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. 2018 Oct 9:9:2343.
doi: 10.3389/fimmu.2018.02343. eCollection 2018.

Extracellular Vesicles From the Dermatophyte Trichophyton interdigitale Modulate Macrophage and Keratinocyte Functions

Tamires Aparecida Bitencourt  1 , Caroline Patini Rezende  2 , Natália Renault Quaresemin  1 , Pedro Moreno  2 , Otavio Hatanaka  2 , Antonio Rossi  1 , Nilce Maria Martinez-Rossi  1 , Fausto Almeida  2
Affiliations

Extracellular Vesicles From the Dermatophyte Trichophyton interdigitale Modulate Macrophage and Keratinocyte Functions

Tamires Aparecida Bitencourt et al. Front Immunol. .

Abstract

The release of biomolecules critically affects all pathogens and their establishment of diseases. For the export of several biomolecules in diverse species, the use of extracellular vesicles (EVs) is considered to represent an alternative transport mechanism, but no study to date has investigated EVs from dermatophytes. Here, we describe biologically active EVs from the dermatophyte Trichophyton interdigitale, a causative agent of mycoses worldwide. EV preparations from T. interdigitale were examined using nanoparticle-tracking analysis, which revealed vesicular structures 20-380 nm in diameter. These vesicles induced the production of proinflammatory mediators by bone marrow-derived macrophages (BMDMs) and keratinocytes in a dose-dependent manner, and an addition of the EVs to BMDMs also stimulated the transcription of the M1-polarization marker iNOS (inducible nitric oxide synthase) and diminished the expression of the M2 markers arginase-1 and Ym-1. The observed M1 macrophages' polarization triggered by EVs was abolished in cells obtained from knockout Toll-like receptor-2 mice. Also, the EVs-induced productions of pro-inflammatory mediators were blocked too. Furthermore, the EVs from T. interdigitale enhanced the fungicidal activity of BMDMs. These results suggest that EVs from T. interdigitale can modulate the innate immune response of the host and influence the interaction between T. interdigitale and host immune cells. Our findings thus open new areas of investigation into the host-parasite relationship in dermatophytosis.

Keywords: Trichophyton interdigitale; extracellular vesicles; fungal infection; innate immunity; keratinocytes; macrophages; nanoparticle-tracking analysis.

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Figures

Figure 1
Figure 1
Nanoparticle-tracking analysis of extracellular vesicles (EVs) produced by T. interdigitale. EVs were purified from T. interdigitale culture supernatants and quantified using a NanoSight NS300. (A) Histogram showing the EV particle-size distribution (EVs × 109/mL vs size in nanometers). (B) Screenshot from video recorded using the NanoSight NS300, showing the distribution of EVs from T. interdigitale.
Figure 2
Figure 2
Extracellular vesicles (EVs) from T. interdigitale induce the production of proinflammatory mediators by bone marrow-derived macrophages (BMDMs). BMDMs from C57BL/6 mice were incubated at 37°C for 48 h with the indicated amounts of EVs isolated from T. interdigitale (x-axis). The medium and LPS (1 μg/mL) plus IFN-γ (2 ng/mL) were used as negative and positive controls, respectively. The culture supernatants were assessed for the concentrations of nitrite (A), TNF-α (B), IL-6 (C), and IL-1β (D). Data are representative of 3 experiments. The results are expressed as means ± SEM and are shown relative to the levels in non-stimulated cells (medium only). *P < 0.05.
Figure 3
Figure 3
Extracellular vesicles (EVs) from T. interdigitale induce the production of proinflammatory mediators by keratinocytes. Human keratinocyte cell line HaCaT cells were incubated at 37°C for 24 h with the indicated amounts of EVs from T. interdigitale (x-axis). The medium and LPS (1 μg/mL) plus IFN-γ (2 ng/mL) were used as negative and positive controls, respectively. The culture supernatants were assessed for the concentrations of nitrite (A), TNF-α (B), IL-8 (C), IL-6 (D), and IL-1β (E). Data are representative of 3 experiments. The results are expressed as means ± SEM and are shown relative to the levels in non-stimulated cells (medium only). *P < 0.05.
Figure 4
Figure 4
Extracellular vesicles (EVs) from T. interdigitale promote classical macrophage activation. Bone marrow-derived macrophages (BMDMs) from C57BL/6 mice were incubated at 37°C for 6 h with EVs (107 particles/mL and with either IFN-γ (2 ng/mL) plus IL-12p40 (50 ng/mL) as M1 inducers (classical activation) or IL-10 plus IL-4 (both at 50 ng/mL) as M2 inducers (alternative activation). The medium was used as the negative control. RNA was extracted and converted into cDNA and real-time PCR was used to evaluate the relative expression of iNOS (A), Ym-1 (B), and arginase-1 (C). Data are representative of 3 experiments. The results are expressed as means ± SEM and are compared to the results obtained for the negative control. *P < 0.05.
Figure 5
Figure 5
Involvement of TLR2 and TLR4 in the proinflammatory cytokine production induced by T. interdigitale EVs. Bone marrow-derived macrophages (BMDMs) obtained from WT, TLR2−/−, and TLR4−/− mice were incubated at 37°C for 48 h with EVs (107 particles/mL). A mixture of LPS (1 μg/mL) plus IFN-γ (2 ng/mL) were used as positive controls for WT and TLR2−/− macrophages; Pam3CSK4 (100 ng/mL) was used as the positive control for TLR4−/− macrophages. The cultures supernatants were assessed for the concentration of TNF-α (A) and IL-6 (B). Data are representative of 3 experiments. The results are expressed as means ± SEM and are shown relative to the levels in non-stimulated cells (medium only). ***P < 0.001, and non-significant differences (n.s.).
Figure 6
Figure 6
Involvement of TLR2 in the activation of bone marrow-derived macrophages (BMDMs) by T. interdigitale EVs. BMDMs from WT, TLR2−/−, and TLR4−/− mice were stimulated for 6 h with EVs (107 particles/mL). As positive control for the M1 activation, the mixture of INF-γ (2 ng/mL) plus IL-12p40 (50 ng/mL) was used. For M2 activation, a mixture of IL-10 plus IL-4 (50 ng/mL both) was used. The medium alone was used as negative control. RNA was extracted and converted into cDNA and real-time PCR was used to evaluate the relative expression of iNOS (A), Ym-1 (B), and arginase-1 (C). Data are representative of 3 experiments. The results are expressed as means ± SEM and are compared to the results obtained for the negative control. ***P < 0.001, and non-significant differences (n.s.).
Figure 7
Figure 7
Extracellular vesicles (EVs) from T. interdigitale enhance fungicidal activity of macrophages. (A) Before the addition of T. interdigitale to bone marrow-derived macrophages (BMDMs; conidia:macrophages = 1:1), the cells were treated for 30 min with EVs (107 particles/mL), IFN-γ (50 ng/mL), or the medium and then plated on glass coverslips; after incubation for 4 h at 37°C, the coverslips were washed with PBS, and the cells were stained with Giemsa and the phagocytic index determined. (B) Alternatively, BMDMs were infected with T. interdigitale (macrophages:conidia = 1:1) for 48 h and then the cells were washed and lysed for the detection of viable intracellular fungal cells based on measuring their CFU. Data are representative of 3 experiments. The results are expressed as means ± SEM and are compared to the results obtained for the negative control. *P < 0.05.
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