Resolvin E1 is a pro-repair molecule that promotes intestinal epithelial wound healing

Significance

Resolvin E1 (RvE1) promotes resolution of inflammation by damping proinflammatory responses and activating restorative pathways. While mechanisms by which RvE1 signaling in immune cells contribute to resolution of inflammation are extensively studied, its role in epithelial signaling and wound repair remains undefined. Intestinal epithelial barrier compromise during an inflammatory event can result in pathogen access 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 targeted immunoresolvent therapeutic agent that not only dampens inflammation but activates prorepair pathways to enhance colonic mucosal wound repair.

Abstract

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 spherocytosis and macrophage production of IL-10. Despite these observations, 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 proliferation and migration through activation of signaling pathways including 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 synthetic 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 mucosal healing in the intestine.

The 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). Resolvin E1 (RvE1), an endogenous lipid mediator derived from omega-3 eicosapentaenoic acid, has been shown to limit inflammation through a number of mechanisms that include modulation of immune cell recruitment, augmentation of phagocytosis, promotion of neutrophil apoptosis, and efferocytosis (5–8). In concert with these anti-inflammatory properties, RvE1 dampens the 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 pathways 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 encapsulated RvE1 in polymeric nanoparticles and demonstrate their therapeutic potential in enhancing colonic mucosal wound repair.

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 mucosal 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 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 injury 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)

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.

RvE1 Activates Reparative Pathways in Intestinal Epithelial Cells.

Efficient repair of epithelial wounds is critical to restore mucosal 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 repair 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 increasing 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 incubation. 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).

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 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.

Since repair of epithelial wounds is dependent on coordinated cellular proliferation and migration, experiments were performed 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; 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-FAK axis were detected. In vitro healing epithelial wounds were incubated with RvE1 for 4 and 8 h. Increased phosphorylation of CREB (serine 133), p70 S6 Kinase (Thr389), and mTOR (Ser2448) was detected (Fig. 2D), in further support of the observed enhanced proliferation in response to RvE1 (Fig. 2C). In addition to RVE1-dependent proproliferative effects, we observed activation of signaling proteins that regulate cell adhesion/migration. Specifically, we observed increased Src phosphorylation (Y416), as well as phosphorylation of focal adhesion kinases (FAK Y397 and Y925) that play important roles in regulation of turnover of cell matrix adhesions and forward cell movement (Fig. 2D). To confirm the involvement of these signaling molecules in mediating RvE1 prorepair effects, we analyzed repair of scratch wounds in the presence of specific inhibitors for Src, CREB, and mTOR. Src inhibition (Dasatinib and PP2) completely 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 promotes 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 downstream 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 intracellular redox-sensitive dye, Hydro-Cy3. RvE1 treatment resulted 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 human 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 identified 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 detachment forces increase linearly with radial position and produce 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.

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.)

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 polyethylene 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 facilitate 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.

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.)

Discussion

Intestinal epithelial damage and compromise of the mucosal barrier are pathognomonic of a number of diseases including inflammatory bowel disease (IBD). Active and coordinated repair responses that promote epithelial cell migration and proliferation are necessary to reestablish mucosal barrier function 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 proresolution responses in immune cells (18). Nevertheless, the prorepair effects of SPMs on epithelial cells remain poorly understood. 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 mediators to restore homeostasis after the inflammatory response by actively damping inflammation and promoting reparative pathways without compromising the immune response (19). RvE1 has been shown to promote resolution of inflammation in model systems of arthritis, multiple sclerosis, bronchial asthma, retinopathies, periodontal diseases, dermatitis, and corneal/conjunctival 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 period that represents a transition between the proinflammatory 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 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 facilitating 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 signaling pathways in IECs that include CREB and mTOR, which have been reported to regulate epithelial proliferation, and ultimately wound repair (31, 32). Other SPMs such as RvD1, RvD2, and MaR1 have also been shown to activate CREB signaling 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 phosphorylation, which has been previously reported to promote tyrosine phosphorylation, and activation of FAK, cytoskeletal reorganization, and focal cell matrix adhesion turnover. RvE1 treatment was observed to activate pSrc-Y⁴¹⁶, as well as result in 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).

Increased SPM generation has been identified in chronic inflammatory 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 increased 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 peritonitis 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 containing 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 innovative therapeutic strategies, such as NPs, to promote resolution of inflammation and repair in chronic inflammatory states.

Methods

Mice.

C57BL/6 were purchased from the Jackson Laboratory.

Human Colonic Enteroids (Colonoids).

Human three-dimensional (3D) colonoids are routinely maintained in the laboratory. 2D epithelial intestinal 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 colonoscope 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. Immunofluorescence was performed following standard immunofluorescence protocols.

Reagents.

The following antibodies were used: FAK (cat. 610088; BD Biosciences); 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), 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 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 London School of Medicine. The experiments were performed with 3 biological replicates.

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 immunofluorescence microscopy, are produced when two labeled proteins are closely apposed within 40 nm, and in this case will indicate active Rac1. In situ 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 dimethylformamide (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.