Jürgen Löffler

Genetic PTX3 Deficiency and Aspergillosis in Stem-Cell Transplantation

BACKGROUND The soluble pattern-recognition receptor known as long pentraxin 3 (PTX3) has a nonredundant role in antifungal immunity. The contribution of single-nucleotide polymorphisms (SNPs) in PTX3 to the development of invasive aspergillosis is unknown. METHODS We screened an initial cohort of 268 patients undergoing hematopoietic stem-cell transplantation (HSCT) and their donors for PTX3 SNPs modifying the risk of invasive aspergillosis. The analysis was also performed in a multicenter study involving 107 patients with invasive aspergillosis and 223 matched controls. The functional consequences of PTX3 SNPs were investigated in vitro and in lung specimens from transplant recipients. RESULTS Receipt of a transplant from a donor with a homozygous haplotype (h2/h2) in PTX3 was associated with an increased risk of infection, in both the discovery study (cumulative incidence, 37% vs. 15%; adjusted hazard ratio, 3.08; P=0.003) and the confirmation study (adjusted odds ratio, 2.78; P=0.03), as well as with defective expression of PTX3. Functionally, PTX3 deficiency in h2/h2 neutrophils, presumably due to messenger RNA instability, led to impaired phagocytosis and clearance of the fungus. CONCLUSIONS Genetic deficiency of PTX3 affects the antifungal capacity of neutrophils and may contribute to the risk of invasive aspergillosis in patients treated with HSCT. (Funded by the European Society of Clinical Microbiology and Infectious Diseases and others.).

Screening for Aspergillus spp. using polymerase chain reaction of whole blood samples from patients with haematological malignancies

Sensitive screening for Aspergillus spp. using polymerase chain reaction (PCR) of whole blood samples in patients with haematological disorders has not been performed to date. In a 2‐year study, 121 patients admitted to the University Hospital of Innsbruck for cancer chemotherapy without clinical signs of fungal infection were prospectively screened for Aspergillus spp. In 28 out of 121 (23%) patients, Aspergillus DNAaemia was detected. Of these patients, 16 (57%) were positive only once for Aspergillus DNA, but positivity was never associated with invasive aspergillosis. PCR positive episodes were short and resolved without antifungal treatment. Five patients (18%) had intermittent PCR positive results. Seven (25%) patients presented at least two consecutive positive PCR results; one of these patients developed invasive aspergillosis and another two were strongly suspected as having aspergillosis. Based on the criteria of the European Organization for Research and Treatment of Cancer case definitions, sensitivity and specificity of serial PCR monitoring were 75% and 96%. Positive PCR results became negative shortly after commencement of antifungal treatment, but the changes did not correlate with clinical responsiveness to treatment in three patients. Our results indicate the potential usefulness of PCR for screening for Aspergillus spp. in patients at risk, but without antifungal treatment.

Induction of ERK‐kinase signalling triggers morphotype‐specific killing of Candida albicans filaments by human neutrophils

Candida albicans is among the most important fungal pathogens in humans. Morphological plasticity has been linked to its pathogenic potential as filamentous forms are associated with tissue invasion and infection. Here we show that human neutrophils discriminate between yeasts and filaments of C. albicans. Whereas filaments induced targeted motility, resulting in the establishment of close contact between neutrophils and fungal cells, yeast forms were largely ignored during coincubation. In transwell assays, C. albicans filaments induced significantly higher migratory activity in neutrophils than yeasts. Neutrophil motility based on actin rearrangement was essential for killing of C. albicans filaments but not involved in killing of yeast forms. Using inhibitors for MAP‐kinase cascades, it was shown that recognition of C. albicans filaments by neutrophils is mediated via the MEK/ERK cascade and independent of JNK or p38 activation. Inhibition of the ERK signalling pathway abolished neutrophil migration induced by C. albicans filaments and selectively impaired the ability to kill this morphotype. These data show that invasive filamentous forms of C. albicans trigger a morphotype‐specific activation of neutrophils, which is strongly dependent on neutrophil motility. Therefore, human neutrophils are capable of sensing C. albicans invasion and initiating an appropriate early immune response.

Human Natural Killer Cells Acting as Phagocytes Against Candida albicans and Mounting an Inflammatory Response That Modulates Neutrophil Antifungal Activity

BACKGROUND Natural killer (NK) cells are innate lymphocytes with potent cytotoxic activity. Whereas activity of NK cells has been demonstrated against the fungal pathogens Aspergillus fumigatus and Cryptococcus neoformans, little was known about their interaction with Candida albicans. METHODS Primary human NK cells were isolated from buffy coats, primed with a cytokine cocktail and used for confrontation assays with C. albicans. Interaction was monitored and quantified using live cell imaging, confocal microscopy, flow cytometry, and enzyme-linked immunosorbent assay. RESULTS Human NK cells actively recognized C. albicans, resulting in degranulation and secretion of granulocyte-macrophage colony-stimulating factor, interferon γ, and tumor necrosis factor α . Uniquely, activation of NK cells was triggered by actin-dependent phagocytosis. Antifungal activity of NK cells against C. albicans could be detected and mainly attributed to secreted perforin. However, NK cells were unable to inhibit filamentation of C. albicans. Human polymorphonuclear neutrophils (PMNs) counteracted the proinflammatory reaction of NK cells by preventing direct contact between NK cells and the fungal pathogen. Activation of PMNs was enhanced in the presence of NK cells, resulting in increased fungicidal activity. CONCLUSIONS Our results show a unique pattern of NK cell interaction with C. albicans, which involves direct proinflammatory activation and modulation of PMN activity. For the first time, phagocytosis of a pathogen is shown to contribute to NK cell activation.

Cytomegalovirus Monitoring by Polymerase Chain Reaction of Whole Blood Samples from Patients Undergoing Autologous Bone Marrow or Peripheral Blood Progenitor Cell Transplantation

Sensitive screening for cytomegalovirus (CMV) by polymerase chain reaction (PCR) following autologous bone marrow or peripheral blood progenitor cell transplantation has not been evaluated. In a three-center study, 98 autograft transplant recipients were prospectively screened for CMV infection by PCR and culture techniques. At a median of 20 days (range, 3-28) after transplantation, 21 (39.6%) of 53 CMV-seronegative patients were PCR positive for CMV, and at a median of 17 days (range, 7-84) after transplantation, 19 (42.2%) of 45 CMV-seropositive patients were PCR positive for CMV. Low-level DNAemia (1-10 fg CMV DNA/mL blood) occurred for 1 week in 31 patients but was never associated with CMV disease. Of 9 patients who presented with at least two consecutive positive PCR results, 1 developed CMV pneumonia. No patients died because of CMV disease. Screening for CMV infection by PCR had a negative predictive value of 100% (as also observed after allogeneic transplantation), but its positive predictive value was significantly lower.

Minireview: host defence in invasive aspergillosis.

Aspergillus is a saprophytic fungus, which mainly becomes pathogenic in immunosuppressed hosts. A failure of host defences results in a diverse set of illnesses, ranging from chronic colonisation, aspergilloma, invasive disease and hypersensitivity. A key concept in immune responses to Aspergillus species is that host susceptibility determines the morphological form, antigenic structure and physical location of the fungus. Traditionally, innate immunity has been considered as a first line of defence and activates adaptive immune mechanisms by the provision of specific signals; innate and adaptive immune responses are intimately linked. The T‐helper cell (TH1) response is associated with increased production of inflammatory cytokines IFN‐γ, IL‐2 and IL‐12 and stimulation of antifungal effector cells. Alternatively, TH2‐type responses are associated with suppression of antifungal effector cell activity, decreased production of IFN‐γ and increased concentrations of IL‐4 and IL‐10, which promote humoral responses to Aspergillus. The host’s defensive capacity is defined by the sum of resistance and tolerance. Resistance displays the ability to limit fungal burden and elimination of the pathogen, and tolerance means the ability to limit host damage caused by immune response.

High Variability between Results of Different In-House Tests for Cytomegalovirus (CMV) Monitoring and a Standardized Quantitative Plasma CMV PCR Assay

ABSTRACT A total of 2,718 blood samples were analyzed in five virological laboratories for the presence of cytomegalovirus (CMV) by in-house tests and one standardized plasma PCR assay. Results from in-house tests showed remarkable variability. Detection of CMV pp65 antigen or DNA from cells was more sensitive than that by plasma CMV PCR assay.

The glycosylphosphatidylinositol-anchored protease Sap9 modulates the interaction of Candida albicans with human neutrophils.

ABSTRACT Human polymorphonuclear neutrophils (PMNs) play a major role in the immune defense against invasive Candida albicans infection. This fungal pathogen produces a set of aspartic proteases that directly contributes to virulence properties such as adhesion, tissue invasion, and immune evasion. We show here that, in contrast to other secreted proteases, the cell surface-associated isoform Sap9 has a major impact on the recognition of C. albicans by PMNs. SAP9 is required for the induction of PMN chemotaxis toward C. albicans filaments, an essential prerequisite of effective PMN activation. Furthermore, deletion of SAP9 leads to a mitigated release of reactive oxygen intermediates (ROI) in human PMNs and decreases C. albicans-induced apoptosis triggered by ROI formation. In confrontation assays, killing of a SAP9 deletion mutant is reduced in comparison to wild-type C. albicans. These data clearly implicate Sap9 protease activity in the initiation of protective innate immunity and suggest novel molecular mechanisms in C. albicans-host interaction leading to neutrophil activation.

Modulation of Glucose Metabolism Inhibits Hypoxic Accumulation of Hypoxia-Inducible Factor-1α (HIF-1α)

Background and Purpose:The hypoxic accumulation of the transcription factor subunit hypoxia-inducible factor-1α (HIF-1α), a potential endogenous hypoxia marker and therapeutic target, has recently been shown to strongly depend on glucose availability. The aim of this study was to investigate the underlying mechanism of this effect.Material and Methods:HIF-1α protein levels were studied by Western blotting in HT 1080 human fibrosarcoma cells and in a hypoxia-responsive element green fluorescent protein (HRE-GFP) reporter assay in stably transfected HT 1080 cells treated with hypoxia (0.1% O2, 12 h) and glycolysis inhibitors 2-deoxyglucose (2-DG) or iodoacetate (IAA). HIF-1α mRNA expression was quantified via real-time polymerase chain reaction (RT-PCR).Results:Both inhibitors drastically reduced hypoxic HIF-1α accumulation (2-DG + hypoxia 2% mean HIF-1α protein level vs. 59% hypoxia alone; IAA + hypoxia 13% mean HIF-1α protein level vs. 96% hypoxia alone), an effect not rescued by the addition of pyruvate and confirmed in an HRE-GFP reporter assay in stably transfected HT 1080 cells. RT-PCR under identical conditions showed no effect of glycolysis inhibition on HIF-1α mRNA levels, suggesting a translational or posttranslational mechanism.Conclusion:The effect of glycolysis modulation on the HIF-1α levels in tumor cells may provide a novel approach to therapeutically target HIF-1α.Hintergrund und Ziel:Die hypoxische Akkumulation des Hypoxia-inducible factor-1α (HIF-1α), eines potentiellen Hypoxiemarkers und therapeutischen Targets, ist in hohem Maße von der Glucoseverfügbarkeit abhängig. Ziel dieser Arbeit war es, den zugrundeliegenden Mechanismus zu untersuchen.Material und Methodik:Das Protein HIF-1α wurde in humanen HT-1080-Fibrosarkomzellen nach Behandlung der Zellen mit den Glykolyseinhibitoren 2-Desoxy-D-Glucose (2-DG) und Iodacetat (IAA) unter hypoxischen Bedingungen (0,1% O2, 12 h) mittels Western-Blot und mit einem HRE-GFP-(„hypoxia-responsive element green fluorescent protein“-)Reporter-Assay in stabil transfizierten HT-1080-Zellen detektiert. Die Expressionsrate der HIF-1α-mRNA wurde mit Real-Time-Polymerase-Kettenreaktion (RTPCR) gemessen.Ergebnisse:Nach Zugabe von Glykolyseinhibitoren zeigte sich eine deutlich verringerte hypoxische HIF-1α-Akkumulation (Abbildungen 1, 2 und 5: 2-DG + Hypoxie: 2% mittleres HIF-1α-Protein-Level vs. 59% Hypoxie ohne 2-DG; IAA + Hypoxie: 13% mittleres HIF-1α-Protein-Level vs. 96% Hypoxie ohne IAA), die durch die Zugabe von Pyruvat nicht rückgängig gemacht werden konnte (Abbildung 3). Diese Ergebnisse wurden mit einem HRE-GFP-Reporter-Assay in stabil transfizierten HT-1080-Zellen bestätigt (Abbildung 4). Die HIF-1α-RT-PCR zeigte keine veränderten Expressionsraten von HIF-1α-mRNA nach Zugabe von IAA bzw. 2-DG (Abbildung 6). Diese Daten deuten auf einen translationalen bzw. posttranslationalen Mechanismus der Inhibition der hypoxischen HIF-1a-Akkumulation durch Glykolyseinhibitoren hin.Schlussfolgerung:Der Einfluss einer Modulation der Glykolyse auf HIF-1α-Level in Tumorzellen könnte einen neuen Ansatz einer HIF-1α-gerichteten Therapie darstellen.

The Fungal Quorum-Sensing Molecule Farnesol Activates Innate Immune Cells but Suppresses Cellular Adaptive Immunity

ABSTRACT Farnesol, produced by the polymorphic fungus Candida albicans, is the first quorum-sensing molecule discovered in eukaryotes. Its main function is control of C. albicans filamentation, a process closely linked to pathogenesis. In this study, we analyzed the effects of farnesol on innate immune cells known to be important for fungal clearance and protective immunity. Farnesol enhanced the expression of activation markers on monocytes (CD86 and HLA-DR) and neutrophils (CD66b and CD11b) and promoted oxidative burst and the release of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α] and macrophage inflammatory protein 1 alpha [MIP-1α]). However, this activation did not result in enhanced fungal uptake or killing. Furthermore, the differentiation of monocytes to immature dendritic cells (iDC) was significantly affected by farnesol. Several markers important for maturation and antigen presentation like CD1a, CD83, CD86, and CD80 were significantly reduced in the presence of farnesol. Furthermore, farnesol modulated migrational behavior and cytokine release and impaired the ability of DC to induce T cell proliferation. Of major importance was the absence of interleukin 12 (IL-12) induction in iDC generated in the presence of farnesol. Transcriptome analyses revealed a farnesol-induced shift in effector molecule expression and a down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor during monocytes to iDC differentiation. Taken together, our data unveil the ability of farnesol to act as a virulence factor of C. albicans by influencing innate immune cells to promote inflammation and mitigating the Th1 response, which is essential for fungal clearance. IMPORTANCE Farnesol is a quorum-sensing molecule which controls morphological plasticity of the pathogenic yeast Candida albicans. As such, it is a major mediator of intraspecies communication. Here, we investigated the impact of farnesol on human innate immune cells known to be important for fungal clearance and protective immunity. We show that farnesol is able to enhance inflammation by inducing activation of neutrophils and monocytes. At the same time, farnesol impairs differentiation of monocytes into immature dendritic cells (iDC) by modulating surface phenotype, cytokine release and migrational behavior. Consequently, iDC generated in the presence of farnesol are unable to induce proper T cell responses and fail to secrete Th1 promoting interleukin 12 (IL-12). As farnesol induced down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, desensitization to GM-CSF could potentially explain transcriptional reprofiling of iDC effector molecules. Taken together, our data show that farnesol can also mediate Candida-host communication and is able to act as a virulence factor. Farnesol is a quorum-sensing molecule which controls morphological plasticity of the pathogenic yeast Candida albicans. As such, it is a major mediator of intraspecies communication. Here, we investigated the impact of farnesol on human innate immune cells known to be important for fungal clearance and protective immunity. We show that farnesol is able to enhance inflammation by inducing activation of neutrophils and monocytes. At the same time, farnesol impairs differentiation of monocytes into immature dendritic cells (iDC) by modulating surface phenotype, cytokine release and migrational behavior. Consequently, iDC generated in the presence of farnesol are unable to induce proper T cell responses and fail to secrete Th1 promoting interleukin 12 (IL-12). As farnesol induced down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, desensitization to GM-CSF could potentially explain transcriptional reprofiling of iDC effector molecules. Taken together, our data show that farnesol can also mediate Candida-host communication and is able to act as a virulence factor.