Statins and sepsis: multiple modifications at multiple levels

Review

Statins and sepsis: multiple modifications at multiple levels Marius Terblanche, Yaniv Almog, Robert S Rosenson, Terry S Smith, Daniel G Hackam Lancet Infect Dis 2007; 7: 358–68 Department of Critical Care Medicine, St Thomas’ Hospital, London, UK (M Terblanche FRCA); Department of Critical Care Medicine (M Terblanche, T S Smith MD), and Division of Clinical Pharmacology and Toxicology (D G Hackam MD), Sunnybrook Health Science Centre, Toronto, Canada; Medical Intensive Care Unit, Soroka University Medical Center, Faculty of Health Sciences, Ben Gurion University, Beer-Sheva, Israel (Y Almog MD); Division of Cardiovascular Medicine, Department of Medicine, University of Michigan, Ann Arbor, MI, USA (R S Rosenson MD); and Institute for Clinical Evaluative Sciences, Toronto, Canada (D G Hackam) Correspondence to: Dr Marius Terblanche, Department of Critical Care Medicine, St Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK. Tel +44 (0)20 7188 3044; fax +44 (0)20 7188 2284; [email protected]

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Sepsis, an infection-induced inflammatory syndrome, is a leading and increasing cause of mortality worldwide. Animal and human observational studies suggest statins may prevent the morbidity and mortality associated with the sepsis syndrome. In this Review, we describe the demonstrated mechanisms through which statins modulate the inflammatory response associated with sepsis. These mechanisms include effects on cell signalling with consequent changes at the transcriptional level, the induction of haem oxygenase, the direct alteration of leucocyte–endothelial cell interaction, and the reduced expression of MHC II. Since statins do not target individual inflammatory mediators, but possibly reduce the overall magnitude of the systemic response, this effect could prove an important distinguishing feature modulating the host response to septic insults. This work establishes the biological plausibility needed for future trials of statins in critical illness.

Introduction

Statins and systemic inflammation

Sepsis is a complex and multifaceted syndrome. In response to an infectious insult, the immune system strives to contain the infection at the point of occurrence. Activation of the innate immune system is associated with the syndrome of sepsis and related organ dysfunction.1 Death frequently follows. Sepsis-associated mortality is a leading cause of death in both the developed and developing world, with reported case fatality rates ranging from 30% to 70%.2–6 In the USA alone an estimated 750 000 patients developed severe sepsis in 1995.7 More than a quarter of these cases (approximately 213 000 patients) died. In the same year the total cost of treating sepsis amounted to approximately US$16·7 billion nationally.7 It is increasingly recognised that interventions attempting to alter the pathogenesis of, and host’s response to, sepsis must be able to modify multiple levels of the inflammatory cascade.1,8–10 When taken individually, results from more than 70 randomised controlled trials of adjuvant mediator therapy have been disappointing.1,11 To date, only one agent has proven successful: recombinant activated protein C.12 However, the very high cost of this agent prevents its use in underresourced health-care settings. Since sepsis potentially represents an inflammatory and hypercoagulable state of the body’s vascular beds, we postulate that statins might be beneficial by preventing or downregulating components of the sepsis syndrome.8 Despite some conflicting reports from other work, a number of human observational studies, reviewed elsewhere, support this concept.13–19 The mechanisms through which statins influence sepsis-associated inflammation are poorly understood. In this Review, we describe the multiple actions of statins in the context of the pathogenesis of sepsis. We outline the link between the mevalonate–cholesterol pathway and inflammatory signalling, and the actions of statins that are independent of the cholesterol pathway. Although many of the effects of statins described in relation to atherosclerosis might also be relevant in sepsis, we focus on the effects demonstrated in animal and human models of inflammation and sepsis.

Several mechanisms of statins, working in concert, simultaneously influence the immune and inflammatory responses associated with sepsis. These mechanisms, demonstrated at the cellular and molecular level, and in both human and animal models of sepsis, are: multiple anti-inflammatory actions (table, figure 1, figure 2), direct activation of haem oxygenase (figure 3), direct interference in leucocyte–endothelial interaction (figure 4), and direct inhibition of MHC II.

Anti-inflammatory actions Statins reduce systemic inflammation by lowering the proinflammatory tendencies of macrophages and neutrophils, by limiting endothelial cell activation, and by enhancing T-helper cell (Th)-1 function. These benefits accrue because of changes in inflammatory signalling, gene transcription, and subsequent changes in the downstream inflammatory mediators (table).

How do statins influence intracellular signalling? Mevalonate is the precursor of many classes of sterol and non-sterol end-products (figure 5). Cholesterol is the main sterol end-product, whereas numerous isoprenoid molecules, including farnesylpyrophosphate and geranylgeranylpyrophosphate, are important non-sterol intermediaries. Farnesylpyrophosphate and geranylgeranylpyrophosphate serve as intracellular lipid attachments for the post-translational modification of a number of proteins. This modification, or isoprenylation, allows the subcellular localisation and intracellular trafficking of membrane-associated proteins such as heterometric G-proteins, haem-a, nuclear lamins, and small GTP-binding proteins.48 GTP-binding proteins have crucial roles in intracellular inflammatory signalling by acting as molecular on/off switches for various protein kinases. They cycle between active and inactive states under the influence of guanine nucleotide exchange factors and GTPase-activating proteins, respectively, and must undergo isoprenylation to enable pathway activation. Although many subfamilies are described, the farnesylated Ras subfamily, and the geranylgeranylated Rho, Rac, and Cdc42 subfamilies are http://infection.thelancet.com Vol 7 May 2007

Review

Effect

Setting/model

Statin

Dose/ concentration

Timing of statin treatment*

Non-atherosclerotic human transgenic mice. Confirmed in human hepatocytes20

Atorvastatin, simvastatin

NR

Pre

Human endothelial cells21

Simvastatin, atorvastatin, lovastatin

0·1–10 μmol/L

In vitro

C-reactive protein-stimulated human vascular endothelial cells22

Fluvastatin

10 μmol/L

2 h pre

Murine RAW 264·7 macrophages stimulated by lipopolysaccharide23

Lovastatin

0·1–30 μmol concentration

In vitro

Human endothelial and vascular smooth muscle cells21

Simvastatin, atorvastatin, lovastatin

0·1–10 μmol/L

In vitro

Activation of PPARγ

Human peripheral monocytes24

Atorvastatin

0·1–10 μmol/L

In vitro

Rapid Akt phosphorylation

Cultured human umbilical vein endothelial cells25

Pravastatin, simvastatin

1 μmol

In vitro

Signal transducer and activator of transcription 1 inhibition

Murine RAW 264·7 macrophages stimulated by intereferon-γ23

Lovastatin

0·1–30 μmol concentration

In vitro

Increased tyrosine kinase, ERK, and p38 MAPK induction causing Murine RAW 264·7 macrophages stimulated by lipopolysaccharide increased COX-2 expression and interferon-γ26

Lovastatin, fluvastatin, atorvastatin, pravastatin

0·1–30 μmol concentration

In vitro

Decreased transcription and activation of PAI-1 promotor

Human saphenous vein endothelial and smooth muscle cells stimulated by PDGF or TGF-β27

Simvastatin

NR

16 h pre

Activation of interleukin-12p40 promoter-luciferase constructs Decreased c-Fos binding to the interleukin-12p40 promoter Induction of c-Jun phosphorylation

Mouse peritoneal macrophages28

Simvastatin

NR

In vitro

Increased CD36 gene transcription

Lipopolysaccharide-stimulated monocytes from human volunteers29

Lovastatin, fluvastatin, atorvastatin

NR

In vitro

Lipopolysaccharide-stimulated CD1 mice30

Cerivastatin

20 mg/kg

12 h and 1 h pre

Lipopolysaccharide-stimulated blood from hypercholesterolaemic adults31

Pravastatin

40 mg/day

7 weeks pre

Human peripheral monocytes24

Atorvastatin

0·1–10 μmol/L

In vitro

C-reactive protein-stimulated human vascular endothelial cells22

Fluvastatin

10 μmol/L

2 h pre

Escherichia coli endotoxin (lipopolysaccharide) in healthy volunteers32

Simvastatin

80 mg/day

4 days pre

Decreased interleukin 1β

Lipopolysaccharide-stimulated CD1 mice30

Cerivastatin

20 mg/kg

12 and 1 h pre

Decreased interleukin 6

Lipopolysaccharide-stimulated and carrageenan-stimulated CD1 mice33

Lovastatin, pravastatin, simvastatin

10 mg/kg

20 h, 12 h, 30 min pre

Lipopolysaccharide-stimulated blood from hypercholesterolemic adults31

Pravastatin

40 mg/day

7 weeks pre

Decreased interleukin 8

Human alveolar macrophages and umbilical cord endothelial cells infected with Chlamydia pneumoniae34

Cerivastatin

NR

In vitro

Increased interleukin 12

Mouse peritoneal macrophages28

Simvastatin

NR

In vitro

Interleukin-1β-stimulated non-atherosclerotic human transgenic mice. Confirmed in human hepatocytes20

Atorvastatin, simvastatin

NR

Pre

Healthy males administered lipopolysaccharide35

Simvastatin

80 mg/day

4 days pre

Lipopolysaccharide-stimulated and carrogeenan-stimulated CD1 mice33

Lovastatin, pravastatin, simvastatin

10 mg/kg

20 h, 12 h, 30 min pre

Mononuclear cells from healthy volunteers stimulated by lipopolysaccharide or inactivated Streptococcus hemolyticus36

Lovastatin, simvastatin

NR

In vitro

Mouse air-pouch model36

Lovastatin, pravastatin

10 mg/kg

20 h, 12 h, 15 min pre

Protein kinases and gene transcription factors Increased NF-IκB

Decreased NFκB

Decreased activator protein 1 nuclear binding Decreased cJUN mRNA expression Decreased hypoxia-inducible factor-1 DNA binding

Cytokines Decreased TNFα

C-reactive protein Decreased plasma C-reactive protein level

Chemokines Decreased MCP1

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Review

(Continued from previous page) Human alveolar macrophages and umbilical cord endothelial cells infected with C pneumoniae34

Cerivastatin

NR

In vitro

Healthy males administered lipopolysaccharide35

Simvastatin

80 mg/day

4 days pre

Decreased mRNA expression of MCP1, MIP1α, MIP1β, CCR1,2,4,5

Human macrophages and endothelial cells stimulated by interferon-γ and TNFα, respectively37

Simvastatin

NR

In vitro

Decreased RANTES

Lipopolysaccharide-stimulated and carrogeenan-stimulated CD1 mice33

Lovastatin, pravastatin, simvastatin

10 mg/kg

20 h, 12 h, 30 min pre

Decreased P-selectin Decreased leucocyte rolling and adhesion Decreased leucocyte transmigration

Intravital microscopy in rat mesentery stimulated by Staphylococcus aureus α-toxin38

Simvastatin

50 vs 100 μg/kg

18 h pre

Decreased monocyte CD11b expression Decreased CD11b-dependent adhesion

Isolated human monocytes and endothelial cells from hypercholesterolaemic patients39

Lovastatin, simvastatin

20–40 mg/day

6 weeks

Decreased CD11a, CD18, VLA4 surface expression

U937 monocytes; interleukin-1β-stimulated human umbilical cord endothelial cells40

Cerivastatin

1 μmol/L

In vitro

Decreased proliferation Increased differentiation Increased adhesion to human umbilical vein endothelial cells

Mono Mac 6 cells41

Lovastatin

10 μM

72 h pre

HLA-DR and CD38 activation downregulated by atorvastatin, but upregulated by simvastatin Superantigen-mediated T-cell activation inhibited by simvastatin, but enhanced by atorvastatin

T cells from healthy volunteers stimulated by staphylococcal enterotoxin B42

Simvastatin, atorvastatin

Simvastatin: 20 mg and 40 mg; atorvastatin: 40 mg

14 day pre

Decreased reactive oxygen species production through NADPH oxidase inactivation

Monocytes from 14 healthy volunteers, 14 septic and 14 non-septic intensive care unit patients43

Simvastatin

1, 5, 10, 20, 40, and 50 μM

Ex vivo

Lipopolysaccharide-stimulated male Wistar rats44

Simvastatin

10, 20, 40, 80 mg/kg

20 min pre

Lipopolysaccharide-stimulated CD1 mice30

Cerivastatin

20 mg/kg

12 and 1 h pre

Decreased inducible NOS

Murine RAW 264·7 macrophages stimulated by lipopolysaccharide and interferon-γ23

Lovastatin, atorvastatin, fluvastatin, pravastatin

0·1–30 μmol concentration

In vitro

Embryonic cardiac myoblasts stimulated by interleukin 1, TNF45

Simvastatin

NR

In vitro

Increased endothelial constitutive NOS expression

Intravital microscopy in rat mesentery stimulated by S aureas α-toxin46

Simvastatin

50 vs 100 μg/kg

18 h pre

Increased prostaglandin E2

Murine RAW 264·7 macrophages stimulated by lipopolysaccharide and interferon-γ26

Lovastatin, fluvastatin, atorvastatin, pravastatin

0·1–30 μmol concentration

In vitro

Adhesion molecules

Leucocytes

Enzymes Decreased nitric oxide production

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the most important in the context of sepsis because of their essential role in intracellular inflammatory signalling.48,49 The biosynthesis of mevalonate is finely regulated and numerous pre-transcriptional and post-transcriptional mechanisms ensure that each cell balances internal and external sources to sustain mevalonate synthesis while avoiding sterol over-accumulation.50 At least two sequential enzymes control this balance: 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and HMG-CoA reductase. The latter is among the most highly regulated enzymes in nature.50 By inhibiting HMG-CoA reductase, statins reduce the availability of farnesylpyrophosphate, geranylgeranylpyrophosphate, and cholesterol. Protein isoprenylation is therefore slowed, but not abolished, since a reduction in 360

prenylated protein concentration reduces the response magnitude of the affected signalling pathways. This is somewhat analogous to reducing the heat under a boiling kettle. In this manner, statins moderate processes involved in cholesterol handling, vascular reactivity, and inflammation.

Intracellular signalling Following transmission of a signal from the extracellular space through the cell membrane, a number of serial or parallel proteins can serve as transducers or regulators of signal propagation (figure 1). The protein kinase system activates the transcription factor nuclear factor kappa B (NFκB).51,52 NFκB most commonly exists as the two polypeptides p50 and p65, and is usually complexed in the http://infection.thelancet.com Vol 7 May 2007

Review

(Continued from previous page) Anti-oxidant Inhibition of neutrophil oxidative burst

E coli endotoxin (lipopolysaccharide) in healthy volunteers32

Simvastatin

80 mg/day

4 days pre

Decreased superoxide anion production

Whole blood from 14 healthy volunteers, 14 septic and 14 non-septic intensive care patients43

Simvastatin

1, 5, 10, 20, 40, and 50 μmol/L

Ex vivo

Human alveolar macrophages and umbilical cord endothelial cells infected with C pneumoniae34

Cerivastatin

NR

In vitro

Human endothelial and vascular smooth muscle cells21

Simvastatin, atorvastatin, lovastatin

0·1–10 μmol/L

In vitro

Human saphenous vein endothelial and smooth muscle cells stimulated by PDGF or TGF-β27

Simvastatin

NR

16 h pre

Increased endothelial constitutive-derived tissue plasminogen activator

Human saphenous vein endothelial and smooth muscle cells stimulated by PDGF or TGF-β27

Simvastatin

NR

16 h pre

Decreased tissue factor Decreased F1·2

Healthy males administered lipopolysaccharide35

Simvastatin

80 mg/day

4 days pre

Increased thrombomodulin expression and functional activity

TNFα-stimulated human umbilical cord endothelial cells and human coronary Ea.hy926 endothelial cells47

Atorvastatin

10 μmol/L

In vitro

Coagulation Decreased PAI-1

NFκB=nuclear factor kappa B. NF-IκB=inhibitor for NFκB. ERK=extracellular signal-regulated kinases. MAPK=mitogen-activated protein kinase. COX-2=cyclooxygenase-2. PPARγ=peroxisome proliferator-activated receptor. PAI-1=plasminogen activator inhibitor 1. PDGF=platelet-derived growth factor. TGF=transforming growth factor. TNF=tumour necrosis factor. MCP=monocytic chemoattractant protein. MIP=macrophage inflammatory protein. RANTES=regulated upon activation, normal T cells expressed and secreted. NOS=nitric oxide synthase. NR=not reported. *Timing of statin treatment in relation to pathogen exposure.

Table: Inflammatory signalling changes and alterations in downstream mediators

cytoplasm with its inhibitor (NF-IκB). Receptor activation causes phosphorylation of NF-IκB, leaving NFκB free to translocate to the cell nucleus. The list of NFκB activators is extensive and includes Gram-positive and Gram-negative bacteria and bacterial products, viruses and viral products, and a variety of cytokines, free radicals, and oxidants. Upon activation, NFκB translocates into the nucleus where it induces the expression of cytokines, chemokines, adhesion molecules, receptors for immune recognition, and enzymes. Neutrophil apoptosis is also counteracted and B-cell and T-cell proliferation at the infection site is promoted. The mitogen-activated protein kinase (MAPK) family is another pathway involved in transcriptional regulation.53 Three members of this family are important: (1) extracellular signal-regulated kinases (ERK; associated with Ras), (2) c-jun N-terminal/stress-activated protein kinase (JNK/ SAPK), and (3) p38. The latter two kinases are associated with Rac and Cdc42. All three kinase pathways are strongly activated by interleukin 1, tissue necrosis factor (TNF) α, lipopolysaccharide, and other cell stressors. ERK activation causes dysregulated cytokine production, whereas JNK activation has an important role in TNF and inducible nitric oxide synthase (NOS) expression by monocytes and macrophages.54,55 In addition to causing the production of inflammatory cytokines, persistent elevation of p38 appears to promote interleukin-10 production, thus inducing a Th2-mediated immunosuppressive state.56 Two further systems should be mentioned. Phosphatidyinositol-3 kinases (PI3K), and the PI3K downstream target kinase Akt (also known as protein kinase B), regulate cellular metabolism, inhibit apoptosis, and enhance the expression of inflammatory genes.57–59 http://infection.thelancet.com Vol 7 May 2007

Activation is associated with increased neutrophil accumulation at sites of infection, enhanced NFκB function, and diminished neutrophil apoptosis.59–64 Peroxisome proliferator-activated receptors (PPARs), a nuclear hormone receptor, regulate the transcription of target genes by binding to specific response elements. PPAR target genes are involved in many crucial physiological and pathological functions, such as cellular differentiation, lipid metabolism, and glucose homeostasis.65 PPARγ appears to be important in macrophage development and function. The inhibition of HMG-CoA reductase by statins influences intracellular inflammatory signalling in many ways (table; figure 1).20–29 MAPK and NFκB-associated signalling are both inhibited leading to a reduction in inflammatory mediators. Both upregulation of NF-IκB and downregulation of NFκB occur. Simvastatin also promotes interleukin-12 gene expression via a c-Fos and c-Jun-based mechanism.28 In this way, Th1 function may be improved. By contrast, a number of statins increase the induction of tyrosine kinase, ERK, and p38 in murine macrophages causing prostaglandin E2 production through increased cyclooxygenase expression.26 The clinical relevance of this is unclear since the effects of eiconasoids in sepsis are still under investigation.66 Akt is rapidly activated in vitro by pravastatin and simvastatin in human endothelial cells.25 Akt activation by simvastatin enhances endothelial constitutive NOS activity and inhibits endothelial cell apoptosis, while promoting angiogenesis. Endothelial function is thereby improved. Finally, although an association has been demonstrated, the anti-inflammatory properties of atorvastatin may not involve PPARγ.24 However, PPARγ gene expression is 361

Review

Interleukin 1, tumour necrosis factor, lipopolysaccharide Statins Mitogens, growth factors

Statins

Statins Receptor complex

Mitogen receptor complex GGPP

GEF FPP

GEF tive Inac DP G Ras-

ve Acti P GT Ras-

Statins Receptor complex

GGPP

Active Rac-GTP

GEF Inactive cdc42-GDP

GAP

Inactive Rac-GDP

Active cdc42-GTP GAP

GEF Inact ive RhoGDP

GAP

GGPP

Activ Rho- e GTP GAP

Mechanism? Raf-1

IKK MEKK1–3

ASK1

p50

p50

p65

p65

I-kB

MEK1/2

I-kB MEK4/7

MEK3/6

ERK1/2

Protein kinase C JNK MAPKAPK2/3

p38

Nucleus

Cytoplasmic kinases Transcription factors

1 c-JUN ELK-

1 SAPc-fos 6 NF-IL 1 K L E -

p53 AP-1

p50 p65

SAP-1

SP1

CHOP ATF2 ECK1

AP-1

p50 p 65 CREB ATF2

Cytokines Chemokines Adhesion molecules Acute phase proteins Enzymes

Figure 1: GTP-binding proteins and intracellular inflammatory signalling Under the influence of various transcription factors, inflammatory gene transcription leads to the production of inflammatory mediators, such as cytokines, chemokines, adhesion molecules, acute phase proteins, and enzymes. A number of cytoplasmic kinase systems, such as NFκB (p50p65), the mitogen activated protein kinase (MAPK) family, and Akt (also known as protein kinase B) serve as transducers or regulators of signal propagation. The small GTP-binding proteins (Rac, Ras, Rho, Cdc42) act as molecular on/off switches for these cytoplasmic kinases. In the active state they are bound to farnesylpyrophosphate and geranylgeranylpyrophosphate. By inhibiting HMG-CoA reductase, statins reduce the availability of farnesylpyrophosphate and geranylgeranylpyrophosphate, thereby inhibiting the ability of Rac, Ras, Rho, and Cdc42 to convert to an active state. The Akt/protein kinase B pathway is not depicted. GEF=guanine nucleotide exchange factor. GAP=GTPase-activating protein. GDP=guanosine diphosphate. FPP=farnesylpyrophosphate. GGPP=geranylgeranylpyrophosphate. IKK=inhibitor κB protein kinases.

inhibited by proinflammatory cytokines and promoted by the anti-inflammatory cytokine interleukin 4.67,68 A statininduced reduction in the intracellular pro-antiinflammatory pressure may therefore be enhanced by PPARγ. The reduction in signalling intensity affects the expression of cytokines, acute phase proteins, chemokines, 362

adhesion molecules, and enzymes, and also modulates the coagulation system and leucocyte function.

Cytokines Statins alter the expression of various cytokines. Whereas levels of TNFα, interleukin 1β, interleukin 6, and interleukin 8 are reduced, interleukin 12 is increased.22,24,28,30–34 http://infection.thelancet.com Vol 7 May 2007

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Since macrophages and endothelial cells stimulated by cytokines produce more proinflammatory cytokines, an overall reduction in these cytokine levels may decrease the systemic proinflammatory state associated with sepsis.

Endothelium Endothelium

C-reactive protein is released by hepatocytes under the influence of interleukin 6 as part of the acute phase response. C-reactive protein facilitates endothelial cell– monocyte interaction, activates complement, and induces tissue factor expression, thereby promoting thrombus formation.69–71 A strong association between C-reactive protein levels and endothelial dysfunction has been demonstrated in atherosclerosis.72,73 Although the role of C-reactive protein in sepsis is not fully understood, C-reactive protein has both proinflammatory and antiinflammatory effects in acute inflammation and appears to be both a non-specific biochemical marker of inflammation and an active immune modulator.74 Since C-reactive protein is associated with organ dysfunction and death in critically ill patients, the C-reactive protein-reducing effects of statins might be clinically important.74 Clinical trials show substantial reductions in levels of C-reactive protein after statin therapy.75 Further evidence suggests that statins inhibit C-reactive protein production induced by interleukin 6, interleukin 1β, and lipopolysaccharide.23,35,76,77

Infection

Microbicidal activity

C-reactive protein Leucocytes

Enzymes T cells Interferon-γ

Chemokines

Apoptosis

eNOS/iNOS COX

Macrophages Cytokines

Neutrophils

Adhesion molecules

NK cells T cells

Reactive oxygen species

Disruption

Platelet activation Coagulation

helium

Endot

T-cell cytokines

Hepatocytes

Inhibited fibrinolysis Increased thrombosis

C-reactive protein

Figure 2: The pleiotropic effects of statin-modified cell signalling in sepsis and inflammatory models Schematic representation of the downstream effects of statin-modified inflammatory cell signalling on the immune response to infection. Red circle denotes a demonstrated effect. Effects are mostly inhibitory, with changes at the transcriptional level leading to the reduced expression of cytokines, chemokines, C-reactive protein, and enzymes. The expression of adhesion molecules by leucocytes and endothelial cells is also reduced. By contrast, activity of endothelial NOS, thrombomodulin, and endothelial-derived tissue plasminogen activator is increased. NK cells=natural killer cells. eNOS=endothelial nitric oxide synthase. iNOS=inducible nitric oxide synthase. COX=cyclooxygenase.

Chemokines Chemokines, such as monocytic chemoattractant protein 1 (MCP1) and regulated upon activation, normal T cells expressed and secreted (RANTES) attract leucocytes to sites of infection or tissue damage. Numerous studies confirm the inhibition of chemokine expression, particularly MCP1, RANTES, and the cytokine interleukin 8, by statins.33–37 Interestingly, lower infection rates and reduced secretion of MCP1 and interleukin 8 were seen in human alveolar macrophages and human umbilical vein endothelial cells incubated with cerivastatin and infected with Chlamydia pneumoniae.34

Iron storage

Fe2+

Ferritin

_ Biliverdin reductase Bilirubin

Biliverdin

?

+ _

Oxidative stress

_

_

_

Carbon monoxide

Inflammation NADPH

O2

Haem

Adhesion molecules Statin treatment inhibits leucocyte movement by reducing P-selectin, CD11b, CD11a, CD18, and VLA4 expression.38–40 Intravital microscopy of rat mesenteric microcirculation treated with simvastatin for 18 h before exposure to Staphylococcus aureus α toxin showed a substantial inhibition of exotoxin-induced leucocyte rolling, adherence, and transmigration.46 These findings were confirmed by an in vitro model mimicking physiological flow conditions.40 Furthermore, lovastatin reduces CD11b expression on monocytes after only 72 h of treatment.39 Since CD11b also binds fibrinogen, factor X, and inactive C3b, lovastatin may have beneficial anticoagulant properties in sepsis.

+

Simvastatin

Haem oxygenase

Statins

p38 Akt

+

Increased HO-promotor activity

Nucleus

+

+

+

HO-mRNA

Figure 3: Haem oxygenase activation Haem oxygenase is involved in haem metabolism and the subsequent generation of carbon monoxide, biliverdin, bilirubin, and ferritin. Simvastatin causes haem oxygenase mRNA expression via the p38 and Akt pathways, whereas several other statins directly increase haem oxygenase promoter activity. HO=haem oxygenase. Fe²⁺=iron.

Enzymes: nitric oxide synthase During sepsis, endothelial constitutive NOS activity decreases rapidly, while a delayed increase in inducible http://infection.thelancet.com Vol 7 May 2007

NOS leads to an overproduction of nitric oxide.78 The effects of nitric oxide overexpression are numerous.79 363

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Pathological levels of inducible NOS expression and nitric oxide synthesis contribute to excessive vasodilatation, loss of systemic vascular resistance, and vascular leak. Furthermore, high doses of nitric oxide are negatively

Statins LFA1

No binding

Coagulation

ICAM1

Figure 4: Interruption of leucocyte–endothelium interaction Leucocyte function-associated antigen 1 (LFA1) is important for lymphocyte binding to endothelial cells and for lymphocyte activation. Various statins directly inhibited this endothelial interaction by attaching to LFA1, thereby blocking its binding to intercellular adhesion molecule 1 (ICAM1). The HIV-1 virus can acquire ICAM1 and consequently increase its infectivity through enhanced binding to host cells.

Acetyl CoA+Acetoacetyl CoA +HMG-CoA synthase HMG-CoA +HMG-CoA reductase

Statins

Mevalonate

Isopenterylpyrophosphate

Geranylpyrophosphate

Farnesylpyrophosphate

GTP-binding proteins

Squalene

Geranylgeranylpyrophosphate

Cholesterol

Plasma low density lipoprotein

Steroid hormones Vitamin D Bile acids Lipoproteins

Figure 5: The cholesterol biosynthesis pathways HMG-CoA reductase inhibition by statins reduces intracellular mevalonate, and consequently cholesterol, levels. Farnesylpyrophosphate and geranylgeranylpyrophosphate are also reduced. These lipids provide the subcellular binding sites for small GTP-binding proteins. CoA=coenzyme A. HMG-CoA=3-hydroxy3-methylglutaryl coenzyme A.

364

inotropic on the myocardium. Whereas physiological concentrations of endothelial constitutive NOS-derived nitric oxide preserve hepatic, splanchnic, and renal microcirculatory flow, pathological levels of nitric oxide in septic shock can induce hepatocyte damage and increase gut epithelial permeability. Finally, high levels of nitric oxide in sepsis may also exert cytotoxic effects, resulting in DNA and membrane phospholipid damage and impaired mitochondrial respiration. Various statins modulate NOS activity, and, consequently, nitric oxide levels by reducing inducible NOS expression and by maintaining or increasing endothelial constitutive NOS production.23,30,44,46 This reduction in the inducible NOS/endothelial constitutive NOS ratio may be clinically important since unselected NOS blockade is associated with increased mortality.80

During sepsis, various factors act simultaneously to produce a procoagulant state.81 First, tissue factor, the main activator of coagulation, is expressed on a number of cells, most notably macrophage and activated endothelial cell surfaces. Second, the anticoagulant regulatory systems are defective—antithrombin levels are low, and protein C becomes ineffective because of low levels and the downregulation of thrombomodulin. Third, the fibrinolytic system, despite rapid activation, is quickly inhibited by the continuous release of plasminogen activator inhibitor (PAI)-1. Lastly, platelets become activated by endotoxin or platelet activation factor. As such, platelets are crucial mediators of the cross-talk between inflammation and coagulation. Statins modulate coagulation through a number of mechanisms: (1) by blunting monocyte tissue factor expression,35 (2) by increasing the expression and functional activity of thrombomodulin while the TNFα-induced downregulation of thrombomodulin is counteracted,47 (3) by reducing PAI-1 levels,21,27 and (4) by increasing endothelial cell-derived tissue plasminogen activator.27 It is known that sepsis is associated with dysregulation of the endothelial thrombomodulin-endothelial protein C receptor pathway and that protein C activation is impaired in septic patients.82,83 By affecting thrombomodulin availability and function, statins might enhance protein C function. This may be relevant since recombinant activated protein C is the only pharmacological treatment shown thus far to reduce mortality in patients with severe sepsis.12 Taken together, these effects indicate that statins may shift the coagulation balance away from the prothrombotic state seen during sepsis.

Leucocytes Lovastatin reduces the proliferation of monocytes/ macrophages and is associated with a less pronounced increase in CD14 and CD11b expression when these cells are stimulated by lipopolysaccharide and TNF.41 Monocytic oxidant activity is also reduced by simvastatin in healthy volunteers, and in septic and non-septic intensive care http://infection.thelancet.com Vol 7 May 2007

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patients.43 Simvastatin and atorvastatin demonstrated opposing in-vivo effects on T cells in healthy volunteers.42 Whereas atorvastatin downregulated HLA-DR and the CD38 marker of activity on peripheral T cells, these molecules were upregulated by simvastatin. Also, simvastatin enhanced, whereas atorvastatin reduced, superantigen-mediated T-cell activation. These effects of simvastatin might explain the reduced mortality of staphylococcal bacteraemia associated with statin treatment.84

Anti-oxidant effects Reactive oxygen species levels are increased in septic patients and it is now clear that oxidative stress is an important factor associated with morbidity and mortality in patients with sepsis and multiorgan failure.85–87 Simvastatin inhibits neutrophil oxidative burst and the production of superoxide anions ex vivo in whole blood from healthy volunteers, and septic and non-septic intensive care patients.32,43 In vitro, simvastatin inhibits phorbol myristate acetate-induced oxygen radical production by monocytes through NADPH oxidase inactivation. Activation of this enzyme, dependent on the isoprenoid group geranylgeraniol (which derives from HMG-CoA) is largely responsible for reactive oxygen species production during sepsis.88,89 Furthermore, cerivastatin inhibits superoxide anion production by human macrophages and endothelial cells.34 The effects described so far accrue because of isoprenoidrelated changes in intracellular signalling. A number of non-isoprenoid mechanisms have also been reported.

Haem oxygenase activation Haem oxygenase is an inducible, heat shock cytoprotective protein involved in haem metabolism, and in the generation of carbon monoxide, biliverdin, bilirubin, and ferritin (figure 3). Various studies suggest that these products have potentially beneficial effects in inflammatory and oxidative stress states. For example, carbon monoxide exhibits antiinflammatory properties, protects against oxidative injury and cell death, and inhibits cell proliferation.90–94 Furthermore, whereas free iron has harmful oxidant effects, a haem oxygenase-induced increase in ferritin has cytoprotective properties.95 Finally, bilirubin may inhibit the oxidation of low-density lipoproteins.96,97 Treatment with simvastatin causes an increase in haem oxygenase in rat and human aortic smooth muscle cells (figure 3).98 These actions occur through the activation of p38 and Akt. Simvastatin and lovastatin, however, also increase haem oxygenase mRNA levels in human umbilical vein endothelial cells in a concentration and timedependent manner by directly upregulating haem oxygenase promoter activity.99 Increased expression of haem oxygenase was found to be associated with a reduction in free radical formation.99 These findings were confirmed in cultured human endothelial cells pretreated with rosuvastatin.100 Additionally, in a murine model of http://infection.thelancet.com Vol 7 May 2007

type A influenza-induced acute lung injury, statin-induced overexpression of exogenous transferred haem oxygenase provided substantial benefits in terms of anti-inflammatory effects, survival, and a reduction of inflammatory cells in the lung.101 Thus, induction of haem oxygenase by statins may provide an additional protective mechanism through the attenuation of sepsis-induced acute lung injury.

Direct interference in lymphocyte–endothelial cell interaction CD18 associates with CD11a to form the lymphocyte function-associated antigen (LFA) 1. LFA1 has a pivotal role in supporting lymphocyte adhesion to intercellular adhesion molecule (ICAM) 1, ICAM2, and ICAM3 on endothelial and other cells. LFA1 is also important for effective T-cell activation by antigen-presenting cells. Numerous statins directly block LFA1 by binding to a novel allosteric site within it.102,103 Lymphocyte–endothelium interaction and T-cell receptor expression is therefore inhibited. Interference with LFA1-mediated leucocyte adhesion may be protective during sepsis since LFAdeficient mice have improved liver sinusoidal perfusion and reduced endotoxin-induced apoptosis and enzyme markers of liver damage compared with normal mice after challenge with lipopolysaccharide and D-galactosamine.104 LFA1 inhibition may also be important in viral infections. For example, statins modify HIV-1 infectivity. Viral loads are reduced and CD4+ cell counts increase in acute infection models and chronically infected HIV-positive patients, possible via a Rho-dependent mechanism.105 It is known that ICAM1, when expressed on HIV-1, can enhance virus attachment to cell membranes several-fold by interacting with LFA1. Interestingly, lovastatin and simvastatin inhibit the viral ICAM1 and host-cell LFA1 interaction directly.106

Direct inhibition of MHC II MHC-II gene expression is highly regulated.107 This tight control directly affects T-lymphocyte action, and thus immune function. Interferon-γ induces class II transactivator (CIITA) promoter IV expression leading to MHC-II expression. It was shown recently that various statins (with atorvastatin the most effective) suppress interferon-γ-stimulated CIITA promoter IV expression in numerous human cells, including endothelial cells and monocytes/macrophages, in a dose-dependent manner.108 This inhibition is highly specific for the inducible form of MHC-II expression, and does not affect MHC class I. Statins may therefore modulate the adaptive immune system through the suppression of MHC-II expression by monocytes/macrophages.

Do these effects translate into benefit? Early data suggest benefit from statin treatment before and, importantly, after septic insult. In mice challenged with lipopolysaccharide, simvastatin pretreatment restored responsiveness to the vasopressor phenylephrine by 365

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Search strategy and selection criteria We identified data for this Review through English-language searches of the Medline, Embase, and Cochrane databases, and from references of relevant articles. Further articles were identified through searches of the extensive files of the authors. Search terms were: “statin”, “HMG coenzyme A reductase inhibitor”, “hydroxymethylglutaryl coenzyme A reductase inhibitor”, “sepsis”, “critical care”, and “intensive care”. We restricted our search to papers focusing on the effects of hydroxymethylglutaryl coenzyme A reductase inhibition on sepsis-associated inflammatory pathways in animal and human models of sepsis. No date or other restrictions were set in these searches.

reducing nitric oxide production.44 Simvastatin similarly completely re-established radial artery responsiveness to norepinephrine and acetylcholine in healthy volunteers receiving Escherichia coli endotoxin.32 Reduced lung vascular leak, myeloperoxidase activity and polymorphonuclear cell count were also demonstrated following simvastatin treatment in a murine model of acute lung injury.108 Mouse survival is improved by statin treatment. CD1 mice rendered septic with lipopolysaccharide showed improved survival (73·3% versus 26·4%; p=0·016) when pretreated with cerivastatin.30 Simvastatin-treated mice rendered septic by cecal ligation and perforation showed improved survival as a result of preservation of cardiac and haemodynamic function, while responsiveness to dobutamine was restored.109 Furthermore, mice rendered septic by cecal ligation and perforation and treated with various statins 6 h and 8 h after the septic insult demonstrated prolonged survival time.110 Although no prospective randomised human trials testing the effects of statins in sepsis exist, a number of observational studies—the largest includes nearly 70 000 patients—point to a potential benefit.13,14,16,19 It is also increasingly recognised that the postulated benefits of statins may be relevant in other high-risk patient groups. Examples include patients admitted to intensive care and those at high risk of developing organ failure caused by aggressive medical treatment.111,112 Furthermore, it was recently postulated that statin therapy may represent a valuable adjuvant treatment for the expected influenza pandemic.113,114

Conclusion We have outlined various mechanisms by which statins directly and indirectly modulate many levels of the immune and inflammatory response associated with systemic inflammatory response syndrome and sepsis. These mechanisms include effects on cell signalling with consequent changes at the transcriptional level, the induction of haem oxygenase, the direct alteration of leucocyte–endothelial cell interaction, and the reduced 366

expression of MHC II. Since statins do not target individual inflammatory mediators, but possibly reduce the overall magnitude of the systemic response, this effect may prove an important distinguishing feature modulating the host response to septic insults. This work establishes the biological plausibility needed for future trials of statins in critical illness. Conflicts of interest MT, YA, TSS, and DGH declare that they have no conflicts of interest. RSR is in receipt of research grants from AstraZeneca, Bristol-Myers Squibb, Merck, and Pfizer, and speaker’s fees from AstraZeneca, Bristol-Myers Squibb, and Merck. Acknowledgments We thank Robert F Fowler, Stephen J Brett, and Mark Griffiths for their kind help and advice in preparing the manuscript. References 1 Marshall JC. Sepsis: current status, future prospects. Curr Opin Crit Care 2004; 10: 250–64. 2 Padkin A, Goldfrad C, Brady AR, Young D, Black N, Rowan K. Epidemiology of severe sepsis occurring in the first 24 hrs in intensive care units in England, Wales, and Northern Ireland. Crit Care Med 2003; 31: 2332–38. 3 Anderson RN, Smith BL. Deaths: leading causes for 2001. Natl Vital Stat Rep 2003; 52: 1–85. 4 Brun-Buisson C, Doyon F, Carlet J, et al. Incidence, risk factors, and outcome of severe sepsis and septic shock in adults. A multicenter prospective study in intensive care units. JAMA 1995; 274: 968–74. 5 Finfer S, Bellomo R, Lipman J, French C, Dobb G, Myburgh J. Adultpopulation incidence of severe sepsis in Australian and New Zealand intensive care units. Intensive Care Med 2004; 30: 589–96. 6 Weycker D, Akhras KS, Edelsberg J, Angus DC, Oster G. Long-term mortality and medical care charges in patients with severe sepsis. Crit Care Med 2003; 31: 2316–23. 7 Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001; 29: 1303–10. 8 Almog Y. Statins, inflammation, and sepsis: hypothesis. Chest 2003; 124: 740–43. 9 Bohrer H, Qiu F, Zimmermann T, et al. Role of NFkappa-B in the mortality of sepsis. J Clin Invest 1997; 100: 972–85. 10 Sevransky JE, Shaked G, Novogrodsky A, et al. Tyrphostin AG 556 improves survival and reduces multiorgan failure in canine Escherichia coli peritonitis. J Clin Invest 1997; 99: 1966–73. 11 Natanson C, Esposito CJ, Banks SM. The sirens’ songs of confirmatory sepsis trials: selection bias and sampling error. Crit Care Med 1998; 26: 1927–31. 12 Bernard GR, Vincent JL, Laterre PF, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699–709. 13 Terblanche M, Almog Y, Rosenson RS, Smith TS, Hackam DG. Statins: panacea for sepsis? Lancet Infect Dis 2006; 6: 242–48. 14 Hackam DG, Mamdani M, Li P, Redelmeier DA. Statins and sepsis in patients with cardiovascular disease: a population-based cohort analysis. Lancet 2006; 367: 413–18. 15 Almog Y, Novack V, Eisinger M, Porath A, Novack L, Gilutz H. The effect of statin therapy on infection-related mortality in patients with atherosclerotic diseases. Crit Care Med 2007; 35: 372–78. 16 Thomsen RW, Hundborg HH, Johnsen SP, et al. Statin use and mortality within 180 days after bacteremia: a population-based cohort study. Crit Care Med 2006; 34: 1080–06. 17 Majumdar SR, McAlister FA, Eurich DT, Padwal RS, Marrie TJ. Statins and outcomes in patients admitted to hospital with community acquired pneumonia: population based prospective cohort study. BMJ 2006; 333: 999. 18 Kruger P, Fitzsimmons K, Cook D, Jones M, Nimmo G. Statin therapy is associated with fewer deaths in patients with bacteraemia. Intensive Care Med 2006; 32: 75–79. 19 Schmidt H, Hennen R, Keller A, et al. Association of statin therapy and increased survival in patients with multiple organ dysfunction syndrome. Intensive Care Med 2006; 32: 1248–51.

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