Resolvin E1 is a pro-repair molecule that promotes
intestinal epithelial wound healing
Miguel Quirosa,1, Darius Feiera, Dorothee Birkla, Rachit Agarwalb, Dennis W. Zhoub, Andrés J. Garcíab,
Charles A. Parkosa, and Asma Nusrata,1
a
Department of Pathology, University of Michigan, Ann Arbor, MI 48109; and bGeorge W. Woodruff School of Mechanical Engineering, Petit Institute for
Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332
Edited by Charles N. Serhan, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, and accepted by Editorial Board Member Ruslan
Medzhitov March 17, 2020 (received for review December 4, 2019)
Resolution of intestinal inflammation and wound repair are active
processes that mediate epithelial healing at mucosal surfaces.
Lipid molecules referred to as specialized proresolving mediators
(SPMs) play an important role in the restorative response. Resolvin
E1 (RvE1), a SPM derived from omega-3 fatty acids, has been
reported to dampen intestinal inflammation by promoting anti-
inflammatory responses including increased neutrophil spherocy-
tosis and macrophage production of IL-10. Despite these observa-
tions, a role for RvE1 in regulating intestinal epithelial cell
migration and proliferation during mucosal wound repair has
not been explored. Using an endoscopic biopsy-based wound
healing model, we report that RvE1 is locally produced in response
to intestinal mucosal injury. Exposure of intestinal epithelial cells
to RvE1 promoted wound repair by increasing cellular prolifera-
tion and migration through activation of signaling pathways in-
cluding CREB, mTOR, and Src-FAK. Additionally, RvE1-triggered
activation of the small GTPase Rac1 led to increased intracellular
reactive oxygen species (ROS) production, cell–matrix adhesion,
and cellular protrusions at the leading edge of migrating cells.
Furthermore, in situ administration of RvE1-encapsulated syn-
thetic targeted polymeric nanoparticles into intestinal wounds
promoted mucosal repair. Together, these findings demonstrate
that RvE1 functions as a prorepair lipid mediator by increasing
intestinal epithelial cell migration and proliferation, and highlight
potential therapeutic applications for this SPM to promote muco-
sal healing in the intestine.
|
Resolvin specialized proresolving mediator | wound healing | repair |
epithelial cells
T he gastrointestinal epithelium forms an important protective
barrier that limits access of luminal antigens to the mucosal
and systemic immune system. Epithelial injury and wounds have
been observed in a number of mucosal disorders that include
acute and chronic inflammatory states (1). Epithelial barrier
disruption results in spatiotemporal recruitment of immune cells
to sites of injury, with consequent release of a complex cascade
of mediators that interact with the epithelium to orchestrate
resolution of inflammation and mucosal repair. Perturbation
in this delicate balance of inflammation, resolution, and repair
contributes to chronic diseases such as inflammatory bowel
disease (2).
In addition to proinflammatory mediators, cells at sites of
mucosal injury release lipid and protein/peptide specialized
proresolvin mediators (SPMs), which have been shown to play
important roles in restoring homeostasis. SPMs are important in
orchestrating active resolution of inflammation and epithelial
repair, which is important in restoring the mucosal barrier (3, 4).
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Resolvin E1 (RvE1), an endogenous lipid mediator derived from
omega-3 eicosapentaenoic acid, has been shown to limit inflam-
mation through a number of mechanisms that include modulation
of immune cell recruitment, augmentation of phagocytosis, pro-
motion of neutrophil apoptosis, and efferocytosis (5–8). In con-
cert with these anti-inflammatory properties, RvE1 dampens the
www.pnas.org/cgi/doi/10.1073/pnas.1921335117
inflammatory response in murine colitis by inhibiting neutrophil
migration (8). However, the contribution of RvE1 in controlling
mucosal epithelial wound repair remains unclear.
Here we show that RvE1 is an immunoresolvent that not only
has anti-inflammatory properties but also activates prorepair path-
ways that promote epithelial cell migration and proliferation, and
ultimately epithelial wound repair. Given the labile nature of lipid
mediators with potential for rapid enzymatic degradation, we en-
capsulated RvE1 in polymeric nanoparticles and demonstrate their
therapeutic potential in enhancing colonic mucosal wound repair.
IMMUNOLOGY AND
INFLAMMATION
Results
Resolvin E1 Is Synthesized in Response to Intestinal Mucosal Injury.
Administration of exogenous RvE1 has been shown to exert anti-
inflammatory effects in models of experimental colitis (6, 8, 9).
However, the spatiotemporal generation of RvE1 during muco-
sal wound repair has not been defined. Thus, we analyzed RvE1
synthesis in healing murine biopsy-induced colonic mucosal
wounds (Fig. 1A). Mucosa on the dorsal aspect of the colon was
injured and wounds harvested at 48 and 72 h postinjury. Healing
wound samples were harvested and analyzed by multiple-
reaction monitoring liquid chromatography mass spectrometry
(MRM-LCMS) to obtain a lipidomic prolife of inflammatory and
Significance
Resolvin E1 (RvE1) promotes resolution of inflammation by
damping proinflammatory responses and activating restorative
pathways. While mechanisms by which RvE1 signaling in im-
mune cells contribute to resolution of inflammation are ex-
tensively studied, its role in epithelial signaling and wound
repair remains undefined. Intestinal epithelial barrier compro-
mise during an inflammatory event can result in pathogen ac-
cess to tissue compartments. Thus, efficient repair of denuded
intestinal mucosal surfaces is vital in restoring homeostasis.
Here we demonstrate that RvE1 promotes intestinal epithelial
cell migration and proliferation leading to wound repair. These
findings suggest that RvE1 has the potential to serve as a tar-
geted immunoresolvent therapeutic agent that not only damp-
ens inflammation but activates prorepair pathways to enhance
colonic mucosal wound repair.
Author contributions: M.Q., A.J.G., C.A.P., and A.N. designed research; M.Q., D.F., D.B.,
and D.W.Z. performed research; M.Q., R.A., A.J.G., and A.N. contributed new reagents/
analytic tools; M.Q. and A.N. analyzed data; and M.Q., A.J.G., C.A.P., and A.N. wrote
the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission. C.N.S. is a guest editor invited by the
Editorial Board.
Published under the PNAS license.
1
To whom correspondence may be addressed. Email: mquirosq@umich.edu or anusrat@
med.umich.edu.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1921335117/-/DCSupplemental.
PNAS Latest Articles | 1 of 6

A B
100
Wounds days
Relative Intensity (%)
2, 3, 4 and
Intact tissue
0
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380
Wound Punch biopsy
MRM
Control
transitions
Q1 Q3 Mean ± SEM Mean
RvE1
RvE2
349 161
333 199
3.6
4.9
±
±
0.8
2.7
6.8
2.0
RvE3 333 201 2.5 ± 0.6 3.5
Fig. 1. RvE1 is produced in the colon in response to mucosal injury. (A)
Consecutive wounds were generated on the dorsal aspect of the distal colon.
Punch biopsies of wounds were harvested for analysis of lipid mediators by
MRM-LCMS. (B) Results are presented as mean ± SEM, n = 3 samples each
composed of biopsies from 3 mice. Values are expressed as pg/100 mg tissue.
* < 0.05, mean SEM.
repair mediators in healing biopsy-induced mucosal wounds (see
SI Appendix, Table S1 for details). Results of LCMS analyses
revealed increased levels of RvE1 in healing wounds compared
with intact mucosal tissue. RvE1 levels peaked at 48 h after in-
jury and returned to baseline values after 72 h postbiopsy
(Fig. 1B). In contrast to RvE1, levels of other closely related
mediators (RvE2 and RvE3) did not show significant changes
after colonic mucosal injury. Interestingly, expression of RvE1
receptor CMKLR1 was also increased in the repairing mucosa
on days 2 and 3 postinjury (SI Appendix, Fig. S1)
RvE1 Activates Reparative Pathways in Intestinal Epithelial Cells.
Efficient repair of epithelial wounds is critical to restore muco-
sal barrier function and dampen the inflammatory response.
Given the observed increase in RvE1 in healing wounds, we
determined whether RvE1 promotes epithelial repair in vitro.
Intestinal epithelial monolayers were scratch-wounded, and re-
pair was monitored by time-lapse video imaging. Dose–response
and time course studies using model human intestinal epithelial
cell (IEC) line SKCO15 revealed RvE1-dependent enhancement
of epithelial wound repair in a dose-dependent fashion with in-
creasing concentrations from 10 to 500 nM, with peak effects
observed at 100 nM (Fig. 2A). Prorepair effects of RvE1 on
epithelial cells were better observed between 8 and 16 h of in-
cubation. Similar effects were observed in a second model IEC
line, T84 (SI Appendix, Figs. S2 and S3). These findings were
confirmed in experiments using primary colonic epithelium
from human colonoids that were cultured and differentiated into
two two-dimensional (2D) monolayers. Analogous to results
obtained with transformed epithelial cell lines, 100 nM RvE1
enhanced primary colonoid wound repair with maximum effects
occurring 24 h after injury (60.1 ± 2.0% control vs. 83.4 ± 2.0
RvE1; P < 0.0001; Fig. 2B).
Since repair of epithelial wounds is dependent on coordinated
cellular proliferation and migration, experiments were per-
formed to explore the effect of RvE1 on IEC proliferation.
Analysis of the incorporation of the thymidine analog EdU
demonstrated that RvE1 increased proliferation of wounded
IEC monolayers (17.3 ± 0.58% RvE1 vs. 10.7 ± 0.7% control;
Downloaded at Interuniversitaire de Medecine on April 16, 2020
P < 0.001; Fig. 2C).
To explore the mechanisms by which RvE1 orchestrates
wound repair, we analyzed signaling pathways that have been
shown to promote epithelial proliferation, migration, and wound
repair. Phosphorylation/activation of proproliferative proteins
CREB and mTOR and migratory signaling proteins in the Src-
2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1921335117
FAK axis were detected. In vitro healing epithelial wounds were
RvE1
incubated with RvE1 for 4 and 8 h. Increased phosphorylation of
291
+H
OH
349
CREB (serine 133), p70 S6 Kinase (Thr389), and mTOR
OH
HO
349=M-H
(Ser2448) was detected (Fig. 2D), in further support of the ob-
331=M-H-H2O
served enhanced proliferation in response to RvE1 (Fig. 2C).
COO- 313=M-H-2H2O
-H +H
305=M-H-CO2
195
223 295=M-H-3H2O
287=M-H-H2O-CO2 In addition to RVE1-dependent proproliferative effects, we ob-
229=291-H 2O-CO2
x5 205=223-H 2O
179=223-CO2
served activation of signaling proteins that regulate cell adhesion/
305
177=195-H 2O
161=223-H 2O-CO2
151=195-CO2
migration. Specifically, we observed increased Src phosphoryla-
195
151 161 179 205 229 287295 313
331
tion (Y416), as well as phosphorylation of focal adhesion kinases
177 223
(FAK Y397 and Y925) that play important roles in regulation of
m/z, Da
turnover of cell matrix adhesions and forward cell movement
48h 72h 96h
(Fig. 2D). To confirm the involvement of these signaling mole-
± SEM Mean ± SEM Mean ± SEM cules in mediating RvE1 prorepair effects, we analyzed repair of
±
±
1.9 *
0.9
3.0
3.9
±
±
0.8
1.7
0.6
0.7
±
±
0.4 *
0.3
scratch wounds in the presence of specific inhibitors for Src,
± 0.3 2.2 ± 1.1 2.0 ± 0.7 CREB, and mTOR. Src inhibition (Dasatinib and PP2) com-
pletely abrogated wound healing, while inhibitors for CREB
(666-15) and mTOR abolished RvE1 triggered increase in
wound repair (SI Appendix, Fig. S4). These results indicate that
RvE1 activates prorepair signaling pathways in IEC and pro-
motes wound healing. As previously shown by us and others,
epithelial intracellular reactive oxygen species (ROS) signaling
promotes oxidative inactivation of regulatory phosphatases,
which in turn increase phosphorylation and activation of down-
stream focal adhesion proteins, such as FAK, which in turn
controls forward cell movement and repair (10–12). Given such
prorepair properties of localized ROS signaling, we analyzed
ROS generation in healing epithelial wounds, using an intra-
cellular redox-sensitive dye, Hydro-Cy3. RvE1 treatment resul-
ted in significantly increased ROS generation (2.0-fold increase
in fluorescence intensity) within 30 min of exposure (Fig. 2E).
These data support an important role for RvE1 in promoting
intestinal epithelial wound repair.
RvE1 Activates Rac1 and Increases Cell Matrix Adhesion. It is well
appreciated that regulation of cell adhesion during tissue repair
is fundamentally important for wound healing. Given results in
Fig. 1, experiments were performed to determine the influence
of RvE1 treatment on intestinal epithelial cell–matrix adhesion.
First, we evaluated whether RvE1 leads to activation of the small
GTPase Rac1, which has been shown to play an important role in
regulating cell matrix adhesion. Spatial Rac1 activity in migrating
epithelial cells was determined by proximity ligation assay (PLA)
between active Rac1 and its effector protein PAK1. This assay
capitalizes on the fact that only active or GTP-bound Rac1 can
bind to PAK1. As shown in Fig. 3A, 8 h of RvE1 exposure in
wounded IEC monolayers (SKCO-15 cells and primary 2D hu-
man colonoids) resulted in increased levels of active Rac1-GTP/
PAK1 at the leading edge of the migrating epithelium. Since we
observed that RvE1 activates ROS signaling, which in turn
promotes oxidative modification and inhibition of phosphatases
that dephosphorylate FAK, we next examined localization of
pFAK-Y861 in epithelial cells that were migrating to heal
wounds. As shown in Fig. 3B, increased pFAK-Y861 was iden-
tified in cells migrating in the presence of RvE1. To determine
whether RvE1 enhances cell–matrix adhesion, we measured the
force required to detach IEC cells from the extracellular matrix
in the presence or absence of RvE1. SKCO-15 cells were seeded
on fibronectin-coated glass coverslips and allowed to adhere for
6 h, followed by exposure to controlled hydrodynamic shear
forces, using a spinning disk device. In this assay, applied de-
tachment forces increase linearly with radial position and pro-
duce a sigmoidal decrease in the number of adherent cells. Cell
adhesion strength is defined as the shear stress that produced
50% detachment of cells. As shown in Fig. 3C, SKCO-15 cells
treated with RvE1 showed significantly increased cell adhesion
strength (111.1 ± 1.83 control vs. 126.9 ± 4.70 RvE1). These
findings show that RvE1 regulates cell–matrix adhesion and
migration of IECs.
Quiros et al.
 A 100 B ** C 25
100 ***

% wound closure

% EdU incorporation
*** 20

% Wound closure
***
***
80
80 15
**
10
Control
60 Resolvin 10 nM 60
Resolvin 50 nM 5
Resolvin 100 nM
Resolvin 500nM
40 40 0
Control RvE1 Control RvE1

D E
2.5 **

2.0

% Fold change
1.5

1.0

0.5

IMMUNOLOGY AND
0.0

INFLAMMATION
Control RvE1

Fig. 2. RvE1 promotes intestinal epithelial wound repair. (A) Wound areas of scratch-wounded SKCO15 IEC monolayers, incubated with increasing con-
centrations (10, 50, 100, and 500 nM) of RvE1 were continuously imaged. Percentage wound closure was calculated by comparison of 0 and 24 h postinjury. (B)
Wounded primary IECs treated with RvE1 100 nM or vehicle for 24 h. (C) Scratch-wounded intestinal epithelial monolayers were treated with RvE1 (100 nM) or
vehicle, and EdU incorporation was determined 24 h postwound ***P < 0.001; mean ± SEM. (D) Immunoblotting was performed on lysates from scratch-
wounded SKCO15 monolayers treated with RvE1 (100 nM) or vehicle for different points. Levels of pP70, pmTOR, pCreb, pSRC (416), and pFAK (Y397, Y925)
were compared with total P70, mTOR, CREB, Src, FAK, and GAPDH to assess activation. Densitometry values are displayed under the phosphorylated protein
blots; values are normalized to total protein and nonwounded cells except for FAK blots, which were normalized to loading control. (E) SKCO15 IECs were
incubated with RvE1 (100 nM) or vehicle for 4 h. ROS generation was detected by confocal microscopy, using the fluorescent hydro-Cy3 dye in scratch-
wounded monolayers adjacent to the wound edge. Quantification was done calculating the fold change increase in pixel counts of RvE1 treatment compared
with vehicle, using ImageJ software. **P < 0.01; ***P < 0.001; mean ± SEM.

Intramucosal Administration of RvE1 Accelerates Intestinal Mucosal
Wound Repair. Because RvE1 has anti-inflammatory and prorepair
properties, we evaluated the effect of RvE1 administration into
murine healing colonic mucosal wounds. Since RvE1 can be rapidly
degraded in tissues, we encapsulated RvE1 into polymeric poly-
ethylene glycol–poly lactic acid-coglycolic acid (PEG-PLGA)
nanoparticles (NPs) that provided sustained and directed release at
the site of injury (Fig. 4A) (13). The NPs used for this experiment
had a range of RvE1 concentration from 5 to 10 nM. RvE1 loading
and size histograms are available in the SI Appendix (SI Appendix,
Fig. S5); NPs were decorated with a collagen IV peptide to facili-
tate enhanced targeting to sites of injury and biotin to detect the
particles after intramucosal injection. Biopsy-induced wounds were
generated in murine colon, and phosphate-buffered saline (PBS),
RvE1, Empty NPs, or RvE1 NPs were administered into wound
beds by a single intramucosal injection 1 d after injury. Localized
NPs delivery was confirmed using fluorescent streptavidin labeling,
as shown in Fig. 4B. Colonic wound closure was analyzed on day 3
after injury (Fig. 4C). Wounds treated with empty NPs had basal
wound closure rates comparable to saline-treated controls. In
contrast, injected RvE1 promoted wound repair, resulting in ∼15%
increased wound closure compared with controls. Furthermore,
RvE1 NP treatment markedly promoted wound repair with ∼35%
more wound closure compared with controls.
Downloaded at Interuniversitaire de Medecine on April 16, 2020
and intestinal homeostasis (14). We and others have shown that
regulated spatiotemporal recruitment of leukocytes that interact
with the repairing epithelium plays an important role in ensuring
timely mucosal repair (15–17). SPMs have been demonstrated to
have important roles in orchestrating resolution of inflammation
in different tissues by triggering anti-inflammatory and pro-
resolution responses in immune cells (18). Nevertheless, the
prorepair effects of SPMs on epithelial cells remain poorly un-
derstood. What sets an SPMs such as RvE1 apart from other
prorepair mediators is their role as immunoresolvant and not as
immune suppressors (4). SPMs function as endogenous media-
tors to restore homeostasis after the inflammatory response by
actively damping inflammation and promoting reparative path-
ways without compromising the immune response (19). RvE1
has been shown to promote resolution of inflammation in model
systems of arthritis, multiple sclerosis, bronchial asthma, reti-
nopathies, periodontal diseases, dermatitis, and corneal/con-
junctival injury (20–26). Resolvin D1, another well-studied SPM,
has been shown to promote corneal epithelial wound healing
through EGF receptor trans activation. Although contribution of
RvE1 in promoting corneal wound repair remains to be defined,
the signaling mediators reported in our study suggest that RvE1
has analogous prorepair effects in the cornea and the gut (27,
28). Temporal analysis of RvE1 levels in biopsy-induced colonic
mucosal wounds revealed maximal increase within 2 d of injury, a

Discussion period that represents a transition between the proinflammatory

Intestinal epithelial damage and compromise of the mucosal
barrier are pathognomonic of a number of diseases including
inflammatory bowel disease (IBD). Active and coordinated re-
pair responses that promote epithelial cell migration and pro-
liferation are necessary to reestablish mucosal barrier function
and restorative phases of mucosal repair when the inflammatory
response has to be actively shut down and restorative pathways
need to be activated. Furthermore, RvE1 has been reported to
down-regulate NF-κB signaling in neutrophils, resulting in their
decreased mobilization, adherence, and polarization of

Quiros et al. PNAS Latest Articles | 3 of 6

 A SKCO-15 2D-colonoids

PLA Rac1/PAK/ Ac n
NT RvE1 NT RvE1

B C 1.0
(+) Resolvin

0.9

Adherent Fraction
0.8
160

Adhesion Strength (dyn/cm2)

0.7

* 0.6

pFAK861, Ac n
0.5
0.4
0.3
0.2
0.1
140 0.0
0 50 100 150 200 250 300 350
Shear Stress (dyn/cm 2)

(-) Resolvin
120 1.0
0.9

Adherent Fraction
0.8
0.7
0.6
0.5

100 0.4
0.3
Control RvE1 0.2
0.1
0.0

NT RvE1 0 50 100 150 200 250

Shear Stress (dyn/cm 2)
300 350

Fig. 3. RvE1 promotes intestinal epithelial cell migration and adhesion. (A) Treatment of scratch-wounded SKCO15 IECs with RvE1 (100 nM) or vehicle for 8 h,
followed by analysis of Rac1 activation using PLA to demonstrate association of Rac1 to PAK. (B) Confocal micrographs of the focal contacts in migrating IECs
at the leading edge of the wound after 8 h treatment with vehicle or RvE1 (100 nM), showing staining of pFAK (Y861) and phalloidin (F-actin). (C) Adhesion
strength measurements of SKCO15 IECs adhering to fibronectin and treated with RvE1 (100 nM) or vehicle for 6 h. Representative adhesion detachment
profiles for each condition. The shear stress for 50% detachment (blue, red solid lines) is a metric for the mean adhesion strength. Higher magnification
images (original magnification, 63×) of the boxed region are shown to the right, *P < 0.05; mean ± SEM. NT, vehicle control. (Scale bar, 20 μm.)

macrophages toward an anti-inflammatory phenotype that leads
to increased phagocytosis and IL-10 expression (9). We have
previously observed that IL-10 plays an important role in facili-
tating intestinal mucosal repair, and our results indicate that
RvE1 might be contributing to this process.
Intestinal epithelial immunomodulatory effects of RvE1, such
as up-regulation of CD55 (decay-accelerating factor), BPI
(bactericidal/permeability-increasing), and alkaline phosphatase
(ALPI) proteins, have been reported (9, 29, 30). CD55 is an anti-
adhesive molecule that promotes clearance of apically adherent
activated neutrophils from the epithelium, BPI protects mucosal
surfaces against gram-negative bacteria and their endotoxins,
and ALPI has been shown to control gram-negative bacterial
growth and neutralization of LPS, thereby protecting against
pathogens. In this study, we observed that RvE1 activates sig-
naling pathways in IECs that include CREB and mTOR, which
have been reported to regulate epithelial proliferation, and ul-
timately wound repair (31, 32). Other SPMs such as RvD1,
RvD2, and MaR1 have also been shown to activate CREB sig-
naling in primary human monocytes, where they have anti-
inflammatory and prorepair effects (33).
In addition to proliferation, remodeling and turnover of
integrin-containing cell matrix contacts play a pivotal role in
wound repair. We observed that RvE1 activates Src phosphor-
ylation, which has been previously reported to promote tyrosine
phosphorylation, and activation of FAK, cytoskeletal re-
organization, and focal cell matrix adhesion turnover. RvE1
treatment was observed to activate pSrc-Y416, as well as result in
Downloaded at Interuniversitaire de Medecine on April 16, 2020
Increased SPM generation has been identified in chronic in-
flammatory diseases such as IBD. Increased RvE1 has been
detected in intestinal mucosal biopsies from individuals with
active IBD (34). RvE1 is an active and specific responder during
active intestinal inflammation. RvE1 is short lived and easily
degraded in an inflammatory environment, which may limit its
bioavailability as therapy to promote repair (35). Thus, to test
the in vivo prorepair properties of RvE1, we encapsulated RvE1
in NPs that were administered into healing colonic mucosal
wounds. Using this approach, we previously demonstrated in-
creased mucosal repair with use of an annexin-1 peptide-
encapsulated NP (36). Similar NPs have been used to promote
resolution of inflammation in murine zymosan-induced perito-
nitis and ischemia-reperfusion injury (37). NPs injected into the
healing mucosa, as presented in our study, localize RvE1 to
specific sites of injury and control its release in a sustained
manner. Whereas our study focused on the colon, NP-mediated
delivery of therapeutic molecules is a promising strategy that can
potentially be used in other epithelial surfaces, such as the skin,
eye, and lung. The development and engineering of NPs con-
taining proresolving compounds may thus establish a modality of
intercellular communication aimed to resolve inflammation and
promote repair of epithelial barriers. Taken together, this study
supports potent prorepair properties of RvE1 encapsulated in
NPs. RvE1 can therefore be used for the development of in-
novative therapeutic strategies, such as NPs, to promote reso-
lution of inflammation and repair in chronic inflammatory states.

phosphorylation of FAK-Y397 and Y925, both of which have been

shown to influence epithelial cell migration. Furthermore, RvE1
treatment increased activation of the small GTPase Rac1, which
functions to promote local ROS generation in concert with
Nox1, thereby modifying phosphatases involved in regulating
focal cell matrix adhesion proteins and cell motility (11).
Methods
Mice. C57BL/6 were purchased from the Jackson Laboratory.
Human Colonic Enteroids (Colonoids). Human three-dimensional (3D) colo-
noids are routinely maintained in the laboratory. 2D epithelial intestinal

4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1921335117 Quiros et al.


A claudin 4 (cat. 364800; Invitrogen). The following reagents were used:
B wounding were analyzed for RvE1 levels at the Queen Mary University
C biological replicates.
80 ***
% wound closure
60 **
40
20
PBS RvE1 Empty RvE1
Nanoparticles
Fig. 4. Intramucosal injections of RvE1 containing NPs promote intestinal
epithelial wound repair. (A) Schematic of targeted NPs encapsulating RvE1
and intramucosal NP injection into the colonic mucosal wounds. (B) Frozen
sections of resealing colonic wounds in mice showing F-actin (Alexa Fluor
488 phalloidin, green) and biotinylated NPs (Streptavidin 555, red). Higher-
magnification image (original magnification, 40×) of the boxed region is
shown to the right. (C) Quantification of wound repair. Data are expressed
as mean ± SEM. **P < 0.01; ***P < 0.0001. (Scale bar, 100 μm.)
monolayers from 3D colonoids were generated as described by Saxena
et al. (38).
Cell Lines and Culture Conditions and IEC Monolayer Wounding In Vitro. Human
IECs (SKCO15, T84) were grown. Wound closure was assessed using a scratch
wound assay, as previously published (11).
In Vivo Wounding of Colonic Mucosa. A high-resolution, miniaturized colo-
noscope system equipped with biopsy forceps (Karl Storz; Germany) was
used to injure the colonic mucosa at 5 to 10 sites along the dorsal artery, and
healing was quantified on days 1 and 3 postinjury.
Immunoblot and Immunofluorescence. For cell lysis, IEC monolayers were
harvested in radioimmunoprecipitation assay (RIPA) buffer. Immunofluo-
rescence was performed following standard immunofluorescence protocols.
Reagents. The following antibodies were used: FAK (cat. 610088; BD Biosci-
ences); pFAK (Y861; cat. PS 1008; Calbiochem); pFAK (Tyr397; cat. 3283), pFAK
(Tyr925; cat. 3284), Src (cat. 2108), pSrc (Tyr416; cat. 2101), p70 S6 Kinase (cat.
9202), p-p70 S6 Kinase (cat. 9209), mTOR (cat. 2983), pmTOR (cat. 2448),
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CREB (cat. 9197), and pCREB (cat. 87G3; Cell Signaling Technology); and
Resolvin E1 (cat. 10007848; Cayman Chemicals); hydrocyanine probe ROSstar
550 (cat. 926-20000; LI-COR Biosciences); and dasatinib (cat. 6793), PP2 (cat.
1407), 666-15 (cat. 5661), torin 1 (cat. 4247), and Rapamycin (cat. 1292; Tocris
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Lipidomic Analysis of RvE1 Levels. From 25 to 30 punch biopsies (3 mm) of
intact tissue or wounded colon from 3 animals on days 1, 2, and 3 after
London Lipid Mediator Unit, William Harvey Research Institute, Barts and The
London School of Medicine. The experiments were performed with 3
Intracellular ROS Generation. Epithelial cells were treated with RvE1 or vehicle
for the indicated times and incubated with 15 μM hydro-Cy3 for 4 h at 37 °C.
Quantification of fluorescence intensity of ROS was determined using
ImageJ software.
Spinning Disk Assay. Cell adhesion strength was measured using the spinning
disk system, as previously described (39).
Proximity Ligation Assay. In situ PLA was used to identify interactions between
Rac1 and PAK. Positive PLA signals, detected as a fluorescent dot by im-
munofluorescence microscopy, are produced when two labeled proteins are
IMMUNOLOGY AND
closely apposed within 40 nm, and in this case will indicate active Rac1. In situ
INFLAMMATION
PLA was performed on frozen tissue sections, fixed at room temperature
in 4% paraformaldehyde, followed by blocking and permeabilization with
3% bovine serum albumin/0.5% Triton X-100 in PBS. DuoLink PLA probes
and reagents (Sigma Aldrich) were used following the manufacturer’s
instructions.
RvE1 Nanoparticles. PLGA-PEG-Maleimide polymer was dissolved in dime-
thylformamide (3 mg/mL), and 2.0 μg of RvE1 (dissolved in ethanol at 0.1 mg/mL)
was added to the polymer solution. Next, 1 mL of this polymer–resolvin
mixture was then added dropwise to 10 mL of nuclease-free water. The NPs
were stirred for 2 h, concentrated by centrifugation using Amicon Ultra-15
centrifugal filter units, and filtered through sterile 0.45-μm syringe filters.
One milligram of collagen IV peptide was then conjugated to the particles,
using maleimide-cysteine reaction for targeting. To facilitate imaging, 1 mg
of 10 k PEG-Biotin-SH was conjugated to particles.
Statistical Analysis. Statistical comparisons were performed by one- or two-
way ANOVA with Bonferroni’s multiple comparison or unpaired two-tailed
Student’s t test, as appropriate. A P value of less than 0.05 was considered
significant.
Data Availability. All data, protocols and materials associated to this paper are
available within the manuscript or SI Appendix.
ACKNOWLEDGMENTS. The authors thank Jesmond Dalli (Queen Mary
University London) for running the Lipidomics analysis in his core and
Giovanna Leoni and Hikaru Nishio for their support in the initial steps of this
project. This work was supported by a Crohn’s and Colitis Foundation Career
Development Award (544599 to M.Q.), the National Science Foundation
Graduate Research Fellowship (DGE-1148903 to D.W.Z.), and the NIH grants
(R01-EB024322, and R01-HL127236 to A.J.G.; DK055679, DK089763, and
DK059888 to A.N.; and DK61739, DK72564, and DK79392 to C.A.P.).
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