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.

Anesthetics isoflurane and sevoflurane attenuate flagellin-mediated inflammation in the lung.

Isoflurane and sevoflurane are volatile anesthetics (VA) widely used in clinical practice to provide general anesthesia. We and others have previously shown that VAs have immunomodulatory effects and may have a significant impact on the progression of disease states. Flagellin is a component of Gram negative bacteria and plays a significant role in the pathophysiology of bacterial pneumonia through its binding to Toll-like Receptor 5 (TLR5). Our results showed that VAs, not an intravenous anesthetic, significantly attenuated the activation of TLR5 and the release of the neutrophil chemoattractant IL-8 from lung epithelial cells. Furthermore, flagellin-induced lung injury was significantly attenuated by VAs by inhibiting neutrophil migration to the bronchoalveolar space. The lungs of cystic fibrosis (CF) patients are highly colonized by Pseudomonas aeruginosa, which causes inflammation. The retrospective study of oxygenation in patients with CF who had received VA versus intravenous anesthesia suggested that VAs might have the protective effect for gas exchange. To understand the interaction between VAs and TLR5, a docking simulation was performed, which indicated that isoflurane and sevoflurane docked into the binding interphase between TLR5 and flagellin.

Inhibitory Effect of Lipoteichoic Acid Derived from Three Lactobacilli on Flagellin-Induced IL-8 Production in Porcine Peripheral Blood Mononuclear Cells.

Probiotics in livestock feed supplements are considered to be an alternative to antibiotics. However, effector molecules responsible for the beneficial roles of probiotics in pigs are in general not well known. Thus, this study demonstrated that a well-known virulence factor, flagellin of Salmonella typhimurium, significantly induced IL-8 production in porcine peripheral blood mononuclear cells, whereas lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria Lactobacillus plantarum, L. casei, and L. rhamnosus GG, effectively inhibited flagellin-induced IL-8 production at mRNA and protein levels. However, the lipoproteins of L. plantarum, L. casei, and L. rhamnosus GG did not suppress flagellin-induced IL-8 production. While D-alanine-deficient L. plantarum LTA inhibited flagellin-induced IL-8 production, L. plantarum LTA deficient in both D-alanine and acyl chains failed to inhibit it; this suggests that the acyl moieties of L. plantarum LTA are essential for inhibiting flagellin-induced IL-8 production. Taken together, L. plantarum LTA plays an important role in improving anti-inflammatory responses of porcine peripheral blood mononuclear cells.

All-Trans Retinoic Acid Enhances Bacterial Flagellin-Stimulated Proinflammatory Responses in Human Monocyte THP-1 Cells by Upregulating CD14.

All-trans retinoic acid (ATRA), an active form of vitamin A, exerts immunomodulatory functions. In this study, we examined the immune potentiating effect of ATRA on bacterial flagellin-induced NF-κB activation and proinflammatory cytokine production in human monocytic cell line THP-1. ATRA treatment significantly enhanced the flagellin-induced NF-κB/AP-1 activity in THP-1 via the RAR/RXR pathway. Similarly, ATRA enhanced the expression and production of TNF-α and IL-1ß in THP-1 cells upon flagellin challenge. The cell surface expression of toll-like receptor 5 (TLR5), which is the receptor for bacterial flagellin, was significantly reduced by ATRA in a concentration- and time-dependent manner. To determine the mechanisms underlying the ATRA-enhanced immune response against bacterial flagellin despite the reduced cell surface expression of TLR5 in ATRA-treated THP-1, we examined the cell surface expression of CD14, which has been proposed to be a TLR co-receptor that enhances the response to microbial components. The cell surface expression of CD14 was significantly enhanced by ATRA treatment, especially in the presence of flagellin. Anti-CD14 antibody treatment prior to ATRA and flagellin treatments completely abolished ATRA-enhanced TNF-α and IL-1ß production. Our results suggest that ATRA enhances flagellin-stimulated proinflammatory responses in human monocyte THP-1 cells by upregulating CD14 in a RAR/RXR-dependent manner.

miR-146a regulates the crosstalk between intestinal epithelial cells, microbial components and inflammatory stimuli.

Regulation of miR-146a abundance and its role in intestinal inflammation and particularly in intestinal epithelial cells (IECs) has been poorly studied. Here we study the relationship between bacterial antigens and inflammatory stimuli, and miR-146a expression using IEC lines and models of colitis (trinitrobenzenesulfonic acid (TNBS), dextran sulfate sodium (DSS) and the CD4 + CD62L + T cell transfer model). Specific bacterial antigens and cytokines (LPS, flagelin and IL-1ß/TNF) stimulate miR-146a expression, while peptidoglycan, muramyldipeptide and CpG DNA have no effect. Overexpression of miR-146a by LPS depends on the activation of the TLR4/MyD88/NF-kB and Akt pathways. Accordingly, the induction of miR-146a is lower in TLR4, but not in TLR2 knock out mice in both basal and colitic conditions. miR-146a overexpression in IECs induces immune tolerance, inhibiting cytokine production (MCP-1 and GROα/IL-8) in response to LPS (IEC18) or IL-1ß (Caco-2). Intestinal inflammation induced by chemical damage to the epithelium (DSS and TNBS models) induces miR-146a, but no effect is observed in the lymphocyte transfer model. Finally, we found that miR-146a expression is upregulated in purified IECs from villi vs. crypts. Our results indicate that miR-146a is a key molecule in the interaction among IECs, inflammatory stimuli and the microbiota.

Pulmonary vascular inflammation: effect of TLR signalling on angiopoietin/TIE regulation.

Increased pulmonary vascular resistance is a critical complication in sepsis. Toll-like receptor (TLR) as well as angiopoietin (ANG) signalling both contribute to the emergence of pulmonary arterial hypertension. We hypothesized that TLR stimulation by bacterial ligands directly affects expression and secretion of ligands and receptors of the angiopoietin/TIE axis. Microvascular endothelial (HPMEC) and smooth muscle cells (SMC) of pulmonary origin were incubated with thrombin and with ligands for TLR2, -4, -5, and -9. Expression and secretion of ANG1, -2, TIE2 and IL-8 were determined using quantitative real-time PCR and ELISA. TLR stimulation had no impact either on the expression of ANG2 and TIE2 in HPMEC or on that of ANG1 in SMC. However, overall levels of both released ANG1 and -2 were halved upon stimulation with the TLR9 ligand CpG, and ANG2 release was significantly enhanced by TLR4 activation when initially provoked by sequentially performed stimulation. Furthermore, enhanced ANG2 activity increased endothelial permeability, as demonstrated in an in vitro transwell assay. We conclude that sole TLR stimulation by bacterial ligands plays no significant role for altered expression and secretion of ANG1, -2 and TIE2 in human pulmonary vascular cells. The interplay between various stimuli is required to induce imbalances between ANG1 and -2.

Activation of AMPK in human fetal membranes alleviates infection-induced expression of pro-inflammatory and pro-labour mediators.

INTRODUCTION: In non-gestational tissues, the activation of adenosine monophosphate (AMP)-activated kinase (AMPK) is associated with potent anti-inflammatory actions. Infection and/or inflammation, by stimulating pro-inflammatory cytokines and matrix metalloproteinase (MMP)-9, play a central role in the rupture of fetal membranes. However, no studies have examined the role of AMPK in human labour. METHODS: Fetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and after preterm pre-labour rupture of membranes (PPROM). AMPK activity was assessed by Western blotting of phosphorylated AMPK expression. To determine the effect of AMPK activators on pro-inflammatory cytokines, fetal membranes were pre-treated with AMPK activators then stimulated with bacterial products LPS and flagellin or viral dsDNA analogue poly(I:C). Primary amnion cells were used to determine the effect of AMPK activators on IL-1ß-stimulated MMP-9 expression. RESULTS: AMPK activity was decreased with term labour. There was no effect of preterm labour. AMPK activity was also decreased in preterm fetal membranes, in the absence of labour, with PROM compared to intact membranes. AMPK activators AICAR, phenformin and A769662 significantly decreased IL-6 and IL-8 stimulated by LPS, flagellin and poly(I:C). Primary amnion cells treated with AMPK activators significantly decreased IL-1ß-induced MMP-9 expression. DISCUSSION: The decrease in AMPK activity in fetal membranes after spontaneous term labour and PPROM indicates an anti-inflammatory role for AMPK in human labour and delivery. The use of AMPK activators as possible therapeutics for threatened preterm labour would be an exciting future avenue of research.

Antigen replacement of domains D2 and D3 in flagellin promotes mucosal IgA production and attenuates flagellin-induced inflammatory response after intranasal immunization.

Targeting early infection in mucosal sites is one of the primary goals for mucosal vaccines so as to prevent pathogen mucosal transmission and infection. The TLR5 agonist flagellin was deemed to be a mucosal adjuvant candidate for clinical usage. However, the high antigenicity of flagellin and the possible inflammatory injury induced by flagellin might restrict its clinical usage. Here HIV-1 p24 protein was selected as an antigen model and we replaced the main antigenicity region domains D2 and D3 of non-pathogenic E.coli-derived flagellin (KF). The derived soluble protein KFD-p24 3D was then compared with KF-p24, which fused p24 directly to the C-terminal of KF. In vitro and ex vivo experiments showed that KFD-p24 3D has lower TLR5 agonist efficacy and less immunocyte-activating efficacy. Interestingly, the production of KF- specific antibody was highly reduced, and KFD-p24 3D induced IgA-biased antibody responses in mucosal sites. Moreover, KFD-p24 3D induced far fewer systemic inflammatory responses and abrogated detectable inflammatory side effects on mice, even at the high dose. The properties of enhanced IgA generation and attenuated inflammatory responses broaden the safe-dose range of KFD-p24 3D flagellin, creating a potentially promising mucosal adjuvant.

Human trophoblast cells modulate endometrial cells nuclear factor κB response to flagellin in vitro.

BACKGROUND: Implantation is a complex process that requires a delicate cooperation between the immune and reproductive system. Any interference in the fine balance could result in embryo loss and infertility. We have recently shown that Toll-like receptor 5 activation results in a decrease of trophoblast cells binding to endometrial cells in an in vitro model of human implantation. However, little is known about the downstream signalling leading to the observed failure in implantation and the factors that modulate this immune response. METHODS AND PRINCIPAL FINDINGS: An in vitro model of embryo implantation was used to evaluate the effect of trophoblasts and flagellin on the activation of NF-κB in endometrial cells and whether TLR5-related in vitro implantation failure is signalled through NF-κB. We generated two different NF-κB reporting cell lines by transfecting either an immortalized endometrial epithelial cell line (hTERT-EECs) or a human endometrial carcinoma cell line (Ishikawa 3-H-12) with a plasmid containing the secreted alkaline phosphatase (SEAP) under the control of five NF-κB sites. The presence of trophoblast cells as well as flagellin increased NF-κB activity when compared to controls. The NF-κB activation induced by flagellin was further increased by the addition of trophoblast cells. Moreover, blocking NF-κB signalling with a specific inhibitor (BAY11-7082) was able to restore the binding ability of our trophoblast cell line to the endometrial monolayer. CONCLUSIONS: These are the first results showing a local effect of the trophoblasts on the innate immune response of the endometrial epithelium. Moreover, we show that implantation failure caused by intrauterine infections could be associated with abnormal levels of NF-κB activation. Further studies are needed to evaluate the target genes through which NF-κB activation after TLR5 stimulation lead to failure in implantation and the effect of the embryo on those genes. Understanding these pathways could help in the diagnosis and treatment of implantation failure cases.

Callose deposition: a multifaceted plant defense response.

Callose deposition in Arabidopsis has emerged as a popular model system to quantify activity of plant immunity. However, there has been a noticeable rise in contradicting reports about the regulation of pathogen-induced callose. To address this controversy, we have examined the robustness of callose deposition under different growth conditions and in response to two different pathogen-associated molecular patterns, the flagellin epitope Flg22 and the polysaccharide chitosan. Based on a commonly used hydroponic culture system, we found that variations in growth conditions have a major impact on the plant's overall capacity to deposit callose. This environmental variability correlated with levels of hydrogen peroxide (H2O2) production. Depending on the growth conditions, pretreatment with abscissic acid stimulated or repressed callose deposition. Despite a similar effect of growth conditions on Flg22- and chitosan-induced callose, both responses showed differences in timing, tissue responsiveness, and colocalization with H2O2. Furthermore, mutant analysis revealed that Flg22- and chitosan-induced callose differ in the requirement for the NADPH oxidase RBOHD, the glucosinolate regulatory enzymes VTC1 and PEN2, and the callose synthase PMR4. Our study demonstrates that callose is a multifaceted defense response that is controlled by distinct signaling pathways, depending on the environmental conditions and the challenging pathogen-associated molecular pattern.

Modulation of defense signal transduction by flagellin-induced WRKY41 transcription factor in Arabidopsis thaliana.

Flagellin, a component of the flagellar filament of Pseudomonas syringae pv. tabaci 6605 (Pta), induces hypersensitive reaction in its non-host Arabidopsis thaliana. We identified the WRKY41 gene, which belongs to a multigene family encoding WRKY plant-specific transcription factors, as one of the flagellin-inducible genes in A. thaliana. Expression of WRKY41 is induced by inoculation with the incompatible pathogen P. syringae pv. tomato DC3000 (Pto) possessing AvrRpt2 and the non-host pathogens Pta within 6-h after inoculation, but not by inoculation with the compatible Pto. Expression of WRKY41 was also induced by inoculation of A. thaliana with an hrp-type three secretion system (T3SS)-defective mutant of Pto, indicating that effectors produced by T3SS in the Pto wild-type suppress the activation of WRKY41. Arabidopsis overexpressing WRKY41 showed enhanced resistance to the Pto wild-type but increased susceptibility to Erwinia carotovora EC1. WRKY41-overexpressing Arabidopsis constitutively expresses the PR5 gene, but suppresses the methyl jasmonate-induced PDF1.2 gene expression. These results demonstrate that WRKY41 may be a key regulator in the cross talk of salicylic acid and jasmonic acid pathways.