Toll-like receptor-2 induced inflammation causes local bone formation and activates canonical Wnt signaling.

It is well established that inflammatory processes in the vicinity of bone often induce osteoclast formation and bone resorption. Effects of inflammatory processes on bone formation are less studied. Therefore, we investigated the effect of locally induced inflammation on bone formation. Toll-like receptor (TLR) 2 agonists LPS from Porphyromonas gingivalis and PAM2 were injected once subcutaneously above mouse calvarial bones. After five days, both agonists induced bone formation mainly at endocranial surfaces. The injection resulted in progressively increased calvarial thickness during 21 days. Excessive new bone formation was mainly observed separated from bone resorption cavities. Anti-RANKL did not affect the increase of bone formation. Inflammation caused increased bone formation rate due to increased mineralizing surfaces as assessed by dynamic histomorphometry. In areas close to new bone formation, an abundance of proliferating cells was observed as well as cells robustly stained for Runx2 and alkaline phosphatase. PAM2 increased the mRNA expression of Lrp5, Lrp6 and Wnt7b, and decreased the expression of Sost and Dkk1. In situ hybridization demonstrated decreased Sost mRNA expression in osteocytes present in old bone. An abundance of cells expressed Wnt7b in Runx2-positive osteoblasts and ß-catenin in areas with new bone formation. These data demonstrate that inflammation, not only induces osteoclastogenesis, but also locally activates canonical WNT signaling and stimulates new bone formation independent on bone resorption.

Synthesis, evaluation and molecular dynamics study of human toll-like receptor 2/6 specific monoacyl lipopeptides as candidate immunostimulants.

TLR2 agonists typified by the S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (Pam2CS) motif have exhibited powerful immunostimulatory activities. Based on simplified monoacyl lipopeptide (Carbamate-linked N-Ac PamCS), we describe interesting SAR investigations where modifications are done to alter the size of substituents on the cysteine amine, introduce ionizable groups to the terminal and insert aromatic substitutions to the aliphatic chain. Our structural modifications have led to a highly specific human TLR2/6 agonist 14a (EC50 = 0.424 nM), which behaves like Pam2CSK4 by inducing NF-κB activation to trigger downstream signaling pathways, such as subsequent phosphorylation of related proteins (p65, p38) and production of key inflammatory cytokines (IL-6, IL-1ß, TNF-α). Importantly, the ability to stimulate enhanced T cell response compared to Carbamate-linked N-Ac PamCS makes compound 14a a further potential candidate immunostimulant.

Activation of Most Toll-Like Receptors in Whole Human Blood Attenuates Platelet Deposition on Collagen under Flow.

Platelets have toll-like receptors (TLRs); however, their function in thrombosis or hemostasis under flow conditions is not fully known. Thrombin-inhibited anticoagulated whole blood was treated with various TLR agonists and then perfused over fibrillar collagen using microfluidic assay at venous wall shear rate (100 s-1). Platelet deposition was imaged with fluorescent anti-CD61. For perfusion of whole blood without TLR agonist addition, platelets rapidly accumulated on collagen and eventually occluded the microchannels. Interestingly, most of the tested TLR agonists (Pam3CKS4, MALP-2, polyinosinic-polycytidylic acid HMW, imiquimod, and CpG oligodeoxynucleotides) strongly reduced platelet deposition on collagen, while only the TLR4 agonist endotoxin lipopolysaccharide (LPS) enhanced deposition. Following 90 sec of deposition under flow of untreated blood, the addition of various TLR-7 agonists (imiquimod, vesatolimod, and GSK2245035) all caused immediate blockade of further platelet deposition. Since TLR signaling can activate nuclear factor-kappaB (NF-κB), the IKK-inhibitor (IKK inhibitor VII) and NF-κB inhibitor (Bay 11-7082) were tested. The IKK/NF-κB inhibitors strongly inhibited platelet deposition under flow. Furthermore, addition of Pam3CSK4 (TLR1/2 ligand), MALP-2 (TLR2/6 ligand), and Imquimod (TLR7 ligand) reduced phosphotidylserine (PS) exposure. Activation of TLR1/2, TLR2/6, TLR3, TLR7, and TLR9 in whole blood reduced platelet deposition under flow on collagen; however, LPS (major Gram negative bacterial pathogenic component) activation of LTR4 was clearly prothrombotic.

Combination of natural polyphenols with a precursor of NAD+ and a TLR2/6 ligand lipopeptide protects mice against lethal γ radiation.

INTRODUCTION: Effective agents that could confer long-term protection against ionizing radiation in vivo would have applications in medicine, biotechnology, and in air and space travel. However, at present, drugs that can effectively protect against lethal ionizing radiations are still an unmet need. OBJECTIVE: To investigate if combinations of natural polyphenols, known for their antioxidant potential, could protect against ionizing radiations. METHODS: Plant-derived polyphenols were screened for their potential ability to confer radioprotection to mice given a lethal whole-body γ radiation (137Cs) dose expected to kill 50% of the animals in 30 days. Telomere and centromere staining, Q-FISH and comet assays were used to investigate chromosomal aberration, micronuclei formation and DNA breaks. Molecular oxidations were investigated by enzyme immunoassays and UPLC-MS/MS. RT-PCR, western blotting and siRNA-induced gene silencing were used to study signaling mechanisms and molecular interactions. RESULTS: The combination of pterostilbene (PT) and silibinin (SIL) was the most effective against γ-irradiation, resulting in 100% of the mice surviving at 30 days and 20% survival at one year. Treatment post γ-irradiation with two potential radiomitigators nicotinamide riboside (NR, a vitamin B3 derivative), and/or fibroblast-stimulating lipoprotein 1 (FSL1, a toll-like receptor 2/6 agonist), did not extend survival. However, the combination of PT, SIL, NR and FSL1 achieved a 90% survival one year post γ-irradiation. The mechanism involves induction of the Nrf2-dependent cellular antioxidant defense, reduction of NF-kB signaling, upregulation of the PGC-1α/sirtuins 1 and 3 axis, PARP1-dependent DNA repair, and stimulation of hematopoietic cell recovery. The pathway linking Nrf2, sirtuin 3 and SOD2 is key to radioprotection. Importantly, this combination did not interfere with X-ray mediated killing of different tumor cells in vivo. CONCLUSION: The combination of the radioprotectors PT and SIL with the radiomitigators NR and FSL1 confer effective, long-term protection against γ radiation in vivo. This strategy is potentially capable of protecting mammals against ionizing radiations.

Identification and immunological evaluation of novel TLR2 agonists through structural optimization of Diprovocim.

As an important family member of Toll-like receptors (TLRs), TLR2 can recognize various pathogen-associated molecular patterns (PAMPs) such as bacteria and viral components. Accumulating evidence demonstrates that TLR2 agonists play a critical role in cancer immunotherapy and infectious diseases. Diprovocim is the most potent small molecule TLR2 agonist known, showing remarkably immune adjuvant activity in mice. However, the further clinical research and development of Diprovocim was hampered because of its structural complexity as well as high molecular weight. Here, we designed and synthesized 21 structurally simplified derivatives of Diprovocim, performed their TLR2 agonistic activities by HEK-Blue hTLR2 SEAP assay, and evaluated the toxicity in two human normal cell lines. Compounds B3-B4 and B9-B12 with excellent TLR2 agonistic activity were found through the structure-activity relationship study. Among them, diastereomer B10 and B12 substituted (S)-2-phenylcyclopropylamide side chain of Diprovocim with simple (R)- and (S)-n-butyl groups exhibited comparable TLR2 agonistic activities with EC50 values of 35 nM and 39 nM, respectively. ELISA and western blot experiments on THP-1 cells showed that B10 and B12 displayed remarkable immunostimulatory activity in the release of various inflammatory cytokines through activating MyD88-dependent NF-κB and MAPK signaling pathways. Importantly, B10 and B12 have less structural complexity and better safety compared to Diprovocim, and the chiral center of right pyrrolidine ring has negligible influence on TLR2 activition. Our study provides simplified Diprovocim derivatives with high agonistic activity, providing a clue to further optimize Diprovocim.

Insights on the antiviral mechanisms of action of the TLR1/2 agonist Pam3CSK4 in hepatitis B virus (HBV)-infected hepatocytes.

OBJECTIVES: Pegylated-interferon-alpha (Peg-IFNα), an injectable innate immune protein, is still used to treat chronically HBV-infected patients, despite its poor tolerability. Peg-IFNα has the advantage over nucleos(t)ide analogues (NAs) to be administrated in finite regimen and to lead to a higher HBsAg loss rate. Yet it would be interesting to improve the efficacy (i.e. while decreasing doses), or replace, this old medicine by novel small molecules/stimulators able to engage innate immune receptors in both HBV replicating hepatocytes and relevant innate immune cells. We have previously identified the Toll-Like-Receptor (TLR)-2 agonist Pam3CSK4 as such a potential novel immune stimulator. The aim of this study was to gain insights on the antiviral mechanisms of action of this agonist in in vitro cultivated human hepatocytes. DESIGN: We used in vitro models of HBV-infected cells, based on both primary human hepatocytes (PHH) and the non-transformed HepaRG cell line to investigate the MoA of Pam3SCK4 and identify relevant combinations with other approved or investigational drugs. RESULTS: We exhaustively described the inhibitory anti-HBV phenotypes induced by Pam3CSK4, which include a strong decrease in HBV RNA production (inhibition of synthesis and acceleration of decay) and cccDNA levels. We confirmed the long-lasting anti-HBV activity of this agonist, better described the kinetics of antiviral events, and demonstrated the specificity of action through the TLR1/2- NF-κB canonical-pathway. Moreover, we found that FEN-1 could be involved in the regulation and inhibitory phenotype on cccDNA levels. Finally, we identified the combination of Pam3CSK4 with IFNα or an investigational kinase inhibitor (called 1C8) as valuable strategies to reduce cccDNA levels and obtain a long-lasting anti-HBV effect in vitro. CONCLUSIONS: TLR2 agonists represent possible assets to improve the rate of HBV cure in patients. Further evaluations, including regulatory toxicity studies, are warranted to move toward clinical trials.

Comparative genomic analysis of five freshwater cyanophages and reference-guided metagenomic data mining.

BACKGROUND: As important producers using photosynthesis on Earth, cyanobacteria contribute to the oxygenation of atmosphere and the primary production of biosphere. However, due to the eutrophication of urban waterbodies and global warming, uncontrollable growth of cyanobacteria usually leads to the seasonal outbreak of cyanobacterial blooms. Cyanophages, a group of viruses that specifically infect and lyse cyanobacteria, are considered as potential environment-friendly agents to control the harmful blooms. Compared to the marine counterparts, only a few freshwater cyanophages have been isolated and genome sequenced to date, largely limiting their characterizations and applications. RESULTS: Here, we isolated five freshwater cyanophages varying in tail morphology, termed Pam1~Pam5, all of which infect the cyanobacterium Pseudanabaena mucicola Chao 1806 that was isolated from the bloom-suffering Lake Chaohu in Anhui, China. The whole-genome sequencing showed that cyanophages Pam1~Pam5 all contain a dsDNA genome, varying in size from 36 to 142 Kb. Phylogenetic analyses suggested that Pam1~Pam5 possess different DNA packaging mechanisms and are evolutionarily distinct from each other. Notably, Pam1 and Pam5 have lysogeny-associated gene clusters, whereas Pam2 possesses 9 punctuated DNA segments identical to the CRISPR spacers in the host genome. Metagenomic data-based calculation of the relative abundance of Pam1~Pam5 at the Nanfei estuary towards the Lake Chaohu revealed that the short-tailed Pam1 and Pam5 account for the majority of the five cyanophages. Moreover, comparative analyses of the reference genomes of Pam1~Pam5 and previously reported cyanophages enabled us to identify three circular and seven linear contigs of virtual freshwater cyanophages from the metagenomic data of the Lake Chaohu. CONCLUSIONS: We propose a high-throughput strategy to systematically identify cyanophages based on the currently available metagenomic data and the very limited reference genomes of experimentally isolated cyanophages. This strategy could be applied to mine the complete or partial genomes of unculturable bacteriophages and viruses. Transformation of the synthesized whole genomes of these virtual phages/viruses to proper hosts will enable the rescue of bona fide viral particles and eventually enrich the library of microorganisms that exist on Earth. Video abstract.

Akkermansia muciniphila phospholipid induces homeostatic immune responses.

Multiple studies have established associations between human gut bacteria and host physiology, but determining the molecular mechanisms underlying these associations has been challenging1-3. Akkermansia muciniphila has been robustly associated with positive systemic effects on host metabolism, favourable outcomes to checkpoint blockade in cancer immunotherapy and homeostatic immunity4-7. Here we report the identification of a lipid from A. muciniphila's cell membrane that recapitulates the immunomodulatory activity of A. muciniphila in cell-based assays8. The isolated immunogen, a diacyl phosphatidylethanolamine with two branched chains (a15:0-i15:0 PE), was characterized through both spectroscopic analysis and chemical synthesis. The immunogenic activity of a15:0-i15:0 PE has a highly restricted structure-activity relationship, and its immune signalling requires an unexpected toll-like receptor TLR2-TLR1 heterodimer9,10. Certain features of the phospholipid's activity are worth noting: it is significantly less potent than known natural and synthetic TLR2 agonists; it preferentially induces some inflammatory cytokines but not others; and, at low doses (1% of EC50) it resets activation thresholds and responses for immune signalling. Identifying both the molecule and an equipotent synthetic analogue, its non-canonical TLR2-TLR1 signalling pathway, its immunomodulatory selectivity and its low-dose immunoregulatory effects provide a molecular mechanism for a model of A. muciniphila's ability to set immunological tone and its varied roles in health and disease.

Evaluation of different combination of pam2CSK4, poly (I:C) and imiquimod enhance immune responses to H9N2 avian influenza antigen in dendritic cells and duck.

Current commercial H9 avian influenza viruses (AIVs) vaccines cannot provide satisfactory antibody titers and protective immunity against AIVs in duck. Toll like receptors (TLR) ligand as AIVs adjuvants can activate dendritic cells to improve immune responses in multiple animals, while the studies were absent in duck. Therefore, we investigated TLR ligands pam2CSK4, poly (I:C) and/or imiquimod enhance immune responses to inactivated H9N2 avian influenza antigen (H9N2 IAIV) in peripheral blood monocyte-derived dendritic cells (MoDCs) and duck. In vitro, we observed that transcription factor NF-κB, Th1/Th2 type cytokines (IFN-γ, IL-2 and IL-6) and the ability of catching H9N2 IAIV antigen were significantly up-regulated when H9N2 IAIV along with TLR ligands (pam2CSK4, poly (I:C) and imiquimod, alone or combination) in duck MoDCs. Also, the best enhancement effects were showed in combination of pam2CSK4, poly (I:C) and imiquimod group, whereas IFN-α showed no significant enhancement in all experimental groups. In vivo, the results demonstrated that the percentages of CD4+/ CD8+ T lymphocytes, the levels of Th1/Th2 type cytokines and H9N2 HI titers were significant enhanced in combination of pam2CSK4, poly (I:C) and imiquimod group. However, pam2CSK4 alone or combining with imiquimod showed no enhancement or additive effects on Th1 cytokines (IFN-γ and IL-2), Th2 cytokines (IL-6) and HI titers in Muscovy duck, respectively. Taken together, our results concluded that not all TLR ligands showed enhancement of immune responses to H9N2 IAIV in duck. The combination of poly (I:C), imiquimod and pam2CSK4 that can be an effectively adjuvant candidate for H9N2 AIVs inactivated vaccine in duck, which provide novel insights in explore waterfowl vaccine.

IL-1ß expression in bone marrow dendritic cells is induced by TLR2 agonists and regulates HSC function.

Hematopoietic stem/progenitor cells (HSPCs) reside in localized microenvironments, or niches, in the bone marrow that provide key signals regulating their activity. A fundamental property of hematopoiesis is the ability to respond to environmental cues such as inflammation. How these cues are transmitted to HSPCs within hematopoietic niches is not well established. Here, we show that perivascular bone marrow dendritic cells (DCs) express a high basal level of Toll-like receptor-1 (TLR1) and TLR2. Systemic treatment with a TLR1/2 agonist induces HSPC expansion and mobilization. It also induces marked alterations in the bone marrow microenvironment, including a decrease in osteoblast activity and sinusoidal endothelial cell numbers. TLR1/2 agonist treatment of mice in which Myd88 is deleted specifically in DCs using Zbtb46-Cre show that the TLR1/2-induced expansion of multipotent HPSCs, but not HSPC mobilization or alterations in the bone marrow microenvironment, is dependent on TLR1/2 signaling in DCs. Interleukin-1ß (IL-1ß) is constitutively expressed in both murine and human DCs and is further induced after TLR1/2 stimulation. Systemic TLR1/2 agonist treatment of Il1r1-/- mice show that TLR1/2-induced HSPC expansion is dependent on IL-1ß signaling. Single-cell RNA-sequencing of low-risk myelodysplastic syndrome bone marrow revealed that IL1B and TLR1 expression is increased in DCs. Collectively, these data suggest a model in which TLR1/2 stimulation of DCs induces secretion of IL-1ß and other inflammatory cytokines into the perivascular niche, which in turn, regulates multipotent HSPCs. Increased DC TLR1/2 signaling may contribute to altered HSPC function in myelodysplastic syndrome by increasing local IL-1ß expression.

Next-Generation Diprovocims with Potent Human and Murine TLR1/TLR2 Agonist Activity That Activate the Innate and Adaptive Immune Response.

The diprovocims, a new class of toll-like receptor (TLR) agonists, bear no similarity to prior TLR agonists, act through a well-defined mechanism (TLR1/TLR2 agonist), exhibit exquisite structure-activity relationships, and display in vivo adjuvant activity. They possess potent and efficacious agonist activity toward human TLR1/TLR2 but modest agonism toward the murine receptor. A manner by which diprovocims can be functionalized without impacting hTLR1/TLR2 activity is detailed, permitting future linkage to antigenic, targeting, or delivery moieties. Improvements in both potency and its low efficacy in the murine system were also achieved, permitting more effective use in animal models while maintaining the hTLR1/TLR2 activity. The prototypical member diprovocim-X exhibits the excellent potency/efficacy of diprovocim-1 in human cells, displays substantially improved potency/efficacy in mouse macrophages, and serves as an adjuvant in mice when coadministered with a nonimmunogenic antigen, indicating stimulation of the adaptive as well as innate immune response.

A MademoiseLLE domain binding platform links the key RNA transporter to endosomes.

Spatiotemporal expression can be achieved by transport and translation of mRNAs at defined subcellular sites. An emerging mechanism mediating mRNA trafficking is microtubule-dependent co-transport on shuttling endosomes. Although progress has been made in identifying various components of the endosomal mRNA transport machinery, a mechanistic understanding of how these RNA-binding proteins are connected to endosomes is still lacking. Here, we demonstrate that a flexible MademoiseLLE (MLLE) domain platform within RNA-binding protein Rrm4 of Ustilago maydis is crucial for endosomal attachment. Our structure/function analysis uncovered three MLLE domains at the C-terminus of Rrm4 with a functionally defined hierarchy. MLLE3 recognises two PAM2-like sequences of the adaptor protein Upa1 and is essential for endosomal shuttling of Rrm4. MLLE1 and MLLE2 are most likely accessory domains exhibiting a variable binding mode for interaction with currently unknown partners. Thus, endosomal attachment of the mRNA transporter is orchestrated by a sophisticated MLLE domain binding platform.

Uterus globulin associated protein 1 (UGRP1) binds podoplanin (PDPN) to promote a novel inflammation pathway during Streptococcus pneumoniae infection.

BACKGROUND: Streptococcus pneumoniae is the major cause of life-threatening infections. Toll-like receptors (TLRs) and NOD-like receptors (NLRs) could recognise S. pneumoniae and regulate the production of pro-inflammatory cytokines. UGRP1, highly expressed in lung, is predominantly secreted in airways. However, the function of UGRP1 in pneumonia is mainly unknown. METHODS AND RESULTS: We showed that upon TLR2/TLR4/NOD2 agonists stimulation or S. pneumoniae infection, treatment with UGRP1 could promote phosphorylation of p65 and enhance IL-6, IL-1ß and TNFα production in macrophages. We further elucidated that after binding with cell-surface receptor PDPN, UGRP1 could activate RhoA to enhance interaction of IKKγ and IKKß, which slightly activated NF-κB to improve expression of TLR2, MyD88, NOD2 and NLRP3. Deletion of UGRP1 or blocking UGRP1 interaction with PDPN protected mice against S. pneumoniae-induced severe pneumococcal pneumonia, and activating RhoA with agonist in UGRP1-deficient mice restored the reduced IL-6 production. CONCLUSION: We demonstrated that UGRP1-PDPN-RhoA signaling could activate NF-κB to promote expression of TLR2, MyD88, NOD2 and NLRP3, which enhanced inflammatory cytokines secretion during S. pneumoniae infection. Antibodies, which could interrupt interaction of UGRP1 and PDPN, are potential therapeutics against S. pneumoniae.

Extracellular Ribosomal RNA Acts Synergistically with Toll-like Receptor 2 Agonists to Promote Inflammation.

Self-extracellular RNA (eRNA), which is released under pathological conditions from damaged tissue, has recently been identified as a new alarmin and synergistic agent together with toll-like receptor (TLR)2 ligands to induce proinflammatory activities of immune cells. In this study, a detailed investigation of these interactions is reported. The macrophage cell line J774 A.1 or C57 BL/6 J wild-type mice were treated with 18S rRNA and different TLR2 agonists. Gene and protein expression of tumor necrosis factor (Tnf)-α; interleukin (Il)-1ß, Il-6; or monocyte chemoattractant protein (Mcp)-1 were analyzed and furthermore in vitro binding studies to TLR2 were performed. The TLR2/TLR6-agonist Pam2 CSK4 (Pam2) together with 18S rRNA significantly increased the mRNA expression of inflammatory genes and the release of TNF-α from macrophages in a TLR2- and nuclear factor kappa B (NF-κB)-dependent manner. The injection of 18S rRNA/Pam2 into mice increased the cytokine levels of TNF-α, IL-6, and MCP-1 in the peritoneal lavage. Mechanistically, 18S rRNA built complexes with Pam2 and thus enhanced the affinity of Pam2 to TLR2. These results indicate that the alarmin eRNA, mainly consisting of rRNA, sensitizes TLR2 to enhance the innate immune response under pathological conditions. Thus, rRNA might serve as a new target for the treatments of bacterial and viral infections.

The transcription factor Hhex regulates inflammation-related genes in microglia.

Microglia have diverse physiological and pathological functions. However, the transcriptional mechanisms remain elusive. Here we sought new transcription factors relevant to microglial functions from the microglial transcriptome of stressed mice and evaluated their roles in primary microglia. TLR2 and TLR4 agonists increased Rel, Atf3, and Cebpb and decreased Hhex in primary microglia as repeated social defeat stress. Although Hhex was not studied in microglia, TLR2 and TLR4 agonists decreased Hhex, and Hhex overexpression attenuated TLR4-increased expression of inflammation-related genes. These findings suggest that Hhex negatively regulates inflammation-related genes in microglia and that TLR2/4 activation reduces Hhex, facilitating TLR4-mediated neuroinflammation.

Evidence of a role for interleukin-6 in anoikis resistance in oral squamous cell carcinoma.

In an endeavour to understand metastasis from oral squamous cell carcinomas, we characterised the metastatic potential of a human tongue derived cell line (SCC-4 cells) and compared this phenotype to pre-cancerous dysplastic oral keratinocyte (DOK) cells derived from human tongue and primary gingival keratinocytes (PGK). We demonstrate that SCC-4 cells constitutively synthesize and release significant amounts of IL-6, a process that is enhanced by the addition of the TLR2/TLR6 agonist, Pam2CSK4. The expression of TLR2/6 and IL-6Ra/gp130 receptors was also confirmed in SCC-4 cells. Cancerous SCC-4 human tongue cells also have a classic EMT profile, unlike precancerous human tongue DOK cells. We also established that IL-6 is driving anoikis resistance in an autocrine fashion and that anti-IL-6 neutralising antibodies, anti-IL-6 receptor antibodies and anti-TLR2 receptor antibodies inhibit anoikis resistance in cancerous SCC-4 human tongue cells. The data suggest a promising role for anti-IL-6 receptor antibody and anti-TLR2 receptor antibody treatment for oral cancer.

Propionate induces cross-tolerance to TLR1/2 and TLR4 agonists in an IFIT-dependent manner.

In this study, we have identified Interferon-stimulated genes (ISGs), especially IFIT1, 2 and 3, as target genes of propionate-induced signalling in the human epithelial cell line A549, the monocytic cell line THP-1 as well as in primary, human peripheral blood-derived macrophages (PBMs). Induction of the IFIT gene family by propionate negatively regulates TLR-induced signalling. Propionate stimulation results in downregulation of pro-inflammatory cytokine and chemokine expression as well as MHC class II expression upon TLR1/2 and TLR4 re-stimulation in A549 and THP-1 cells as well as in PBMs, demonstrating that propionate-induced signalling is involved in the induction of TLR cross-tolerance. Signalling pathway analysis clearly demonstrates that propionate-induced IFIT expression is mediated by FFAR2 in a Gαq/11 signalling pathway-dependent manner. Furthermore, propionate-induced IFIT expression is dependent on IFN type I and/or type III-mediated signalling since pre-treatment of A549 cells with Ruxolitinib, a specific JAK1/2 tyrosine kinase inhibitor, prior to stimulation with propionate, inhibited the upregulation of IFIT1 expression. The hypo-responsiveness towards TLR1/2 and TLR4 agonists seems to be mediated by different members of the IFIT gene family in a cell type-specific manner. Collectively, our data indicate that propionate-induced signalling controls pro-inflammatory responses by activation of IFN type I and/or type III-induced and IFIT-mediated counter-regulatory mechanisms in order to protect against exacerbating inflammatory reactions.

Toll-like receptor 2 and 6 agonist fibroblast-stimulating lipopeptide increases expression and secretion of CXCL1 and CXCL2 by uveal melanocytes.

Fibroblast-stimulating lipopeptide (FSL-1) can activate Toll-like receptor 2 and 6 (TLR2/6), which recognize relevant molecules from gram-positive pathogens, fungus, and mycoplasma, and elevates the expression of CXCL1 and CXCL2, neutrophil chemoattractants, in certain types of cells. This effect has not previously been reported in the uveal melanocytes (UM). This study was designed to test the hypothesis that FSL-1 can induce the expression and secretion of CXCL1 and CXCL2 via activation of TLR2/6 in cultured human UM and producing an acute non-infectious uveitis reaction in the mouse. Flow cytometry and fluorescent immunostaining were used to measure the effect of FSL-1 on the expression of TLR2/6 in UM. Real time PCR and ELISA analysis were used to assess the ability of FSL-1 to elevate CXCL1/CXCL2 levels in cell lysates and conditioned media of UM, respectively. Flow cytometry measured phosphorylated MAPK and activated NF-κB signals in UM, with and without FSL-1 treatment. ELISA analysis tested the impact of various signal inhibitors (NF-κB, p38 MAPK, JNK1/2 and ERK1/2) and TLR2/6 antagonists on FSL-1-induced CXCL1/CXCL2 levels in cultured UM. The effects of neutralizing antibodies to TLR2 on FSL-1-induced mouse uveitis were tested in an experimental animal model. FSL-1 induced the expression of TLR2/6 proteins in cultured UM. FSL-1 significantly elevated the CXCL1 and CXCL2 proteins and mRNA levels in cultured UM time- and dose-dependently. FSL-1 mainly activated NF-κB, JNK, and expression of TLR2. FSL-1-induced expression of CXCL1 and CXCL2 was blocked by NF-κB, JNK, ERK inhibitors and TLR2 antagonists. Intravitreal injection of FSL-1 induced acute non-infectious mouse uveitis, which was significantly reduced in severity by a TLR2 antagonist. These results suggest that UM may play a role in the immune reaction, which targets invading pathogens, especially gram-positive bacteria. On the other hand, an excessive reaction to molecules from gram-positive bacteria may promote an inflammatory state of non-infectious uveitis.

Platelet-Rich Fibrin Decreases the Inflammatory Response of Mesenchymal Cells.

Chronic inflammation is a pathological process where cells of the mesenchymal lineage become a major source of inflammatory mediators. Platelet-rich fibrin (PRF) has been shown to possess potent anti-inflammatory activity in macrophages, but its impact on mesenchymal cells has not been investigated. The aim of this study was, therefore, to expose mesenchymal cells to inflammatory cytokines together with lysates generated from liquid platelet-poor plasma (PPP), the cell-rich buffy coat layer (BC; concentrated-PRF or C-PRF), and the remaining red clot layer (RC), following centrifugation of blood. Heating PPP generates an albumin gel (Alb-gel) that when mixed back with C-PRF produces Alb-PRF. Membranes prepared from solid PRF were also subjected to lysis. We report here that lysates of PPP, BC, and PRF decreased the cytokine-induced expression of interleukin 6 (IL6) and nitric oxide synthase (iNOS) in the bone marrow-derived ST2 cells. Consistently, PPP, BC, and PRF greatly decreased the phosphorylation and nuclear translocation of p65 in ST2 cells. The inflammatory response caused by Pam3CSK4 was reduced accordingly. Moreover, PPP, BC, and PRF reduced the enhanced expression of inflammatory mediators IL6 and iNOS in 3T3-L1 pre-adipocyte mesenchymal cells, and iNOS and CCL5 in murine calvarial cells. Surprisingly, PRF lysates were not effective in reducing the inflammatory response of human gingival fibroblasts and HSC2 epithelial cells. The data from the present study suggest that both liquid PRF and solid PRF exert potent anti-inflammatory activity in murine mesenchymal cells.

NK Cell-Dependent Antibody-Mediated Immunotherapy Is Improved In Vitro and In Vivo When Combined with Agonists for Toll-like Receptor 2 in Head and Neck Cancer Models.

The immunosuppressive character of head and neck cancers may explain the relatively low response rates to antibody therapy targeting a tumor antigen, such as cetuximab, and anti-PD-1 checkpoint inhibition. Immunostimulatory agents that overcome tumor-derived inhibitory signals could augment therapeutic efficacy, thereby enhancing tumor elimination and improving patient survival. Here, we demonstrate that cetuximab treatment combined with immunostimulatory agonists for Toll-like receptor (TLR) 2 induces profound immune responses. Natural killer (NK) cells, isolated from healthy individuals or patients with head and neck cancer, harbored enhanced cytotoxic capacity and increased tumor-killing potential in vitro. Additionally, combination treatment increased the release of several pro-inflammatory cytokines and chemokines by NK cells. Tumor-bearing mice that received cetuximab and the TLR2 ligand Pam3CSK4 showed increased infiltration of immune cells into the tumors compared to mice that received cetuximab monotherapy, resulting in a significant delay in tumor growth or even complete tumor regression. Moreover, combination treatment resulted in improved overall survival in vivo. In conclusion, combining tumor-targeting antibody-based immunotherapy with TLR stimulation represents a promising treatment strategy to improve the clinical outcomes of cancer patients. This treatment could well be applied together with other therapeutic strategies such as anti-PD-(L)1 checkpoint inhibition to further overcome immunosuppression.