Journal: Frontiers in Immunology

Article Title: Neutrophil-Specific Knockdown of β2 Integrins Impairs Antifungal Effector Functions and Aggravates the Course of Invasive Pulmonal Aspergillosis

doi: 10.3389/fimmu.2022.823121

Figure Lengend Snippet: Infection with IPA caused an impaired survival (A) and an aggravated course of the disease (B) in CD18 Ly6G cKO as compared to CD18 fl/fl mice. CD18 fl/fl and CD18 Ly6G cKO mice were infected i.t. with A fumigatus (each 10 7 conidia/mouse) in 2 independent experiments. (A) Survival was monitored daily for 2 weeks and is presented in a Kaplan-Meier survival curve. In parallel settings PMN were depleted in some mice via injection of an anti-Gr-1 antibody one day before infection. Data show the cumulative results of two independent experiments with a total of 12 (CD18 fl/fl ) and 13 (CD18 Ly6G cKO) mice/group. 5 mice/group received an anti-Gr-1 antibody in order to deplete PMN in these mice. All Gr-1 depleted mice died within the first days after IPA infection, whereas all non-depleted CD18 fl/fl mice survived. By contrast, some non-depleted CD18 Ly6G cKO mice (n=2) deceased within the first week after IPA infection. (B) The clinical course of IPA of monitoring was assessed in CD18 fl/fl (n=7) and CD18 Ly6G cKO mice (n=8) for 14 days. Parameters comprised breathing, reaction to pain overall appearance, hypothermia, strong weight loss, motoric disabilities and apathy (each 0-2).

Article Snippet: Mice were anesthetized with 14.5% Ketamin (50mg/ml)/5.7% Xylazin (0.2%) and were subsequently challenged with 10 7 A. fumigatus conidia (strain ATCC 46645) applied intratracheally as described ( , ).

Techniques: Infection, Injection

Journal: Frontiers in Immunology

Article Title: Neutrophil-Specific Knockdown of β2 Integrins Impairs Antifungal Effector Functions and Aggravates the Course of Invasive Pulmonal Aspergillosis

doi: 10.3389/fimmu.2022.823121

Figure Lengend Snippet: CD18 Ly6G cKO mice show a higher pulmonary fungal burden. Histopathological analysis of H&E and Grocott-stained lungs derived from IPA-infected mice 24h upon A.fumigatus inoculation revealed a higher fungal burden and a stronger lung damage (i.e., hyaline membranes, fibrin-exudate within the alveoli) in CD18 Ly6G cKO mice (A) . Cellular inflammation did not show significant genotype-dependent differences. Representative examples of histological analysis are shown in (B) (Magnification 10x). Data in (A) denote results of histopathological analysis of n=9-10 mice/genotype. We further observed higher CFU counts in serial dilutions of lung homogenates (1:500) after incubation for 24h on Sabouraud-4% Glucose agar plates (C) . Data show the mean ± SEM of 6 mice/group. Statistically significant differences between groups are indicated (*p<0.05, **p<0.005, ***p<0.001).

Article Snippet: Mice were anesthetized with 14.5% Ketamin (50mg/ml)/5.7% Xylazin (0.2%) and were subsequently challenged with 10 7 A. fumigatus conidia (strain ATCC 46645) applied intratracheally as described ( , ).

Techniques: Staining, Derivative Assay, Infection, Incubation

Journal: Frontiers in Immunology

Article Title: Neutrophil-Specific Knockdown of β2 Integrins Impairs Antifungal Effector Functions and Aggravates the Course of Invasive Pulmonal Aspergillosis

doi: 10.3389/fimmu.2022.823121

Figure Lengend Snippet: The BAL fluid of A. fumigatus infected CD18 Ly6G cKO mice contains lower levels of proinflammatory cytokines. CD18 fl/fl and CD18 Ly6G cKO mice were infected i.t. with A. fumigatus . On the next day, mice were euthanized, and cytokines in BAL fluid were analyzed. Data denote the mean ± SEM of 6-10 samples analyzed per group. Statistically significant differences between groups are indicated (* p < 0.05).

Article Snippet: Mice were anesthetized with 14.5% Ketamin (50mg/ml)/5.7% Xylazin (0.2%) and were subsequently challenged with 10 7 A. fumigatus conidia (strain ATCC 46645) applied intratracheally as described ( , ).

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Neutrophil-Specific Knockdown of β2 Integrins Impairs Antifungal Effector Functions and Aggravates the Course of Invasive Pulmonal Aspergillosis

doi: 10.3389/fimmu.2022.823121

Figure Lengend Snippet: CD18 Ly6G cKO mice infected with A. fumigatus are characterized by elevated lung infiltration of PMN, but not in spleens and blood (n=10/genotype) (A) . Elevated PMN counts have also been found in Cytospins (bars depict the mean ± SEM of n=8 cytospins/genotype; cell infiltration has been assessed using a scoring system; 0=missing - 3=strongest infiltration) (B) . Here, we could additionally observe higher numbers of mononuclear cells (B) . This is consistent with the finding of higher macrophage counts in the BALF of CD18 fl/fl mice as compared to CD18 Ly6G cKO mice observed in FACS-analysis (B) . Assessment of PMN apoptosis revealed a stronger expression of apoptosis marker Annexin V in PMN derived from BALF and blood of CD18 Ly6G cKO mice (C) . Bars depict the mean ± SEM of the relative cell counts found in n=8 cytospins/genotype. Legend in A applies to all panels. Statistically significant differences between groups are indicated (*p<0.05)

Article Snippet: Mice were anesthetized with 14.5% Ketamin (50mg/ml)/5.7% Xylazin (0.2%) and were subsequently challenged with 10 7 A. fumigatus conidia (strain ATCC 46645) applied intratracheally as described ( , ).

Techniques: Infection, Expressing, Marker, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Neutrophil-Specific Knockdown of β2 Integrins Impairs Antifungal Effector Functions and Aggravates the Course of Invasive Pulmonal Aspergillosis

doi: 10.3389/fimmu.2022.823121

Figure Lengend Snippet: Phagocytosis of inert particles and fungal conidia is less effective in PMN of CD18 Ly6G cKO mice. Freshly isolated PMN were co-incubated either with nanoparticles (NP), microBeads (A) or with GFP-fluorescent A. fumigatus conidia (AFC) at 37°C with indicated ratios (B) . Simultaneous co-incubation at 4°C served to differentiate mere adhesion from temperature-dependent binding. After 30 min and 60 min the frequency of either Cy5 positive NP, PE-positive microBeads (A) , or GFP-positive PMN (B) was determined by flow cytometry. Data represent the mean ± SEM of 3 samples analyzed/group. Exemplary flow cytometry data depicting the diminished uptake of GFP-fluorescent conidia by PMN from CD18 Ly6G cKO mice are shown in (C) . Statistically significant differences between groups are indicated (*p<0.05, **p<0.005).

Article Snippet: Mice were anesthetized with 14.5% Ketamin (50mg/ml)/5.7% Xylazin (0.2%) and were subsequently challenged with 10 7 A. fumigatus conidia (strain ATCC 46645) applied intratracheally as described ( , ).

Techniques: Isolation, Incubation, Binding Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Neutrophil-Specific Knockdown of β2 Integrins Impairs Antifungal Effector Functions and Aggravates the Course of Invasive Pulmonal Aspergillosis

doi: 10.3389/fimmu.2022.823121

Figure Lengend Snippet: Impaired oxidative and non-oxidative effector functions of CD18-deficient PMN. We could observe that the formation of NET after 3h of incubation (A) and ROS-generation (B) were significantly lower in PMN derived from CD18 Ly6G cKO mice, particularly after stimulation with A. fumigatus conidia. Data represent the mean ± SEM of 3 samples analyzed/group. Statistically significant differences between groups are indicated (*p<0.05)

Article Snippet: Mice were anesthetized with 14.5% Ketamin (50mg/ml)/5.7% Xylazin (0.2%) and were subsequently challenged with 10 7 A. fumigatus conidia (strain ATCC 46645) applied intratracheally as described ( , ).

Techniques: Incubation, Derivative Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: New Claims for Wild Carrot ( Daucus carota subsp. carota ) Essential Oil

doi: 10.1155/2016/9045196

Figure Lengend Snippet: Antifungal activity (MIC and MLC) of Daucus carota subsp. carota essential oil for Candida spp., Cryptococcus neoformans , dermatophyte, and Aspergillus strains.

Article Snippet: The antifungal properties of the essential oil were tested against three Candida reference strains ( C. albicans ATCC 10231, C. tropicalis ATCC 13803, and C. parapsilosis ATCC 90018) and two clinical strains ( C. krusei H9 and C. guilliermondii MAT23); one Cryptococcus neoformans reference strain ( C. neoformans CECT 1078); four dermatophyte strains ( Trichophyton rubrum CECT 2794, T. mentagrophytes var. interdigitale CECT 2958, T. verrucosum CECT 2992, and Microsporum gypseum CECT 2908); the remaining dermatophytes were clinically isolated ( T. mentagrophytes FF7, M. canis FF1, and Epidermophyton floccosum FF9); two reference Aspergillus strains ( A. niger ATCC 16404 and A. fumigatus ATCC 46645); and one Aspergillus strain was from a clinical origin ( A. flavus F44).

Techniques: Activity Assay

Journal: Microbes and infection

Article Title: The influenza A virus promotes fungal growth of Aspergillus fumigatus via direct interaction in vitro.

doi: 10.1016/j.micinf.2023.105264

Figure Lengend Snippet: Fig. 1. Fungal growth of A. fumigatus is increased during 8 þ 16 h and 16 þ 10 h of coinfection with IAV on Calu-3 cells. Calu-3 cells were infected with IAV (H1N1, 0.5 MOI) for 8 or 16 h or left uninfected and were subsequently superinfected with conidia of A. fumigatus (10 MOI) for further 16 or 10 h or were left uninfected again. (A) Overview of the experimental setup. (B) For immunofluorescence microscopy, IAV (H1N1) infection was detected by an IAV-NP-specific antibody and an AlexaFluor® 647-conjugated donkey anti- mouse secondary antibody (red). GFP-expressing A. fumigatus is displayed in green and nuclei of Calu-3 cells were stained with Hoechst 33342 (blue). All images were taken with an Axio Observer.Z1 microscope (Zeiss) at 200 magnification. Scale bars represent 100 mm. (C) Fluorescence images were used to quantify fungal growth (mm2 per fungus) after 16 þ 10 h of coinfection. (D) Viral titers of IAV were analyzed by standard plaque assay at the indicated times. (E) Expression levels of mRNA encoding viral proteins (IAV-M1, IAV-NP and IAV-PB1) were analyzed by qRT-PCR. Diagrams display the n-fold expression levels compared to the IAV-infected samples. (F) Western blot analysis was performed to evaluate protein expression. Antibodies against IAV-PB1 and IAV-HA were used to analyze viral infection. GFP-expression was detected to demonstrate infection by A. fumigatus (Afu-GFP).

Article Snippet: Green fluorescent protein (GFP)-expressing A. fumigatus (parental strain: ATCC 46645) [16], A. fumigatuswildtype (wt; ATCC 46645) and A. fumigatus CEA10 (CBS 144.89) were grown on Sabouraud-2 % dextrose broth at 37 C. After seven days, the fungal mycelium with formed conidia was transferred to a fresh tube and vortexed together with PBS-Tween 20.

Techniques: Infection, Microscopy, Expressing, Staining, Fluorescence, Plaque Assay, Quantitative RT-PCR, Western Blot

Journal: Microbes and infection

Article Title: The influenza A virus promotes fungal growth of Aspergillus fumigatus via direct interaction in vitro.

doi: 10.1016/j.micinf.2023.105264

Figure Lengend Snippet: Fig. 2. Purified viral particles of IAV induce hyphal growth of A. fumigatus in a host cell-free environment. (A, B) A. fumigatus was stimulated with a cytokine mixture (10 ng ml1 IFN-b, IL-6 and TNF-a, respectively) for 10 h or left untreated. (C, D) To analyze the effect of IAV on A. fumigatus in a host cell-free system, conidia of A. fumigatus were incubated with purified IAV (H1N1)-particles at the indicated concentrations for 6, 8 or 10 h. (E, F) The efficiency to induce fungal growth after 10 h of stimulation was also examined for other IAV strains (5 107 IAV per sample), namely H1N1pdm09 and H3N2. (G, H) Differences in induction of hyphal growth, between infectious and non-infectious IAV-particles, were examined by coincubating conidia of A. fumigatus with infectious or UV-inactivated, purified IAV (H1N1)-particles (5 107 IAV per sample) for 10 h. (A, D, E, G) Fluorescence images show GFP-expressing fungi of A. fumigatus stimulated for 10 h with the indicated substances. All fluorescence images show one representative example out of three in- dependent experiments. Images were taken with an Axio Observer.Z1 microscope (Zeiss) at 200 magnification. Scale bars represent 100 mm. (B, C, F, H) Fungal growth (mm2 per fungus) was quantified using the fluorescence images. Diagrams show the results of three independent experiments including duplicates. Statistical significance was evaluated by paired t-test (B) or one-way ANOVA followed by Tukey's multiple comparisons test (C, F, H) (***p 0.001, **p 0.01, *p 0.05).

Article Snippet: Green fluorescent protein (GFP)-expressing A. fumigatus (parental strain: ATCC 46645) [16], A. fumigatuswildtype (wt; ATCC 46645) and A. fumigatus CEA10 (CBS 144.89) were grown on Sabouraud-2 % dextrose broth at 37 C. After seven days, the fungal mycelium with formed conidia was transferred to a fresh tube and vortexed together with PBS-Tween 20.

Techniques: Incubation, Fluorescence, Expressing, Microscopy

Journal: Microbes and infection

Article Title: The influenza A virus promotes fungal growth of Aspergillus fumigatus via direct interaction in vitro.

doi: 10.1016/j.micinf.2023.105264

Figure Lengend Snippet: Fig. 3. Purified IAV particles bind on the fungal surface of A. fumigatus. To demonstrate an interaction between A. fumigatus (A) or A. fumigatus wt (B) and IAV, conidia of A. fumigatus were grown for 9 h and then coincubated with purified IAV (H1N1)-particles (5 107 IAV per sample) for 1 h or were left untreated. (A) In immunofluorescence microscopy, IAV-particles were stained with anti-NP mouse primary antibodies and AlexaFluor®-647 conjugated donkey anti-mouse secondary antibodies (red). GFP-expressing fungi of A. fumigatus are shown in green. (B) Scanning electron microscopy (SEM) images were taken with 4000 and 20,000 magnification. Arrows point at unknown vesi- cles on the surface of A. fumigatus wt with a size of around 180 nm in diameter (i) and vesicles with a size of 90e100 nm only detected in IAV-coincubated samples (ii). Scale bars represent 5 mm (Mag.: 4000 ), 500 nm (Mag.: 20,000 ) and 200 nm (image section). (C, D) To investigate whether the IAV-HA is involved in the interaction with A. fumigatus hyphae, purified IAV (H1N1)-particles were incubated with antibodies against HA (10 mg ml1) for 30 min, on ice. Then, conidia of A. fumigatus were coincubated with the virus- antibody-mixture (5 107 IAV per sample) or the respective controls for 10 h. (C) Immunofluorescence images show GFP-expressing fungi of A. fumigatus. (D) Fungal growth (mm2

Article Snippet: Green fluorescent protein (GFP)-expressing A. fumigatus (parental strain: ATCC 46645) [16], A. fumigatuswildtype (wt; ATCC 46645) and A. fumigatus CEA10 (CBS 144.89) were grown on Sabouraud-2 % dextrose broth at 37 C. After seven days, the fungal mycelium with formed conidia was transferred to a fresh tube and vortexed together with PBS-Tween 20.

Techniques: Microscopy, Staining, Expressing, Electron Microscopy, Incubation, Virus

Journal: Microbes and infection

Article Title: The influenza A virus promotes fungal growth of Aspergillus fumigatus via direct interaction in vitro.

doi: 10.1016/j.micinf.2023.105264

Figure Lengend Snippet: Fig. 4. IAV protein- and mRNA expression levels are slightly reduced during the first replication cycle in the presence of A. fumigatus. Calu-3 cells were infected with 5 MOI (4, 8 and 10 h) or 1 MOI (16 h) IAV (H1N1) or were left uninfected. After 30 min, supernatant was exchanged with fresh medium with or without conidia of A. fumigatus at 10 MOI (4, 8, 10 h) or 5, 10 and 20 MOI (16 h) and the samples were incubated for the indicated periods. (A) Overview of the experimental setup. (B) Immunofluorescence images show samples after 10 h of coinfection. IAV infection was detected by an IAV-NP-specific antibody and an AlexaFluor® 647-conjugated donkey anti-mouse secondary antibody (red). GFP- expressing A. fumigatus is visible in green, and nuclei of Calu-3 cells were stained with Hoechst 33342 (blue). All images were taken with an Axio Observer.Z1 microscope (Zeiss) and a 200 magnification. Scale bars represent 100 mm. (C) Fluorescence images were also used to quantify fungal growth (mm2 per fungus) after 10 h of coinfection. (D) Viral titers of IAV were analyzed by standard plaque assay at the indicated time points. (E) mRNA expression levels encoding for viral proteins (IAV-M1, IAV-NP and IAV-PB1) were analyzed by qRT-PCR. Diagrams display the n-fold expression levels compared to the IAV-infected samples. (F) Western blot analysis was performed to evaluate protein expression. Antibodies against IAV-PB1 and IAV-HA were used to analyze viral infection. GFP-expression represents the infection by A. fumigatus (Afu-GFP). The ERK2 signal was used as loading control. Immunofluorescence (B) and Western blot images (F) show one representative example out of three independent experiments, each. (CeE) Diagrams show the results of three independent experiments including three replicates (D) or duplicates (C, E). Statistical significance was evaluated by paired t-test (CeE) or one-way ANOVA followed by Tukey's multiple comparisons test (D, right panel, 16 h) (****p 0.0001, ***p 0.001, **p 0.01).

Article Snippet: Green fluorescent protein (GFP)-expressing A. fumigatus (parental strain: ATCC 46645) [16], A. fumigatuswildtype (wt; ATCC 46645) and A. fumigatus CEA10 (CBS 144.89) were grown on Sabouraud-2 % dextrose broth at 37 C. After seven days, the fungal mycelium with formed conidia was transferred to a fresh tube and vortexed together with PBS-Tween 20.

Techniques: Expressing, Infection, Incubation, Staining, Microscopy, Fluorescence, Plaque Assay, Quantitative RT-PCR, Western Blot, Control

Journal: Microbes and infection

Article Title: The influenza A virus promotes fungal growth of Aspergillus fumigatus via direct interaction in vitro.

doi: 10.1016/j.micinf.2023.105264

Figure Lengend Snippet: Fig. 5. IAV-induced cytokine expression and cell death are inhibited in A. fumigatus coinfected samples. (A) The mRNA levels of IP-10, IL-6, IL-8, MxA and TRAIL were analyzed after 8 h of coinfection with or without IAV (H1N1, 1 MOI) and A. fumigatus (10 MOI). Diagrams display the n-fold expression levels compared to the uninfected control. (B) Cytokine release was analyzed in the supernatants of 16 h coinfected Calu-3 cells by flow cytometric measurements (LegendPlex™). Diagrams show expression levels of IFN-a2, IL-6, IL-8, IL- 18, IL-23, IL-33, MCP-1 and TNF-a. (C) Furthermore, cell death was examined after 8 þ 16 h, 16 þ 10 h and 16 h of coinfection, by measuring the LDH availability in the supernatants. (D) As a second cell death marker, PARP cleavage was detected by Western blot assay after 16 h of coinfection. ERK2 signal was used as loading control. Images show one representative example out of three different experiments. (AeC) Diagrams show the results of three independent experiments including duplicates. Significance was evaluated by one-way ANOVA followed by Tukey's multiple comparison test (****p 0.0001, ***p 0.001,**p 0.01, *p 0.05).

Article Snippet: Green fluorescent protein (GFP)-expressing A. fumigatus (parental strain: ATCC 46645) [16], A. fumigatuswildtype (wt; ATCC 46645) and A. fumigatus CEA10 (CBS 144.89) were grown on Sabouraud-2 % dextrose broth at 37 C. After seven days, the fungal mycelium with formed conidia was transferred to a fresh tube and vortexed together with PBS-Tween 20.

Techniques: Expressing, Control, Marker, Western Blot, Comparison

Journal: Microbes and infection

Article Title: The influenza A virus promotes fungal growth of Aspergillus fumigatus via direct interaction in vitro.

doi: 10.1016/j.micinf.2023.105264

Figure Lengend Snippet: Fig. 6. Overview of the proposed interplay between IAV, A. fumigatus und lung epithelial cells during coinfection in vitro. (A) Red arrows indicate the effect of IAV on lung epithelial cells and A. fumigatus (Afu). Blue arrows indicate the effect of IAV-induced host cell response on A. fumigatus. (B) The influence of A. fumigatus on IAV and lung epithelial cells is represented by green arrows. (A) Single infection of lung epithelial cells with IAV leads to viral replication, which results in the release of progeny virus particles, cytokines and chemokines and induction of cell death. During fungal infection, conidia of A. fumigatus start to germinate and to grow hyphae. Under coinfection conditions, extracellular virus particles provoke increased growth of A. fumigatus by interacting with the fungal surface. Additionally, released cytokines and chemokines from the host cell also have an impact on the fungal growth. (B) However, the presence of the fungus results in lower IAV titers, less released cytokines and chemokines and inhibition of IAV-induced cell death.

Article Snippet: Green fluorescent protein (GFP)-expressing A. fumigatus (parental strain: ATCC 46645) [16], A. fumigatuswildtype (wt; ATCC 46645) and A. fumigatus CEA10 (CBS 144.89) were grown on Sabouraud-2 % dextrose broth at 37 C. After seven days, the fungal mycelium with formed conidia was transferred to a fresh tube and vortexed together with PBS-Tween 20.

Techniques: In Vitro, Infection, Virus, Inhibition