Characterization of cellular immune response and innate immune signaling in human and nonhuman primate primary mononuclear cells exposed to Burkholderia mallei.

Burkholderia pseudomallei infection causes melioidosis and is often characterized by severe sepsis. Although rare in humans, Burkholderia mallei has caused infections in laboratory workers, and the early innate cellular response to B. mallei in human and nonhuman primates has not been characterized. In this study, we examined the primary cellular immune response to B. mallei in PBMC cultures of non-human primates (NHPs), Chlorocebus aethiops (African Green Monkeys), Macaca fascicularis (Cynomolgus macaque), and Macaca mulatta (Rhesus macaque) and humans. Our results demonstrated that B. mallei elicited strong primary pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1ß, and IL-6) equivalent to the levels of B. pseudomallei in primary PBMC cultures of NHPs and humans. When we examined IL-1ß and other cytokine responses by comparison to Escherichia coli LPS, African Green Monkeys appears to be most responsive to B. mallei than Cynomolgus or Rhesus. Characterization of the immune signaling mechanism for cellular response was conducted by using a ligand induced cell-based reporter assay, and our results demonstrated that MyD88 mediated signaling contributed to the B. mallei and B. pseudomallei induced pro-inflammatory responses. Notably, the induced reporter activity with B. mallei, B. pseudomallei, or purified LPS from these pathogens was inhibited and cytokine production was attenuated by a MyD88 inhibitor. Together, these results show that in the scenario of severe hyper-inflammatory responses to B. mallei infection, MyD88 targeted therapeutic intervention may be a successful strategy for therapy.

A small molecule that mimics the BB-loop in the Toll interleukin-1 (IL-1) receptor domain of MyD88 attenuates staphylococcal enterotoxin B-induced pro-inflammatory cytokine production and toxicity in mice.

Toxic shock syndrome (TSS) is a clinical consequence of the profound amplification of host pro-inflammatory cytokine signaling that results from staphylococcal enterotoxin (SE) exposure. We recently reported that MyD88(-/-) mice were resistant to SEA or SEB toxic shock and displayed reduced levels of pro-inflammatory cytokines in their serum. Here we report that SEB stimulation of total mononuclear cells up-regulated MyD88 in monocytes and T cells. Further, MyD88 gene silencing in primary human cells using siRNA prevented SEB or SEB plus lipopolysaccharide (LPS) induction of interleukin-1ß (IL-1ß) transcriptional activation, suggesting that MyD88-mediated signaling is an essential component of SEB toxicity. We synthesized small molecules that mimic the conserved BB-loop in the Toll/IL-1 receptor (TIR) domain of MyD88. In primary human cells, these mimetics attenuated SEB-induced pro-inflammatory cytokine production. SEB stimulation of primary cells with mimetic affected newly synthesized MyD88 and downstream signaling components. Furthermore, LPS-induced MyD88 signaling was likewise inhibited in a cell-based reporter assay. More importantly, administration of mimetic reduced cytokine responses and increased survivability in a murine SEB challenge model. Collectively, these results suggest that MyD88 BB-loop mimetics interfere with SEB-induced pro-inflammatory signaling and toxicity, thus offering a potential approach in the therapy of toxic shock.

Author Correction: Discovery of small molecule inhibitors of MyD88-dependent signaling pathways using a computational screen.

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Small Molecule Analogues of the parasitic worm product ES-62 interact with the TIR domain of MyD88 to inhibit pro-inflammatory signalling.

ES-62 is a protein secreted by the parasitic worm Acanthocheilonema viteae that is anti-inflammatory by virtue of covalently attached phosphorylcholine. Previously we have reported that drug-like Small Molecule Analogues (SMAs) of its phosphorylcholine moiety can mimic ES-62 in protecting against disease development in certain mouse models of autoimmune and allergic conditions, due to them causing partial degradation of the TLR/IL-1R adaptor MyD88. We have now taken a molecular modelling approach to investigating the mechanism underlying this effect and this predicts that the SMAs interact directly with the MyD88 TIR domain. Further support for this is provided by assay of LPS-induced MyD88/NF-κB-driven secreted alkaline phosphatase (SEAP) reporter activity in commercially-available stably transfected (TLR4-MD2-NF-κB-SEAP) HEK293 cells, as SMA12b-mediated inhibition of such SEAP activity is blocked by its pre-incubation with recombinant MyD88-TIR domain. Direct binding of SMA12b to the TIR domain is also shown to inhibit homo-dimerization of the adaptor, an event that can explain the observed degradation of the adaptor and inhibition of subsequent downstream signalling. Thus, these new data identify initial events by which drug-like ES-62 SMAs, which we also demonstrate are able to inhibit cytokine production by human cells, homeostatically maintain "safe" levels of MyD88 signalling.

Rational design of peptide derivatives for inhibition of MyD88-mediated toll-like receptor signaling in human peripheral blood mononuclear cells and epithelial cells exposed to Francisella tularensis.

Small molecules were developed to attenuate proinflammatory cytokines resulting from activation of MyD88-mediated toll-like receptor (TLR) signaling by Francisella tularensis. Fifty-three tripeptide derivatives were synthesized to mimic a key BB-loop region involved in toll-like/interleukin-1 receptor recognition (TIR) domain interactions. Compounds were tested for inhibition of TNF-α, IFN-γ, IL-6, and IL-1ß in human peripheral blood mononuclear cells (PBMCs) and primary human bronchial epithelial cells exposed to LPS extracts from F. tularensis. From 53 compounds synthesized and tested, ten compounds were identified as effective inhibitors of F. tularensisLPS-induced cytokines. Compound stability testing in the presence of human liver microsomes and human serum resulted in the identification of tripeptide derivative 7 that was a potent, stable, and drug-like small molecule. Target corroboration using a cell-based reporter assay and competition experiments with MyD88 TIR domain protein supported that the effect of 7 was through MyD88 TIR domain interactions. Compound 7 also attenuated proinflammatory cytokines in human peripheral blood mononuclear cells and bronchial epithelial cells challenged with a live vaccine strain of F. tularensis at a multiplicity of infection of 1:5. Small molecules that target TIR domain interactions in MyD88-dependent TLR signaling represent a promising strategy toward host-directed adjunctive therapeutics for inflammation associated with biothreat agent-induced sepsis.

Structure-Based Design and Synthesis of a Small Molecule that Exhibits Anti-inflammatory Activity by Inhibition of MyD88-mediated Signaling to Bacterial Toxin Exposure.

Both Gram-positive and Gram-negative pathogens or pathogen-derived components, such as staphylococcal enterotoxins (SEs) and endotoxin (LPS) exposure, activate MyD88-mediated pro-inflammatory cellular immunity for host defense. However, dysregulated MyD88-mediated signaling triggers exaggerated immune response that often leads to toxic shock and death. Previously, we reported a small molecule compound 1 mimicking BB-loop structure of MyD88 was capable of inhibiting pro-inflammatory response to SEB exposure in mice. In this study, we designed a dimeric structure compound 4210 covalently linked with compound 1 by a non-polar cyclohexane linker which strongly inhibited the production of pro-inflammatory cytokines in human primary cells to SEB (IC50 1-50 µm) or LPS extracted from Francisella tularensis, Escherichia coli, or Burkholderia mallei (IC50 10-200 µm). Consistent with cytokine inhibition, in a ligand-induced cell-based reporter assay, compound 4210 inhibited Burkholderia mallei or LPS-induced MyD88-mediated NF-kB-dependent expression of reporter activity (IC50 10-30 µm). Furthermore, results from a newly expressed MyD88 revealed that 4210 inhibited MyD88 dimer formation which is critical for pro-inflammatory signaling. Importantly, a single administration of compound 4210 in mice showed complete protection from lethal toxin challenge. Collectively, these results demonstrated that compound 4210 inhibits toxin-induced inflated pro-inflammatory immune signaling, thus displays a potential bacterial toxin therapeutic.

Discovery of small molecule inhibitors of MyD88-dependent signaling pathways using a computational screen.

In this study, we used high-throughput computational screening to discover drug-like inhibitors of the host MyD88 protein-protein signaling interaction implicated in the potentially lethal immune response associated with Staphylococcal enterotoxins. We built a protein-protein dimeric docking model of the Toll-interleukin receptor (TIR)-domain of MyD88 and identified a binding site for docking small molecules. Computational screening of 5 million drug-like compounds led to testing of 30 small molecules; one of these molecules inhibits the TIR-TIR domain interaction and attenuates pro-inflammatory cytokine production in human primary cell cultures. Compounds chemically similar to this hit from the PubChem database were observed to be more potent with improved drug-like properties. Most of these 2(nd) generation compounds inhibit Staphylococcal enterotoxin B (SEB)-induced TNF-α, IFN-γ, IL-6, and IL-1ß production at 2-10 µM in human primary cells. Biochemical analysis and a cell-based reporter assay revealed that the most promising compound, T6167923, disrupts MyD88 homodimeric formation, which is critical for its signaling function. Furthermore, we observed that administration of a single dose of T6167923 completely protects mice from lethal SEB-induced toxic shock. In summary, our in silico approach has identified anti-inflammatory inhibitors against in vitro and in vivo toxin exposure with promise to treat other MyD88-related pro-inflammatory diseases.

Cytotoxicity of oxidized low-density lipoprotein in cultured RPE cells is dependent on the formation of 7-ketocholesterol.

PURPOSE: To determine which components present in oxidized LDL are responsible for the cytotoxicity associated with its internalization by culture ARPE19 cells. METHODS: ARPE19 cells were grown in 24-well and 96-well plates. Cell viability was measured by MTT and/or adenosine triphosphate (ATP) content. LDL was oxidized with Cu(+2) and oxysterol content analyzed by a novel HPLC method. RESULTS: OxLDL showed increased cytotoxicity with prolonged oxidation. Analysis of the oxLDL showed a predominance of the 7-oxygenated products, 7 alpha-hydroxycholesterol (7 alpha HCh), 7 beta-hydroxycholesterol (7 beta HCh), and 7-ketocholesterol (7kCh). Addition of these oxysterols to the ARPE19 cell in free form indicated that 7kCh is the most cytotoxic of the oxysterols but at physiologically unrealistic concentrations. Partitioning of individual oxysterols into nonoxidized LDL at concentrations similar to those found in the oxLDL also indicated that 7kCh is the most cytotoxic of the oxysterols. Transition metals are tightly bound by LDL and play an important role in the oxidation of LDL, but do not seem to enhance its cytotoxicity directly. CONCLUSIONS: Prolonged oxidation of LDL increases the levels of 7kCh due to further oxidation of 7 alpha HCh and 7 beta HCh. The formation of 7KCh seems to be responsible for most of the cytotoxicity associated with oxLDL internalization in ARPE19 cells.

Retinal capillary pericyte proliferation and c-Fos mRNA induction by prostaglandin D2 through the cAMP response element.

PURPOSE: Cycloxygenase inhibitors have been shown to prevent angiogenesis in some circumstances, suggesting that growth of capillary pericytes or endothelial cells may be regulated by prostaglandins (PGs). The present study tests the effects of PGs on the growth of human retinal capillary pericytes. METHODS: Cell growth was assayed by formazan formation and 5-bromo-2'-deoxyuridine (BrdU) incorporation. The expression of mRNAs corresponding to c-fos, PG receptors, and VEGF was examined by RT-PCR. Signal transduction was evaluated by immunoblot analysis using phosphospecific antibodies against mitogen-activated protein kinases (MAPKs) and cAMP response element-binding protein (CREB). Synthesis of cAMP was inhibited with the adenyl cyclase inhibitor SQ22536. A reporter gene (luciferase) assay was conducted using the expression vector pSVOADelta5' containing the 379-bp c-fos promoter with and without a mutation in cAMP response element (CRE). RESULTS. PGD2 treatment induced c-fos mRNA, stimulated pericyte growth, and increased expression of VEGF mRNA. PGE2 and -F(2alpha) had similar effects on c-fos induction and pericyte growth, whereas PGI2 was ineffective. RT-PCR confirmed that mRNAs corresponding to the receptors for PGD2, -E2, -F(2alpha), and -I(2) were expressed in human retinal pericytes. Stimulation by PGD2 led to phosphorylation of CREB, but had negligible effect on phosphorylation of p44/42 MAPK. The adenylyl cyclase inhibitor inhibited CREB activation and c-fos induction by PGD2. In a reporter gene assay, c-fos induction occurred only with wild-type c-fos promoter. Mutation in CRE eliminated the response to PGD2. CONCLUSIONS: PGD2 promotes the growth of retinal capillary pericytes by signaling through cAMP and CREB. The findings underscore the importance of PGs in the growth of human retinal capillary pericytes and raise the possibility that PGs may play a role in proliferative retinopathies.

Therapeutic inhibition of pro-inflammatory signaling and toxicity to staphylococcal enterotoxin B by a synthetic dimeric BB-loop mimetic of MyD88.

Staphylococcal enterotoxin B (SEB) exposure triggers an exaggerated pro-inflammatory cytokine response that often leads to toxic shock syndrome (TSS) associated with organ failure and death. MyD88 mediates pro-inflammatory cytokine signaling induced by SEB exposure and MyD88(-/-) mice are resistant to SEB intoxication, suggesting that MyD88 may be a potential target for therapeutic intervention. We targeted the BB loop region of the Toll/IL-1 receptor (TIR) domain of MyD88 to develop small-molecule therapeutics. Here, we report that a synthetic compound (EM-163), mimic to dimeric form of BB-loop of MyD88 attenuated tumor necrosis factor (TNF)- α, interferon (IFN)-γ, interleukin (IL)-1ß, IL-2 and IL-6 production in human primary cells, whether administered pre- or post-SEB exposure. Results from a direct binding assay, and from MyD88 co-transfection/co-immunoprecipitation experiments, suggest that EM-163 inhibits TIR-TIR domain interaction. Additional results indicate that EM-163 prevents MyD88 from mediating downstream signaling. In an NF-kB-driven reporter assay of lipopolysaccharide-stimulated MyD88 signaling, EM-163 demonstrated a dose-dependent inhibition of reporter activity as well as TNF-α and IL-1ß production. Importantly, administration of EM-163 pre- or post exposure to a lethal dose of SEB abrogated pro-inflammatory cytokine responses and protected mice from toxic shock-induced death. Taken together, our results suggest that EM-163 exhibits a potential for therapeutic use against SEB intoxication.

Activation of MyD88 signaling upon staphylococcal enterotoxin binding to MHC class II molecules.

Ligands binding to Toll-like receptor (TLR), interleukin 1 receptor (IL-1R), or IFN-γR1 are known to trigger MyD88-mediated signaling, which activates pro-inflammatory cytokine responses. Recently we reported that staphylococcal enterotoxins (SEA or SEB), which bind to MHC class II molecules on APCs and cross link T cell receptors, activate MyD88- mediated pro-inflammatory cytokine responses. We also reported that MyD88(-/-) mice were resistant to SE- induced toxic shock and had reduced levels of serum cytokines. In this study, we investigated whether MHC class II- SE interaction by itself is sufficient to activate MyD88 in MHC class II(+) cells and induce downstream pro-inflammatory signaling and production of cytokines such as TNF-α and IL-1ß. Here we report that human monocytes treated with SEA, SEB, or anti-MHC class II monoclonal antibodies up regulated MyD88 expression, induced activation of NF-kB, and increased expression of IL-1R1 accessory protein, TNF-α and IL-1ß. MyD88 immunoprecipitated from cell extracts after SEB stimulation showed a greater proportion of MyD88 phosphorylation compared to unstimulated cells indicating that MyD88 was a component of intracellular signaling. MyD88 downstream proteins such as IRAK4 and TRAF6 were also up regulated in monocytes after SEB stimulation. In addition to monocytes, primary B cells up regulated MyD88 in response to SEA or SEB stimulation. Importantly, in contrast to primary B cells, MHC class II deficient T2 cells had no change of MyD88 after SEA or SEB stimulation, whereas MHC class II-independent activation of MyD88 was elicited by CpG or LPS. Collectively, these results demonstrate that MHC class II utilizes a MyD88-mediated signaling mechanism when in contact with ligands such as SEs to induce pro-inflammatory cytokines.