Maria Gambuzza

Bacterial recognition by TLR7 in the lysosomes of conventional dendritic cells

Little is known of how and where bacterial recognition triggers the induction of type I interferon. Whether the type of recognition receptor used in these responses is determined by the subcellular location of bacteria is not understood. Here we show that phagosomal bacteria such as group B streptococcus, but not cytosolic bacteria, potently induced interferon in conventional dendritic cells by a mechanism that required Toll-like receptor 7, the adaptor MyD88 and the transcription factor IRF1, all of which localized together with bacterial products in degradative vacuoles bearing lysosomal markers. Thus, this cell type–specific recognition pathway links lysosomal recognition of bacterial RNA with a robust, host-protective interferon response.

Type I IFN Signaling Is Crucial for Host Resistance against Different Species of Pathogenic Bacteria

It is known that host cells can produce type I IFNs (IFN-αβ) after exposure to conserved bacterial products, but the functional consequences of such responses on the outcome of bacterial infections are incompletely understood. We show in this study that IFN-αβ signaling is crucial for host defenses against different bacteria, including group B streptococci (GBS), pneumococci, and Escherichia coli. In response to GBS challenge, most mice lacking either the IFN-αβR or IFN-β died from unrestrained bacteremia, whereas all wild-type controls survived. The effect of IFN-αβR deficiency was marked, with mortality surpassing that seen in IFN-γR-deficient mice. Animals lacking both IFN-αβR and IFN-γR displayed additive lethality, suggesting that the two IFN types have complementary and nonredundant roles in host defenses. Increased production of IFN-αβ was detected in macrophages after exposure to GBS. Moreover, in the absence of IFN-αβ signaling, a marked reduction in macrophage production of IFN-γ, NO, and TNF-α was observed after stimulation with live bacteria or with purified LPS. Collectively, our data document a novel, fundamental function of IFN-αβ in boosting macrophage responses and host resistance against bacterial pathogens. These data may be useful to devise alternative strategies to treat bacterial infections.

Haemophilus influenzae Porin Induces Toll-Like Receptor 2-Mediated Cytokine Production in Human Monocytes and Mouse Macrophages

ABSTRACT The production of proinflammatory cytokines is likely to play a major pathophysiological role in meningitis and other infections caused by Haemophilus influenzae type b (Hib). Previous studies have shown that Hib porin contributes to signaling of the inflammatory cascade. We examined here the role of Toll-like receptors (TLRs) and the TLR-associated adaptor protein MyD88 in Hib porin-induced production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Hib porin-induced TNF-α and IL-6 production was virtually eliminated in macrophages from TLR2- or MyD88-deficient mice. In contrast, macrophages from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, which are defective in TLR4 function, responded normally to Hib porin. Moreover anti-TLR2 antibodies but not anti-TLR4 antibodies significantly reduced Hib porin-stimulated TNF-α and IL-6 release from the human monocytic cell line THP-1. These data indicate that the TLR2/MyD88 pathway plays an essential role in Hib porin-mediated cytokine production. These findings may be useful in the development of alternative therapies aimed at reducing excessive inflammatory responses during Hib infections.

Bacteroides fragilis-Derived Lipopolysaccharide Produces Cell Activation and Lethal Toxicity via Toll-Like Receptor 4

ABSTRACT Bacteroides fragilis, which is part of the normal intestinal flora, is a frequent cause of serious disease, especially in diabetic and surgical patients. In these conditions, B. fragilis lipopolysaccharide (LPS) is likely to play a major pathophysiologic role. B. fragilis LPS is structurally different from classical enterobacterial LPS, whose biological activities are mediated by Toll-like receptor 4 (TLR4) activation. The ability of B. fragilis LPS to activate TLR4 and TLR2 was investigated here, since evidence on this issue is scarce and controversial. Each of four different protein-free B. fragilis LPS preparations could induce interleukin-8 responses in cells cotransfected with TLR4/CD14/MD2 but not TLR4/CD14 alone. Two of the preparations also induced cytokine production in cells cotransfected with TLR2/CD14 or in peritoneal macrophages from TLR4 mutant C3H/HeJ mice. Both of these activities, however, were lost after repurification with a modified phenol reextraction procedure. Importantly, all preparations could induce endotoxic shock in TLR2-deficient mice, but not in TLR4 mutant C3H/HeJ mice. Consistent with these findings, anti-TLR4 and anti-CD14, but not anti-TLR2, antibodies could inhibit B. fragilis LPS-induced cytokine production in human monocytes. Collectively, these results indicate that B. fragilis LPS signals via a TLR4/CD14/MD2-dependent pathway, and it is unable to activate TLR2. Moreover, our data document the occurrence of TLR2-activating contaminants even in highly purified B. fragilis LPS preparations. This may explain earlier contradictory findings on the ability of B. fragilis LPS to activate cells in the absence of functional TLR4. These data may be useful to devise strategies to prevent the pathophysiologic changes observed during B. fragilis sepsis and to better understand structure-activity relationships of LPS.

IFN-alpha/beta signaling is required for polarization of cytokine responses toward a protective type 1 pattern during experimental cryptococcosis

The antiviral activities of type I IFNs have long been established. However, comparatively little is known of their role in defenses against nonviral pathogens. We examined here the effects of type I IFNs on host resistance against the model pathogenic yeast Cryptococcus neoformans. After intratracheal or i.v. challenge with this fungus, most mice lacking either the IFN-α/β receptor (IFN-α/βR) or IFN-β died from unrestrained pneumonia and encephalitis, while all wild-type controls survived. The pulmonary immune response of IFN-α/βR−/− mice was characterized by increased expression of IL-4, IL-13, and IL-10, decreased expression of TNF-α, IFN-γ, inducible NO synthetase, and CXCL10, and similar levels of IL-12 mRNA, compared with wild-type controls. Histopathological analysis showed eosinophilic infiltrates in the lungs of IFN-α/βR−/− mice, although this change was less extensive than that observed in similarly infected IFN-γR-deficient animals. Type I IFN responses could not be detected in the lung after intratracheal challenge. However, small, but statistically significant, elevations in IFN-β levels were measured in the supernatants of bone marrow-derived macrophages or dendritic cells infected with C. neoformans. Our data demonstrate that type I IFN signaling is required for polarization of cytokine responses toward a protective type I pattern during cryptococcal infection.

Antiidiotypic DNA vaccination induces serum bactericidal activity and protection against group B meningococci

No vaccine is available for preventing infections by serogroup B Neisseria meningitidis (MenB), which accounts for a major portion of meningococcal cases in developed countries, because of the poor immunogenicity of the capsular polysaccharide (CP) even after protein conjugation. We have previously induced anticapsular antibodies by immunization with a single chain variable fragment (scFv), which mimics a protective CP epitope. This surrogate antigen, however, was ineffective at inducing serum bactericidal activity, an accepted marker of protection in humans. Serum bactericidal activity was consistently achieved by immunizing mice with the scFv-encoding gene. Immunization with vectors without a secretory signal sequence before the scFv resulted in markedly higher bactericidal activity relative to those with such a sequence. The induced antibodies were capsule specific, as shown by complete inhibition of bactericidal activity by purified MenB CP and by resistance to killing of MenA or MenC. Moreover, these antibodies were predominantly of the IgG2a isotype, reflecting a T helper type 1 response. Administration of sera from scFv gene–vaccinated animals protected infant rats against MenB bacteremia. These data illustrate the potential of vaccination with genes encoding capsular mimics in providing protection against MenB and other encapsulated bacteria.

Age-related sensitivity of neonatal mice to toxicity induced by heat-killed group B streptococci.

Newborns are highly susceptible to infections by group B streptococci (GBS), a relatively infrequent cause of serious disease in adults. Several immunological defects, including decreased opsono-phagocytosis, can, at least partially, account for the increased susceptibility of neonates to GBS infections. We have previously demonstrated that tumor necrosis factor alpha (TNF-α) plays an important pathophysiologic role in neonatal models of GBS disease1. This study was undertaken to test the hypothesis that neonatal age is associated with altered sensitivity to TNF-α-dependent toxic activities of GBS products.