Mouse Toll-like Receptor Research Articles

A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation.

The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.

Computational Analysis of Interaction between Mycobacterial Antigens Rv0679c and Rv0180c with Toll like Receptors of Human and Mouse

Toll like receptors (TLRs) play a key role in the innate immune response to infectious agents. The Mycobacterium tuberculosis bacilli and its antigens interaction with TLR-2-1 and TLR-4-MD2 were shown to activate intracellular signalling which determines the outcome of the disease. Present study focused on two new antigens Rv0679c and Rv0180c of M.tb H37Rv and their interactions with human and mouse TLRs using computational approach. Structures of Rv0679c and Rv0180c antigens of M.tb were generated using I-TASSER and docked with TLR-2-1 and TLR-4-MD2 complexes of human and mouse using Cluspro2.0. Rv0180c antigen has better binding energy to both human and mouse TLR-2-1 and TLR-4-MD2 compared to Rv0679c and other mycobacterial antigens. The Rv0679c and Rv0180c antigens have better binding energy to TLR 4-MD2 complex compared to TLR-2-1 in both human and mouse. Both antigens has better binding energy to mouse TLRs compared to human except where Rv0180c has better binding to human TLR-2-1. Our findings suggest that Rv0180c might be the preferred for binding to TLRs than Rv0679c and within TLRs, TLR-4MD2 for interaction. General Terms Invasive mechanism of mycobacterial antigens using TLRs

Distinctive Recognition of Flagellin by Human and Mouse Toll-Like Receptor 5.

Toll-like receptor 5 (TLR5) is a receptor of the innate immune system that recognizes flagellin from certain bacterial species and triggers an inflammatory response. The Salmonella dublin flagellin in complex with zebrafish TLR5 has been crystallized previously. In the present study, we extrapolate the structure of this complex using structure-guided mutagenesis to determine the recognition modes of human and mouse TLR5 receptors and demonstrate species-specific differences in flagellin recognition. In general, the recognition mode of the mouse receptor can be said to be more robust in comparison to that of the human receptor. All-atom molecular dynamics simulation showed differences between the two receptors within the primary binding region. Using a functional motility assay, we show that although the highly conserved area of the flagellin analyzed in this study encompasses key structural requirements for flagella formation, a direct correlation between immune recognition and structure on the level of amino acid residues is not observed.

261. Gain-of-Function Effect Augments Therapeutic Efficacy of CpG-STAT3 Anti-Sense Oligonucleotide Against Castration-Resistant Prostate Cancers

The treatment for castration-resistant prostate cancers (CRPC) remains a clinical challenge. STAT3 expression level correlates with poor prognosis in prostate cancer patients and resistance to hormone therapy. In addition to oncogenic role of STAT3 in cancer cells, it is also commonly activated in the prostate tumor microenvironment. Therefore, STAT3 is a promising target targeting STAT3 is expected to generate dual effects; anti-tumor activity and anti-immunosuppression in CRPC. Targeting transcriptions factors for cancer therapy has not been successful due to the lack of enzymatic activity. Recent advances in oligonucleotide-based therapy using anti-sense oligonucleotide (ASO) targeting STAT3 had shown promising results in phase I clinical trials in various human cancers, including liver metastasis. However, the limitation fo these reagents remained targeting We previously demonstrated that Toll-like Receptor 9 (TLR9) is up-regulated in prostate cancer stem-like cells in the late-stage prostate cancer. STAT3 signaling is activated in TLR9+ prostate cancer cells and tumor associated immune cells. Here, we describe a new molecule that enables specific delivery of STAT3 ASO to TLR9+ tumor and immune by linking STAT3 ASO to CpG ODN, TLR9 ligand. CpG-STAT3 ASO was quickly and efficiently internalized by human and mouse prostate cancer cell lines showing cytoplasmic localization after 15 minutes after incubation in vitro. Human and mouse TLR9+ immune cells; DCs, Macrophages and B cells were able to efficiently uptake CpG-STAT3 ASO in vitro. In a biodistribution experiment, CpG-STAT3 ASOCy3 was effectively internalized by TLR9+ myeloid cells in various organs including lymph node and bone marrow. Treatment with CpG-STAT3 ASO conjugates in vitro decreased the expression of STAT3 in TLR9+ human (DU145 and LNCaP-TLR9) and mouse (RM1 and RM9) prostate cancer cells more efficiently than unconjugated STAT3 ASO. Notably, CpG-STAT3 ASO showed significantly augmented cytotoxicity in prostate cancer cells compared to STAT3 ASO. In a preliminary anti-tumor efficacy study using syngeneic mouse model, repeated intra-tumoral injections of CpG-STAT3 ASO reduced the tumor growth of RM9. In contrast, the inhibitory effect of STAT3 ASO was transient leading to tumor relapse after the initial treatments. Noteworthy, CpG-STAT3 ASO reduced tumor growth and STAT3 expression in the untreated tumors in the contra-lateral site. Moreover, CpG-STAT3 ASO, but not the unconjugated STAT3 ASO, reduced activation of STAT3 in infiltrated myeloid-derived suppressor cells (MDSC) (CD11b+Gr1+) in both tumors and the percentage of T reg (CD4+FoxP3+) cells in the tumor draining lymph nodes. These results implicate that only when combined with concomitant TLR9 stimulation STAT3 inhibition can systemically overcome tumor immune tolerance. Overall, these results suggest that CpG-STAT3 ASO conjugate holds a great promise for an anti-cancer therapy of CRPC by simultaneously targeting STAT3 signaling in TLR9+ prostate cancer cells and tumor-associated immune cells.

Discovery and Structure-Activity Relationships of the Neoseptins: A New Class of Toll-like Receptor-4 (TLR4) Agonists.

Herein, we report studies leading to the discovery of the neoseptins and a comprehensive examination of the structure-activity relationships (SARs) of this new class of small-molecule mouse Toll-like receptor 4 (mTLR4) agonists. The compounds in this class, which emerged from screening an α-helix mimetic library, stimulate the immune response, act by a well-defined mechanism (mouse TLR4 agonist), are easy to produce and structurally manipulate, exhibit exquisite SARs, are nontoxic, and elicit improved and qualitatively different responses compared to lipopolysaccharide, even though they share the same receptor.

Serum-resistant CpG-STAT3 decoy for targeting survival and immune checkpoint signaling in acute myeloid leukemia

AbstractTargeting oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) in acute myeloid leukemia (AML) can reduce blast survival and tumor immune evasion. Decoy oligodeoxynucleotides (dODNs), which comprise STAT3-specific DNA sequences are competitive inhibition of STAT3 transcriptional activity. To deliver STAT3dODN specifically to myeloid cells, we linked STAT3dODN to the Toll-like receptor 9 (TLR9) ligand, cytosine guanine dinucleotide (CpG). The CpG-STAT3dODN conjugates are quickly internalized by human and mouse TLR9+ immune cells (dendritic cells, B cells) and the majority of patients' derived AML blasts, including leukemia stem/progenitor cells. Following uptake, CpG-STAT3dODNs are released from endosomes, and bind and sequester cytoplasmic STAT3, thereby inhibiting downstream gene expression in target cells. STAT3 inhibition in patients' AML cells limits their immunosuppressive potential by reduced arginase expression, thereby partly restoring T-cell proliferation. Partly chemically modified CpG-STAT3dODNs have >60 hours serum half-life which allows for IV administration to leukemia-bearing mice (50% effective dose ∼ 2.5 mg/kg). Repeated administration of CpG-STAT3dODN resulted in regression of human MV4-11 AML in mice. The antitumor efficacy of this strategy is further enhanced in immunocompetent mice by combining direct leukemia-specific cytotoxicity with immunogenic effects of STAT3 blocking/TLR9 triggering. CpG-STAT3dODN effectively reduced Cbfb/MYH11/Mpl AML burden in various organs and eliminated leukemia stem/progenitor cells, mainly through CD8/CD4 T-cell–mediated immune responses. In contrast, small-molecule Janus kinase 2/STAT3 inhibitor failed to reproduce therapeutic effects of cell-selective CpG-STAT3dODN strategy. These results demonstrate therapeutic potential of CpG-STAT3dODN inhibitors with broad implications for treatement of AML and potentially other hematologic malignancies.

Successful Therapy of Murine Visceral Leishmaniasis with Astrakurkurone, a Triterpene Isolated from the Mushroom Astraeus hygrometricus, Involves the Induction of Protective Cell-Mediated Immunity and TLR9.

In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 μg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo.

Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use.

Toll-like receptors (TLRs) are a major class of pattern recognition receptors, which mediate the responses of innate immune cells to microbial stimuli. To systematically determine the roles of proteins in canonical TLR signaling pathways, we conducted an RNA interference (RNAi)-based screen in human and mouse macrophages. We observed a pattern of conserved signaling module dependencies across species, but found notable species-specific requirements at the level of individual proteins. Among these, we identified unexpected differences in the involvement of members of the interleukin-1 receptor-associated kinase (IRAK) family between the human and mouse TLR pathways. Whereas TLR signaling in mouse macrophages depended primarily on IRAK4 and IRAK2, with little or no role for IRAK1, TLR signaling and proinflammatory cytokine production in human macrophages depended on IRAK1, with knockdown of IRAK4 or IRAK2 having less of an effect. Consistent with species-specific roles for these kinases, IRAK4 orthologs failed to rescue signaling in IRAK4-deficient macrophages from the other species, and only mouse macrophages required the kinase activity of IRAK4 to mediate TLR responses. The identification of a critical role for IRAK1 in TLR signaling in humans could potentially explain the association of IRAK1 with several autoimmune diseases. Furthermore, this study demonstrated how systematic screening can be used to identify important characteristics of innate immune responses across species, which could optimize therapeutic targeting to manipulate human TLR-dependent outputs.

Structure of the mouse Toll-like receptor 13 ectodomain in complex with a conserved sequence from bacterial 23S ribosomal RNA.

Toll-like receptor 13 (TLR13) recognizes a conserved 10-nucleotide sequence from bacterial 23S ribosomal RNA, and binding of TLR13 to the target rRNA molecule triggers immune responses. Recently, the crystal structure of the TLR13 ectodomain bound by a 13-nucleotide single-stranded RNA (ssRNA13) was determined by using the initial phases provided by a cryo-electron microscopy map of TLR13 in complex with a 25-nucleotide ssRNA (ssRNA25). This structural snapshot describes a unique method for solving the crystal structure of the TLR13-ssRNA13 complex based on medium-resolution reconstruction of TLR13-ssRNA25 cryo-electron microscopy images.

Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2

Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential to promote TLR2-dependent bone loss as is reported in experimental periodontitis following oral infection with P. gingivalis. These results also support the conclusion that serine dipeptide lipids are involved in alveolar bone loss in chronic periodontitis.

Toll-Like Receptor Gene Expression during Trichinella spiralis Infection.

In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T (Treg) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and Treg cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/TIRAP-/- MEF cells, and quite substantially decreased in TRIF-/- MEF cells. In contrast, IL-10 and TGF-β expression levels were not elevated in MyD88/TIRAP-/- MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and Treg cell mediated immune responses, although additional data are needed to convincingly prove this observation.

A novel class of small molecule agonists with preference for human over mouse TLR4 activation (VAC3P.1056)

Abstract The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.

Toll-like receptor 8 signaling facilitates West Nile virus infection in mice (VIR9P.1161)

Abstract West Nile virus (WNV) is a neurotropic virus that can cause encephalitis, meningitis or possible death in humans. Human and mouse Toll-like receptor 7 (h/mTLR7) and human Toll-like receptor 8 (hTLR8) detect viral single-stranded (ss)RNA motifs. Importantly, mTLR7 recognizes WNV, as TLR7-/- mice are susceptible to lethal WNV infection. However, the role of TLR8 in WNV pathogenesis is not clear. In this study, we found that mice deficient in TLR8 (TLR8-/-) infected with WNV (2,000 plaque forming units) had improved percentage survival (65%) compared to wild-type (WT) controls (25%), which was coupled to reduced viral burden in the brains of TLR8-/- mice. These data suggest that TLR8 signaling facilities WNV infection in mice. Further mechanistic studies showed that expression of TLR7 and the interferon-induced anti-viral gene Isg-56 (IFIT-1) were significantly increased in TLR8-/- mice whole-brain samples and mixed neuronal cultures following WNV infection, compared to WT controls. These results indicate that TLR8 signaling may down-regulate TLR7 and Isg-56 expression, thus promoting WNV-induced neuroinvasive disease.

Evaluation of four α-diketones for toll-like receptor-4 (TLR-4) activation in a human transfected cell line

Toll-like receptor-4 (TLR-4) activity is upregulated in persons with fibrotic lung diseases secondary to chronic inflammatory conditions like Crohn's disease and rheumatoid arthritis. We hypothesized that α-diketones associated with fixed obstructive lung disease may activate TLR-4. We utilized a human embryonic kidney cell assay (HEK293) with human TLR-4 receptors to test for potential activation effects of 2,3-butandeione, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione at test concentrations of 1, 10, 100, and 1000 µM. The assay detects NF-κB-induced expression of secreted alkaline phosphatase measured after 16 h incubation by a UV-VIS Spectrometer at 650 nm. Escherichia coli K12 lipopolysaccharide (LPS) at 0.5 ng/mL served as a positive control and was added with each test compound to evaluate combined effects. None of the tested α-diketones were found to exhibit cytotoxicity, agonism, or synergistic effects with LPS in the human TLR-4 assay up to 1000 µM. Screening of 2,3-butanedione for agonist activity using the HEK assay with mouse TLR-4 receptors exhibited cytotoxicity at 1000 µM, but no agonist activity. We conclude that the tested α-diketones at relatively high concentrations in vitro do not exhibit TLR-4 agonist or synergistic activity and, therefore, apparently do not directly induce inflammasome activation through this pathway in humans or mice.

Activation of c-Src: A hub for exogenous pro-oxidant-mediated activation of Toll-like receptor 4 signaling

To study the role of c-Src kinase in pro-oxidant-induced stimulation of Toll-like receptor 4 (TLR4), we used lipopolysaccharide from Escherichia coli K12 (LPS-EK) and monophosphoryl lipid A, as TLR4-specific agonists and positive controls, and SIN-1 and potassium peroxychromate as pro-oxidant sources. We used the HEK-Blue mTLR4 cell line, which is stably transfected with mouse TLR4 and expresses optimized SEAP reporter under the control of a promoter inducible by NF-κB transcription factor. The level of SEAP released due to TLR4 stimulation was a measure of NF-κB activation. Treatment with either the pro-oxidants or LPS-EK increased SEAP release and TNF-α production in these cells. These treatments also increased intracellular reactive oxygen species accumulation, with an enhanced production of nitric oxide and TBARS to confirm oxidant stress in these cells. Pretreatment with c-Src kinase inhibitors, PP2 and Ca-pY, which act by different mechanisms, decreased these parameters. Pretreatment with SSG, a c-Src activator, enhanced the effects promoted by LPS-EK and pro-oxidants and rescued cells from the PP2- and Ca-pY-induced effects. Curiously, pro-oxidants, but not TLR4 agonist, increased the ratio of TNF-α to IL-10 released, suggesting that pro-oxidants can initiate and maintain an imbalance of TNF-α production over IL-10. To different degrees, both pro-oxidants and TLR4 agonist increased formation of c-Src complexes with TLR4 and IκB-α as coimmunoprecipitates. Both pro-oxidants and TLR4 agonist increased c-Src phosphorylation of the Tyr42 residue in IκB-α, but the pro-oxidant-induced effect was more robust and much longer lasting. Taken together, these studies provide a mechanism whereby c-Src assumes a central role in pro-oxidant-induced NF-κB activation in TLR4 signaling. Pro-oxidant-induced activation of TLR4 through c-Src/NF-κB/IκB-α coupling provides a basis for a molecular dissection of the initiation and maintenance of sterile inflammation that may serve as a “pathophysiologic primer” for many diseases.

Toll-Like Receptors and Malaria – Sensing and Susceptibility

Toll-Like Receptors (TLRs) are important mediators of the innate immune response to pathogens, including malaria. Of the ten human and twelve mouse TLRs, TLR2, TLR4, TLR7 and TLR9 are known to detect malarial antigens and induce anti-malarial immune responses. Multiple immune cell populations express TLRs, and much has been done to elucidate the TLR-mediated immune response to malaria infections, in particular the involvement of TLRs in severe malaria pathogenesis. Here we review the role TLRs play in parasite detection, immune response, and severe malaria, with a focus on recent findings. Furthermore, the use of TLR ligands as malarial vaccine adjuvants is discussed, as this could have great potential in improving efficacy of vaccine candidates

Bacterial lipodipeptide, Lipid 654, is a microbiome-associated biomarker for multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of unknown etiology. Infectious agents have been suggested to have a role as environmental factors in MS, but this concept remains controversial. Recently, gastrointestinal commensal bacteria have been implicated in the pathogenesis of autoimmune diseases, but mechanisms underlying the relationship of human systemic autoimmunity with the commensal microbiome have yet to be identified. Consistent with the lack of understanding of pathogenic mechanisms and relevant environmental factors in MS, no blood biomarkers have been identified that distinguish MS patients from healthy individuals. We recently identified a unique gastrointestinal and oral bacteria-derived lipodipeptide, Lipid 654, which is produced by commensal bacteria and functions as a human and mouse Toll-like receptor 2 ligand. Using multiple-reaction-monitoring mass spectrometry, a critical approach in targeted lipidomics, we now report that Lipid 654 can be recovered in the serum of healthy individuals. Most interestingly, we find that Lipid 654 is expressed at significantly lower levels in the serum of patients with MS compared with both healthy individuals and patients with Alzheimer's disease. These results thus identify for the first time a potential mechanism relating the gastrointestinal and oral commensal microbiome to a human systemic autoimmune disease. In addition, these results also identify a potential etiologic environmental factor and novel clinically relevant serum biomarker for MS.

Conformationally Constrained Lipid A Mimetics for Exploration of Structural Basis of TLR4/MD-2 Activation by Lipopolysaccharide

Recognition of the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, by the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex is essential for the control of bacterial infection. A pro-inflammatory signaling cascade is initiated upon binding of membrane-associated portion of LPS, a glycophospholipid Lipid A, by a coreceptor protein MD-2, which results in a protective host innate immune response. However, activation of TLR4 signaling by LPS may lead to the dysregulated immune response resulting in a variety of inflammatory conditions including sepsis syndrome. Understanding of structural requirements for Lipid A endotoxicity would ensure the development of effective anti-inflammatory medications. Herein, we report on design, synthesis, and biological activities of a series of conformationally confined Lipid A mimetics based on β,α-trehalose-type scaffold. Replacement of the flexible three-bond β(1→6) linkage in diglucosamine backbone of Lipid A by a two-bond β,α(1↔1) glycosidic linkage afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated Lipid A mimetics based on a β–GlcN(1↔1)α–GlcN scaffold selectively block the LPS binding site on both human and murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory signaling, thereby serving as antisepsis drug candidates. In contrast to their natural counterpart lipid IVa, conformationally constrained Lipid A mimetics do not activate mouse TLR4. The structural basis for high antagonistic activity of novel Lipid A mimetics was confirmed by molecular dynamics simulation. Our findings suggest that besides the chemical structure, also the three-dimensional arrangement of the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic effects on TLR4.

Toll-like receptor 4-mediated nuclear factor kappa B activation is essential for sensing exogenous oxidants to propagate and maintain oxidative/nitrosative cellular stress.

The mechanism(s) by which cells can sense exogenous oxidants that may contribute to intracellular oxidative/nitrosative stress is not clear. The objective of this study was to determine how cells might respond to exogenous oxidants to potentially initiate, propagate and/or maintain inflammation associated with many human diseases through NF-κB activation. First, we used HEK-Blue cells that are stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2. These cells also express optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. These cells were challenged with their respective receptor-specific ligands, different pro-oxidants and/or inhibitors that act at different levels of the receptor signaling pathways. A neutralizing antibody directed against TLR4 inhibited responses to both TLR4-specific agonist and a prooxidant, which confirmed that both agents act through TLR4. We used the level of SEAP released into the culture media due to NF-κB activation as a measure of TLR4 or TLR2 stimulation. Pro-oxidants evoked increased release of SEAP from HEK-Blue mTLR4 cells at a much lower concentration compared with release from the HEK-Blue mTLR2 cells. Specific TLR4 signaling pathway inhibitors and oxidant scavengers (anti-oxidants) significantly attenuated oxidant-induced SEAP release by TLR4 stimulation. Furthermore, a novel pro-oxidant that decays to produce the same reactants as activated phagocytes induced inflammatory pain responses in the mouse orofacial region with increased TLR4 expression, and IL-1β and TNFα tissue levels. EUK-134, a synthetic serum-stable scavenger of oxidative species decreased these effects. Our data provide in vitro and related in vivo evidence that exogenous oxidants can induce and maintain inflammation by acting mainly through a TLR4-dependent pathway, with implications in many chronic human ailments.

Differences in both Toll-like receptor 4 and MD-2 account for the host-specific recognition of Bordetella pertussis lipid A (P1240)

Abstract The lipid A portion of lipopolysaccharide (LPS) activates innate immunity through the Toll-like receptor 4 (TLR4)/MD-2 complex on host cells. Variation in lipid A has significant consequences for TLR4 activation and thus may be a means by which pathogenic bacteria modulate host immunity. We have previously seen that strain BP338 of Bordetella pertussis modifies its lipid A by the addition of glucosamine moieties which promote TLR4 activation in human macrophages. In the absence of glucosamine modification, TLR4 activation is attenuated. This effect is host-specific: glucosamine modification does not affect mouse TLR4 activation. We hypothesized that the species-specific effect is due to differences in TLR4, and used inter-species chimeric receptors to test this hypothesis. We found that the middle 330 amino acids of TLR4 were sufficient to generate species-specific responses to B. pertussis lipid A, dependent on co-expression of the matching species of MD-2. Based on the published crystal structure of TLR4/MD-2 in complex with E. coli LPS, we narrowed our search to the interface where TLR4/MD-2 complexes dimerize for activation, and used site-directed mutagenesis to identify specific amino acid residues in TLR4 and MD-2 that promote activation in response to B. pertussis lipid A. Taken together, we have demonstrated that charged amino acids in TLR4 and MD-2 contribute to host-specific immune responses to lipid A variants from B. pertussis.