Immune Reconstitution After Allogeneic Hematopoietic Stem Cell Transplantation and Association With Occurrence and Outcome of Invasive Aspergillosis.

BACKGROUND: Invasive aspergillosis (IA) remains a leading cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). To date, no reliable immunological biomarkers for management and outcome of IA exist. Here, we investigated reconstitution of antifungal immunity in patients during the first 12 months after HSCT and correlated it with IA. METHODS: Fifty-one patients were included, 9 with probable/proven IA. We determined quantitative and qualitative reconstitution of polymorphonuclear (PMN), CD4, CD8, and natural killer (NK) cells against Aspergillus fumigatus over 5 time points and compared the values to healthy donors. RESULTS: Absolute CD4 and CD8 cell counts, antigen-specific T-cell responses, and killing capacity of PMN against A. fumigatus were significantly decreased in all patients over 12 months. In patients with probable/proven IA, reactive oxygen species (ROS) production tended to be lower compared to patients without IA, and absolute NK-cell counts remained below 200 cells/µL. Patients with well-controlled IA showed significantly higher ROS production and NK-cell counts compared to patients with poor outcome. CONCLUSIONS: This study highlights the importance of functional PMN, T-cell, and NK-cell immunity for the outcome of IA. Larger multicenter studies should address the potential use of NK-cell counts for the management of antifungal therapy.

T and B cells are not required for clearing Staphylococcus aureus in systemic infection despite a strong TLR2-MyD88-dependent T cell activation.

Staphylococcus aureus infection elicits through its mature lipoproteins an innate immune response by TLR2-MyD88 signaling, which improves bacterial clearing and disease outcome. The role of dendritic cells (DCs) and T cells in this immune activation and the function of T and B cells in defense against S. aureus infection remain unclear. Therefore, we first evaluated DC and T cell activation after infection with S. aureus wild type (WT) and its isogenic mutant, which is deficient in lipoprotein maturation, in vitro. Lipoproteins in viable S. aureus contributed via TLR2-MyD88 to activation of DCs, which promoted the release of IFN-γ and IL-17 in CD4(+) T cells. This strong effect was independent of superantigens and MHC class II. We next evaluated the function of T cells and their cytokines IFN-γ and IL-17 in infection in vivo. Six days after systemic murine infection IFN-γ, IL-17, and IL-10 production in total spleen cells were MyD88-dependent and their levels increased until day 21. The comparison of CD3(-/-), Rag2(-/-), and C57BL/6 mice after infection revealed that IFN-γ and IL-17 originated from T cells and IL-10 originated from innate immune cells. Furthermore, vaccination of mice to activate T and B cells did not improve eradication of S. aureus from organs. In conclusion, S. aureus enhances DC activation via TLR2-MyD88 and thereby promotes T(H)1 and T(H)17 cell differentiation. However, neither T cells and their MyD88-regulated products, IFN-γ and IL-17, nor B cells affected bacterial clearing from organs and disease outcome.

TLR2 enhances NADPH oxidase activity and killing of Staphylococcus aureus by PMN.

Toll-like receptors play an essential role in the detection of invading pathogens. TLR2 is expressed in high concentrations on neutrophils and has been implicated as a critical mediator inducing host antimicrobial defenses against Gram-positive bacteria. Neutrophil responses induced via TLR2 are likely to have important clinical consequences, since Gram-positive organisms, such as Staphylococcus aureus, are an increasingly important source of severe infections. In the present study, we report that TLR2 has a central role in killing of S. aureus by murine PMN via enhancement of NADPH oxidase activity. PMN from TLR2-deficient mice showed a similar inability to kill S. aureus in vitro and under in vivo-like conditions as PMN with a non-functional NADPH oxidase. This defect in killing by TLR2-deficient PMN was not related to phagocytosis but caused by delayed and reduced NADPH oxidase-mediated production of superoxide anion in response to S. aureus and other Gram-positive bacteria. The cause of this was independent of PI3K- and p38 signaling. The TLR2-enhanced induction of superoxide was a defect in proper NADPH oxidase assembly. We hypothesize that early activation of TLR2-signaling may enhance p47(phox) phosphorylation subsequent to phagocytosis-mediated phosphorylation. Summarized, these data demonstrate a novel role of TLR2 in the killing of S. aureus by ensuring a rapid activation of the NADPH oxidase complex.

Lipoproteins in Staphylococcus aureus mediate inflammation by TLR2 and iron-dependent growth in vivo.

Lipoproteins (Lpp) are ligands of TLR2 and signal by the adaptor MyD88. As part of the bacterial cell envelope, Lpp are mainly involved in nutrient acquisition for Staphylococcus aureus. The impact of Lpp on TLR2-MyD88 activation for S. aureus in systemic infection is unknown. S. aureus strain SA113 deficient in the enzyme encoded by the prolipoprotein diacylglyceryl transferase gene (Deltalgt), which attaches the lipid anchor to pro-Lpp, was used to study benefits and costs of Lpp maturation. Lpp in S. aureus induced early and strong cytokines by TLR2-MyD88 signaling in murine peritoneal macrophages. Lpp contributed via TLR2 to pathogenesis of sepsis in C57BL/6 mice with IL-1beta, chemokine-mediated inflammation, and high bacterial numbers. In the absence of MyD88-mediated inflammation, Lpp allowed bacterial clearing from liver devoid of infiltrating cells, but still conferred a strong growth advantage in mice, which was shown to rely on iron uptake and storage in vitro and in vivo. With iron-restricted bacteria, the Lpp-related growth advantage was evident in infection of MyD88(-/-), but not of C57BL/6, mice. On the other hand, iron overload of the host restored the growth deficit of Deltalgt in MyD88(-/-), but not in immunocompetent C57BL/6 mice. These results indicate that iron acquisition is improved by Lpp of S. aureus but is counteracted by inflammation. Thus, lipid anchoring is an evolutionary advantage for S. aureus to retain essential proteins for better survival in infection.

Toll-like receptor 2 deficiency delays pneumococcal phagocytosis and impairs oxidative killing by granulocytes.

Phagocytosis and killing of Streptococcus pneumoniae was compared in blood-derived wild-type (WT) and Toll-like receptor 2 (TLR2)-deficient (TLR2-/-) polymorphonuclear leukocytes (PMN). Phagocytosis of green fluorescent protein-transformed pneumococci was delayed in TLR2-/- PMN. These cells exhibited also a lower oxidative bactericidal activity against S. pneumoniae than WT PMN, suggesting that TLR2 modulates bacterial clearance in PMN.

Toll-like receptor-2 deficiency is associated with enhanced brain TNF gene expression during pneumococcal meningitis.

TNF is a marker of disease activity in bacterial meningitis. To investigate TNF modulation by Toll-like receptor-2 (TLR2), we studied temporal and anatomical expression patterns of TLR2 and TNF in a pneumococcal meningitis model in wild type (wt) and TLR2(-/-) mice. We show by in situ hybridization that transcripts of TLR2 and of the comolecules CD14, MD-2, TLR1/6 strongly increased and colocalized with TNF in CD45-positive infiltrating cells in the ventricles, corpus callosum and the meninges. TNF gene and protein expression was stronger in TLR2(-/-) than wt brains and associated with increased IkappaB expression suggesting that TLR2 is controlling inflammation via TNF regulation.

The ability of biofilm formation does not influence virulence of Staphylococcus aureus and host response in a mouse tissue cage infection model.

The virulence of Staphylococcus aureus Sa113 (SA113) and an isogenic ica deletion mutant (ica-), deficient in the production of polysaccharide intercellular adhesin (PIA), which is crucial for biofilm formation, was compared in a mouse tissue cage infection model. The minimal infective doses for the induction of persistent tissue infections in C57BL/6 mice were 10(3) CFU for both SA113 and the ica- mutant. Bacterial growth, initial adherence to surfaces within the implants and the course of inflammation including growth-dependent host TNF and MIP-2 release, influx of phagocytes and an accumulation of dead leukocytes were similar as well. Since SA113 expressed PIA in vivo, we could demonstrate that PIA and the lack of biofilm formation did not influence the capacity of S. aureus to induce persistent infections and did not modulate host responses in the mouse tissue cage model.

Generation and functional characterization of a clonal murine periportal Kupffer cell line from H-2Kb -tsA58 mice.

Murine Kupffer cells (KCs) are heterogeneous and survive only for a short time in vitro. Here, a clonal, murine KC line was generated from transgenic mice, expressing the thermolabile mutant tsA58 of the Simian virus 40 large T antigen under the control of the H-2K(b) promoter. Thirty-three degrees Celsius and 37 degrees C but not 39 degrees C have been permissive for growth of the clone; it required conditioned media from hepatocytes and endothelial cells for proliferation. In contrast to primary cells, the cells of the clone were uniform, survived detachment, and could therefore be analyzed by cytofluorimetry. The clone, as primary KCs, constitutively expressed nonspecific esterase, peroxidase, MOMA-2, BM8, scavenger receptor A, CD14, and Toll-like receptor 4 (TLR4); the antigen-presenting molecules CD40, CD80, and CD1d; and endocytosed dextran-fluorescein isothiocyanate. It lacked complement, Fc receptors, F4/80 marker, and the phagosomal coat protein tryptophan aspartate-containing coat protein (TACO). The clone exhibited CD14- and TLR4/MD2-independent, plasma-dependent lipopolysaccharide (LPS) binding, Escherichia coli and Streptococcus pneumoniae phagocytosis, and LPS- and interferon-gamma-induced NO production but no tumor necrosis factor alpha, interleukin (IL)-6, or IL-10 release. The large size, surface-marker expression, and capacity to clear gram-negative and -positive bacteria indicate that the clone was derived from the periportal, large KC subpopulation. The clone allows molecular studies of anti-infective and immune functions of KCs.

Microheterogeneity of anti-myelin-associated glycoprotein antibodies.

Antibodies to the myelin-associated glycoprotein (MAG) are implicated in the pathogenesis of an acquired demyelinating polyneuropathy. We studied IgM affinity to MAG in 18 patients with anti-MAG antibodies. Binding of sera was tested for anti-MAG immunoreactivity in central nervous system (CNS) by ELISA and in CNS and peripheral nervous system (PNS) by Western blot analysis. Furthermore, immunohistochemical characterization of IgM binding on sural nerve tissue was investigated using the indirect peroxidase method. Western blot analysis revealed that all sera detected MAG in central myelin, but only eight in peripheral myelin. Anti-MAG-IgM-ELISA-titers correlated significantly (p<0.0001) with PNS-Western blot results. By indirect immunoperoxidase immunohistochemistry, 12 sera stained myelin sheaths, while 6 sera showed no staining. These results demonstrate considerable variations in antibody binding strength to MAG between PNS myelin and CNS myelin. The relevance of these differences for the pathogenesis of the neuropathy and clinical impairment remains to be demonstrated.