Tumor Microenvironment-Regulating Immunosenescence-Independent Nanostimulant Synergizing with Near-Infrared Light Irradiation for Antitumor Immunity.

The combination of photothermal therapy (PTT) and toll-like receptor (TLR)-mediated immunotherapy can elicit antitumor immunity and modulate the immunosuppressive tumor microenvironment (TME). Unlike other TLRs, TLR-5 is a promising target for immune activation, as its expression is well-maintained even during immunosenescence. Here, we developed a unique tumor microenvironment-regulating immunosenescence-independent nanostimulant consisting of TLR-5 adjuvant Vibrio vulnificus flagellin B (FlaB) conjugated onto the surface to an IR 780-loaded hyaluronic acid-stearylamine (HIF) micelles. These HIF micelles induced immune-mediated cell death via PTT when irradiated with a near-infrared laser. In comparison with PTT alone, the combination of in situ-generated tumor-associated antigens produced during PTT and the immune adjuvant FlaB demonstrated enhanced vaccine-like properties and modulated the TME by suppressing immune-suppressive regulatory cells (Tregs) and increasing the fraction of CD103+ migratory dendritic cells, which are responsible for trafficking tumor antigens to draining lymph nodes (DLNs). This combinatorial strategy (i.e., applying a TLR-5 adjuvant targeted to immunosenescence-independent TLR-5 and the in situ photothermal generation of tumor-associated antigens) is a robust system for next-generation immunotherapy and could even be applied in elderly patients, thus broadening the clinical scope of immunotherapy strategies.

Mitigation of Radiation-Induced Epithelial Damage by the TLR5 Agonist Entolimod in a Mouse Model of Fractionated Head and Neck Irradiation.

Radiation treatment of head and neck cancer frequently causes severe collateral damage to normal tissues including mouth mucosa, salivary glands and skin. This toxicity limits the radiation dose that can be delivered and affects the patient's quality of life. Previous studies in mice and nonhuman primates showed that entolimod, a toll-like receptor 5 (TLR5) agonist derived from bacterial flagellin, effectively reduced radiation damage to hematopoietic and gastrointestinal tissues in both total-body and local irradiation scenarios, with no protection of tumors. Here, using a mouse model, we analyzed the efficacy of entolimod administered before or after irradiation in reducing damage to normal tissues. Animals received local fractionated radiation to the head and neck area, thus modeling radiotherapy of head and neck cancer. Tissue damage was evaluated through histomorphological examination of samples collected at different time points up to four weeks, mice were exposed locally to five daily fractions of 5, 6 or 7 Gy. A semiquantitative scoring system was used to assess the severity of observed pathomorphological changes. In this model, radiation damage was most severe in the lips, tongue and skin, moderate in the upper esophagus and minor in salivary glands. The kinetics of injury appearance and recovery of normal morphology varied among tissues, with maximal damage to the tongue, esophagus and salivary glands developing at earlier times (days 8-11 postirradiation) relative to that of lip and skin mucosa (days 11-15 postirradiation). While both tested regimens of entolimod significantly reduced the extent of radiation damage and accelerated restoration of normal structure in all tissues analyzed, administration of entolimod 1 h after each irradiation was more effective than treatment 30 min before irradiation. These results support the potential clinical use of entolimod as an adjuvant for improving the therapeutic index of head and neck cancer radiotherapy by reducing the radiation toxicity in normal tissues.

Fragment Pharmacophore-Based Screening: An Efficient Approach for Discovery of New Inhibitors of Toll-Like Receptor 5.

AIM AND OBJECTIVE: Rheumatoid Arthritis (RA) is a progressing autoimmune inflammatory disease of joint, hallmarked by inflammation, pain and atrophy of bones. Toll-like receptor 5 (TLR5) is a novel inflammatory mediator in RA, and TLR5 inhibitors are speculated to have a therapeutic potential for the treatment of RA. MATERIAL AND METHOD: Here we applied fragment pharmacophore-based virtual screening to identify novel TLR5 ligands. RESULTS: Among compounds collected from Otava peptidomimetic compounds, Maybridge fragment and ZINC libraries, 3355 compounds were selected for docking into the flagellin-binding site of TLR5. 16 compounds with the required interaction, critical amino acid residues and the binding free energies <-7 kcal/mol were identified as potential TLR5 inhibitors, one of which was followed up by molecular dynamics simulation. CONCLUSION: These compounds open a possibility to discover novel TLR5 inhibitors for the treatment of RA.

Pyrimidine Triazole Thioether Derivatives as Toll-Like Receptor†5 (TLR5)/Flagellin Complex Inhibitors.

Protein-protein interactions have been regarded as "undruggable" despite their importance in many biological processes. The complex formed between host toll-like receptor 5 (TLR5) and flagellin, a globular protein that is the main component of a bacterial flagellum, plays a vital role in a number of pathogen defenses, immunological diseases and cancers. Through high-throughput screening, we identified two hits with a common pharmacophore, which were used to successfully develop a series of small-molecule probes as novel inhibitors of flagellin binding to TLR5. In a multitude of assays, 4-((4-benzyl-5-(pyridin4yl)-4H-1,2,4-triazol-3-yl)thio)pyrido[3',2':4,5]thieno[3,2-d]pyrimidine (TH1020) was identified as a potent antagonist of TLR5 signaling with promising activity (IC50 =0.85±0.12 µm) and specificity. Furthermore, TH1020 was shown to repress the expression of downstream TNF-α signaling pathways mediated by the TLR5/flagellin complex formation. Based on molecular docking simulation, TH1020 is suggested to compete with flagellin and disrupt its association with TLR5. TH1020 provides a much-needed molecular probe for studying this important protein-protein interaction and a lead compound for identifying novel therapeutics targeting TLR5.

mTORC1 Regulates Flagellin-Induced Inflammatory Response in Macrophages.

Bacterial flagellin triggers inflammatory responses. Phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) regulate the production of pro- and anti-inflammatory cytokines that are induced by extrinsic antigens, but the function of mTORC1 in flagellin-induced inflammatory response is unknown. The purpose of this study was to examine the role and the mechanism of PI3K/Akt/mTOR pathway in flagellin-induced cytokine expression in mouse macrophages. We observed that flagellin upregulated TNF-α time- and dose-dependently. Flagellin stimulated rapid (<15 min) PI3K/Akt/mTOR phosphorylation that was mediated by TLR5. Inhibition of PI3K with LY294002 and wortmannin, and of mTORC1 with rapamycin decreased flagellin-induced TNF-α and IL-6 expression and cell proliferation. The activation of NF-κB p65 and STAT3 was regulated by mTORC1 via degradation of IκBα and phosphorylation of STAT3 in response to flagellin, respectively. Thus, the PI3K/Akt/mTORC1 pathway regulates the innate immune response to bacterial flagellin. Rapamycin is potential therapy that can regulate host defense against pathogenic infections.

Odanacatib, A Cathepsin K-Specific Inhibitor, Inhibits Inflammation and Bone Loss Caused by Periodontal Diseases.

BACKGROUND: Periodontitis is a bacteria-induced inflammatory disease mainly affecting periodontal tissues, leading to periodontal inflammation, bone breakdown, and loss of the tooth. The main obstacle for treating periodontitis effectively is the difficulty in finding a target that can inhibit bone loss and inflammation simultaneously. Recent studies showed that cathepsin K (CTSK) might have functions in the immune system besides its role in osteoclasts. Thus, targeting CTSK would have a potential therapeutic effect in both the bone system and the immune system during the progression of periodontitis. METHODS: In the current study, a small molecular inhibitor (odanacatib [ODN]) is explored to inhibit the function of CTSK in a bacteria-induced periodontitis mouse model. RESULTS: The application of ODN decreased the number of osteoclasts, macrophages, and T cells, as well as the expression of Toll-like receptors (TLRs) in the periodontitis lesion area. Furthermore, lack of CTSK inhibited the expression of TLR4, TLR5, and TLR9 and their downstream cytokine signaling in the gingival epithelial cells in periodontitis lesions, demonstrating that the innate immune response was inhibited in periodontitis. CONCLUSION: The present results show that inhibition of CTSK can prevent bone loss and the immune response during the progression of periodontitis, indicating that CTSK is a promising target for treating inflammatory diseases such as periodontitis by affecting both osteoclasts and the immune system.

The TLR2 ligand FSL-1 and the TLR5 ligand Flagellin mediate pro-inflammatory and pro-labour response via MyD88/TRAF6/NF-κB-dependent signalling.

PROBLEM: Toll-like receptors (TLRs) 2 and 5 induce inflammation via the adapter proteins myeloid differentiation factor 88 (MyD88) and TNFR-associated factor 6 (TRAF6) and the transcription factor nuclear factor-kappa B (NF-κB). The aims of this study were to determine the effects of the TLR5 ligand flagellin and the TLR2 ligand FSL-1 on pro-inflammatory and pro-labour mediators in human fetal membranes and myometrium, and to establish whether their actions are dependent on MyD88, TRAF6 and NF-κB. METHOD OF STUDY: Tissue explants were performed to determine the effect of flagellin and FSL-1 on pro-labour mediators in fetal membranes and myometrium. siRNA knockdown was performed in primary amnion and myometrium cells to determine the role of MyD88, TRAF6 and NF-κB. RESULTS: Flagellin and FSL-1 increased pro-inflammatory cytokines (IL-6 and IL-8), MMP-9 expression and activity, and COX-2 expression and prostaglandin release. siRNA knockdown of TLR2 decreased FSL-1 induced production of IL-6, IL-8, COX-2, prostaglandins and MMP-9; similarly, siRNA knockdown of TLR5 decreased flagellin induced production of these pro-labour mediators. The effects of flagellin and FSL-1 are mediated by MyD88 and TRAF6, as siRNA knockdown of MyD88 and TRAF6 decreased flagellin and FSL-1 induced pro-labour mediators. Additionally, the effects of flagellin and FSL-1 are mediated via NF-κB, as flagellin and FSL-1 increased NF-κB transcriptional activity, and the NF-κB inhibitor BAY 11-7082 attenuated flagellin and FSL-1 induced expression and secretion of pro-labour mediators. CONCLUSION: TLR2 engagement by the synthetic lipoprotein FSL-1 and TLR5 engagement by bacterial flagellin enhances pro-inflammatory and pro-labour mediators in human fetal membranes and myometrium via MyD88/TRAF6/NF-κB.

TLR5 signaling enhances the proliferation of human allogeneic CD40-activated B cell induced CD4hiCD25+ regulatory T cells.

Although diverse functions of different toll-like receptors (TLR) on human natural regulatory T cells have been demonstrated recently, the role of TLR-related signals on human induced regulatory T cells remain elusive. Previously our group developed an ex vivo high-efficient system in generating human alloantigen-specific CD4(hi)CD25(+) regulatory T cells from naïve CD4(+)CD25(-) T cells using allogeneic CD40-activated B cells as stimulators. In this study, we investigated the role of TLR5-related signals on the generation and function of these novel CD4(hi)CD25(+) regulatory T cells. It was found that induced CD4(hi)CD25(+) regulatory T cells expressed an up-regulated level of TLR5 compared to their precursors. The blockade of TLR5 using anti-TLR5 antibodies during the co-culture decreased CD4(hi)CD25(+) regulatory T cells proliferation by induction of S phase arrest. The S phase arrest was associated with reduced ERK1/2 phosphorylation. However, TLR5 blockade did not decrease the CTLA-4, GITR and FOXP3 expressions, and the suppressive function of CD4(hi)CD25(+) regulatory T cells. In conclusion, we discovered a novel function of TLR5-related signaling in enhancing the proliferation of CD4(hi)CD25(+) regulatory T cells by promoting S phase progress but not involved in the suppressive function of human CD40-activated B cell-induced CD4(hi)CD25(+) regulatory T cells, suggesting a novel role of TLR5-related signals in the generation of induced regulatory T cells.

Toll-like receptor 5 engagement induces interleukin-17C expression in intestinal epithelial cells.

The family of interleukin-17 (IL-17) cytokine is the essential inflammatory mediator that influences the pathophysiology of various inflammatory diseases. Many studies focused on investigating the expression, signaling, and biological impacts of IL-17A and IL-17F, and the neutralization of these cytokines exhibited some promising results in clinical trials. In contrast, the expression resources and physiological relevance of IL-17C remained to be studied. In this study, through a microarray approach conducted with nontransformed human colonic epithelial cells (NCM460), we found that bacterial flagellin stimulation elicited potent IL-17C mRNA expression. We also confirmed that IL-17C protein production was strongly induced by flagellin in these cells. Flagellin-induced IL-17C expression was also observed in human colon adenocarcinoma cells such as DLD-1 and HT-29, indicating that IL-17C could be a signature inflammatory cytokine from intestinal epithelial cells in response to flagellin. Since inhibited in TLR5-, or MyD88- or TRIF-silenced cells, flagellin-induced IL-17C expression was specifically mediated by TLR5 and, subsequently, MyD88 and TRIF adaptor molecules. Furthermore, in line with inflammatory nature of IL-17, we found that IL-17C expression was substantially enhanced in the intestinal tissues from Ulcerative colitis patients. Given the facts that TLR5 is a key pattern recognition receptor which mediates microbial recognition in the intestinal epithelium and IL-17C turned out to be a unique member of the IL-17 family expressed in intestinal epithelial cells on TLR5 activation, our study may provide an important clue on understanding how intestinal microbes would contribute to an inflammatory program in the gut.

Airway epithelial expression of TLR5 is downregulated in healthy smokers and smokers with chronic obstructive pulmonary disease.

The TLRs are important components of the respiratory epithelium host innate defense, enabling the airway surface to recognize and respond to a variety of insults in inhaled air. On the basis of the knowledge that smokers are more susceptible to pulmonary infection and that the airway epithelium of smokers with chronic obstructive pulmonary disease (COPD) is characterized by bacterial colonization and acute exacerbation of airway infections, we assessed whether smoking alters expression of TLRs in human small airway epithelium, the primary site of smoking-induced disease. Microarrays were used to survey the TLR family gene expression in small airway (10th to 12th order) epithelium from healthy nonsmokers (n = 60), healthy smokers (n = 73), and smokers with COPD (n = 36). Using the criteria of detection call of present (P call) ≥ 50%, 6 of 10 TLRs (TLRs 1-5 and 8) were expressed. Compared with nonsmokers, the most striking change was for TLR5, which was downregulated in healthy smokers (1.4-fold, p < 10⁻¹°) and smokers with COPD (1.6-fold, p < 10⁻¹¹). TaqMan RT-PCR confirmed these observations. Bronchial biopsy immunofluorescence studies showed that TLR5 was expressed mainly on the apical side of the epithelium and was decreased in healthy smokers and smokers with COPD. In vitro, the level of TLR5 downstream genes, IL-6 and IL-8, was highly induced by flagellin in TLR5 high-expressing cells compared with TLR5 low-expressing cells. In the context that TLR5 functions to recognize pathogens and activate innate immune responses, the smoking-induced downregulation of TLR5 may contribute to smoking-related susceptibility to airway infection, at least for flagellated bacteria.

Flagella from five Cronobacter species induce pro-inflammatory cytokines in macrophage derivatives from human monocytes.

Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314-6025 pg/ml), TNF-α (39-359 pg/ml), and IL-10 (2-96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95-100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria.

Pseudomonas evades immune recognition of flagellin in both mammals and plants.

The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.

Induction of human ß-defensin-2 in pulmonary epithelial cells by Legionella pneumophila: involvement of TLR2 and TLR5, p38 MAPK, JNK, NF-κB, and AP-1.

Legionella pneumophila is an important causative agent of severe pneumonia in humans. Human alveolar epithelium is an effective barrier for inhaled microorganisms and actively participates in the initiation of innate host defense. Induction of antimicrobial peptide human ß-defensin-2 (hBD-2) by various stimuli in epithelial cells has been reported. However, the mechanisms by which bacterial infections enhance hBD-2 expression remain poorly understood. In this study, we investigated the effect of the pulmonary pathogen L. pneumophila on induction of hBD-2 in human pulmonary epithelial cells. Infection with L. pneumophila markedly increased hBD-2 production, and the response was attenuated in Toll-like receptor (TLR) 2 and TLR5 transient knockdown cells. Furthermore, pretreatment with SB-202190 (an inhibitor of p38 MAPK) and JNK II (an inhibitor of c-Jun NH(2)-terminal kinase), but not U0126 (an inhibitor of ERK), reduced L. pneumophila-induced hBD-2 release in A549 cells. L. pneumophila-induced hBD-2 liberation was mediated via recruitment of NF-κB and AP-1 to the hBD-2 gene promoter. Additionally, we showed that exo- and endogenous hBD-2 elicited a strong antimicrobial effect towards L. pneumophila. Together, these results suggest that L. pneumophila induces hBD-2 release in A549 cells, and the induction seems to be mediated through TLR2 and TLR5 as well as activation of p38 MAPK, JNK, NF-κB, and AP-1.

IgA and IgG antibody responses following systemic immunization of cattle with native H7 flagellin differ in epitope recognition and capacity to neutralise TLR5 signalling.

Systemic immunization of cattle with H7 flagellin results in induction of both H7-specific IgA and IgG antibodies but only partially protects against subsequent colonization with Escherichia coli O157:H7. Recent studies indicate that anti-flagellin antibodies directed against TLR5 binding domains located in the conserved N- and C-terminal domains of flagellin can neutralise TLR5 activation and impair vaccine efficacy. In the current study we determined whether systemic immunization of cattle with H7 flagellin induces antibodies capable of interfering with flagellin-mediated TLR5 activation. Both anti-H7 IgG1 and IgG2 but not IgA antibodies recognised epitopes within the conserved N- and C-terminal domains of H7 flagellin, and purified H7-specific IgG but not IgA was capable of inhibiting H7-mediated TLR5 activation in vitro. These results suggest that (i) IgA and IgG isotypes originated from different populations of B cells and (ii) systemically induced H7-specific IgG but not IgA may impair innate immune responses to E. coli O157:H7 via neutralisation of TLR5 activation and subsequently reduce vaccine efficacy.

Innate immunity mediated by TLR5 as a novel antiinflammatory target for cystic fibrosis lung disease.

Novel therapies to target lung inflammation are predicted to improve the lives of people with cystic fibrosis (CF) but specific antiinflammatory targets have not been identified. The goal of this study was to establish whether TLR5 signaling is the key molecular pathway mediating lung inflammation in CF, and to determine whether strategies to inhibit TLR5 can reduce the damaging inflammatory response. The innate immune responses were analyzed in both airway epithelial cells and primary PBMCs from CF patients and matched controls. Additionally, 151 clinical isolates of Pseudomonas aeruginosa from CF patients were assessed for motility and capacity to activate TLR5. Blood and airway cells from CF patients produced significantly more proinflammatory cytokine than did control cells following exposure to the CF pathogens P. aeruginosa and Burkholderia cepacia complex (p < 0.001). Stimulation with pure TLR ligands demonstrated that TLR signaling appears to mediate the excessive cytokine production occurring in CF. Using complementary approaches involving both neutralizing Ab targeting TLR5 and flagellin-deficient bacteria, we established that inhibition of TLR5 abolished the damaging inflammatory response generated by CF airway cells following exposure to P. aeruginosa (p < 0.01). The potential therapeutic value of TLR5 inhibition was further supported by our demonstration that 75% of clinical isolates of P. aeruginosa retained TLR5 activating capacity during chronic CF lung infection. These studies identify the innate immune receptor TLR5 as a novel antiinflammatory target for reducing damaging lung inflammation in CF.