Limits...
Exosome-delivered microRNAs modulate the inflammatory response to endotoxin.

Alexander M, Hu R, Runtsch MC, Kagele DA, Mosbruger TL, Tolmachova T, Seabra MC, Round JL, Ward DM, O'Connell RM - Nat Commun (2015)

Bottom Line: However, recent evidence suggests that microRNAs can be transferred between cells and mediate target gene repression.We find that endogenous miR-155 and miR-146a, two critical microRNAs that regulate inflammation, are released from dendritic cells within exosomes and are subsequently taken up by recipient dendritic cells.Following uptake, exogenous microRNAs mediate target gene repression and can reprogramme the cellular response to endotoxin, where exosome-delivered miR-155 enhances while miR-146a reduces inflammatory gene expression.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology and Immunology, Department of Pathology, University of Utah, 4280 JMRB, 15 North Medical Drive East, Salt Lake City, Utah 84112, USA.

ABSTRACT
MicroRNAs regulate gene expression posttranscriptionally and function within the cells in which they are transcribed. However, recent evidence suggests that microRNAs can be transferred between cells and mediate target gene repression. We find that endogenous miR-155 and miR-146a, two critical microRNAs that regulate inflammation, are released from dendritic cells within exosomes and are subsequently taken up by recipient dendritic cells. Following uptake, exogenous microRNAs mediate target gene repression and can reprogramme the cellular response to endotoxin, where exosome-delivered miR-155 enhances while miR-146a reduces inflammatory gene expression. We also find that miR-155 and miR-146a are present in exosomes and pass between immune cells in vivo, as well as demonstrate that exosomal miR-146a inhibits while miR-155 promotes endotoxin-induced inflammation in mice. Together, our findings provide strong evidence that endogenous microRNAs undergo a functional transfer between immune cells and constitute a mechanism of regulating the inflammatory response.

No MeSH data available.


Related in: MedlinePlus

Functional transfer of miR-155 via exosomes in vitro.(a) Schematic of the exosome transfer experiment. (b) qRT–PCR was used to measure relative miR-155 levels in miR-155−/− BMDCs that received either Wt or miR-155−/− exosomes derived from BMDCs treated with or without GW4869 (n=5). (c,d) mRNA levels of miR-155 targets, BACH1 and SHIP1, from the same experiment shown in b as measured by qRT–PCR (n=5). (e) Representative western blottings of SHIP1 and β-actin in miR-155−/− BMDCs given either Wt or miR-155−/− exosomes. (f) Protein levels of SHIP1 were quantified using ImageJ software (n=2). (g) Relative miR-155 levels in Wt BMDCs given either Wt or miR-155−/− exosomes as quantified by qRT–PCR (n=6). (h,i) BACH1 and SHIP1 mRNA levels were measured in the same experiment shown in g as quantified by qRT–PCR (n=6). (j) qRT–PCR was used to quantify HO1 mRNA levels during the experiment in b (n=5). (k) Western blotting for AGO2 and β-actin from miR-155−/− BMDCs given Wt or miR-155−/− exosomes. On the left is the input (whole-cell lysate), the middle is from the pan-AGO pulldown where one-third of input was used and the right is the IgG pulldown where one-third of the input was used. (l) Relative miR-155 levels were quantified via qRT–PCR in the same experiment shown in k. (m) miR-146a levels were quantified using qRT–PCR during the experiment in k. Levels in l,m are plotted as Ago:IgG. Dotted line separates input from pull-down groups. Data represent two independent experiments and are presented as the mean±s.d. (error bars). *P<0.05; **P<0.01, Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4557301&req=5

f2: Functional transfer of miR-155 via exosomes in vitro.(a) Schematic of the exosome transfer experiment. (b) qRT–PCR was used to measure relative miR-155 levels in miR-155−/− BMDCs that received either Wt or miR-155−/− exosomes derived from BMDCs treated with or without GW4869 (n=5). (c,d) mRNA levels of miR-155 targets, BACH1 and SHIP1, from the same experiment shown in b as measured by qRT–PCR (n=5). (e) Representative western blottings of SHIP1 and β-actin in miR-155−/− BMDCs given either Wt or miR-155−/− exosomes. (f) Protein levels of SHIP1 were quantified using ImageJ software (n=2). (g) Relative miR-155 levels in Wt BMDCs given either Wt or miR-155−/− exosomes as quantified by qRT–PCR (n=6). (h,i) BACH1 and SHIP1 mRNA levels were measured in the same experiment shown in g as quantified by qRT–PCR (n=6). (j) qRT–PCR was used to quantify HO1 mRNA levels during the experiment in b (n=5). (k) Western blotting for AGO2 and β-actin from miR-155−/− BMDCs given Wt or miR-155−/− exosomes. On the left is the input (whole-cell lysate), the middle is from the pan-AGO pulldown where one-third of input was used and the right is the IgG pulldown where one-third of the input was used. (l) Relative miR-155 levels were quantified via qRT–PCR in the same experiment shown in k. (m) miR-146a levels were quantified using qRT–PCR during the experiment in k. Levels in l,m are plotted as Ago:IgG. Dotted line separates input from pull-down groups. Data represent two independent experiments and are presented as the mean±s.d. (error bars). *P<0.05; **P<0.01, Student's t-test.

Mentions: With the knowledge that miR-155 can be transferred between BMDCs, we wanted to determine whether exosomes are sufficient for this transfer and whether transfer could result in knockdown of target mRNAs. To specifically investigate the impact of exosomally transferred miRNA without the effects of other factors that are released from BMDCs, we purified exosomes away from other components in the conditioned medium using differential centrifugation and washing. Next, the exosomes were re-suspended in fresh medium and administered to recipient cells. Wt (1 × 106) or miR-155−/− BMDCs produced ∼5 × 108 exosomes in 24 h (Supplementary Fig. 2). Exosomes isolated from the supernatant of both Wt and miR-155−/− BMDCs treated with GW4869 or dimethylsulfoxide vehicle control were transferred to miR-155−/− receipient BMDCs. miR-155−/− recipient BMDCs were incubated with donor exosomes for 24 h, to allow time for miRNA transfer and knockdown of miRNA targets (Fig. 2a). Using qRT–PCR, we detected increased miR-155 levels and decreased mRNA levels of miR-155 targets BACH1 and SHIP1 when cells were treated with Wt exosomes (Fig. 2b–d). These changes were prevented if the exosomes were derived from miR-155−/− BMDCs, or if the Wt donor cells were pretreated with GW4869. SHIP1 protein levels were also decreased in miR-155−/− BMDCs that received Wt exosomes (Fig. 2e,f). Exosome delivery of miR-155 brought its levels in the miR-155−/− recipient cells to ∼20% of Wt miR-155 levels (Supplementary Fig. 3a,b). Furthermore, we also looked at the relative expression of a separate miRNA, miR-425, which has been previously seen to be released in exosomes30, as a control. The levels of miR-425 increased with exosome delivery and were roughly the same in Wt and knockout groups (Supplementary Fig. 3c,d).


Exosome-delivered microRNAs modulate the inflammatory response to endotoxin.

Alexander M, Hu R, Runtsch MC, Kagele DA, Mosbruger TL, Tolmachova T, Seabra MC, Round JL, Ward DM, O'Connell RM - Nat Commun (2015)

Functional transfer of miR-155 via exosomes in vitro.(a) Schematic of the exosome transfer experiment. (b) qRT–PCR was used to measure relative miR-155 levels in miR-155−/− BMDCs that received either Wt or miR-155−/− exosomes derived from BMDCs treated with or without GW4869 (n=5). (c,d) mRNA levels of miR-155 targets, BACH1 and SHIP1, from the same experiment shown in b as measured by qRT–PCR (n=5). (e) Representative western blottings of SHIP1 and β-actin in miR-155−/− BMDCs given either Wt or miR-155−/− exosomes. (f) Protein levels of SHIP1 were quantified using ImageJ software (n=2). (g) Relative miR-155 levels in Wt BMDCs given either Wt or miR-155−/− exosomes as quantified by qRT–PCR (n=6). (h,i) BACH1 and SHIP1 mRNA levels were measured in the same experiment shown in g as quantified by qRT–PCR (n=6). (j) qRT–PCR was used to quantify HO1 mRNA levels during the experiment in b (n=5). (k) Western blotting for AGO2 and β-actin from miR-155−/− BMDCs given Wt or miR-155−/− exosomes. On the left is the input (whole-cell lysate), the middle is from the pan-AGO pulldown where one-third of input was used and the right is the IgG pulldown where one-third of the input was used. (l) Relative miR-155 levels were quantified via qRT–PCR in the same experiment shown in k. (m) miR-146a levels were quantified using qRT–PCR during the experiment in k. Levels in l,m are plotted as Ago:IgG. Dotted line separates input from pull-down groups. Data represent two independent experiments and are presented as the mean±s.d. (error bars). *P<0.05; **P<0.01, Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557301&req=5

f2: Functional transfer of miR-155 via exosomes in vitro.(a) Schematic of the exosome transfer experiment. (b) qRT–PCR was used to measure relative miR-155 levels in miR-155−/− BMDCs that received either Wt or miR-155−/− exosomes derived from BMDCs treated with or without GW4869 (n=5). (c,d) mRNA levels of miR-155 targets, BACH1 and SHIP1, from the same experiment shown in b as measured by qRT–PCR (n=5). (e) Representative western blottings of SHIP1 and β-actin in miR-155−/− BMDCs given either Wt or miR-155−/− exosomes. (f) Protein levels of SHIP1 were quantified using ImageJ software (n=2). (g) Relative miR-155 levels in Wt BMDCs given either Wt or miR-155−/− exosomes as quantified by qRT–PCR (n=6). (h,i) BACH1 and SHIP1 mRNA levels were measured in the same experiment shown in g as quantified by qRT–PCR (n=6). (j) qRT–PCR was used to quantify HO1 mRNA levels during the experiment in b (n=5). (k) Western blotting for AGO2 and β-actin from miR-155−/− BMDCs given Wt or miR-155−/− exosomes. On the left is the input (whole-cell lysate), the middle is from the pan-AGO pulldown where one-third of input was used and the right is the IgG pulldown where one-third of the input was used. (l) Relative miR-155 levels were quantified via qRT–PCR in the same experiment shown in k. (m) miR-146a levels were quantified using qRT–PCR during the experiment in k. Levels in l,m are plotted as Ago:IgG. Dotted line separates input from pull-down groups. Data represent two independent experiments and are presented as the mean±s.d. (error bars). *P<0.05; **P<0.01, Student's t-test.
Mentions: With the knowledge that miR-155 can be transferred between BMDCs, we wanted to determine whether exosomes are sufficient for this transfer and whether transfer could result in knockdown of target mRNAs. To specifically investigate the impact of exosomally transferred miRNA without the effects of other factors that are released from BMDCs, we purified exosomes away from other components in the conditioned medium using differential centrifugation and washing. Next, the exosomes were re-suspended in fresh medium and administered to recipient cells. Wt (1 × 106) or miR-155−/− BMDCs produced ∼5 × 108 exosomes in 24 h (Supplementary Fig. 2). Exosomes isolated from the supernatant of both Wt and miR-155−/− BMDCs treated with GW4869 or dimethylsulfoxide vehicle control were transferred to miR-155−/− receipient BMDCs. miR-155−/− recipient BMDCs were incubated with donor exosomes for 24 h, to allow time for miRNA transfer and knockdown of miRNA targets (Fig. 2a). Using qRT–PCR, we detected increased miR-155 levels and decreased mRNA levels of miR-155 targets BACH1 and SHIP1 when cells were treated with Wt exosomes (Fig. 2b–d). These changes were prevented if the exosomes were derived from miR-155−/− BMDCs, or if the Wt donor cells were pretreated with GW4869. SHIP1 protein levels were also decreased in miR-155−/− BMDCs that received Wt exosomes (Fig. 2e,f). Exosome delivery of miR-155 brought its levels in the miR-155−/− recipient cells to ∼20% of Wt miR-155 levels (Supplementary Fig. 3a,b). Furthermore, we also looked at the relative expression of a separate miRNA, miR-425, which has been previously seen to be released in exosomes30, as a control. The levels of miR-425 increased with exosome delivery and were roughly the same in Wt and knockout groups (Supplementary Fig. 3c,d).

Bottom Line: However, recent evidence suggests that microRNAs can be transferred between cells and mediate target gene repression.We find that endogenous miR-155 and miR-146a, two critical microRNAs that regulate inflammation, are released from dendritic cells within exosomes and are subsequently taken up by recipient dendritic cells.Following uptake, exogenous microRNAs mediate target gene repression and can reprogramme the cellular response to endotoxin, where exosome-delivered miR-155 enhances while miR-146a reduces inflammatory gene expression.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology and Immunology, Department of Pathology, University of Utah, 4280 JMRB, 15 North Medical Drive East, Salt Lake City, Utah 84112, USA.

ABSTRACT
MicroRNAs regulate gene expression posttranscriptionally and function within the cells in which they are transcribed. However, recent evidence suggests that microRNAs can be transferred between cells and mediate target gene repression. We find that endogenous miR-155 and miR-146a, two critical microRNAs that regulate inflammation, are released from dendritic cells within exosomes and are subsequently taken up by recipient dendritic cells. Following uptake, exogenous microRNAs mediate target gene repression and can reprogramme the cellular response to endotoxin, where exosome-delivered miR-155 enhances while miR-146a reduces inflammatory gene expression. We also find that miR-155 and miR-146a are present in exosomes and pass between immune cells in vivo, as well as demonstrate that exosomal miR-146a inhibits while miR-155 promotes endotoxin-induced inflammation in mice. Together, our findings provide strong evidence that endogenous microRNAs undergo a functional transfer between immune cells and constitute a mechanism of regulating the inflammatory response.

No MeSH data available.


Related in: MedlinePlus