measuring-phagocytosis-aspergillus-fumigatus-conidia-human-leukocytes

Measuring Phagocytosis of <em>Aspergillus fumigatus</em> 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 ...

Jena University Hospital

An 11-year-old male patient suffering mixed connective tissue disease with life-threatening pulmonary arterial hypertension, progressive heart failure (New York Heart Association class III-IV), skin ulcers, Raynaud's phenomenon and arthritis, showing no improvement after intensive immunosuppressive therapy or high dose steroids, was treated with immunoadsorption onto protein A. With a combined therapy of low-dose cortisone and bosentan and 22 sessions of immunoadsorption, his condition improved significantly and he continues in clinical remission. At the time of writing no further immunosuppressive therapy or immunoadsorption had been necessary. The patient is now 15 years old and healthy with an age-based constitution comparable to the normal population.

A case report of successful treatment with immunoadsorption onto protein A in mixed connective tissue disease in childhood.

Immunological problems like preformed donor specific antibodies (DSA) or high degree of immunization complicate the transplantation (TX) and can limit the therapeutic success. Essential pillars of antihumoral therapy are the extracorporeal procedures like therapeutic plasma exchange (TPE) and high plasma volume immunoadsorption (IA). Both extracorporeal treatments have the ability to remove pre-existing or newly formed antibodies quickly and effectively. In the last 5 years a total of 27 patients, 12 heart transplant (HTX) patients, 3 patients after combined lung and heart transplantation (Lu/HTX) and 12 lung transplant (LuTX) patients were treated for prevention (6 patients) or in case of rejection (20 patients) with TPE, cascade filtration (CF) or IA. 6 HTX recipients and 5 LuTX recipients were tested positive for HLA-ab either donor specific or de novo HLA-ab. Altogether 7 patients were tested positive for non-HLA-ab. 6 patients were treated prior to TX with 1-3 TPEs. After TX we treated 24 patients initially with TPE and performed on average 4 TPEs (1-11) for antibody elimination. 22 of 27 treated patients survived with stable graft function. The extracorporeal procedures we performed are qualified for rescue therapy of antibody mediated rejection (AMR).

Therapeutic apheresis in transplantation medicine, experience with cardiac and lung transplantation in Jena.

Monocytes are an important constituent of the innate immune system. Therefore, manipulating gene expression of primary human monocytes is a crucial mean to study and characterize the functions of targeted proteins in monocytes. Gene silencing by transfection of cells with small interfering RNA (siRNA) leading to the degradation of the corresponding mRNA and thus to reduced target protein levels is an important tool to investigate gene and protein function of interest. However, non-viral transfection of primary monocytes is challenging because siRNA uptake by these suspended cells is tricky, and the individual cells vary among different donors and do not proliferate. Here, we describe a procedure for efficient non-viral transfection of primary human monocytes isolated from peripheral blood, which maintains cell viability and cell functions, such as responsiveness to stimuli like LPS and IL-10. Nucleofection was used as an electroporation technique that enables efficient introduction of siRNA and silencing of target genes. Using a modification of our previously published protocol for the fast-proliferating THP-1 monocytic cell line, we transfected primary human monocytes with siRNA targeting 5-lipoxygenase (5-LO). In fact, we successfully downregulated 5-LO mRNA resulting in reduced protein levels and enzymatic activity. 

A procedure for efficient non-viral siRNA transfection of primary human monocytes using nucleofection.

5-Lipoxygenase (5-LO) catalyzes the initial steps in the biosynthesis of proinflammatory leukotrienes. Upon cell activation, 5-LO translocates to the nuclear membrane where arachidonic acid is transferred by 5-LO-activating protein (FLAP) to 5-LO for metabolism. Although previous data indicate association of 5-LO with FLAP, the in situ assembly of native 5-LO/FLAP complexes remains elusive. Here, we show time-resolved 5-LO/FLAP colocalization by immunofluorescence microscopy and in situ 5-LO/FLAP interaction by proximity ligation assay at the nuclear membrane of Ca(2+)-ionophore A23187-activated human monocytes and neutrophils in relation to 5-LO activity. Although 5-LO translocation and product formation is completed within 1.5-3 min, 5-LO/FLAP interaction is delayed and proceeds up to 30 min. Though monocytes and neutrophils contain comparable amounts of 5-LO protein, neutrophils produce 3-5 times higher levels of 5-LO products due to prolonged activity, accompanied by delayed 5-LO nuclear membrane translocation. Arachidonic acid seemingly acts as adaptor for 5-LO/FLAP assembly, whereas FLAP inhibitors (MK886, 100 nM; BAY X 1005, 3 µM) disrupt the complex. We conclude that FLAP may regulate 5-LO activity in 2 ways: first by inducing an initial flexible association for efficient 5-LO product synthesis, followed by the formation of a tight 5-LO/FLAP complex that terminates 5-LO activity.

Time-resolved in situ assembly of the leukotriene-synthetic 5-lipoxygenase/5-lipoxygenase-activating protein complex in blood leukocytes.

5-Lipoxygenase (5-LO) catalyzes the first two steps in leukotriene (LT) biosynthesis. Because LTs play pivotal roles in allergy and inflammation, 5-LO represents a valuable target for anti-inflammatory drugs. Here, we investigated the molecular mechanism, the pharmacological profile, and the in vivo effectiveness of the novel 1,2-benzoquinone-featured 5-LO inhibitor RF-22c. Compound RF-22c potently inhibited 5-LO product synthesis in neutrophils and monocytes (IC50⩾22nM) and in cell-free assays (IC50⩾140nM) without affecting 12/15-LOs, cyclooxygenase (COX)-1/2, or arachidonic acid release, in a specific and reversible manner, supported by molecular docking data. Antioxidant or iron-chelating properties were not evident for RF-22c and 5-LO-regulatory cofactors like Ca(2+) mobilization, ERK-1/2 activation, and 5-LO nuclear membrane translocation and interaction with 5-LO-activating protein (FLAP) were unaffected. RF-22c (0.1mg/kg; i.p.) impaired (I) bronchoconstriction in ovalbumin-sensitized mice challenged with acetylcholine, (II) exudate formation in carrageenan-induced paw edema, and (III) zymosan-induced leukocyte infiltration in air pouches. Taken together, RF-22c is a highly selective and potent 5-LO inhibitor in intact human leukocytes with pronounced effectiveness in different models of inflammation that warrants further preclinical analysis of this agent as anti-inflammatory drug.

The 5-lipoxygenase inhibitor RF-22c potently suppresses leukotriene biosynthesis in cellulo and blocks bronchoconstriction and inflammation in vivo.

Cysteinyl leukotrienes (cys-LTs) cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) yielding the unstable leukotriene A (LTA) that is subsequently conjugated with glutathione (GSH) by LTC synthase (LTCS). Cys-LT receptor antagonists and LTCS inhibitors have been developed, but only the former have reached the market. High structural homology to related enzymes and lack of convenient test systems due to instability of added LTA have hampered the development of LTCS inhibitors. We present smart cell-free and cell-based assay systems based on in situ-generated LTA that allow studying LTCS activity and investigating LTCS inhibitors. Co-incubations of microsomes from HEK293 cells expressing LTCS with isolated 5-LOX efficiently converted exogenous AA to LTC (~1.3μg/200μg protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTCS with Ca-ionophore A23187 and 20μM AA resulted in strong LTC formation (~250ng/10 cells). MK-886, a well-known 5-LOX activating protein (FLAP) inhibitor that also acts on LTCS, consistently inhibited LTC formation in all assay types (IC=3.1-3.5μM) and we successfully confirmed TK04a as potent LTCS inhibitor in these assay systems (IC=17 and 300nM, respectively). We demonstrated transcellular LTC biosynthesis between neutrophils or 5-LOX-expressing HEK293 cells that produce LTA from AA and HEK293 cells expressing LTCS that transform LTA to LTC. In conclusion, our assay approaches are advantageous as the substrate LTA is generated in situ and are suitable for studying enzymatic functionality of LTCS including site-directed mutations and evaluation of LTCS inhibitors.

Development of smart cell-free and cell-based assay systems for investigation of leukotriene C synthase activity and evaluation of inhibitors.

The pro-inflammatory leukotrienes (LTs) are formed from arachidonic acid (AA) in activated leukocytes, where 5-lipoxygenase (5-LO) translocates to the nuclear envelope to assemble a functional complex with the integral nuclear membrane protein 5-LO-activating protein (FLAP). FLAP, a MAPEG family member, facilitates AA transfer to 5-LO for efficient conversion, and LT biosynthesis critically depends on FLAP. Here we show that the novel LT biosynthesis inhibitor BRP-187 prevents the 5-LO/FLAP interaction at the nuclear envelope of human leukocytes without blocking 5-LO nuclear redistribution. BRP-187 inhibited 5-LO product formation in human monocytes and polymorphonuclear leukocytes stimulated by lipopolysaccharide plus N-formyl-methionyl-leucyl-phenylalanine (IC=7-10nM), and upon activation by ionophore A23187 (IC=10-60nM). Excess of exogenous AA markedly impaired the potency of BRP-187. Direct 5-LO inhibition in cell-free assays was evident only at >35-fold higher concentrations, which was reversible and not improved under reducing conditions. BRP-187 prevented A23187-induced 5-LO/FLAP complex assembly in leukocytes but failed to block 5-LO nuclear translocation, features that were shared with the FLAP inhibitor MK886. Whereas AA release, cyclooxygenases and related LOs were unaffected, BRP-187 also potently inhibited microsomal prostaglandin E synthase-1 (IC=0.2μM), another MAPEG member. In vivo, BRP-187 (10mg/kg) exhibited significant effectiveness in zymosan-induced murine peritonitis, suppressing LT levels in peritoneal exudates as well as vascular permeability and neutrophil infiltration. Together, BRP-187 potently inhibits LT biosynthesis in vitro and in vivo, which seemingly is caused by preventing the 5-LO/FLAP complex assembly and warrants further preclinical evaluation.

BRP-187: A potent inhibitor of leukotriene biosynthesis that acts through impeding the dynamic 5-lipoxygenase/5-lipoxygenase-activating protein (FLAP) complex assembly.

Vitamin A and vitamin D are essential nutrients with a wide range of pleiotropic effects in humans. Beyond their well-documented roles in cellular differentiation, embryogenesis, tissue maintenance and bone/calcium homeostasis, both vitamins have attracted considerable attention due to their association with-immunological traits. Nevertheless, our knowledge of their immunomodulatory potential during infection is restricted to single gene-centric studies, which do not reflect the complexity of immune processes. In the present study, we performed a comprehensive RNA-seq-based approach to define the whole immunomodulatory role of vitamins A and D during infection. Using human monocytes as host cells, we characterized the differential role of both vitamins upon infection with three different pathogens: Aspergillus fumigatus, Candida albicans and Escherichia coli. Both vitamins showed an unexpected ability to counteract the pathogen-induced transcriptional responses. Upon infection, we identified 346 and 176 immune-relevant genes that were regulated by atRA and vitD, respectively. This immunomodulatory activity was dependent on the inflammatory stimulus, allowing us to distinguish regulatory patterns which were specific for each stimulatory setting. Moreover, we explored possible direct and indirect mechanisms of vitamin-mediated regulation of the immune response. Our findings highlight the importance of vitamin-monitoring in critically ill patients. Moreover, our results underpin the potential of atRA and vitD as therapeutic options for anti-inflammatory treatment.

Differential Effects of Vitamins A and D on the Transcriptional Landscape of Human Monocytes during Infection.

Pharmacological interference with vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, is considered a potential strategy for cancer therapy. Macrophages are critically involved in tumor progression and may occur as pro-tumoral M2 phenotype, whereas classically-activated M1 can inhibit tumor development for example by releasing tumor-suppressing molecules, including tumor necrosis factor (TNF)α. Here, we show that targeting V-ATPase by selective inhibitors such as archazolid upregulates the expression and secretion of TNFα in lipopolysaccharide (LPS)- or LPS/interferon (INF)γ-activated M1-like macrophages derived from human blood monocytes. In contrast, archazolid failed to elevate TNFα production from uncommitted (M0) or interleukin (IL)-4-treated M2-like macrophages. Secretion of other relevant cytokines (i.e., IL-1β, IL-6, IL-10) or chemokines (i.e. IL-8 and monocyte chemotactic protein-1) from M1 was not affected by archazolid. Though V-ATPase inhibitors elevated the lysosomal pH in M1 comparable to chloroquine or ammonium chloride, the latter agents suppressed TNFα secretion. Archazolid selectively increased TNFα mRNA levels, which was abolished by dexamethasone. Interestingly, archazolid enhanced the phosphorylation and nuclear translocation of the p65 subunit of NFκB and stimulated phosphorylation of SAPK/JNK. In a microfluidically-supported human tumor biochip model, archazolid-treated M1 significantly reduced tumor cell viability. Together, our data show that V-ATPase inhibition selectively upregulates TNFα production in classically-activated macrophages along with NFκB and SAPK/JNK activation. Such increased TNFα release caused by V-ATPase inhibitors may contribute to tumor suppression in addition to direct targeting cancer cells.

Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase.

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.

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

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.

Article	Year
A case report of successful treatment with immunoadsorption onto protein A in mixed connective tissue disease in childhood. Therapeutic apheresis and dialysis : official peer-reviewed journal of the International Society for Apheresis, the Japanese Society for Apheresis, the Japanese Society for Dialysis Therapy\| PubMed ID: 18789123	2008
Therapeutic apheresis in transplantation medicine, experience with cardiac and lung transplantation in Jena. Atherosclerosis. Supplements\| PubMed ID: 23357138	2013
A procedure for efficient non-viral siRNA transfection of primary human monocytes using nucleofection. Journal of immunological methods\| PubMed ID: 25891792	2015
Time-resolved in situ assembly of the leukotriene-synthetic 5-lipoxygenase/5-lipoxygenase-activating protein complex in blood leukocytes. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 26396238	2016
The 5-lipoxygenase inhibitor RF-22c potently suppresses leukotriene biosynthesis in cellulo and blocks bronchoconstriction and inflammation in vivo. Biochemical pharmacology\| PubMed ID: 27157409	2016
Development of smart cell-free and cell-based assay systems for investigation of leukotriene C synthase activity and evaluation of inhibitors. Biochimica et biophysica acta\| PubMed ID: 27477678	2016
BRP-187: A potent inhibitor of leukotriene biosynthesis that acts through impeding the dynamic 5-lipoxygenase/5-lipoxygenase-activating protein (FLAP) complex assembly. Biochemical pharmacology\| PubMed ID: 27592027	2016
Differential Effects of Vitamins A and D on the Transcriptional Landscape of Human Monocytes during Infection. Scientific reports\| PubMed ID: 28094291	2017
Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase. Biochemical pharmacology\| PubMed ID: 28189727	2017
Human MAIT cells are rapidly activated by Aspergillus spp. in an APC-dependent manner. European journal of immunology\| PubMed ID: 30059139	2018
Fast and Quantitative Evaluation of Human Leukocyte Interaction with Aspergillus fumigatus Conidia by Flow Cytometry. Cytometry. Part A : the journal of the International Society for Analytical Cytology\| PubMed ID: 30450827	2019

Silke Rummler

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