; while accumulation of lipid rafts, which is the critical step for signaling, was blocked by flavonoids. The intake of dietary flavonoids reduces acute and chronic inflammation due to blocking receptor accumulation and signaling cascades, and would assist individuals at highrisk from life-style related diseases.