Cell wall fraction of Enterococcus hirae ameliorates TNF-alpha-induced barrier impairment in the human epithelial tight junction

Aims: The evaluation of the effects of Enterococcus hirae, an intestinal bacterium in the adjacent mucosa (mucosal bacterium), on tumour necrosis factor‐alpha (TNF‐α)‐induced barrier impairment in human epithelial Caco‐2 cells. Methods and Results: The filter‐grown Caco‐2 monolayers were used as an intestinal epithelial model system. In Caco‐2 cells, heat‐killed E. hirae ATCC 9790^T suppressed the TNF‐α‐induced barrier impairment and increase in interleukin‐8 (IL‐8) secretion, but lipase‐ and mutanolysin‐treated E. hirae ATCC 9790^T did not have these effects. It was demonstrated that lipoteichoic acid (LTA) from E. hirae ATCC 9790^T is responsible for Caco‐2 cells’ recovery from TNF‐α‐induced impairments. In addition, Caco‐2 cells had the same response to Toll‐like receptor 2 (TLR2) ligand, Pam₃Cys‐Ser‐(Lys)₄ as they did to LTA. Increased expression of zonula occludens‐1 was observed by the addition of E. hirae ATCC 9790^T to TNF‐α‐treated Caco‐2 cells, and decreased expression of myosin light chain kinase was observed by the addition of LTA and Pam₃Cys‐Ser‐(Lys)₄; this, in turn, led to barrier enforcement. Conclusions: Enterococcus hirae ATCC 9790^T cell wall fractions, such as LTA, protect against intestinal impairment by regulation of epithelial tight junction via TLR2 signalling. Significance and Impact of the Study: Enterococcus hirae could be useful in the treatment of inflammatory bowel disease, as well as other intestinal disorders.     

Effects of the TLR2 agonists MALP-2 and Pam3Cys in isolated mouse lungs

Background

Gram-positive and Gram-negative bacteria are main causes of pneumonia or acute lung injury. They are recognized by the innate immune system via toll-like receptor-2 (TLR2) or TLR4, respectively. Among all organs, the lungs have the highest expression of TLR2 receptors, but little is known about the pulmonary consequences of their activation. Here we studied the effects of the TLR2/6 agonist MALP-2, the TLR2/1 agonist Pam₃Cys and the TLR4 agonist lipopolysaccharide (LPS) on pro-inflammatory responses in isolated lungs.

Methodology/Principal Findings

Isolated perfused mouse lungs were perfused for 60 min or 180 min with MALP-2 (25 ng/mL), Pam₃Cys (160 ng/mL) or LPS (1 µg/mL). We studied mediator release by enzyme linked immunosorbent assay (ELISA), the activation of mitogen activated protein kinase (MAPK) and AKT/protein kinase B by immunoblotting, and gene induction by quantitative polymerase chain reaction. All agonists activated the MAPK ERK1/2 and p38, but neither JNK or AKT kinase. The TLR ligands upregulated the inflammation related genes Tnf, Il1β, Il6, Il10, Il12, Ifng, Cxcl2 (MIP-2α) and Ptgs2. MALP-2 was more potent than Pam₃Cys in inducing Slpi, Cxcl10 (IP10) and Parg. Remarkable was the strong induction of Tnc by MALP2, which was not seen with Pam₃Cys or LPS. The growth factor related genes Areg and Hbegf were not affected. In addition, all three TLR agonists stimulated the release of IL-6, TNF, CXCL2 and CXCL10 protein from the lungs.

Conclusions/Significance

TLR2 and TLR4 activation leads to similar reactions in the lungs regarding MAPK activation, gene induction and mediator release. Several genes studied here have not yet been appreciated as targets of TLR2-activation in the lungs before, i.e., Slpi, tenascin C, Parg and Traf1. In addition, the MALP-2 dependent induction of Tnc may indicate the existence of TLR2/6-specific pathways.     

Pro‐inflammatory capacity of classically activated monocytes relates positively to muscle mass and strength

In mice, monocytes that exhibit a pro‐inflammatory profile enter muscle tissue after muscle injury and are crucial for clearance of necrotic tissue and stimulation of muscle progenitor cell proliferation and differentiation. The aim of this study was to test if pro‐inflammatory capacity of classically activated (M1) monocytes relates to muscle mass and strength in humans. This study included 191 male and 195 female subjects (mean age 64.2 years (SD 6.4) and 61.9 ± 6.4, respectively) of the Leiden Longevity Study. Pro‐inflammatory capacity of M1 monocytes was assessed by ex vivo stimulation of whole blood with Toll‐like receptor (TLR) 4 agonist lipopolysaccharide (LPS) and TLR‐2/1 agonist tripalmitoyl‐S‐glycerylcysteine (Pam₃Cys‐SK₄), both M1 phenotype activators. Cytokines that stimulate M1 monocyte response (IFN‐γ and GM‐CSF) as well as cytokines that are secreted by M1 monocytes (IL‐6, TNF‐α, IL‐12, and IL‐1β) were measured. Analyses were adjusted for age, height, and body fat mass. Upon stimulation with LPS, the cytokine production capacity of INF‐γ, GM‐CSF, and TNF‐α was significantly positively associated with lean body mass, appendicular lean mass and handgrip strength in men, but not in women. Upon stimulation with Pam₃Cys‐SK₄, IL‐6; TNF‐α; and Il‐1β were significantly positively associated with lean body mass and appendicular lean in women, but not in men. Taken together, this study shows that higher pro‐inflammatory capacity of M1 monocytes upon stimulation is associated with muscle characteristics and sex dependent.     

Mal connects TLR2 to PI3Kinase activation and phagocyte polarization

The recognition of bacterial lipoproteins by toll‐like receptor (TLR) 2 is pivotal for inflammation initiation and control in many bacterial infections. TLR2‐dependent signalling is currently believed to essentially require both adaptor proteins MyD88 (m yeloid d ifferentiation primary response gene 88) and Mal/TIRAP (M yD88‐a dapter‐l ike/TI R‐domain‐containing a daptor p rotein). TLR2‐dependent, but MyD88‐independent responses have not been described yet. We report here on a novel‐signalling pathway downstream of TLR2, which does not adhere to the established model. On stimulation of the TLR2/6 heterodimer with diacylated bacterial lipoproteins, Mal directly interacts with the regulatory subunit of phosphoinositide 3‐kinase (PI3K), p85α, in an inducible fashion. The Mal–p85α interaction drives PI3K‐dependent phosphorylation of Akt, phosphatidylinositol(3,4,5)P3 (PIP₃) generation and macrophage polarization. MyD88 is not essential for PI3K activation and Akt phosphorylation; however, cooperates with Mal for PIP₃ formation and accumulation at the leading edge. In contrast to TLR2/6, TLR2/1 does not require Mal or MyD88 for Akt phosphorylation. Hence, Mal specifically connects TLR2/6 to PI3K activation, PIP₃ generation and macrophage polarization.     

Distinctive probiotic features share common TLR2‐dependent signalling in intestinal epithelial cells

The underlying mechanisms of probiotics and postbiotics are not well understood, but it is known that both affect the adaptive and innate immune responses. In addition, there is a growing concept that some probiotic strains have common core mechanisms that provide certain health benefits. Here, we aimed to elucidate the signalization of the probiotic bacterial strains Lactobacillus paragasseri K7, Limosilactobacillus fermentum L930BB, Bifidobacterium animalis subsp. animalis IM386 and Lactiplantibacillus plantarum WCFS1. We showed in in vitro experiments that the tested probiotics exhibit common TLR2‐ and TLR10‐dependent downstream signalling cascades involving inhibition of NF‐κB signal transduction. Under inflammatory conditions, the probiotics activated phosphatidylinositol 3‐kinase (PI3K)/Akt anti‐apoptotic pathways and protein kinase C (PKC)‐dependent pathways, which led to regulation of the actin cytoskeleton and tight junctions. These pathways contribute to the regeneration of the intestinal epithelium and modulation of the mucosal immune system, which, together with the inhibition of canonical TLR signalling, promote general immune tolerance. With this study we identified shared probiotic mechanisms and were the first to pinpoint the role of anti‐inflammatory probiotic signalling through TLR10.     

A Network of Hydrogen Bonds on the Surface of TLR2 Controls Ligand Positioning and Cell Signaling

TLR2 is a pattern recognition receptor that functions in association with TLR1 or TLR6 to mediate innate immune responses to a variety of conserved microbial products. In the present study, the ectodomain of TLR2 was extensively mutated, and the mutants were assessed for their ability to bind and to mediate cellular responses to triacylated lipopeptide Pam₃CSK₄. This analysis provides evidence that the recently published crystal structure of the TLR2-TLR1-Pam₃CSK₄ complex represents a functional signal-inducing complex. Furthermore, we report that extended H-bond networks on the surface of TLR2 are critical for signaling in response to Pam₃CSK₄ and to other di- and tri-acylated TLR2-TLR6 and TLR2-TLR1 ligands. Based on this finding, we suggest a dynamic model for TLR2-mediated recognition of these ligands in which TLR2 fluctuates between a conformation that is more suitable for binding of the fatty acyl moieties of the ligands and a conformation that favors, via a specific orientation of the ligand head group, formation of a signal-inducing ternary complex.     

Role of JAK2–STAT3 in TLR2‐mediated tissue factor expression

Tissue factor (TF) is a core protein with an essential function in the coagulation cascade that maintains the homeostasis of the blood vessels. TF not only participates in neointima formation, but also causes the development of atherosclerosis. This study investigated the mechanism regulating TF expression in macrophages using Pam₃CSK₄, a TLR2 ligand. Pam₃CSK₄ induced TF expression in two types of macrophages (Raw264.7 and BMDM), but not in TLR2 KO mice derived BMDM. Pam₃CSK₄ induced TF expression was inhibited by pretreatment with pan‐JAK inhibitor or JAK2 inhibitor AG490. JAK2 knock‐down by siRNA inhibited Pam₃CSK₄ induced TF expression. Pam₃CSK₄ stimulated STAT3 phosphorylation (S727), while STAT3 knock‐down by siRNA reduced Pam₃CSK₄ induced TF expression. These results suggest that Pam₃CSK₄ induced TF expression is regulated by the JAK2–STAT3 signaling pathway. Pam₃CSK₄, unlike increased TF expression, significantly decreased RGS2 expression, while RGS2 overexpression decreased Pam₃CSK₄ induced TF expression. Inhibition of TF by RGS2 WT did not occur in mutants with flawed RGS domains. We also investigated the correlation between RGS2 and STAT3 phosphorylation. RGS2 knock‐down elevated Pam₃CSK₄ induced STAT3 phosphorylation, but RGS2 overexpression had the opposite effect on STAT3 phosphorylation. These results suggest that, while Pam₃CSK₄ induced TF expression is regulated by JAK2–STAT3 signaling, RGS2 is a negative regulator targeted to STAT3. J. Cell. Biochem. 114: 1315–1321, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

S1PR3 is essential for phosphorylated fingolimod to protect astrocytes against oxygen‐glucose deprivation‐induced neuroinflammation via inhibiting TLR2/4‐NFκB signalling

Fingolimod (FTY720) is used as an immunosuppressant for multiple sclerosis. Numerous studies indicated its neuroprotective effects in stroke. However, the mechanism remains to be elucidated. This study was intended to investigate the mechanisms of phosphorylated FTY720 (pFTY720), which was the principle active molecule in regulating astrocyte‐mediated inflammatory responses induced by oxygen‐glucose deprivation (OGD). Results demonstrated that pFTY720 could protect astrocytes against OGD‐induced injury and inflammatory responses. It significantly decreased pro‐inflammatory cytokines, including high mobility group box 1 (HMGB1) and tumour necrosis factor‐α (TNF‐α). Further, studies displayed that pFTY720 could prevent up‐regulation of Toll‐like receptor 2 (TLR2), phosphorylation of phosphoinositide 3‐kinase (PI3K) and nuclear translocation of nuclear factor kappa B (NFκB) p65 subunit caused by OGD. Sphingosine‐1‐phosphate receptor 3 (S1PR3) knockdown could reverse the above change. Moreover, administration of TLR2/4 blocker abolished the protective effects of pFTY720. Taken together, this study reveals that pFTY720 depends on S1PR3 to protect astrocytes against OGD‐induced neuroinflammation, due to inhibiting TLR2/4‐PI3K‐NFκB signalling pathway.     

Salidroside suppressing LPS‐induced myocardial injury by inhibiting ROS‐mediated PI3K/Akt/mTOR pathway in vitro and in vivo

The purpose of the present study was to investigate the effect of salidroside (Sal) on myocardial injury in lipopolysaccharide (LPS)‐induced endotoxemic in vitro and in vivo. SD rats were randomly divided into five groups: control group, LPS group (15 mg/kg), LPS plus dexamethasone (2 mg/kg), LPS plus Sal groups with different Sal doses (20, 40 mg/kg). Hemodynamic measurement and haematoxylin and eosin staining were performed. Serum levels of creatine kinase (CK), lactate dehydrogenase, the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH‐px), glutathione, tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) were measured after the rats were killed. iNOS, COX‐2, NF‐κB and PI3K/Akt/mTOR pathway proteins were detected by Western blot. In vitro, we evaluated the protective effect of Sal on rat embryonic heart‐derived myogenic cell line H9c2 induced by LPS. Reactive oxygen species (ROS) in H9c2 cells was measured by flow cytometry, and the activities of the antioxidant enzymes CAT, SOD, GSH‐px, glutathione‐S‐transferase, TNF‐α, IL‐6 and IL‐1β in cellular supernatant were measured. PI3K/Akt/mTOR signalling was examined by Western blot. As a result, Sal significantly attenuated the above indices. In addition, Sal exerts pronounced cardioprotective effect in rats subjected to LPS possibly through inhibiting the iNOS, COX‐2, NF‐κB and PI3K/Akt/mTOR pathway in vivo. Furthermore, the pharmacological effect of Sal associated with the ROS‐mediated PI3K/Akt/mTOR pathway was proved by the use of ROS scavenger, N‐acetyl‐l ‐cysteine, in LPS‐stimulated H9C2 cells. Our results indicated that Sal could be a potential therapeutic agent for the treatment of cardiovascular disease.     

Bruton's tyrosine kinase together with PI 3-kinase are part of Toll-like receptor 2 multiprotein complex and mediate LTA induced Toll-like receptor 2 responses in macrophages

Lipoteichoic acid (LTA) of Gram-positive bacteria initiates innate immune responses via Toll-like receptor-2 (TLR2), resulting in the activation of intracellular signaling and production of inflammatory cytokines in macrophages. Although Bruton's tyrosine kinase (Btk) is biologically important molecule implicated in immune regulation and recently in TLR signaling its importance for LTA-TLR2 mediated responses has not been evaluated. In this study, we detected Btk in the LTA signaling complex with TLR2 and PI 3-kinase (PI3K). The constitutive interaction of these proteins was mediated via PI3K Src homology (SH3) -domain. Both Btk and PI3K were activated by LTA stimulation and the LTA induced cytokine expression was differentially modulated by these kinases. LTA induced the activation of nuclear factor kappaB (NFkappaB), however, only Btk inhibition affected the LTA induced Ser536 phosphorylation and DNA-binding of NFkappaB. In conclusion, our results demonstrate that Btk and PI3K occupy important roles in TLR2-induced activation of macrophages, resulting in selective regulation of cytokines.     

Blockade of the PI‐3K signalling pathway by the Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces macrophages to synthesize and secrete pro‐inflammatory cytokines

The Aggregatibactor actinomycetemcomitans cytolethal distending toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes; these toxic effects are due to the active subunit, CdtB, which functions as a phosphatidylinositol‐3,4,5‐triphosphate (PIP3) phosphatase. We now extend our investigation and demonstrate that Cdt is able to perturb human macrophage function. THP‐1‐ and monocyte‐derived macrophages were found not to be susceptible to Cdt‐induced apoptosis. Nonetheless, the toxin was capable of binding to macrophages and perturbing PI‐3K signalling resulting in decreased PIP3 levels and reduced phosphorylation of Akt and GSK3β; these changes were accompanied by concomitant alterations in kinase activity. Exposure of monocytes and macrophages to Cdt resulted in pro‐inflammatory cytokine production including increased expression and release of IL‐1β, TNFα and IL‐6. Furthermore, treatment of cells with either TLR‐2, ‐3 or ‐4 agonists in the presence of Cdt resulted in an augmented pro‐inflammatory response relative to agonist alone. GSK3β inhibitors blocked the Cdt‐induced pro‐inflammatory cytokine response suggesting a pivotal role for PI‐3K blockade, concomitant decrease in GSK3β phosphorylation and increased kinase activity. Collectively, these studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt‐producing organisms.     

Syk negatively regulates TLR4-mediated IFNβ and IL-10 production and promotes inflammatory responses in dendritic cells

BackgroundWhile Syk has been shown to associate with TLR4, the immune consequences of Syk–TLR interactions and related molecular mechanisms are unclear.MethodsGain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting.ResultsSyk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNβ and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3β. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNβ and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr–Akt) or constitutively active GSK3β (GSK3 S9A). Moreover, neither inhibition of PI3K–Akt signaling nor neutralization of de novo synthesized IFNβ could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKβ and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses.ConclusionsSyk negatively regulates TLR4-mediated production of IFNβ and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K–Akt and NF-κB signaling pathways.General significanceSyk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.     

Hepatitis C VLPs Delivered to Dendritic Cells by a TLR2 Targeting Lipopeptide Results in Enhanced Antibody and Cell-Mediated Responses

Although many studies provide strong evidence supporting the development of HCV virus-like particle (VLP)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. In this study, we have evaluated the use of an anionic self-adjuvanting lipopeptide containing the TLR2 agonist Pam₂Cys (E₈Pam₂Cys) to enhance the immunogenicity of VLPs containing the HCV structural proteins (core, E1 and E2) of genotype 1a. While co-formulation of this lipopeptide with VLPs only resulted in marginal improvements in dendritic cell (DC) uptake, its ability to concomitantly induce DC maturation at very small doses is a feature not observed using VLPs alone or in the presence of an aluminium hydroxide-based adjuvant (Alum). Dramatically improved VLP and E2-specific antibody responses were observed in VLP+E₈Pam₂Cys vaccinated mice where up to 3 doses of non-adjuvanted or traditionally alum-adjuvanted VLPs was required to match the antibody titres obtained with a single dose of VLPs formulated with this lipopeptide. This result also correlated with significantly higher numbers of specific antibody secreting cells that was detected in the spleens of VLP+E₈Pam₂Cys vaccinated mice and greater ability of sera from these mice to neutralise the binding and uptake of VLPs by Huh7 cells. Moreover, vaccination of HLA-A2 transgenic mice with this formulation also induced better VLP-specific IFN-γ-mediated responses compared to non-adjuvanted VLPs but comparable levels to that achieved when coadministered with complete freund’s adjuvant. These results suggest overall that the immunogenicity of HCV VLPs can be significantly improved by the addition of this novel adjuvant by targeting their delivery to DCs and could therefore constitute a viable vaccine strategy for the treatment of HCV.     

Lipopolysaccharides can protect mesenchymal stem cells (MSCs) from oxidative stress-induced apoptosis and enhance proliferation of MSCs via Toll-like receptor(TLR)-4 and PI3K/Akt

Apoptosis of implanted mesenchymal stem cells (MSCs) limits the efficiency of MSC therapy. Recent studies showed the ligands of Toll-like receptors (TLRs) could control the function of these cells. We have investigated the effect of lipopolysaccharides (LPS), a ligand of TLR4, on the survival of MSCs and explored the roles of TLR4 and PI3K/Akt. H₂O₂/serum deprivation(H₂O₂/SD) induced apoptosis of MSCs but LPS-preconditioning (1.0 μg/ml) protected MSCs from H₂O₂/SD-induced apoptosis and promoted their proliferation. Western blotting showed that 1.0 μg/ml LPS enhanced phosphorylation of both Akt at Ser 473 and nuclear factor-kappa B (NF-κB) p65 at Ser 536. However, the protective effects of LPS on survival were not observed in TLR4^lps-del MSCs. The results suggest appropriate treatments with LPS can protect MSCs from oxidative stress-induced apoptosis and improve the survival of MSCs via the TLR4 and PI3K/Akt pathway.     

Cardiotoxin III suppresses hepatocyte growth factor–stimulated migration and invasion of MDA‐MB‐231 cells

The hepatocyte growth factor (HGF)/c‐Met signalling pathway is deregulated in most cancers and associated with a poor prognosis in breast cancer. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. In this study, we use HGF as an invasive inducer to investigate the effect of CTX III on MDA‐MB‐231 cells. When cells were treated with non‐toxic doses of CTX III, CTX III inhibited the HGF‐promoted cell migration and invasion. CTX III significantly suppressed the HGF‐induced c‐Met phosphorylation and downstream activation of phosphatidylinositol 3‐kinase (PI3k)/Akt and extracellular signal‐regulated kinase (ERK) 1/2. Additionally, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an upstream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by HGF. This effect was paralleled by a significant reduction in phosphorylation of IκBα kinase and IκBα and nuclear translocation of nuclear factor κB (NF‐κB) as well as a reduction of matrix metalloproteinase‐9 (MMP‐9) activity. Furthermore, the c‐Met inhibitor PHA665752 inhibited HGF‐induced MMP‐9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occurs downstream of c‐Met activation. Taken together, these findings suggest that CTX III inhibits the HGF‐induced invasion and migration of MDA‐MB‐231 cells via HGF/c‐Met‐dependent PI3K/Akt, ERK1/2 and NF‐κB signalling pathways, leading to the downregulation of MMP‐9 expression. Copyright © 2014 John Wiley & Sons, Ltd.