Alan W. Varley

Shield as Signal: Lipopolysaccharides and the Evolution of Immunity to Gram-Negative Bacteria

According to the innate immunity concept [1], animals defend themselves from microbes by recognizing pathogen-associated molecular patterns. To detect many Gram-negative bacteria, animals use the CD14–MD-2–TLR4 receptor mechanism to recognize the lipid A moiety of the cell wall lipopolysaccharide (LPS). Lipid A is a glucosamine disaccharide that carries phosphates at positions 1 and 4′ and usually has four primary (glucosamine-linked) hydroxyacyl chains and one or more secondary acyl chains. Gram-negative bacteria produce numerous variations on this basic structure, yet sensitive LPS recognition and pro-inflammatory signaling by human TLR4 occur only when lipid A has both phosphates and is hexaacyl, with two secondary acyl chains. What might bacteria derive from producing this type of lipid A, and what do animals gain from recognizing it? A survey of diverse lipid A structures found that the best-recognized configuration is produced by most of the aerobic or facultatively anaerobic Gram-negative bacteria that can live in the gastrointestinal and upper respiratory tracts. We hypothesize that the CD14–MD-2–TLR4 mechanism evolved to recognize not just pathogens, but also many of the commensals (normal flora) and colonizers that can inhabit the bodys most vulnerable surfaces. Producing this lipid A structure seems to favor bacterial persistence on host mucosae, whereas recognizing it allows the host to kill invading bacteria within subepithelial tissues and prevent dissemination. A conserved host lipase can then limit the inflammatory response by removing a key feature of the lipid A signal, the secondary acyl chains.

A Host Lipase Detoxifies Bacterial Lipopolysaccharides in the Liver and Spleen

Much of the inflammatory response of the body to bloodborne Gram-negative bacteria occurs in the liver and spleen, the major organs that remove these bacteria and their lipopolysaccharide (LPS, endotoxin) from the bloodstream. We show here that LPS undergoes deacylation in the liver and spleen by acyloxyacyl hydrolase (AOAH), an endogenous lipase that selectively removes the secondary fatty acyl chains that are required for LPS recognition by its mammalian signaling receptor, MD-2-TLR4. We further show that Kupffer cells produce AOAH and are required for hepatic LPS deacylation in vivo. AOAH-deficient mice did not deacylate LPS and, whereas their inflammatory responses to low doses of LPS were similar to those of wild type mice for ∼3 days after LPS challenge, they subsequently developed pronounced hepatosplenomegaly. Providing recombinant AOAH restored LPS deacylating ability to Aoah-/- mice and prevented LPS-induced hepatomegaly. AOAH-mediated deacylation is a previously unappreciated mechanism that prevents prolonged inflammatory reactions to Gram-negative bacteria and LPS in the liver and spleen.

Host Inactivation of Bacterial Lipopolysaccharide Prevents Prolonged Tolerance Following Gram-Negative Bacterial Infection

A transient state of tolerance to microbial molecules accompanies many infectious diseases. Such tolerance is thought to minimize inflammation-induced injury, but it may also alter host defenses. Here we report that recovery from the tolerant state induced by Gram-negative bacteria is greatly delayed in mice that lack acyloxyacyl hydrolase (AOAH), a lipase that partially deacylates the bacterial cell-wall lipopolysaccharide (LPS). Whereas wild-type mice regained normal responsiveness within 14 days after they received an intraperitoneal injection of LPS or Gram-negative bacteria, AOAH-deficient mice had greatly reduced proinflammatory responses to a second LPS injection for at least 3 weeks. In contrast, LPS-primed Aoah- knockout mice maintained an anti-inflammatory response, evident from their plasma levels of interleukin-10 (IL-10). LPS-primed Aoah-knockout mice experiencing prolonged tolerance were highly susceptible to virulent E. coli challenge. Inactivating LPS, an immunostimulatory microbial molecule, is thus important for restoring effective host defenses following Gram-negative bacterial infection in animals.

Physiologically responsive gene therapy

The goal of physiologically responsive gene therapy is to allow a hosts endogenous regulatory mechanisms to control the production of therapeutic proteins (effectors). Ideally, effector production would be switched on in response to specific signals, stay within therapeutic limits and be switched off when no longer needed. In this way, the unwanted consequences of constitutive, high-level effector expression could be avoided. While recent studies have shown that transgenes can be regulated within animal hosts, they have also highlighted significant problems that require much further research.

Hepatic uptake and deacylation of the LPS in bloodborne LPS-lipoprotein complexes

Much evidence indicates that bacterial LPS (endotoxin) is removed from the bloodstream mainly by the liver, yet the hepatic uptake mechanisms remain uncertain and controversial. In plasma, LPS can be either ‘free’ (as aggregates, bacterial membrane fragments or loosely bound to albumin, CD14, or other proteins) or ‘bound’ (complexed with lipoproteins). Whereas most free LPS is taken up by Kupffer cells (KCs), lipoprotein-bound LPS has seemed to be cleared principally by hepatocytes. Here, we compared the liver’s ability to take up and deacylate free LPS aggregates and the LPS in preformed LPS-high density lipoprotein (HDL) complexes. In mice examined from 1 h to 7 d after a small amount of fluorescent (FITC-)LPS was injected into a lateral tail vein, we found FITC-LPS almost entirely within, or adjacent to, KCs. As expected, FITC-LPS complexed with HDL (FITC-LPS-HDL) disappeared more slowly from the circulation and a smaller fraction of the injected dose of FITC-LPS was found in the liver. Unexpectedly, the FITC-LPS injected as FITC-LPS-HDL complexes was also found within sinusoids, adjacent to, or within, KCs. In other experiments, we found that both free and HDL-bound radiolabeled LPS underwent enzymatic deacylation by acyloxyacyl hydrolase (AOAH), the LPS-inactivating enzyme that is principally produced within the liver by KCs. Our observations suggest that KCs and AOAH play important roles in clearing and catabolizing both free LPS and the LPS in circulating LPS-HDL complexes.

ABCA1 promotes the efflux of bacterial LPS from macrophages and accelerates recovery from LPS-induced tolerance

Macrophages play important roles in both lipid metabolism and innate immunity. We show here that macrophage ATP-binding cassette transporter A1 (ABCA1), a transporter known for its ability to promote apolipoprotein-dependent cholesterol efflux, also participates in the removal of an immunostimulatory bacterial lipid, lipopolysaccharide (LPS). Whereas monocytes require an exogenous lipoprotein acceptor to remove cell-associated LPS, macrophages released LPS in the absence of an exogenous acceptor by a mechanism that was driven, in part, by endogenous apolipoprotein E (apoE). Agents that increased ABCA1 expression increased LPS efflux from wild-type but not ABCA1-deficient macrophages. Preexposure of peritoneal macrophages to LPS for 24 h increased the expression of ABCA1 and increased LPS efflux with a requirement for exogenous apolipoproteins due to suppression of endogenous apoE production. In contrast, LPS preconditioning of ABCA1-deficient macrophages significantly decreased LPS efflux and led to prolonged retention of cell-surface LPS. Although the initial response to LPS was similar in wild-type and ABCA1-deficient macrophages, LPS-induced tolerance was greater and more prolonged in macrophages that lacked ABCA1. Our results define a new role for macrophage ABCA1 in removing cell-associated LPS and restoring normal macrophage responsiveness.

Overproduction of acyloxyacyl hydrolase by macrophages and dendritic cells prevents prolonged reactions to bacterial lipopolysaccharide in vivo.

Although recognition of lipopolysaccharide (LPS) by the myeloid differentiation factor 2-Toll-like receptor 4 complex is important for triggering protective inflammatory responses in animals, terminating many of these responses requires LPS inactivation by a host lipase, acyloxyacyl hydrolase (AOAH). To test whether endogenously produced recombinant AOAH can modulate responses to LPS and gram-negative bacteria, we engineered transgenic mice that overexpress AOAH in dendritic cells and macrophages, cell types that normally produce it. Transgenic mice deacylated LPS more rapidly than did wild-type controls. They also were protected from LPS-induced hepatosplenomegaly, recovered more quickly from LPS-induced weight loss, and were more likely to survive when challenged with live Escherichia coli. Constitutive overexpression of AOAH in vivo hastened recovery from LPS exposure without interfering with the normal acute inflammatory response to this important microbial signal molecule. Our results suggest that the extent to which macrophages and dendritic cells produce AOAH may influence the outcome of many gram-negative bacterial diseases.

Prolonged hepatomegaly in mice that cannot inactivate bacterial endotoxin

Transient hepatomegaly often accompanies acute bacterial infections. Reversible, dose‐dependent hepatomegaly also occurs when animals are given intravenous infusions of bacterial lipopolysaccharide (LPS). We found that recovery from LPS‐induced hepatomegaly requires a host enzyme, acyloxyacyl hydrolase (AOAH), that inactivates LPS. When we challenged Aoah−/− mice with low doses of LPS or Gram‐negative bacteria, their livers remained enlarged (as much as 80% above normal) many weeks longer than did the livers of Aoah+/+ animals. When compared with livers from LPS‐primed Aoah+/+ mice, LPS‐primed Aoah−/− livers had (1) more numerous and larger Kupffer cells, (2) intrasinusoidal leukocyte aggregates and activated sinusoidal endothelial cells, and (3) sustained production of interleukin (IL)‐10 and messenger RNAs (mRNAs) for tumor necrosis factor (TNF), IL‐10, and IRAK‐M. Depleting Kupffer cells decreased the liver enlargement by ≈40%, whereas depletion of neutrophils, dendritic cells, natural killer (NK) cells, NK‐T cells, or B cells had no effect. Pretreatment with dexamethasone almost completely prevented prolonged hepatomegaly in Aoah−/− mice, whereas neutralizing TNF or interleukin‐1β was only partially effective. In contrast, an antagonistic antibody to the IL‐10 receptor increased LPS‐induced hepatomegaly by as much as 50%. Conclusion: our findings suggest that persistently active LPS induces Kupffer cells to elaborate mediators that promote the accumulation of leukocytes within enlarged sinusoids. Large increases in IL‐10 and several other modulatory molecules are unable to prevent prolonged hepatomegaly in mice that cannot inactivate LPS. The striking findings in this mouse model should encourage studies to find out how AOAH contributes to human liver physiology and disease. (HEPATOLOGY 2011;)

Distinctive structural features are shared by human, lapine, and murine acyloxyacyl hydrolases

Human acyloxyacyl hydrolase is an unusual lipase, found in phagocytic cells, that removes acyl chains from bacterial lipopolysaccharides (LPS) and glycerolipids. It is a heterodimer in which two glycosylated peptides are linked by disulfide bonding. The large subunit contains the active site serine, while the smaller subunit has striking sequence similarity to the saposins, peptide cofactors for several sphingolipid hydrolases. Since rabbits and mice are widely used for studies of LPS—animal interactions, we asked if murine and lapine AOAHs resemble the human enzyme. We report here that murine and lapine AOAHs share the distinctive features of the human AOAH primary sequence and have similar affinity for LPS. The structure of this unusual lipase appears to have been highly conserved.