SCIENCE ADVANCES | RESEARCH ARTICLEHEALTH AND MEDICINE

Exosome-based delivery of super-repressor IB
relieves sepsis-associated organ damage and mortality
Hojun Choi1,2*, Youngeun Kim3*, Amin Mirzaaghasi1, Jaenyoung Heo3, Yu Na Kim3,
Ju Hye Shin4, Seonghun Kim5, Nam Hee Kim6, Eunae Sandra Cho6, Jong In Yook6,
Tae-Hyun Yoo5, Eunjoo Song7, Pilhan Kim2,7,8, Eui-Cheol Shin2†, Kyungsoo Chung4†,
Kyungsun Choi3†, Chulhee Choi1,2,3

INTRODUCTION

Sepsis is a systemic inflammatory syndrome caused by activation of
the innate immune system due to acute microbial infection. It continues to be the main cause of mortality in intensive care units, withan estimated 400,000 to 600,000 patients developing sepsis annuallyin the US and Europe (1). On September 13 of each year, “World
Sepsis Day” is published by the Global Sepsis Alliance. Sepsis is aserious disease that has become a major social issue. Previously,
Xigris (Eli Lilly) was introduced in 2001 as the only U.S. Food andDrug Administration–approved sepsis treatment; however, it waswithdrawn from the market in October 2011 due to bleeding-relatedside effects and lack of efficacy (2). Currently, no sepsis-specifictreatments are available for clinical usage. The development ofeffective alternative therapies for sepsis is urgently needed.
Uncontrolled inflammation is a prominent feature of the septic
response (3, 4). Host-pathogen interactions mediated by Toll-likereceptors stimulate the production of proinflammatory cytokines,
chemokines, and immune-activating molecules (5). This proinflammatory response is followed by a compensatory immunosuppressive response that involves various quantitative and functional
defects of immune cells. Pathogen-induced cellular modification isaccompanied by marked changes in host gene expression (6), with
nuclear factor B (NF-B) transcription factors playing a pivotal
1

Department of Bio and Brain Engineering, KAIST, Daejeon 34141, Republic of Korea.
Graduate School of Medical Science and Engineering, KAIST, Daejeon 34141,
Republic of Korea. 3ILIAS Biologics Inc., Daejeon 34014, Republic of Korea. 4Divisionof Pulmonology, Department of Internal Medicine, Yonsei University College ofMedicine, Seoul 03722, Republic of Korea. 5Department of Internal Medicine, Yonsei
University College of Medicine, Seoul 03722, Republic of Korea. 6Department ofOral Pathology, Oral Cancer Research Institute, Yonsei University College of Dentistry,Seoul 03722, Republic of Korea. 7IVIM Technology, Daejeon 34051, Republic ofKorea. 8Graduate School of Nanoscience and Technology, KAIST, Daejeon 34141,
Republic of Korea.
*These authors contributed equally to this work.
†Corresponding author. Email: kchoi@iliasbio.com (K.Cho.); euicheols@kaist.ac.kr(E.-C.S.); chungks@yuhs.ac (K.Chu.)
2

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role in modulating these changes. Several cytokines under the regulatory control of NF-B, including tumor necrosis factor– (TNF-)and interleukin-1 (IL-1), can induce further activation of this transcriptional factor, leading to potentiation of the inflammatory
response and septic shock. In addition, NF-B is involved in theapoptotic response, primarily through enhancing the transcriptionof antiapoptotic genes such as Bcl-xL, A1, and A20 (7, 8). Inflammation associated with NF-B activation can, thus, be exacerbated by
two interacting mechanisms, increased expression of proinflammatory mediators and prolongation of the life spans of cell populations
such as neutrophils, which are activated to produce proinflammatory
molecules and participate directly in acute inflammatory processes.
Recent studies in animal models of sepsis induced either by lipopolysaccharide (LPS; endotoxin model) or cecal ligation andpuncture (CLP; polymicrobial model) have demonstrated that NF-B
inhibitors with diverse chemical properties and mechanisms ofaction protect animals from septic lethality (9–12). Although morethan 700 inhibitors of NF-B have been reported (13), no NF-B
blocker has been approved for human use to date. Various steroids
and nonsteroid anti-inflammatory drugs have been found to block
NF-B, but their effects are highly pleiotropic and lack specificity.
Novel therapeutic strategies aimed at specific inhibition of key elementsin the NF-B activation pathway have been under development in recent years, and expectations are high regarding their potential efficacy
as sepsis treatments. Genetic constructs expressing an engineered
inhibitor of nuclear factor B (IB) protein without sites forphosphorylation, super-repressor IB (srIB), have also been used.
IB mutation at specific phosphorylation sites (Ser32 and Ser36
replaced to Ala) results in a dominant active form of IB with aprolonged half-life. These srIBs lead to a stable cytoplasmic pool ofIB, thereby preventing nuclear NF-B activation (14, 15).
Here, we used exosomes as a therapeutic carrier to deliver
srIB to a therapeutic target. Exosomes have been recognized as
potent therapeutic vehicles for transferring various proteins and regulatory genes to target cells (16–18). They function as nonimmunogenic
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As extracellular vesicles that play an active role in intercellular communication by transferring cellular materials torecipient cells, exosomes offer great potential as a natural therapeutic drug delivery vehicle. The inflammatory responses in various disease models can be attenuated through introduction of super-repressor IB (srIB), which isthe dominant active form of IB and can inhibit translocation of nuclear factor B into the nucleus. An optogenetically engineered exosome system (EXPLOR) that we previously developed was implemented for loading alarge amount of srIB into exosomes. We showed that intraperitoneal injection of purified srIB-loaded exosomes (Exo-srIBs) attenuates mortality and systemic inflammation in septic mouse models. In a biodistribution
study, Exo-srIBs were observed mainly in the neutrophils, and in monocytes to a lesser extent, in the spleens andlivers of mice. Moreover, we found that Exo-srIB alleviates inflammatory responses in monocytic THP-1 cells andhuman umbilical vein endothelial cells.

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SCIENCE ADVANCES | RESEARCH ARTICLE
nanovesicles and can protect their cargo from serum proteases andimmune responses (19). Loading of soluble proteins into exosomes
has been enabled by a technology called EXPLOR, which exploits thenatural exosome biogenesis process and reversible protein-protein
interactions controlled by optogenetics (20). To load specific target
proteins into exosomes, we generated a human embryonic kidney(HEK) 293T cell line that stably expresses two recombinant proteins,
CIBN-EGFP-CD9 and srIB-mCherry-­CRY2 (Fig. 1A). Consistent
with previous studies, EXPLOR technology was used to produce
srIB-loaded exosomes. We designated the srIB loading exosomes
as Exo-srIB, and exosomes generated from intact HEK293T cells
as Exo-Naïve. By applying Exo-srIB to sepsis models for therapeutic
purposes, we ameliorated sepsis-induced organ injury and inhibitedthe secretion of proinflammatory cytokines, thereby improving theoverall survival of patients with sepsis.

trate the exosomes. Each type of exosome isolated through sequential purification was analyzed via nanoparticle tracking analysis (NTA),
which characterizes the size and concentration of exosomes (fig.
S1B). Particles with diameters from 30 to 120 nm accounted forover 80% of all particles, with a mean size of 101 nm, which is consistent with the characteristic size range (30 to 120 nm) of exosomes.
In addition, transmission electron microscopy (TEM) showed intact cup-shaped membrane vesicles with sizes corresponding to the
NTA results obtained from Exo-Naïve and Exo-srIB (Fig. 1B). To
further characterize the exosomes in our preparations, we investigated two common exosome markers, the tetraspanins CD63 and
TSG101, through Western blotting. The presence of CD63 and
TSG101 was observed in the samples, whereas a Golgi-derived contaminant, GM130, was only detected in the cell lysate (Fig. 1C). This
analysis of the exosome preparations showed that they exhibit thecharacteristics of exosomes. Furthermore, we observed robust loadingof srIB-mCherry-CRY2 (130 kDa) into exosomes isolated from srIB
stable cell lines.

RESULTSA

CIBN-EGFP-CD9
srI κB-mCherry-CRY2

CIBN

Exo-srIB improves survival and ameliorates acute organ
injury in septic mice
We investigated whether intraperitoneal injection with Exo-srIB
protected against endotoxic shock. Animals received a single intraperitoneal injection with a lethal dose of LPS (40 mg/kg body weight for
C57BL/6, and 20 mg/kg body weight for BALB/c), followed by asingle intraperitoneal injection of Exo-srIB (6 hours apart). Compared with control C57BL/6 mice, which showed 100% mortality

EGFP CD9

srIκB mCherry CRY2

Light onLight offsrIκB

mCherry

Exosome
Naïve

Naïve

Exo-Naïve

srIκB

Producing cellCmCherryB

srIκB-mCherry-CRY2

IB: IκBαEndogenous IκBα

IB: GFP

CIBN-EGFP-CD9

Exo-srIκB

IB: CD63
IB: TSG101
100 nm

100 nm

IB: GM130
IB: GAPDH

Fig. 1. Generation and characterization of engineered exosomes. (A) Schematic of DNA constructs used for the production of Exo-srIB (top). Schematic showing
fusion proteins and their proposed activities (bottom). (B) Morphological characterization of Exo-Naïve and Exo-srIB through transmission electron microscopy.
(C) HEK293T cells that stably express mCherry or srIB, and exosomes from these HEK293T cells, were lysed and immunoblotted against the indicated proteins. IB, immunoblot.
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Characterization and analysis of srIB-loaded exosomes
To produce sufficient exosomes, we collected culture medium fromeach cell type. Exosomes were isolated using a combination of tangential flow filtration (TFF) and size exclusion chromatography(SEC) (fig. S1A). To reduce the risk of loading the SEC column with
impurities that would exceed its binding capacity, the samples were
subjected to diafiltration and concentration through TFF. After
TFF, the concentrated medium was loaded onto an SEC column forfurther purification. A second TFF was then performed to concen-

SCIENCE ADVANCES | RESEARCH ARTICLE
The animals survived the 7-day monitoring period, indicating that
Exo-srIB provides lasting protection against sepsis mortality(Fig. 2A, bottom). To further investigate whether Exo-srIB treatment alleviated the inflammation induced by sepsis, enzyme-linked
immunosorbent assay (ELISA) was used to determine the concentrations of key inflammation factors in the serum. We observed significant reductions in the expression levels of the proinflammatory
cytokines TNF-, IL-1, and IL-6, as well as the chemokine carbon
tetrachloride (CCL4), in the Exo-srIB treatment group with
LPS-induced sepsis (Fig. 2B, top). We also observed that serum levels

due to LPS-induced sepsis, mice treated with Exo-srIB were remarkably resistant to LPS-induced mortality; most of the mice were
rescued from sepsis and showed prolonged survival (Fig. 2A, top).
Exo-srIB–mediated effects were associated with modest protection from the LPS-induced temperature drop (fig. S2A). We also
developed a mouse model of LPS-induced sepsis using BALB/c mice.
Exo-srIB treatment significantly improved the survival rate of LPS-­
induced sepsis in BALB/c mice (Fig. 2A, middle). We next examined
the role of Exo-srIB in survival using a model of CLP-induced
sepsis as a standardized murine model of intra-abdominal sepsis.
PBS

Exo-Naïve

100

Exo-srIκB

B

Sham
LPS/CLP + PBS

C57BL/6

80
60

20
0

20

40

60

80

100

IL-1β (pg/ml)

60
40
20

*
0

20

†

†

LPS

BALB/c

80

0

*

**

40

60

**
††

**

CCL4 (pg/ml)

0

TNF-α (pg/ml)**

IL-6 (pg/ml)

40Time after LPS (hours)
Survival rate (%)

LPS/CLP + Exo-Naïve
LPS/CLP + Exo-srIκB

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Survival rate (%)

ATime after LPS (hours)

60

CLP

40
20
0

*
0

24

48

72

96

120

144

CCL4 (pg/ml)*

C57BL/6

80

TNF-α (pg/ml)

Survival rate (%)

100

†

168

Time after CLP (hours)
ShamC

CLP + PBS

D
PBS

Exo-Naïve

CLP + Exo-Naïve

CLP + Exo-srIκB

LPS

Kidney injury score

KidneyCLP

Exo-srIκB

C57BL/6

C57BL/6

BALB/cFig. 2. The protective effects of Exo-srIB in endotoxemia and CLP-induced sepsis. (A) Survival curves of phosphate-buffered saline (PBS)–, Exo-Naïve–, and Exo-­srIB–treated septic mice. LPS C57BL/6 mice (n = 5 to 6 per group), LPS BALB/c mice (n = 10 per group), and CLP C57BL/6 mice (n = 14 to 15 per group). **P < 0.01 and*P < 0.05 compared with the PBS-treated sepsis group. (B) Levels of TNF-, IL-6, IL-1, and CCL4/macrophage inflammatory protein-1 in the plasma of exosome-treatedmice were measured 24 hours after LPS injection or CLP. **P < 0.01 and *P < 0.05 compared with the PBS-treated sepsis group. †P < 0.05 compared with the Exo-Naïve–treated sepsis group. (C) Representative images of cortical tubular cells in kidney sections from sham, CLP with PBS, CLP with Exo-Naïve, and CLP with Exo-srIB mice.
Normal brush border (*) of proximal tubules or loss of brush border (○), chromatin condensation (white arrows), denuded basement membrane (white arrow heads), andvacuolization (yellow arrows). Scale bars, 100 M. (D) Pathological kidney injury scores of representative kidney samples of each group. *P < 0.05 compared with the
PBS-treated sepsis group. †P < 0.05 compared with the Exo-Naïve–treated sepsis group. GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Biodistribution of exosomes after LPS injection
Next, we investigated the changes in exosome biodistribution in the
LPS-injected septic mouse model. The in vivo biodistribution was
analyzed using a custom-made intravital video-rate laser scanningconfocal microscopy system (22). mCLING fluorescence dye–labeled
exosomes were intravenously injected into LysMgfp/+ transgenic
C57BL/6 mice that had also been injected with anti-Ly6G antibody
to visualize neutrophils [green fluorescent protein (GFP) andAlexa Fluor 555] and macrophages (GFP). We observed thatmost exosomes were taken up by neutrophils and macrophages
rapidly, i.e., within a few minutes of intravenous exosome injection(Fig. 3A and movie S1).
Exosomes were delivered mainly to the neutrophils of the liver(Fig. 3B), and we observed increased recruitment of neutrophils
and macrophages, as well as increased uptake of exosomes, in the
spleens of LPS-induced mice (Fig. 3C). mCLING-labeled exosomes
were also delivered to neutrophils in the kidneys of LPS-injected
mice (fig. S3A). These observations suggest that therapeutic exosomes
were successfully delivered to target neutrophils and macrophages
within 30 min in the septic mouse model.

cells are widely used to represent monocytes in cell culture models.
When THP-1 cells were stimulated with LPS, treatment with Exo-­srIB led to decreased secretion of the inflammatory cytokines
TNF- and monocyte chemoattractant protein-1 (MCP-1), which
are under NF-B transcriptional control, compared with Exo-Naïve
(Fig. 4C). We also compared the effects of Exo-srIB with those of
the commonly used NF-B inhibitor JSH-23, which interferes withthe binding of NF-B to its target DNA (25). Our data showed that
Exo-srIB and JSH-23 had comparable effects with respect to inhibiting LPS-induced NF-B activation and cytokine production in
THP-1 cells (Fig. 4C, lane 4 versus lane 6).
Sepsis is characterized by monocyte adherence to the endothelium.
Under normal conditions, endothelial cells are quiescent and do notinteract with monocytes. However, in an inflammatory environment, activated endothelial cells express adhesion molecules, suchas intercellular cell adhesion molecule-1 (ICAM-1), which bind tomonocytes. The expression of adhesion molecules is widely considered to be activated during the development of sepsis (26). To
explore the effect of Exo-srIB on the expression of cell adhesion
molecules in human umbilical vein endothelial cells (HUVECs), we
first examined whether Exo-srIBs are internalized in the cells.
mCLING-labeled Exo-srIBs were detectable in HUVECs, withmaximum internalization observed after 24 hours (Fig. 4D). We
found that ICAM-1 expression in HUVECs was significantly inhibitedby Exo-srIB treatment (Fig. 4E). IL-8 and MCP-1 also triggered
the migration and adhesion of monocytes to the vascular endothelium and led to extravasation of these cells into the surrounding
tissues (27). Expression of IL-8 and MCP-1 has been observed previously in various tissues during acute inflammation caused bybacterial and viral infections and is associated with severe sepsis(28). We found that Exo-srIB suppressed LPS-induced IL-8 and
MCP-1 production (fig. S4A) by inhibiting NF-B transcriptional
activity in HUVECs (fig. S4B). These results suggest that Exo-srIB
reduces the NF-B–mediated inflammatory response during sepsis.
DISCUSSION

The complex pathophysiology of sepsis, which involves multiple
organ failure, hinders the development of therapeutic agents thatcan effectively treat all manifestations of sepsis. Nevertheless, thegeneral consensus is that sepsis is initiated through strong activation of the innate immune system and mediated by the activation ofpattern recognition receptors by pathogens, leading to activation of
the complement system, the coagulation system, and the vascularendothelium (29). This process is followed by an immunosuppressive state, which, in turn, results in a failure to return to normal
homeostasis. Therefore, innovative immunomodulatory agents that
can target the root cause of sepsis are urgently needed. Here, we
Exo-srIB alleviates inflammation associated with sepsis
report the effect of Exo-srIB on resolving inflammatory responses
Next, we identified the target specificity and efficiency of Exo-srIB and reducing organ damage in a septic mouse model through inhiin vitro. Exo-srIB, but not Exo-Naïve, significantly blocked bition of NF-B activity. We used a recently developed technology
TNF-–induced NF-B activation in an HEK293 cell stably ex- that can load functional proteins into exosomes (EXPLOR) to genpressing an NF-B luciferase reporter gene (Fig. 4A). Increasing erate an immunosuppressive exosome loaded with srIB (20). srIB
doses of Exo-srIB decreased NF-B reporter activity in a dose-­ is a nondegradable form of IB that prevents NF-B from entering
dependent manner, indicating that srIB released from exosomes the nucleus and acting as a transcription factor (14, 15).
prevents NF-B transcriptional activity (Fig. 4B).
Through systemic delivery of Exo-srIB in a septic mouse model,
Among all sepsis-responsive cells, monocytes/macrophages play we demonstrated that exosomes are delivered primarily to neutrothe most critical role in promoting the immune response (23), and phils and macrophages, which are key players in the overwhelming
depletion of these cells in septic mice increases mortality (24). THP-1 inflammatory response of sepsis. Our results showed that Exo-srIB

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4 of 9Downloaded from http://advances.sciencemag.org/ on April 8, 2020of TNF- and CCL4 in the CLP group were significantly higher
than those in sham-operation control mice but were not elevated inmice treated with both CLP and Exo-srIB (Fig. 2B, bottom). These
results suggest that Exo-srIB alleviates inflammation induced by
LPS or CLP and protects septic mice against the acute inflammatory
process.
Numerous studies have shown that acute kidney injury (AKI) isa frequent and serious complication of sepsis, occurring in 50% ormore cases, and is associated with very high mortality (21). To
determine the role of Exo-srIB in CLP-induced AKI in the septic
mouse model, renal histological examination was performed. In
comparison with those that underwent a sham operation, the renal
tubular cells of mice treated with phosphate-buffered saline (PBS)
and Exo-Naive mice showed significant damage in terms of vacuolization, loss of the brush border, and nuclear condensation of
tubular epithelial cells (Fig. 2C). Notably, Exo-srIB treatment
markedly decreased damage to tubular areas, preserving proximal
tubules and demonstrating the therapeutic potential of Exo-srIB
for sepsis-induced renal injury. These results are consistent with thosefor LPS-induced AKI (fig. S2B). We further quantified the severityof histological kidney damage and found that Exo-srIB treatment
significantly reduced the injury score by 50% in both sepsis models(Fig. 2D). Exo-srIB ameliorated degenerative changes in the renal
tubules, although some damaged areas were present. These datasuggest the critical importance of Exo-srIB in improving renal
physiological structure and function in septic mice.

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A

B

C Exosome

30

60

90

(min)

LPS LysMgfp/+

Sham LysMgfp/+

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Fig. 3. The biodistribution of exosomes in LPS-injected mice. (A) Intravital imaging of mCLING-labeled exosome (red) uptake into neutrophils (LysMgfp/+, green; Ly6G+,
blue) in the liver of sham mice. (B) Representative time-lapse imaging of mCLING-labeled exosomes (red) inside the Ly6G+ neutrophil cells (green) of the liver in LPS-treated
C57BL/6 mice. (C) Sequential images of flowing mCLING-labeled exosomes (red) inside the spleen of sham- and LPS-treated LysMgfp/+ mice. Elapsed time is indicated.
Magenta, autofluorescence. Scale bars, 50 m.

treatment significantly reduced proinflammatory markers such as
TNF-, IL-1, and IL-6, but not the anti-inflammatory cytokine
IL-10 (fig. S2D), in two mouse sepsis models. Even when exosomes
were injected 1 hour after LPS injection, we observed a significant
therapeutic effect of Exo-srIB. Considered together with our otherfindings, these results show that Exo-srIB treatment could be
administered during the early phase of sepsis, before conversion
to the immunosuppressive state.
Growing evidence has shown that proinflammatory cytokines
play a prominent role in sepsis-induced AKI (30). In the present
study, we successfully established a murine model of sepsis-induced
AKI through CLP surgery or LPS injection. Histopathological examChoi et al., Sci. Adv. 2020; 6 : eaaz6980

8 April 2020

ination showed that the glomerular structure was destroyed, renal
tubular epithelial cells were degenerated, and severe intracellularedema and congestion occurred within the renal tubule in thesepsis-induced group. However, treatment with Exo-srIB reduced
the severity of lesions and limited renal injury and the infiltration of
inflammatory cells.
Numerous studies have suggested that neutrophil tissue infiltration is a key process in sepsis (31, 32). For example, depletion ofneutrophils or inhibition of neutrophil recruitment through targeting specific adhesion molecules has been repeatedly shown to protect against tissue injury in sepsis (33, 34). TNF- and IL-1 aremajor mediators of the expression of chemokines such as MCP-1
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A(ratio to control value)

NF-κB luciferase activity(ratio to control value)

NF-κB luciferase activity

BCE
Exo-srlκB

PE-A mean

Exo-srlκB

Fig. 4. Inhibitory effect of Exo-srIB on NF-B signaling in vitro. (A) HEK293–
NF-B–luciferase cells (2 × 104 cells) were cultured either with Exo-Naïve or with
Exo-srIB (2 × 105 particles). After 24 hours, the cells were treated with TNF-
(0.5 ng/ml) for an additional 18 hours. Luciferase activities were measured and normalized. (B) Exo-srIB dose-dependently repressed NF-B activation in NF-B–­
luciferase cells. (C) THP-1 cells (5 × 105 cells) were stimulated with LPS (1 g/ml) andthen treated with Exo-srIBs (5 × 106 particles). The supernatants were collected
and assayed for the production of TNF- and MCP-1. JSH-23 (50 M) was used as
the positive control. **P < 0.01. (D) Immunofluorescence of HUVECs incubated with
mCLING-labeled Exo-srIB. Representative images are shown. Nuclei were labeledwith Hoechst. (E) HUVECs were stimulated with LPS (300 ng/ml) for 24 hours. Cells
were harvested into a single-cell suspension and assessed through flow cytometry
using specific phycoerythrin (PE)–conjugated antibodies against human ICAM-1.
DMSO, dimethyl sulfoxide. *P < 0.05; **P < 0.01; ***P < 0.001.
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MATERIALS AND METHODS

Animals
All animal experiments were performed in accordance with standard guidelines for the care and use of laboratory animals and were
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Dand IL-8, which are important chemoattractants for neutrophils
and monocytes/macrophages during inflammation. These chemokines could initiate local infiltration of monocytes/macrophages after
their activation. In HUVECs, Exo-srIB could attenuate LPS-induced
release of MCP-1 and IL-8, in turn inhibiting the infiltration of
monocytes/macrophages. Our data are consistent with the results obtained from in vivo studies. We found that treatment with Exo-srIB
greatly decreased neutrophil infiltration in the spleen, kidney, andliver of septic animal models (fig. S2C). In light of the importantrole of neutrophils in the pathophysiology of sepsis (31, 32), thetissue-protective effect of Exo-srIB might be related, at least in thekidney, to decreased infiltration of neutrophils. The spleen showeda higher level of neutrophils compared with other organs, such as
the kidney and liver, suggesting that proinflammatory cytokines may
arise in the spleen and subsequently affect other organs.
Most genes encoding proinflammatory cytokines are under thecontrol of NF-B, which is one of the most important transcription
factors in the LPS-induced septic inflammation response (35). In
this study, we examined the expression of NF-B–responsive reporter
proteins and measured p65 translocation in the presence and absence of Exo-srIB treatment. As expected, our results showed that
Exo-srIBs inhibit NF-B signal activation by blocking nuclear
translocation of NF-B subunit p65.
Current conventional strategies to deliver the therapeutic proteins include enveloping the proteins within synthetic nanoparticles(36). The most preferred protein delivery systems are liposomes
and polymeric nanoparticles (PNPs). A liposome is a synthetic
vesicle with a phospholipid membrane that self-assembles into various sizes and shapes in an aqueous environment. A PNP is a solid
colloidal particle with the size between 10 and 1000 nm and is madeup of biodegradable polymers, in which the cargo proteins can beentrapped, encapsulated, or attached to a nanoparticle matrix (37).
However, liposomes tend to fuse or aggregate with each other,resulting in immature release of cargo over time (38). PNPs may
have better stability than liposomes, but their biocompatibility andlong-term potential safety are still a concern. In addition, encapsulating proteins to liposomes and PNPs requires generation of synthetic
or recombinant proteins and ex vivo encapsulation processes, whereas
EXPLOR technology requires generation and maintenance of stable
cells that can produce cargo-loaded exosomes via natural exosome
biogenesis. In addition, exosomes have many desirable features ofan ideal protein delivery system, such as biocompatibility, minimal
or no inherent toxicity, long half-life in the circulation, and intrinsic
ability to target tissues (39).
In conclusion, we used exosomes as a mechanism for therapeutic
delivery of immunosuppressive proteins in a septic mouse model.
Exosomes are a promising vehicle for intracellular delivery of srIB
and constitute a new option for sepsis therapy; no methods of delivering immunosuppressive proteins directly into the target cells in vivo
have been established previously. Our data demonstrate that Exo-srIB
acts as an inhibitor of NF-B and can directly counteract the overwhelming inflammatory response, thus ameliorating the proinflammatory cytokine storm and subsequent organ damage.

SCIENCE ADVANCES | RESEARCH ARTICLE
approved by our Institutional Animal Care and Use Committee.
C56BL/6, C57BL/6N, and BALB/c mice were purchased from
Orientbio (Seong-Nam, Republic of Korea). LysMgfp/+ mice intrinsically expressing GFP in neutrophils and macrophages were provided
by M. Kim (University of Rochester, Rochester, NY, USA).
LPS endotoxemia model
To generate the LPS endotoxemia mouse model, LPS derived from
Escherichia coli (Sigma-Aldrich, Milwaukee, WI, USA) was injected
into male mice.

Intravital imaging
A laser scanning intravital confocal microscope (IVIM Technology,
Daejeon, South Korea) was used to visualize the biodistribution andantiseptic effects of Exo-srIB. During intravital imaging, mouse
body temperature was maintained at 37°C using a homoeothermic
controller. Mice were anaesthetized with intramuscular injectionsof Zoletil (30 mg/kg) and xylazine (10 mg/kg). To access the internalorgans, including the liver, spleen, and kidney, a small incision of
10 mm was made in both the skin and peritoneum. Exposed organs
were kept hydrated through repeated application of saline during
imaging. The biodistribution of exosomes was visualized throughwide-area z-stack imaging before and after the injection of Exo-Naïve
or Exo-srIB. To fluorescently label neutrophils of C57BL/6N micein vivo, anti-Ly6G antibody (BD Bioscience, San Jose, CA, USA)
conjugated with Alexa Fluor 555 (A20009, Thermo Fisher Scientific,
Waltham, MA, USA) was injected intravenously. Naïve or srIB
exosomes labeled with mCLING ATTO 647N (Synaptic System,
Göttingen, Germany) were intravenously injected via a tail veincatheter or 31-gauge insulin syringe. To generate the sepsis model,
Choi et al., Sci. Adv. 2020; 6 : eaaz6980

8 April 2020

Histopathologic assessment of kidney injury score
Formalin-fixed and paraffin-embedded kidney tissue samples were
stained with hematoxylin and eosin and examined to assess thedegree of kidney injury. To examine histologic changes in an objective manner, cortical areas were subjected to kidney injury evaluation by two pathologists (E.S.C. and J.I.Y.) in a blinded manner.
Kidney injury was scored according to the percentage of damaged
tubules among total tubules: 0, normal; 1, <25% damage; 2, 25 to
50% damage; 3, 50 to 75% damage; 4, 75 to 90% damage; and 5,>90% damage. Histological criteria for injured tubules were loss ofthe tubular brush border, vacuolization, chromatin condensation,and denuded tubular basement membrane. Renal tubules in fiverandomly selected high-power fields, typically including 50 to 100
tubules, were evaluated and scored.
Cell culture
HUVECs were purchased from the American Type Culture Collection(ATCC; Manassas, VA, USA) and cultured in F-12 K medium (ATCC)
containing 10% fetal bovine serum (FBS; Atlas Biologicals, Fort Worth,
CO, USA), heparin (Sigma-Aldrich), endothelial cell growth supplement(BD Biosciences), and 1% penicillin/streptomycin (Thermo Fisher
Scientific). HUVECs between the third and sixth passages were usedin all experiments. Human monocytes (THP-1) were purchased from
ATCC and cultured in RPMI 1640 medium (WELGENE, Daegu,
Republic of Korea) with 10% FBS, 1% penicillin/streptomycin, and0.05 mM 2-mercaptoethanol (Sigma-Aldrich).
Confocal microscopy
For exosome uptake analysis, exosomes were diluted in 0.5 ml ofDulbecco’s PBS (DPBS). Subsequently, the suspension was incubatedwith 647N-labeled mCLING ATTO (Synaptic System) according to
the manufacturer’s instructions. Then, a pellet of the mixture wasobtained through centrifugation, and 300 l of the resuspended pellet
was incubated with the HUVECs. After 24 hours, cells were washedwith DPBS and fixed in 4% paraformaldehyde solution. Hoechst
was used for nuclear staining. Images were recorded using a Zeiss
710 confocal microscope (Zeiss, Oberkochen, Germany).
Luciferase assay
HEK293 cells were stably transfected with a luciferase reporter construct regulated under the NF-B response element (SL-0012, Signosis,
Santa Clara, CA, USA) and grown according to the manufacturer’s
instructions. Cells were treated with exosomes for 24 hours at 37°C,
and luciferase activity was measured (E1501, Promega, Madison,
WI, USA) using a SpectraMax ID3 microplate reader (Molecular
Devices, Sunnyvale, CA, USA).
Western blotting
Western blotting was performed as described in a previous study(20). Antibodies targeting the following proteins were used: IB
(CST4812, Cell Signaling Technology, Danvers, MA, USA), p65(CST6956S, Cell Signaling Technology), CD9 (NBP2-22187, Novus
Biologicals, Centennial, CO, USA), CD63 (EXOAB-CD63A-1, SBI,
Tokyo, Japan), TSG101 (ab228013, Abcam, Cambridge, UK), GM130(ab52649, Abcam), glyceraldehyde-3-phosphate dehydrogenase(GAPDH; sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA,
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High-grade CLP sepsis model and treatment regiment
Male C57BL/6 mice purchased from Orientbio (Seongnam-si,
Gyeonggi-do, Republic of Korea) were subjected to high-grade CLP
at 9 to 10 weeks of age using a previously described procedure withminor modification (40). All mice were housed, and experiments
were performed in a specific pathogen–free area of Yonsei Biomedical
Research Institute, Yonsei University College of Medicine, which
was certified by the Association for Assessment and Accreditationof Laboratory Animal Care International. Mice were anesthetizedthrough intraperitoneal injection of a mixture of ketamine (80 mg/kg)
and xylazine (10 mg/kg) prior to all procedures. After anesthesia,
the peritoneum was opened in a sterile manner, and the cecum wasligated using 4-0 black silk thread 1 cm from the distal end andpunctured with a 23-gauge needle. After puncturing, the needle wasremoved, and a small amount of stool was extruded through bothpunctures to ensure patency. Then, the cecum was placed back into
the abdominal cavity, and the abdominal incision was closed with
6-0 nylon sutures, with stainless steel removable wound clips usedon the skin. After this procedure, 1 ml of prewarmed saline per 20-g
body weight was administered subcutaneously. Sham-treated mice
underwent the same procedure except for the ligation and punctureof the cecum. After this procedure, 1.0 × 109 particles of Exo-Naïve
or Exo-srIB were administered to mice via intraperitoneal injection
at 0, 6, 12, and 18 hours. As a control, an equivalent volume of PBS
was injected in the same manner. We assessed the animals every
2 hours during the initial 48 hours after CLP and then every 4 hours
for 5 days. Samples were collected to evaluate the outcome of theprocedure within 18 hours after CLP.

high-dose LPS (Sigma-Aldrich, St. Louis, MO, USA) was intravenously injected 1 to 16 hours before imaging.

SCIENCE ADVANCES | RESEARCH ARTICLE
USA), histone H3 (ab1791, Abcam), mCherry (ab125096, Abcam),and GFP (CST2555, Cell Signaling Technology).

Flow cytometry
The levels of surface markers expressed on HUVECs were assessedusing flow cytometry. The cells were separated and harvested
through centrifugation, labeled with phycoerythrin (PE)–conjugatedantibodies specific for human ICAM-1 (BD Biosciences) on ice for
30 min in the dark, and then washed extensively. All samples wereanalyzed with a BD Celesta flow cytometer (BD Biosciences). The
data were analyzed using BD FACSDiva software. PE-conjugatedantibodies specific to IgG1 (BD Biosciences) were used as theisotype control.
Isolation of exosomes
To produce Exo-srIB, the stable cells were seeded into T175 flasks.
After 1 day, the medium was carefully removed, the cells wererinsed with PBS, and exosome-depleted medium was added. Then,the cells were exposed to continuous blue light illumination from a
460-nm light emitting diode in a CO2 incubator. After 72 hours, thecell culture supernatant was harvested and centrifuged at 1000g for
15 min to remove cells and cell debris and then filtered through a
0.22-m polyethersulfone filter to remove large particles. The exosomes were isolated using molecular weight cutoff–based membrane
filtration. The isolated exosomes were purified through SEC.
Transmission electron microscopy
Extracellular vesicles (EVs) were evaluated morphologically throughnegative staining. First, 5 l of EV suspended in PBS was loaded
onto glow-discharged carbon-coated copper grids (Electron Microscopy Sciences, Hatfield, PA, USA). After sample adsorption for 3 to 5 s,
the grid was blotted with filter paper and stained with 2% uranyl
acetate. Next, samples were dried for 20 s using a dryer. EVs wereviewed with Tecnai G2 Retrofit (FEI Company, Hillsboro, OR, USA)at a voltage of 200 kV.
Nanoparticle trafficking analysis
A Zetaview instrument (PMX120, Particle Metrix, München,
Germany) was used to analyze EV particle numbers and size distribution. Particle number was calculated from the rate of Brownian
motion, and size was determined using the two-dimensional Stokes-­
Choi et al., Sci. Adv. 2020; 6 : eaaz6980

8 April 2020

Statistical analysis
Values are presented as mean ± SEM. Statistical differences between
means were determined using the unpaired two-tailed Student’s
t test or Mann-Whitney test, as appropriate. Kaplan-Meier survival
plots and log-rank tests were used to compare survival results
among treatment groups. Statistical significance was set at P < 0.05 or
P < 0.01.
SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/6/15/eaaz6980/DC1
View/request a protocol for this paper from Bio-protocol.

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Enzyme-linked immunosorbent assays
The TNF-, IL-8, and MCP-1 levels in supernatants collected from
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Acknowledgments: We thank all members of ILIAS Corporation for the discussion andtechnical support. Funding: This research was supported by Basic Science Research Program
through the National Research Foundation (NRF) of Korea funded by the Ministry of Education(NRF- 2017R1D1A1B03034177) and a faculty research grant of Yonsei University College ofMedicine (6-2019-0078). Author contributions: K.Cho., C.C., K.Chu., and E.-C.S. conceived anddesigned the project. E.-C.S., K.Chu., K.Cho., J.I.Y., T.-H.Y., and P.K. supervised and developed
the study. H.C. and Y.K. wrote the manuscript. H.C., Y.K., A.M., J.H., Y.N.K., J.H.S., S.K., N.H.K.,
E.S., and K.Chu. performed the experiments and analyzed the data. Competing interests: C.C.,
K.Cho., and H.C. are inventors of a patent related to this work filed by ILIAS Biologics Inc. (no.
KR 10-1877010). E.-C.S., K.Chu., J.I.Y., and T.-H.Y. are scientific advisory board members of ILIAS
Biologics Inc. C.C. is the founder and shareholder, and K.Cho., H.C., and Y.K. are minor
shareholders of ILIAS Biologics Inc. P.K. is the founder and shareholder of IVIM Technology. Allother authors declare that they have no competing interests. Data and materials
availability: All data needed to evaluate the conclusions in the paper are present in the paper
and/or the Supplementary Materials. Additional data related to this paper may be requested
from the authors.
Submitted 1 October 2019
Accepted 13 January 2020
Published 8 April 2020
10.1126/sciadv.aaz6980
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Exosome-based delivery of super-repressor IκBα relieves sepsis-associated organ damage
and mortality
Hojun Choi, Youngeun Kim, Amin Mirzaaghasi, Jaenyoung Heo, Yu Na Kim, Ju Hye Shin, Seonghun Kim, Nam Hee Kim,
Eunae Sandra Cho, Jong In Yook, Tae-Hyun Yoo, Eunjoo Song, Pilhan Kim, Eui-Cheol Shin, Kyungsoo Chung, Kyungsun
Choi and Chulhee Choi

Sci Adv 6 (15), eaaz6980.
DOI: 10.1126/sciadv.aaz6980

http://advances.sciencemag.org/content/6/15/eaaz6980

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