Preventive nasal administration of flagellin restores antimicrobial effect of gentamicin and protects against a multidrug-resistant strain of Pseudomonas aeruginosa.

The increasing prevalence of multidrug-resistant Pseudomonas aeruginosa (PA) is a significant concern for chronic respiratory disease exacerbations. Host-directed drugs, such as flagellin, an agonist of toll-like receptor 5 (TLR5), have emerged as a promising solution. In this study, we evaluated the prophylactic intranasal administration of flagellin against a multidrug-resistant strain of PA (PAMDR) in mice and assessed the possible synergy with the antibiotic gentamicin (GNT). The results indicated that flagellin treatment before infection decreased bacterial load in the lungs, likely due to an increase in neutrophil recruitment, and reduced signs of inflammation, including proinflammatory cytokines. The combination of flagellin and GNT showed a synergistic effect, decreasing even more the bacterial load and increasing mice survival rates, in comparison to mice pre-treated only with flagellin. These findings suggest that preventive nasal administration of flagellin could restore the effect of GNT against MDR strains of PA, paving the way for the use of flagellin in vulnerable patients with chronic respiratory diseases.

Mesenchymal stem cells preconditioned with a TLR5 agonist enhanced immunoregulatory effect through M2 macrophage polarization in a murine graft-versus-host disease model.

Graft-versus-host disease (GVHD) is a common complication following hematopoietic stem cell transplantation and can be life-threatening. Mesenchymal stem cells (MSCs), adult stem cells with immunomodulatory properties, have been used as therapeutic agents in a variety of ways and have demonstrated efficacy against acute GVHD (aGVHD); however, variability in MSC pro- and anti-inflammatory properties and the limitation that they only exhibit immunosuppressive effects at high levels of inflammation have prevented their widespread clinical use. The outcomes of GVHD treated with MSCs in the clinic have been variable, and the underlying mechanisms remain unclear. Therefore, the unique biological effects of Toll-like receptor 5 (TLR5) agonists led us to compare and validate the efficacy of MSCs primed with KMRC011, a TLR5 agonist. KMRC011 is a stimulant that induces the secretion of cytokines, which play an important role in immune regulation. In this study, we found that MSCs pretreated with KMRC011 increased the secretion of immunosuppressive cytokines indoleamine 2,3-dioxygenase (IDO) and cyclooxygenase-2 (COX2) and increased the expression of M2 macrophage polarizing cytokines macrophage colony-stimulating factor (M-CSF) and interleukin 10 (IL-10) in vitro. We investigated the immunosuppressive effects of TLR5 agonist (KMRC011)-primed MSCs on lymphocytes and their preventive and therapeutic effects on an in vivo mouse aGVHD model. In vitro experiments showed that KMRC011-primed MSCs had enhanced immunosuppressive effects on lymphocyte proliferation. In vivo experiments showed that KMRC011-primed MSCs ameliorated GVHD severity in a mouse model of induced GVHD disease. Finally, macrophages harvested from the spleens of mice treated with KMRC011-primed MSCs showed a significant increase in the anti-inflammatory M2 phenotype. Overall, the results suggest that KMRC011-primed MSCs attenuated GVHD severity in mice by polarizing macrophages to the M2 phenotype and increasing the proportion of anti-inflammatory cells, opening new horizons for GVHD treatment.

The GM-CSF Released by Airway Epithelial Cells Orchestrates the Mucosal Adjuvant Activity of Flagellin.

The TLR5 agonist flagellin is a potent adjuvant and is currently being developed for use in vaccines. The mechanisms that drive flagellin's activity are influenced by its administration route. Previous studies showed that lung structural cells (especially epithelial cells lining the conducting airways) are pivotal for the efficacy of intranasally administered flagellin-containing vaccines. In this study, we looked at how the airway epithelial cells (AECs) regulate the flagellin-dependent stimulation of Ag-specific CD4+ T cells and the Ab response in mice. Our results demonstrate that after sensing flagellin, AECs trigger the release of GM-CSF in a TLR5-dependent fashion and the doubling of the number of activated type 2 conventional dendritic cells (cDC2s) in draining lymph nodes. Furthermore, the neutralization of GM-CSF reduced cDC2s activation. This resulted in lower of Ag-specific CD4+ T cell count and Ab titers in mice. Our data indicate that during pulmonary immunization, the GM-CSF released by AECs orchestrates the cross-talk between cDC2s and CD4+ T cells and thus drives flagellin's adjuvant effect.

Boosting the IL-22 response using flagellin prevents bacterial infection in cigarette smoke-exposed mice.

The progression of chronic obstructive pulmonary disease (COPD), a lung inflammatory disease being the fourth cause of death worldwide, is marked by acute exacerbations. These episodes are mainly caused by bacterial infections, frequently due to Streptococcus pneumoniae. This susceptibility to infection involves a defect in interleukin (IL)-22, which plays a pivotal role in mucosal defense mechanism. Administration of flagellin, a Toll-like receptor 5 (TLR-5) agonist, can protect mice and primates against respiratory infections in a non-pathological background. We hypothesized that TLR-5-mediated stimulation of innate immunity might improve the development of bacteria-induced exacerbations in a COPD context. Mice chronically exposed to cigarette smoke (CS), mimicking COPD symptoms, are infected with S. pneumoniae, and treated in a preventive and a delayed manner with flagellin. Both treatments induced a lower bacterial load in the lungs and blood, and strongly reduced the inflammation and lung lesions associated with the infection. This protection implicated an enhanced production of IL-22 and involved the recirculation of soluble factors secreted by spleen cells. This is also associated with higher levels of the S100A8 anti-microbial peptide in the lung. Furthermore, human mononuclear cells from non-smokers were able to respond to recombinant flagellin by increasing IL-22 production while active smoker cells do not, a defect associated with an altered IL-23 production. This study shows that stimulation of innate immunity by a TLR-5 ligand reduces CS-induced susceptibility to bacterial infection in mice, and should be considered in therapeutic strategies against COPD exacerbations.

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.

Increased expression of interleukin 36 in chronic rhinosinusitis and its contribution to chemokine secretion and increased epithelial permeability.

BACKGROUND: IL-36 family, a recently reported member of the IL-1 cytokine family, plays an essential role in nonspecific innate immune response to infection. This study aims at investigating the expression of IL-36 family members (α, ß, and γ) in normal and inflammatory sinus mucosa of patients with chronic rhinosinusitis (CRS), their effects on chemokine secretion and on the barrier function of epithelial and endothelial cells, and the effect of Toll-like receptors on the expression of IL-36 in epithelial cells. MATERIAL AND METHODS: The expression of IL-36 family in normal and inflammatory sinus mucosa, the production of chemokines or the expression levels of IL-36 family in epithelial cells treated with IL-36 family members or stimulated with TLR3, TLR4, TLR5, or TLR7/8 agonists were measured with real time PCR, ELISA, immunohistochemistry, or Western blot. The epithelial and endothelial permeability, and transendothelial leukocyte migration were investigated using cultured epithelial and endothelial cells. RESULTS: IL-36α, IL-36ß, and IL-36γ were localized in epithelial cells of sinonasal mucosa. Their levels increased in inflammatory mucosa of CRS patients and are up-regulated by TLR3, TLR4, or TLR5 agonists. IL-36α, or IL-36γ induced CXCL1, CXCL2, and CXCL3 production. Epithelial and endothelial permeability, transendothelial leukocyte migration were increased in cells treated with IL-36α, IL-36ß, or IL-36γ. CONCLUSIONS: These results suggest that IL-36α, IL-36ß, and IL-36γ localized in superficial epithelium may act as a responder to microbial and nonmicrobial elements through TLR and subsequently produce CXC chemokines, playing an interplay between innate and adaptive immune response.

CBLB502, a Toll-like receptor 5 agonist, offers protection against radiation-induced male reproductive system damage in mice.

CBLB502, a Toll-like receptor (TLR)5 agonist derived from Salmonella flagellin, was shown to protect mammalian hematopoietic and gastrointestinal systems from acute irradiation syndrome and to stimulate regeneration. To explore whether CBLB502 can improve testicular injuries caused by irradiation, mice were intraperitoneally injected with 0.2 mg/kg CBLB502 or vehicle control 30 min prior to applying 5.0 Gy ionizing radiation (IR). We observed these mice for the following 120 days and determined that CBLB502 pretreatment alleviated IR-induced oxidative stress, alleviated the distorted architecture of seminiferous tubules, reversed the decline of sperm quantity and quality, and helped recover male mouse fertility. Additionally, CBLB502 efficiently reduced DNA damage and chromosomal aberrations in IR-treated mice and their offspring. Due to the suppression of p53-dependent apoptosis, in IR-treated mice, CBLB502 was shown to significantly activate the nuclear factor kappa B (NFκB) pathway and reduce the apoptotic rate in association with an increase in anti-apoptotic B-cell lymphoma 2 levels and a decrease in the levels of DNA repair protein and proliferating cell nuclear antigen. Moreover, an IR-induced reduction in serum testosterone and superoxide dismutase levels and an increase in malondialdehyde levels were considerably reversed in CBLB502-pretreated mice. No significant reverse effects were found in Tlr5 knockout mice, suggesting that protection of the testis against IR by CBLB502 is primarily dependent on the TLR5 signaling pathway. Our results may help further investigations into potential CBLB502 applications for the protection of the male reproductive system during radiotherapy.

Evaluation of immunogenicity and protective effect of DNA vaccine encoding surface antigen1 (SAG1) of Toxoplasma gondii and TLR-5 ligand as a genetic adjuvant against acute toxoplasmosis in BALB/c mice.

AIMS: Toxoplasma gondii is an obligate intracellular, protozoan that causes a high incidence of serious zoonotic parasitic disease in humans. In the present study the immune-protective efficacy of a DNA vaccine encoding SAG1 in combination with a gene sequence encoding FliC of Salmonella typhimurium (Toll-like receptor 5 agonist) was evaluated against acute T. gondii infection in mice. METHODS AND RESULTS: Ninety-nine female inbred BALB/c mice were divided into nine groups of 11 mice and were immunized intramuscularly three times at three-week intervals (days 0, 21 and 42) and challenged with virulent T. gondii RH strain 4 weeks later. The immunization of pVAX1-SAG1 administered with pVAX1-fliC in mice indicated specific humoral responses, with higher IgG antibody titers and a mixed IgG1/IgG2a response than in other groups (with a predominance of IgG2a over IgG2b and IgG1). Also, the cellular immune response elicited high levels of IFN-γ and IL-12 cytokines and low levels of IL-4 production compared to traditional adjuvants. Furthermore, the mice vaccinated with pVAX1-SAG1+pVAX1-fliC survived for slightly longer after the last immunization and challenge with the T. gondii. CONCLUSION: This investigation indicated that cocktail DNA vaccine encoded SAG1 gene of T. gondii and FliC can protect against acute toxoplasmosis.

Repeated injection of KMRC011, a medical countermeasure for radiation, can cause adverse health effects in cynomolgus monkeys.

High-dose radiation-induced tissue damage is a major limiting factor in the medical application of nuclear technology. Herein, we tested 28-day repeated-dose toxicity of KMRC011, an agonist of toll-like receptor (TLR) 5, which is being developed as a medical countermeasure for radiation, using cynomolgus monkeys. KMRC011 (0.01, 0.02 or 0.04 mg/kg/day) was intramuscularly injected once daily for 4 weeks, and each two monkeys in both control and 0.04 mg/kg/day group were observed for an additional 2-week recovery period. There were no dose-related toxicological changes in mortality, clinical observations, body weight, food consumption, ophthalmological findings, electrocardiographs, coagulation, serum chemistry, organ weights, or urinalysis and urine chemistry. Although treatment-related changes, such as increased white blood cells, increased absolute and relative neutrophils, decreased relative lymphocytes and inflammatory lesions, were noted in the maximum dose group, these findings were not observed after the 2-week recovery period. Further, we considered that the kidneys and heart may be target organs of TLR5 agonists, as well as the spleen, and that autophagic signals can be triggered in tissue damage and the repair process. Importantly, accumulation of p62 protein, an indicator of autophagy, and a decrease of caveolin-1 protein, a regulator of TLR5 protein half-life, were found in both tissues from the highest dose group. Therefore, we conclude that the no-observed-adverse-effect level for KMRC011 may be greater than 0.04 mg/kg/day in male and female monkeys. Additionally, we propose that further studies are needed to identify the molecular signals, which are related to KMRC011-induced adverse effects.

Toll-like receptor-5 agonist, entolimod, suppresses metastasis and induces immunity by stimulating an NK-dendritic-CD8+ T-cell axis.

Activation of an anticancer innate immune response is highly desirable because of its inherent ability to generate an adaptive antitumor T-cell response. However, insufficient safety of innate immune modulators limits clinical use to topical applications. Toll-like receptor 5 (TLR5) agonists are favorably positioned as potential systemic immunotherapeutic agents because of unusual tissue specificity of expression, uniquely safe profile of induced cytokines, and antitumor efficacy demonstrated in a number of animal models. Here, we decipher the molecular and cellular events underlying the metastasis suppressive activity of entolimod, a clinical stage TLR5 agonist that activates NF-κB-, AP-1-, and STAT3-driven immunomodulatory signaling pathways specifically within the liver. Used as a single agent in murine colon and mammary metastatic cancer models, entolimod rapidly induces CXCL9 and -10 that support homing of blood-borne CXCR3-expressing NK cells to the liver predominantly through an IFN-γ signaling independent mechanism. NK cell-dependent activation of dendritic cells is followed by stimulation of a CD8(+) T-cell response, which exert both antimetastatic effect of entolimod and establishment of tumor-specific and durable immune memory. These results define systemically administered TLR5 agonists as organ-specific immunoadjuvants, enabling efficient antitumor vaccination that does not depend on identification of tumor-specific antigens.

Helicobacter pylori flagellin: TLR5 evasion and fusion-based conversion into a TLR5 agonist.

Helicobacter pylori is a flagellated bacterium of the Epsilonproteobacteria class that causes peptic ulcers. Flagellin is a primary structural protein that assembles into the flagellar filament. Flagellins from bacteria that belong to the Gammaproteobacteria and Firmicutes groups are detected by Toll-like receptor 5 (TLR5) in the host, triggering the innate immune response, and thus have been studied for the development of vaccines against diverse infections through fusion with protein antigens. However, H. pylori flagellin (hFlg) does not stimulate TLR5, allowing H. pylori to evade TLR5-mediated immune surveillance. The unresponsiveness of TLR5 to hFlg, along with the tendency of the hFlg protein to precipitate, limits the utility of hFlg for H. pylori vaccine development. Here, we report a soluble hFlg derivative protein that activates TLR5. We performed expression and purification screens with full-length and fragment hFlg proteins and identified the hypervariable domains as the soluble part of hFlg. The hypervariable domains of hFlg were engineered into a TLR5 agonist through fusion with the TLR5-activating Bacillus subtilis flagellin. Furthermore, based on comparative sequence and mutation analyses, we reveal that hFlg evolved to evade TLR5 detection by modifying residues that correspond to a TLR5-activation hot spot.

Antigenic and functional profiles of a Lawsonia intracellularis protein that shows a flagellin-like trait and its immuno-stimulatory assessment.

The obligate intracellular Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), is an economically important disease in the swine industry. Due to extreme difficulty of in vitro culture of the pathogen, molecular characterization of protein components of LI that are targets of the immune system, is difficult; thus, the scientific evidence to drive the development of preventive measures is lacking. In this work, we investigated the antigenic and functional characteristics of a putative flagellar-associated protein, LI0570, using in silico computational approaches for epitope prediction and an in vitro protein-based molecular assay. The amino acid sequence of LI0570 exhibited similarities to flagellar-associated proteins in four different bacterial strains. The presence of B cell linear confirmative epitopes of the protein predicted by a bioinformatics tool was validated by western blot analysis using anti-LI mouse hyperimmune serum, which implied that LI0570 induced production of antigen-specific antibodies in vivo. Further, TLR5-stimulating activity and IL-8 cytokine expression produced via downstream signaling were observed in HEK-Blue™-hTLR5 cells stimulated with LI0570. This result indicates that the LI0570 protein can trigger an innate immune response followed by a T-cell-related adaptive immune response in an infected host. Collectively, the data presented here support that the LI0570 protein which shows the antigenic potential could be a useful component of a recombinant vaccine against PE, providing progress toward an effective prevention strategy.

Toll-like receptor 5 agonist CBLB502 induces radioprotective effects in vitro.

CBLB502 derived from Salmonella flagellin is a novel agonist of Toll-like receptor 5 (TLR5). It has been shown that CBLB502 can exert high radioprotective efficacy on mice and primates from both GI and hematopoietic syndromes during whole-body irradiation with low toxicity and immunogenicity. However, no effective system has been used to investigate the protective effect of CBLB502 against irradiation and the related mechanism in vitro. In this study, we investigated the radioprotective properties of CBLB502 in HEK293-N-T cells constitutively expressing human TLR5 and NF-κB-dependent luciferase. HEK293-N-T cells were treated with different doses of CBLB502 prior to 60Co-γ ray irradiation. After irradiation, cell viability was real-time measured for 4 days by using the real-time cell analysis system. We found that CBLB502 was capable of efficiently maintaining the survival rate of irradiated HEK293-N-T cells. Then apoptotic cell death and cell cycle were detected by flow cytometry. The results showed that CBLB502 pre-treatment could reduce the apoptosis and promote the recovery of irradiated HEK293-N-T cells from G2-phase arrest in a dose-dependent manner. Our data indicated that CBLB502 has a direct radioprotective effect in vitro via anti-apoptosis and promotes cell cycle recovery. The method developed here could be an effective in vitro system to screen other TLR5-target radioprotectants like CBLB502.

Intestinal expression of Fas and Fas ligand is upregulated by bacterial signaling through TLR4 and TLR5, with activation of Fas modulating intestinal TLR-mediated inflammation.

TLRs play an important role in mediating intestinal inflammation and homeostasis. Fas is best studied in terms of its function in apoptosis, but recent studies demonstrate that Fas signaling may mediate additional functions such as inflammation. The role of Fas, and the Fas ligand (FasL), in the intestine is poorly understood. The aim of this study was to evaluate potential cross-talk between TLRs and Fas/FasL system in intestinal epithelial cells (IECs). IECs were stimulated with TLR ligands, and expression of Fas and FasL was investigated. Treatment with TLR4 and TLR5 ligands, but not TLR2 and 9 ligands, increased expression of Fas and FasL in IECs in vitro. Consistent with this finding, expression of intestinal Fas and FasL was reduced in vivo in the epithelium of TLR4 knockout (KO), 5KO, and germ-free mice, but not in TLR2KO mice. Modulating Fas signaling using agonistic anti-Fas augmented TLR4- and TLR5-mediated TNF-α and IL-8 production by IECs. In addition, suppression of Fas in IECs reduced the ability of TLR4 and TLR5 ligands and the intestinal pathogens Salmonella typhimurium and Listeria monocytogenes to induce the expression of IL-8. In conclusion, this study demonstrates that extensive cross-talk in IECs occurs between the Fas and TLR signaling pathways, with the FasL/Fas system playing a role in TLR-mediated inflammatory responses in the intestine.

Central role of liver in anticancer and radioprotective activities of Toll-like receptor 5 agonist.

Vertebrate Toll-like receptor 5 (TLR5) recognizes bacterial flagellin proteins and activates innate immune responses to motile bacteria. In addition, activation of TLR5 signaling can inhibit growth of TLR5-expressing tumors and protect normal tissues from radiation and ischemia-reperfusion injuries. To understand the mechanisms behind these phenomena at the organismal level, we assessed nuclear factor kappa B (NF-κB) activation (indicative of TLR5 signaling) in tissues and cells of mice treated with CBLB502, a pharmacologically optimized flagellin derivative. This identified the liver and gastrointestinal tract as primary CBLB502 target organs. In particular, liver hepatocytes were the main cell type directly and specifically responding to systemic administration of CBLB502 but not to that of the TLR4 agonist LPS. To assess CBLB502 impact on other pathways, we created multireporter mice with hepatocytes transduced in vivo with reporters for 46 inducible transcription factor families and found that along with NF-κB, CBLB502 strongly activated STAT3-, phenobarbital-responsive enhancer module (PREM), and activator protein 1 (AP-1-) -driven pathways. Livers of CBLB502-treated mice displayed induction of numerous immunomodulatory factors and massive recruitment of various types of immune cells. This led to inhibition of growth of liver metastases of multiple tumors regardless of their TLR5 status. The changed liver microenvironment was not, however, hepatotoxic, because CBLB502 induced resistance to Fas-mediated apoptosis in normal liver cells. Temporary occlusion of liver blood circulation prevented CBLB502 from protecting hematopoietic progenitors in lethally irradiated mice, indicating involvement of a factor secreted by responding liver cells. These results define the liver as the key mediator of TLR5-dependent effects in vivo and suggest clinical applications for TLR5 agonists as hepatoprotective and antimetastatic agents.

A Toll-Like Receptor 5 Agonist Improves the Efficacy of Antibiotics in Treatment of Primary and Influenza Virus-Associated Pneumococcal Mouse Infections.

Prophylactic intranasal administration of the Toll-like receptor 5 (TLR5) agonist flagellin protects mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might improve the therapeutic index of antibiotics for the treatment of Streptococcus pneumoniae respiratory infections in mice. Intranasal administration of flagellin was combined with either oral administration of amoxicillin or intraperitoneal injection of trimethoprim-sulfamethoxazole to treat S. pneumoniae-infected animals. Compared with standalone treatments, the combination of antibiotic and flagellin resulted in a lower bacterial load in the lungs and greater protection against S. pneumoniae dissemination and was associated with an early increase in neutrophil infiltration in the airways. The antibiotic-flagellin combination treatment was, however, not associated with any exacerbation of inflammation. Moreover, combination treatment was more efficacious than standalone antibiotic treatments in the context of post-influenza virus pneumococcal infection. Lastly, TLR5 signaling was shown to be mandatory for the efficacy of the combined antibacterial therapy. This report is the first to show that combining antibiotic treatment with the stimulation of mucosal innate immunity is a potent antibacterial strategy against pneumonia.

Intestinal epithelial suppressor of cytokine signaling 3 enhances microbial-induced inflammatory tumor necrosis factor-α, contributing to epithelial barrier dysfunction.

A single layer of intestinal epithelial cells (IEC) lines the entire gastrointestinal tract and provides the first line of defense and barrier against an abundance of microbial stimuli. IEC homeostasis and repair are mediated through microbe-sensing Toll-like receptor (TLR)-induced inflammatory pathways. Increasing evidence supports a role of suppressor of cytokine signaling 3 (SOCS3) as a modulator of IEC turnover, balancing controlled repair and replenishment with excessive IEC proliferation predisposing to dysplasia and cancer. Our data indicate that SOCS3 can limit microbial-induced IEC repair, potentially through promoting tumor necrosis factor-α (TNF-α) and limiting TNFR2 expression. Activation of TLR5 signaling pathways, compared with other TLR, increases TNF-α mRNA in a dose-dependent manner and SOCS3 enhances TLR5-induced TNF-α. We also show that flagellin promotes transcription of TNFR2 and that SOCS3 limits this expression, presenting a mechanism of SOCS3 action. Our data also support the role of microbial ligands in epithelial wound healing and suggest that a functional consequence of increased TNF-α is reduced wound healing. These results provide further evidence to support the regulatory role of epithelial SOCS3 in intestinal health and suggest that the increased expression of SOCS3 observed in IBD may serve to perpetuate "inflammation" by promoting TNF-α production and limiting epithelial repair in response to commensal microflora.

Anti-metastatic immunotherapy based on mucosal administration of flagellin and immunomodulatory P10.

Current therapies against malignant melanoma generally fail to increase survival in most patients, and immunotherapy is a promising approach as it could reduce the dosage of toxic therapeutic drugs. In the present study, we show that an immunotherapeutic approach based on the use of the Toll-like receptor (TLR)-5 ligand flagellin (Salmonella Typhimurium FliCi) combined with the major histocompatibility complex class II-restricted P10 peptide, derived from the Paracoccidioides brasiliensis gp43 major surface protein, reduced the number of lung metastasis in a murine melanoma model. Compounds were administered intranasally into C57Bl/6 mice intravenously challenged with syngeneic B16F10-Nex2 melanoma cells, aiming at the local (pulmonary) immune response modulation. Along with a marked reduction in the number of lung nodules, a significant increase in survival was observed. The immunization regimen induced both local and systemic proinflammatory responses. Lung macrophages were polarized towards a M1 phenotype, lymph node cells, and splenocytes secreted higher interleukin-12p40 and interferon (IFN)-γ levels when re-stimulated with tumor antigens. The protective effect of the FliCi+P10 formulation required TLR-5, myeloid differentiation primary response gene 88 and IFN-γ expression, but caspase-1 knockout mice were only partially protected, suggesting that intracellular flagellin receptors are not involved with the anti-tumor effect. The immune therapy resulted in the activation of tumor-specific CD4(+) T lymphocytes, which conferred protection to metastatic melanoma growth after adoptive transfer. Taken together, our results report a new immunotherapeutic approach based on TLR-5 activation and IFN-γ production capable to control the metastatic growth of B16F10-Nex2 melanoma, being a promising alternative to be associated with chemotherapeutic drugs for an effective anti-tumor responses.

Toll-like receptors -4 and -5 in oral and cutaneous squamous cell carcinomas.

BACKGROUND: Oral squamous cell carcinoma (OSCC) has a worse prognosis than cutaneous squamous cell carcinoma (CSCC). Toll-like receptor- 4 (TLR-4) and TLR-5 are transmembrane proteins that recognize endogenous and microbial agents. Their activation has been connected to cancer invasion. OBJECTIVE: The aim was to study the expression of TLR-4 and TLR-5 in OSCC and CSCC samples, and the effects of TLR-5 ligand flagellin on the proliferation, migration, and invasion of different mucocutaneous cell lines in vitro. METHODS: Samples of early-stage tumors (T1-T2N0M0) from 63 patients with OSCC and CSCC were obtained, in addition to eight normal mucosa and skin tissues from healthy subjects. Oral-cavity-derived highly aggressive HSC-3, less invasive SAS, and HPV-transformed benign IHGK as well as C-ha-ras-transformed (HaCat) skin carcinoma II-4 and non-invasive A5 cell lines were used. Flagellin-induced mucocutaneous cell lines were compared by using BrdU-proliferation, scratch migration, and myoma organotypic invasion assays. RESULTS: TLR-4 expression was similar in OSCC and CSCC tumors. TLR-5 was more abundant in OSCC than in CSCC samples. Flagellin induced the proliferation of SAS, II-4 and A5, migration of IHGK, II-4 and A5, and the invasion of II-4 cells. It had no effect on HSC-3 cells. CONCLUSIONS: Flagellin, a TLR-5 agonist, induced the migration and invasion of less aggressive mucocutaneous cell lines, but it had no effect on the most invasive oral carcinoma cells. The more aggressive clinical behavior of OSCC compared to CSCC may partially be related to the differences in the expression of TLR-5 in these malignancies.