Pathogenesis of sepsis: new concepts and implications for future treatment

Severe sepsis and septic shock are important causes of death in intensive care units. Although our understanding of the pathogenesis of inflammation and sepsis has improved, until recently this has not translated into clinical benefit. Several new treatment approaches have given encouraging results. Evidence suggests that the way forward is to develop pathogen specific regimens rather than assume that one treatment fits all.

Summary points

• Bacterial cell walls, endotoxins, and exotoxins are powerful activators of innate and acquired immune responses

• Molecules expressed by pathogens interact with Toll-like receptors on immune cells, activating the immune response

• Cytokines are important in the pathogenesis of sepsis

• Susceptibility to sepsis may be due to inherited or acquired mutations of innate immune genes

• Severe sepsis and septic shock are clinical manifestations of a dysregulated immune response to invasive pathogens

• Adjunctive therapy with low dose steroids, activated protein C or early supportive care can reduce mortality from severe sepsis and septic shock

• Pathogen recognition receptors (such as Toll-like receptors) and mediators of sepsis (such as macrophage migration inhibitory factor) might be novel targets for treatment

Sources and methods

We selected articles for this review by searching Medline using the keywords sepsis, therapy, and Toll-like receptors. We concentrated on publications on the pathogenesis of sepsis and treatment of septic shock. As the number of references that could be cited was limited, we have often referenced review articles rather than original publications.

Epidemiology and importance of severe sepsis and septic shock

Severe sepsis and septic shock are life threatening complications of infections and the most common cause of death in intensive care units. However, a lack of widely accepted definitions of these complications has made it difficult to obtain accurate estimates of their frequency. A study published by the Centers for Disease Control in the United States indicated that the incidence of septicaemia had increased from 73.6 per 100 000 patients in 1979 to 175.9 per 100 000 patients in 1987.¹ Recent US and European surveys have estimated that severe sepsis accounts for 2-11% of all admissions to hospital or intensive care units.²

Although Gram negative infections were predominant in the 1960s and early 1970s, Gram positive infections have increased in the past two decades and now account for about half of cases of severe sepsis.³ Fungal infections are also increasing in many countries. Despite better supportive care, the hospital mortality from severe sepsis and septic shock (30% and over 60%, respectively) has not changed much over recent decades.

Innate immune responses to microbial products

The innate immune system is the first line of defence against infection and is activated when a pathogen crosses the host's natural defence barriers.⁴ It consists of soluble elements (the alternative and mannan-binding lectin pathways of the complement system, acute phase proteins, and cytokines) and cellular elements (monocytes, macrophages, neutrophils, dendritic cells, and natural killer cells). Innate immune responses must be tightly regulated as unbalanced inflammatory and immune reactions can result in either uncontrolled microbial growth or devastating inflammatory responses with tissue injury, vascular collapse, and multiorgan failure.

Detection of invading microorganisms is mediated by pattern recognition receptors expressed on the surface of innate immune cells (figure). Pattern recognition receptors recognise structures common to many microbial pathogens. These structures are called pathogen associated molecular patterns and include endotoxins (lipopolysaccharide), peptidoglycan, lipoteichoic acid, lipopeptides, flagellin, mannan, and viral RNA. The structures are essential for survival of the microorganisms and therefore do not undergo major mutations.

When a pathogen associated molecular pattern binds to a pattern recognition receptor, it activates several intracellular signalling pathways resulting in the activation of transcription factors (NF-κB, AP-1, Fos, Jun). The transcription factors control the expression of immune response genes and the release of numerous effector molecules, such as cytokines. Cytokines have an essential role in orchestrating the innate and acquired immune responses to an invading pathogen.⁵

Bacterial sepsis

Gram negative bacilli (mainly Escherichia coli, Klebsiella species, and Pseudomonas aeruginosa) and Gram positive cocci (mainly staphylococci and streptococci) are the commonest microbes isolated from patients with severe sepsis and septic shock.³ Fungi, mostly Candida, account for only about 5% of all cases of severe sepsis.

Gram negative sepsis

Most cases of Gram negative sepsis are caused by Enterobacteriaceae such as E coli and Klebsiella species. Pseudomonas aeruginosa is the third commonest cause. Gram negative infections usually occur in the lung, abdomen, bloodstream, or urinary tract.

Lipopolysaccharide is an important component of the outer membrane of Gram negative bacteria and has a pivotal role in inducing Gram negative sepsis.⁶ Lipopolysaccharide binding protein in host cells binds to lipopolysaccharide in the bacteria and transfers it to CD14.⁷ CD14 is a protein anchored in the outer leaflet of the plasma membrane, although it also exists as a soluble plasma protein that attaches lipopolysaccharide to CD14-negative cells, such as endothelial cells. CD14 is located in the extracellular space and therefore cannot induce cellular activation without a transmembrane signal transducing coreceptor.

A series of remarkable investigations have recently led to the identification of Toll-like receptor 4 (TLR4) as the coreceptor for lipopolysaccharide. Toll receptors were first discovered in Drosophila, where they were found to have a role in the defence of flies against fungi and Gram positive bacteria.⁸ Toll-like receptors were then identified in other species. Human Toll-like receptors, like their homologues in insects and other mammalian species, are type I transmembrane proteins with an extracellular leucine rich repeat domain and an intracellular domain homologous to the interleukin 1 receptor. Genetic studies in mice showed that mutations in the Tlr4 gene were linked to resistance to lipopolysaccharide, providing evidence that TLR4 was an essential component of the lipopolysaccharide receptor complex.⁹ MD-2, a secreted protein associated with the extracellular domain of TLR4, has also recently been shown to have an important role in responsiveness to lipopolysaccharide.¹⁰

Gram positive sepsis

Staphylococci (mainly Staph aureus and coagulase-negative staphylococci) and streptococci (Strep pyogenes, viridans streptococci, Strep pneumoniae) are the commonest causes of Gram positive sepsis. They are usually responsible for infections of skin and soft tissue, infections associated with intravascular devices, primary bloodstream infections, or respiratory infections. Gram positive organisms can cause sepsis by at least two mechanisms: by producing exotoxins that act as superantigens (see definition below) and by components of their cell walls stimulating immune cells.¹¹

Superantigens are molecules that bind to MHC class II molecules of antigen presenting cells and to Vβ chains of T cell receptors. In doing so, they activate large numbers of T cells to produce massive amounts of proinflammatory cytokines. Staphylococcal enterotoxins, toxic shock syndrome toxin-1, and streptococcal pyrogenic exotoxins are examples of bacterial superantigens.

Gram positive bacteria without exotoxins can also induce shock, probably by stimulating innate immune responses through similar mechanisms to those in Gram negative sepsis. Indeed, Toll-like receptor 2 (TLR2) has been shown to mediate cellular responses to heat killed Gram positive bacteria and their cell wall structures (peptidoglycan, lipoproteins, lipoteichoic acid, and phenol soluble modulin).¹²

Pathways to sepsis

The clinical manifestations of sepsis produced by different Gram positive and Gram negative bacteria vary. For example, the clinical pictures of streptococcal toxic shock syndrome and meningococcaemia are very different. In addition, E coli urosepsis follows a more benign course than nosocomial pneumonia due to P aeruginosa. Moreover, Gram positive and Gram negative sepsis result in different expression and release of pro-inflammatory mediators, such as the cytokine tumour necrosis factor-α.¹³ These observations suggest that there are specific host immune responses to each pathogen mediated by various sets of pathogen associated molecular patterns and pattern recognition receptors.

Toll-like receptors

Of the 10 human Toll-like receptors identified so far, seven interact with microbial motifs (table 1).⁴ For example, TLR2 binds components of the cell wall of Gram positive bacteria as well as ligands derived from other pathogens, TLR5 is the receptor for bacterial flagellin,¹⁴ and TLR9 is required for cellular activation by unmethylated CpG motifs of bacterial DNA.¹⁵ Cooperation between Toll-like receptors is necessary to respond to certain pathogens, such as Gram positive bacteria and yeast (zymosan).¹⁶ Several signal transducing pathways are activated after microbial ligands bind to Toll-like receptors (figure).¹⁷

Table 1.

 Ligands for human Toll-like receptors and their sources

Receptors	Ligands	Source of ligand
TLR1-TLR2	Soluble factors released by live bacteria	Neisseria meningitidis
TLR2-TLRX	Lipoproteins	Several bacterial species
	Lipoteichoic acid	Staphylococcus aureus
	Lipoarabinomannan	Mycobacteria
	Phosphatidylinositol dimannoside	Staph aureus
	Glycosylphosphatidylinositol anchors	Trypanosoma cruzi
	Endotoxin (LPS)	Leptospira interrogans, Porphyromonas gingivalis
TLR2-TLR6	Peptidoglycan	Gram positive bacteria
	Soluble phenol modulin	Staph aureus
	Zymosan	Yeasts
	Macrophage activating lipopeptide-2 (MALP-2)	Mycoplasma fermentans
TLR3	Double-stranded RNA	Virus
TLR4	Endotoxin (lipopolysaccharide)	Gram negative bacteria
	Taxol	Plants
TLR5	Flagellin	Flagellated bacteria
TLR7	Imidazoquinoline antiviral compounds (imiquimod and R-848)	Chemical compounds
TLR9	Unmethylated GpG DNA	Bacteria

The fact that different microbial products bind to different Toll-like receptors, the existence of receptor specific signalling pathways, and the idea of differential expression of Toll-like receptors by tissues and organs strongly suggest that the innate immune system is tailored in a pathogen and tissue specific manner. Expression of immune genes and host responses to infections will vary depending on the structural and biochemical composition of the invading pathogen. If confirmed, these hypotheses point to the need to develop pathogen specific approaches to treatment.

Other soluble and membrane associated proteins have recently been shown to be involved in recognising bacteria or microbial products. These include peptidoglycan recognition proteins and triggering receptor expressed on myeloid cells (TREM-1). Additional information on these proteins is available on bmj.com

Adjunctive therapies for sepsis

Numerous adjunctive treatments (that is, other than antibiotics and supportive care) for severe sepsis and septic shock have been tested in clinical trials (table 2). These include neutralisation of microbial toxins such as lipopolysaccharide, non-specific anti-inflammatory and immunosuppressive drugs, neutralisation of pro-inflammatory cytokines, and correction of abnormalities in coagulation. The results have been mixed,¹⁸ although several recent clinical trials have given encouraging results.

Table 2.

 Selected antibacterial, anti-inflammatory, and immunomodulating adjunctive therapies investigated in patients with severe sepsis and septic shock

Type of therapy	Target (s)	Agents
Neutralisation of microbial toxins	Endotoxin	Anti-endotoxin antibodies, anti-lipid A antibodies, lipopolysaccharide analogues, lipopolysaccharide removal
Non-specific anti-inflammatory and immunomodulating drugs	Multiple inflammatory and immune mediators	High dose corticosteroids, low dose corticosteroids, pentoxifylline, immunoglobulins, interferon gamma
Inhibition of specific mediators	Pro-inflammatory cytokines:
	Tumour necrosis factor	Anti-tumour necrosis factor antibodies, soluble tumour necrosis factor receptors
	Interleukin-1	Interleukin-1 receptor antagonist
	Phospholipid components:
	Phospholipase A2	Phospholipase A2 inhibitor
	Cyclo-oxygenase	Ibuprofen
	Thromboxane	Dazoxiben, ketoconazole
	Platelet activating factor	Platelet activating factor antagonists, platelet activating factor acetylhydrolase
	Oxygen free radicals	N-acetylcysteine, selenium
	Nitric oxide	N-methyl-l-arginine
	Bradykinin	Bradykinin antagonist
Correction of coagulopathy	Coagulation cascade	Antithrombin III, tissue factor pathway inhibitor, activated protein C
Other		Prostaglandin E1, granulocyte colony stimulation factor

Coagulation abnormalities, especially disseminated intravascular coagulation, are common in patients with sepsis and microvascular thrombosis. The ensuing tissue damage may have an important role in the pathophysiology of organ dysfunction. Treatment with activated protein C, a protein that has anti-thrombotic, pro-fibrinolytic, and anti-inflammatory effects, reduces mortality from severe sepsis at the price of a slight increase in bleeding events.¹⁹

Glucocorticoids exert broad metabolic and immunomodulating effects and have been used to treat several inflammatory diseases. Although high doses of steroids have no clinical benefit,¹⁸ a recent multicentre trial found that a seven day course of low doses of hydrocortisone and fludrocortisone reduced mortality in patients with septic shock and relative adrenal insufficiency.²⁰ Finally, two studies of supportive care, one focusing on early therapy with fluids, vasopressors, and transfusions and the other on meticulous control of glycaemia with insulin, have shown reduced mortality in patients with severe sepsis and septic shock.²¹,²²

Future treatment strategies

Microbial drugs and pattern recognition molecules

Designing new drugs to neutralise microbial products or block their interaction with specific receptor on immune cells is an attractive concept. Potential targets include lipolysaccharide binding protein, CD14, TLR4, and MD-2 for Gram negative sepsis, and CD14, TLR2, and TLR6 for Gram positive sepsis. Monoclonal antibodies against CD14 are being evaluated in phase II studies. Several intracellular signalling molecules, such as MyD88 and the mitogen-activated protein kinase are other possible therapeutic targets. However, inactivating molecules that are pivotal to innate immunity can be harmful, as shown by the increased sensitivity to bacterial sepsis in mice with mutations of the Tlr4 gene.²³ Careful selection of patients with severe infections associated with a high probability of death will therefore be essential.

Macrophage migration inhibitory factor

Macrophage migration inhibitory factor is a cytokine that has recently been shown to be important in innate immunity and sepsis.²⁴ It is constitutively expressed in large amounts by immune, endocrine, and epithelial cells and is rapidly released after exposure to microbial products and pro-inflammatory cytokines. Macrophage migration inhibitory factor regulates innate immune responses to endotoxin and Gram negative bacteria by modulating the expression of TLR4, enabling macrophages and other cells at the front line of defences to respond quickly.²⁵ High levels of macrophage migration inhibitory factor have been detected in patients with inflammatory and infectious diseases, including severe sepsis and septic shock.²⁶

Immunoneutralisation of macrophage migration inhibitory factor or deletion of the Mif gene protects mice against lethal endotoxaemia, Gram positive toxic shock syndromes, and experimental bacterial peritonitis. Conversely, mice injected with macrophage migration inhibitory factor together with live bacteria or microbial toxins have increased death rates.^26–28 This factor thus has the potential to endanger life when expressed in excess during sepsis. Development of drugs to block the production of macrophage migration inhibitory factor or inhibit its function may help treat severe sepsis and other inflammatory diseases.

High mobility group protein 1

High mobility group protein 1, a protein previously known as DNA binding protein regulating gene transcription and stabilising nucleosome formation, has recently been described as a “late” mediator of inflammation and sepsis.²⁹ Patients with septic or haemorrhagic shock have raised serum concentrations of high mobility group protein 1, and concentrations are associated with patient's outcome. Use of polyclonal antibodies to block high mobility group protein 1 in mice protects them against lipopolysaccharide induced acute lung injury and lethal endotoxaemia.³⁰

Genetic studies of susceptibility to sepsis

Several gene polymorphisms have been associated with increased susceptibility to sepsis (see bmj.com for more information). Testing for polymorphisms of important genes may help to identify people who are at increased risk of sepsis when exposed to virulent bacteria and who may benefit from targeted immunomodulatory therapies.

Figure.

Interaction between bacterial products and pattern recognition receptors expressed on immune cells. Components of bacterial cell walls (such as lipopolysaccharide, peptidoglycan, lipoteichoic acid, flagellin, and unmethylated CpG DNA sequences) interact with specific Toll-like receptors (TLR) expressed on immune cells. The receptors then activate intracellular signalling pathways and transcription factors resulting in expression of the gene for immune response