Transcriptional Responses in Mouse Models of Invasive Pulmonary Aspergillosis
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ABSTRACT: Abstract From manuscript: Incidences of invasive pulmonary aspergillosis, an infection caused predominantly by Aspergillus fumigatus, have increased due to the growing number of immunocompromised individuals. While A. fumigatus is reliant upon deficiencies in the host to facilitate invasive disease, the distinct mechanisms that govern the host-pathogen interaction remain enigmatic, particularly in the context of distinct immune modulating therapies. To gain insights into these mechanisms, RNA-Seq technology was utilized to sequence RNA derived from lungs of 2 clinically relevant, but immunologically distinct murine models of IPA on days 2 and 3 post inoculation when infection is established and active disease present. Our findings identify notable differences in host gene expression between the chemotherapeutic and steroid models at the interface of immunity and metabolism. RT-qPCR verified model specific and nonspecific expression of 23 immune-associated genes. Deep sequencing facilitated identification of highly expressed fungal genes. We utilized sequence similarity and gene expression to categorize the A. fumigatus putative in vivo secretome. RT-qPCR suggests model specific gene expression for nine putative fungal secreted proteins. Our analysis identifies contrasting responses by the host and fungus from day 2 to 3 between the two models. These differences may help tailor the identification, development, and deployment of host- and/or fungal-targeted therapeutics.
Project description:In lung diseases caused by the major mould pathogen Aspergillus fumigatus the pulmonary epithelium is destroyed by invasive growth of fungal hyphae, a process thought to require fungal proteases. Here we show that the A. fumigatus pH-responsive transcription factor PacC governs expression of secreted proteases during invasive lung infections and is required for epithelial invasion and pathogenicity. In addition, A. fumigatus M-NM-^TpacC mutants aberrantly remodel the fungal cell wall during infection. This study defines distinct PacC-mediated mechanisms of host damage during pulmonary aspergillosis. ch1: treatment protocol Temporal transcriptional profiling of ATCC46645 strain and isogenic M-NM-^TpacC Aspergillus fumigatus mutant during murine infection
Project description:In lung diseases caused by the major mould pathogen Aspergillus fumigatus the pulmonary epithelium is destroyed by invasive growth of fungal hyphae, a process thought to require fungal proteases. Here we show that the A. fumigatus pH-responsive transcription factor PacC governs expression of secreted proteases during invasive lung infections and is required for epithelial invasion and pathogenicity. In addition, A. fumigatus ΔpacC mutants aberrantly remodel the fungal cell wall during infection. This study defines distinct PacC-mediated mechanisms of host damage during pulmonary aspergillosis. ch1: treatment protocol
Project description:Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. We illustrate by modeling the active, but so far neglected, major role of alveolar epithelial cells in phagocytosis and cytokine release as well as the importance of fungal growth states for virulence.
Hence, we discovered that germination speed is a key virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. We proved this by linking measured germination kinetics of four Aspergillus spp. with their cytotoxicity against epithelial cells in silico and in vitro.Furthermore, we could reveal by modeling and ex vivo measurements, that epithelial cells are not only important phagocytes to clear conidia, but also potent mediators of cytokine release.
In conclusion, our findings illustrate an underestimated role of epithelial cells in invasive aspergillosis. Further, our model affirms the importance of neutrophils and underlines that the role of macrophages in invasive aspergillosis remains elusive.
We expect that our model will contribute to improvement of treatment protocols by focusing on
the critical components of immune response to fungi but also fungal virulence.
Project description:Invasive aspergillosis (IA) is a devastating opportunistic infection and its treatment constitutes a considerable burden for the health care system. Immunocompromised patients are at an increased risk for IA, which is mainly caused by the species Aspergillus fumigatus. An early and reliable diagnosis is required to initiate the appropriate antifungal therapy. However, diagnostic sensitivity and accuracy still needs to be improved, which can be achieved at least partly by the definition of new biomarkers. Besides the direct detection of the pathogen by the current diagnostic methods, the analysis of the host response is a promising strategy towards this aim. Following this approach, we sought to identify new biomarkers for IA. For this purpose, we analyzed gene expression profiles of haematological patients and compared profiles of patients suffering from IA with non-IA patients. Based on microarray data, we applied a comprehensive feature selection using a random forest classifier. We identified the transcript coding for the S100 calcium-binding protein B (S100B) as a potential new biomarker for the diagnosis of IA. Considering the expression of this gene, we were able to classify samples from patients with IA with 82.3% sensitivity and 74.6% specificity. Moreover, we validated the expression of S100B in a real-time RT-PCR assay and we also found a down-regulation of S100B in A. fumigatus stimulated DCs. An influence on the IL1B and CXCL1 downstream levels was demonstrated by this S100B knockdown. In conclusion, this study covers an effective feature selection revealing a key regulator of the human immune response during IA. S100B may represent an additional diagnostic marker that in combination with the established techniques may improve the accuracy of IA diagnosis. The gene expression data of patients with invasive aspergillosis (IA) was compared to patients without IA. 23 samples of 8 IA patients, 13 samples from 7 unclassified patients and 1 patient with possible invasive fungal disease (IFD), and 9 control samples from 8 healthy donors were generated. Patients were classified according to the current EORTC/MSG criteria.
Project description:Treatment of post-transplant patients with immunosuppressive drugs targeting the calcineurin-NFAT pathway, such as Cyclosporine A or Tacrolimus, are commonly associated with a higher incidence of opportunistic infections, such as Aspergillus fumigatus, which can lead to severe life-threating conditions. A component of the A. fumigatus cell wall, β-glucan, is recognized by dendritic cells via the Dectin-1 receptor, triggering downstream signaling that leads to calcineurin-NFAT binding, NFAT translocation, and transcription of NFAT-regulated genes. Here, we address the question of whether calcineurin signaling in CD11c-expressing cells, such as DCs, has a specific role in the innate control of A. fumigatus. Impairment of calcineurin in CD11c-expressing cells (CD11ccrecnb1loxP) significantly increased susceptibility to systemic A. fumigatus infection and to intranasal infection in irradiated mice undergoing bone marrow transplant. Global expression profiling of bone marrow-derived DCs identified calcineurin-regulated processes in the immune response to infection, including expression of pentraxin-3, an important anti-fungal defense protein. These results suggest that calcineurin inhibition directly impairs important immunoprotective functions of myeloid cells, as shown by the higher susceptibility of CD11ccrecnbloxP mice in models of systemic and invasive pulmonary aspergillosis, including after allogeneic bone marrow transplantation. These findings are relevant to the clinical management of transplant patients with severe Aspergillus infections.
Project description:Aspergillus fumigatus is an opportunistic fungal pathogen of the respiratory system that may cause invasive infection or an allergic response. Monocytoid dendritic cells (moDCs) are a known recruited cell population during fungal colonization and are believed to be a critical player in balancing innate versus adaptive immune responses. We conducted an RNA-Seq time course analysis of moDCs challenged against A. fumigatus, to gain an appreciation of gene expression changes. We concurrently utilized Nlrx1 deficient moDCs as we have recently shown the importance of Nlrx1 during invasive pulmonary aspergillosis and shown the absence of Nlrx1 results in a robust shift towards a detrimental Th2 response. Our findings suggest a decision tree by moDCs in response to A. fumigatus viable conidia that is absent in Nlrx1-/- moDCs.
Project description:Comprehensive proteomic analysis of the protein expression landscape of bronchoalveolar lavage fluid during invasive pulmonary aspergillosis in murine and human samples. 38 murine BALF samples (10 Aspergillus fumigatus infected mice without immunosuppression and without invasive pulmonary aspergillosis (IPA), 19 immunosuppressed and infected mice with IPA and 9 immunosuppressed animals without infection) were analysed for their global protein expression. In addition, 54 human BALF specimen from patients with probable IPA (23 samples), proven IPA (4 cases) and 27 control samples from patients with unrelated pulmonary diseases were analysed for their global protein composition. Host responses and Aspergillus fumigatus-specific proteins detectable in BALF were studied.
Project description:Invasive aspergillosis (IA) is a devastating opportunistic infection and its treatment constitutes a considerable burden for the health care system. Immunocompromised patients are at an increased risk for IA, which is mainly caused by the species Aspergillus fumigatus. An early and reliable diagnosis is required to initiate the appropriate antifungal therapy. However, diagnostic sensitivity and accuracy still needs to be improved, which can be achieved at least partly by the definition of new biomarkers. Besides the direct detection of the pathogen by the current diagnostic methods, the analysis of the host response is a promising strategy towards this aim. Following this approach, we sought to identify new biomarkers for IA. For this purpose, we analyzed gene expression profiles of haematological patients and compared profiles of patients suffering from IA with non-IA patients. Based on microarray data, we applied a comprehensive feature selection using a random forest classifier. We identified the transcript coding for the S100 calcium-binding protein B (S100B) as a potential new biomarker for the diagnosis of IA. Considering the expression of this gene, we were able to classify samples from patients with IA with 82.3% sensitivity and 74.6% specificity. Moreover, we validated the expression of S100B in a real-time RT-PCR assay and we also found a down-regulation of S100B in A. fumigatus stimulated DCs. An influence on the IL1B and CXCL1 downstream levels was demonstrated by this S100B knockdown. In conclusion, this study covers an effective feature selection revealing a key regulator of the human immune response during IA. S100B may represent an additional diagnostic marker that in combination with the established techniques may improve the accuracy of IA diagnosis.
Project description:Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections including invasive aspergillosis. We aimed to understand molecular targets of AMB in Aspergillus fumigatus (Afu) by genomic approaches. Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections including invasive aspergillosis. We aimed to understand molecular targets of AMB in Aspergillus fumigatus (Afu) by microarray and proteomic methods. Keywords: Aspergillus fumigatus treated with amphotericin B for 24 hours Experiment was performed in dye swap manner from two different biological replicates
Project description:Fungal infections remain a tremendous source of global morbidity and mortality, with more than 1 billion individuals affected by fungal infections each year. Invasive infections are particularly dangerous and kill numbers on par with tuberculosis or malaria. Aspergillus fumigatus is a major source of invasive fungal infections in humans, and is particularly dangerous to the immunocompromised. In many cases, research into invasive fungal infections is limited by a lack of model systems. In this study we hypothesized that cultured, differentiated human PLB-985 neutrophil-like cells could serve as model system to study the host-pathogenesis of A. fumigatus. We tested this hypothesis by defining the phagocytosis and intracellular trafficking of A. fumigatus conidia by PLB-985 cells, the production of neutrophil extracellular traps, and by characterizing extracellular vesicles produced in response to infection. As part of this analysis, we performed a detailed LC-MS/MS-based proteomic comparison of extracellular vesicles isolated by two different methods, centrifugation-based isolation or size-exclusion chromatography. We find numerous similarities between primary neutrophils and PLB-985 cells that suggest differentiated PLB-985 cells can serve as a tractable in vitro model system for further study of many aspects of A. fumigatus pathogenesis.