The alveolar microenvironment of patients infected with human immunodeficiency virus does not modify alveolar macrophage interactions with Streptococcus pneumoniae.

We tested the hypothesis that HIV infection results in activation of alveolar macrophages and that this might be associated with impaired defense against pneumococcus. We compared alveolar macrophages and lymphocytes in 131 bronchoalveolar lavage samples from HIV-infected and healthy controls using inflammatory gene microarrays, flow cytometry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA) to determine the pattern of macrophage activation associated with HIV infection and the effect of this activation on defense against pneumococcus. We used gamma interferon (IFN-γ) priming to mimic the cellular milieu in HIV-infected lungs. InnateDB and BioLayout 3D were used to analyze the interactions of the upregulated genes. Alveolar macrophages from HIV-infected adults showed increased gene expression and cytokine production in a classical pattern. Bronchoalveolar lavage from HIV-infected subjects showed excess CD8(+) lymphocytes with activated phenotype. Toll-like receptor 4 (TLR4) expression was increased in macrophages from HIV-infected subjects, but function was similar between the groups; lung lavage fluid did not inhibit TLR function in transfected HeLa cells. Alveolar macrophages from HIV-infected subjects showed normal binding and internalization of opsonized pneumococci, with or without IFN-γ priming. Alveolar macrophages from HIV-infected subjects showed classical activation compared to that of healthy controls, but this does not alter macrophage interactions with pneumococci

The glycopeptide vancomycin does not enhance toll-like receptor 2 (TLR2) activation by Streptococcus pneumoniae

OBJECTIVES: The exposure of Streptococcus pneumoniae to cell-wall-active antibiotics in vivo and in vitro results in the release of bacterial components that can induce proinflammatory activation of human cells via toll-like receptor 2 (TLR2). The aim of this study was to compare the activation of human TLR2 pathways after exposure of S. pneumoniae to faropenem, cefotaxime and vancomycin.MATERIALS AND METHODS: Streptococcus pneumoniae D39 was exposed to cefotaxime, faropenem or vancomycin for 6 h during lag or early log phase growth. IL-8 promoter activity of HeLa cells was measured using a dual luciferase reporter plasmid system. HeLa cells were transfected with an expression vector containing TLR2/CD14, or empty vector/CD14 and IL-8 promoter activity was measured using luminescence. Cells were stimulated with antibiotic-treated bacteria, untreated bacteria or medium-only controls.RESULTS: Lag phase S. pneumoniae treated at sub-MIC (1/8 MIC) cefotaxime or faropenem induced 11-fold and 8-fold increases, respectively, in TLR2-mediated IL-8 promoter activity when compared with untreated bacteria. Early log MIC cefotaxime or faropenem-treated bacteria also enhanced TLR2 activation by 3-fold and 4-fold, respectively, when compared with untreated bacteria. Vancomycin treatment had no effect on TLR2 induction at any growth stage or MIC ratio tested.CONCLUSIONS: beta-Lactam antibiotics induce surface changes and release of cell wall structures from bacteria that are proinflammatory via TLR2, but the glycopeptide vancomycin does not.</p

Penicillin enhances the toll-like receptor 2-mediated proinflammatory activity of Streptococcus pneumoniae

The Streptococcus pneumoniae cell-wall components peptidoglycan and lipoteichoic acid activate Toll-like receptor 2 (TLR2), which transduces an inflammatory response. After exposure to penicillin, type 2 S. pneumoniae strain D39, but not the isogenic autolysin-deficient mutant AL2, induced significantly enhanced interleukin-8 promoter activity in TLR2-transfected HeLa cells. Lag-phase D39 exhibited enhanced TLR2 activation after exposure to penicillin at levels below the minimum inhibitory concentration (MIC); in contrast, early log-phase S. pneumoniae were most active when exposed to the MIC. This enhancement was not ablated by heat treatment but was attenuated by autolysin inhibitors. The antimicrobial activity of moxifloxacin and erythromycin was not associated with TLR2 activation by S. pneumoniae. These data show that penicillin treatment of S. pneumoniae releases proinflammatory cell-wall components that activate TLR2 and that this activity is dependent on autolysin, the growth phase of the organism, and the antibiotic concentration.</p

Induction of pro-inflammatory cytokine release by human macrophages during exposure of Streptococcus pneumoniae to penicillin is influenced by minimum inhibitory concentration ratio

beta-Lactam antibiotics cause release of pro-inflammatory bacterial cell wall structures. We determined the effect of penicillin treatment of Streptococcus pneumoniae on the induction of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) genes by human macrophages and the influence of antibiotic concentration and bacterial growth phase upon this induction. Gene expression was measured by real-time polymerase chain reaction (PCR) and protein was measured by enzyme-linked immunosorbent assay (ELISA). Treatment of lag phase S. pneumoniae with one-eighth minimum inhibitory concentration (MIC) penicillin resulted in enhanced expression of TNF-alpha messenger RNA (mRNA), but not TNF-alpha protein at 6h compared with untreated bacteria. IL-1beta mRNA and protein were not affected by these bacteria. MIC treatment of lag or early log phase bacteria induced both protein and mRNA for IL-1beta. Bacteria exposed to concentrations of penicillin that cause lysis (MIC) or no lysis with morphological changes (sub-MIC) induce differential patterns of pro-inflammatory cytokine expression by human macrophages.</p

Pneumolysin-induced CXCL8 production by nasopharyngeal epithelial cells is dependent on calcium flux and MAPK activation via Toll-like receptor 4

The natural niche of Streptococcus pneumoniae is the nasopharyngeal mucosa and nasopharyngeal carriage of pneumococci is widely prevalent. Pneumolysin (Ply), a pore-forming protein produced by S. pneumonia, may be important in driving the innate immune response of the nasopharynx. We studied the Ply-induced production of CXCL8 by nasopharyngeal cells and further analysed the mechanism of this induction. Detroit nasopharyngeal cells were stimulated with supernatants derived from bacterial cultures of Ply-deficient, wild-type pneumococci and recombinant Ply, and CXCL8 measured by ELISA. The role of MAP kinase family members in Ply-induced CXCL8 production was analysed using specific inhibitors, NF-κB activity was measured by immunoblot and Ply-mediated TLR4 activation analysed by a CXCL8 promotor luciferase assay. Ply significantly increased production of CXCL8 in Detroit and primary nasal cells. Flow cytometric analysis showed that Detroit cells express cell surface TLR4. CXCL8 production was dependent on changes in the intracellular Ca2+ levels and also by NF-κB via activation of TLR4, and MAP kinase signalling. Ply induces production of CXCL8 by nasopharyngeal cells using signalling mechanisms involving Ca2+ mobilisation and activation of MAPK and NF-κB via TLR4. This may be important in regulating nasopharyngeal immunity against pneumococcal colonization

Superantigen recognition by HLA class II on monocytes up-regulates toll-like receptor 4 and enhances proinflammatory responses to endotoxin

The devastating systemic effects of bacterial superantigens may be explained by powerful proinflammatory synergy with lipopolysaccharide (LPS). However, the mechanism underlying this phenomenon remains unclear and has never been investigated in humans. Specifically, there is no known link between superantigen-induced immune effects and the pattern recognition of LPS at toll-like receptor 4 (TLR4). Here we show that bacterial superantigens induce rapid transcription and increased membrane expression of TLR4 in primary human monocytes by ligation of major histocompatibility complex (MHC) class II. We also demonstrate that superantigens are solely responsible for monocyte TLR4 up-regulation induced by products from Gram-positive bacteria. In parallel with enhanced TLR4 expression, priming of purified monocytes or mixed peripheral blood mononuclear cells with superantigens significantly enhanced the induction of proinflammatory cytokines by known TLR4 ligands. Staphylococcal enterotoxin A constructs containing targeted mutations were used to demonstrate a requirement for MHC class II ligation in both TLR4 up-regulation and enhanced responses to endotoxin. In contrast to results from animal models, superantigen-endotoxin interaction was not dependent on T-cell receptor ligation by superantigen or interferon gamma production. Pattern recognition of bacterial superantigens by MHC class II receptors may exacerbate the proinflammatory response of monocytes to Gram-negative infection or endotoxin by up-regulation of TLR4.</p

Proinflammatory activation of Toll-like receptor-2 during exposure of penicillin-resistant Streptococcus pneumoniae to beta-lactam antibiotics

Objectives: Disease caused by penicillin-resistant Streptococcus pneumoniae (PRSP) is associated with more suppurative complications than disease caused by penicillin-susceptible S. pneumoniae (PSSP). Exposure of S. pneumoniae to beta-lactam antibiotics enhances the proinflammatory activation of human cells by pneumococci via Toll-like receptor-2 (TLR2). To test the hypothesis that penicillin resistance influences cellular TLR2 activation by beta-lactam-exposed pneumococci, we compared TLR2 induction by PSSP (MIC 0.06 mg/L) and a high-level PRSP clinical isolate (159; MIC 16 mg/L) following exposure to penicillin and cefotaxime. Methods: Both organisms were treated with penicillin or cefotaxime at and around the MIC. TLR2 signalling was measured as relative IL-8 promoter activation in transfected HeLa cells. Results: On exposure to penicillin, log-phase PSSP and PRSP induced TLR2-proinflammatory activation at levels significantly higher than unexposed bacteria, and maximal in each case at the MIC. Transformants containing low-affinity penicillin-binding proteins (PBP) 2x, 1a and 2b exhibited stepwise resistance to cefotaxime and penicillin. TLR2 activation following penicillin treatment was dependent on an abnormal cell wall (PBP1a and 2x) and autolysis (PBP2b). High affinity PBP2x was required for this effect to be observed in log-phase pneumococci exposed to cefotaxime at the MIC. Cefotaxime-mediated TLR2 activation was not observed in lag-phase transformants exposed to sub-lethal concentrations. Conclusions: These data show that PRSP have similar TLR2-proinflammatory effects to PSSP when exposed to beta-lactam antibiotics but the antibiotic concentration relative to the MIC is critical. This has implications for treatment of pneumococcal disease when tissue concentrations of antibiotic are close to the MIC

Activation of toll-like receptor 2 (TLR2) and TLR4/MD2 by Neisseria is independent of capsule and lipooligosaccharide (LOS) sialylation but varies widely among LOS from different strains

Lipooligosaccharide (LOS) structure and capsular polysaccharide of Neisseria meningitidis each greatly influence the virulence of the organism and the quality of host innate immune responses. In this study, we found that production of the proinflammatory cytokine tumor necrosis factor (TNF) by a human monocyte-derived cell line (THP-1) exposed to strains of N. meningitidis lacking capsule and/or with truncated LOS was similar to that elicited by the isogenic wild-type strain. These mutants also exhibited no difference in induction of the interleukin-8 (IL-8) promoter in a transfected HeLa cell system of Toll-like receptor 2 (TLR2) and TLR4/MD2 signaling. However, purified LOS from diverse strains of Neisseria (both N. meningitidis and N. gonorrhoeae) caused widely variant levels of IL-8 promoter induction in cells expressing MD2 that correlated with the production of TNF from THP-1 cells. These data suggest that although modification of the oligosaccharide chain of LOS and/or absence of capsule do not affect cell signaling mediated by TLR4/MD2, fine-structural differences in the LOS do influence signaling through TLR4/MD2 and, through this pathway, influence some of the proinflammatory responses elicited by Neisseria.</p

Increased surface toll-like receptor 2 expression in superantigen shock

Superantigens up-regulate monocyte surface TLR2 expression through major histocompatibility complex class II signaling. Enhanced surface TLR2 expression may be a specific feature of patients with S. pyogenes-induced shock. Importantly, intensity of TLR2 signaling is not necessarily coupled to TLR2 expression when ligand concentrations are low or after onset of critical illnes