Selective impairment of TLR-mediated innate immunity in human newborns: neonatal blood plasma reduces monocyte TNF-alpha induction by bacterial lipopeptides, lipopolysaccharide, and imiquimod, but preserves the response to R-848.

Newborns are at increased risk of overwhelming infection, yet the mechanisms underlying this susceptibility are incompletely defined. In this study we report a striking 1- to 3-log decrease in sensitivity of monocytes in human neonatal cord blood, compared with monocytes in adult peripheral blood, to the TNF-alpha-inducing effect of multiple TLR ligands, including bacterial lipopeptides (BLPs), LPS, and the imidazoquinoline compound, imiquimod. In marked contrast, TNF-alpha release in response to R-848, a TLR ligand that is a congener of imiquimod, was equivalent in newborn and adult blood. Differences in ligand-induced TNF-alpha release correlated with divergent ligand-induced changes in monocyte TNF-alpha mRNA levels. Newborn and adult monocytes did not differ in basal mRNA or protein expression of TLRs or mRNA expression of functionally related molecules. Newborn monocytes demonstrated diminished LPS-induced, but equivalent R-848-induced, phosphorylation of p38 mitogen-activated protein kinase and altered BLP- and LPS-induced acute modulation of cognate receptors, suggesting that the mechanism accounting for the observed differences may be localized proximal to ligand recognition by surface TLRs. Remarkably, newborn plasma conferred substantially reduced BLP-, LPS-, and imiquimod-induced TNF-alpha release on adult monocytes without any effect on R-848-induced TNF-alpha release, reflecting differences in a plasma factor(s) distinct from soluble CD14. Impaired response to multiple TLR ligands may significantly contribute to immature neonatal immunity. Conversely, relative preservation of responses to R-848 may present unique opportunities for augmenting innate and acquired immunity in the human newborn.

Critical role of the complement system in group B streptococcus-induced tumor necrosis factor alpha release.

Group B Streptococcus (GBS) is a major cause of newborn sepsis and meningitis and induces systemic release of tumor necrosis factor alpha (TNF-alpha), believed to play a role in morbidity and mortality. While previous studies have shown that GBS can induce TNF-alpha release from monocytes and macrophages, little is known about the potential modulating effect of plasma or serum on GBS-induced TNF-alpha release, and there are conflicting reports as to the host receptors involved. In a human whole-blood assay system, GBS type III COH-1 potently induced substantial monocyte TNF-alpha release in adult peripheral blood and, due to a higher concentration of monocytes, 10-fold-greater TNF-alpha release in newborn cord blood. Remarkably, GBS-induced TNF-alpha release from human monocytes was enhanced approximately 1000-fold by heat-labile serum components. Experiments employing C2-, C3-, or C7-depleted serum demonstrated that C3 activation via the alternative pathway is crucial for potent GBS-induced TNF-alpha release. Accordingly, whole blood from C3-deficient mice demonstrated significantly reduced GBS-induced TNF-alpha release. Preincubation with human serum enhanced the TNF-alpha-inducing activity of GBS in a C3- and factor B-dependent manner, implying deposition of complement components via the alternative pathway. GBS-induced TNF-alpha release was inhibited by monoclonal antibodies directed against each of the components of CR3 and CR4: the common integrin beta subunit CD18 and the alpha subunits CD11b (of CR3) and CD11c (of CR4). Blood derived from CR3 (CD11b/CD18)-deficient mice demonstrated a markedly diminished TNF-alpha response to GBS. We conclude that the ability of plasma and serum to greatly amplify GBS-induced TNF-alpha release reflects the activity of the alternative complement pathway that deposits fragments on GBS and thereby enhances CR3- and CR4-mediated monocyte activation.

Cytotoxic effects of streptolysin o and streptolysin s enhance the virulence of poorly encapsulated group a streptococci.

Although the toxicity of streptolysin O (SLO) and streptolysin S (SLS) in purified group A streptococci (GAS) has been established, the effect of these molecules in natural infection is not well understood. To identify whether biologically relevant concentrations of SLO and SLS were cytotoxic to epithelial and phagocytic cells that the bacteria would typically encounter during human infection and to characterize the influence of cell injury on bacterial pathogenesis, we derived GAS strains deficient in SLO or SLS in the background of an invasive GAS M3 isolate and determined their virulence in in vitro and in vivo models of human disease. Whereas bacterial production of SLO resulted in lysis of both human keratinocytes and polymorphonuclear leukocytes, GAS expression of SLS was associated only with keratinocyte injury. Expression of SLO but not SLS impaired polymorphonuclear leukocyte killing of GAS in vitro, but this effect could only be demonstrated in the background of acapsular organisms. In mouse invasive soft-tissue infection, neither SLO or SLS expression significantly influenced mouse survival. By contrast, in a mouse model of bacterial sepsis after intraperitoneal inoculation of GAS, SLO expression enhanced the virulence of both encapsulated and acapsular GAS, whereas SLS expression increased the virulence only of acapsular GAS. We conclude that the cytotoxic effects of SLO protect GAS from phagocytic killing and enhance bacterial virulence, particularly of strains that may be relatively deficient in hyaluronic acid capsule. Compared to SLO, SLS in this strain background has a more modest influence on GAS pathogenicity and the effect does not appear to involve bacterial resistance to phagocytosis.

NAD+-glycohydrolase acts as an intracellular toxin to enhance the extracellular survival of group A streptococci.

Group A streptococci (GAS) produce several secreted products that are thought to enhance pathogenicity by facilitating spread of the organisms through host tissues. Two such products, streptolysin O (SLO) and NAD+-glycohydrolase, appear to be functionally linked, in that SLO is required for transfer of NAD+-glycohydrolase into epithelial cells. However, the effects of NAD+-glycohydrolase on host cells are largely unexplored. We now report that SLO-mediated delivery of NAD+-glycohydrolase to the cytoplasm of human keratinocytes results in major changes in host cell biology that enhance GAS pathogenicity. We derived isogenic mutant strains deficient in the expression of SLO, NAD+-glycohydrolase or both proteins in the background of a virulent, M-type 3 strain of GAS. All three mutant strains were internalized by human keratinocytes more rapidly and in higher numbers than were organisms from the wild-type strain. Association of the mutant strains with keratinocytes also resulted in reduced cytotoxicity and reduced keratinocyte apoptosis compared with wild-type GAS. These results support a model in which NAD+-glycohydrolase contributes to GAS pathogenesis by modulating host cell signalling pathways to inhibit GAS internalization, to augment SLO-mediated cytotoxicity and to induce keratinocyte apoptosis. We conclude that NAD+-glycohydrolase is a novel type of bacterial toxin that acts intracellularly in the infected host to enhance the survival and proliferation of an extracellular pathogen.