Virus Mimetic Poly (I:C)-Primed Airway Exosome-like Particles Enter Brain and Induce Inflammatory Cytokines and Mitochondrial Reactive Oxygen Species in Microglia

Abstract

Viral infections induce extracellular vesicles (EVs) containing viral material and inflammatory factors. Exosomes can easily cross the blood-brain barrier during respiratory tract infection and transmit the inflammatory signal to the brain; however, such a hypothesis has no experimental evidence. The study investigated whether exosome-like vesicles (ELVs) from virus mimetic poly (I:C)-primed airway cells enter the brain and interact with brain immune cells microglia. Airway cells were isolated from Wistar rats and BALB/c mice; microglial cell cultures-from Wistar rats. ELVs from poly (I:C)-stimulated airway cell culture medium were isolated by precipitation, visualised by transmission electron microscopy, and evaluated by nanoparticle analyser; exosomal markers CD81 and CD9 were determined by ELISA. For in vitro and in vivo tracking, particles were loaded with Alexa Fluor 555-labelled RNA. Intracellular reactive oxygen species (ROS) were evaluated by DCFDA fluorescence and mitochondrial superoxide-by MitoSOX. ELVs from poly (I:C)-primed airway cells entered the brain within an hour after intranasal introduction, were internalised by microglia and induced intracellular and intramitochondrial ROS production. There was no ROS increase in microglial cells was after treatment with ELVs from airway cells untreated with poly (I:C). In addition, poly (I:C)-primed airway cells induced inflammatory cytokine expression in the brain. The data indicate that ELVs secreted by virus-primed airway cells might enter the brain, cause the activation of microglial cells and neuroinflammation.

Keywords: airway cell exosomes; microglia; mitochondria; reactive oxygen species; viral infection.

Figure A1

Representative images of the primary mouse (a) and rat (b) airway cell culture images 7 d after plating before the treatment with poly (I:C) and exosome collection. The cells on the bottom are fibroblasts, and the small cells organised in colonies on top of the fibroblasts are epithelial cells.

Figure A2

AF555-RNR retention by Exo-Spin collumns efficiency. The sample fluorescence data are presented as averages of 3 esperiments of 2 technical replicates; **** means statistically significant difference when p < 0.0001.

Figure A3

Representative images of viability evaluation of airway exosome-treated microglial cultures. The cells were stained with Hoechst33342 to visualise all nuclei blue and with propidium iodide to detect dying cells with lost plasma membrane integrity (red fluorescence of the nuclei).

Figure 1

The experimental design of the study. Primary airway cells of rodent origin were treated with poly (I:C), and their ELVs were applied on microglial cell cultures and intranasally to mice. Microglial cells in vitro and in vivo were investigated for particle internalisation, and cultured microglia were analysed for intracellular and intramitochondrial ROS production.

Figure 2

Poly (I:C)-treated rat (a–c) and mouse (d–f) airway cell ELV morphology, particle size distribution and specific markers. Representative transmission electron microscopy images (a,d), dynamic light scattering nanoparticle analysis data of three exosome samples (b,e) and tetraspanin CD63 and CD9 content per total exosome preparation protein determined by ELISA (c,f).

Figure 3

ELVs from poly (I:C)-treated airway cells in mouse brain coronal slices from central section after 0, 1, 3, and 5 h following intranasal introduction (a). In (b)—quantitative evaluation of—AFF555 fluorescence intensity in brain slice micrographs 3 h after intranasal administration of the stained particles. The data are expressed as averages ± standard deviation of 3 independent experiments that involved three animals in each experimental group; the fluorescence intensity was evaluated in 12 images for each separate animal sample. PIC-Exo is for poly (I:C)-primed airway ELVs, Exo—not primed airway ELVs, and controls brains from mice without treatment. * indicates statistically significant difference compared to the Control when p < 0.001, ^—compared to Exo or PIC-Exo, respectively, after one hour, when p < 0.05. In (c,d), there are ELVs in the olfactory bulb, prefrontal cortex and hippocampus slices, respectively, one hour after intranasal introduction. Particles loaded with RNR-conjugated AF555 are red, and nuclei are stained blue with DAPI. Scale bar 100 μm.

Figure 4

Poly (I:C)-treated airway ELV internalisation by microglia in mouse brain 2 h after intranasal introduction. In (a), a representative brain slice image, where particles loaded with RNR-conjugated AF555 are red, nuclei are stained blue with DAPI, and microglial cells are green, visualised by immunostaining against TMEM119. White arrows indicate microglial cell bodies with colocalised ELVs (upper image row, MERGED), and particles colocalising with microglial processes is visible in the zoomed ROI image (lower image row). The scale bar is 20 μm. Colocalisation analysis in seven small ROIs indicated by the white lines was performed by ImageJ plugin JACoP; cytofluorograms, Mander’s and Pearson’s coefficient values are presented in (b). Results of colocalisation analysis in full-size images are shown in (c), n = 15. M1—Mander’s overlapping coefficient showing the red fluorescence fraction overlapping the green, M1—the green fraction overlapping the red; PCC—Pearson’s correlation coefficient. Exo—images of the slices from brains treated with ELVs from control airway cells; PIC-Exo—from poly (I:C)-treated airway cells.

Figure 5

Poly (I:C)-treated airway cell ELV internalisation by microglia in and in pure microglial (a) and mixed rat (b) glial cultures 30 min after treatment. Particles loaded with RNR-conjugated AF555 are red, nuclei are stained blue with Hoechst33342, and microglial cells are green, stained with AF488-conjugated isolectin B4. The scale bar in (a) is 10 μm and in (b)—50 μm.

Figure 6

Intramitochondrial and cytoplasmic reactive oxygen species (ROS) formation in microglia after treatment with ELVs from poly (I:C)-primed airway cells. Cytoplasmic ROS were detected by DCFDA assay and mitochondrial superoxide—by MitoSOX^TM fluorescence. (a)—representative images and quantitative evaluation of MitoSOX fluorescence in cultured microglia. For positive control of the assay, 100 μM Antimycin A was used. The scale bar is 100 μm. (b)—representative images and quantitative evaluation of 2,7-dichlorofluorescein (derived from DCFDA) fluorescence in microglial cultures. For positive control of the assay, 1 μg/mL poly (I:C) was used. The scale bar is 100 μm. The quantitative data of ROS-dependent fluorescence intensity in micrographs are presented as percentage of untreated control and expressed as averages ± standard deviation of 3 experiments in 3 biological replicates. *** indicates statistically significant difference when p < 0.001; **—p < 0.01; *—p < 0.05.

Figure 7

Expression of mRNA of inflammation markers after poly (I:C)-primed airway ELV treatment in (a) brain tissue and (b) cultured rat microglial cells. Data are given as log2 Fold Change (2^−ΔΔCT) calculated from control specimens treated with unprimed ELVs. Statistical differences of markers expression between control specimens and specimens treated with poly (I:C)-primed airway ELVs calculated applying unpaired t-test; p < 0.05 considered significant. Bar plot represent mean of 3 experiments and whiskers—standard deviation; ns—not significant. Ifna is for interferon-alpha gene, Ifng—interferon-gamma, Ccl5—chemokine (C-C motif) ligand 5, or Rantes, Il1b—interleukin-1-beta, Tnf—tumour necrosis factor-alpha, Ptgs2—prostaglandin-endoperoxide synthase-2, Il6—interleukin-6, Ifnb1—interferon beta-1.