Identification and immunological evaluation of novel TLR2 agonists through structure optimization of Pam3CSK4

Toll-like receptor 2 (TLR2) is a bridge between innate immunity and adaptive immunity. TLR2 agonists have been exploited as potential vaccine adjuvants and antitumor agents. However, no TLR2 agonists have been approved by FDA up to now. To discover drug-like TLR2 selective agonists, a novel series of Pam₃CSK₄ derivatives were designed based on the crystal structure of hTLR2-hTLR1-Pam₃CSK₄ complex, synthesized and evaluated for their immune-stimulatory activities. Among them, 35c was identified as a murine-specific TLR2 agonist, while 35f was a human-specific TLR2 agonist. Besides, 35d (human and murine TLR2 agonist) showed TLR2 agonistic activity comparable to Pam₃CSK₄, which included: elevated IL-6 expression level (EC₅₀ = 83.08 ± 5.94 nM), up-regulated TNF-α and IL-6 mRNA expression and promoted maturation of DCs through activating the NF-κB signaling pathway. TLRs antibodies test showed that 35a and 35d were TLR2/1 agonists, while 35f was a TLR2/6 agonist.     

A Network of Hydrogen Bonds on the Surface of TLR2 Controls Ligand Positioning and Cell Signaling

TLR2 is a pattern recognition receptor that functions in association with TLR1 or TLR6 to mediate innate immune responses to a variety of conserved microbial products. In the present study, the ectodomain of TLR2 was extensively mutated, and the mutants were assessed for their ability to bind and to mediate cellular responses to triacylated lipopeptide Pam₃CSK₄. This analysis provides evidence that the recently published crystal structure of the TLR2-TLR1-Pam₃CSK₄ complex represents a functional signal-inducing complex. Furthermore, we report that extended H-bond networks on the surface of TLR2 are critical for signaling in response to Pam₃CSK₄ and to other di- and tri-acylated TLR2-TLR6 and TLR2-TLR1 ligands. Based on this finding, we suggest a dynamic model for TLR2-mediated recognition of these ligands in which TLR2 fluctuates between a conformation that is more suitable for binding of the fatty acyl moieties of the ligands and a conformation that favors, via a specific orientation of the ligand head group, formation of a signal-inducing ternary complex.     

TLR2 stimulates ABCA1 expression via PKC-η and PLD2 pathway

ATP-binding cassette transporter A1 (ABCA1) is a membrane-bound protein that regulates cardiovascular disease including atherosclerosis by the efflux of excess cholesterol from cells and by suppression of inflammation. Using a mouse macrophage cell line Raw264.7, we studied the importance of toll-like receptor 2 (TLR2) on ABCA1 expression and the signaling pathway responsible for TLR2-mediated ABCA1 expression. Interestingly, our data demonstrated that treatment of macrophages with TLR2 agonist Pam₃CSK₄ significantly increased ABCA1 mRNA and protein levels. We found that ABCA1 induction is myeloid differentiation primary response gene 88 (MyD88)-dependent as well as TLR2-dependent. ABCA1 induction upon Pam₃CSK₄ is controlled by protein kinase C-η (PKC-η) and phospholipase D2 (PLD2). Furthermore, direct treatment of dioctanoyl phosphatidic acid (diC₈PA) into cells also induced ABCA1 mRNA and protein indicating that PLD2-mediated PA involve in the TLR2-stimulated ABCA1 expression. Cumulatively, these results demonstrate for the first time that activation of PKC-η and PLD2 signaling pathway is an important mechanism for regulation of TLR2-induced ABCA1 expression.     

Crosstalk between Smad2/3 and specific isoforms of ERK in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes

This study investigated the roles of ERK1 and ERK2 in transforming growth factor‐β1 (TGF‐β1)‐induced tissue inhibitor of metalloproteinases‐3 (TIMP‐3) expression in rat chondrocytes, and the specific roles of ERK1 and ERK2 in crosstalk with Smad2/3 were investigated to demonstrate the molecular mechanism of ERK1/2 regulation of TGF‐β1 signalling. To examine the interaction of specific isoforms of ERK and the Smad2/3 signalling pathway, chondrocytes were infected with LV expressing either ERK1 or ERK2 siRNA and stimulated with or without TGF‐β1. At indicated time‐points, TIMP‐3 expression was determined by real‐time PCR and Western blotting; p‐Smad3, nuclear p‐Smad3, Smad2/3, p‐ERK1/2 and ERK1/2 levels were assessed. And then, aggrecan, type II collagen and the intensity of matrix were examined. TGF‐β1‐induced TIMP‐3 expression was significantly inhibited by ERK1 knock‐down, and the decrease in TIMP‐3 expression was accompanied by a reduction of p‐Smad3 in ERK1 knock‐down cells. Knock‐down of ERK2 had no effect on neither TGF‐β1‐induced TIMP‐3 expression nor the quantity of p‐Smad3. Moreover, aggrecan, type II collagen expression and the intensity of matrix were significantly suppressed by ERK1 knock‐down instead of ERK2 knock‐down. Taken together, ERK1 and ERK2 have different roles in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes. ERK1 instead of ERK2 can regulate TGF‐β/Smad signalling, which may be the mechanism through which ERK1 regulates TGF‐β1‐induced TIMP‐3 expression.     

Systemic stimulation of TLR2 impairs neonatal mouse brain development

Background

Inflammation is associated with perinatal brain injury but the underlying mechanisms are not completely characterized. Stimulation of Toll-like receptors (TLRs) through specific agonists induces inflammatory responses that trigger both innate and adaptive immune responses. The impact of engagement of TLR2 signaling pathways on the neonatal brain is still unclear. The aim of this study was to investigate the potential effect of a TLR2 agonist on neonatal brain development.

Methodology/Principal Findings

Mice were injected intraperitoneally (i.p.) once a day from postnatal day (PND) 3 to PND11 with endotoxin-free saline, a TLR2 agonist Pam₃CSK₄ (5 mg/kg) or Lipopolysaccharide (LPS, 0.3 mg/kg). Pups were sacrificed at PND12 or PND53 and brain, spleen and liver were collected and weighed. Brain sections were stained for brain injury markers. Long-term effects on memory function were assessed using the Trace Fear Conditioning test at PND50. After 9 days of Pam₃CSK₄ administration, we found a decreased volume of cerebral gray matter, white matter in the forebrain and cerebellar molecular layer that was accompanied by an increase in spleen and liver weight at PND12. Such effects were not observed in Pam3CSK4-treated TLR 2-deficient mice. Pam3CSK4-treated mice also displayed decreased hippocampus neuronal density, and increased cerebral microglia density, while there was no effect on caspase-3 or general cell proliferation at PND12. Significantly elevated levels of IL-1β, IL-6, KC, and MCP-1 were detected after the first Pam3CSK4 injection in brain homogenates of PND3 mice. Pam₃CSK₄ administration did not affect long-term memory function nor the volume of gray or white matter.

Conclusions/Significance

Repeated systemic exposure to the TLR2 agonist Pam₃CSK₄ can have a short-term negative impact on the neonatal mouse brain.     

TLR4‐dependent induction of vascular adhesion molecule‐1 in rheumatoid arthritis synovial fibroblasts: Roles of cytosolic phospholipase A2α/cyclooxygenase‐2

Lipopolysaccharide (LPS)/Toll‐like receptor 4 (TLR4)‐mediated signaling pathways have caught the attention of strategies designed for rheumatoid arthritis (RA). In this study, we identified that cPLA₂α acted as a modulator of LPS‐induced VCAM‐1 expression and THP‐1 (human acute monocytic leukemia cell line) adherence. Treatment of RA synovial fibroblasts (RASFs) with LPS, a TLR4 agonist, promoted the VCAM‐1 expression and THP‐1 adherence which were decreased by pretreatment with a selective cytosolic phospholipase A₂ (cPLA₂) inhibitor (AACOCF₃), implying the involvement of cPLA₂α in these responses. This notion was further confirmed by knockdown of cPLA₂α expression by transfection with cPLA₂α small interfering RNA (siRNA) leading to a decrease in VCAM‐1 expression and THP‐1 adherence induced by LPS. Subsequently, the LPS‐stimulated cPLA₂α phosphorylation was attenuated by pretreatment with a MEK1/2 inhibitor (U0126), suggesting that LPS‐stimulated cPLA₂α phosphorylation and activity are mediated through an ERK‐dependent mechanism. Moreover, COX‐2‐derived PGE₂ production appeared to involve in LPS‐induced VCAM‐1 expression which was attenuated by pretreatment with selective COX‐2 inhibitors (NS‐398 and celecoxib), transfection with COX‐2 siRNA, or PGE₂ receptor antagonists. In addition, pretreatment with ecosapentaenoic acid (EPA), a substrate competitor of arachidonic acid (AA), also blocked LPS‐induced VCAM‐1 mRNA and protein expression, and THP‐1 adherence. Collectively, these results suggest that LPS‐induced VCAM‐1 expression and adhesion of THP‐1 cells are mediated through the TLR4/ERK/cPLA₂α phosphorylation and COX‐2 expression/PGE₂ synthesis in RASFs. J. Cell. Physiol. 223: 480–491, 2010. © 2010 Wiley‐Liss, Inc.     

Evaluation of an Intranasal Virosomal Vaccine against Respiratory Syncytial Virus in Mice: Effect of TLR2 and NOD2 Ligands on Induction of Systemic and Mucosal Immune Responses

Introduction

RSV infection remains a serious threat to newborns and the elderly. Currently, there is no vaccine available to prevent RSV infection. A mucosal RSV vaccine would be attractive as it could induce mucosal as well as systemic antibodies, capable of protecting both the upper and lower respiratory tract. Previously, we reported on a virosomal RSV vaccine for intramuscular injection with intrinsic adjuvant properties mediated by an incorporated lipophilic Toll-like receptor 2 (TLR2) ligand. However, it has not been investigated whether this virosomal RSV vaccine candidate would be suitable for use in mucosal immunization strategies and if additional incorporation of other innate receptor ligands, like NOD2-ligand, could further enhance the immunogenicity and protective efficacy of the vaccine.

Objective

To explore if intranasal (IN) immunization with a virosomal RSV vaccine, supplemented with TLR2 and/or NOD2-ligands, is an effective strategy to induce RSV-specific immunity.

Methods

We produced RSV-virosomes carrying TLR2 (Pam₃CSK₄) and/or NOD2 (L18-MDP) ligands. We tested the immunopotentiating properties of these virosomes in vitro, using TLR2- and/or NOD2-ligand-responsive murine and human cell lines, and in vivo by assessing induction of protective antibody and cellular responses upon IN immunization of BALB/c mice.

Results

Incorporation of Pam₃CSK₄ and/or L18-MDP potentiates the capacity of virosomes to activate (antigen-presenting) cells in vitro, as demonstrated by NF-κB induction. In vivo, incorporation of Pam₃CSK₄ in virosomes boosted serum IgG antibody responses and mucosal antibody responses after IN immunization. While L18-MDP alone was ineffective, incorporation of L18-MDP in Pam₃CSK₄-carrying virosomes further boosted mucosal antibody responses. Finally, IN immunization with adjuvanted virosomes, particularly Pam₃CSK₄/L18-MDP-adjuvanted-virosomes, protected mice against infection with RSV, without priming for enhanced disease.

Conclusion

Mucosal immunization with RSV-virosomes, supplemented with incorporated TLR2- and/or NOD2-ligands, represents a promising approach to induce effective and safe RSV-specific immunity.     

PRR signaling during <Emphasis Type="Italic">in vitro</Emphasis> macrophage differentiation from progenitors modulates their subsequent response to inflammatory stimuli

Toll-like receptor (TLR) agonists drive hematopoietic stem and progenitor cells (HSPCs) to differentiate along the myeloid lineage in vitro and also in vivo following infection. In this study, we used an in vitro model of HSPC differentiation to investigate the functional consequences (cytokine production) that exposing HSPCs to various pathogen-associated molecular patterns (PAMPs) and Candida albicans cells have on the subsequently derived macrophages. Mouse HSPCs (Lin^– cells) were cultured with GM-CSF to induce macrophage differentiation in the presence or absence of the following pattern recognition receptor (PRR) agonists: Pam₃CSK₄ (TLR2 ligand), LPS (TLR4 ligand), depleted zymosan (which only activates Dectin-1), or inactivated C. albicans yeasts (which activate several PRRs, mainly TLR2 and Dectin-1). Our data show that only pure TLR2 ligand exposure (transient and continuous) impacts the inflammatory function of GM-CSF-derived macrophages, because Pam₃CSK₄-exposed HSPCs generate macrophages with a diminished ability to produce inflammatory cytokines. Interestingly, the Pam₃CSK₄-induced tolerance of macrophages (by transient exposure of HSPCs) is reinforced by subsequent exposure to C. albicans cells in GM-CSF-derived macrophages; however, the induced tolerance is partially reversed in M-CSF-derived macrophages. Therefore, the ability of macrophages to produce inflammatory cytokines is extremely dependent on how the HSPCs from which they are derived receive and integrate multiple microenvironmental signals (PRR ligands and/or CSFs).     

Empty liposomes induce antitumoral effects associated with macrophage responses distinct from those of the TLR1/2 agonist Pam3CSK4 (BLP)

Liposomes are frequently used in cancer therapy to encapsulate and apply anticancer drugs. Here, we show that a systemic treatment of mice bearing skin tumors with empty phosphatidylcholine liposomes (PCL) resulted in inhibition of tumor growth, which was similar to that observed with the synthetic bacterial lipoprotein and TLR1/2 agonist Pam₃CSK₄ (BLP). Both compounds led to a substantial decrease of macrophages in spleen and in the tumor-bearing skin. Furthermore, both treatments induced the expression of typical macrophage markers in the tumor-bearing tissue. As expected, BLP induced the expression of the M1 marker genes Cxcl10 and iNOS, whereas PCL, besides inducing iNOS, also increased the M2 marker genes Arg1 and Trem2. In vitro experiments demonstrated that neither PCL nor BLP influenced proliferation or survival of tumor cells, whereas both compounds inhibited proliferation and survival and increased the migratory capacity of bone marrow-derived macrophages (BMDM). However, in contrast to BLP, PCL did not activate cytokine secretion and induced a different BMDM phenotype. Together, the data suggest that similar to BLP, PCL induce an antitumor response by influencing the tumor microenvironment, in particular by functional alterations of macrophages, however, in a distinct manner from those induced by BLP.     

TLR2/1 and sphingosine 1-phosphate modulate inflammation,myofibroblast differentiation and cell migration in fibroblasts

Dermal fibroblasts are important regulators of inflammatory and immune responses in the skin. The aim of the present study was to elucidate the interaction between two key players in inflammation, Toll-like receptors (TLRs) and sphingosine 1-phosphate (S1P), in normal human fibroblasts in the context of inflammation, fibrosis and cell migration. We demonstrate that TLR2 ligation strongly enhances the production of the pro-inflammatory cytokines IL-6 and IL-8. S1P significantly induces pro-inflammatory cytokines time- and concentration-dependently via S1P receptor (S1PR)2 and S1PR3. The TLR2/1 agonist Pam₃CSK₄ and S1P (> 1 μM) or TGF-β markedly upregulate IL-6 and IL-8 secretion. Pam₃CSK₄ and S1P alone promote myofibroblast differentiation as assessed by significant increases of α-smooth muscle actin and collagen I expression. Importantly, costimulation with S1P (> 1 μM) induces differentiation into myofibroblasts. In contrast, Pam₃CSK₄ and low S1P concentrations (< 1 μM) accelerate cell migration. These results suggest that TLR2/1 signaling and S1P cooperate in pro-inflammatory cytokine production and myofibroblast differentiation and promote cell migration of skin fibroblasts in a S1P-concentration dependent manner. Our findings provide significant insights into how infectious stimuli or danger signals and sphingolipids contribute to dermal inflammation which may be relevant for skin tissue repair after injury or disease.     

High mobility group box protein 1 (HMGB1)-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor

The nuclear protein high mobility group box protein 1 (HMGB1) promotes inflammation upon extracellular release. HMGB1 induces proinflammatory cytokine production in macrophages via Toll-like receptor (TLR)-4 signaling in a redox-dependent fashion. Independent of its redox state and endogenous cytokine-inducing ability, HMGB1 can form highly immunostimulatory complexes by interaction with certain proinflammatory mediators. Such complexes have the ability to enhance the induced immune response up to 100-fold, compared with induction by the ligand alone. To clarify the mechanisms for these strong synergistic effects, we studied receptor requirements. Interleukin (IL)-6 production was assessed in supernatants from cultured peritoneal macrophages from mice each deficient in one of the HMGB1 receptors (receptor for advanced glycation end products [RAGE], TLR2 or TLR4) or from wild-type controls. The cultures were stimulated with the TLR4 ligand lipopolysaccaride (LPS), the TLR2 ligand Pam₃CysSerLys₄ (Pam₃CSK₄), noninflammatory HMGB1 or each TLR ligand in complex with noninflammatory HMGB1. The activity of the HMGB1-TLR ligand complexes relied on engagement of the same receptor as for the noncomplexed TLR ligand, since HMGB1-LPS complexes used TLR4 and HMGB1-Pam₃CSK₄ complexes used TLR2. Deletion of any of the intracellular adaptor molecules used by TLR2 (myeloid differentiation factor-88 [MyD88], TIR domain–containing adaptor protein [TIRAP]) or TLR4 (MyD88, TIRAP, TIR domain–containing adaptor-inducing interferon-β [TRIF], TRIF-related adaptor molecule [TRAM]) had similar effects on HMGB1 complex activation compared with noncomplexed LPS or Pam₃CSK₄. This result implies that the enhancing effects of HMGB1-partner molecule complexes are not regulated by the induction of additional signaling cascades. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.     

Site-specific effect of polar functional group-modification in lipids of TLR2 ligands for modulating the ligand immunostimulatory activity

Toll-like receptor 2 (TLR2), a member of the TLR innate immune receptor family, recognizes lipoproteins from bacteria and modulates the immune response by inducing the expression of various cytokines. TLR2 has a large hydrophobic pocket that recognizes long fatty acyl groups on TLR2 ligands. However, few studies have focused on the property of the hydrophobic TLR2 pocket. Based on the X-ray crystal structure of TLR2, small polar regions were found in the hydrophobic TLR2 pocket. Interactions between the polar residues and ligands were explored here by designing and synthesizing a Pam₂CSK₄ derivative of the TLR2 ligands, containing an amide group within the lipid moiety. We evaluated the binding affinities and immunomodulatory activities of these ligands. Results suggested that the amide groups in the lipid chain interacted with the polar residues in the hydrophobic lipid-binding pocket of TLR2.     

Effect of the antimicrobial peptide LL-37 on Toll-like receptors 2-, 3- and 4-triggered expression of IL-6, IL-8 and CXCL10 in human gingival fibroblasts

The antimicrobial peptide LL-37 is known to have a potent LPS-neutralizing activity in monocytes and macrophages. Recently, LL-37 in gingival crevicular fluids is suggested to be the major protective factor preventing infection of periodontogenic pathogens. In this study, we tried to address the effect of LL-37 on proinflammatory responses of human gingival fibroblasts (HGFs) stimulated with Toll-like receptor (TLR)-stimulant microbial compounds. LL-37 potently suppressed LPS-induced gene expression of IL6, IL8 and CXCL10 and intracellular signaling events, degradation of IRAK-1 and IκBα and phosphorylation of p38 MAPK and IRF3, indicating that the LPS-neutralizing activity is also exerted in HGFs. LL-37 also suppressed the expression of IL6, IL8 and CXCL10 induced by the TLR3 ligand poly(I:C). LL-37 modestly attenuated the expression of IL6 and IL8 induced by the TLR2/TLR1 ligand Pam₃CSK₄, but did not affect the expression induced by the TLR2/TLR6 ligand MALP-2. Interestingly, LL-37 rather upregulated the expression of IL6, IL8 and CXCL10 induced by another TLR2/TLR6 ligand FSL-1. Thus, the regulatory effect of LL-37 is differently exerted towards proinflammatory responses of HGFs induced by different microbial stimuli, which may lead to unbalanced proinflammatory responses of the gingival tissue to infection of oral microbes.