The Development of a Highly Potent and Selective Human Toll-like Receptor 2 Agonist: Synthesis and Biological Evaluation of CaLGL-1 and Its Derivatives.

Toll-like receptor 2 (TLR2) plays a crucial role in detecting microbial pathogen-associated molecular patterns, offering potential applications as an adjuvant for vaccines and antitumor therapies. Here, we present the gram-scale synthesis of CaLGL-1 and its derivatives, natural products known for activating mouse TLR2 (EC50 = 3.2 µM). This synthesis involves a streamlined six-step reaction sequence utilizing oxidant-promoted acetalization, effectively preserving the acid-sensitive glycosidic bond for maintaining the compounds' functional integrity. Our structure-activity relationship studies identified R-7d as a potent human TLR2 activator. It demonstrated subnanomolar activity (EC50 = 116 pM) in human THP-1 cells, comparable to that of diprovocim (EC50 = 110 pM). Experiments revealed that R-7d enhances NF-kB promoter activation through TLR2/TLR1 heterodimers rather than TLR2/TLR6. The discovery of R-7d as a robust human TLR2 agonist opens up new possibilities for combination therapies.

TLR1/2 Agonist Enhances Reversal of HIV-1 Latency and Promotes NK Cell-Induced Suppression of HIV-1-Infected Autologous CD4+ T Cells.

The complete eradication of human immunodeficiency virus type 1 (HIV-1) is blocked by latent reservoirs in CD4+ T cells and myeloid lineage cells. Toll-like receptors (TLRs) can induce the reversal of HIV-1 latency and trigger the innate immune response. To the best of our knowledge, there is little evidence showing the "killing" effect of TLR1/2 agonists but only a small "shock" potential. To identify a new approach for eradicating the HIV latent reservoir, we evaluated the effectiveness of SMU-Z1, a novel small-molecule TLR1/2 agonist, in the "shock-and-kill" strategy. The results showed that SMU-Z1 could enhance latent HIV-1 transcription not only ex vivo in peripheral blood mononuclear cells from aviremic HIV-1-infected donors receiving combined antiretroviral therapy but also in vitro in cells of myeloid-monocytic origin targeting the NF-κB and mitogen-activated protein kinase pathways. Interestingly, the activation marker CD69 was significantly upregulated in natural killer (NK) cells, B cells, and monocytes 48 h after SMU-Z1 treatment. Furthermore, SMU-Z1 was able to activate T cells without global T cell activation, as well as increasing NK cell degranulation and gamma interferon (IFN-γ) production, which further block HIV-1-infected CD4+ lymphocytes. In summary, the present study found that SMU-Z1 can both enhance HIV-1 transcription and promote NK cell-mediated inhibition of HIV-1-infected autologous CD4+ T cells. These findings indicate that the novel TLR1/2 agonist SMU-Z1 is a promising latency-reversing agent (LRA) for eradication of HIV-1 reservoirs. IMPORTANCE Multiple in vivo studies showed that many LRAs used in the shock-and-kill approach could activate viral transcription but could not induce killing effectively. Therefore, a dual-function LRA is needed for elimination of HIV-1 reservoirs. We previously developed a small-molecule TLR1/2 agonist, SMU-Z1, and demonstrated that it could upregulate NK cells and CD8+ T cells with immune adjuvant and antitumor properties in vivo. In the present study, SMU-Z1 could activate innate immune cells without global T cell activation, induce production of proinflammatory and antiviral cytokines, and enhance the cytotoxic function of NK cells. We showed that SMU-Z1 displayed dual potential ex vivo in the shock of exposure of latently HIV-1-infected cells and in the kill of clearance of infected cells, which is critical for effective use in combination with therapeutic vaccines or broadly neutralizing antibody treatments aimed at curing AIDS.

Design, Synthesis, and Structure-Activity Relationship of N-Aryl-N'-(thiophen-2-yl)thiourea Derivatives as Novel and Specific Human TLR1/2 Agonists for Potential Cancer Immunotherapy.

The previous virtual screening of ten million compounds yielded two novel nonlipopeptide-like chemotypes as TLR2 agonists. Herein, we present the chemical optimization of our initial hit, 1-phenyl-3-(thiophen-2-yl)urea, which resulted in the identification of SMU-C80 (EC50 = 31.02 ± 1.01 nM) as a TLR2-specific agonist with a 370-fold improvement in bioactivity. Mechanistic studies revealed that SMU-C80, through TLR1/2, recruits the adaptor protein MyD88 and triggers the NF-κB pathway to release cytokines such as TNF-α and IL-1ß from human, but not murine, cells. To the best of our knowledge, it is the first species-specific TLR1/2 agonist reported until now. Moreover, SMU-C80 increased the percentage of T, B, and NK cells ex vivo and activated the immune cells, which suppressed cancer cell growth in vitro. In summary, we obtained a highly efficient and specific human TLR1/2 agonist that acts through the MyD88 and NF-κB pathway, facilitating cytokine release and the simultaneous activation of immune cells that in turn affects the apoptosis of cancer cells.

Pam3CSK4-CDGSF Augments Antitumor Immunotherapy by Synergistically Activating TLR1/2 and STING.

Cyclic dinucleotides (CDNs), agonists of stimulator of interferon genes (STING), are promising agents for immunotherapy. However, the application of CDNs has been limited by their instability and low transmembrane efficiency. Here, we introduced a conjugated adjuvant of STING and TLR1/2, Pam3CSK4-CDGSF. Conjugating CDGSF with Pam3CSK4 increased the stability and intracellular delivery. In addition, by synergistically activating the STING and TLR pathways, Pam3CSK4-CDGSF was able to enhance immune activation. Both humoral and cellular immune responses were triggered by Pam3CSK4-CDGSF plus OVA (V4), and tumor growth was significantly inhibited after V4 administration. More importantly, V4 can also boost the antigen-specific CD8+ T cell response for cancer cell killing. Thus, the conjugated STING and TLR1/2 agonist Pam3CSK4-CDGSF can serve as a potent adjuvant for vaccine construction to augment antitumor immunotherapy.

TLR1/2 and 5 induce elevated cytokine levels from rheumatoid arthritis monocytes independent of ACPA or RF autoantibody status.

OBJECTIVE: RA is an autoimmune inflammatory joint disease. Both RF and ACPA are associated with more progressive disease and higher levels of systemic inflammation. Monocyte activation of toll-like receptors (TLRs) by endogenous ligands is a potential source of increased production of systemic cytokines. RA monocytes have elevated TLRs, some of which are associated with the disease activity score using 28 joints (DAS28). The aim of this study was to measure TLR-induced cytokine production from monocytes, stratified by autoantibody status, to assess if their capacity to induce cytokines is related to autoantibody status or DAS28. METHODS: Peripheral blood monocytes isolated from RA patients and healthy controls were stimulated with TLR1/2, TLR2/6, TLR4, TLR5, TLR7, TLR8 and TLR9 ligands for 18 h before measuring IL-6, TNFα and IL-10. Serum was used to confirm the autoantibody status. Cytokine levels were compared with RF, ACPA and DAS28. RESULTS: RA monocytes demonstrated significantly increased IL-6 and TNFα upon TLR1/2 stimulation and IL-6 and IL-10 upon TLR5 activation. TLR7 and TLR9 activation did not induce cytokines and no significant differences were observed between RA and healthy control monocytes upon TLR2/6, TLR4 or TLR8 activation. When stratified by ACPA or RF status there were no correlations between autoantibody status and elevated cytokine levels. However, TLR1/2-induced IL-6 did correlate with DAS28. CONCLUSIONS: Elevated TLR-induced cytokines in RA monocytes were not related to ACPA or RF status. However, TLR1/2-induced IL-6 was associated with disease activity.

Structural Basis of TLR2/TLR1 Activation by the Synthetic Agonist Diprovocim.

Diprovocim is a recently discovered exceptionally potent, synthetic small molecule agonist of TLR2/TLR1 and has shown significant adjuvant activity in anticancer vaccination against murine melanoma. Since Diprovocim bears no structural similarity to the canonical lipopeptide ligands of TLR2/TLR1, we investigated how Diprovocim interacts with TLR2/TLR1 through in vitro biophysical, structural, and computational approaches. We found that Diprovocim induced the formation of TLR2/TLR1 heterodimers as well as TLR2 homodimers in vitro. We determined the crystal structure of Diprovocim in a complex with a TLR2 ectodomain, which revealed, unexpectedly, two Diprovocim molecules bound to the ligand binding pocket formed between two TLR2 ectodomains. Extensive hydrophobic interactions and a hydrogen-bonding network between the protein and Diprovocim molecules are observed within the defined ligand binding pocket and likely underlie the high potency of Diprovocim. Our work shed first light into the activation mechanism of TLR2/TLR1 by a noncanonical agonist. The structural information obtained here may be exploited to manipulate TLR2/TLR1-dependent signaling.

Diprovocims: A New and Exceptionally Potent Class of Toll-like Receptor Agonists.

A screen conducted with nearly 100000 compounds and a surrogate functional assay for stimulation of an immune response that measured the release of TNF-α from treated human THP-1 myeloid cells differentiated along the macrophage line led to the discovery of the diprovocims. Unique to these efforts and of special interest, the screening leads for this new class of activators of an immune response came from a compound library designed to promote cell-surface receptor dimerization. Subsequent comprehensive structure-activity relationship studies improved the potency 800-fold over that of the screening leads, providing diprovocim-1 and diprovocim-2. The diprovocims act by inducing cell-surface toll-like receptor (TLR)-2 dimerization and activation with TLR1 (TLR1/TLR2 agonist), bear no structural similarity to any known natural or synthetic TLR agonist, and are easy to prepare and synthetically modify, and selected members are active in both human and murine systems. The most potent diprovocim (3, diprovocim-1) elicits full agonist activity at extraordinarily low concentrations (EC50 = 110 pM) in human THP-1 cells, being more potent than the naturally derived TLR1/TLR2 agonist Pam3CSK4 or any other known small molecule TLR agonist.

Adjuvant effect of the novel TLR1/TLR2 agonist Diprovocim synergizes with anti-PD-L1 to eliminate melanoma in mice.

Successful cancer immunotherapy entails activation of innate immune receptors to promote dendritic cell (DC) maturation, antigen presentation, up-regulation of costimulatory molecules, and cytokine secretion, leading to activation of tumor antigen-specific cytotoxic T lymphocytes (CTLs). Here we screened a synthetic library of 100,000 compounds for innate immune activators using TNF production by THP-1 cells as a readout. We identified and optimized a potent human and mouse Toll-like receptor (TLR)1/TLR2 agonist, Diprovocim, which exhibited an EC50 of 110 pM in human THP-1 cells and 1.3 nM in primary mouse peritoneal macrophages. In mice, Diprovocim-adjuvanted ovalbumin immunization promoted antigen-specific humoral and CTL responses and synergized with anti-PD-L1 treatment to inhibit tumor growth, generating long-term antitumor memory, curing or prolonging survival of mice engrafted with the murine melanoma B16-OVA. Diprovocim induced greater frequencies of tumor-infiltrating leukocytes than alum, of which CD8 T cells were necessary for the antitumor effect of immunization plus anti-PD-L1 treatment.

Alteration of the immune status of umbilical cord mesenchymal stem cells stimulated by TLR1/2 agonist, Pam3Csk.

Mesenchymal stem cells (MSCs) have been widely used in clinical trials due to their multiple differentiation ability, low immunogenicity and immunosuppressant effects on immune response. However, accumulating evidence has indicated that MSCs may stimulate in vivo immune responses and result in the disappearance of MSCs following engrafting. Toll­like receptors (TLRs) are important in immune response induction against invaded pathogens, however, the function of TLRs in regulating the immune status of MSCs has been seldom reported. The present stimulated umbilical cord (UC) MSCs by treatment with the TLR1/2 agonist, Pam3Csk, the to determine whether activation of TLR1/2 signaling alters the immune status of UCMSCs. The results indicated that activation of TLR1/2 increased the proliferation of peripheral blood mononuclear cells (PBMCs) and the production of lactate dehydrogenase in a PBMC­MSC co­culture system. The study also demonstrated that Pam3Csk induced the secretion of pro­inflammatory molecules, and increased the expression levels of cytokine and chemokines in UCMSCs. Flow cytometry analysis indicated that the levels of surface co­stimulators, CD80 and CD86, were increased on UCMSCs in the presence of Pam3Csk, whereas activation of TLR1/2 exerted no observable effect on the differentiation abilities of UCMSCs. The results of the current study indicated that activation of TLR1/2 signaling may alter the immune status of UCMSCs, however, further mechanistic research is required in future studies.

Staphylococcus aureus Inhibits IL-8 Responses Induced by Pseudomonas aeruginosa in Airway Epithelial Cells.

Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are major respiratory pathogens and can concurrently colonize the airways of patients with chronic obstructive diseases, such as cystic fibrosis (CF). Airway epithelial cell signalling is critical to the activation of innate immune responses. In the setting of polymicrobial colonization or infection of the respiratory tract, how epithelial cells integrate different bacterial stimuli remains unknown. Our study examined the inflammatory responses to PA and SA co-stimulations. Immortalised airway epithelial cells (Beas-2B) exposed to bacteria-free filtrates from PA (PAF) induced a robust production of the neutrophil chemoattractant IL-8 while bacteria-free filtrates from SA (SAF) had a minimal effect. Surprisingly, co-stimulation with PAF+SAF demonstrated that SAF strongly inhibited the PAF-driven IL-8 production, showing that SAF has potent anti-inflammatory effects. Similarly SAF decreased IL-8 production induced by the TLR1/TLR2 ligand Pam3CysSK4 but not the TLR4 ligand LPS nor TLR5 ligand flagellin in Beas-2B cells. Moreover, SAF greatly dampened TLR1/TLR2-mediated activation of the NF-κB pathway, but not the p38 MAPK pathway. We observed this SAF-dependent anti-inflammatory activity in several SA clinical strains, as well as in the CF epithelial cell line CFBE41o-. These findings show a novel direct anti-inflammatory effect of SA on airway epithelial cells, highlighting its potential to modulate inflammatory responses in the setting of polymicrobial infections.

Systemic injection of TLR1/2 agonist improves adoptive antigen-specific T cell therapy in glioma-bearing mice.

Adoptive immunotherapy is an attractive strategy for glioma treatment. However, some obstacles still need be overcome. In this study, GL261-bearing mice treated with adoptively transferred antigen-specific T cells and systemic injection of bacterial lipoprotein (BLP), a TLR1/2 agonist, got a long-term survival and even immune protection. By analyzing adoptive T cells, it was found that BLP maintained T cell survival, proliferation and anti-tumor efficacy in the brains of tumor-bearing hosts. Moreover, tumor microenvironment was modified by up-regulating IFN-γ-secreting CD8+ T cells and down-regulating MDSC, which might be related with high CXCL10 and low CCL2 expression. In addition, TLR2 deficiency abrogated therapeutic effect with increased MDSC accumulation and decreased IFN-γ-secreting CD8+ T cells in the brains. Thus, the systemic injection of BLP could improve the adoptive T cell therapy by maintaining T cell persistence, modifying the tumor microenvironment and even inducing systemic anti-tumor immunity, which might offer a clinically promising immunotherapeutic strategy for glioma.

Toll-like receptor (TLR)-1/2 triggering of multiple myeloma cells modulates their adhesion to bone marrow stromal cells and enhances bortezomib-induced apoptosis.

In multiple myeloma (MM), the malignant plasma cells usually localize to the bone marrow where they develop drug resistance due to adhesion to stromal cells and various environmental signals. Hence, modulation of this interaction is expected to influence drug sensitivity of MM cells. Toll-like receptor (TLR) ligands have displayed heterogeneous effects on B-cell malignancies and also on MM cells in a few recent studies, but effects on adhesion and drug sensitivity of myeloma cells in the context of bone marrow stromal cells (BMSCs) have never been investigated. In the present study, we explored the modulatory effects of TLR1/2 ligand (Pam3CSK4) on adhesion of human myeloma cells to BMSCs. It is shown that TLR1/2 triggering has opposite effects in different HMCLs on their adhesion to BMSCs. Fravel, L363, UM-6, UM-9 and U266 showed increased adhesion to BMSC in parallel with an increased surface expression of integrin molecules α4 and αVß3. OPM-1, OPM-2 and NCI-H929 showed a dose-dependent decrease in adhesion upon TLR activation following a downregulation of ß7 integrin expression. Importantly, TLR1/2 triggering increased cytotoxic and apoptotic effects of bortezomib in myeloma cells independent of the effect on stromal cell adhesion. Moreover, the apoptosis-enhancing effect of Pam3CSK4 paralleled induction of cleaved caspase-3 protein in FACS analysis suggesting a caspase-dependent mechanism. Our findings uncover a novel role of TLR activation in MM cells in the context of bone marrow microenvironment. Stimulation of TLR1/2 bypasses the protective shield of BMSCs and may be an interesting strategy to enhance drug sensitivity of multiple myeloma cells.

Dendritic-cell exosomes cross-present Toll-like receptor-ligands and activate bystander dendritic cells.

Dendritic cells (DCs) are the major sentinel, antigen-presenting and regulatory components of the immune system. One of the central DC functions is to rapidly sense and alert host immune system of a pathogen invasion. In the present study, we investigated the role of DC exosomes (DCex) in this sentinel function. We demonstrated that DCex could bind bacterial Toll-like-receptor ligands (TLR-Ls), and acquire their ability to strongly activate bystander DCs. Consequently, bystander DCs enhance the expression of transmembrane tumor necrosis factor, secretion of proinflammatory cytokines and cross-talk with natural killer cells leading to the elevated secretion of IFNγ. These findings newly show that DCex can bind and cross-present TLR-Ls to innate-immunity effector cells, and indicate a potent mechanism to systemically alert the host immune system of pathogen invasion. They also suggest a potential novel strategy to generate effective vaccines by binding TLR-L-immune adjuvants to DCex.

Toll-like receptor agonists promote prolonged triglyceride storage in macrophages.

Macrophages in infected tissues may sense microbial molecules that significantly alter their metabolism. In a seeming paradox, these critical host defense cells often respond by increasing glucose catabolism while simultaneously storing fatty acids (FA) as triglycerides (TAG) in lipid droplets. We used a load-chase strategy to study the mechanisms that promote long term retention of TAG in murine and human macrophages. Toll-like receptor (TLR)1/2, TLR3, and TLR4 agonists all induced the cells to retain TAG for ≥3 days. Prolonged TAG retention was accompanied by the following: (a) enhanced FA uptake and FA incorporation into TAG, with long lasting increases in acyl-CoA synthetase long 1 (ACSL1) and diacylglycerol acyltransferase-2 (DGAT2), and (b) decreases in lipolysis and FA ß-oxidation that paralleled a prolonged drop in adipose triglyceride lipase (ATGL). TLR agonist-induced TAG storage is a multifaceted process that persists long after most early pro-inflammatory responses have subsided and may contribute to the formation of "lipid-laden" macrophages in infected tissues.

Empty liposomes induce antitumoral effects associated with macrophage responses distinct from those of the TLR1/2 agonist Pam3CSK 4 (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 Pam(3)CSK(4) (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.

Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro.

BACKGROUND: Plasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-α secretions (ELISA). RESULTS: At first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68-75%) was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NP>chitosan DNA NP=DNA unloaded chitosan NP>control (culture medium). CONCLUSIONS: Chitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-α levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).

A novel synthetic lipopeptide is allergy-protective by the induction of LPS-tolerance.

BACKGROUND: Exposure to the environment of traditional farms can protect children from some allergic disease. Due to this exposure, TLR2 expression in these children is increased. TLR2 ligands derived from gram-positive bacteria are found in the dust of these farms. OBJECTIVES: We proved whether a synthetic lipopeptide binding to the TLR1/2 heterodimer is able to protect from allergic disease in two different murine models of allergy. We also investigated the immunological mechanisms underlying the protective properties of the lipopeptide. METHODS: We synthesized a lipopeptide derived from a germination lipoprotein of Bacillus cereus (LPGerD). We evaluated the immunomodulatory activity of LPGerD in a murine model of systemic sensitization (OVA/Alum) and in a model in which mice were sensitized with OVA pulsed bone-marrow-derived dendritic cells (BMDCs) via the airways. Furthermore, the induction of LPS tolerance was studied. RESULTS: Treatment of mice with LPGerD in a mouse model of asthma led to protection against sensitization and airway inflammation. Similarly, bone-marrow-derived dendritic cells (BMDCs) pre-treated with LPGerD were not able to prime mice for allergic immune response. We observed that pre-treatment with LPGerD led to the induction of a LPS-tolerant state in BMDCs. These cells secreted markedly lower amounts of pro-inflammatory cytokines upon LPS stimulation. Furthermore, we observed an up-regulation of IRAK-M mRNA in BMDCs pre-treated with LPGerD. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that induction of a LPS-tolerant state in antigen-presenting cells (APCs) may contribute to the protective effect of a farming environment. TLR2 agonists similar to those appearing in cowshed dust extracts, such as our synthetic LPGerD, lead to the ignorance of the LPS stimulus, which is important for the activation of APCs to mount a Th2 immune response. This substance might be a promising candidate for allergy-preventive treatments as LPGerD had only low pro-inflammatory characteristics.

The Toll-like receptor 1/2 agonists Pam(3) CSK(4) and human ß-defensin-3 differentially induce interleukin-10 and nuclear factor-κB signalling patterns in human monocytes.

Human ß-defensin 3 (hBD-3) activates antigen-presenting cells through Toll-like receptors (TLRs) 1/2. Several TLR1/2 agonists have been identified but little is known about how they might differentially affect cellular activation. We compared the effects of hBD-3 with those of another TLR1/2 agonist, Pam(3) CSK(4) , in human monocytes. Monocytes incubated with hBD-3 or Pam(3) CSK(4) produced interleukin-6 (IL-6), IL-8 and IL-1ß, but only Pam(3) CSK(4) induced IL-10. The IL-10 induction by Pam(3) CSK(4) caused down-modulation of the co-stimulatory molecule, CD86, whereas CD86 expression was increased in monocytes exposed to hBD-3. Assessment of signalling pathways linked to IL-10 induction indicated that mitogen-activated protein kinases were activated similarly by hBD-3 or Pam(3) CSK(4) , whereas the non-canonical nuclear factor-κB pathway was only induced by Pam(3) CSK(4) . Our data suggest that the lack of non-canonical nuclear factor-κB signalling by hBD-3 could contribute to the failure of this TLR agonist to induce production of the anti-inflammatory cytokine, IL-10, in human monocytes.

TLR1/TLR2 agonist induces tumor regression by reciprocal modulation of effector and regulatory T cells.

Using TLR agonists in cancer treatment can have either beneficial or detrimental effects. Therefore, it is important to determine their effect on the tumor growth and understand the underlying mechanisms in animal tumor models. In this study, we report a general immunotherapeutic activity of a synthetic bacterial lipoprotein (BLP), a TLR1/TLR2 agonist, on established lung carcinoma, leukemia, and melanoma in mice. Systemic treatment of 3LL tumor-bearing mice with BLP, but not LPS, led to a dose-dependent tumor regression and a long-lasting protective response against tumor rechallenge. The BLP-mediated tumor remission was neither mediated by a direct tumoricidal activity nor by innate immune cells, because it lacked therapeutic effect in immunodeficient SCID mice. Instead, BLP treatment reduced the suppressive function of Foxp3(+) regulatory T cells (Tregs) and enhanced the cytotoxicity of tumor-specific CTL in vitro and in vivo. Furthermore, adoptive cotransfer of BLP-pretreated but not untreated CTL and Tregs from wild-type but not from TLR2(-/-) mice was sufficient to restore antitumor immunity in SCID mice by reciprocally modulating Treg and CTL function. These results demonstrate that the TLR1/TLR2 agonist BLP may have a general tumor therapeutic property involving reciprocal downregulation of Treg and upregulation of CTL function. This property may play an important role in the development of novel antitumor strategies.

Human TLRs 10 and 1 share common mechanisms of innate immune sensing but not signaling.

TLRs are central receptors of the innate immune system that drive host inflammation and adaptive immune responses in response to invading microbes. Among human TLRs, TLR10 is the only family member without a defined agonist or function. Phylogenetic analysis reveals that TLR10 is most related to TLR1 and TLR6, both of which mediate immune responses to a variety of microbial and fungal components in cooperation with TLR2. The generation and analysis of chimeric receptors containing the extracellular recognition domain of TLR10 and the intracellular signaling domain of TLR1, revealed that TLR10 senses triacylated lipopeptides and a wide variety of other microbial-derived agonists shared by TLR1, but not TLR6. TLR10 requires TLR2 for innate immune recognition, and these receptors colocalize in the phagosome and physically interact in an agonist-dependent fashion. Computational modeling and mutational analysis of TLR10 showed preservation of the essential TLR2 dimer interface and lipopeptide-binding channel found in TLR1. Coimmunoprecipitation experiments indicate that, similar to TLR2/1, TLR2/10 complexes recruit the proximal adaptor MyD88 to the activated receptor complex. However, TLR10, alone or in cooperation with TLR2, fails to activate typical TLR-induced signaling, including NF-kappaB-, IL-8-, or IFN-beta-driven reporters. We conclude that human TLR10 cooperates with TLR2 in the sensing of microbes and fungi but possesses a signaling function distinct from that of other TLR2 subfamily members.