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.

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.

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.

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.

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.

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.

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.

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. In both wild-type and mutant LOS·MD-2 complexes, one of the six fatty acyl chains of LOS is more susceptible to paramagnetic attenuation, suggesting protrusion of that fatty acyl chain from the hydrophobic pocket of MD-2, independent of association with TLR4. These findings indicate that re-orientation of the aromatic side chain of Phe(126) is induced by binding of hexaacylated E, preceding interaction with TLR4. This re-arrangement of Phe(126) may act as a "hydrophobic switch," driving agonist-dependent contacts needed for TLR4 dimerization and activation.

Expression of functional D299G.T399I polymorphic variant of TLR4 depends more on coexpression of MD-2 than does wild-type TLR4.

Two missense variants (D299G and T399I) of TLR4 are cosegregated in individuals of European descent and, in a number of test systems, result in reduced responsiveness to endotoxin. How these changes within the ectodomain (ecd) of TLR4 affect TLR4 function is unclear. For both wild-type and D299G.T399I TLR4, we used endotoxinCD14 and endotoxinMD-2 complexes of high specific radioactivity to measure: 1) interaction of recombinant MD-2TLR4 with endotoxinCD14 and TLR4 with endotoxinMD-2; 2) expression of functional MD-2TLR4 and TLR4; and 3) MD-2TLR4 and TLR4-dependent cellular endotoxin responsiveness. Both wild-type and D299G.T399I TLR4(ecd) demonstrated high affinity (K(d) approximately 200 pM) interaction of endotoxinCD14 with MD-2TLR4(ecd) and endotoxinMD-2 with TLR4(ecd). However, levels of functional TLR4 were reduced up to 2-fold when D299G.T399I TLR4 was coexpressed with MD-2 and >10-fold when expressed without MD-2, paralleling differences in cellular endotoxin responsiveness. The dramatic effect of the D299G.T399I haplotype on expression of functional TLR4 without MD-2 suggests that cells expressing TLR4 without MD-2 are most affected by these polymorphisms.

LBP/BPI proteins and their relatives: conservation over evolution and roles in mutualism.

LBP [LPS (lipopolysaccharide)-binding protein] and BPI (bactericidal/permeability-increasing protein) are components of the immune system that have been principally studied in mammals for their involvement in defence against bacterial pathogens. These proteins share a basic architecture and residues involved in LPS binding. Putative orthologues, i.e. proteins encoded by similar genes that diverged from a common ancestor, have been found in a number of non-mammalian vertebrate species and several non-vertebrates. Similar to other aspects of immunity, such as the activity of Toll-like receptors and NOD (nucleotide-binding oligomerization domain) proteins, analysis of the conservation of LBPs and BPIs in the invertebrates promises to provide insight into features essential to the form and function of these molecules. This review considers state-of-the-art knowledge in the diversity of the LBP/BPI proteins across the eukaryotes and also considers their role in mutualistic symbioses. Recent studies of the LBPs and BPIs in an invertebrate model of beneficial associations, the Hawaiian bobtail squid Euprymna scolopes' alliance with the marine luminous bacterium Vibrio fischeri, are discussed as an example of the use of non-vertebrate models for the study of LBPs and BPIs.

Novel roles of lysines 122, 125, and 58 in functional differences between human and murine MD-2.

The MD-2/TLR4 complex provides a highly robust mechanism for recognition and response of mammalian innate immunity to Gram-negative bacterial endotoxins. Despite overall close structural and functional similarity, human (h) and murine (m) MD-2 show several species-related differences, including the ability of hMD-2, but not mMD-2, to bind endotoxin (E) in the absence of TLR4. Wild-type mMD-2 can support TLR4-dependent cell activation by E only when mMD-2 and mTLR4 are coexpressed in the same cell. However, replacement of Glu122, Leu125, and/or Asn58 of mMD-2 with the corresponding residues (lysines) of hMD-2 was sufficient to yield soluble extracellular MD-2 that reacted with monomeric E . sCD14 complex to form extracellular monomeric E . MD-2 that activated cells expressing TLR4 without MD-2. Moreover, in contrast to wild-type mMD-2, double and triple mMD-2 mutants also supported E-triggered signaling in combination with human TLR4. Conversely, a K125L mutant of hMD-2 reacted with E . CD14 and activated TLR4 only when coexpressed with TLR4, and not when secreted without TLR4. These findings reveal novel roles of lysines 122, 125, and 58 in human MD-2 that contribute to the functional differences between human and murine MD-2 and, potentially, to differences in the sensitivity of humans and mice to endotoxin.

Neutrophil bleaching of GFP-expressing staphylococci: probing the intraphagosomal fate of individual bacteria.

Successful host defense against bacteria such as Staphylococcus aureus (SA) depends on a prompt response by circulating polymorphonuclear leukocytes (PMN). Stimulated PMN create in their phagosomes an environment inhospitable to most ingested bacteria. Granules that fuse with the phagosome deliver an array of catalytic and noncatalytic antimicrobial peptides, while activation of the NADPH oxidase at the phagosomal membrane generates reactive oxygen species within the phagosome, including hypochlorous acid (HOCl), formed by the oxidation of chloride by the granule protein myeloperoxidase in the presence of H(2)O(2). In this study, we used SA-expressing cytosolic GFP to provide a novel probe of the fate of SA in human PMN. PMN bleaching of GFP in SA required phagocytosis, active myeloperoxidase, H(2)O(2) from the NADPH oxidase, and chloride. Not all ingested SA were bleached, and the number of cocci within PMN-retaining fluorescent GFP closely correlated with the number of viable bacteria remaining intracellularly. The percent of intracellular fluorescent and viable SA increased at higher multiplicity of infection and when SA presented to PMN had been harvested from the stationary phase of growth. These studies demonstrate that the loss of GFP fluorescence in ingested SA provides a sensitive experimental probe for monitoring biochemical events within individual phagosomes and for identifying subpopulations of SA that resist intracellular PMN cytotoxicity. Defining the molecular basis of SA survival within PMN should provide important insights into bacterial and host properties that limit PMN antistaphylococcal action and thus contribute to the pathogenesis of staphylococcal infection.

Evidence of a specific interaction between new synthetic antisepsis agents and CD14.

Synthetic molecules derived from natural sugars with a positively charged amino group or ammonium salt and two lipophilic chains have been shown to inhibit TLR4 activation in vitro and in vivo. To characterize the mechanism of action of this class of molecules, we investigated possible interactions with the extracellular components that bind and shuttle endotoxin [lipopolysaccharide (LPS)] to TLR4, namely, LBP, CD14, and MD-2. Molecules that inhibited TLR4 activation inhibited LBP.CD14-dependent transfer of endotoxin monomers derived from aggregates of tritiated lipooligosaccharide ([(3)H]LOS) from Neisseria meninigitidis to MD-2.TLR4, resulting in a reduced level of formation of a ([(3)H]LOS.MD-2.TLR4(ECD))(2) (M(r) approximately 190000) complex. This effect was due to inhibition of the transfer of [(3)H]LOS from aggregates in solution to sCD14 with little or no effect on [(3)H]LOS shuttling from [(3)H]LOS.sCD14 to MD-2. These compounds also inhibited transfer of the [(3)H]LOS monomer from full-length CD14 to a truncated, polyhistidine-tagged CD14. Dose-dependent inhibition of the transfer of [(3)H]LOS between the two forms of CD14 was observed with each of three different synthetic compounds that inhibited TLR4 activation but not by another structurally related analogue that lacked TLR4 antagonistic activity. Saturation transfer difference (STD) NMR data showed direct binding to CD14 by the synthetic TLR4 antagonist mediated principally through the lipid chains of the synthetic compound. Taken together, our findings strongly suggest that these compounds inhibit TLR4 activation by endotoxin by competitively occupying CD14 and thereby reducing the level of delivery of activating endotoxin to MD-2.TLR4.

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.

MD-2-dependent pulmonary immune responses to inhaled lipooligosaccharides: effect of acylation state.

Endotoxins represent one of the most potent classes of microbial immunoactive components that can cause pulmonary inflammation. The aim of this study was to compare the inflammatory potency of two types of Neisseria meningitidis endotoxins (lipooligosaccharides) in lungs: wild type (hexaacylated, LOS(wt)) and mutant type (pentaacylated, LOS(msbB)), and to determine the importance of MD-2 in endotoxin responses in lungs in vivo. Endotoxin-normoresponsive mice (BALB/c) were exposed to selected doses of penta- and hexaacylated lipooligosaccharides (LOS) by nasal aspiration. Cellular and cytokine/chemokine inflammatory responses in bronchoalveolar lavage were measured at 1-, 4-, 8-, 16-, 24-, and 48-hour time points. MD-2-null mice were exposed to one dose of hexaacylated LOS and inflammatory responses were measured after 4 and 24 hours. Inhalation of hexaacylated LOS resulted in strong inflammatory responses, while pentaacylated LOS was much less potent in inducing increases of neutrophils, TNF-alpha, macrophage inflammatory protein-1 alpha, IL-6, granulocyte colony-stimulating factor, and IL-1 beta concentration in bronchoalveolar lavage. Similar kinetics of inflammatory responses in lungs were found in both types of endotoxin exposures. Inhalation of hexaacylated LOS in MD-2-null mice resulted in significantly lower numbers of neutrophils in bronchoalveolar lavage than in normoresponsive mice. Markedly lower inflammatory potency of pentaacylated LOS was observed compared with hexaacylated LOS. Hyporesponsiveness in MD-2-null mice after nasal aspiration of wild-type LOS indicate its essential role in airway responsiveness to endotoxin.

Wall teichoic acid deficiency in Staphylococcus aureus confers selective resistance to mammalian group IIA phospholipase A(2) and human beta-defensin 3.

Wall teichoic acids (WTAs) and membrane lipoteichoic acids (LTAs) are the major polyanionic polymers in the envelope of Staphylococcus aureus. WTAs in S. aureus play an important role in bacteriophage attachment and bacterial adherence to certain host cells, suggesting that WTAs are exposed on the cell surface and could also provide necessary binding sites for cationic antimicrobial peptides and proteins (CAMPs). Highly cationic mammalian group IIA phospholipase A(2) (gIIA PLA(2)) kills S. aureus at nanomolar concentrations by an action(s) that depends on initial electrostatic interactions, cell wall penetration, membrane phospholipid (PL) degradation, and activation of autolysins. A tagO mutant of S. aureus that lacks WTA is up to 100-fold more resistant to PL degradation and killing by gIIA PLA(2) and CAMP human beta-defensin 3 (HBD-3) but has the sensitivity of the wild type (wt) to other CAMPs, such as Magainin II amide, hNP1-3, LL-37, and lactoferrin. In contrast, there is little or no difference in either gIIA PLA(2) activity toward cell wall-depleted protoplasts of the wt and tagO strains of S. aureus or in binding of gIIA PLA(2) to wt and tagO strains. Scanning and transmission electron microscopy reveal increased surface protrusions in the S. aureus tagO mutant that might account for reduced activity of bound gIIA PLA(2) and HBD-3 toward the tagO mutant. In summary, the absence of WTA in S. aureus causes a selective increase in bacterial resistance to gIIA PLA(2) and HBD-3, the former apparently by reducing access and/or activity of bound antibacterial enzyme to the bacterial membrane.

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.

Regulation of interactions of Gram-negative bacterial endotoxins with mammalian cells.

Host defense against many invading Gram-negative bacteria (GNB) depends on innate immune recognition of endotoxin (lipopolysaccharides, LPS), unique surface glycolipids of GNB. Host responses to endotoxin must be highly sensitive but self-limited. In mammals, optimal sensitivity is achieved by ordered interactions of endotoxin with several different extracellular and cell surface proteins-the LPS-binding protein (LBP), CD14, MD-2, and Toll-like receptor (TLR) 4-reflecting the requirement for specific protein-endotoxin and protein-protein interactions. This complex reaction pathway also provides many ways to attenuate endotoxin-driven inflammation and can explain how differences in endotoxin structure, either intrinsic among GNB or induced by metabolic remodeling, can alter host responsiveness and thus the outcome of host-GNB interactions. Major goals of our research are to better understand: (1) the structural bases of specific host-endotoxin interactions; (2) functional diversity among host endotoxin-binding proteins; and (3) how the actions of various endotoxin-binding proteins are regulated to permit optimal host responses to GNB infection. In addition, the identification of a water-soluble endotoxin:MD-2 complex that, depending on the structure of endotoxin or MD-2, has potent TLR4 agonist or antagonist properties suggests novel pharmacologic approaches to immuno-modulation.

The bactericidal/permeability-increasing protein (BPI) in infection and inflammatory disease.

Gram-negative bacteria (GNB) and their endotoxin present a constant environmental challenge. Endotoxins can potently signal mobilization of host defenses against invading GNB but also potentially induce severe pathophysiology, necessitating controlled initiation and resolution of endotoxin-induced inflammation to maintain host integrity. The bactericidal/permeability-increasing protein (BPI) is a pluripotent protein expressed, in humans, mainly neutrophils. BPI exhibits strong antimicrobial activity against GNB and potent endotoxin-neutralizing activity. BPI mobilized with neutrophils in response to invading GNB can promote intracellular and extracellular bacterial killing, endotoxin neutralization and clearance, and delivery of GNB outer membrane antigens to dendritic cells. Tissue expression by dermal fibroblasts and epithelia could further amplify local levels of BPI and local interaction with GNB and endotoxin, helping to constrain local tissue infection and inflammation and prevent systemic infection and systemic inflammation. This review article focuses on the structural and functional properties of BPI with respect to its contribution to host defense during GNB infections and endotoxin-induced inflammation and the genesis of autoantibodies against BPI that can blunt BPI activity and potentially contribute to chronic inflammatory disease.