Biochemical transformation of bacterial lipopolysaccharides by acyloxyacyl hydrolase reduces host injury and promotes recovery.

Animals can sense the presence of microbes in their tissues and mobilize their own defenses by recognizing and responding to conserved microbial structures (often called microbe-associated molecular patterns (MAMPs)). Successful host defenses may kill the invaders, yet the host animal may fail to restore homeostasis if the stimulatory microbial structures are not silenced. Although mice have many mechanisms for limiting their responses to lipopolysaccharide (LPS), a major Gram-negative bacterial MAMP, a highly conserved host lipase is required to extinguish LPS sensing in tissues and restore homeostasis.

Detecting lipopolysaccharide in the cytosol of mammalian cells: Lessons from MD-2/TLR4.

Proinflammatory immune responses to Gram-negative bacterial lipopolysaccharides (LPS) are crucial to innate host defenses but can also contribute to pathology. How host cells sensitively detect structural features of LPS was a mystery for years, especially given that a portion of the molecule essential for its potent proinflammatory properties-lipid A-is buried in the bacterial membrane. Studies of responses to extracellular and vacuolar LPS revealed a crucial role for accessory proteins that specifically bind LPS-rich membranes and extract LPS monomers to generate a complex of LPS, MD-2, and TLR4.

Diverse pro-inflammatory endotoxin recognition systems of mammalian innate immunity.

In humans and other mammals, recognition of endotoxins-abundant surface lipopolysaccharides (LPS) of Gram-negative bacteria-provides a potent stimulus for induction of inflammation and mobilization of host defenses. The structurally unique lipid A region of LPS is the principal determinant of this pro-inflammatory activity. This region of LPS is normally buried within the bacterial outer membrane and aggregates of purified LPS, making even more remarkable its picomolar potency and the ability of discrete variations in lipid A structure to markedly alter the pro-inflammatory activity of LPS.

Cutting Edge: Mitochondrial Assembly of the NLRP3 Inflammasome Complex Is Initiated at Priming.

The NLRP3 inflammasome is activated in response to microbial and danger signals, resulting in caspase-1-dependent secretion of the proinflammatory cytokines IL-1β and IL-18. Canonical NLRP3 inflammasome activation is a two-step process requiring both priming and activation signals. During inflammasome activation, NLRP3 associates with mitochondria; however, the role for this interaction is unclear.

The phospholipid-repair system LplT/Aas in Gram-negative bacteria protects the bacterial membrane envelope from host phospholipase A attack.

Secretory phospholipases A (sPLAs) are potent components of mammalian innate-immunity antibacterial mechanisms. sPLA enzymes attack bacteria by hydrolyzing bacterial membrane phospholipids, causing membrane disorganization and cell lysis. However, most Gram-negative bacteria are naturally resistant to sPLA Here we report a novel resistance mechanism to mammalian sPLA in , mediated by a phospholipid repair system consisting of the lysophospholipid transporter LplT and the acyltransferase Aas in the cytoplasmic membrane.

High-affinity caspase-4 binding to LPS presented as high molecular mass aggregates or in outer membrane vesicles.

Caspases of the non-canonical inflammasome (caspases -4, -5, and -11) directly bind endotoxin (LOS/LPS) and can be activated in the absence of any co-factors. Models of LPS-induced caspase activation have postulated that 1:1 binding of endotoxin monomers to caspase trigger caspase oligomerization and activation, analogous to that established for endotoxin-induced activation of MD-2/TLR4. However, using metabolically radiolabeled LOS and LPS, we now show high affinity and selective binding of caspase-4 to high molecular mass aggregates of purified endotoxin and to endotoxin-rich outer membrane vesicles without formation of 1:1 endotoxin:caspase complexes.

Modification of sample processing for the Limulus amebocyte lysate assay enhances detection of inflammogenic endotoxin in intact bacteria and organic dust.

The pro-inflammatory potency and causal relationship with asthma of inhaled endotoxins have underscored the importance of accurately assessing the endotoxin content of organic dusts. The Limulus amebocyte lysate (LAL) assay has emerged as the preferred assay, but its ability to measure endotoxin in intact bacteria and organic dusts with similar sensitivity as purified endotoxin is unknown. We used metabolically radiolabeled Neisseria meningitidis and both rough and smooth Escherichia coli to compare dose-dependent activation in the LAL with purified endotoxin from these bacteria and shed outer membrane (OM) blebs.

Evidence Suggesting That Francisella tularensis O-Antigen Capsule Contains a Lipid A-Like Molecule That Is Structurally Distinct from the More Abundant Free Lipid A.

Francisella tularensis, the Gram-negative bacterium that causes tularemia, produces a high molecular weight capsule that is immunologically distinct from Francisella lipopolysaccharide but contains the same O-antigen tetrasaccharide. To pursue the possibility that the capsule of Francisella live vaccine strain (LVS) has a structurally unique lipid anchor, we have metabolically labeled Francisella with [14C]acetate to facilitate highly sensitive compositional analysis of capsule-associated lipids. Capsule was purified by two independent methods and yielded similar results.

Molecular Basis of the Functional Differences between Soluble Human Versus Murine MD-2: Role of Val135 in Transfer of Lipopolysaccharide from CD14 to MD-2.

Myeloid differentiation factor 2 (MD-2) is an extracellular protein, associated with the ectodomain of TLR4, that plays a critical role in the recognition of bacterial LPS. Despite high overall structural and functional similarity, human (h) and murine (m) MD-2 exhibit several species-related differences. hMD-2 is capable of binding LPS in the absence of TLR4, whereas mMD-2 supports LPS responsiveness only when mMD-2 and mTLR4 are coexpressed in the same cell.

Structural and functional features of a developmentally regulated lipopolysaccharide-binding protein.

Unlabelled: Mammalian lipopolysaccharide (LPS) binding proteins (LBPs) occur mainly in extracellular fluids and promote LPS delivery to specific host cell receptors. The function of LBPs has been studied principally in the context of host defense; the possible role of LBPs in nonpathogenic host-microbe interactions has not been well characterized. Using the Euprymna scolopes-Vibrio fischeri model, we analyzed the structure and function of an LBP family protein, E.

Molecular determinants of bacterial sensitivity and resistance to mammalian Group IIA phospholipase A2.

Group IIA secretory phospholipase A2 (sPLA(2)-IIA) of mammalian species is unique among the many structurally and functionally related mammalian sPLA(2) in their high net positive charge and potent (nM) antibacterial activity. Toward the Gram-positive bacteria tested thus far, the global cationic properties of sPLA(2)-IIA are necessary for optimal binding to intact bacteria and penetration of the multi-layered thick cell wall, but not for the degradation of membrane phospholipids that is essential for bacterial killing. Various Gram-positive bacterial species can differ as much as 1000-fold in sPLA(2)-IIA sensitivity despite similar intrinsic enzymatic activity of sPLA(2)-IIA toward the membrane phospholipids of various bacteria.

Purified monomeric ligand.MD-2 complexes reveal molecular and structural requirements for activation and antagonism of TLR4 by Gram-negative bacterial endotoxins.

A major focus of work in our laboratory concerns the molecular mechanisms and structural bases of Gram-negative bacterial endotoxin recognition by host (e.g., human) endotoxin-recognition proteins that mediate and/or regulate activation of Toll-like receptor (TLR) 4.

Tetraacylated lipid A and paclitaxel-selective activation of TLR4/MD-2 conferred through hydrophobic interactions.

LPS exerts potent immunostimulatory effects through activation of the TLR4/MD-2 receptor complex. The hexaacylated lipid A is an agonist of mouse (mTLR4) and human TLR4/MD-2, whereas the tetraacylated lipid IVa and paclitaxel activate only mTLR4/MD-2 and antagonize activation of the human receptor complex. Hydrophobic mutants of TLR4 or MD-2 were used to investigate activation of human embryonic kidney 293 cells by different TLR4 agonists.

Identification of single nucleotide polymorphisms in hematopoietic cell transplant patients affecting early recognition of, and response to, endotoxin.

Hematopoietic cell transplant (HCT) is a life-saving therapy for many malignant and non-malignant bone marrow diseases. Associated morbidities are often due to transplant-related toxicities and infections, exacerbated by regimen-induced immune suppression and systemic incursion of bacterial products. Patients undergoing myeloablative conditioning for HCT become endotoxemic and display blood/plasma changes consistent with lipopolysaccharide (LPS)-induced systemic innate immune activation.

Metabolic labeling to characterize the overall composition of Francisella lipid A and LPS grown in broth and in human phagocytes.

A hallmark of Francisella tularensis, a highly virulent Gram-negative bacterium, is an unusual LPS that possesses both structural heterogeneity and characteristics that may contribute to innate immune evasion. However, none of the methods yet employed has been sufficient to determine the overall LPS composition of Francisella. We now demonstrate that metabolic labeling of francisellae with [(14)C]acetate, combined with fractionation of [(14)C]acetate-labeled lipids by ethanol precipitation rather than hot phenol-water extraction, permits a more sensitive and quantitative appraisal of overall compositional heterogeneity in lipid A and LPS.

The TLR4 antagonist Eritoran protects mice from lethal influenza infection.

There is a pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury caused by chemical or microbial insults is secondary to the generation of host-derived, oxidized phospholipid that potently stimulates Toll-like receptor 4 (TLR4)-dependent inflammation. Subsequently, we reported that Tlr4(-/-) mice are highly refractory to influenza-induced lethality, and proposed that therapeutic antagonism of TLR4 signalling would protect against influenza-induced acute lung injury.

Radioiodination of an endotoxin·MD-2 complex generates a novel sensitive, high-affinity ligand for TLR4.

A purified complex of metabolically labeled [(3)H]lipooligosaccharide (LOS) and recombinant human myeloid differentiation factor 2 (MD-2), [(3)H]LOS·MD-2, has been used to demonstrate pM affinity binding interactions with soluble TLR4 ectodomain (TLR4ecd). For measurement of the binding parameters of membrane-bound TLR4, we took advantage of the stability of endotoxin·MD-2 and tyrosine(s) present on the surface of MD-2 to radioiodinate LOS·MD-2. Radioiodinated LOS·MD-2 generated a reagent with an estimated 1:1 molar ratio of [(125)I] to sMD-2 with 20-fold higher specific radioactivity and TLR4-activating properties comparable to metabolically-labeled LOS·MD-2.

IFN-α and lipopolysaccharide upregulate APOBEC3 mRNA through different signaling pathways.

APOBEC3 (A3) proteins are virus-restriction factors that provide intrinsic immunity against infections by viruses like HIV-1 and mouse mammary tumor virus. A3 proteins are inducible by inflammatory stimuli, such as LPS and IFN-α, via mechanisms that are not fully defined. Using genetic and pharmacological studies on C57BL/6 mice and cells, we show that IFN-α and LPS induce A3 via different pathways, independently of each other.

Respiratory syncytial virus fusion protein-induced toll-like receptor 4 (TLR4) signaling is inhibited by the TLR4 antagonists Rhodobacter sphaeroides lipopolysaccharide and eritoran (E5564) and requires direct interaction with MD-2.

Unlabelled: Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2.

NMR studies of hexaacylated endotoxin bound to wild-type and F126A mutant MD-2 and MD-2·TLR4 ectodomain complexes.

Host response to invasion by many gram-negative bacteria depends upon activation of Toll-like receptor 4 (TLR4) by endotoxin presented as a monomer bound to myeloid differentiation factor 2 (MD-2). Metabolic labeling of hexaacylated endotoxin (LOS) from Neisseria meningitidis with [(13)C]acetate allowed the use of NMR to examine structural properties of the fatty acyl chains of LOS present in TLR4-agonistic and -antagonistic binary and ternary complexes with, respectively, wild-type or mutant (F126A) MD-2 ± TLR4 ectodomain. Chemical shift perturbation indicates that Phe(126) affects the environment and/or position of each of the bound fatty acyl chains both in the binary LOS·MD-2 complex and in the ternary LOS·MD-2·TLR4 ectodomain complex.

High-mobility group nucleosome-binding protein 1 acts as an alarmin and is critical for lipopolysaccharide-induced immune responses.

Alarmins are endogenous mediators capable of promoting the recruitment and activation of antigen-presenting cells (APCs), including dendritic cells (DCs), that can potentially alert host defense against danger signals. However, the relevance of alarmins to the induction of adaptive immune responses remains to be demonstrated. In this study, we report the identification of HMGN1 (high-mobility group nucleosome-binding protein 1) as a novel alarmin and demonstrate that it contributes to the induction of antigen-specific immune responses.

Bactericidal/permeability-increasing protein (rBPI21) and fluoroquinolone mitigate radiation-induced bone marrow aplasia and death.

Identification of safe, effective treatments to mitigate toxicity after extensive radiation exposure has proven challenging. Only a limited number of candidate approaches have emerged, and the U.S.