Trichoderma stromaticum spores induce autophagy and downregulate inflammatory mediators in human peripheral blood-derived macrophages

Abstract

Trichoderma spp. are usually considered safe and normally used as biocontrol and biofertilization. Safety for human health is evaluated by several tests that detect various effects such as allergenicity, toxicity, infectivity, and pathogenicity. However, they do not evaluate the effects of the agent upon the immune system. The aim of this study was to investigate the interaction between T. stromaticum spores and mammalian cells to assess the immunomodulatory potential of the spores of this fungus. First, mouse macrophage cell line J774 and human macrophages were exposed to fungal spores and analyzed for structural features, through scanning and transmission electron microscopy. Then, various analysis were performed in human macrophages as to their effect in some functional and molecular aspects of the immune system through immunocytochemistry, flow cytometry and gene expression assays. We demonstrated that T. stromaticum spores induces autophagy and autophagy-related genes (ATGs) and downmodulate inflammatory mediators, including ROS, NLRP3, the cytokines IL-1β, IL-18, IL-12 and IL-10, as well as TLR2, TLR4, miR-146b and miR-155, which may lead to an augmented susceptibility to pathogens. Our study shows the extension of damages the biofungicide Tricovab® can cause in the innate immune response. Further studies are necessary to elucidate other innate and adaptive immune responses and, consequently, the safety of this fungus when in contact with humans.

Keywords: Autophagy, ROS, Inflammasomes; Biofungicide, Trichoderma; microRNAs.

Graphical abstract

Fig. 1

Morphological characteristics of T. stromaticum. (A) Macroscopic appearance of spores in culture; (B) Optical microscopy – Magnification 1000x, (C) Spores accessed by scanning EM displaying surface edges, mainly longitudinally oriented.

Fig. 2

Effects of interaction between T. stromaticum spores and J774 cells analyzed with TEM. (A) untreated J774 cells showing their typical ultrastructure; (B - D) J774 cells exposed to the fungal spores for 24h showing that spores (S) were completely phagocytosed; (B) Note the presence of spores with electron-lucent or washed-out cytoplasm, (arrowhead); (C) spores were surrounded by membranes (thin arrows) and endoplasmic reticulum cisternae (thick arrow), which were eventually discontinued (inset, arrowheads); (D) some spores were completely free within the phagocyte cytoplasm; (E) of Myelin-like figures (MF) and autophagosomes (AF- in the cytoplasm of the phagocytes after T. stromaticum spore exposure during 24h; Autophagy triggering was analyzed by MDC staining (F and G). Control cells not exposed to T. stromaticum (F) presented faint and diffuse staining whereas macrophages exposed to spores (G) displayed intense punctate labelling, which was significantly (* = p<0.05) more frequent than in controls (H). The bars represent the mean ± SD of three independent experiments. S - T. stromaticum spores; LD - Lipid Droplets; M – Mitochondria; N – nucleus

Fig. 3

Phagocytic and viability of macrophages exposed to T. stromaticum spores. Macrophages exposed to fungal spores for 24h showing that cells (M) ingested spores (thick arrows); (A) Light microscopy- note that some macrophages were also attached to lymphocytes (L); (B) SEM shows a monocyte tightly adhered to a lymphocyte and phagocytosing T. stromaticum spore. Note that monocyte displays pseudopodia both in the phagocytic cup, ingesting the spores (thin arrows) and attaching to the substrate (arrowheads); (C) TEM shows that T. stromaticum spores were internalized by macrophage after 12h. (D) Determination of cell viability (10⁵ cells/well) after interaction for 18 hours at different spore densities (0, 10⁴, 10⁵ or 10⁶/well) using the MTT assay. The results are presented as mean of three independent experiments in triplicate with confidence interval of 95%;

Fig. 4

Detection of autophagy marker LC3 in human macrophages before and after exposure to T. stromaticum spores. (C-F) Immunoperoxidase using anti-LC3 antibody. Cells (A) not exposed to spores; (B) exposed to 100 ng rapamycin; exposed to spores for (C) 30 min; (D) 3h; (E) 6 h (F) 24h. (G) Percentage of cells stained with antibody anti-LC3. Data represent the mean ± SD of 3 independent experiments.

Fig. 5

Relative expression of autophagy related genes. Human macrophages obtained from human peripheral blood were cultivated without treatment (untreated control), exposed to beads (Negative control), exposed rampamicin (Positive control) and exposed to T. stromaticum spores for 3h and 24h and the gene expression analysis was evaluated by quantitative PCR. (A) BECLIN 1, (B) ATG9, (C) p62, (D) GABARAP, (E) GABARAP L1, (F) GABARAP L2 gene expression values. The relative expression was calculated using the 2^-∆∆Ct methodology and values were normalized with the GAPDH gene. Data is represented by mean ± SD of three independent experiments by ANOVA

Fig. 6

Detection of ROS production and transcriptional levels of inflammasome related genes in human macrophages after T. stromaticum exposure. Human macrophages exposed to PMA, T. stromaticum spores and spores + PMA were incubated with the Dihydroethidium (DHE), a probe that enters in the cellular cytoplasm and once oxidized emits fluorescence. Fluorescence was detected by flow cytometry after 30min (B-E), 3h, (G-J) 6h (L-O) or 24h (Q-T) of stimuli, summarized on panels F, K, P and U. The gate A represents the percentage of DHE-positive cells during acquisition of 20,000 events by Flow Cytometry. Data is shown as mean + standard deviation; ANOVA followed by Bonferroni post-test. (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001. Data is representative of three different experiments. Expression of the genes NLRP3 (V), IL-1β (W) and IL-18 (X) in human macrophages exposed or not to T. stromaticum spores during 30min, 3h, 6h and 24h were obtained by qPCR using three independent experiments. The relative expression was calculated using the 2^-∆∆Ct methodology and values were normalized with the GAPDH gene. Data is represented by mean + SD by ANOVA with Bonferroni post-test. Bars represent mean + SE (n=5 replicates/group). (*) p≤0.05 and (***) p≤0.001 compared to control group.

Fig. 7

Relative levels of PRRs and cytokines in human macrophages exposed to T. stromaticum spores. Human macrophages cultured in the presence of T. stromaticum spores had the levels of (A) TLR2, (B) TLR4, (C) CLEC7A, (D) IL10, (E) IL12, and (F) TNFα expression evaluated after 12 hours of exposure by qPCR. Results represent the mean of two independent assays together, with confidence interval of 95%. Asterisks indicate significant differences relative to the control (p < 0.05), by Tukey test analysis.

Fig. 8

Relative levels of miRNAs and PRRs in macrophages exposed to T. stromaticum spores. Macrophages cultured in the presence of T. stromaticum spores had the levels of (A) miR-146a, (B) miR-146b, (C) miR155, expression evaluated after 12 hours of exposure, by qPCR. Results represent the mean of three independent assays in duplicate together with confidence interval of 95%., by Tukey test analysis. (D) Correlation matrix between levels of all genes analyzed in this study and miRNAs. Circles and corresponding sizes represent the significance level. Colors represent the directionality of the correlation (blue - positive correlations; red - negative correlations).