Resolvin E1 Is a Pro-Repair Molecule That Promotes Intestinal Epithelial Wound Healing

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

aDepartment 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 inflammatory response in murine colitis by inhibiting neutrophil processes that mediate epithelial healing at mucosal surfaces. migration (8). However, the contribution of RvE1 in controlling Lipid molecules referred to as specialized proresolving mediators mucosal epithelial wound repair remains unclear. (SPMs) play an important role in the restorative response. Resolvin Here we show that RvE1 is an immunoresolvent that not only E1 (RvE1), a SPM derived from omega-3 fatty acids, has been has anti-inflammatory properties but also activates prorepair path- reported to dampen intestinal inflammation by promoting anti- ways that promote epithelial cell migration and proliferation, and inflammatory responses including increased neutrophil spherocy- ultimately epithelial wound repair. Given the labile nature of lipid tosis and macrophage production of IL-10. Despite these observa- mediators with potential for rapid enzymatic degradation, we en- tions, a role for RvE1 in regulating intestinal epithelial cell capsulated RvE1 in polymeric nanoparticles and demonstrate their migration and proliferation during mucosal wound repair has therapeutic potential in enhancing colonic mucosal wound repair. not been explored. Using an endoscopic biopsy-based wound healing model, we report that RvE1 is locally produced in response Results to intestinal mucosal injury. Exposure of intestinal epithelial cells Resolvin E1 Is Synthesized in Response to Intestinal Mucosal Injury. to RvE1 promoted wound repair by increasing cellular prolifera- Administration of exogenous RvE1 has been shown to exert anti- tion and migration through activation of signaling pathways in- inflammatory effects in models of experimental colitis (6, 8, 9). cluding CREB, mTOR, and Src-FAK. Additionally, RvE1-triggered However, the spatiotemporal generation of RvE1 during muco- IMMUNOLOGY AND INFLAMMATION activation of the small GTPase Rac1 led to increased intracellular sal wound repair has not been defined. Thus, we analyzed RvE1 reactive oxygen species (ROS) production, cell–matrix adhesion, synthesis in healing murine biopsy-induced colonic mucosal and cellular protrusions at the leading edge of migrating cells. wounds (Fig. 1A). Mucosa on the dorsal aspect of the colon was Furthermore, in situ administration of RvE1-encapsulated syn- injured and wounds harvested at 48 and 72 h postinjury. Healing thetic targeted polymeric nanoparticles into intestinal wounds wound samples were harvested and analyzed by multiple- promoted mucosal repair. Together, these findings demonstrate reaction monitoring liquid chromatography mass spectrometry that RvE1 functions as a prorepair lipid mediator by increasing intestinal epithelial cell migration and proliferation, and highlight Significance potential therapeutic applications for this SPM to promote mucosal healing in the intestine. Resolvin E1 (RvE1) promotes resolution of inflammation by damping proinflammatory responses and activating restorative Resolvin | specialized proresolving mediator | wound healing | repair | pathways. While mechanisms by which RvE1 signaling in im- epithelial cells mune cells contribute to resolution of inflammation are ex- tensively studied, its role in epithelial signaling and wound he gastrointestinal epithelium forms an important protective repair remains undefined. Intestinal epithelial barrier compro- Tbarrier that limits access of luminal antigens to the mucosal mise during an inflammatory event can result in pathogen ac- and systemic immune system. Epithelial injury and wounds have cess to tissue compartments. Thus, efficient repair of denuded been observed in a number of mucosal disorders that include intestinal mucosal surfaces is vital in restoring homeostasis. acute and chronic inflammatory states (1). Epithelial barrier Here we demonstrate that RvE1 promotes intestinal epithelial disruption results in spatiotemporal recruitment of immune cells cell migration and proliferation leading to wound repair. These to sites of injury, with consequent release of a complex cascade findings suggest that RvE1 has the potential to serve as a tar- of mediators that interact with the epithelium to orchestrate geted immunoresolvent therapeutic agent that not only damp- resolution of inflammation and mucosal repair. Perturbation ens inflammation but activates prorepair pathways to enhance in this delicate balance of inflammation, resolution, and repair colonic mucosal wound repair. contributes to chronic diseases such as inflammatory bowel disease (2). Author contributions: M.Q., A.J.G., C.A.P., and A.N. designed research; M.Q., D.F., D.B., In addition to proinflammatory mediators, cells at sites of 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 mucosal injury release lipid and protein/peptide specialized the paper. proresolvin mediators (SPMs), which have been shown to play The authors declare no competing interest. important roles in restoring homeostasis. SPMs are important in This article is a PNAS Direct Submission. C.N.S. is a guest editor invited by the orchestrating active resolution of inflammation and epithelial Editorial Board. repair, which is important in restoring the mucosal barrier (3, 4). Published under the PNAS license. Resolvin E1 (RvE1), an endogenous lipid mediator derived from See online for related content such as Commentaries. omega-3 eicosapentaenoic acid, has been shown to limit inflam- 1To whom correspondence may be addressed. Email: [email protected] or anusrat@ mation through a number of mechanisms that include modulation med.umich.edu. of immune cell recruitment, augmentation of phagocytosis, pro- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ motion of neutrophil apoptosis, and efferocytosis (5–8). In con- doi:10.1073/pnas.1921335117/-/DCSupplemental. cert with these anti-inflammatory properties, RvE1 dampens the First published April 16, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.1921335117 PNAS | April 28, 2020 | vol. 117 | no. 17 | 9477–9482 Downloaded by guest on September 24, 2021 CREB and mTOR and migratory signaling proteins in the Src- A B RvE1 FAK axis were detected. In vitro healing epithelial wounds were 100 OH 291 349 incubated with RvE1 for 4 and 8 h. Increased phosphorylation of +H HO OH 349=M-H CREB (serine 133), p70 S6 Kinase (Thr389), and mTOR Wounds days 331=M-H-H2O - 313=M-H-2H O D COO 2 -H +H (Ser2448) was detected (Fig. 2 ), in further support of the ob- 2, 3, 4 and 305=M-H-CO2 195 295=M-H-3H O Intact tissue 223 2 C 287=M-H-H2O-CO2 served enhanced proliferation in response to RvE1 (Fig. 2 ). 229=291-H2O-CO2 x 5 205=223-H2O In addition to RVE1-dependent proproliferative effects, we ob- 179=223-CO2

Relative Intensity (%) 177=195-H2O 161=223-H O-CO 305 2 2 served activation of signaling proteins that regulate cell adhesion/ 151=195-CO2 331 195 151 161 179 205 229 287 313 migration. Specifically, we observed increased Src phosphoryla- 223 295 0 177 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 tion (Y416), as well as phosphorylation of focal adhesion kinases Wound Punch biopsy m/z, Da (FAK Y397 and Y925) that play important roles in regulation of MRM Control 48h 72h 96h turnover of cell matrix adhesions and forward cell movement transitions Q1 Q3 Mean ± SEM Mean ± SEM Mean ± SEM Mean ± SEM (Fig. 2D). To confirm the involvement of these signaling mole- RvE1 349 161 3.6 ± 0.8 6.8 ± 1.9 * 3.0 ± 0.8 0.6 ± 0.4 * RvE2 333 199 4.9 ± 2.7 2.0 ± 0.9 3.9 ± 1.7 0.7 ± 0.3 cules in mediating RvE1 prorepair effects, we analyzed repair of RvE3 333 201 2.5 ± 0.6 3.5 ± 0.3 2.2 ± 1.1 2.0 ± 0.7 scratch wounds in the presence of specific inhibitors for Src, Fig. 1. RvE1 is produced in the colon in response to mucosal injury. (A) CREB, and mTOR. Src inhibition (Dasatinib and PP2) com- Consecutive wounds were generated on the dorsal aspect of the distal colon. pletely abrogated wound healing, while inhibitors for CREB Punch biopsies of wounds were harvested for analysis of lipid mediators by (666-15) and mTOR abolished RvE1 triggered increase in MRM-LCMS. (B) Results are presented as mean ± SEM, n = 3 samples each wound repair (SI Appendix, Fig. S4). These results indicate that composed of biopsies from 3 mice. Values are expressed as pg/100 mg tissue. RvE1 activates prorepair signaling pathways in IEC and pro- * < 0.05, mean SEM. motes wound healing. As previously shown by us and others, epithelial intracellular reactive oxygen species (ROS) signaling (MRM-LCMS) to obtain a lipidomic prolife of inflammatory and promotes oxidative inactivation of regulatory phosphatases, repair mediators in healing biopsy-induced mucosal wounds (see which in turn increase phosphorylation and activation of down- SI Appendix stream focal adhesion proteins, such as FAK, which in turn , Table S1 for details). Results of LCMS analyses – revealed increased levels of RvE1 in healing wounds compared controls forward cell movement and repair (10 12). Given such with intact mucosal tissue. RvE1 levels peaked at 48 h after in- prorepair properties of localized ROS signaling, we analyzed jury and returned to baseline values after 72 h postbiopsy ROS generation in healing epithelial wounds, using an intra- (Fig. 1B). In contrast to RvE1, levels of other closely related cellular redox-sensitive dye, Hydro-Cy3. RvE1 treatment resul- mediators (RvE2 and RvE3) did not show significant changes ted in significantly increased ROS generation (2.0-fold increase E after colonic mucosal injury. Interestingly, expression of RvE1 in fluorescence intensity) within 30 min of exposure (Fig. 2 ). receptor CMKLR1 was also increased in the repairing mucosa These data support an important role for RvE1 in promoting on days 2 and 3 postinjury (SI Appendix, Fig. S1) intestinal epithelial wound repair.

RvE1 Activates Reparative Pathways in Intestinal Epithelial Cells. RvE1 Activates Rac1 and Increases Cell Matrix Adhesion. It is well Efficient repair of epithelial wounds is critical to restore muco- appreciated that regulation of cell adhesion during tissue repair sal barrier function and dampen the inflammatory response. is fundamentally important for wound healing. Given results in Given the observed increase in RvE1 in healing wounds, we Fig. 1, experiments were performed to determine the influence – determined whether RvE1 promotes epithelial repair in vitro. of RvE1 treatment on intestinal epithelial cell matrix adhesion. Intestinal epithelial monolayers were scratch-wounded, and re- First, we evaluated whether RvE1 leads to activation of the small pair was monitored by time-lapse video imaging. Dose–response GTPase Rac1, which has been shown to play an important role in and time course studies using model human intestinal epithelial regulating cell matrix adhesion. Spatial Rac1 activity in migrating cell (IEC) line SKCO15 revealed RvE1-dependent enhancement epithelial cells was determined by proximity ligation assay (PLA) of epithelial wound repair in a dose-dependent fashion with in- between active Rac1 and its effector protein PAK1. This assay creasing concentrations from 10 to 500 nM, with peak effects capitalizes on the fact that only active or GTP-bound Rac1 can observed at 100 nM (Fig. 2A). Prorepair effects of RvE1 on bind to PAK1. As shown in Fig. 3A, 8 h of RvE1 exposure in epithelial cells were better observed between 8 and 16 h of in- wounded IEC monolayers (SKCO-15 cells and primary 2D hu- cubation. Similar effects were observed in a second model IEC man colonoids) resulted in increased levels of active Rac1-GTP/ line, T84 (SI Appendix, Figs. S2 and S3). These findings were PAK1 at the leading edge of the migrating epithelium. Since we confirmed in experiments using primary colonic epithelium observed that RvE1 activates ROS signaling, which in turn from human colonoids that were cultured and differentiated into promotes oxidative modification and inhibition of phosphatases two two-dimensional (2D) monolayers. Analogous to results that dephosphorylate FAK, we next examined localization of obtained with transformed epithelial cell lines, 100 nM RvE1 pFAK-Y861 in epithelial cells that were migrating to heal enhanced primary colonoid wound repair with maximum effects wounds. As shown in Fig. 3B, increased pFAK-Y861 was iden- occurring 24 h after injury (60.1 ± 2.0% control vs. 83.4 ± 2.0 tified in cells migrating in the presence of RvE1. To determine RvE1; P < 0.0001; Fig. 2B). whether RvE1 enhances cell–matrix adhesion, we measured the Since repair of epithelial wounds is dependent on coordinated force required to detach IEC cells from the extracellular matrix cellular proliferation and migration, experiments were per- in the presence or absence of RvE1. SKCO-15 cells were seeded formed to explore the effect of RvE1 on IEC proliferation. on fibronectin-coated glass coverslips and allowed to adhere for Analysis of the incorporation of the thymidine analog EdU 6 h, followed by exposure to controlled hydrodynamic shear demonstrated that RvE1 increased proliferation of wounded forces, using a spinning disk device. In this assay, applied de- IEC monolayers (17.3 ± 0.58% RvE1 vs. 10.7 ± 0.7% control; tachment forces increase linearly with radial position and pro- P < 0.001; Fig. 2C). duce a sigmoidal decrease in the number of adherent cells. Cell To explore the mechanisms by which RvE1 orchestrates adhesion strength is defined as the shear stress that produced wound repair, we analyzed signaling pathways that have been 50% detachment of cells. As shown in Fig. 3C, SKCO-15 cells shown to promote epithelial proliferation, migration, and wound treated with RvE1 showed significantly increased cell adhesion repair. Phosphorylation/activation of proproliferative proteins strength (111.1 ± 1.83 control vs. 126.9 ± 4.70 RvE1). These

9478 | www.pnas.org/cgi/doi/10.1073/pnas.1921335117 Quiros et al. Downloaded by guest on September 24, 2021 25 A 100 B ** C 100 *** *** 20 *** *** 80 80 15 ** 10 60 Control Resolvin 10 nM 60 Resolvin 50 nM 5 % EdU incorporation

% wound closure Resolvin 100 nM % Wound closure % Wound Resolvin 500nM 40 40 0 Control RvE1 Control RvE1 D E

2.5 **

2.0

1.5

1.0

% Fold change % Fold change 0.5

0.0 Control RvE1

Fig. 2. RvE1 promotes intestinal epithelial wound repair. (A) Wound areas of scratch-wounded SKCO15 IEC monolayers, incubated with increasing concentrations (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 IMMUNOLOGY AND INFLAMMATION 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.

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

Quiros et al. PNAS | April 28, 2020 | vol. 117 | no. 17 | 9479 Downloaded by guest on September 24, 2021 A SKCO-15 2D-colonoids Acn

NT RvE1 NT RvE1 PLA Rac1/PAK/ PLA Rac1/PAK/

B C (+) Resolvin 1.0 0.9 0.8 )

2 160 0.7 0.6 * 0.5 0.4 0.3 0.2 Adherent Fraction Adherent

Acn 0.1

, 140 0.0 0 50 100 150 200 250 300 350 Shear Stress (dyn/cm2)

120 (-) Resolvin 1.0 pFAK861 0.9 0.8 0.7 0.6 0.5 0.4 Adhesion Strength (dyn/cm 100 0.3 0.2 Control RvE1 Fraction Adherent 0.1 0.0 RvE1 0 50 100 150 200 250 300 350 NT Shear Stress (dyn/cm2)

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.)

need to be activated. Furthermore, RvE1 has been reported to functions to promote local ROS generation in concert with down-regulate NF-κB signaling in neutrophils, resulting in their Nox1, thereby modifying phosphatases involved in regulating decreased mobilization, adherence, and polarization of macro- focal cell matrix adhesion proteins and cell motility (11). phages toward an anti-inflammatory phenotype that leads to Increased SPM generation has been identified in chronic in- increased phagocytosis and IL-10 expression (9). We have pre- flammatory diseases such as IBD. Increased RvE1 has been viously observed that IL-10 plays an important role in facilitating detected in intestinal mucosal biopsies from individuals with intestinal mucosal repair, and our results indicate that RvE1 active IBD (34). RvE1 is an active and specific responder during might be contributing to this process. active intestinal inflammation. RvE1 is short lived and easily Intestinal epithelial immunomodulatory effects of RvE1, such degraded in an inflammatory environment, which may limit its as up-regulation of CD55 (decay-accelerating factor), BPI bioavailability as therapy to promote repair (35). Thus, to test (bactericidal/permeability-increasing), and alkaline phosphatase the in vivo prorepair properties of RvE1, we encapsulated RvE1 (ALPI) proteins, have been reported (9, 29, 30). CD55 is an anti- in NPs that were administered into healing colonic mucosal adhesive molecule that promotes clearance of apically adherent wounds. Using this approach, we previously demonstrated in- activated neutrophils from the epithelium, BPI protects mucosal creased mucosal repair with use of an annexin-1 peptide- surfaces against gram-negative bacteria and their endotoxins, encapsulated NP (36). Similar NPs have been used to promote and ALPI has been shown to control gram-negative bacterial growth and neutralization of LPS, thereby protecting against resolution of inflammation in murine zymosan-induced perito- pathogens. In this study, we observed that RvE1 activates sig- nitis and ischemia-reperfusion injury (37). NPs injected into the naling pathways in IECs that include CREB and mTOR, which healing mucosa, as presented in our study, localize RvE1 to have been reported to regulate epithelial proliferation, and ul- specific sites of injury and control its release in a sustained timately wound repair (31, 32). Other SPMs such as RvD1, manner. Whereas our study focused on the colon, NP-mediated RvD2, and MaR1 have also been shown to activate CREB sig- delivery of therapeutic molecules is a promising strategy that can naling in primary human monocytes, where they have anti- potentially be used in other epithelial surfaces, such as the skin, inflammatory and prorepair effects (33). eye, and lung. The development and engineering of NPs con- In addition to proliferation, remodeling and turnover of taining proresolving compounds may thus establish a modality of integrin-containing cell matrix contacts play a pivotal role in intercellular communication aimed to resolve inflammation and wound repair. We observed that RvE1 activates Src phosphor- promote repair of epithelial barriers. Taken together, this study ylation, which has been previously reported to promote tyrosine supports potent prorepair properties of RvE1 encapsulated in phosphorylation, and activation of FAK, cytoskeletal re- NPs. RvE1 can therefore be used for the development of in- organization, and focal cell matrix adhesion turnover. RvE1 novative therapeutic strategies, such as NPs, to promote reso- 416 treatment was observed to activate pSrc-Y , as well as result in 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 Methods treatment increased activation of the small GTPase Rac1, which Mice. C57BL/6 were purchased from the Jackson Laboratory.

9480 | www.pnas.org/cgi/doi/10.1073/pnas.1921335117 Quiros et al. Downloaded by guest on September 24, 2021 9202), p-p70 S6 Kinase (cat. 9209), mTOR (cat. 2983), pmTOR (cat. 2448), A CREB (cat. 9197), and pCREB (cat. 87G3; Cell Signaling Technology); and claudin 4 (cat. 364800; Invitrogen). The following reagents were used: 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 Bioscience).

Lipidomic Analysis of RvE1 Levels. From 25 to 30 punch biopsies (3 mm) of B intact tissue or wounded colon from 3 animals on days 1, 2, and 3 after wounding were analyzed for RvE1 levels at the Queen Mary University London Lipid Mediator Unit, William Harvey Research Institute, Barts and The C London School of Medicine. The experiments were performed with 3 80 *** biological replicates.

Intracellular ROS Generation. Epithelial cells were treated with RvE1 or vehicle 60 ** 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. 40 Spinning Disk Assay. Cell adhesion strength was measured using the spinning % wound closure disk system, as previously described (39). 20 PBS RvE1 Empty RvE1 Proximity Ligation Assay. In situ PLA was used to identify interactions between Nanoparticles Rac1 and PAK. Positive PLA signals, detected as a fluorescent dot by immunofluorescence microscopy, are produced when two labeled proteins are closely apposed within 40 nm, and in this case will indicate active Rac1. In situ Fig. 4. Intramucosal injections of RvE1 containing NPs promote intestinal PLA was performed on frozen tissue sections, fixed at room temperature epithelial wound repair. (A) Schematic of targeted NPs encapsulating RvE1 in 4% paraformaldehyde, followed by blocking and permeabilization with and intramucosal NP injection into the colonic mucosal wounds. (B) Frozen 3% bovine serum albumin/0.5% Triton X-100 in PBS. DuoLink PLA probes

sections of resealing colonic wounds in mice showing F-actin (Alexa Fluor and reagents (Sigma Aldrich) were used following the manufacturer’s IMMUNOLOGY AND INFLAMMATION 488 phalloidin, green) and biotinylated NPs (Streptavidin 555, red). Higher- instructions. magnification image (original magnification, 40×) of the boxed region is shown to the right. (C) Quantification of wound repair. Data are expressed RvE1 Nanoparticles. PLGA-PEG-Maleimide polymer was dissolved in dime- as mean ± SEM. **P < 0.01; ***P < 0.0001. (Scale bar, 100 μm.) 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 Human Colonic Enteroids (Colonoids). Human three-dimensional (3D) colo- centrifugal filter units, and filtered through sterile 0.45-μm syringe filters. noids are routinely maintained in the laboratory. 2D epithelial intestinal One milligram of collagen IV peptide was then conjugated to the particles, monolayers from 3D colonoids were generated as described by Saxena using maleimide-cysteine reaction for targeting. To facilitate imaging, 1 mg et al. (38). of 10 k PEG-Biotin-SH was conjugated to particles. Cell Lines and Culture Conditions and IEC Monolayer Wounding In Vitro. Human IECs (SKCO15, T84) were grown. Wound closure was assessed using a scratch Statistical Analysis. Statistical comparisons were performed by one- or two- ’ wound assay, as previously published (11). 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. 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 Data Availability. All data, protocols and materials associated to this paper are healing was quantified on days 1 and 3 postinjury. available within the manuscript or SI Appendix.

Immunoblot and Immunofluorescence. For cell lysis, IEC monolayers were ACKNOWLEDGMENTS. The authors thank Jesmond Dalli (Queen Mary harvested in radioimmunoprecipitation assay (RIPA) buffer. Immunofluo- 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 rescence was performed following standard immunofluorescence protocols. project. This work was supported by a Crohn’s and Colitis Foundation Career Development Award (544599 to M.Q.), the National Science Foundation Reagents. The following antibodies were used: FAK (cat. 610088; BD Biosci- Graduate Research Fellowship (DGE-1148903 to D.W.Z.), and the NIH grants ences); pFAK (Y861; cat. PS 1008; Calbiochem); pFAK (Tyr397; cat. 3283), pFAK (R01-EB024322, and R01-HL127236 to A.J.G.; DK055679, DK089763, and (Tyr925; cat. 3284), Src (cat. 2108), pSrc (Tyr416; cat. 2101), p70 S6 Kinase (cat. DK059888 to A.N.; and DK61739, DK72564, and DK79392 to C.A.P.).

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