SIRS and group-B streptococcal sepsis in newborns: Pathogenesis and perspectives in adjunctive therapy

Seminars in Fetal & Neonatal Medicine (2006) 11, 333e342

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / s i n y

SIRS and group-B streptococcal sepsis in newborns: Pathogenesis and perspectives in adjunctive therapy Philipp Henneke*, Reinhard Berner Zentrum fu¨r Kinderheilkunde und Jugendmedizin, Albert-Ludwigs Universita¨t Freiburg, Mathildenstr. 1, 79106 Freiburg, Germany

KEYWORDS Neonatal sepsis; Group B Streptococcus; Toll-like receptors

Summary Clinical signs of systemic inflammation and suspected systemic infection are common in neonatal medicine. Yet, causative infectious organisms can only infrequently be isolated. In previously healthy infants at low risk of sepsis, group B streptococcus (GBS) is the most common isolate. In vitro and in vivo data suggest that immune cells from newborn infants have impaired antimicrobial properties against GBS. In contrast large amounts of inflammatory mediators are formed upon GBS challenge and Toll-like receptors (TLR) are critical host molecules in this context. Thus, the immune balance tilts towards inflammation, SIRS and sepsis. Adjunctive therapy of neonatal sepsis needs to adjust the inflammatory response without further impairing bacterial clearance. This article summarises the pathophysiological events leading to sepsis and suggests molecular targets for adjunctive therapy. ª 2006 Elsevier Ltd. All rights reserved.

Innate immunity and the systemic inflammatory response syndrome Any living organism, however simple or complex, is constantly challenged by contact with environmental microorganisms. Moreover, in mammals a symbiotic coexistence with microorganisms is part of the survival strategy. For example, the average human body consists of about 1013 cells, but contains 10 times as many microorganisms (about * Corresponding author. Tel.: C49 761 270 4300; fax: C49 761 270 4454. E-mail address: [email protected] (P. Henneke).

1014) in the gut. The innate immune system, which comprises monocytes, granulocytes, macrophages and the complement system, carries the major burden of containing microorganisms at the mucosal sites and instructing an inflammatory response upon invasion by microorganisms.1 Since basic components of innate immunity are shared between different kingdoms such as plants, insects and animals, it is now generally accepted that the evolution of innate immunity was a prerequisite for evolution altogether. In the healthy host, a targeted, local immune response destroys invasive bacteria without measurable systemic inflammatory activity in the blood.2 Hence, local immunity is per se non-phlogistic. If, however, local immunity does not succeed in eliminating the invading bacteria, systemic inflammation will ensue.3

1744-165X/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.siny.2006.03.003

334 Since generalised inflammation manifests as a rather non-specific state (fever, respiratory and cardiovascular impairment, changes in leukocyte count), the term systemic inflammatory response syndrome (SIRS) was coined to cover a clinical state that results from stressors such as trauma, burn or infection.4 If the SIRS criteria are met and clinical signs of infection are observed, the diagnosis of sepsis can be made. The degree of compromise of the cardiovascular function determines the severity of sepsis, which may proceed to severe sepsis and finally septic shock (Table 1). The strength of this consensus is the precise definition of SIRS, including clear-cut limits for body temperature, heart rate, respiratory rate and white blood cell count. Since these criteria are age-dependent, diagnostic criteria for children with SIRS were suggested in 1994.5 Finally, a broad consensus on the particular definitions of SIRS, infection and sepsis in children has been agreed upon.6 Table 2 summarises the age-specific clinical variables that define SIRS in the first year of life. Even though this consensus now provides a valuable tool for the paediatrician to define neonatal SIRS and sepsis, some inherent problems remain. Birth and perinatal cardiovascular adaptation are major stressors. Accordingly, many newborns, in particular preterm infants, develop SIRS. Due to difficulties in specimen collection, such as small blood volumes available, the true incidence of sepsis among infants with SIRS can only be estimated. Some authors calculate that the true incidence of early-onset sepsis is fivefold higher than the recorded sepsis incidence in the same patient population.7 Furthermore, fever, an almost inevitable prerequisite of sepsis in older children, is a notoriously poor parameter in newborns due to immature thermoregulation. Due to these uncertainties the vast majority of newborn infants with SIRS suffers from ‘suspected or proven infection’ (Table 1) and therefore fulfills the criteria for sepsis. Since failure to immediately treat a septic newborn might have devastating consequences for the entire life to come, too many infants are treated with antibiotics. As an example, almost 50% of very preterm infants receive antibiotic therapy immediately postnatally, but bacteria can be cultured from only 2% of these infants.8 Accordingly, SIRS and suspected sepsis are very common entities in newborn infants. In this article, we summarise the important molecular events within the host-pathogen interaction and the innate immune response that render newborn infants particularly susceptible to systemic inflammation.

P. Henneke, R. Berner Table 1 Definitions of SIRS, sepsis, severe sepsis and septic shock in children6,70 SIRS

2 of the following criteria, one of which must be abnormal temperature or leukocyte count 1) Core temperature of >38.5  C or <36  C 2) Tachycardia: Mean heart rate >2 SD above normal for age in the absence of external stimulus, chronic drugs, or painful stimuli; or unexplained persistent elevation over a 0.5- to 4-h time period OR for children <1 year old Bradycardia: mean heart rate <10th percentile for age in the absence of external vagal stimulus, b-blocker drugs, or congenital heart disease; or otherwise unexplained persistent depression over a 0.5-h time period 3) Tachypnoea: mean respiratory rate >2 SD above normal for age or mechanical ventilation for acute process not related to underlying neuromuscular disease or general anaesthesia 4) Leukocyte count: elevated or depressed for age (not secondary to chemotherapy-induced leukopenia) or >10% immature neutrophils

Sepsis

SIRS plus signs of infection

Severe sepsis (1 symptom)

Sepsis plus a. Impaired consciousness b. Oliguria c. Lactate [ d. Hypoxemia e. Arterial hypotension (reversible <60 min)

Septic shock

Severe sepsis plus catecholamines are required for cardiovascular resuscitation

Multi-organ failure

Altered organ function in an acutely ill patient: homeostasis that cannot be maintained without intervention with or without SIRS

SIRS, systemic inflammatory response syndrome.

Neonatal sepsis For newborn infants, their innate immune system is their most important armour. First, newborn infants are in transition from the sterile intrauterine environment to a coexistence with microorganisms, which probably facilitates expansion and invasion of potentially pathogenic bacteria. Second, the capacity of the adaptive immune response in newborn and particularly preterm infants is determined largely by the placental transfer of maternal antibodies, since neonatal antibody formation is significantly impaired as a result of decreased IgG synthesis and

constraints in the VH gene repertoire.9 A significant transplacental transfer of maternal antibodies begins in the third trimester of pregnancy. Thus preterm infants are not protected by sufficient amounts of specific antibodies. Accordingly, the success or failure of neonatal immunity to eliminate bacteria that cross the mucosal epithelial lining and to prevent sepsis depends on the competence of the newborn’s innate immune system. Neonatal sepsis comprises early-onset sepsis (EOS) and late-onset sepsis (LOS). EOS develops within the first 6 days of life. However, more than 90% of EOS manifests within the

Neonatal SIRS and Sepsis Table 2

335

SIRS: Age-specific vital signs and laboratory values in the first year of life6

Age

Heart rate (beats/min)

Respiratory rate (breaths/min)

Leukocyte count (103/mm3)

Systolic blood pressure (mmHg)

0e7 day 7e28 day 2e12 months

<100 or >180 <100 or >180 <90 or >180

>50 >40 >34

>34 >19.5 or <5 >17.5 or <5

<65 <75 <100

SIRS, systemic inflammatory response syndrome.

first 48 h of life. Accordingly, some authors limit EOS to the first 72 h after birth.10 EOS may present in all the forms listed in Table 1, from SIRS developing several days after birth to irreversible septic shock at birth. High awareness for a clinical entity that initially presents with minor nonspecific signs such as temperature instability, but might progress within hours to a life threatening disease, has decreased the lethality of EOS by over 80%.11 LOS develops between the second postnatal week and the end of the third month of life. Similar to EOS, LOS is feared for its dramatic development within hours. In many cases, nonspecific gastrointestinal symptoms are early warning signs. About 30 years ago, group B Streptococcus (GBS) emerged as the dominant pathogen of neonatal sepsis and continues to be the single most important cause of earlyneonatal sepsis in newborn infants despite the recent implementation of intra-partum antibiotic prophylaxis.12e14 GBS is a common coloniser of the intestinal tract in 15% of normal adults and of the cervical mucosa in about 20% of healthy pregnant women. GBS is found at the dermal and mucosal surfaces in approximately half of the infants born to colonised women. One to two percent of GBS-colonised newborns will eventually develop sepsis due to this organism.12,13 Despite striking advances in neonatal medicine, GBS sepsis still carries a mortality of 5e10%.12,13 Beyond sepsis, GBS causes bacterial meningitis in both EOS and LOS, however it is relatively more common in LOS (about 40% of cases),15 with one GBS serotype (III) causing over 80% of cases.13,15 Accordingly, GBS is the third most frequent cause of bacterial meningitis in countries with a routine immunisation program against Haemophilus influenzae b.12 Around 50% of all infants with GBS meningitis exhibit a varying degree of long term neurodevelopmental sequelae.13 Despite the persistently important role of GBS in neonatal sepsis, Escherichia coli has outnumbered GBS as the leading cause of EOS in very preterm infants in some regions of the world. This development is probably due, in part, to the increasing usage of perinatal antibiotic prophylaxis, which reduces the incidence of GBS sepsis.12 Furthermore, nosocomial infections are increasing in newborn infants and affect over 20% of very low birth weight infants at least once per hospital stay.16 Coagulase-negative staphylococci are currently the most common cause of late onset nosocomial sepsis in very preterm infants.16 This probably results from the combination of increased prematurity and longer hospital stays with devices such as percutaneous catheters paving the way for infections with bacteria from the skin flora.16,17 Despite these important developments in microbial epidemiology, GBS remains the paradigmatic organism in both EOS and LOS for several reasons. First, GBS has the

capacity to affect healthy newborn infants. Second, the clinical course of GBS sepsis and meningitis is dramatic with a high morbidity and mortality due to an excessive inflammatory response. Thus, the following sections predominantly explore the virulence of GBS and the innate neonatal immune mechanisms targeting this organism.

Group B Streptococcus: virulence and specific neonatal immunity Neonatal exposure to GBS during birth is very common and results in the colonisation of one in 10 infants. Most infants appear to successfully control GBS at the dermal and mucosal surfaces. Translocation of bacteria across mucosal surfaces can be assumed to occur frequently, usually leaving the immunological homeostasis of the host undisturbed.3 In most cases of GBS sepsis, newborn infants aspirate GBS that colonises the birth canal. GBS infections in infants delivered by caesarian section with membranes intact rarely occur. However, newborns with GBS sepsis have excessively high cord blood levels of proinflammatory cytokines, indicating that the infection and inflammatory process started prior to birth. In the neonatal lung, GBS can proliferate to enormous densities, as demonstrated in newborn primates with neonatal GBS pneumonia (109e1011 colony-forming units (CFU)/g lung tissue).18 Notably, mice with a targeted deletion of the surfactant protein (SP)-A gene exhibit decreased phagocytosis and bacterial killing of alveolar macrophages upon intratracheal instillation of GBS.19 In a baboon model, concentrations of SP-A increase with gestational age and are inversely correlated to the risk of lung infection in newborn infants.20 Hence low concentrations of surfactant might promote GBS multiplication in the lung. Newborn mice are exquisitely sensitive to GBS dissemination. Thirty GBS organisms injected subcutaneously represent the LD50 in newborn mice, while a similarly effective dose is approximately 1 000 000-fold higher in adult mice of the same strain.2 Hence, host factors that underlie postnatal maturation and directly influence the elimination of GBS from tissue are likely to contribute significantly to GBS sepsis in newborns. For effective infection, the following sequence of events is critical. First, GBS has to adhere to the pulmonary epithelium of the lung and to invade the epithelial cells in order to reach the subepithelial space. Establishment of a pulmonary GBS infection requires evasion of local host immune mechanisms such as directly antibacterial activities by epithelial cells and resident phagocytes. Next, GBS has to disseminate via the bloodstream without being

336 eliminated by circulating monocytes. Finally, GBS will trigger the potentially detrimental systemic inflammatory response. Accordingly, first line immune mechanisms have to eliminate microbial structures before a significant local or even systemic inflammatory reaction develops. The weakly- to non-phlogistic sequence of events has to be accomplished by phagocytes that exhibit directed cellular movements (chemotaxis), engulfment (phagocytosis) and finally destruction of the microorganism. In the following sections, we will expand on each of the above sequential events.

GBS attachment and invasion GBS has adapted particularly well to its ecological niche. GBS adheres best at low pH values as is found at the vaginal epithelial mucosa.21 Molecules that mediate interactions at the interphase between bacteria and epithelial cells are lipoteichoic acid (LTA) and the surface proteins Rib, C5a peptidase and laminin-binding protein on the side of GBS, and the extracellular host components fibronectin, fibrinogen and laminin, which interact with integrins expressed on epithelial cells. Subsequent to attachment, GBS has to translocate across the epithelial barrier to invade the host. The endocytotic uptake and entry into epithelial cells involves, among others, the GBS surface proteins alpha C protein, C5a peptidase and the host factors fibronectin and phosphatidyl-inositol 3-kinase. Cellular invasion appears to be significantly associated with the virulence of the organism, since GBS isolates from infants with bloodstream infections exceed colonising strains with respect to their ability to invade epithelial cells and to trigger inflammatory cytokines in phagocytes.22

Chemotaxis of neonatal phagocytes Chemotaxis in itself is a stunning example of a complex immune process that requires a high degree of coordination on a single cell level. First, a phagocyte needs to adhere to matrix in order to gain traction. Then, a leading edge comprising so-called lamellopodia of polymerised actin and disassembled myosin filaments has to be established. Simultaneously, the cell has to pull up its rear end in a myosin II-dependent process that requires the breaking of cell-matrix contacts. On the global level, cell movement directed towards inflammatory stimuli was shown to be impaired in both preterm and term neonates, with neutrophils from newborn infants migrating at about half the speed of adult cells.23 Moreover, the total mass of neutrophils, which constitutes the most critical source of chemotaxis, is decreased in newborn infants and small numbers of circulating granulocytes are a hallmark of neonatal sepsis.24 Accordingly, adjunctive sepsis therapy with granulocyte colony stimulating factor (G-CSF) has been attempted. However, while application of G-CSF does increase the total neutrophil count in preterm infants, it appears to be without effect on the outcome of neonatal sepsis.24 Apparently, granulocyte dysfunction is due to several distinct deficiencies at the cellular level in preterm and normal term infants (although the deficiencies are less obvious in the latter). Rolling adhesion to activated endothelium and transmigration through endothelial monolayers are

P. Henneke, R. Berner decreased in cells from newborn infants compared with those from adults.25 This might be due, in part, to an impaired expression of the adhesion molecule L-selectin in newborns and particularly in those born preterm. L-Selectin controls neutrophil rolling along the endothelium as an early step in the recruitment of neutrophils to sites of bacterial invasion and localised inflammation. Furthermore, formation of lamellopodia and directed movements towards a stimulus are impaired in neonatal cells.25 Neonatal neutrophils exhibit an imperfect bipolar shape change due to an impaired ability to rapidly polymerase monomeric G-actin to form microtubules. Notably, neutrophil chemotaxis undergoes quite rapid maturation in newborn infants resulting in near normal values 10 days postnatally, although this process takes longer in very preterm infants (for a review, see Carr, 2000).24 It is tempting to speculate that a deficiency in chemotaxis contributes to the development of EOS. With respect to GBS it has been shown that this organism induces chemokines such as interleukin (IL)8 and leukotriene B4 together with complement factors, in particular C3b, C3d,g and C5a, which, in turn, indirectly induce chemotaxis by neutrophils.26,27 It seems important that preterm infants have very low levels of circulating native complement proteins. However, they generate remarkable amounts of activated complement products such as C3a and -b during sepsis.28 Whereas GBS clearly induces complement and chemokines that in turn promote chemotaxis, a GBS substructure and a phagocyte receptor that directly induce chemotaxis to GBS have not been described.

Phagocytosis by neonatal phagocytes Chemotaxis, engulfment and digestion of bacteria are interdigitating processes accomplished by the cellular actin motor. Chemotaxis enables the phagocyte to establish a direct interphase with the microorganism. Phagocytosis, the binding and ingestion of particles such as GBS, includes recognition by cell surface receptors, the subsequent formation of actin-rich membrane extensions around the particle, phagosome formation and phagosome maturation into a phagolysosome. Recognition of bacteria as foreign material can be indirect via opsonisation with antibodies or complement components. Accordingly, low antibody and complement concentrations in neonatal serum might negatively affect both the chemotaxis and phagocytosis in newborn infants. Notably, the potent chemotactic complement component C5a is targeted by a specific GBS serine protease, the C5a peptidase, which enables GBS immune evasion by inhibition of chemotaxis and phagocytosis.29 Furthermore, the GBS C protein b-antigen increases binding of the complement factor H that inhibits the alternative pathway.30 Opsonised particles are recognised by phagocytes primarily via (1) the b2-integrin CD11b/CD18 and (2) two classes of Fcg receptors (FcgRII and III). Whereas opsonisation with complement is critical for CD11b/CD18mediated phagocytosis, specific antibodies do not seem to be absolutely necessary in this context.31,32 Notably, both resting and stimulated phagocytes of preterm infants, but not term infants, exhibit an impaired surface expression of CD11b/CD18.33 The clinical significance of low CD11b expression remains to be elucidated. Similar to CD11b,

Neonatal SIRS and Sepsis

TLR2/6

GBS

Inflammatory response to GBS It is now generally accepted that TLRs are the key phagocyte receptors. TLRs sense traces of microbial substructures and instruct the cellular host response. In humans, 10 distinct TLRs are expressed ubiquitously in many tissues, albeit in varying compositions. TLRs sense and discriminate spurious amounts of microbial substructures, such as lipoproteins from Gram-negative bacteria, spirochetes and Mycoplasmataceae (TLR2), lipopolysaccharide (LPS) from Gram-negative bacteria (TLR4), flagellin (TLR5) and hypomethylated bacterial DNA (TLR9).1 TLRs contain an intracellular domain with homology to the IL-1-receptor, the ‘Toll-/Interleukin-1-receptor-resistance (TIR)-Domain.’ Upon TLR activation, several intracellular proteins (MAL/TIRAP, TRIF, TRAM, TOLLIP) that partially contain TIR domains themselves, form an intracellular

TLRX MyD88 p38

Intracellular killing of GBS Cord blood granulocytes and monocytes are severely impaired in their capability for killing intracellular GBS, but not for E. coli and Streptococcus pyogenes as compared to adult cells.34 In general, bacterial killing can be categorised into oxygen-dependent and oxygen-independent mechanisms, with the latter comprising enzymatic (e.g. lysozyme, cathepsin G, elastase, 14 kDa phospholipase A2) and non-enzymatic, mostly membrane active mechanisms (defensins, cathelicidins, lactoferrin, bactericidal/permeability-increasing protein).35 Unfortunately, little is known about the antibacterial killing properties of neonatal phagocytes by enzymatic and oxidative mechanisms. In various animal models of invasive GBS infections, oxygen radical species are critical for killing of GBS,36 which is counteracted by the manganese-dependent superoxide dismutase (SodA) of GBS.37 Furthermore, interaction of antibody opsonised GBS with Fc receptors on phagocytes mediates intracellular killing of GBS via oxygen radicals.38 In neutrophils from very preterm infants the intracellular oxidative burst in response to opsonised GBS was found to be markedly reduced compared with cells from adults.39 In the absence of specific antibodies, GBS cell walls induce oxygen radicals via a distinct pathway that involves the Toll-like receptor (TLR) adapter protein myeloid primary response protein (MyD) 88, but not nuclear factor (NF)kB.32 Furthermore, the mitogen-activated protein kinase p38 is crucial in this context (Fig. 1).40 Notably, costimulation of alveolar macrophages by granulocyte-macrophage colony stimulating factor (GM-CSF) and GBS is a prerequisite for proper antibacterial superoxide and hydrogen peroxide formation by mouse alveolar macrophages and GBS clearance from the lung, as evidenced in GM-CSFÿ/ÿ mice.41 Moreover, these mice exhibited an increased cytokine response by alveolar macrophages.41

GBS

CD11b/CD18 CD14

FcgRIII, which is involved in neutrophil granule exocytosis, is expressed in lower densities on neutrophils from preterm neonates compared with cells from term neonates or adults.24 Moreover, Fc receptor mediated phagocytosis is likely to be further impaired in preterm infants as it relies on the synthesis of specific immunoglobulins that are lacking in these patients.

337

JNK

H2O2 NF-κB, AP-1 EGR-1 nucleus

Figure 1 Schematic view of intracellular killing of group B Streptococcus (GBS). TLR, Toll-like receptor; JNK, c-Jun kinase; MyD88, myeloid primary response protein; p38, mitogenactivated protein kinase; NF-kB, Nuclear factor-kappa B; AP-1, Activator-protein-1; EGR-1, early growth response factor-1.

adapter.1 This adapter multimer and downstream kinases, such as the four IL-1 receptor associated kinases (IRAKs), direct the signalling cascade and discretely modulate cellular activation by mechanisms that still await full elucidation.42 Specific data on TLR expression and function in newborn infants and other fetal or newborn mammals are very limited. Expression of TLR4 and MyD88 on human peripheral phagocytes increases from fetal through postnatal life and correlates with tumor necrosis factor (TNF) formation.43,44 However, the biological significance of age-dependent differences in TLR4 expression are unclear, since low TLR4 copy numbers are sufficient for a full LPS response.45 However, neonatal immune cells exert a different TLRrestricted response compared with adult cells in some instances. Most, but not all, studies found that cord blood mononuclear cells from newborn infants exhibited an inferior inflammatory cytokine response, particularly concerning TNF, compared with cells from normal adults upon ligation of some purified TLR-ligands (TLRs 2, 4, 7).22,44,46 The reduced inflammatory response might be explained in part by excessive anti-inflammatory activity in neonatal plasma that can be transferred to adult monocytes.44,46 IL-10 constitutes an attractive candidate for this activity, since the reduced ability of neonatal phagocytes to mount a pro-inflammatory cytokine response is partially a consequence of an exaggerated IL-10 formation and neonatal IL 10ÿ/ÿ mouse macrophages secrete ‘adult concentrations’ of IL-6 and TNF. This observation corresponds to the notion that a low inflammatory capability of the fetus is a prerequisite for the child to survive as a graft in the mother’s womb. In concordance with this, a reduced inflammatory cytokine response of neonatal leukocytes to various microbial stimuli has been suggested to render infants susceptible to infection. However, several observations in both septic newborns and in vitro are in conflict with the concept of the newborn infant as a globally immunodeficient or tolerant human being.

338 First, newborn infants with GBS sepsis suffer from all the consequences of hyper-inflammation and levels of inflammatory cytokines such as IL-1b and IL-8 are exceptionally high in septic newborn infants with a trend to even higher values in very immature infants.47 Second, in contrast to isolated microbial structures such as LPS, GBS stimulates neonatal monocytes at least as potently as adult cells.48 If the magnitude of the cytokine response correlated negatively with the susceptibility to sepsis, as the hypothesis on the ‘immunodeficient newborn’ suggests, newborn infants would be resistant to GBS. Since the cytokine response to GBS, but not isolated substructures, is preserved in neonatal phagocytes, it seems essential to understand the basic mechanisms of GBS recognition and the subsequent immune response in order to prepare a custom-tailored anti-GBS strategy.

Inflammatory substructures of the GBS cell wall GBS expresses at least two, and most probably more, molecular patterns that result in inflammatory activation of phagocytes. First, direct interaction of macrophages and monocytes with cell wall material from inactivated GBS elicits a powerful inflammatory stimulus that exceeds the response to similar preparations of virulent S. pneumoniae by at least 100-fold.32 Second, GBS releases an inflammatory factor during growth that is functionally clearly distinct from the cell wall-mediated activity.49 Surprisingly, inactivated GBS organisms were found not to significantly activate TLR2 or other candidate TLRs (1, 2, 4, 6, and 9) that were identified as receptors for other Gram-positive bacteria. However, since GBS-mediated inflammatory signalling was entirely dependent on the common TLR adapter protein MyD88, a TLR beyond the TLRs that have been connected to stimulation by other Gram-positive bacteria seems to be involved in this process.32 Downstream from MyD88, the mitogen activated protein (MAP) kinases p38 and c-Jun, the transcription factors NF-kB and c-Jun/activator protein-1 (Fig. 1),40 as well as phosphatidyl inositol 3-kinase, Ets-like kinase1 (ELK-1), early growth response factor-1 (EGR-1) and the c-AMP response element are critical signalling intermediates in the cytokine response to GBS (see Fig. 1; 32,40,50; S. Kenzel & P. Henneke, unpub. obs.). Accordingly, significant evidence in vitro and in vivo supports the notion that GBS is recognised by the TLR system, although the specific cognate TLR has yet to be identified. What are the molecular entities in the GBS cell wall that potently activate phagocytes? The backbone of the GBS cell wall is a macromolecular network of LTA, peptidoglycan (PGN) with cross-linked surface proteins and covalently linked type- and group-specific capsular polysaccharides. LTA resembles the potent Gram-negative endotoxin LPS in some respects. Both are membrane-anchored via glycolipids and their polysaccharide chains extend into the PGN layer of the cell wall. However, whereas TLR4 is the cognate receptor for LPS, LTA is recognised by TLR2.32 TLR6 is an essential co-receptor for LTA from GBS and Saphylococcus aureus,32 although TLR1 has been suggested by other authors.51 Notably, GBS LTA is of low inflammatory potency and the interaction of TLR2 with GBS LTA appears to be non-essential for the recognition of the GBS cell wall.50

P. Henneke, R. Berner Although not covalently linked, LTA is in closest proximity to PGN. PGN consists of repeating disaccharide units and is further organised as a macromolecule via cross-linking of short attached peptides (‘wall peptides’) with other peptides attached to neighbouring glycan strands. PGN needs to be locally digested by endogenous murein hydrolases in order to permit polar growth of the bacterial chain. It is tempting to speculate that PGN exposed at sites of enzymatic cleavage interacts with pattern recognition receptors such as TLRs. In accordance with this, PGN from S. aureus has been shown to activate immune cells via the cell wall anchored glycoprotein CD14 and TLR2.52 Despite these intriguing data, several questions about the immunogenicity of PGN exist. Additional purification of PGN from S. aureus that removed lipid contaminants abrogated TLR2 activity, but not interaction with a novel intracellular receptor (NOD2). Interestingly, the only highly purified streptococcal PGN preparation that was examined (from S. pneumoniae) did not activate NOD2 either, in striking contrast to PGN from S. aureus, Listeria and E. coli.51 Data on specific recognition of PGN from GBS are missing, thus the role of PGN in the innate immune response elicited by GBS is not known. PGN is covalently linked to two distinct capsular carbohydrate species expressed by GBS, type-specific and groupspecific polysaccharide. Nine antigenically and partially chemically distinct type-specific polysaccharides of GBS are associated with human infection.53 Clearly, sialic acid residues of type-specific polysaccharide contribute to GBS virulence by inhibiting C3 opsonisation and activation of the alternative complement pathway.54 Furthermore, sialic acid substituents of GBS capsular polysaccharide mimic the human Lewis X antigen, thus making GBS capsular polysaccharide a poor antigen. The role of type-specific polysaccharide in inflammation appears to be minor, since it exhibits only weak TNF-a-inducing properties and acapsular GBS mutants are normal with respect to the induction of inflammatory cytokines. In contrast to type-specific polysaccharide, group-specific polysaccharide from GBS is a more potent inflammatory stimulus that engages the surface protein CD14 and heterodimeric b2-integrins.55

Inflammatory toxins released by GBS GBS releases a functionally homogenous inflammatory factor that is essentially recognised by the cell surface molecules TLR2, TLR6 and CD14 on phagocytes (Fig. 1)49 and appears to be critical in a neonatal mouse model of GBS sepsis.2,32,56 Several interesting molecules are exported or liberated during growth of GBS, e.g. CAMP-factor, a pore-forming co-haemolysin for the beta-toxin of S. aureus, the protein of cell wall separation B (PCSB), enolase, fructose bisphosphonate aldolase, heat shock protein 70 and surface immunogenic protein.57 However, their role, if any, in the activation of innate immune cells has not been thoroughly investigated. The membrane-anchored LTA can be isolated from GBS supernatant. However, as outlined above, LTA from GBS is a relatively weak stimulus for phagocytes. If GBS-supernatant is purified for TLR2 activity by size exclusion chromatography, resulting fractions are 10 000-fold more potent than their LTA content (P. Henneke, unpub. obs.). Further

Neonatal SIRS and Sepsis

339

interesting candidates are the more than 30 putatively amino terminally lipidated proteins encoded in the GBS genome (http://www.mrc-lmb.cam.ac.uk/genomes/dolop/) that have not been analysed for their inflammatory potency. Another interesting extracellular GBS product is bhaemolysin, which is encoded by a single gene (cylE ) but has not yet been purified to homogeneity.58 In various in vitro and in vivo models, expression of b-haemolysin is associated with bacterial invasion, cytolytic injury, inflammatory activation of phagocytes (nitric oxide) and resistance to oxidative killing of GBS. In rodent models of GBS arthritis, sepsis and pneumonia, b-haemolytic GBS strains were more virulent than non-haemolytic mutant strains.58 Summarising the discussion above, a single substructure of GBS that dominates the inflammatory response has not yet been identified. This currently hampers the development of a sepsis therapy that targets specific virulence factors of GBS. In contrast, TLR2, JNK and TNF are attractive host cell targets that have been successfully investigated as therapeutic targets in GBS sepsis models.2,40

Polymorphisms in the innate immune system: role in neonatal sepsis The functionally transient incompetence to clear a GBS infection appears to be widespread in early life since GBS affected 0.6% of all infants before widespread usage of antibiotic therapy. Hence it is tempting to speculate on genetic polymorphisms as one molecular component of sepsis pathogenesis. Despite this, we know little about how the over 8 million known single nucleotide

Table 3

polymorphisms affect gene expression and protein function. Still, the body of research on common alterations in genes encoding molecules of the innate immune system and their role in sepsis susceptibility and outcome is growing. Interesting genes that encode proteins in the pathways that are potentially relevant for the course of GBS sepsis in newborn infants and that exhibit polymorphisms are CD14 (-159C/T), FCgRIIa, mannose binding lectin, TLR2 and plasminogen activator inhibitor type 1 (PAI-1). However, a clear association between single mutations in any of these inflammatory genes and the development of sepsis has yet to be established. Five studies are available that have investigated the contribution of genetic alterations in inflammatory genes to neonatal sepsis (Table 3). Two studies found a modest risk for sepsis in very preterm infants correlated to the polymorphism in the IL-6 promoter (-174G), but this association could not be confirmed by a larger, more recent study.59e61 Unfortunately, all available studies were performed in preterm infants who are at increased risk for nosocomial sepsis. As yet, no studies on the specific genetic susceptibility to GBS sepsis have been published.

Clinical studies on sepsis therapy in newborn infants According to the pathomechanisms outlined, anti-inflammatory interventions in SIRS and sepsis can be expected to improve outcome in EOS and LOS. Currently, few clinical intervention studies in newborn infants are available that allow conclusions to be drawn for the treatment of infants affected with SIRS and sepsis.

Studies on the role of polymorphisms in inflammatory genes in neonatal sepsis

Gene

Patient (numbers)

Inclusion criteria

Differences between groups

Reference

TNF IL-1b IL-4 IL-6 IL-10

Sepsis (33) Controls (35)

Birth weight <1500 g

No

71

IL-1ra

Sepsis (34) Controls (61)

Birth weight <2500 g

No

72

CD14

Sepsis (50)

Birth weight <1500 g

NOD2 OR Z 3.2 (CI Z 1.0e10.4), P Z 0.052

59

TLR4 NOD2

Controls (356) IL-6 OR Z 1.9 (CI Z 1.0e3.9), P Z 0.039

L-6 (174GG) Mannose-binding lectin IL-6 (174GG)

Sepsis (51)

GA  32 wks

OR Z 2.3 (CI Z 1.1e4.5), P Z 0.021

60

Birth weight <1500 g

No

61

Controls (126) IL-6 (174GG)

Sepsis (417) Controls (789)

SIRS, systemic inflammatory response syndrome; TNF, tumor necrosis factor; IL, interleukin; TLR, Toll-like receptor; NOD, novel intracellular receptor; OR, odds ratio; CI, confidence interval; GA, gestational age.

340 Adjunctive sepsis therapy with pentoxifylline successfully reduced all-cause mortality by >80% in neonatal sepsis in a placebo-controlled, double-blind study.62 However, this was a single study enrolling a limited number of patients. Confirmation from larger, multi-centre trials is warranted. Other adjunctive strategies including immunoglobulins, granulocytes, G-CSF/GM-CSF have yet to be shown to improve outcome. A large trial that aims to investigate the effect of intravenous immunoglobulins in 5000 babies with suspected serious infections is supposed to finish enrollment in 2006.63,64 In adults with severe sepsis, the so-called early goaldirected therapy that is tailored to rapidly adjust haemodynamic parameters and oxygen delivery, the strict control of the glucose metabolism and use of low-dose steroids have all been shown to be effective.65,66 Similar strategies (e.g. aggressive fluid resuscitation with 40e60 ml/kg, maintenance of blood glucose <150 mg/dl) have been suggested for paediatric patients by the Surviving Sepsis Campaign, but have not been thoroughly investigated in children, especially not in newborns.67 A large trial on the effect of activated protein C in children with sepsis (the RESOLVE trial) had to be stopped because of increased adverse events, although the study data have not been published (http:// www.mrc-lmb.cam.ac.uk/genomes/dolop/). Accordingly, although adjunctive sepsis treatment with activated protein C is clearly effective in adults, it currently seems not to be appropriate for use in children and infants. With respect to causative therapy, the essential role of early antibiotic sepsis therapy tailored to cover the likely organisms (such as GBS, E. coli, Enterococcus, Listeria etc.) is beyond question. However, studies of appropriate size and quality comparing prophylactic versus selective use in infants at risk, or of different antibiotic regimens (such as the widespread combination of b-lactam acntibiotics with aminoglycosides) are not available.68,69

Concluding remarks The systemic inflammatory response syndrome (SIRS) is a rather non-specific sequel to stress that can be frequently observed in newborn infants, although the definitive diagnosis can be made in only a minority of cases. Group B streptococcus (GBS) is a common coloniser of normal newborn infants and can be assumed to occasionally translocate across mucosal surfaces. Since GBS is a very potent inflammatory stimulus in vitro and in vivo, a tight balance between a sufficient local proinflammatory response and a non-phlogistic, immediate removal of invading bacteria is critical for the successful outcome of the infant. GBS elicits a powerful inflammatory cytokine response in neonatal phagocytes primarily via engagement of the Toll-like receptor system. In contrast, several components of the clearance apparatus, such as directed movement to the microorganism, expression of adhesion molecules, phagocytosis and the intracellular killing of GBS, are deficient in newborn, particularly preterm, infants. The neonatal innate immune system appears to be deficient in the initial compartmentalisation of inflammation by rapidly eliminating GBS at the site of microbial invasion. Thus, the immune balance tilts towards inflammation, SIRS and sepsis. A goal worth pursuing is to further define minute events at the

P. Henneke, R. Berner interphase between GBS and host cells since they might serve as targets for successful adjunctive GBS therapy. Currently, however, prophylaxis and effective antimicrobial therapy of GBS remain the cornerstones in the management of neonatal SIRS and sepsis.

Acknowledgements This work was supported, in part, by the Deutsche Forschungsgemeinschaft (He 3127/2-1.) and by the National Institutes of Health (AI52455). We are grateful to Julia Wennekamp for careful review of the manuscript.

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