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,||,1
* Department of Biomedical Research, TNO-Quality of Life, Leiden; Departments of
General Internal Medicine,
Vascular Surgery,
Infectious Diseases, and
|| Cardiology, Leiden University Medical Center, Leiden, The Netherlands;
¶ Department of Internal Medicine, Division of Infectious Diseases, UT Southwestern Medical Center, Dallas, Texas, USA; and
** Department of Medical Microbiology and Infectious Diseases, Erasmus Medical Center, Rotterdam, The Netherlands
1Correspondence: Leiden University Medical Center, Department Endocrinology and Metabolism, C4-R81, Albinusdreef 2, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail: p.c.n.rensen{at}lumc.nl
SPECIFIC AIMS
Lipoproteins such as high-density lipoprotein (HDL) have been shown to bind lipopolysaccharide (LPS) and to protect against sepsis. Since septic HDL is virtually devoid of apolipoprotein CI (apoCI), we hypothesized that apoCI may have a protective function in sepsis. The aim of our study was to evaluate whether apoCI can affect the biological response to LPS and reduce mortality in Gram-negative sepsis.
PRINCIPAL FINDINGS
1. ApoCI avidly binds LPS via the conserved LPS-binding motif KVKEKLK
Sequence alignment analysis showed that apoCI contains a KVKEKLK motif in its C-terminal domain (apoCI48–54) that is highly homologous to the LPS-binding sequence of established LPS-binding proteins. ApoCI fully disaggregated LPS micelles already at a 1:1 molar ratio, and the LPS-deaggregating properties of plasma were dependent on the apoCI content. We found that apoCI directly bound to LPS via the positively charged Lys residues within the KVKEKLK motif, since replacement of Lys residues by Ala residues (AVAEALA) largely reduced the binding of apoCI to LPS.
2. ApoCI prevents the clearance of LPS by the liver and spleen
Incubation of radiolabeled mutant LPS (ReLPS) or wild-type (WT) LPS (wtLPS) with apoCI dose-dependently reduced the uptake of LPS by the liver and spleen on intravenous injection. In fact, at molar equilibrium, the clearance of LPS from serum was almost completely abolished, and LPS was fully recovered with HDL. The LPS-binding motif within apoCI (KVKEKLK) was crucially involved in redirecting the metabolic fate of LPS, since the apoCI mutant (AVAEALA) was much less efficient in preventing the serum clearance of ReLPS and wtLPS.
3. ApoCI stimulates the LPS-induced proinflammatory response
Incubation of LPS with apoCI before injection into mice enhanced the LPS-induced production of tumor necrosis factor-
(TNF-) by 3.8-fold as compared with injection of LPS alone. Likewise, injection of LPS into apoCI-deficient (apoc1–/–), WT, and human apoCI-transgenic (APOC1) mice resulted in an apoCI-dependent increase in plasma TNF- levels (Fig. 1
A). In vitro studies in RAW 264.7 mouse macrophages showed that apoCI could directly stimulate the TNF- response to ReLPS (Fig. 1B
) and wtLPS (Fig. 1C
), whereas apoCIII was hardly effective (Fig. 1B,C
). ApoCI did not stimulate the TNF- response to the non-lipopolysaccharide stimulus zymosan (Fig. 1D
). Again, the LPS-binding domain KVKEKLK is crucial for the enhanced response to LPS, since the apoCI mutant was ineffective in this regard.
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4. ApoCI increases the inflammatory response to Klebsiella pneumoniae and protects against fatal sepsis
Apoc1–/–, WT, and APOC1 mice were challenged intrapulmonally with Klebsiella pneumoniae, an established procedure leading to Gram-negative bacterial pneumonia, sepsis, and eventually death. At 24 h after inoculation, the apoCI status of the mice positively correlated with markers of the inflammatory response, i.e., the plasma levels of TNF-, macrophage migration inhibitory factor (MIF), and E-selectin (Fig. 2
A–C). At 48 h after inoculation, the number of circulating Klebsiella pneumoniae was strongly and dose-dependently decreased by apoCI. APOC1 mice displayed a 25-fold lower bacterial count as compared with apoc1–/– mice (Fig. 2D
). Accordingly, apoCI protected mice against fatal sepsis. After bacterial inoculation in WT mice, a gradual loss of animals occurred, with 39% survival after 2 wk. Whereas apoc1–/– mice were more susceptible to death after sepsis (only 16% survival), expression of human apoCI in APOC1 mice improved survival (53% survival; Fig. 2E
). Together, these data indicate that elevated apoCI levels are associated with an enhanced inflammatory response to LPS, a more efficient bacterial killing, and that apoCI can prevent mice from septic death.
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CONCLUSIONS AND SIGNIFICANCE
We discovered by sequence alignment analysis that one of the highly conserved intervals within the C-terminal domain of apoCI appears to contain the LPS-binding motif KVKEKLK (apoCI48–54), which is identical between mice and humans. Indeed, human apoCI avidly bound to LPS in vitro, and the resulting complex was resistant to dissociation in the blood despite the abundance of potentially destabilizing plasma transfer factors (e.g., LPS-binding protein and phospholipids transfer protein). As a consequence, on intravenous injection in mice, apoCI markedly prolonged the residence time of LPS in serum by association with long-circulating HDL, reaching a maximum effect already at an apoCI = 1:1 LPS molar ratio. Importantly, the LPS-monomerizing capacity of murine plasma was increased by the presence of moderate levels of human apoCI (i.e., 35 mg/dl in APOC1 mice as compared with 10 mg/dl in human plasma). Reciprocally, the monomerization of LPS was decreased in the absence of apoCI, indicating that the endogenous plasma level of apoCI per se can have a profound impact on the LPS distribution in vivo.
Physiological amounts of both exogenous and endogenous apoCI strongly enhanced the TNF- production on intravenous LPS administration in mice. We showed that apoCI can directly and specifically stimulate the LPS-induced TNF- production by macrophages in vitro. The increased apoCI-induced inflammatory response to LPS in vivo may thus be caused by a prolonged residence of LPS in the serum and/or direct activation of macrophages.
ApoCI was able to affect the serum clearance and TNF--stimulating properties of both full-length wtLPS (composed of the toxic lipid A moiety, as well as the inner core, outer core, and O-antigen) and truncated ReLPS (merely composed of the lipid A moiety and some KDO sugars), indicating that the lipid A/KDO moiety of the LPS molecule contains the crucial elements for interaction with apoCI. Mutation of lysine residues to alanine residues within the KVKEKLK domain of apoCI markedly reduced these LPS-modulating effects of apoCI without affecting the helical structure, indicating that this highly conserved domain within the C-terminal helix of apoCI is indeed responsible for LPS binding. Taken together, these data suggest that the interaction between LPS and apoCI is likely to involve electrostatic interaction between the lysine residues within apoCI and electronegative elements (presumably phosphate groups) within the lipid A moiety of LPS. Since lipid A is the common determinant of LPS molecules from all bacterial species, apoCI is likely to bind a wide array of WT and mutant LPS molecules. The fact that apoCI is able to enhance the binding of LPS to HDL further indicates that such an electrostatic interaction between apoCI and LPS can still occur on the lipoprotein surface.
Since sepsis has initially been regarded as an excessive systemic proinflammatory response to infections as largely mediated by TNF-, anti-inflammatory strategies, for example, aimed to neutralize TNF-, have been widely used as potential therapeutic tools for the treatment of sepsis. However, although such approaches have been successful in inhibiting LPS-induced toxicity and mortality, a recent metaregression analysis of 22 clinical sepsis trials with anti-inflammatory agents, including 9 trials with anti-TNF- antibodies, showed that beneficial therapeutic effects of anti-inflammatory therapies could only be demonstrated in patients with a high risk of death, whereas anti-inflammatory agents were harmful in those patients with a low mortality risk. Therefore, we hypothesized that the observed apoCI-mediated increased inflammatory reaction to LPS may enhance survival from Gram-negative infections as a result of a timely and effective host response.
As a model for human sepsis caused by Gram-negative bacteria, we applied an experimental pneumonia model in which a local Klebsiella pneumoniae infection causes a lethal sepsis. The protective role of LPS-induced Toll-like receptor 4 (TLR4) signaling in this septic model has been conclusively established, as TLR4-deficiency shortened survival together with an enhanced bacterial outgrowth. Consistent with our finding that apoCI directly stimulated the LPS-induced TNF- response by macrophages in vitro, apoCI expression in mice also dose-dependently increased the initial proinflammatory response toward Klebsiella pneumoniae, as judged from increased plasma inflammation markers (TNF-, MIF, and E-selectin) reflecting an activation of macrophages. These effects were accompanied by lower bacterial counts in plasma and reduced mortality resulting from sepsis, confirming that an increased early inflammatory response effectuates an efficient antibacterial response.
Based on our present data, we propose the following model for the protective effects of apoCI in Gram-negative sepsis (Fig. 3
). On the entry and proliferation of bacteria in the blood, LPS is released into the plasma and binds to apoCI, which involves the interactionbetween the LPS-binding motif KVKEKLK within apoCI and presumably phosphate groups within lipid A, the shared moiety of all LPS species. Although apoCI may bind LPS in the lipid-free and lipid-bound state, the resulting apoCI-LPS complex is mainly associated with HDL, which is the main carrier of apoCI in mice and humans. ApoCI effectively presents the LPS to responsive cells such as macrophages (the mechanistic basis of which is under current investigation), leading to a rapid and enhanced production of proinflammatory cytokines, among which are TNF- and MIF. These cytokines are essential for effective eradication of the bacterial infection thereby preventing infection-related mortality. Therefore, plasma apoCI protects against fatal sepsis by effectuating an early and adequate antibacterial response. We speculate that apoCI may provide a therapeutic handle in the ongoing search for strategies that are aimed to prevent or treat sepsis at an early stage.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5639fje
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, n=29), and APOC1 (
, n=19) mice (i.e., including mice described above and a second set of mice) were inoculated intratracheally with same dose of Klebsiella pneumoniae or saline (
, WT mice, n=6) and their 2-wk survival was assessed. *P < 0.05, **P < 0.01 (log-rank test, Graph Pad Software Inc.).