The Lipid Raft-Associated Protein Stomatin Is Required for Accumulation of Dectin-1 in the Phagosomal Membrane and for Full Activity of Macrophages against Aspergillus fumigatus.

Alveolar macrophages belong to the first line of defense against inhaled conidia of the human-pathogenic fungus Aspergillus fumigatus. In lung alveoli, they contribute to phagocytosis and elimination of conidia. As a counterdefense, conidia have a gray-green pigment that enables them to survive in phagosomes of macrophages for some time. Previously, we showed that this conidial pigment interferes with the formation of flotillin-dependent lipid raft microdomains in the phagosomal membrane, thereby preventing the formation of functional phagolysosomes. Besides flotillins, stomatin is a major component of lipid rafts and can be targeted to the membrane. However, only limited information on stomatin is available, in particular on its role in defense against pathogens. To determine the function of this integral membrane protein, a stomatin-deficient macrophage line was generated by CRISPR/Cas9 gene editing. Immunofluorescence microscopy and flow cytometry revealed that stomatin contributes to the phagocytosis of conidia and is important for recruitment of the ß-glucan receptor dectin-1 to both the cytoplasmic membrane and phagosomal membrane. In stomatin knockout cells, fusion of phagosomes and lysosomes, recruitment of the vATPase to phagosomes, and tumor necrosis factor alpha (TNF-α) levels were reduced when cells were infected with pigmentless conidia. Thus, our data suggest that stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. IMPORTANCE Stomatin is an integral membrane protein that contributes to the uptake of microbes, e.g., spores of the human-pathogenic fungus Aspergillus fumigatus. By generation of a stomatin-deficient macrophage line by advanced genetic engineering, we found that stomatin is involved in the recruitment of the ß-glucan receptor dectin-1 to the phagosomal membrane of macrophages. Furthermore, stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. The data provide new insights on the important role of stomatin in the immune response against human-pathogenic fungi.

The TLR-NF-kB axis contributes to the monocytic inflammatory response against a virulent strain of Lichtheimia corymbifera, a causative agent of invasive mucormycosis.

Invasive mucormycosis (IM) is a life-threatening infection caused by the fungal order Mucorales, its diagnosis is often delayed, and mortality rates range from 40-80% due to its rapid progression. Individuals suffering from hematological malignancies, diabetes mellitus, organ transplantations, and most recently COVID-19 are particularly susceptible to infection by Mucorales. Given the increase in the occurrence of these diseases, mucormycosis has emerged as one of the most common fungal infections in the last years. However, little is known about the host immune response to Mucorales. Therefore, we characterized the interaction among L. corymbifera-one of the most common causative agents of IM-and human monocytes, which are specialized phagocytes that play an instrumental role in the modulation of the inflammatory response against several pathogenic fungi. This study covered four relevant aspects of the host-pathogen interaction: i) The recognition of L. corymbifera by human monocytes. ii) The intracellular fate of L. corymbifera. iii) The inflammatory response by human monocytes against the most common causative agents of mucormycosis. iv) The main activated Pattern-Recognition Receptors (PRRs) inflammatory signaling cascades in response to L. corymbifera. Here, we demonstrate that L. corymbifera exhibits resistance to intracellular killing over 24 hours, does not germinate, and inflicts minimal damage to the host cell. Nonetheless, viable fungal spores of L. corymbifera induced early production of the pro-inflammatory cytokine IL-1ß, and late release of TNF-α and IL-6 by human monocytes. Moreover, we revealed that IL-1ß production predominantly depends on Toll-like receptors (TLRs) priming, especially via TLR4, while TNF-α is secreted via C-type lectin receptors (CTLs), and IL-6 is produced by synergistic activation of TLRs and CTLs. All these signaling pathways lead to the activation of NF-kB, a transcription factor that not only regulates the inflammatory response but also the apoptotic fate of monocytes during infection with L. corymbifera. Collectively, our findings provide new insights into the host-pathogen interactions, which may serve for future therapies to enhance the host inflammatory response to L. corymbifera.

Human mucosal-associated invariant T cells respond to Mucorales species in a MR1-dependent manner.

Activation of mucosal-associated invariant T cells (MAIT cells) by certain bacteria, viruses, and yeast is well studied, but the activation potential of filamentous moulds from the order Mucorales is not known. Here, we show a rapid response of human MAIT cells against the Mucorales species Mucor circinelloides, Rhizopus arrhizus, and Rhizopus microsporus. This activation included upregulation of CD69 and degranulation marked by increased CD107a expression, while intracellular perforin and granzyme A expression were reduced. Furthermore, blocking of the antigen-presenting molecule major histocompatibility complex class I-related abrogated MAIT cell activation demonstrating a T cell receptor-dependent stimulation by Mucorales.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Fast and Quantitative Evaluation of Human Leukocyte Interaction with Aspergillus fumigatus Conidia by Flow Cytometry.

Systemic infections with the opportunistic mold Aspergillus fumigatus are a great threat to immunocompromised patients such as transplant recipients. Immunological research on A. fumigatus involves the measurement of phagocytosis of fungal conidia (spores) by human phagocytes. Here, we present a fast and flexible way to analyze phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC) labeling of conidia prior to co-incubation with human leukocytes and an anti-FITC counterstaining step postincubation to allow the discrimination of internalized and adherent conidia. In contrast to many other protocols, this method can be combined with further surface marker analyses. We sought to determine phagocytosis rates of A. fumigatus conidia in different stages and after several incubation times using this method. Moreover, we provide an example of application by comparing phagocytosis of A. fumigatus mutants to the wild type. © 2018 International Society for Advancement of Cytometry.

Human MAIT cells are rapidly activated by Aspergillus spp. in an APC-dependent manner.

Mucosal associated invariant T cells (MAIT cells) are innate-like T cells (TC) which are known to be activated by several bacteria and viruses. However, activation of MAIT cells by moulds, such as the opportunistic human pathogen Aspergillus, is not well described. Stimulation of human PBMC with A. fumigatus, A. flavus, or A. terreus conidia revealed that in contrast to conventional CD4+ and CD8+ TC, MAIT cells responded already after 4 h of coincubation with upregulation of CD69. Furthermore, concurrent increase of CD107a expression and reduced intracellular expression of cytolytic proteins like perforin and granzyme indicated degranulation of intracellular vesicles. MAIT cell activation only occurred in the presence of APC and was dependent on cell-cell contact as separation of TC and APC abrogated MAIT cell activation. Furthermore, we observed that MAIT cell activation by moulds requires presentation of riboflavin metabolites and depends on TCR engagement as antibody blocking of MR1, the antigen presenting molecule for MAIT cells, prevented upregulation of CD69 and CD107a. In summary, we could demonstrate that MAIT cells are activated by Aspergillus conidia in a TCR-dependent manner by APC. These findings reveal MAIT cells as an interesting new target in antifungal defense.

Fast and efficient multitransgenic modification of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) represent a prime cell source for pharmacological research and regenerative therapies because of their extensive expansion potential and their ability to differentiate into essentially all somatic lineages in vitro. Improved methods to stably introduce multiple transgenes into hPSCs will promote, for example, their preclinical testing by facilitating lineage differentiation and purification in vitro and the subsequent in vivo monitoring of respective progenies after their transplantation into relevant animal models. To date, the establishment of stable transgenic hPSC lines is still laborious and time-consuming. Current limitations include the low transfection efficiency of hPSCs via nonviral methods, the inefficient recovery of genetically engineered clones, and the silencing of transgene expression. Here we describe a fast, electroporation-based method for the generation of multitransgenic hPSC lines by overcoming the need for any preadaptation of conventional hPSC cultures to feeder-free conditions before genetic manipulation. We further show that the selection for a single antibiotic resistance marker encoded on one plasmid allowed for the stable genomic (co-)integration of up to two additional, independent expression plasmids. The method thereby enables the straightforward, nonviral generation of valuable multitransgenic hPSC lines in a single step. Practical applicability of the method is demonstrated for antibiotic-based lineage enrichment in vitro and for sodium iodide symporter transgene-based in situ cell imaging after intramyocardial cell infusion into explanted pig hearts.

Promoter and lineage independent anti-silencing activity of the A2 ubiquitous chromatin opening element for optimized human pluripotent stem cell-based gene therapy.

Epigenetic silencing of retroviral transgene expression in pluripotent stem cells (PSC) and their differentiated progeny constitutes a major roadblock for PSC-based gene therapy. As ubiquitous chromatin opening elements (UCOEs) have been successfully employed to stabilize transgene expression in murine hematopoietic and pluripotent stem cells as well as their differentiated progeny, we here investigated UCOE activity in their human counterparts to establish a basis for future clinical application of the element. To this end, we demonstrate profound anti-silencing activity of the A2UCOE in several human iPS and ES cell lines including their progeny obtained upon directed cardiac or hematopoietic differentiation. We also provide evidence for A2UCOE activity in murine iPSC-derived hepatocyte-like cells, thus establishing efficacy of the element in cells of different germ layers. Finally, we investigated combinations of the A2UCOE with viral promoter/enhancer elements again demonstrating profound stabilization of transgene expression. In all these settings the effect of the A2UCOE was associated with strongly reduced promoter DNA-methylation. Thus, our data clearly support the concept of the A2UCOE as a generalized strategy to prevent epigenetic silencing in PSC and their differentiated progeny and strongly favors its application to stabilize transgene expression in PSC-based cell and gene therapy approaches.

Directing cardiomyogenic differentiation of human pluripotent stem cells by plasmid-based transient overexpression of cardiac transcription factors.

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) possess a high potential for regenerative medicine. Previous publications suggested that viral transduction of a defined set of transcription factors (TFs) known to play pivotal roles in heart development also increases cardiomyogenesis in vitro upon overexpression in mouse or human ES cells. To circumvent issues associated with viral approaches such as insertional mutagenesis, we have established a transient transfection system for straightforward testing of TF combinations. Applying this method, the transfection efficiency and the temporal pattern of transgene expression were extensively assessed in hPSCs by quantitative real time-polymerase chain reaction (qRT-PCR), TF-specific immunofluorescence analysis, and flow cytometry. Testing TF combinations in our approach revealed that BAF60C, GATA4, and MESP1 (BGM) were most effective for cardiac forward programming in human induced pluripotent stem cell lines and human ES cells as well. Removal of BAF60C slightly diminished formation of CM-like cells, whereas depletion of GATA4 or MESP1 abolished cardiomyogenesis. Each of these TFs alone had no inductive effect. In addition, we have noted sensitivity of CM formation to cell density effects, which highlights the necessity for cautious analysis when interpreting TF-directed lineage induction. In summary, this is the first report on TF-induced cardiomyogenesis of hPSCs applying a transient, nonintegrating method of cell transfection.