Through Toll-like Receptors Agonists Research Articles

SARS-CoV-2 Envelope Protein Tolerizes Macrophage Response to Secondary Inflammatory Stimuli

Abstract Exposure of innate immune cells to bacteria or viruses may result in epigenetic rewiring that either enhances or weakens innate immune responses to a secondary challenge. One primary mechanism of innate immune cell function is through toll-like receptors (TLRs) that sense a variety of bacterial and viral molecules and cause immune activation. SARS-CoV-2 Envelope protein (E protein) has been shown to mediate innate immune activation, through engagement of TLR2. In this study, we determined the secretome of chemokines, cytokines and growth factors from monocytes in response to E protein stimulation and compared this response with other viral antigens and TLR agonists. Next, we stimulated monocytes with E protein followed by secondary stimulation with lipopolysaccharide (LPS) on monocyte-derived macrophages (MDM) 1 week later. Reduced expression of CCL3, CCL4, CCL5, CXCL10, TNF-A, and IL-12 upon secondary LPS or autologous E protein stimulation were statistically reflecting a more tolerant MDM state that is imprinted after E protein exposure. Finally, we demonstrated that immunizing neonatal mice with SARS-CoV-2 E protein induced proinflammatory cytokine secretion and lung tissue inflammation pathology and demonstrated a long-term impact on immune cell function and secondary response to LPS. Thus, E protein induces macrophage proinflammatory networks that lead to tissue inflammation, but also induce a more tolerant state after return to a resting state that could impact the ability to respond to secondary infection. Further understanding of the molecular pathways that drive this immune activation and tolerance could identify treatment targets to restore immunity after SARS-CoV-2 infection.

Abstract P3072: Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (iPSC-CMs) Respond To Inflammatory Stimuli Through Innate Immune Responses

Introduction: Cells respond to inflammatory stimuli in settings of sterile and pathologic cardiac inflammation. Nuclear factor Kappa B (NFKB) is a transcriptional mediator of innate immune responses. Lipopolysaccharide (LPS) promotes NFKB activation through toll-like receptors (TLR) to initiate cell autonomous processes that resist infection. High-mobility box group 1 (HMGB1) is a chromatin associated protein released upon nuclear rupture that similarly promotes cytokine release in response to injury. Few studies have interrogated multiple inflammatory stimuli and their impacts on iPSC-CM function. We examined innate immune activation using distinct TLR agonists and assessed functional responses in both 2D iPSC-CM monoculture and 3D collagen-based engineered heart tissues. Methods and Results: We measured electric field potential duration (Nanion CardioExcyte 96) of iPSC-CM monolayers after exposure to LPS and HMGB1. We observed a four fold increase in downstroke velocity after exposure to HMGB1 (512.7±126.9 V/s; p=0.0002 n=12) and a 30% decrease in downstroke velocity after LPS treatment (-188.9±61.4 V/s; p=0.004 n=12). This response was associated with nuclear localization of phosphorylated NFKB. Collagen-based engineered heart tissues demonstrated contractile defects in response to 48h of exposure to TLR agonists. A 50% reduction in fractional shortening was observed following HMGB1 treatment (-0.0060±0.0009; p<0.0001 n=6) and an 80% reduction was associated with LPS exposure (-0.0088±0.0006; p<0.0001 n=6). To identify gene expression regulatory regions associated with this response, we examined transcriptional activity of three candidate enhancer regions implicated in cytokine production in iPSC-CMs. These regions demonstrated a 0.5- to 20-fold increase in enhancer activity in iPSC-CMs. One enhancer demonstrated a 22% increase in activity following 48h of LPS, consistent with a direct role for NFKB in innate immune activation in human iPSC-CMs; this enhancer regulates a gene with increased expression in dilated cardiomyopathy. Conclusion: These data indicate that cardiomyocytes respond directly to inflammatory stimuli and demonstrate the utility of 2D and 3D human tissue culture models for cardiac inflammation.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Responses of increasingly complex intestinal epithelium in vitro models to bacterial toll-like receptor agonists

The intestine fulfills roles in the uptake of nutrients and water regulation and acts as a gatekeeper for the intestinal microbiome. For the latter, the intestinal gut barrier system is able to respond to a broad range of bacterial antigens, generally through Toll-like receptor (TLR) signaling pathways. To test the capacity of various in vitro intestinal models, we studied IL-8 secretion, as a marker of pro-inflammatory response through the TLR pathway, in a Caco-2 monoculture, Caco-2/HT29-MTX di-culture, Caco-2/HT29-MTX/HMVEC-d tri-culture and in a HT29-p monoculture in response to exposure to various TLR agonists. Twenty-one-day-old differentiated cells in Transwells were exposed to Pam3CSK4 (TLR1/2), lipopolysaccharide (TLR4), single-stranded RNA (TLR7/8), Poly(i:C) (TLR3) and flagellin (TLR5) for 24 h. In all systems IL-8 secretion was increased in response to flagellin exposure, with HT29-p cells also responding to Poly(I:C) exposure. All other agonists did not induce an IL-8 response in the tested in vitro models, indicating that the specific TLRs are either not present or not functional in these models. This highlights the need for careful selection of in vitro models when studying intestinal immune responses and the need for improved in vitro models that better recapitulate intestinal immune responses.

Platelets modulate multiple markers of neutrophil function in response to in vitro Toll-like receptor stimulation.

IntroductionIn addition to their role in facilitating leukocyte-mediated inflammation, platelets can dampen leukocyte pro-inflammatory responses in some contexts. Consequently, platelets are increasingly appreciated as regulators of inflammation. Together, platelets and neutrophils play a role in inflammation through Toll-like receptor (TLR) expression, although we do not fully understand how platelets shape neutrophil responses to TLR stimulation. Here, we aimed to determine the extent to which platelets can modulate neutrophil function in response to in vitro stimulation with TLR4, TLR2/1, and TLR2/6 agonists.MethodsNeutrophils from 10 healthy individuals were cultured alone or with autologous platelets. Neutrophils ± platelets were left unstimulated or were stimulated with 1 or 100 ng/mL lipopolysaccharide (LPS; a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist) and fibroblast-stimulating lipopeptide (FSL)-1 (a TLR2/6 agonist). Neutrophil activation and phagocytic activity were assessed by flow cytometry, and elastase and interleukin-8 secretion were assessed by ELISA.ResultsThe addition of platelets attenuated neutrophil CD66b and CD11b expression in response to various doses of Pam3CSK4 and FSL-1. Furthermore, platelet co-culture was associated with higher CD62L expression (indicating reduced CD62L shedding) in response to these TLR agonists. Platelets also reduced elastase secretion in unstimulated cultures and in response to low-dose TLR stimulation. Conversely, platelet co-culture increased neutrophil phagocytosis in unstimulated cultures and in response to low-dose Pam3CSK4 and FSL-1. Platelets also increased IL-8 secretion in response to low-dose LPS.ConclusionPlatelets are complex immunomodulators that can attenuate some, and simultaneously augment other, neutrophil functions. This modulation can occur both in the absence and presence of TLR stimulation.

Ability of TLR agonists to upregulate Brucella abortus strain RB51 mediated protection in a murine respiratory model

Brucella abortus is a Gram-negative intracellular zoonotic bacterium that causes infertility and abortion in cattle, and undulant fever in humans. Its low dose of infectivity, ability to be aerosolized, and ease of genetic modification, makes it a bioterror concern. The overall goal is to generate a safe and effective vaccine for humans. One candidate vaccine is Brucella abortus strain RB51, which was approved for use in cattle, and provides protection by initiating a strong T-helper 1 (CD4 Th1) response. Based on a model for aerosol exposure, mice were vaccinated intranasally (IN) with strain RB51 and challenged IN with B. abortus strain 2308. However, strain RB51 did not protect. Protection against Brucella is mediated through Toll Like Receptors (TLRs) 2, 4 and 9. The addition of TLR 2 or TLR 4, and a trend with TLR9 agonists, when combined with IN strain RB51 vaccination, significantly increased bacterial clearance in the lung after strain 2308 challenge. Therefore, for this study, we hypothesized that combining TLR agonists 2, 4, and 9 with strain RB51 IN would enhance protection and clearance in the lung against strain 2308 challenge (IN), by activating the DC1 and CD4 Th1 and CD8 immune response. This study showed that protection was not enhanced by combining all TLR agonists. The group with non-significantly greater clearance was strain RB51 and TLR 2 and 4 agonists. Additional studies are warranted to further define the differential mechanisms and endpoints of protection and chronic infection.

Impaired CD8(+) T cell responses upon Toll-like receptor activation in common variable immunodeficiency.

BackgroundInfections caused by bacteria or viruses are frequent in common variable immunodeficiency (CVID) patients due to antibody deficiencies, which may be associated with altered T cell function. CVID patients are frequently in contact with pathogen-associated molecular patterns (PAMPs), leading to the activation of innate immunity through Toll-like receptors (TLR) affecting T cell activation. We evaluated the effect of TLR activation on T cells in CVID patients undergoing intravenous immunoglobulin (IVIg) replacement using synthetic ligands.MethodsExpression of exhaustion, activation and maturation markers on T cells from peripheral blood as well as regulatory T cells and follicular T cells in peripheral blood mononuclear cells (PBMCs) from CVID and healthy individuals were evaluated by flow cytometry. PBMCs cultured with TLR agonists were assessed for intracellular IFN-γ, TNF, IL-10, IL-17a or IL-22 secretion as monofunctional or polyfunctional T cells (simultaneous cytokine secretion) by flow cytometry.ResultsWe found increased expression of the exhaustion marker PD-1 on effector memory CD4+ T cells (CD45RA−CCR7−) in the peripheral blood and increased expression of CD38 in terminally differentiated CD8+ T cells (CD45RA+CCR7−). Furthermore, a decreased frequency of naïve regulatory T cells (CD45RA+Foxp3low), but not of activated regulatory T cells (CD45RA−Foxp3high) was detected in CVID patients with splenomegaly, the non-infectious manifestation in this CVID cohort (43.7 %). Moreover, the frequency of peripheral blood follicular helper T cells (CD3+CD4+CXCR5+PD-1+ICOS+) was similar between the CVID and control groups. Upon in vitro TLR3 activation, a decreased frequency of CD8+ T cells secreting IFN-γ, IL-17a or IL-22 was detected in the CVID group compared to the control group. However, a TLR7/TLR8 agonist and staphylococcal enterotoxin B induced an increased Th22/Tc22 (IL-22+, IFN-γ−, IL-17a−) response in CVID patients. Both TLR2 and TLR7/8/CL097 activation induced an increased response of CD4+ T cells secreting three cytokines (IL-17a, IL-22 and TNF)in CVID patients, whereas CD8+ T cells were unresponsive to these stimuli.ConclusionThe data show that despite the unresponsive profile of CD8+ T cells to TLR activation, CD4+ T cells and Tc22/Th22 cells are responsive, suggesting that activation of innate immunity by TLRs could be a strategy to stimulate CD4+ T cells in CVID.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0900-2) contains supplementary material, which is available to authorized users.

Integration of innate into adaptive immune responses in ZAP-70–positive chronic lymphocytic leukemia

AbstractThe crucial dependence of chronic lymphocytic leukemia (CLL) cells on signals derived from the B cell receptor (BCR) has encouraged the development of new inhibitors, which interfere with BCR signaling and demonstrate clinical benefits in nearly all patients. In addition, signaling through Toll-like receptor (TLR) 9 of the innate immune system has been shown to further contribute to the activation of CLL cells. However, responses to TLR9 engagement are not uniform, but diametrically opposed with cell death in some patients and cell proliferation in others. We now provide evidence that heterogeneous responses to TLR agonists are related to differences in the ability of CLL cells to activate the BCR-associated kinase Syk. Notably, expression of ZAP-70 appears to be of crucial importance for TLR9-mediated activation of Syk. We show that the activation of Syk provides an antiapoptotic signal, which is independent of Mcl-1, Bcl-2, and Bcl-XL, but related to the degradation of the proapoptotic Bim. Mechanistically, TLR9-mediated antiapoptotic signals in ZAP-70–positive CLL trigger secretion of immunoglobulin M, which then serves as (auto-) antigen for a prosurvival BCR signal. Thus, our data show that single activation of the innate immune receptor TLR9 is sufficient to fully engage BCR signaling in ZAP-70–positive CLL, protecting malignant cells from apoptosis. We conclude that the integration of TLR signaling into an adaptive immune response can further promote survival of CLL cells and may contribute to the unfavorable prognosis of ZAP-70–positive CLL.

A Blunted Response to the Anti-Inflammatory Cytokine IL-10 Contributes to Excessive TNF Production in Myeloproliferative Neoplasm

Rationale:Tumor Necrosis Factor-alpha (TNF) is elevated in myeloproliferative neoplasm (MPN) and plays a key role in expansion of the JAK2V617F neoplastic clone. A high TNF environment, as is the case in MPN patients, gives TNF resistant JAK2V617F mutant cells a selective advantage over their TNF sensitive non-mutant counterparts resulting in expansion of the neoplastic clone. To efficiently target TNF production therapeutically it is necessary to identify the mechanism driving this excessive TNF production. TNF is classically produced by monocytes after stimulation through Toll-like receptors (TLR), crucial pattern recognition receptors for microbial products. Because TLR signaling plays an integral role in inflammation and TNF production, we hypothesized that exaggerated signaling of the TLR pathway is the mechanism by which TNF is overproduced in MPN.Results:We compared the response of peripheral blood monocytes from MPN versus normal controls to the TLR agonists R848 (TLR7/8), LPS (TLR4), or zymosan (TLR2). After stimulation with each of these TLR agonists for 24 hours, CD14+ monocytes from MPN patients (n=18) produced increased amounts of TNF (measured by ELISA) as compared to normal controls (n=10) at all concentrations tested (p<0.05). The increased TNF production in MPN monocytes could not be explained by a higher fraction of inflammatory (CD16+ CD14+) monocytes in MPN. We next used phosflow to detect whether MPN patients have abnormal activation of downstream signaling molecules following stimulation with TLR agonists. At early time points (15min) following stimulation, MPN patients (n=6) and normal controls (n=6) phosphorylated p38 and ERK1/2 equally. At later time points (2hrs) MPN patients maintained phosphorylation of p38 and ERK1/2, whereas in normal controls phosphorylation of p38 and ERK1/2 returned to baseline (p<0.05).This suggested that the negative feedback regulation of TLR signaling may be defective in MPN patients resulting in prolonged TNF production after stimulation. To test this, we compared the tempo of TNF production following LPS stimulation in MPN versus normal monocytes using intracellular flow cytometry as well as ELISA. Monocytes from normal controls (n=12) cease production of TNF by 9 hours post LPS stimulation, whereas MPN monocytes (n=12) continue to produce TNF at 18 hours (p<0.05).LPS stimulation induces a negative feedback loop culminating in the production of IL-10, an anti-inflammatory cytokine which serves to dampen TNF production. We found that MPN monocytes produce abundant IL-10 in response to LPS stimulation. Next we measured the ability of IL-10 to dampen TNF production in LPS stimulated monocytes comparing MPN patients versus normal controls. Addition of low concentrations of rhIL-10 (1ng/ml) to LPS stimulated monocytes reduced TNF production by 50% in normal controls (n=8), whereas it reduced TNF production by only 10-20% in MPN (n=5). However, higher concentrations of IL-10 (>10ng/ml) was able to dampen TNF production in LPS stimulated MPN monocytes by 50%.Conclusions:MPN monocytes produce excessive amounts of TNF in response to TLR ligation due to a defect in the negative regulatory feedback loop which normally serves to dampen TNF production following TLR ligation. We have localized this defect to a blunted response to the anti-inflammatory cytokine IL-10. MPN monocytes are less responsive to the anti-inflammatory actions of IL-10 at low concentrations but these effects can be restored by increasing the concentration of IL-10. Together, these data suggest that administration of IL-10 may reduce excessive inflammatory cytokine production in MPN. DisclosuresNo relevant conflicts of interest to declare.

Toll-like receptor signaling is functional in immune cells of the endangered Tasmanian devil

Devil facial tumour disease (DFTD) is a fatally transmissible cancer that threatens the Tasmanian devil population. As Tasmanian devils do not produce an immune response against DFTD cells, an effective vaccine will require a strong adjuvant. Activation of innate immune system cells through toll-like receptors (TLRs) could provide this stimulation. It is unknown whether marsupials, including Tasmanian devils, express functional TLRs. We isolated RNA from peripheral blood mononuclear cells and, with PCR, detected transcripts for TLRs 2, 3, 4, 5, 6, 7, 8, 9, 10 and 13. Stimulation of the mononuclear cells with agonists to these TLRs increased the expression of downstream TLR signaling products (IL1α, IL6, IL12A and IFNβ). Our data provide the first evidence that TLR signaling is functional in the mononuclear cells of the Tasmanian devil. Future DFTD vaccination trials will incorporate TLR agonists to enhance the immune response against DFTD.

Bacterial lipopolysaccharide augments febrile-range hyperthermia-induced heat shock protein 70 expression and extracellular release in human THP1 cells.

Sepsis, a devastating and often lethal complication of severe infection, is characterized by fever and dysregulated inflammation. While infections activate the inflammatory response in part through Toll-like receptors (TLRs), fever can partially activate the heat shock response with generation of heat shock proteins (HSPs). Since extracellular HSPs, especially HSP70 (eHSP70), are proinflammatory TLR agonists, we investigated how exposure to the TLR4 agonist, bacterial lipopolysaccharide (LPS) and febrile range hyperthermia (FRH; 39.5°C) modify HSP70 expression and extracellular release. Using differentiated THP1 cells, we found that concurrent exposure to FRH and LPS as well as TLR2 and TLR3 agonists synergized to activate expression of inducible HSP72 (HSPA1A) mRNA and protein via a p38 MAP kinase-requiring mechanism. Treatment with LPS for 6 h stimulated eHSP70 release; levels of eHSP70 released at 39.5°C were higher than at 37°C roughly paralleling the increase in intracellular HSP72 in the 39.5°C cells. By contrast, 6 h exposure to FRH in the absence of LPS failed to promote eHSP70 release. Release of eHSP70 by LPS-treated THP1 cells was inhibited by glibenclamide, but not brefeldin, indicating that eHSP70 secretion occurred via a non-classical protein secretory mechanism. Analysis of eHSP70 levels in exosomes and exosome-depleted culture supernatants from LPS-treated THP1 cells using ELISA demonstrated similar eHSP70 levels in unfractionated and exosome-depleted culture supernatants, indicating that LPS-stimulated eHSP70 release did not occur via the exosome pathway. Immunoblot analysis of the exosome fraction of culture supernatants from these cells showed constitutive HSC70 (HSPA8) to be the predominant HSP70 family member present in exosomes. In summary, we have shown that LPS stimulates macrophages to secrete inducible HSP72 via a non-classical non-exosomal pathway while synergizing with FRH exposure to increase both intracellular and secreted levels of inducible HSP72. The impact of increased macrophage intracellular HSP70 levels and augmented secretion of proinflammatory eHSP70 in the febrile, infected patient remains to be elucidated.

Stimulation of Innate Immune Cells Induced by Probiotics: Participation of Toll-Like Receptors

Objective: The present work aimed to study the functionality of macrophages from different locations (peritoneum, spleen and Peyer´s patches) when they were stimulated with probiotics microorganisms: Lactobacillus casei CRL 431 and Lactobacillus paracasei CNCM I-1518 or a Probiotic Fermented Milk (PFM) through Toll-Like Receptors (TLRs), prior challenged with agonists or antagonists of TLRs. Methods: BALB/c mice received in the drinking water, the probiotic bacteria (L. casei CRL 431 and L. paracasei CNCM I-1518) or the PFM. We focused our investigation mainly on the phagocytic activity of macrophages from peritoneum, spleen and Peyer’s patches and cytokine production were evaluated prior challenged with TLR2 and TLR4 agonists or antagonists. The microbicidal activity of macrophages and against an infection with Salmonella typhimurium was also studied. To assess the role of TLR in probiotic stimulation, we evaluated the phagocytic activity, cytokine production and Immunoglobin G (IgG) anti-OVA in C57BL/6 knockout mice to MyD88, TLR2 and TLR4. Results: In BALB/c mice, the best effect in the phagocytosis assay was obtained with the probiotic bacteria L. casei CRL 431, this effect was reinforced with TLR2 agonist. The production of different cytokines (IL-10 and IL-6) was improved with the probiotic treatments and this production was ameliorated with TLRs agonists. The antimicrobial activity was increased with L. casei CRL 431 and L. paracasei CNCM I-1518, TLR2 and TLR4 antagonists had a negative effect on microbicidal activity. The administration of probiotic bacteria or PFM improved the host response against S. typhimurium controlling the infection during the first hours post-infection. In C57BL/6 knockout mice, phagocytic activity was significantly diminished in comparison to wild type mice and the probiotic bacteria or PFM administration was not able to improve this activity. The IL-10 production was increased at a concentration of 108 cells/ml of L. casei CRL 431 in TLR2-/- and TLR4-/-, but not in MyD88-/- mice. The administration of probiotic bacteria or PFM did not play a stimulating effect in the systemic immune response against to OVA antigen in knockout mice. Conclusions: Probiotics modulate the different signaling pathways of innate immune cells through the TLRs. The macrophages activation depends on location of them and that different probiotic strains of Lactobacilli can evoke different intensity of responses. The data suggest that probiotic not only promote a major expression of TLRs but also use these receptors via the innate immune cells as macrophages to stimulate and modulate the immune response.

Exaggerated Response to Toll-like Receptor Agonist Contributes to Excessive TNF Production in Myeloproliferative Neoplasm

Rationale:Tumor Necrosis Factor-alpha (TNF) is elevated in myeloproliferative neoplasm (MPN) and plays a key role in expansion of the JAK2V617F neoplastic clone. A high TNF environment, as is the case in MPN patients, gives TNF resistant JAK2V617F mutant cells a selective advantage over their TNF sensitive non-mutant counterparts resulting in expansion of the neoplastic clone. To efficiently target TNF production therapeutically it is necessary to identify the mechanism driving this excessive TNF production. TNF is classically produced by monocytes after stimulation through Toll-like receptors (TLR), crucial pattern recognition receptors for microbial products. Because TLR signaling plays an integral role in inflammation and TNF production we hypothesized that exaggerated signaling of the TLR pathway is the mechanism by which TNF is overproduced in MPN. To test this hypothesis we quantified TLR responses in monocytes from MPN patients and normal controls.Results:We compared the response of peripheral blood monocytes from MPN versus normal controls to the TLR agonists R848 (TLR7/8), LPS (TLR4), or zymosan (TLR2). After stimulation with each of these TLR agonists for 24 hours, CD14+ monocytes from MPN patients (n=18) produced increased amounts of TNF (measured by ELISA) as compared to normal controls (n=10) at all concentrations tested (p<0.05). The increased TNF production in MPN monocytes could not be explained by a higher fraction of inflammatory (CD16+ CD14+) monocytes in MPN. We next used phosflow to detect whether MPN patients have abnormal activation of downstream signaling molecules following stimulation with TLR agonists. At early time points (15min) following stimulation, MPN patients (n=6) and normal controls (n=6) phosphorylated p38 and ERK1/2 equally. At later time points (2hrs) MPN patients maintained phosphorylation of p38 and ERK1/2, whereas in normal controls phosphorylation of p38 and ERK1/2 returned to baseline (p<0.05). These data suggest that the excessive production of TNF in MPN patients may be due either to persistent activation of signaling following TLR stimulation or failure to repress the activation state of these two proteins.TLR ligation induces a negative feedback loop culminating in the production of IL-10, an anti-inflammatory cytokine which serves to dampen TNF production. We next used intracellular flow cytometry to determine whether the excessive TNF production in MPN is due to a failure to dampen TNF production over time. Monocytes from normal controls (n=12) cease production of TNF by 9 hours post LPS stimulation, whereas MPN monocytes (n=8) continue producing TNF (p<0.05). This continued TNF production by MPN monocytes is coincident with a failure to produce the anti-inflammatory cytokine IL-10 in response to TLR ligation (p<0.05).Conclusions:We find that MPN monocytes produce excessive amounts of TNF in response to TLR ligation. This excessive production of TNF is due to a defect in the negative regulatory feedback loop which normally serves to dampen TNF production. Targeting the TLR pathway therapeutically in MPN may serve to reduce TNF production and neutralize the selective advantage of the JAK2V617F neoplastic clone, ultimately leading to a reduction in the JAK2V617F allele burden. DisclosuresNo relevant conflicts of interest to declare.

Toll gates: An emerging therapeutic target

Innate immune system forms the first line of defense against microbial infections, as it exerts an immediate response. Innate immunity works through Toll-like receptors (TLRs) which functions as primary sensors of pathogens. TLR activates multiple signaling cascades leading to the induction of genes responsible for the release of inflammatory cytokines and type I interferon. Thus, they induce antimicrobial responses and also have an instructive role in adaptive immunity. However, TLR-mediated inflammation is said to be responsible for many of the destructive host responses in inflammatory diseases like periodontitis. Hence, therapeutics targeting TLRs are being used to treat disease such as HIV, Hepatitis B, asthma etc. Recently, synthetic TLR agonists are tried as novel vaccine adjuvant in treating periodontal diseases. This paper reviews the scope of TLR-based therapeutics in treating periodontitis.

TLR3, TLR4 and TLRs7-9 Induced Interferons Are Not Impaired in Airway and Blood Cells in Well Controlled Asthma.

Defective Rhinovirus induced interferon-β and interferon-λ production has been reported in bronchial epithelial cells from asthmatics but the mechanisms of defective interferon induction in asthma are unknown. Virus infection can induce interferon through Toll like Receptors (TLR)3, TLR7 and TLR8. The role of these TLRs in interferon induction in asthma is unclear. This objective of this study was to measure the type I and III interferon response to TLR in bronchial epithelial cells and peripheral blood cells from atopic asthmatics and non-atopic non-asthmatics. Bronchial epithelial cells and peripheral blood mononuclear cells from atopic asthmatic and non-atopic non-asthmatic subjects were stimulated with agonists to TLR3, TLR4 & TLRs7–9 and type I and III interferon and pro-inflammatory cytokine, interleukin(IL)-6 and IL-8, responses assessed. mRNA expression was analysed by qPCR. Interferon proteins were analysed by ELISA. Pro-inflammatory cytokines were induced by each TLR ligand in both cell types. Ligands to TLR3 and TLR7/8, but not other TLRs, induced interferon-β and interferon-λ in bronchial epithelial cells. The ligand to TLR7/8, but not those to other TLRs, induced only type I interferons in peripheral blood mononuclear cells. No difference was observed in TLR induced interferon or pro-inflammatory cytokine production between asthmatic and non-asthmatic subjects from either cell type. TLR3 and TLR7/8,, stimulation induced interferon in bronchial epithelial cells and peripheral blood mononuclear cells. Interferon induction to TLR agonists was not observed to be different in asthmatics and non-asthmatics.

S119 Roles of TLR3, TLR4- and TLRs7–9 in Interferon Induction in Bronchial Epithelial Cells and Peripheral Blood Mononuclear Cells from Asthmatic and Non-Asthmatic Subjects

IntroductionDefective rhinovirus (RV) induced interferon (IFN)-β and IFN-λ production has been reported in primary human bronchial epithelial cells (HBECs) and peripheral blood mononuclear cells (PBMCs) from asthmatics. The mechanisms of...

The TLR2 agonists lipoteichoic acid and Pam3CSK4 induce greater pro-inflammatory responses than inactivated Mycobacterium butyricum

The innate immune system can recognize pathogen-associated molecular patterns (PAMP) through toll-like receptors (TLRs). TLR stimulation by TLR-ligands (TLR-L) induces several genes that can regulate the immune response. In this study, we compared the ability of diverse TLR2-L to activate professional antigen presenting cells (pAPCs). We found that in comparison to whole non-replicating microorganism Mycobacterium butyricum, the smaller components; lipoteichoic acid and Pam3CSK4 significantly enhanced the expression of several pro-inflammatory mediators. These included IL-6, TNF-α and nitric oxide both at the mRNA and the protein levels. Moreover, the higher response was associated with a differential activation of nuclear transcription factor kappa-B (NF-κB) by the diverse TLR2-L. However, all three ligands enhanced antigen cross-presentation and T cell induction after virus infection to the same extent. In conclusion, the data highlight the potential for small components of TLR agonists to induce superior inflammatory immune responses than whole microbial preparation in the field of vaccine studies.

Bacterial toll-like receptor agonists induce sequential NF-κB-mediated leukotriene B4 and prostaglandin E2 production in chicken heterophils

Studies of the response of the primary avian polymorphonuclear leukocyte, the heterophil, to microbe associated molecular patterns (MAMPs) through toll-like receptors (TLR) has concentrated on the activation of the respiratory burst, release of intracellular granules, and the induction of cytokine and chemokine expression. Virtually no studies have been described on the role of lipid mediators, leukotrienes and prostaglandins, as effectors of the avian inflammatory response. We have previously shown that flagellin (FLG), the bacterial lipoprotein mimic palmitoly-3-cysteine–serine–lysine-4 (PAM), and unmethylated CpG motifs of bacteria DNA (CpG) are all potent activators of the avian innate immune system. In the present studies, we hypothesized that FLG, PAM, and CpG are also capable of eliciting the production of these lipid mediators of inflammation by avian heterophils. Compared to non-stimulated control heterophils, all three TLR agonists were potent inducers (3–5-fold increase) of a rapid production (30min) of leukotriene B4 (LTB4) followed by a later release (60–120min) of prostaglandin (PGE2) by the heterophils. LTB4 and PGE2 production were derived from lipoxygenase-5 (5-LO) and cyclooxygenase-2 (COX-2) enzymatic activities, respectively, as the selective 5-LO (caffeic acid) and COX-2 (NS-398) inhibitors eliminated LTB4 and PGE2 production from the MAMP-stimulated heterophils. These results demonstrate that both the lipoxygenase and cycloxygenase pathways are operational in avian heterophils in response to bacterial MAMPs. Treatment of heterophils with either FLG, PAM, or CpG also induced a significant increase in DNA binding by NF-κB family members’ p50, c-Rel, and RelB. Additionally, the production of LTB4 and PGE2 were inhibited following treatment of heterophils with the specific pharmacologic inhibitor of NF-κB (Bay 11–7086), thus suggesting that TLR pathway activation of NF-κB controls LTB4 and PGE2 production. This the first report of the production of lipid mediators of inflammation by avian heterophils in response to PAMPs. Since FLG, lipoproteins, and bacterial CpG DNA are abundant during bacterial infections, these data support their role in the inflammatory response mediated by avian heterophils.

Toll-like receptor cross-talk in human monocytes regulates CC-chemokine production, antigen uptake and immune cell recruitment

Chemokines production in monocytes/macrophages is crucial in modulating immune responses generated through Toll-like receptor (TLR)-mediated recognition of microbes. During microbial onset, multiple pathogen-associated structures can be present at infection sites, and simultaneously trigger different TLRs. We report here that TLR3, TLR4 and TLR8 engagement induce CCL1, CCL2 and CCL4 production in freshly isolated monocytes. While differentiating cells maintain the capacity to secrete CCL2 and CCL4, CCL1 is no longer induced at later differentiation stages. Although different pairs of TLR agonists have been described to synergistically induce cytokine production in different cell types, agonist combinations cooperate in reducing CCL1 and CCL2, but not CCL4 secretion in freshly isolated monocytes, and fail to rescue CCL1 production at later differentiation stages. The effects of single, but not combined, TLR engagement on chemokine expression mostly occur at transcriptional level, and are IL-10 independent. Conversely, inhibition of CCL1 secretion upon combined TLR engagement is partially rescued by blocking IL-23. A different chemotactic activity of monocyte-conditioned medium on blood mononuclear cells as well as antigen uptake capacity of TLR agonist activated monocytes parallel the regulated production of chemokines. Overall, these findings indicate that simultaneous engagement of TLRs may lead to different patterns of chemokine expression depending on cellular differentiation state, chemokine, and TLR agonist combination. These different responses may be relevant for the distinct but complementary functions of monocytes and macrophages in the immune response, and may have important implications for the therapeutic manipulation of the innate immune system.

Aberrant O-GlcNAcylation characterizes chronic lymphocytic leukemia

O-linked N-Acetylglucosamine (O-GlcNAc) post-translational modifications originate from the activity of the hexosamine pathway, and are known to affect intracellular signaling processes. As aberrant responses to microenvironmental signals are a feature of chronic lymphocytic leukemia (CLL), O-GlcNAcylated protein levels were measured in primary CLL cells. In contrast to normal circulating and tonsillar B cells, CLL cells expressed high levels of O-GlcNAcylated proteins, including p53, c-myc and Akt. O-GlcNAcylation in CLL cells increased following activation with cytokines and through toll-like receptors (TLRs), or after loading with hexosamine pathway substrates. However, high baseline O-GlcNAc levels were associated with impaired signaling responses to TLR agonists, chemotherapeutic agents, B cell receptor crosslinking and mitogens. Indolent and aggressive clinical behavior of CLL cells were found to correlate with higher and lower O-GlcNAc levels, respectively. These findings suggest that intracellular O-GlcNAcylation is associated with the pathogenesis of CLL, which could potentially have therapeutic implications.