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. We review recent progress in understanding how this enzyme, acyloxyacyl hydrolase (AOAH), transforms LPS from stimulus to inhibitor, reduces tissue injury and death from infection, prevents prolonged post-infection immunosuppression, and keeps stimulatory LPS from entering the bloodstream. We also discuss how AOAH may increase sensitivity to pulmonary allergens. Better appreciation of how host enzymes modify LPS and other MAMPs may help prevent tissue injury and hasten recovery from infection.

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. These insights provided means to understand better both the remarkable host sensitivity to LPS and the means whereby specific LPS structural features are discerned. More recently, the noncanonical inflammasome, consisting of caspases-4/5 in humans and caspase-11 in mice, has been demonstrated to mediate responses to LPS that has reached the host cytosol. Precisely how LPS gains access to cytosolic caspases-and in what form-is not well characterized, and understanding this process will provide crucial insights into how the noncanonical inflammasome is regulated during infection. Herein, we briefly review what is known about LPS detection by cytosolic caspases-4/5/11, focusing on lessons derived from studies of the better-characterized TLR4 system that might direct future mechanistic questions.

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. Two recognition systems-MD-2/TLR4 and "LPS-sensing" cytosolic caspases-together confer LPS responsiveness at the host cell surface, within endosomes, and at sites physically accessible to the cytosol. Understanding how the lipid A of LPS is delivered and recognized at these diverse sites is crucial to understanding how the magnitude and character of the inflammatory responses are regulated.

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-1ß 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. In this article, we show that mouse NLRP3 and caspase-1 independently interact with the mitochondrial lipid cardiolipin, which is externalized to the outer mitochondrial membrane at priming in response to reactive oxygen species. An NLRP3 activation signal is then required for the calcium-dependent association of the adaptor molecule ASC with NLRP3 on the mitochondrial surface, resulting in inflammasome complex assembly and activation. These findings demonstrate a novel lipid interaction for caspase-1 and identify a role for mitochondria as supramolecular organizing centers in the assembly and activation of the NLRP3 inflammasome.

The phospholipid-repair system LplT/Aas in Gram-negative bacteria protects the bacterial membrane envelope from host phospholipase A2 attack.

Secretory phospholipases A2 (sPLA2s) are potent components of mammalian innate-immunity antibacterial mechanisms. sPLA2 enzymes attack bacteria by hydrolyzing bacterial membrane phospholipids, causing membrane disorganization and cell lysis. However, most Gram-negative bacteria are naturally resistant to sPLA2 Here we report a novel resistance mechanism to mammalian sPLA2 in Escherichia coli, mediated by a phospholipid repair system consisting of the lysophospholipid transporter LplT and the acyltransferase Aas in the cytoplasmic membrane. Mutation of the lplT or aas gene abolished bacterial lysophospholipid acylation activity and drastically increased bacterial susceptibility to the combined actions of inflammatory fluid components and sPLA2, resulting in bulk phospholipid degradation and loss of colony-forming ability. sPLA2-mediated hydrolysis of the three major bacterial phospholipids exhibited distinctive kinetics and deacylation of cardiolipin to its monoacyl-derivative closely paralleled bacterial death. Characterization of the membrane envelope in lplT- or aas-knockout mutant bacteria revealed reduced membrane packing and disruption of lipid asymmetry with more phosphatidylethanolamine present in the outer leaflet of the outer membrane. Moreover, modest accumulation of lysophospholipids in these mutant bacteria destabilized the inner membrane and rendered outer membrane-depleted spheroplasts much more sensitive to sPLA2 These findings indicated that LplT/Aas inactivation perturbs both the outer and inner membranes by bypassing bacterial membrane maintenance mechanisms to trigger specific interfacial activation of sPLA2 We conclude that the LplT/Aas system is important for maintaining the integrity of the membrane envelope in Gram-negative bacteria. Our insights may help inform new therapeutic strategies to enhance host sPLA2 antimicrobial activity.

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. Labeled [14C]-3-OH-fatty acids were used to quantify the endotoxin content of the samples. Purified meningococcal and E. coli endotoxins and OM blebs displayed similar specific activity in the LAL assay to the purified LPS standard. In contrast, intact bacteria exhibited fivefold lower specific activity in the LAL assay but showed similar MD-2-dependent potency as purified endotoxin in inducing acute airway inflammation in mice. Pre-treatment of intact bacteria and organic dusts with 0.1 M Tris-HCl/10 mM EDTA increased by fivefold the release of endotoxin. These findings demonstrate that house dust and other organic dusts should be extracted with Tris/EDTA to more accurately assess the endotoxin content and pro-inflammatory potential of these environmental samples.

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. Thus, our findings demonstrate markedly different endotoxin recognition properties of caspase-4 from that of MD-2/TLR4 and strongly suggest that activation of caspase-4 (and presumably caspase-5 and caspase-11) are mediated by interactions with activating endotoxin-rich membrane interfaces rather than by endotoxin monomers.

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. Autoradiographic and immunologic analysis confirmed that this purified material was largely devoid of low molecular weight LPS and of the copious amounts of free lipid A that the Francisellae accumulate. Chemical hydrolysis yielded [14C]-labeled free fatty acids characteristic of Francisella lipid A but with a different molar ratio of 3-OH C18:0 to 3-OH C16:0 and different composition of non-hydroxylated fatty acids (mainly C14:0 rather than C16:0) than that of free Francisella lipid A. Mild acid hydrolysis to induce selective cleavage of KDO-lipid A linkage yielded a [14C]-labeled product that partitioned during Bligh/Dyer extraction and migrated during thin-layer chromatography like lipid A. These findings suggest that the O-antigen capsule of Francisella contains a covalently linked and structurally distinct lipid A species. The presence of a discrete lipid A-like molecule associated with capsule raises the possibility that Francisella selectively exploits lipid A structural heterogeneity to regulate synthesis, transport, and stable bacterial surface association of the O-antigen capsular layer.

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. Previously, charged residues at the edge of the LPS binding pocket have been attributed to this difference. In this study, site-directed mutagenesis was used to explore the hydrophobic residues within the MD-2 binding pocket as the source of functional differences between hMD-2 and mMD-2. Whereas decreased hydrophobicity of residues 61 and 63 in the hMD-2 binding pocket retained the characteristics of wild-type hMD-2, a relatively minor change of valine to alanine at position 135 completely abolished the binding of LPS to the hMD-2 mutant. The mutant, however, retained the LPS binding in complex with TLR4 and also cell activation, resulting in a murine-like phenotype. These results were supported by the molecular dynamics simulation. We propose that the residue at position 135 of MD-2 governs the dynamics of the binding pocket and its ability to accommodate lipid A, which is allosterically affected by bound TLR4.

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. scolopes LBP1 (EsLBP1), and provide evidence for its role in triggering a symbiont-induced host developmental program. Previous studies showed that, during initial host colonization, the LPS of V. fischeri synergizes with peptidoglycan (PGN) monomer to induce morphogenesis of epithelial tissues of the host animal. Computationally modeled EsLBP1 shares some but not all structural features of mammalian LBPs that are thought important for LPS binding. Similar to human LBP, recombinant EsLBP1 expressed in insect cells bound V. fischeri LPS and Neisseria meningitidis lipooligosaccharide (LOS) with nanomolar or greater affinity but bound Francisella tularensis LPS only weakly and did not bind PGN monomer. Unlike human LBP, EsLBP1 did not bind N. meningitidis LOS:CD14 complexes. The eslbp1 transcript was upregulated ~22-fold by V. fischeri at 24 h postinoculation. Surprisingly, this upregulation was not induced by exposure to LPS but, rather, to the PGN monomer alone. Hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) and immunocytochemistry (ICC) localized eslbp1 transcript and protein in crypt epithelia, where V. fischeri induces morphogenesis. The data presented here provide a window into the evolution of LBPs and the scope of their roles in animal symbioses. IMPORTANCE: Mammalian lipopolysaccharide (LPS)-binding protein (LBP) is implicated in conveying LPS to host cells and potentiating its signaling activity. In certain disease states, such as obesity, the overproduction of this protein has been a reliable biomarker of chronic inflammation. Here, we describe a symbiosis-induced invertebrate LBP whose tertiary structure and LPS-binding characteristics are similar to those of mammalian LBPs; however, the primary structure of this distantly related squid protein (EsLBP1) differs in key residues previously believed to be essential for LPS binding, suggesting that an alternative strategy exists. Surprisingly, symbiotic expression of eslbp1 is induced by peptidoglycan derivatives, not LPS, a pattern converse to that of RegIIIγ, an important mammalian immunity protein that binds peptidoglycan but whose gene expression is induced by LPS. Finally, EsLBP1 occurs along the apical surfaces of all the host's epithelia, suggesting that it was recruited from a general defensive role to one that mediates specific interactions with its symbiont.

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. d-alanylation of wall- and lipo-teichoic acids in Staphylococcus aureus and sortase function in Streptococcus pyogenes increase bacterial resistance to sPLA(2)-IIA by up to 100-fold apparently by affecting translocation of bound sPLA(2)-IIA to the cell membrane. Action of the sPLA(2)-IIA and other related sPLA(2) against Gram-negative bacteria is more dependent on cationic properties of the enzyme near the amino-terminus of the protein and collaboration with other host defense proteins that produce alterations of the unique Gram-negative bacterial outer membrane that normally represents a barrier to sPLA(2)-IIA action. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.

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. Here, we review studies of wild-type and variant monomeric endotoxin.MD-2 complexes first produced and characterized in our laboratories. These purified complexes have provided unique experimental reagents, revealing both quantitative and qualitative determinants of TLR4 activation and antagonism. This review is dedicated to the memory of Dr. Theresa L. Gioannini (1949-2014) who played a central role in many of the studies and discoveries that are reviewed.

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. We show that each of the hydrophobic residues F438 and F461, which are located on the convex face of leucine-rich repeats 16 and 17 of the mTLR4 ectodomain, are essential for activation of with lipid IVa and paclitaxel, which, although not a structural analog of LPS, activates cells expressing mTLR4/MD-2. Both TLR4 mutants were inactive when stimulated with lipid IVa or paclitaxel, but retained significant activation when stimulated with LPS or hexaacylated lipid A. We show that the phenylalanine residue at position 126 of mouse MD-2 is indispensable only for activation with paclitaxel. Its replacement with leucine or valine completely abolished activation with paclitaxel while preserving the responsiveness to lipid IVa and lipid A. This suggests specific interaction of paclitaxel with F126 because its replacement with leucine even augmented activation by lipid A. These results provide an insight into the molecular mechanism of TLR4 activation by two structurally very different 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. Herein, we addressed whether patients scheduled for HCT display differences in recognition/response to LPS ex vivo traceable to specific single nucleotide polymorphisms (SNPs). Two SNPs of LPS binding protein (LBP) were associated with changes in plasma LBP levels, with one LBP SNP also associating with differences in efficiency of extraction and transfer of endotoxin to myeloid differentiation factor-2 (MD-2), a step needed for activation of TLR4. None of the examined SNPs of CD14, bactericidal/permeability-increasing protein (BPI), TLR4 or MD-2 were associated with corresponding protein plasma levels or endotoxin delivery to MD-2, but CD14 and BPI SNPs significantly associated with differences in LPS-induced TNF-α release ex vivo and infection frequency, respectively. These findings suggest that specific LBP, CD14 and BPI SNPs might be contributory assessments in studies where clinical outcome may be affected by host response to endotoxin and bacterial infection.

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 majority of lipid A of different francisellae strains grown in diverse bacteriologic media and within human phagocytes accumulated as very hydrophobic species, including free lipid A, with <10% of the lipid A molecules substituted with O-Ag polysaccharides. The spectrum of lipid A and LPS species varied in a medium- and strain-dependent fashion, and growth in THP-1 cells yielded lipid A species that were not present in the same bacteria grown in brain heart infusion broth. In summary, metabolic labeling with [(14)C]acetate greatly facilitates assessment of the effect of genotypic and/or environmental variables on the synthesis and accumulation of lipid A and LPS by Francisella, including during growth within the cytosol of infected host cells.

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. Here we report that therapeutic administration of Eritoran (also known as E5564)-a potent, well-tolerated, synthetic TLR4 antagonist-blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titres. CD14 and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2. Thus, Eritoran blockade of TLR signalling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other infections.

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. LOS·MD-2[(125)I] and [(3)H]LOS·MD-2 have similar affinities for soluble (FLAG) TLR4ecd and for membrane-bound TLR4 in HEK293T/TLR4 cells. In a similar dose-dependent manner, sMD-2 and LOS·MD-2 inhibit LOS·MD-2[(125)I] binding to TLR4 indicating the pM affinity binding of LOS·MD-2[(125)I] is agonist-independent. LOS·MD-2[(125)I] allowed measurement of low levels of cell-surface human or murine TLR4 expressed by stable cell lines (2000-3000 sites/cell) and quantitatively measures low levels of 'MD-2-free' TLR4 (est. 250 molecules/cell) in cells co-expressing TLR4 and MD-2. Occupation of 50-100 TLR4/cell by LOS·MD-2 is sufficient to trigger measurable TLR4-dependent cell activation. LOS·MD-2[(125)I] provides a powerful reagent to measure quantitatively functional human and murine cell-surface TLR4, including in cells where surface TLR4 is potentially functionally significant but not detectable by other methods.

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. IFN-α positively regulates mouse APOBEC3 (mA3) mRNA expression through IFN-αR/PKC/STAT1 and negatively regulates mA3 mRNA expression via IFN-αR/MAPKs-signaling pathways. Interestingly, LPS shows some variation in its regulatory behavior. Although LPS-mediated positive regulation of mA3 mRNA occurs through TLR4/TRIF/IRF3/PKC, it negatively modulates mA3 mRNA via TLR4/MyD88/MAPK-signaling pathways. Additional studies on human peripheral blood mononuclear cells reveal that PKC differentially regulates IFN-α and LPS induction of human A3A, A3F, and A3G mRNA expression. In summary, we identified important signaling targets downstream of IFN-αR and TLR4 that mediate A3 mRNA induction by both LPS and IFN-α. Our results provide new insights into the signaling targets that could be manipulated to enhance the intracellular store of A3 and potentially enhance A3 antiviral function in the host.