Extracellular Vesicles Secreted by TDO2-Augmented Fibroblasts Regulate Pro-inflammatory Response in Macrophages

Figures

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  FIGURE 1. Transwell co-culture of bone marrow-derived macrophages. (A–D) Gene expression of inflammatory genes after co-culturing of cells with nHDF or nHDFTDO2 cells (n = 3 triplicates from three independent experiments). (E) Representative image of western blot detection of iNOS in BMDM lysates after overnight co-culture with nHDF or nHDFTDO2 cells and pooled data (n = 2 biological replicates). (F) Analysis was done using one-way ANOVA with Sidak’s multiple comparison test. Error bars represent standard deviation. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

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  FIGURE 2. nHDF and nHDFTDO2 derived extracellular vesicles share a similar phenotype. (A) Nanosight NS300 analysis of 15-day, serum-starved, conditioned medium, from nHDF and nHDFTDO2 cultures (n = 3 technical replicates). (B,C) Particle concentration and particle diameter as observed by NTA (n = 3 triplicates from three independent experiments). (D) Western blot of common EV markers. (E) Total RNA was extracted from 1.0e10 EVs and quantified using Qubit (n = 4 from two biological replicates). (F) EV protein content was analyzed by micro-BCA from 1.0e10 particles per sample (n = 4 from two biological replicates). (G) miRNA sequencing aligned 2,169 miRNA sequences with annotated miRNA. (H) Ratio of RNA species found during sequencing. Sequencing data were derived from three biological replicates from each nHDF and nHDFTDO2 derived EV.

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  FIGURE 3. nHDFTDO2-EVs show anti-inflammatory function in bone marrow-derived macrophages. (A–D) Gene expression of inflammatory genes in BMDM cells after overnight treatment with nHDF or nHDFTDO2 EVs results graphed as log2 fold change versus untreated cells (not shown) (n = 3 triplicates from three independent experiments). (E) Representative image of western blot detection of iNOS in BMDM cell lysates (n = 2 from biological replicates). (F) Quantification of iNOS western blot images. (G) ELISA quantification of IL-6 in the cell culture conditioned medium of BMDMs treated overnight with nHDF-EV or nHDFTDO2-EVs (n = 3 triplicates from three independent experiments). (H) Proliferation of BMDM cells analyzed by colorimetric BrdU incorporation assay (n = 8 technical replicates from a single experiment). (I) Results of modified Bowden-chamber assay for cell migration (n = 3, 10 technical replicates; images from three independent experiments). (A–H) Analysis was done using one-way ANOVA with Sidek’s multiple comparisons test. (I) Analysis was done using an unpaired, two-tailed, t-test. All error bars represent standard deviation. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

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  FIGURE 4. RNA sequencing of EV-treated bone marrow-derived macrophages (BMDMs) reveals activation of inflammatory pathways by nHDF-EV. Differential expression analysis was calculated from n=3 from two biological replicates. (A) Volcano plot visualizing the changes in gene expression in BMDM cells after treatment with nHDF-EVs compared to the untreated group. (B) Volcano plot visualizing the changes in gene expression in BMDM cells after treatment with nHDFTDO2-EVs compared to the untreated group. (C) Principal component plot of the top 300 differentially expressed genes (p < 0.001). (D) Volcano plot visualizing the changes in gene expression in BMDM cells after treatment with nHDFTDO2-EVs compared to the nHDF-EV treated group. All volcano plots use −log10 adjusted p-values and log2 fold-changes. Genes with adjusted p-values less than 0.001 and fold-changes greater than twofold are highlighted. (E–J) Log2 fold change gene expression changes in inflammatory markers when BMDMs are treated with nHDF-EV vs nHDFTDO2-EV. Log2FC values calculated from FPKM count values, n=3, error bars represent standard deviation, and starred plots indicate p < 0.001.

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  FIGURE 5. Neonatal human fibroblasts transduced to overexpress TDO2 increase ejection fraction in a 3-week mouse model of MI. (A) In an acute model of MI, BL6 mice underwent MI and hearts were injected with nHDF cells (n = 10) and nHDFTDO2 cells (n = 8). (B) Treatment with nHDFTDO2 cells increased ejection fraction in mice 21-day post-infarct. (C) Representative B-mode images of systole and diastole illustrating the ventricular-volume tracing method used to measure ejection fraction. M-mode images of left ventricular contraction. Improved contraction in the left ventricular wall after treatment with nHDFTDO2 cells is observed by the inward contraction of the left ventricular wall (yellow arrow). (D) Average change in left ventricular end-diastolic volumes. (E) Average change in left ventricular end-systolic volume. Statistical analysis was done using an unpaired t-test. **p < 0.01.