Proteomic Characterization, Biodistribution, and Functional Studies of Immune-Therapeutic Exosomes: Implications for Inflammatory Lung Diseases

Abstract

Dendritic cell (DC)-derived exosomes (DC EXO), natural nanoparticles of endosomal origin, are under intense scrutiny in clinical trials for various inflammatory diseases. DC EXO are eobiotic, meaning they are well-tolerated by the host; moreover, they can be custom-tailored for immune-regulatory or -stimulatory functions, thus presenting attractive opportunities for immune therapy. Previously we documented the efficacy of immunoregulatory DCs EXO (regDCs EXO) as immunotherapy for inflammatory bone disease, in an in-vivo model. We showed a key role for encapsulated TGFβ1 in promoting a bone sparing immune response. However, the on- and off-target effects of these therapeutic regDC EXO and how target signaling in acceptor cells is activated is unclear. In the present report, therapeutic regDC EXO were analyzed by high throughput proteomics, with non-therapeutic EXO from immature DCs and mature DCs as controls, to identify shared and distinct proteins and potential off-target proteins, as corroborated by immunoblot. The predominant expression in regDC EXO of immunoregulatory proteins as well as proteins involved in trafficking from the circulation to peripheral tissues, cell surface binding, and transmigration, prompted us to investigate how these DC EXO are biodistributed to major organs after intravenous injection. Live animal imaging showed preferential accumulation of regDCs EXO in the lungs, followed by spleen and liver tissue. In addition, TGFβ1 in regDCs EXO sustained downstream signaling in acceptor DCs. Blocking experiments suggested that sustaining TGFβ1 signaling require initial interaction of regDCs EXO with TGFβ1R followed by internalization of regDCs EXO with TGFβ1-TGFβ1R complex. Finally, these regDCs EXO that contain immunoregulatory cargo and showed biodistribution to lungs could downregulate the main severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target receptor, ACE2 on recipient lung parenchymal cells via TGFβ1 in-vitro. In conclusion, these results in mice may have important immunotherapeutic implications for lung inflammatory disorders.

Keywords: COVID-19; dendritic cells; exosomes; immune therapy; lung diseases.

Figure 1

(A) Nano-tracking analysis to determine EXO number and size distribution in nm. (B) Transmission electron microscopy (TEM) to visualize EXO shape and size. (C) Zeta potential analysis to determine EXO surface charge. regDCs EXO (Left), iDCs EXO (middle) and stimDCs EXO (right).

Figure 2

(A) Venn diagram showing the overlap of proteins between regDCs EXO, iDCs EXO and StimDCs EXO. 1278 proteins were overlapped, whereas 859, 1,054, and 634 proteins were unique to regDCs EXO, stimDCs EXO, and iDCs EXO, respectively. (B) Heat map showing differential expression in the overlapped proteins. The dots are color coded with red and blue indicating upregulation and downregulation, respectively.

Figure 3

KEGG pathway enrichment analysis of the uniquely expressed proteins listed on their percentage (upper panel) and level of significance (lower panel) in (A) regDCs EXO, (B) iDCs EXO, and (C) stimDCs EXO.

Figure 4

KEGG pathway enrichment analysis of (A) non-differentially expressed and (B) differentially expressed proteins listed on their percentage (upper panel) and level of significance (lower panel).

Figure 5

(A) Identification of exosomal markers tetraspanins and ESCRT complex related proteins and (B) DCs markers, immune-stimulatory/inhibitory molecules and pro/anti-inflammatory cytokines in DCs EXO.

Figure 6

Identification of integrins and chemotactic factors in DCs EXO.

Figure 7

Gene ontology (GO) enrichment analysis of the uniquely expressed proteins listed on their percentage (upper panel) and level of significance (lower panel) in (A) regDCs EXO, (B) iDCs EXO, and (C) stimDCs EXO.

Figure 8

Gene ontology (GO) enrichment of proteins (A) non differentially expressed and (B) differentially expressed proteins between DCs exosomes subsets, listed on their percentage (upper panel) and level of significance (lower panel).

Figure 9

Biodistribution of IV administrated EXO at 3 h and 24 h time points. SPECT CT live animal in vivo imaging of free In-111 (left) or In-111-labeled exosomes (right) in mice after (A) 3 h and (B) 24 h of IV administration. Radioactivity in lung, liver, spleen, and lymph nodes, relative to total, when free radiolabels or bound to DC EXO, expressed as % determined using SPECT CT images after (C) 3 h and (D) 24 h of EXO IV injection. N = 3; *P < 0.05 by two-way ANOVA, followed by Tukey’s multiple comparisons.

Figure 10

Early and sustained pSMAD2/3 signaling by uptake of regDC EXO with TGFβRI: (A) Immunoblot of Psmad2/3 and total smad2/3 in recipient DCs co-cultured for 24 h with reg DCS EXO +/- TGFβ1R inhibitor SB431542. Loading control was GAPDH (B) Immunoblot of Psmad2/3 and total smad2/3 in recipient DCs co-cultured for 1 and 24 h with reg DC EXO or iDC EXO +/- cytochalasin D. Loading control was B-actin. (C) Uptake of Dil labeled EXO (red) by recipient DCs, DAPI (blue), Alexa Fluor 680 phalloidin (violet) for FActin, Alexa flour 488 (green)-mouse anti-TGFβR1, visualized under confocal microscopy. Dil-DCs EXO or no EXO were added to recipient DCs at a 10:1 EXO : DC ratio (24 h shown). Results shown are representative of three independent experiments.

Figure 11

regDCs EXO are taken up by acceptor PBTECs, inhibiting ACE2 expression in vitro. (A) Uptake of Dil labeled EXO (red) by PBTECs, counterstained with nuclear stain DAPI (blue), phalloidin (Alex flour 647) for cell membrane and visualized under confocal microscopy. ACE2 mRNA expression (B) and flow cytometry scattergrams showing ACE2 positive cells percentage (C) in PBTECs treated or not treated with iDCs or regDCs EXO in the presence or absence of TGFβR1 inhibitor SB431542. (D) representative bar graph of (C). Results shown are representative of three independent experiments (*P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparisons).