Dendritic cell derived exosomes loaded with immunoregulatory cargo reprogram local immune responses and inhibit degenerative bone disease in vivo

Abstract

Chronic bone degenerative diseases represent a major threat to the health and well-being of the population, particularly those with advanced age. This study isolated exosomes (EXO), natural nano-particles, from dendritic cells, the "directors" of the immune response, to examine the immunobiology of DC EXO in mice, and their ability to reprogram immune cells responsible for experimental alveolar bone loss in vivo. Distinct DC EXO subtypes including immune-regulatory (regDC EXO), loaded with TGFB1 and IL10 after purification, along with immune stimulatory (stimDC EXO) and immune "null" immature (iDCs EXO) unmodified after purification, were delivered via I.V. route or locally into the soft tissues overlying the alveolar bone. Locally administrated regDC EXO showed high affinity for inflamed sites, and were taken up by both DCs and T cells in situ. RegDC EXO-encapsulated immunoregulatory cargo (TGFB1 and IL10) was protected from proteolytic degradation. Moreover, maturation of recipient DCs and induction of Th17 effectors was suppressed by regDC EXO, while T-regulatory cell recruitment was promoted, resulting in inhibition of bone resorptive cytokines and reduction in osteoclastic bone loss. This work is the first demonstration of DC exosome-based therapy for a degenerative alveolar bone disease and provides the basis for a novel treatment strategy.

Keywords: (MeSH): Dendritic Cells; alveolar Bone loss; exosomes; inflammation; periodontitis.

Figure 1.

RegDCs EXO protect encapsulated immunoregulatory cargo (TGFB1 and IL10) from proteolytic degradation. (A) Nano-tracking analysis to determine EXO number and size distribution in nm. (B) Transmission electron microscopy (TEM) to visualize EXO shape and immuno-gold TEM to detect EXO marker tetraspanin CD63, showing unstained (left) and positive staining (right)(arrows). (C) Western blotting to detect other EXO-related markers including CD81, TSG101, GRP94 and B-actin in donor DCs and EXO (left) and anti/pro-inflammatory cytokines including TGFB1, IL10, IL6, IL1B and TNF and the costimulatory molecule CD86 as well as EXO associated proteins ALIX and TSG101 in DCs EXO subsets (right). (D) TGFB1 and IL10 content of lysed EXO and in regDCs EXO supernatant by ELISA. (E) TGFB1 and IL10 content of non-lysed EXO (transmembrane domain) by ELISA. (F) Immunogold TEM to detect luminal and transmembrane TGFB1 in regDCs EXO. (G) regDCs EXO (left) or equivalent concentration of free TGFB1 and IL10 (right) were treated with trypsin (upper panel) or proteinase-K (lower panel) and incubated in control buffer (1hour at 37°C) and analysed by western blotting to detect the levels of TGFB1, IL10 and exosomal markers TSG101 (upper panel) and CD81 (lower panel).

Figure 2.

LPS/inflammation induced miRNAs and mRNA are down regulated in regDCs EXO in vitro. (A) Colocalization of TGFB1 (back arrow) and EXO (red arrow) in multivesicular bodies in regDCs. (B) miRNA analysis of LPS/inflammation induced miRNAs in EXO. (C) IL6mRNA expression in EXO by PCR.

Figure 3.

DC EXO uptake by acceptor DCs, with EXO subtype determining maturation and cytokine profile of acceptor DCs in vitro. (A) Uptake of Dil labelled EXO (red) by bone marrow derived DCs, counterstained with nuclear stain DAPI (blue), phalloidin (green) for cell membrane and visualized under confocal microscopy. (B) Flow cytometry scattergrams showing % of (top panel) CD11c MHCII and (bottom panel) CD11c CD86 double positive cells in DCs treated or not treated with EXO (C) Bar graphs showing % of (left panel) CD11c MHCII and (right panel) CD11c CD86 double positive cells in DCs treated or not treated with each EXO subtype. (D) mRNA expression in DCs treated or not treated with EXO of IL6 (left panel), IL12 (middle panel) and IL23 (right panel). (E) Western blotting analysis of levels of phosphorylation of TGFB1and IL10 transcription factors, SMAD2/3 and STAT3 respectively and MHCII in DCs treated with or without EXO. (B) Flow cytometry scattergrams (top panels) showing % of double positive CD11c MHCII cells in DCs treated with or without regDCs EXO in presence or absence of neutralizing antibodies against TGFB1 and or IL10 and (bottom panel) representative bar graph. Results shown are representative of three independent experiments (* P<0.05 by one-way ANOVA followed by Tukeys multiple comparisons).

Figure 4.

RegDCs EXO increase acceptor DC resistance to LPS mediated maturation and lower antigen presenting ability. Flow cytometry histograms showing in vitro influence of EXO subtypes on MHCII (A) and CD86 (B) expression on acceptor DCs challenged by LPS (left). Results presented as median fluorescent intensity measurements in representative bar graph (right). (C) IL-6 mRNA expression in acceptor DCs by PCR analysis. (D) Flow cytometry histograms showing proliferation of splenic ovalbumin specific CD4+T – cells, labelled with CFSE, after coculture with ovalbumin – pulsed DCs treated with or without EXO subtypes. Results shown are representative of three independent experiments (* P<0.05 by one-way ANOVA followed by Tukeys multiple comparisons).

Figure 5.

RegDCs EXO uptake by CD4T-cells, promoting TGFB1 dependent Tregs induction while stim DCs EXO induce T-helper17 response in vitro. (A) Uptake of Dil labelled EXO (red) by splenic CD4T-cells, showing DAPI-labelled nuclei, and phalloidin (green) labelled cell membrane, as visualized under confocal microscopy. (B) Flowcytometry scattergrams showing in vitro influence of EXO on induction of T-regulatory cells, as measured by % of double positive CD25 and Foxp3 cells in gated CD4T-cell population, stimulated with antiCD3/CD28 antibodies. (C) Summary bar graph of flow cytometry data. (D) Western blotting analysis of Foxp3 expression in acceptor CD4T-cells. (E) Flow cytometry analysis showing proliferation % of CFSE-labelled splenic CD4+T-cells, as expressed by histograms (left panel) and bar graphs (right panel), after stimulation with anti-CD3/CD28 in presence or absence of EXO subtypes. (F) Flow cytometry analysis of CD4+IL-17+T cells %, as expressed by histograms (left) and bar graph (right). (G) ELISA of IL17 expression in supernatant of T cells treated with EXO subtypes. Results shown are representative of three independent experiments (* P<0.05 by one-way ANOVA followed by Tukeys multiple comparisons).

Figure 6.

Locally administrated exosomes showed higher affinity and slower clearance from periodontal tissues in inflammatory alveolar bone loss model. (A) SPECT CT live animal in vivo imaging of free In-111 (left) or In-111-labelled exosomes (right) in mice after 24h of IV administration. (B) Local delivery of free In-111 (left) or In-111-labelled exosomes (right) by injection in the palatal gingiva at the right side of maxilla was utilized. (C) Radioactivity in maxilla, relative to total, when free or bound to DC EXO, expressed as % determined using SPECT CT images. (D) Radioactivity in maxilla, relative to total, when free or bound to DC EXO, expressed as %, in post-mortem isolated maxilla, determined by gamma counter. Mice were subjected to ligature placement to induce inflammatory bone loss prior to imaging. Yellow arrows delineate maxilla, white arrows liver, spleen and other non-oral sites.

Figure 7.

RegDCs EXO co-localize with gingival acceptor DCs, inhibiting maturation in alveolar bone loss model. DIL (Red) labelled EXO were injected into the palatal gingiva of the upper right second molar of inflammatory alveolar bone loss model at days −2, 0 and 2 relatives to ligature placement. At day 9 gingival tissues were harvested and dissected for cell isolation. Immunofluorescence labelling of cells was performed using Alexa Flour 488-labelled anti CD11c to identify dendritic cells, counterstained with nuclear stain DAPI and visualized under confocal microscopy. (A) Co-localization of EXO (red) with nucleus (DAPI) and dendritic cells (green). (B) Flow cytometry analysis of MHCII+ cells % in CD11c+DCs gate (top histograms) and CD86+cells % in MHCII+ CD11c+DCs gate (bottom histograms) in gingival cells isolated from six tested groups. (C) Bar graphs of flow cytometry data from (B) showing statistically significant differences in the groups. (D) Representative immunohistochemical staining of CD86+presumptive DCs (arrows) in lamina propria of gingival tissue sections of (left) no EXO-treated +ligature and (right) regDCs EXO-treated +ligature groups. (E) Bar graph of immunohistochemical staining data from (D) showing statistically significant differences in the groups. N=5 in each group; * P<0.05 by one-way ANOVA, followed by Tukeys multiple comparisons.

Figure 8.

RegDCs EXO interact with gingival CD4T-cells, inducing Tregs and inhibiting Th17 recruitment in vivo. DIL (red) labelled EXO were injected into palatal gingiva of the upper right second molar at days −2, 0 and 2 relatives to ligature placement. At day 9 gingival tissue were harvested and dissected for cell isolation. Immunofluorescence labelling of cells were performed with Dylight 488 labelled-anti-CD4 (green) to identify CD4-T cells and DAPI for nucleus and visualized under confocal microscopy. (A) Co-localization of EXO with nucleus (DAPI) and CD4T-cell (green). (B) Scattergrams from flow cytometry analysis of CD25+Foxp3+Tregs % (top panels) and CD4+IL-17A+ Th17 cells % (bottom panels), gated on CD4+and CD3+lymphocytes, respectively, in the gingival cells isolated from the six tested groups. (C) (Left) Bar graphs of CD25+Foxp3+Tregs % (upper panel) and CD4+IL-17A+ Th17 cells % (lower panel) in all groups from (B), and (Right) mRNA levels of (top) Foxp3, (middle) CTLA4 and (bottom) IL-10 in: no EXO vs regDCs EXO-treated groups + ligature. Representative immunohistochemical staining and bar graphs of (D) Foxp3 and (E) IL-17A in lamina propria of gingival tissue sections of (left) no EXO-treated +ligature group and (right) regDCs EXO-treated +ligature group. White arrows delineate positive staining. N=5 in each group; * P<0.05 by one-way ANOVA, followed by Tukeys multiple comparisons.

Figure 9.

RegDCs EXO inhibit RANK-L, TNFa and TRAP+ osteoclast induction. mRNA expression of RANKL (A) and TNFa (B) in gingival tissue of ligature groups that received indicated EXO subtypes or no EXO. Representative immunohistochemical stained sections and bar graphs showing expression of (C) RANKL and (D) TNFa in lamina propria of gingival tissue sections treated with no EXO + ligature or regDCs EXO + ligature. (E) Representative Trap+staining of periodontal tissue sections in EXO subtypes + ligature or no EXO and no ligature or ligature alone treated groups. (F) Bar graph showing quantification of Trap positive multinucleated cell per bone surface area in interdental area, three slides per animal. N=5 in each group; * P<0.05 by one-way ANOVA followed by Tukeys multiple comparisons.

Figure 10.

RegDCs EXO inhibit, stimDC EXO promote inflammatory bone loss. (A) Representative microCT generated 3-D images of upper right maxilla with teeth. (B) Bar graphs of alveolar bone volume change % based on quantification of the whole 3-D alveolar bone volume around upper second molar (ligature placement site) by micro CT and normalization to the measurement obtained from the contralateral side (no ligature) which served as baseline, followed by normalization to the alveolar bone volume around the upper right second molar of group that did not receive ligature or exosomes (N=5 in each group; * P<0.05 by one-way ANOVA followed by Tukeys multiple-comparisons). (C) Histological sections showing distinct levels of alveolar bone in furcation area and interdental bone (arrows) of upper right second molar of tested groups.