The new generation synthetic reconstituted surfactant CHF5633 suppresses LPS-induced cytokine responses in human neonatal monocytes

BackgroundNew generation synthetic surfactants represent a promising alternative in the treatment of respiratory distress syndrome in preterm infants. CHF5633, a new generation reconstituted agent, has demonstrated biophysical effectiveness in vitro and in vivo. In accordance to several well-known surfactant preparations, we recently demonstrated anti-inflammatory effects on LPS-induced cytokine responses in human adult monocytes. The present study addressed pro- and anti-inflammatory effects of CHF5633 in human cord blood monocytes.MethodsPurified neonatal CD14⁺ cells, either native or simultaneously stimulated with E. coli LPS, were exposed to CHF5633. TNF-α, IL-1β, IL-8 and IL-10 as well as TLR2 and TLR4 expression were analyzed by means of real-time quantitative PCR and flow cytometry.ResultsCHF5633 did not induce pro-inflammation in native human neonatal monocytes and did not aggravate LPS-induced cytokine responses. Exposure to CHF5633 led to a significant decrease in LPS-induced intracellular TNF-α protein expression, and significantly suppressed LPS-induced mRNA and intracellular protein expression of IL-1β. CHF5633 incubation did not affect cell viability, indicating that the suppressive activity was not due to toxic effects on neonatal monocytes. LPS-induced IL-8, IL-10, TLR2 and TLR4 expression were unaffected.ConclusionOur data confirm that CHF5633 does not exert unintended pro-apoptotic and pro-inflammatory effects in human neonatal monocytes. CHF5633 rather suppressed LPS-induced TNF-α and IL-1β cytokine responses. Our data add to previous work and may indicate anti-inflammatory features of CHF5633 on LPS-induced monocyte cytokine responses.     

A TLR4/MD2 fusion protein inhibits LPS-induced pro-inflammatory signaling in hepatic stellate cells

Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. In injured liver they are the main extracellular matrix protein producing cell type and further perpetuate hepatic injury by secretion of pro-inflammatory mediators. Since LPS-mediated signaling through toll-like receptor 4 (TLR4) has been identified as key fibrogenic signal in HSCs we aimed to test TLR4 as potential target of therapy via ligand-binding soluble receptors. Incubation of human HSCs with a fusion protein between the extracellular domain of TLR4 and MD2 which binds LPS inhibited LPS-induced NFκB and JNK activation. TLR4/MD2 abolished LPS-induced secretion of IL-6, IL-8, MCP1, and RANTES in HSCs. In addition, TLR4/MD2 fused to human IgG-Fc neutralized LPS activity. Since TLR4 mutant mice are resistant to liver fibrosis, the TLR4/MD2 soluble receptor might represent a new therapeutic molecule for liver fibrogenesis in vivo.     

Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia

Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium.     

TLR4-independent and PKR-dependent interleukin 1 receptor antagonist expression upon LPS stimulation

Dendritic cells (DCs) induce innate immune responses by recognizing bacterial LPS through TLR4 receptor complexes. In this study, we compared gene expression profiles of TLR4 knockout (TLR4^neg) DCs and wild type (TLR4^pos) DCs after stimulating with LPS. We found that the expression of various inflammatory genes by LPS were TLR4-independent. Among them, interleukin 1 receptor antagonist (IL-1rn) was of particular interest since IL-1rn is a potent natural inhibitor of proinflammatory IL-1. Using RT-PCR, real-time PCR, immunoblotting and ELISA, we demonstrated that IL-1rn was induced by DCs stimulated with LPS in the absence of TLR4. 2-Aminopurine, a pharmacological PKR inhibitor, completely abrogated LPS-induced expression of IL-1rn in TLR4^neg DCs, suggesting that LPS-induced TLR4-independent expression of IL-1rn might be mediated by PKR pathways. Considering that IL-1rn is a physiological inhibitor of IL-1, TLR4-independent and PKR-dependent pathways might be crucial in counter-balancing proinflammatory effector functions of DCs resulted from TLR4-dependent activation by LPS.     

Molecular Modeling-Based Evaluation of hTLR10 and Identification of Potential Ligands in Toll-Like Receptor Signaling

Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogens based on distinct molecular signatures. The human (h)TLR1, 2, 6 and 10 belong to the hTLR1 subfamilies, which are localized in the extracellular regions and activated in response to diverse ligand molecules. Due to the unavailability of the hTLR10 crystal structure, the understanding of its homo and heterodimerization with hTLR2 and hTLR1 and the ligand responsible for its activation is limited. To improve our understanding of the TLR10 receptor-ligand interaction, we used homology modeling to construct a three dimensional (3D) structure of hTLR10 and refined the model through molecular dynamics (MD) simulations. We utilized the optimized structures for the molecular docking in order to identify the potential site of interactions between the homo and heterodimer (hTLR10/2 and hTLR10/1). The docked complexes were then used for interaction with ligands (Pam₃CSK₄ and PamCysPamSK₄) using MOE-Dock and ASEDock. Our docking studies have shown the binding orientations of hTLR10 heterodimer to be similar with other TLR2 family members. However, the binding orientation of hTLR10 homodimer is different from the heterodimer due to the presence of negative charged surfaces at the LRR11-14, thereby providing a specific cavity for ligand binding. Moreover, the multiple protein-ligand docking approach revealed that Pam₃CSK₄ might be the ligand for the hTLR10/2 complex and PamCysPamSK_4, a di-acylated peptide, might activate hTLR10/1 hetero and hTLR10 homodimer. Therefore, the current modeled complexes can be a useful tool for further experimental studies on TLR biology.     

Anthocyanins isolated from Hibiscus syriacus L. attenuate lipopolysaccharide-induced inflammation and endotoxic shock by inhibiting the TLR4/MD2-mediated NF-κB signaling pathway

BackgroundHibiscus syriacus L. has been used as a medicinal plant in many Asian countries. However, anti-inflammatory activity of H. syriacus L. remains unknown.PurposeThis study was aimed to investigating the anti-inflammatory effect of anthocyanin fractions from the H. syriacus L. variety Pulsae (PS) on the lipopolysaccharide (LPS)-induced inflammation and endotoxic shock.Study design and methodsMTT assay and flow cytometry analysis were performed to determine cytotoxicity of PS. RT-PCR, western blotting, and ELISA were conducted to evaluate the expression of proinflammatory mediators and cytokines. Molecular docking study predicted the binding scores and sites of PS to TLR4/MD2 complex. Immunohistochemical assay was conducted to evaluate the binding capability of PS to TLR4/MD2 and nuclear translocation of NF-κB p65. A zebrafish endotoxic shock model was used to evaluate anti-inflammatory activity of PS in vivo.ResultsPS suppressed LPS-induced nitric oxide and prostaglandin E₂ secretion concomitant with the downregulation of inducible nitric oxide synthase and cyclooxygenase-2 expression. Furthermore, PS inhibited the production of proinflammatory cytokines such as TNF-α, IL-6, and IL-12 in LPS-stimulated RAW 264.7 macrophages. Additionally, molecular docking data showed that PS mostly fit into the hydrophobic pocket of MD2 and bound to TLR4. In particular, apigenin-7-O-glucoside powerfully bound to MD2 and TLR4 via hydrogen bonding. Additionally, immunohistochemistry assay revealed that PS inhibited LPS-induced TLR4 dimerization or expression on the cell surface, which consequently decreased MyD88 recruitment and IRAK4 phosphorylation, resulting in the inhibition of NF-κB activity. PS also attenuated LPS-mediated mortality and abnormality in zebrafish larvae and diminished the recruitment of neutrophils and macrophages at the inflammatory site accompanied by the low levels of proinflammatory mediators and cytokines.ConclusionPS might be a novel immunomodulator for the effective treatment of LPS-mediated inflammatory diseases.     

Identification and immunological evaluation of novel TLR2 agonists through structure optimization of Pam3CSK4

Toll-like receptor 2 (TLR2) is a bridge between innate immunity and adaptive immunity. TLR2 agonists have been exploited as potential vaccine adjuvants and antitumor agents. However, no TLR2 agonists have been approved by FDA up to now. To discover drug-like TLR2 selective agonists, a novel series of Pam₃CSK₄ derivatives were designed based on the crystal structure of hTLR2-hTLR1-Pam₃CSK₄ complex, synthesized and evaluated for their immune-stimulatory activities. Among them, 35c was identified as a murine-specific TLR2 agonist, while 35f was a human-specific TLR2 agonist. Besides, 35d (human and murine TLR2 agonist) showed TLR2 agonistic activity comparable to Pam₃CSK₄, which included: elevated IL-6 expression level (EC₅₀ = 83.08 ± 5.94 nM), up-regulated TNF-α and IL-6 mRNA expression and promoted maturation of DCs through activating the NF-κB signaling pathway. TLRs antibodies test showed that 35a and 35d were TLR2/1 agonists, while 35f was a TLR2/6 agonist.     

Effect of the antimicrobial peptide LL-37 on Toll-like receptors 2-, 3- and 4-triggered expression of IL-6, IL-8 and CXCL10 in human gingival fibroblasts

The antimicrobial peptide LL-37 is known to have a potent LPS-neutralizing activity in monocytes and macrophages. Recently, LL-37 in gingival crevicular fluids is suggested to be the major protective factor preventing infection of periodontogenic pathogens. In this study, we tried to address the effect of LL-37 on proinflammatory responses of human gingival fibroblasts (HGFs) stimulated with Toll-like receptor (TLR)-stimulant microbial compounds. LL-37 potently suppressed LPS-induced gene expression of IL6, IL8 and CXCL10 and intracellular signaling events, degradation of IRAK-1 and IκBα and phosphorylation of p38 MAPK and IRF3, indicating that the LPS-neutralizing activity is also exerted in HGFs. LL-37 also suppressed the expression of IL6, IL8 and CXCL10 induced by the TLR3 ligand poly(I:C). LL-37 modestly attenuated the expression of IL6 and IL8 induced by the TLR2/TLR1 ligand Pam₃CSK₄, but did not affect the expression induced by the TLR2/TLR6 ligand MALP-2. Interestingly, LL-37 rather upregulated the expression of IL6, IL8 and CXCL10 induced by another TLR2/TLR6 ligand FSL-1. Thus, the regulatory effect of LL-37 is differently exerted towards proinflammatory responses of HGFs induced by different microbial stimuli, which may lead to unbalanced proinflammatory responses of the gingival tissue to infection of oral microbes.     

Immuno-Stimulatory Activity of Escherichia coli Mutants Producing Kdo2-Monophosphoryl-Lipid A or Kdo2-Pentaacyl-Monophosphoryl-Lipid A

Lipid A is the active center of lipopolysaccharide which also known as endotoxin. Monophosphoryl-lipid A (MPLA) has less toxicity but retains potent immunoadjuvant activity; therefore, it can be developed as adjuvant for improving the strength and duration of the immune response to antigens. However, MPLA cannot be chemically synthesized and can only be obtained by hydrolyzing lipopolysaccharide (LPS) purified from Gram-negative bacteria. Purifying LPS is difficult and time-consuming and can damage the structure of MPLA. In this study, Escherichia coli mutant strains HWB01 and HWB02 were constructed by deleting several genes and integrating Francisella novicida gene lpxE into the chromosome of E. coli wild type strain W3110. Compared with W3110, HWB01 and HWB02 synthesized very short LPS, Kdo₂-monophosphoryl-lipid A (Kdo₂-MPLA) and Kdo₂-pentaacyl-monophosphoryl-lipid A (Kdo₂-pentaacyl-MPLA), respectively. Structural changes of LPS in the outer membranes of HWB01 and HWB02 increased their membrane permeability, surface hydrophobicity, auto-aggregation ability and sensitivity to some antibiotics, but the abilities of these strains to activate the TLR4/MD-2 receptor of HKE-Blue hTLR4 cells were deceased. Importantly, purified Kdo₂-MPLA and Kdo₂-pentaacyl-MPLA differed from wild type LPS in their ability to stimulate the mammalian cell lines THP-1 and RAW264.7. The purification of Kdo₂-MPLA and Kdo₂-pentaacyl-MPLA from HWB01 and HWB02, respectively, is much easier than the purification of LPS from W3110, and these lipid A derivatives could be important tools for developing future vaccine adjuvants.     

Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

We set out to gain deeper insight into the potential of antibody light chain variable domains (V_Ls) as immunotherapeutics. To this end, we generated a naïve human V_L phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (V_Hs), we isolated a diversity of V_L domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating V_L repertoires are a rich source of non-aggregating domains. In addition, the V_Ls demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human V_Hs revealed that the V_Ls had similar overall profiles with respect to melting temperature (T_m), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of V_Ls did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their T_ms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of V_Ls. Human V_Ls and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered V_Ls may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin.     

Leptospiral Hemolysins Induce Proinflammatory Cytokines through Toll-Like Receptor 2-and 4-Mediated JNK and NF-κB Signaling Pathways

Background

Infection with pathogenic Leptospira species causes serious systemic inflammation in patients. Although a few leptospiral proinflammatory molecules have been identified, Leptospira likely encodes other unidentified strong inflammation stimulators. The pathogenic L. interrogans genome encodes numerous putative hemolysin genes. Since hemolysins from other bacteria can cause inflammatory reactions, we hypothesized that leptospiral hemolysins may function as proinflammatory stimulators that contribute to the strong inflammation associated with Leptospira infection.

Methodology/Principal Findings

We first used cytokine protein microarrays for systematic analysis of serum cytokine profiles in leptospirosis patients and leptospire-infected mice. We found that IL-1β, IL-6 and TNF-α were the main proinflammatory cytokines in the sera of both the patients and the mice. We then analyzed eight putative hemolysins in L. interrogans strain Lai. The results showed that five of them, Sph1, Sph2, Sph3, HlpA and TlyA were secreted and had hemolytic activity. More importantly, these five hemolysins induced the strong production of IL-1β, IL-6 and TNF-α in human and mouse macrophages (although a bit lower in the latter). Furthermore, blockade of TLR2 or TLR4 with either antibodies or inhibitors of the NF-κB or JNK signaling pathways significantly reduced the production of hemolysin-induced IL-1β, IL-6 and TNF-α. Macrophages isolated from TLR2-, TLR4-or double TLR2-and 4-deficient mice also confirmed that the leptospiral hemolysins that induce proinflammatory cytokines are both TLR2-and TLR4-dependent.

Conclusions/Significance

Our findings demonstrate that L. interrogans secretes many hemolysins that function as powerful inducers of proinflammatory cytokines through both TLR2-and TLR4-dependent JNK and NF-κB pathways.     

IL-27 enhances LPS-induced proinflammatory cytokine production via upregulation of TLR4 expression and signaling in human monocytes

IL-27, which is produced by activated APCs, bridges innate and adaptive immunity by regulating the development of Th cells. Recent evidence supports a role for IL-27 in the activation of monocytic cells in terms of inflammatory responses. Indeed, proinflammatory and anti-inflammatory activities are attributed to IL-27, and IL-27 production itself is modulated by inflammatory agents such as LPS. IL-27 primes LPS responses in monocytes; however, the molecular mechanism behind this phenomenon is not understood. In this study, we demonstrate that IL-27 priming results in enhanced LPS-induced IL-6, TNF-α, MIP-1α, and MIP-1β expression in human primary monocytes. To elucidate the molecular mechanisms responsible for IL-27 priming, we measured levels of CD14 and TLR4 required for LPS binding. We determined that IL-27 upregulates TLR4 in a STAT3- and NF-κB-dependent manner. Immunofluorescence microscopy revealed enhanced membrane expression of TLR4 and more distinct colocalization of CD14 and TLR4 upon IL-27 priming. Furthermore, IL-27 priming enhanced LPS-induced activation of NF-κB family members. To our knowledge, this study is the first to show a role for IL-27 in regulating TLR4 expression and function. This work is significant as it reveals new mechanisms by which IL-27 can enhance proinflammatory responses that can occur during bacterial infections.     

Human αS1-casein induces IL-8 secretion by binding to the ecto-domain of the TLR4/MD2 receptor complex

Background

The milk protein α_S1-casein was recently reported to induce secretion of proinflammatory cytokines via Toll-like receptor 4 (TLR4). In this study, α_S1-casein was identified as binder of theTLR4 ecto domain.

Lycopene suppresses proinflammatory response in lipopolysaccharide-stimulated macrophages by inhibiting ROS-induced trafficking of TLR4 to lipid raft-like domains

We recently showed that lycopene inhibited lipopolysaccharide (LPS)-induced productions of nitric oxide (NO) and interleukin-6 (IL-6) in murine RAW264.7 macrophages by mechanisms related to inhibition of ERK and nuclear factor-κB. Since the assembly of Toll-like receptor 4 (TLR4) in lipid rafts is a key element in LPS induced signaling, we investigated whether this process would be influenced by lycopene. We found that pretreatment of RAW264.7 cells with lycopene inhibited LPS-induced recruitment of TLR4 into fractions — enriched with lipid raft marker. By the methods of immunoprecipitation and immunoblotting, we also found that lycopene inhibited the subsequent formation of the complex of TLR4 with its adaptors including myeloid differentiation primary-response protein 88 and TIR domain–containing adaptor-inducing IFN-β. We also found that the lycopene induced inhibition was associated with reduced formation of reactive oxygen species (ROS), which was an upstream mechanism for the effects of lycopene, because treating the cells with the antioxidant N-acetyl-l-cysteine and NADPH oxidase inhibitor diphenyleneiodonium chloride significantly inhibited LPS-induced recruitment of TLR4 into lipid raft-like domains as well as the production of proinflammatory molecule NO and IL-6. Thus, our findings suggest that lycopene may prevent LPS-induced TLR4 assembly into lipid rafts through reducing intracellular ROS level.     

Oxidatively modified low density lipoprotein (LDL) inhibits TLR2 and TLR4 cytokine responses in human monocytes but not in macrophages

Inflammation characterized by the expression and release of cytokines and chemokines is implicated in the development and progression of atherosclerosis. Oxidatively modified low density lipoproteins, central to the formation of atherosclerotic plaques, have been reported to signal through Toll-like receptors (TLRs), TLR4 and TLR2, in concert with scavenger receptors to regulate the inflammatory microenvironment in atherosclerosis. This study evaluates the role of low density lipoproteins (LDL) and oxidatively modified LDL (oxmLDL) in the expression and release of proinflammatory mediators IκBζ, IL-6, IL-1β, TNFα, and IL-8 in human monocytes and macrophages. Although standard LDL preparations induced IκBζ along with IL-6 and IL-8 production, this inflammatory effect was eliminated when LDL was isolated under endotoxin-restricted conditions. However, when added with TLR4 and TLR2 ligands, this low endotoxin preparation of oxmLDL suppressed the expression and release of IL-1β, IL-6, and TNFα but surprisingly spared IL-8 production. The suppressive effect of oxmLDL was specific to monocytes as it did not inhibit LPS-induced proinflammatory cytokines in human macrophages. Thus, TLR ligand contamination of LDL/oxmLDL preparations can complicate interpretations of inflammatory responses to these modified lipoproteins. In contrast to providing a proinflammatory function, oxmLDL suppresses the expression and release of selected proinflammatory mediators.     

Corticotrophin-Releasing Factor (CRF) and the Urocortins Are Potent Regulators of the Inflammatory Phenotype of Human and Mouse White Adipocytes and the Differentiation of Mouse 3T3L1 Pre-Adipocytes

Chronic activation of innate immunity takes place in obesity and initiated by the hypertrophic adipocytes which obtain a pro-inflammatory phenotype. The corticotrophin-releasing factor (CRF) family of neuropeptides and their receptors (CRF₁ and CRF₂) affect stress response and innate immunity. Adipose tissue expresses a complete CRF system. The aim of this study was to examine the role of CRF neuropeptides in the immune phenotype of adipocytes assessed by their expression of the toll-like receptor-4 (TLR4), the production of inflammatory cytokines IL-6, TNF-α and IL-1β, chemokines IL-8, monocyte attractant protein-1 (MCP-1) and of the adipokines adiponectin, resistin and leptin. Our data are as follows: (a) CRF, UCN2 and UCN3 are expressed in human white adipocytes as well as CRFR_1a, CRFR_2a and CRFR_2b but not CRFR_2c. 3T3L1 pre-adipocytes and differentiated adipocytes expressed both CRF₁ and CRF₂ receptors and UCN3, while UCN2 was detected only in differentiated adipocytes. CRF₂ was up-regulated in mouse mature adipocytes. (b) CRF₁ agonists suppressed media- and LPS-induced pre-adipocyte differentiation while CRF₂ receptor agonists had no effect. (c) In mouse pre-adipocytes, CRF₂ agonists suppressed TLR4 expression and the production of IL-6, CXCL1 and adiponectin while CRF₁ agonists had no effect. (d) In mature mouse adipocytes LPS induced IL-6 and CXCL1 production and suppressed leptin. (e) In human visceral adipocytes LPS induced IL-6, TNF-α, IL-8, MCP-1 and leptin production and suppressed adiponectin and resistin. (f) In mouse mature adipocytes CRF₁ and CRF₂ agonists suppressed basal and LPS-induced production of inflammatory cytokines, TLR4 expression and adiponectin production, while in human visceral adipocytes CRF and UCN1 suppressed basal and LPS-induced IL-6, TNF-α, IL-8 and MCP-1 production. In conclusion, the effects of the activation of CRF₁ and CRF₂ may be significant in ameliorating the pro-inflammatory activity of adipocytes in obesity.     

Leishmania (Viannia) braziliensis amastigotes induces the expression of TNFα and IL-10 by human peripheral blood mononuclear cells in vitro in a TLR4-dependent manner

While the role of Toll-like receptors (TLRs) has been investigated in murine models of tegumentary leishmaniasis caused by Leishmania (Viannia) braziliensis, the interaction between TLRs and Leishmania sp. has not been investigated in human cells. The aim of this study was to evaluate the involvement of TLR4 in cytokine production of human peripheral blood mononuclear cells (PBMCs) induced by L. braziliensis, and whether the parasite alters the expression of TLR4 on monocytes/macrophages. Amastigote forms were obtained from mice lesions and PBMCs were isolated from healthy donors. PBMCs were cultured in absence or presence of IFNγ, TLR4 neutralizing antibodies, natural antagonist of TLR4 (Bartonella LPS), TLR4 agonist (E. coli LPS), and amastigote forms. The concentrations of tumor necrosis factor (TNFα) and interleukin 10 (IL-10) were assayed by ELISA and TLR4 expression by flow cytometry. Amastigotes forms of L. braziliensis induced TNFα and IL-10 production only in IFNγ-primed PBMCs. The TNFα and IL-10 production was inhibited by TLR4 neutralization, both with anti-TLR4 antibodies and Bartonella LPS. Interestingly, addition of E. coli LPS further increased TNFα but not IL-10 production induced by L. braziliensis amastigotes. Amastigotes of L. braziliensis strongly reduced membrane TLR4 expression on monocytes/macrophages, apparently by internalization after the infection. The present study reveals that TLR4 drives the production of TNFα and IL-10 induced by L. braziliensis amastigotes and that the parasites decrease TLR4 expression on monocyte surface.     

Anti-Inflammatory Activity of a Novel Family of Aryl Ureas Compounds in an Endotoxin-Induced Airway Epithelial Cell Injury Model

Background

Despite our increased understanding of the mechanisms involved in acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS), there is no specific pharmacological treatment of proven benefit. We used a novel screening methodology to examine potential anti-inflammatory effects of a small structure-focused library of synthetic carbamate and urea derivatives in a well established cell model of lipopolysaccharide (LPS)-induced ALI/ARDS.

Methodology/Principal Findings

After a pilot study to develop an in vitro LPS-induced airway epithelial cell injury model, a library of synthetic carbamate and urea derivates was screened against representative panels of human solid tumor cell lines and bacterial and fungal strains. Molecules that were non-cytotoxic and were inactive in terms of antiproliferative and antimicrobial activities were selected to study the effects on LPS-induced inflammatory response in an in vitro cell culture model using A549 human alveolar and BEAS-2B human bronchial cells. These cells were exposed for 18 h to LPS obtained from Escherichia coli, either alone or in combination with the test compounds. The LPS antagonists rhein and emodin were used as reference compounds. The most active compound (CKT0103) was selected as the lead compound and the impact of CKT0103 on pro-inflammatory IL-6 and IL-8 cytokine levels, expression of toll-like receptor-4 (TLR4) and nuclear factor kappa B inhibitor alpha (IκBα) was measured. CKT0103 significantly inhibited the synthesis and release of IL-6 and IL-8 induced by LPS. This suppression was associated with inhibition of TLR4 up-regulation and IκBα down-regulation. Immunocytochemical staining for TLR4 and IκBα supported these findings.

Conclusions/Significance

Using a novel screening methodology, we identified a compound – CKT0103 – with potent anti-inflammatory effects. These findings suggest that CKT0103 is a potential target for the treatment of the acute phase of sepsis and sepsis-induced ALI/ARDS.