Identification of TLR2 Signalling Mechanisms Which Contribute to Barrett’s and Oesophageal Adenocarcinoma Disease Progression

Chronic inflammation plays an important role in the pathogenesis of oesophageal adenocarcinoma (EAC) and its only known precursor, Barrett’s oesophagus (BE). Recent studies have shown that oesophageal TLR2 levels increase from normal epithelium towards EAC. TLR2 signalling is therefore likely to be important during EAC development and progression, which requires an inflammatory microenvironment. Here, we show that, in response to TLR2 stimulation, BE organoids and early-stage EAC cells secrete pro-inflammatory cytokines and chemokines which recruit macrophages to the tumour site. Factors secreted from TLR2-stimulated EAC cells are shown to subsequently activate TLR2 on naïve macrophages, priming them for inflammasome activation and inducing their differentiation to an M2/TAM-like phenotype. We identify the endogenous TLR2 ligand, HMGB1, as the factor secreted from EAC cells responsible for the observed TLR2-mediated effects on macrophages. Our results indicate that HMGB1 signalling between EAC cells and macrophages creates an inflammatory tumour microenvironment to facilitate EAC progression. In addition to identifying HMGB1 as a potential target for early-stage EAC treatment, our data suggest that blocking TLR2 signalling represents a mechanism to limit HMGB1 release, inflammatory cell infiltration and inflammation during EAC progression.

Macrophage cytokine responses to commensal Gram-positive Lactobacillus salivarius strains are TLR2-independent and Myd88-dependent

The mechanisms through which cells of the host innate immune system distinguish commensal bacteria from pathogens are currently unclear. Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) expressed by host cells which recognize microbe-associated molecular patterns (MAMPs) common to both commensal and pathogenic bacteria. Of the different TLRs, TLR2/6 recognize bacterial lipopeptides and trigger cytokines responses, especially to Gram-positive and Gram-negative pathogens. We report here that TLR2 is dispensable for triggering macrophage cytokine responses to different strains of the Gram-positive commensal bacterial species Lactobacillus salivarius. The L. salivarius UCC118 strain strongly upregulated expression of the PRRs, Mincle (Clec4e), TLR1 and TLR2 in macrophages while downregulating other TLR pathways. Cytokine responses triggered by L. salivarius UCC118 were predominantly TLR2-independent but MyD88-dependent. However, macrophage cytokine responses triggered by another Gram-positive commensal bacteria, Bifidobacterium breve UCC2003 were predominantly TLR2-dependent. Thus, we report a differential requirement for TLR2-dependency in triggering macrophage cytokine responses to different commensal Gram-positive bacteria. Furthermore, TNF-α responses to the TLR2 ligand FSL-1 and L. salivarius UCC118 were partially Mincle-dependent suggesting that PRR pathways such as Mincle contribute to the recognition of MAMPs on distinct Gram-positive commensal bacteria. Ultimately, integration of signals from these different PRR pathways and other MyD88-dependent pathways may determine immune responses to commensal bacteria at the host-microbe interface.

Bacterial Lipoteichoic Acid Attenuates Toll-Like Receptor Dependent Dendritic Cells Activation and Inflammatory Response

Toll-like receptor (TLR) signaling is an indispensable factor in immune cells activation. Many TLR ligands have been identified, and were characterized the immunological functions such as inflammatory cytokine production in immune cells. However, the anti-inflammatory response in TLR ligand-mediated manner is poorly understood. In this report, we show that bacterial lipoteichoic acid (LTA), which is a TLR2 ligand from gram-positive bacteria including Staphylococcus aureus (S. aureus), suppresses TLR-mediated inflammatory response in dendritic cells (DCs). The TLR ligand-induced Tumor Necrosis Factor-alpha (TNF-α) production was suppressed in the bone marrow derived dendritic cells (BMDCs) by co-treatment of LTA. The cellular activation, which was characterized as upregulations of CD80, CD86 and major histocompatibility complex II (MHC II) expression, was also suppressed in the TLR ligand stimulated BMDCs in the presence of LTA. While LTA itself didn’t induced both TNF-α production and upregulation of cell surface markers. The LTA mediated immunosuppressive function was abolished by TLR2 blocking in lipopolysaccharide (LPS)-stimulated BMDCs. Furthermore, LTA also showed the immunosuppressive function in the generation of IFN-γ+CD4+ T (Th1) cells by attenuation of antigen presenting activity in the BMDCs. In the imiquimod (IMQ)-induced acute skin inflammation, LTA suppressed the inflammation by downregulation of the activation in skin accumulated DCs. Thus, LTA is a TLR2 dependent immunological suppressor against inflammatory response induced by other TLR ligands in the DCs.

Combination of Innate Immune Modulators as Vaccine Adjuvants in Mice

The development of new, effective, and safe vaccines necessarily requires the identification of new adjuvant(s) to enhance the potency and longevity of antigen-specific immune responses. In the present study, we compare the antibody-mediated and cell-mediated immune (CMI) responses within groups of mice vaccinated subcutaneously with ovalbumin (OVA; as an experimental antigen) plus polyphosphazene (an innate immune modulator), Polyinosinic:polycytidylic acid (poly-I:C; (an RNA mimetic) and glycopeptide ARC5 (which is a Toll-like receptor (TLR), TLR2 ligand and PAM3CSK4 analogue) formulated together in a soluble vaccine. We also investigated the effect of a polymeric nanoparticle of ARC4 and ARC7 (which are a novel muramyl dipeptide analogue and a monophosophoryl lipid A (MPLA) analogue, respectively) plus OVA +/− ARC5 as a subcutaneous vaccine in mice. OVA+ARC4/ARC7 nanoparticle +/− ARC5 triggered a robust and balanced Th1/Th2-type humoral response with significant anti-OVA IgA in serum, and significant interferon (IFN)-γ and interleukin (IL)-17 production in splenocytes after 35 days relative to the controls. Formulation of OVA with ARC4/ARC7 nanoparticles should be investigated for inducing protective immunity against infectious pathogens in mice and other species.

A novel phosphoglycerol serine-glycine lipodipeptide of Porphyromonas gingivalis is a TLR2 ligand

Porphyromonas gingivalis is a Gram-negative anaerobic periodontal microorganism strongly associated with tissue-destructive processes in human periodontitis. Following oral infection with P. gingivalis, the periodontal bone loss in mice is reported to require the engagement of Toll-like receptor 2 (TLR2). Serine-glycine lipodipeptide or glycine aminolipid classes of P. gingivalis engage human and mouse TLR2, but a novel lipid class reported here is considerably more potent in engaging TLR2 and the heterodimer receptor TLR2/TLR6. The novel lipid class, termed Lipid 1256, consists of a diacylated phosphoglycerol moiety linked to a serine-glycine lipodipeptide previously termed Lipid 654. Lipid 1256 is approximately 50-fold more potent in engaging TLR2 than the previously reported serine-glycine lipid classes. Lipid 1256 also stimulates cytokine secretory responses from peripheral blood monocytes and is recovered in selected oral and intestinal Bacteroidetes organisms. Therefore, these findings suggest that Lipid 1256 may be a microbial TLR2 ligand relevant to chronic periodontitis in humans.

Recombinant protein rMBP-NAP restricts tumor progression by triggering antitumor immunity in mouse metastatic lung cancer

Recombinant Helicobacter pylori neutrophil-activating protein fused with maltose-binding protein (rMBP-NAP), a potential TLR2 ligand, was reported to possess immunomodulatory effects on in situ tumors in our previous study. In the present work, we attempt to elucidate the effect of rMBP-NAP at the local immune modulation in B16-F10-induced metastatic lung cancer. Our results demonstrated that growth of B16-F10 melanoma metastases in the lung was significantly arrested after rMBP-NAP treatment, along with marked reduction in metastatic lung nodules and significant increase in survival. The treatment induced both local and systemic immune responses, which were associated with higher influx of CD4+/CD8+ T cells and drove toward Th1-like and cytotoxic immune environment. Moreover, rMBP-NAP also showed significant anti-angiogenic activity by reducing vascularization in lung tumor sections. rMBP-NAP could induce antitumor immunity through activating Th1 cells and producing pro-inflammatory cytokines, which are responsible for the effective cytotoxic immune response against cancer progression. Our findings indicate that rMBP-NAP might be a novel antitumor therapeutic strategy.

Time-lapse monitoring of TLR2 ligand internalization with newly developed fluorescent probes

Bacterial lipopeptide fluorescent probes were developed as TLR2 ligands, and their time-lapse monitoring of cellular internalization was performed.