Birch Pollen Induces Toll-Like Receptor 4-Dependent Dendritic Cell Activation Favoring T Cell Responses

Seasonal exposure to birch pollen (BP) is a major cause of pollinosis. The specific role of Toll-like receptor 4 (TLR4) in BP-induced allergic inflammation and the identification of key factors in birch pollen extracts (BPE) initiating this process remain to be explored. This study aimed to examine (i) the importance of TLR4 for dendritic cell (DC) activation by BPE, (ii) the extent of the contribution of BPE-derived lipopolysaccharide (LPS) and other potential TLR4 adjuvant(s) in BPE, and (iii) the relevance of the TLR4-dependent activation of BPE-stimulated DCs in the initiation of an adaptive immune response. In vitro, activation of murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs by BPE or the equivalent LPS (nLPS) was analyzed by flow cytometry. Polymyxin B (PMB), a TLR4 antagonist and TLR4-deficient BMDCs were used to investigate the TLR4 signaling in DC activation. The immunostimulatory activity of BPE was compared to protein-/lipid-depleted BPE-fractions. In co-cultures of BPE-pulsed BMDCs and Bet v 1-specific hybridoma T cells, the influence of the TLR4-dependent DC activation on T cell activation was analyzed. In vivo immunization of IL-4 reporter mice was conducted to study BPE-induced Th2 polarization upon PMB pre-treatment. Murine and human DC activation induced by either BPE or nLPS was inhibited by the TLR4 antagonist or by PMB, and abrogated in TLR4-deficient BMDCs compared to wild-type BMDCs. The lipid-free but not the protein-free fraction showed a reduced capacity to activate the TLR4 signaling and murine DCs. In human DCs, nLPS only partially reproduced the BPE-induced activation intensity. BPE-primed BMDCs efficiently stimulated T cell activation, which was repressed by the TLR4 antagonist or PMB, and the addition of nLPS to Bet v 1 did not reproduce the effect of BPE. In vivo, immunization with BPE induced a significant Th2 polarization, whereas administration of BPE pre-incubated with PMB showed a decreased tendency. These findings suggest that TLR4 is a major pathway by which BPE triggers DC activation that is involved in the initiation of adaptive immune responses. Further characterization of these BP-derived TLR4 adjuvants could provide new candidates for therapeutic strategies targeting specific mechanisms in BP-induced allergic inflammation.

Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages

TLRs, including TLR4, play a crucial role in inflammatory-based diseases, and TLR4 has been identified as a therapeutic target for pharmacological intervention. In previous studies, we investigated the potential of FP7, a novel synthetic glycolipid active as a TLR4 antagonist, to inhibit haematopoietic and non-haematopoietic MyD88-dependent TLR4 pro-inflammatory signalling. The main aim of this study was to investigate the action of FP7 and its derivative FP12 on MyD88-independent TLR4 signalling in THP-1 derived macrophages. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 and FP12 on TRIF-dependent TLR4 functional activity in response to LPS and other endogenous TLR4 ligands in THP-1 macrophages. A different kinetic in the inhibition of endotoxin-driven TBK1, IRF3 and STAT1 phosphorylation was observed using different LPS chemotypes. Following activation of TLR4 by LPS, data revealed that FP7 and FP12 inhibited TBK1, IRF3 and STAT1 phosphorylation which was associated with down-regulation IFN-β and IP-10. Specific blockage of the IFN type one receptor showed that these novel molecules inhibited TRIF-dependent TLR4 signalling via IFN-β pathways. These results add novel information on the mechanism of action of monosaccharide FP derivatives. The inhibition of the TRIF-dependent pathway in human macrophages suggests potential therapeutic uses for these novel TLR4 antagonists in pharmacological interventions on inflammatory diseases.

Chronic inflammation of middle ear cholesteatoma promotes its recurrence via a paracrine mechanism

Background Cholesteatoma disease is an expanding lesion in the middle ear. Hearing loss and facial paralysis alongside with other intracranial complications are found. No pharmaceutical treatment is available today and recurrence after surgical extraction occurs. We investigated possible TLR4-based mechanisms promoting recurrence and explore possible treatments strategies. Methods We isolated fibroblasts and epidermal stem cells from cholesteatoma tissue and healthy auditory canal skin. Subsequently, their expression under standard culture conditions and after stimulation with LPS was investigated by RT-qPCR. Cell metabolism and proliferation were analysed upon LPS treatment, with and without TLR4 antagonist. An indirect co-culture of fibroblasts and epidermal stem cells isolated from cholesteatoma tissue was utilized to monitor epidermal differentiation upon LPS treatment by RT-qPCR and immunocytochemistry. Results Under standard culture conditions, we detected a tissue-independent higher expression of IL-1β and IL-8 in stem cells, an upregulation of KGF and IGF-2 in both cell types derived from cholesteatoma and higher expression of TLR4 in stem cells derived from cholesteatoma tissue. Upon LPS challenge, we could detect a significantly higher expression of IL-1α, IL-1β, IL-6 and IL-8 in stem cells and of TNF-a, GM-CSF and CXCL-5 in stem cells and fibroblasts derived from cholesteatoma. The expression of the growth factors KGF, EGF, EREG, IGF-2 and HGF was significantly higher in fibroblasts, particularly when derived from cholesteatoma. Upon treatment with LPS the metabolism was elevated in stem cells and fibroblasts, proliferation was only enhanced in fibroblasts derived from cholesteatoma. This could be reversed by the treatment with a TLR4 antagonist. The cholesteatoma fibroblasts could be triggered by LPS to promote the epidermal differentiation of the stem cells, while no LPS treatment or LPS treatment without the presence of fibroblasts did not result in such a differentiation. Conclusion We propose that cholesteatoma recurrence is based on TLR4 signalling imprinted in the cholesteatoma cells. It induces excessive inflammation of stem cells and fibroblasts, proliferation of perimatrix fibroblasts and the generation of epidermal cells from stem cells thru paracrine signalling by fibroblasts. Treatment of the operation site with a TLR4 antagonist might reduce the chance of cholesteatoma recurrence.

CD4+CD25+Foxp3+ regulatory T cells regulate immune balance in unexplained recurrent spontaneous abortion via the Toll-like receptor 4/nuclear factor-κB pathway

Objective The present study aimed to evaluate the effects of cluster of differentiation (CD)4+CD25+ forkhead box p3 (Foxp3)+ regulatory T cells (Tregs) on unexplained recurrent spontaneous abortion (URSA) and the associated mechanisms. Methods The proportion of CD4+CD25+Foxp3+ Tregs and inflammatory cytokine concentrations in the peripheral blood of women with URSA were measured by flow cytometry and enzyme-linked immunosorbent assay, respectively. CBA/JxDBA/2J mating was used to establish an abortion-prone mouse model and the model mice were treated with the Toll-like receptor 4 (TLR4) antagonist E5564 and the TLR4 agonist lipopolysaccharide. Results The proportion of CD4+CD25+Foxp3+ Tregs was decreased and the inflammatory response was increased in women with URSA. In the abortion-prone mouse model, E5564 significantly increased the proportion of CD4+CD25+Foxp3+ Tregs, decreased the inflammatory response, and increased Foxp3 mRNA and protein expression. Lipopolysaccharide had adverse effects on the abortion-prone model. Conclusions These data suggest that CD4+CD25+Foxp3+ Tregs regulate immune homeostasis in URSA via the TLR4/nuclear factor-κB pathway, and that the TLR4 antagonist E5564 may be a novel and potential drug for treating URSA.

Mechanism of Toll-like receptor 4 on chronic post-thoracotomy pain via caspase-1 activation

Objective: Post-operative chronic post-thoracotomy pain (CPTP) has been linked to restrictions in mobility and daily activities. However, its potential causes and optimal therapy have not been well characterized. Here, the purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) in CPTP rats and its underlying mechanism. Methods: Initially, rat models of CPTP were established. Then, the mechanical withdrawal threshold (MWT) was measured after intrathecal injection of TLR4 antagonist (LPS-RS), TLR4 agonist (LPS-PG), or caspase-1 inhibitor (Ac-YVAD-CMK) in CPTP rats. Levels of TNF-α, IL-6 and IL-1β in the spinal dorsal horn (SDH) were measured by ELISA. TLR4 and caspase-1 were located by immunofluorescence double staining. TLR4 and caspase-1 levels were assessed by qRT-PCR and Western blot. Results: TLR4 and caspase-1 were up-regulated in SDH of CPTP rats. Compared with Sham and non-CPTP groups, MWT was effectively decreased while TNF-α, IL-6 and IL-1β in SDH were increased in CPTP group. Moreover, intrathecal injection of TLR4 antagonist or caspase-1 inhibitor significantly elevated MWT expression and reduced levels of TNF-α, IL-6 and IL-1β in SDH. Additionally, high expression of TLR4 promoted mechanical hyperalgesia and inflammatory response, while intrathecal injection of a mixture of caspase-1 inhibitor and TLR4 agonist reversed the alleviation of caspase-1 inhibitor on the mechanical hyperalgesia and inflammatory response. TLR4 and caspase-1 were co-located in neurons. Conclusion: TLR4 aggravated CPTP in rats by mediating activation of caspase-1 in SDH.

Role of toll-like receptor 4 on tissue factor regulation in human monocytes

Background Inflammation and coagulation play a pivotal role in the pathogenesis of acute coronary events and an extensive cross-talk links the two systems, whereby inflammation activates coagulation and coagulation affects inflammatory activity. Infact, pro-inflammatory stimuli can induce tissue factor (TF) expression, the principal initiator of the clotting cascade, in circulating monocytes and activate pathways leading to thrombin generation.In turn, TF may bind cellular receptors which may affect the production and release of inflammatory mediators. According to our recent results, proprotein convertase subtilisin/kexin9 (PCSK9) and Gamma-Glutamyltransferase (GGT), two molecules involved in the pathogenesis of cardiovascular disease, are able to up-regulate TF expression in monocytes by activating NFkB pathway but the mechanism and the receptor involved in this biological response is unknown. One plausible possibility is that both molecules bind a Toll-like Receptor (TLR)4 located on membranes of human and cultured monocytes,activating the TLR4/MyD88-NFkB pathway and eventually leading to stimulation of TF expression. Aim To assess whether both molecules are able to bind to TLR4 located on the surface of human monocytes and whether this specific binding involves the TLR4/MyD88-NFkB pathway on TF modulation. Methods THP-1 cells, a human monocytic cell line derived from an acute monocytic leukemia patient, was used as in vitro model. The cells were stimulated with human (h) recombinant (r) PCSK9 (5 μg/ml) and hrGGT (1 μg/ml) or pre-incubated with BAY-117082 (BAY, 10–5M) a NFκB inhibitor, CLI-095 (3x10–6M), a highly Myd88/TLR4 signaling specific inhibitor and LPS-RS (1 μg/ml) a TLR4 antagonist. TF procoagulant activity (PCA), was assessed by 1-stage clotting assay and the results expressed by ρg/mL of active protein. Experimental series were carried out in endotoxin-free conditions, in order to exclude lipopolysaccharide (LPS)-dependent immune responses. Results hrPCSK9 and hrGGT stimulated TF expression (PCA: from 50±20 to 120±20, n=10, p<0.01) and (PCA: from 190±140 to 460±360, n=15, p<0.001) respectively, an effect down-regulated by BAY,a NFκB inhibitor (PCA by hrPCSK9: −71±23%, n=5, p<0.01; PCA by hrGGT: −90±21%, n=7, p<0.001). CLI-095, a TLR4 inhibitor (PCA by hrPCSK9: −86±26%, n=3, p<0.05; and PCA by GGT: −89±10%, n=5, p<0.001).LPS-RS, a TLR4 antagonist, (PCA by hrPCSK9: −74±25%, n=3, p<0.05; PCA by hrGGT: −70±17%, n=5, p<0.001) abolished both PCSK9 and GGT-induced TF expression. Conclusions These data are the first demonstration of a direct role of PCSK9 and GGT as active mediators of inflammatory-based thrombotic diseases. The possible mechanism of action involves recognition of two proteins by TLR4 on monocytes membrane surface, lead to activation of the transcription factor NFκB. Further studies will be needed to better understand the regulatory mechanisms underlying this complex set of biological responses that bind TLR4 modulation and TF expression. Funding Acknowledgement Type of funding source: None

Chronic inflammation of middle ear cholesteatoma promotes its recurrence via a paracrine mechanism

BackgroundCholesteatoma disease is an expanding lesion in the middle ear. Hearing loss and facial paralysis alongside with other intracranial complications are found. No pharmaceutical treatment is available today and recurrence after surgical extraction occurs. We investigated possible TLR4-based mechanisms promoting recurrence and explore possible treatments strategies.MethodsWe isolated fibroblasts and epidermal stem cells from cholesteatoma tissue and healthy auditory canal skin. Subsequently, their expression under standard culture conditions and after stimulation with LPS was investigated by RT-qPCR. Cell metabolism and proliferation were analysed upon LPS treatment, with and without TLR4 antagonist. An indirect co-culture of fibroblasts and epidermal stem cells isolated from cholesteatoma tissue was utilized to monitor epidermal differentiation upon LPS treatment by RT-qPCR and immunocytochemistry.ResultsUnder standard culture conditions, we detected a tissue-independent higher expression of IL-1β and IL-8 in stem cells, an upregulation of KGF and IGF-2 in both cell types derived from cholesteatoma and higher expression of TLR4 in stem cells derived from cholesteatoma tissue. Upon LPS challenge, we could detect a significantly higher expression of IL-1α, IL-1β, IL-6 and IL-8 in stem cells and of TNF-a, GM-CSF and CXCL-5 in stem cells and fibroblasts derived from cholesteatoma. The expression of the growth factors KGF, EGF, EREG, IGF-2 and HGF was significantly higher in fibroblasts, particularly when derived from cholesteatoma. Upon treatment with LPS the metabolism was elevated in stem cells and fibroblasts, proliferation was only enhanced in fibroblasts derived from cholesteatoma. This could be reversed by the treatment with a TLR4 antagonist. The cholesteatoma fibroblasts could be triggered by LPS to promote the epidermal differentiation of the stem cells, while no LPS treatment or LPS treatment without the presence of fibroblasts did not result in such a differentiation.ConclusionWe propose that cholesteatoma recurrence is based on TLR4 signalling imprinted in the cholesteatoma cells. It induces excessive inflammation of stem cells and fibroblasts, proliferation of perimatrix fibroblasts and the generation of epidermal cells from stem cells thru paracrine signalling by fibroblasts. Treatment of the operation site with a TLR4 antagonist might reduce the chance of cholesteatoma recurrence.

Stem Cell-Induced Inflammation in Cholesteatoma Is Inhibited by the TLR4 Antagonist LPS-RS

Cholesteatoma is a severe non-cancerous lesion of the middle ear characterized by massive inflammation, tissue destruction, and an abnormal growth of keratinized squamous epithelium. We recently demonstrated the presence of pathogenic stem cells within cholesteatoma tissue, unfortunately their potential roles in regulating disease-specific chronic inflammation remain poorly understood. In the presented study, we utilized our established human in vitro cholesteatoma stem cell model for treatments with lipopolysaccharides (LPS), tumor necrosis factor α (TNFα), and the TLR4-antagonist LPS from R. sphaeroides (LPS-RS) followed by qPCR, western blot, and immunocytochemistry. Middle ear cholesteatoma stem cells (ME-CSCs) showed a significantly increased expression of TLR4 accompanied by a significantly enhanced LPS-dependent pro-inflammatory gene expression pattern of TNFα, IL-1α, IL-1ß, IL-6, and IL-8 compared to non-pathogenic control cells. LPS-dependent pro-inflammatory gene expression in ME-CSCs was driven by an enhanced activity of NF-κB p65 leading to a TNFα-mediated feed-forward-loop of pro-inflammatory NF-κB target gene expression. Functional inactivation of TLR4 via the TLR4-antagonist LPS-RS blocked chronic inflammation in ME-CSCs, resulting in a nearly complete loss of IL-1ß, IL-6, and TNFα expression. In summary, we determined that ME-CSCs mediate the inflammatory environment of cholesteatoma via TLR4-mediated NF-κB-signaling, suggesting a distinct role of ME-CSCs as drivers of cholesteatoma progression and TLR4 on ME-CSCs as a therapeutic target.