Endotoxin Detoxification

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

During the 10 years of its existence, the AHDL has worked to solidify and extend our observations that uncovered a previously unappreciated aspect of innate immunity: the requirement that a potent microbial stimulus, lipopolysaccharide (LPS), be inactivated by a host enzyme before homeostasis can be restored following Gram-negative bacterial diseases. We discovered the enzyme, acyloxyacyl hydrolase (AOAH), purified the low-abundance protein to homogeneity, cloned its cDNA, made knockout and transgenic mice, and studied the enzymes properties for almost 30 years before moving to the NIAID in 2009. We showed, with confirmation by others, that AOAH is required for mice to recover from parenteral exposure to LPS; none of the many better-known LPS inhibitors (immunoglobulins, BPI, lipoproteins, etc.) can compensate for the absence of AOAH in Aoah-/- mice. At NIH we showed that AOAH effectively inactivates LPS before it can enter the bloodstream from a subcutaneous tissue inoculation site and that bioactive LPS can persist for many weeks in the peritoneal cavity of an AOAH-/- mouse. Since staff scientist Mingfang Lu returned to China in 2014 we have collaborated with her and shown that AOAH can also inactivate oxidized phospholipids (manuscript in preparation, see below), prevent atherosclerosis in LPS-challenged mice (manuscript in revision, JCI), and influence pulmonary allergy by inactivating intestinal LPS (published by Lu lab in 2018). Encouraged by leadership to study a topic more closely related to the LCIMs interests, we embarked on a study of the pathogenesis of lipid-laden macrophages in CGD. We found that LPS induces fat retention to the same extent in both normal and CGD mouse macrophages and characterized the enzymology involved; in a follow-up study we made the surprising finding that the major determinant of triglyceride retention was the pH of the medium, not LPS or even the oxygen concentration. This prompted us to investigate the impact of low extracellular pH (pHe) on macrophage longevity, realizing that tissue macrophages often encounter an acidic environment and manage to survive and recover. This investigation led us to study mitochondrial fission, oxidative phosphorylation, the utilization of lactate and glutamine, and conversion to a M2 phenotype; the experiments have been finished and a manuscript is nearing submission. This work was done largely at NIH with the help of a student from the Lu-Fudan lab who visited NIH for 3 months in 2018-2019. The absence of a metabolomics resource slowed our progress. We learned early on that AOAH can also act on phospholipids and that certain phospholipids compete with LPS for uptake by cells. In 2017 we began to study the enzymes role in inactivating oxidized phosphatidylcholine (oxPL). These studies have slowly progressed both in vitro and in vivo. Michael Goodwin in the Barry lab helped us with MS analysis of the reaction products while the Lu lab in Shanghai tested AOAHs ability to inactivate oxPL in 4 animal models. The results are convincing and the paper is already on-line while we revise it for final publication in eLIFE. This is potentially a major discovery, since oxidized phospholipids potently activate inflammasomes and their inactivation by host enzymes is not well understood. In addition, we have shown that AOAH can prevent the development of arterial foam cells (atherosclerosis) in mice that receive low doses of LPS and a high fat diet over a period of weeks. This seminal work has been accepted for publication in iSCIENCE.