NIL10: A New IL10-Receptor Binding Nanoparticle That Induces Cardiac Protection in Mice and Pigs Subjected to Acute Myocardial Infarction through STAT3/NF-κB Activation

(1) Background: Early response after acute myocardial infarction (AMI) prevents extensive cardiac necrosis, in which inflammation resolution, including expression of anti-inflammatory interleukin-10 (IL-10), may play a key role. (2) Methods: We synthesized NIL10, a micelle-based nanoparticle, to target IL-10 receptor in mice and pigs subjected to AMI. (3) Results: Administration of NIL10 induced cardiac protection of wild-type and IL-10 knockout mice and pigs subjected to AMI. Cardiac protection was not induced in IL-10-receptor null mice, as shown by a significant recovery of cardiac function, in which inflammatory foci and fibrosis were strongly reduced, together with the finding that resolving M2-like macrophage populations were increased after day 3 of reperfusion. In addition, anti-inflammatory cytokines, including IL-4, IL-7, IL-10, IL-13, IL-16, and IL-27 were also elevated. Mechanistically, NIL10 induced activation of the IL-10 receptor/STAT-3 signaling pathway, and STAT3-dependent inhibition of nuclear translocation of pro-inflammatory NF-ĸB transcription factor. (4) Conclusions: Taken together, we propose using NIL10 as a novel therapeutic tool against AMI-induced cardiac damage.


Introduction
Cardiovascular diseases, including acute myocardial infarction (AMI), are the most frequent cause of death in the world [1]. AMI remains the commonest origin of heart failure (HF), which conditions long-term life quality and may result in the death of the patient [2][3][4].
Following an ischemic event, the lack of oxygen and nutrients in the heart triggers an inflammatory response. Necrotic cardiomyocytes release signals that stimulate innate immune signaling, then cell mobilization to the damaged area begins, and, finally, anti-inflammatory signaling leads to the replacement of necrotic myocardial tissue by the formation of a fibrotic scar [5]. Even though inflammation is a critical component of tissue healing, currently, more contributions point towards a prolonged inflammatory response
The paramagnetic nanoparticles were prepared by the lipid film hydration method. In brief, a film was prepared by rotary evaporation of Gd-DTPA-bis (GdDTPA-BSA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000), DSPE-PEG2000-maleimide, and Rhodamine-PE, in a molar ratio 50:39:1:10, dissolved in a mixture of chloroform/methanol (4:1 v/v). The lipid film was hydrated with HEPES buffer, pH 6.7, and 150 mM NaCl, and the solution was rotated at 65 • C for 1 h. IT9302 and the corresponding scrambled peptides were modified by adding a terminal cysteine residue (Figure 1) to bind to the maleimide moiety at a molar ratio of 1:40 (micelle: peptide). Uncoupled peptides were separated with centrifuge concentrators of 100 kDa. The number of peptide molecules bound per micelle was calculated by HPLC as described [26], estimating 4 ± 2 and 5 ± 1 as the number of peptides present in NIL10 and NIL10SC, respectively. Physical and chemical properties, including Zeta-potential, nanoparticle size (calculated as hydrodynamic diameter with dynamic laser light scattering (DSL, Malvern Zetasizer)), nanoparticle morphology (visualized with transmission microscopy (TEM)), and longitudinal and transverse relaxivities, were calculated. Longitudinal relaxivities were evaluated by using inversion recovery sequences with 15 inversion times. Transverse relaxivities were calculated from spin-echo images and different echo times. In both cases, a series of images with different T1 and T2 weighting were generated. The r1 and r2 values were estimated from the slope of longitudinal and transverse relaxation rates vs. Gd-DTPA or nanoparticle.

Peptide and Nanoprobe Composition
Peptide composition of IT9302 and scrambled: IT9302: CAYMTMKIRN. IT9302scrambled: CANYRMITKM The paramagnetic nanoparticles were prepared by the lipid film hydration method. In brief, a film was prepared by rotary evaporation of Gd-DTPA-bis (GdDTPA-BSA), 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[ methoxy (polyethylene glycol)-2000] (DSPE-PEG2000), DSPE-PEG2000-maleimide, and Rhodamine-PE, in a molar ratio 50:39:1:10, dissolved in a mixture of chloroform/methanol (4:1 v/v). The lipid film was hydrated with HEPES buffer, pH 6.7, and 150 mM NaCl, and the solution was rotated at 65 °C for 1 h. IT9302 and the corresponding scrambled peptides were modified by adding a terminal cysteine residue ( Figure 1) to bind to the maleimide moiety at a molar ratio of 1:40 (micelle: peptide). Uncoupled peptides were separated with centrifuge concentrators of 100 kDa. The number of peptide molecules bound per micelle was calculated by HPLC as described [26], estimating 4 ± 2 and 5 ± 1 as the number of peptides present in NIL10 and NIL10SC, respectively. Physical and chemical properties, including Zeta-potential, nanoparticle size (calculated as hydrodynamic diameter with dynamic laser light scattering (DSL, Malvern Zetasizer)), nanoparticle morphology (visualized with transmission microscopy (TEM)), and longitudinal and transverse relaxivities, were calculated. Longitudinal relaxivities were evaluated by using inversion recovery sequences with 15 inversion times. Transverse relaxivities were calculated from spin-echo images and different echo times. In both cases, a series of images with different T1 and T2 weighting were generated. The r1 and r2 values were estimated from the slope of longitudinal and transverse relaxation rates vs. Gd-DTPA or nanoparticle.

Animal Studies
All the surgical procedures were performed in the Experimental Surgery Department of the Hospital Universitario La Paz in Madrid (Spain). The procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes Twenty male Yorkshire pigs (30 ± 4 kg) were housed 1 week preceding the surgery to avoid unease or stress associated with the new environment. Prior to the surgical intervention, animals were anesthetized with 10 mg/kg intramuscular ketamine and 0.5 mg/kg midazolam. Anesthesia was induced by inhaled sevoflurane and maintained with continuous infusion of 2 mL/kg/h propofol, 50 µg/kg/h fentanyl, and 10 µg/kg/h diazepam. After intubation and mechanical ventilation with 100% oxygen saturation, 5000 IU of heparin and 2 mg/kg/h amiodarone was administered to avoid blood clotting of catheters and malignant cardiac arrhythmias, respectively. Preceding the complete occlusion, hearts were submitted to ischemic preconditioning by blocking the LAD for short periods (1, 3, and 5 min each). Ischemia/reperfusion was produced by occluding LAD for 45 min using a JL3 6F catheter and an angioplasty balloon. The complete myocardial ischemia was confirmed by ST-segment elevation. Then, the balloon was removed to reopen the artery. Animals received NIL10 or NIL10SC (0.1 mg/kg) 24 h postischemia. Blood was extracted before and after the procedure, 3 days postischemia, and at the final point. After 7 days of reperfusion, animals were sacrificed to extract the heart, spleen, kidney, lung, liver, and pancreas. A portion was included in 4% formalin and the rest was immediately frozen at −80 • C for protein analyses.

Murine Model of Coronary Ischemia/Reperfusion
Wild-type, IL-10 knock-out, and IL-10RA knock-out C57/BL6 male mice were anesthetized with 3% sevoflurane and oxygen inhalation with a flow rate of 0.4 L/min until loss of righting reflex. Endotracheal intubation was performed using an intubation cannula for artificial ventilation (tidal volume: 260 µL/stroke, ventilation rate: 130 strokes per minute). The fourth left intercostal space was opened and widened using mouse chest retractors. The left ventricle was then exposed, and the LAD was occluded for 30 min by using a 6-0 silk suture and 1 mm tube. Reperfusion was performed by ligation release, and after the procedure, the chest was closed, negative pressure restored, and the skin sutured. At the reperfusion time of 24 h, NIL10 was intravenously administrated. Animals were sacrificed on days 3 and 7 postprocedure to extract the organs and process the samples. NIL10SC-treated animals were included in the assays as control, in which the same procedure was performed.

Echocardiography
Vivid Q ultrasound system from GE Healthcare (General Electric, Chicago, IL, USA), equipped with a 1.9-4 MHz scan head was used to determine LV function. Parasternal long-and short-axis-view images of the heart were taken prior to the surgery, at the end of ischemia, and at the endpoint to determine LV function worsening and recovery. The parameters studied using the onsite software cardiac package were: systolic and diastolic interventricular septum thickness (IVS), systolic and diastolic left-ventricle internal diameter (LVID), systolic and diastolic left-ventricle posterior wall thickness (LVPW), leftventricle ejection fraction (LVEF), left ventricle shortening fraction (LVFS), heart rate (HR), and cardiac output (CO). Data acquisition and analysis were performed by using the onsite cardiac software VividQ ultrasound equipment, and the same operator analyzed all samples to avoid interobserver biases.

Histology
Evans blue/TTC staining in pigs was performed as described [27]. In brief, 7 days after surgery, the LAD was re-occluded through balloon inflation at the same location as day 0. Then, a 5F fenestrated pigtail catheter was inserted through the second femoral artery and placed into the left ventricle to inject 200 mL of 5% Evans blue solution to distribute the compound across the entire cardiovascular system, excepting the blood-deprived area of the heart. After 2 min, the animal was sacrificed, and the heart was extracted and frozen at −20 • C for 24 h. Then, 0.8 cm heart slices were incubated in 1% TTC solution for 20 min at 37 • C and in 10% paraformaldehyde solution. The necrotic area was calculated in reference to the area at risk to avoid intervariability biasing.
Heart morphology was visualized by hematoxylin/eosin staining and collagen deposition was detected by Masson's Trichrome staining both in mice and pigs.

Confocal Microscopy
Rhodamine containing NIL10 nanoparticles were visualized in 5 µm heart sections mounted in PBS buffer containing Hoechst for nuclei visualization by confocal microscopy.
RAW 264.7 cell monolayers were incubated with anti-NFκB primary antibody diluted 1:500 in PBS 1.5% BSA for 1 h. They were then washed and incubated with the corresponding goat anti-rabbit IgG Alexa Fluor 488 secondary antibody diluted 1:1000 in PBS 1.5% BSA for 1 h. Cells were washed three times with PBS and mounted in PBS media containing Hoechst for nuclei visualization. Images were taken using a Leica TCS SP5 confocal microscope. At least three different fields per condition were obtained.

Immunoblotting
Protein lysates from healthy and necrotic areas of mouse and pig hearts were extracted with RIPA buffer to measure the levels of MMP-9, IL-10R, P-IL-10R, STAT3, and P-STAT3, while and P-IκB-α, and NOS2 were assayed in RAW247 cells. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 3% BSA in 25 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.4 (T-TBS), washed and incubated with the corresponding primary antibodies diluted 1:1000 for one hour at room temperature. Membranes were subsequently washed 3 times with T-TBS and then incubated with horseradish peroxidase-conjugated secondary antibodies 1:3000 for an hour for detection of proteins by chemiluminescence. The levels of GAPDH and β-Tubulin were used as loading controls.

Blood Collection and Plasma Isolation
Animal blood samples were collected in sodium citrate (363086) and EDTA tubes (367861) (BD Vacutainer, Franklin Lakes, NJ, USA) from a retro-orbital bleed in mice or from femoral venous in pigs. Blood samples were centrifuged at 400× g for 10 min, and the plasma was stored at −20 • C.

Cytokine and Chemokine Determinations
We detected a total of 40 human cytokines, chemokines, and acute phase proteins simultaneously from the serum of pigs and mice at the times indicated, by using the Proteome Profiler Cytokine Array Kit (RD Systems, Minneapolis, MN, USA), a membranebased antibody array for the parallel determination of the relative levels of selected human cytokines and chemokines, as described [9].

Single-Cell Suspension for Flow Cytometry
Whole mouse hearts were mashed in complete DMEM (10% FBS, penicillin/streptomycin) through a 100 µm cell strainer. ACK Lysing Buffer was added to the single-cell suspension and spun down at 350 g for 5 min. The ACK was washed out with 50 mL of washing buffer (1% FBS), and cells were incubated with 1:100 anti-CD68 and anti-CD206 antibodies. All flow cytometry samples were assayed in a MACSQuant Analyzer Flow Cytometer, analyzed with the MACSQuantify Software and results were graphed with the Graphpad Prism software package.

Statistical Analysis
All values were given as mean ± SD. Significance is reported at the 5% level. Whenever comparisons were made with common control, the significance of differences was tested by Dunnett's modification of the t-test.

NIL10 Improves Cardiac Function in Mice Subjected to IR
To test whether NIL10, a PEGylated nanoparticle conjugated with analog IL-10 peptide IT9302 ( Figure 1A-C for physicochemical details), binds to the IL-10 receptor, we incubated RAW 264.7 macrophages with 1 µg/mL NIL10 (a nanoparticle conjugated with specific peptide IT9302) or NIL10SC (the same nanoparticle in which IT9302 was substituted by a scrambled peptide (see methods for details)), detecting rhodamine-containing nanoparticles by confocal microscopy (Figure 2A). Colocalization with the IL-10R (as detected by immunohistofluorescence (FITC, green)) was positive in cells incubated with NIL10 (rhodamine, red. Figure 2B), as detected by confocal microscopy (merged panel, yellow and colocalization pixels panel). No binding was found in cells incubated with NIL10SC, as NIL10SC is conjugated with a peptide that does not bind to IL-10R ( Figure 2C). Cell viability was not affected at the dosage of NIL10 tested.
suspension and spun down at 350 g for 5 min. The ACK was washed out with 50 mL of washing buffer (1% FBS), and cells were incubated with 1:100 anti-CD68 and anti-CD206 antibodies. All flow cytometry samples were assayed in a MACSQuant Analyzer Flow Cytometer, analyzed with the MACSQuantify Software and results were graphed with the Graphpad Prism software package.

Statistical Analysis
All values were given as mean ± SD. Significance is reported at the 5% level. Whenever comparisons were made with common control, the significance of differences was tested by Dunnett's modification of the t-test.

NIL10 Improves Cardiac Function in Mice Subjected to IR
To test whether NIL10, a PEGylated nanoparticle conjugated with analog IL-10 peptide IT9302 ( Figure 1A-C for physicochemical details), binds to the IL-10 receptor, we incubated RAW 264.7 macrophages with 1 μg/mL NIL10 (a nanoparticle conjugated with specific peptide IT9302) or NIL10SC (the same nanoparticle in which IT9302 was substituted by a scrambled peptide (see methods for details)), detecting rhodamine-containing nanoparticles by confocal microscopy (Figure 2A). Colocalization with the IL-10R (as detected by immunohistofluorescence (FITC, green)) was positive in cells incubated with NIL10 (rhodamine, red. Figure 2B), as detected by confocal microscopy (merged panel, yellow and colocalization pixels panel). No binding was found in cells incubated with NIL10SC, as NIL10SC is conjugated with a peptide that does not bind to IL-10R ( Figure  2C). Cell viability was not affected at the dosage of NIL10 tested.  We intravenously administered NIL10 and NIL10SC at dosages of 0.1 mg/kg, 1 mg/kg, and 10 mg/kg, and analyzed the tissue distribution by confocal microscopy in the heart, liver, kidney, pancreas, and spleen of healthy animals at times 7, 14, 21, and 28 days after injection. We found a faint accumulation of NIL10 in the heart and kidney by day 7 after injection of 1 mg/kg and 10 mg/kg (Supplementary Figure S1). On the other hand, Kaplan-Meier curves of healthy mice injected with 1, 10, and 100 mg/kg indicated no mortality linked to the lowest dose, whereas the dosages of 10 and 100 mg/kg were lethal for 20% and 75% of animals, respectively, after 30 days of testing (Supplementary Figure S2).
After selecting a dosage of 1 mg/kg, we proceeded to inject 1 mg/kg of either NIL10 or NIL10SC into mice 24 h after IR ( Figure 3A,B). The result was that NIL10 improved the LVEF by days 3 and 7 after IR when compared to NIL10SC (41.5% ± 4.33 vs. 68% ± 7.02 and 65.6% ± 5.11, respectively. Figure 3C). Proof of concept was obtained by performing the same assay in IL-10 null mice, in which NIL10 also exhibited a significant degree of cardioprotection; administration of NIL10SC did not show any improvement ( Figure 3D). By contrast, in IL-10-receptor-deficient mice subjected to IR, NIL10 had no effect ( Figure 3E), suggesting that NIL10 induces cardiac protection through activation of the IL-10 receptor signaling pathway. Colocalization of NIL10 (B) (rhodamine, red) or NIL10SC (C) with IL-10R (FICT, green) in confocal microscopy sections of RAW 264.7 cells incubated with NIL10 and anti-IL-10R specific antibody. Merged panel show the co-localization of both signals. Nuclei were stained with Hoechst. Right panel: colocalization analysis with Image J co-localization plugin software, in which white dots correspond to co-localization (n = 3).
We intravenously administered NIL10 and NIL10SC at dosages of 0.1 mg/kg, 1 mg/kg, and 10 mg/kg, and analyzed the tissue distribution by confocal microscopy in the heart, liver, kidney, pancreas, and spleen of healthy animals at times 7, 14, 21, and 28 days after injection. We found a faint accumulation of NIL10 in the heart and kidney by day 7 after injection of 1 mg/kg and 10 mg/kg (Supplementary Figure S1). On the other hand, Kaplan-Meier curves of healthy mice injected with 1, 10, and 100 mg/kg indicated no mortality linked to the lowest dose, whereas the dosages of 10 and 100 mg/kg were lethal for 20% and 75% of animals, respectively, after 30 days of testing (Supplementary Figure S2).
After selecting a dosage of 1 mg/kg, we proceeded to inject 1 mg/kg of either NIL10 or NIL10SC into mice 24 h after IR ( Figure 3A,B). The result was that NIL10 improved the LVEF by days 3 and 7 after IR when compared to NIL10SC (41.5% ± 4.33 vs. 68% ± 7.02 and 65.6% ± 5.11, respectively. Figure 3C). Proof of concept was obtained by performing the same assay in IL-10 null mice, in which NIL10 also exhibited a significant degree of cardioprotection; administration of NIL10SC did not show any improvement ( Figure 3D). By contrast, in IL-10-receptor-deficient mice subjected to IR, NIL10 had no effect ( Figure  3E), suggesting that NIL10 induces cardiac protection through activation of the IL-10 receptor signaling pathway.

Administration of NIL10 Reduces Necrosis and Fibrosis in Mouse Hearts Subjected to IR
To shed light on the cardioprotective effect of NIL10, Hematoyilin/eosin (H/E) staining of heart sections, isolated on days 3 and 7 after IR, showed a significant reduction in the inflammatory foci and the extension of myocardial necrosis in response to NIL10 when compared to NIL10SC (Figure 4). Likewise, the levels of the necrosis marker, matrix metalloproteinase 9 (MMP9), were markedly reduced in the necrotic areas of NIL10 mice ( Figure 5A), including the extension of fibrosis, whereas in the NIL10SC group, fibrotic lesions were widespread in the hearts by day 7 after IR ( Figure 5B). knockout mice. n = 10 mice/group. Results are expressed as mean ± SD. * p < 0.01 NIL10 vs. NIL10SC day 7. # p < 0.05 NIL10 vs. NIL10SC day 3. (E) LVEF in IL-10 receptor knockout mice. n = 10 mice/group. Results are expressed as mean ± SD.

Administration of NIL10 Reduces Necrosis and Fibrosis in Mouse Hearts Subjected to IR
To shed light on the cardioprotective effect of NIL10, Hematoyilin/eosin (H/E) staining of heart sections, isolated on days 3 and 7 after IR, showed a significant reduction in the inflammatory foci and the extension of myocardial necrosis in response to NIL10 when compared to NIL10SC (Figure 4). Likewise, the levels of the necrosis marker, matrix metalloproteinase 9 (MMP9), were markedly reduced in the necrotic areas of NIL10 mice (Figure 5A), including the extension of fibrosis, whereas in the NIL10SC group, fibrotic lesions were widespread in the hearts by day 7 after IR. (Figure 5B).

NIL10 Improves Cardiac Function in Pigs Subjected to IR
To test whether NIL10 may also induce cardiac protection in large animals, we injected 1 mg/kg of either NIL10 or NIL10SC into pigs subjected to IR by angioplasty balloon inflation of the left anterior descending coronary artery ( Figure 3A). As shown by Evans blue/TTC staining of left ventricle sections ( Figure 6A), we found a significant reduction in the necrotic area between the NIL10 and NIL10SC groups by 47% after 7 days of IR (NIL10SC 55 ± 7.34 vs. NIL10 26.48 ± 4.32) (Figure 6B), which contributes to explain the improvement in cardiac function as evidenced by the recovery of LVEF in animals injected with NIL10 ( Figure 6C). As in mice, HE and Masson's trichrome staining of heart sections from pigs injected with NIL10 indicated a marked reduction of myocardial necrosis (Figure 6D) and fibrosis ( Figure 6E) when compared with the NIL10SC group.

NIL10 Improves Cardiac Function in Pigs Subjected to IR
To test whether NIL10 may also induce cardiac protection in large animals, we injected 1 mg/kg of either NIL10 or NIL10SC into pigs subjected to IR by angioplasty balloon inflation of the left anterior descending coronary artery ( Figure 3A). As shown by Evans blue/TTC staining of left ventricle sections ( Figure 6A), we found a significant reduction in the necrotic area between the NIL10 and NIL10SC groups by 47% after 7 days of IR (NIL10SC 55 ± 7.34 vs. NIL10 26.48 ± 4.32) (Figure 6B), which contributes to explain the improvement in cardiac function as evidenced by the recovery of LVEF in animals injected with NIL10 ( Figure 6C). As in mice, HE and Masson's trichrome staining of heart sections from pigs injected with NIL10 indicated a marked reduction of myocardial necrosis ( Figure 6D) and fibrosis ( Figure 6E) when compared with the NIL10SC group.

NIL10 Has an Impact on Macrophage Polarization
The difference in the number and intensity of inflammatory foci led us to study whether NIL10 could have an impact on macrophage polarization. M2-resolving macro-

NIL10 Has an Impact on Macrophage Polarization
The difference in the number and intensity of inflammatory foci led us to study whether NIL10 could have an impact on macrophage polarization. M2-resolving macrophages increased in NIL10 and NIL10SC at days 3 and 7, respectively ( Figure 7A), suggesting that NIL10 accelerate macrophage polarization, and further supports the severity of inflammation found in the group of NIL10SC. of inflammation found in the group of NIL10SC.
We also assessed the level of 40 cytokines and chemokines in plasma collected from mice and pigs after 3 days of IR (Supplementary Figure S3). We identified differences in the expression of a significant number of cytokines, highlighting the presence of four mouse (5 pig) expression clusters [28], depending on whether the animals have been injected with NIL10 or NIL10SC. This highlighted that, in both species, we identified a marked increase in the expression of anti-inflammatory cytokines IL-4, -10, -13, -16, and -27, together with IL-5, and IL-7 in pigs, in response to NIL10 administration ( Figure 7B,C). Taken together, these results suggest that NIL10 may induce cardiac protection by, at least, macrophage polarization towards inflammation resolution.  We also assessed the level of 40 cytokines and chemokines in plasma collected from mice and pigs after 3 days of IR (Supplementary Figure S3). We identified differences in the expression of a significant number of cytokines, highlighting the presence of four mouse (5 pig) expression clusters [28], depending on whether the animals have been injected with NIL10 or NIL10SC. This highlighted that, in both species, we identified a marked increase in the expression of anti-inflammatory cytokines IL-4, -10, -13, -16, and -27, together with IL-5, and IL-7 in pigs, in response to NIL10 administration ( Figure 7B,C). Taken together, these results suggest that NIL10 may induce cardiac protection by, at least, macrophage polarization towards inflammation resolution.

NIL10 Induces Phosphorylation of STAT3 in Pigs Subjected to IR
Cardiac exposure to ischemia triggers activation of specific pro-inflammatory transcription factors, of which NF-κB plays a major role [29]. Polarization of immune cells through an inflammatory resolving state implies the activation of specific signaling cascades, highlighting the contribution of IL-10-induced JNK signaling pathway that activates STAT3 transcriptional regulation of anti-inflammatory pathways, including prevention of NF-κB nuclear translocation [30].
NIL10 led to an accumulation of phosphorylated IL-10 receptor subunit IL-10RA and phospho-STAT3 in mice ( Figure 8A,B) and in pigs subjected to IR ( Figure 8C,D), indicative that NIL10, but not NIL10SC, induces receptor-agonist complex activation through the IL-10/JNK-STAT3 signaling pathway.

NIL10 Induces Phosphorylation of STAT3 in Pigs Subjected to IR
Cardiac exposure to ischemia triggers activation of specific pro-inflammatory transcription factors, of which NF-κB plays a major role [29]. Polarization of immune cells through an inflammatory resolving state implies the activation of specific signaling cascades, highlighting the contribution of IL-10-induced JNK signaling pathway that activates STAT3 transcriptional regulation of anti-inflammatory pathways, including prevention of NF-κB nuclear translocation [30].
NIL10 led to an accumulation of phosphorylated IL-10 receptor subunit IL-10RA and phospho-STAT3 in mice ( Figure 8A,B) and in pigs subjected to IR ( Figure 8C,D), indicative that NIL10, but not NIL10SC, induces receptor-agonist complex activation through the IL-10/JNK-STAT3 signaling pathway.  , and total IL-10-RA in the healthy (H) and necrotic (N) areas of mouse hearts at day 7 after IR (p < 0.04 NIL10 vs. NIL10SC necrotic areas). (B) Immunoblot detection of P-STAT3, and total STAT3 in the same protein extracts. n = 6 mice/group. Results are expressed as Mean ± SD (p < 0.05 NIL10 vs. NIL10SC necrotic areas). (C) Immunoblot detection as described in A. of pig hearts at day 7 after IR (p < 0.05 NIL10 vs. NIL10SC necrotic areas). (D) Immunoblot detection of P-STAT3, and total STAT3 in the same protein extracts. n = 6 pigs/group. Results are expressed as Mean ± SD (* p < 0.005 NIL10 vs. NIL10SC necrotic areas).
The anti-inflammatory effect of NIL10 was further investigated in RAW 264.7 macrophages stimulated with 500 µM LPS. Nuclear translocation of pro-inflammatory NF-kB transcription factor was prevented by NIL10 ( Figure 9A), at least by suppressing phosphorylation of the NF-κB cytoplasmic sequestering IkB-α ( Figure 9B), required for IkB-α proteasomemediated proteolytic degradation.

Discussion
In the current work, we showed that NIL10, a novel anti-inflammatory nanoparticle, induces cardiac protection after acute myocardial infarction following cardiac ischemia/reperfusion. Administration of NIL10 24 h after IR attenuated interstitial fibrosis and To further validate the anti-inflammatory effect of NIL10, we stimulated NF-kB nuclear translocation with 500 µM LPS in RAW 264.7 cells co-incubated with 5 µM of STAT3specific pharmacological inhibitor STATTIC. This prevented the NIL10-induced cytoplasmic localization of NF-kB in LPS-treated cells, thus indicating that NIL10 inhibits NF-kB nuclear translocation at least, by promoting STAT3 activation ( Figure 9C). The expression of the readout inducible nitric oxide synthase (iNOS) by pro-inflammatory signals was also assayed. As shown, iNOS was expressed by stimulating RAW 264.7 macrophages with 500 µM LPS and significantly inhibited by co-incubation with NIL10. The effect was reversed in the presence of STATTIC (Figure 9 lower panels). Taken together, NIL10 may act as an anti-inflammatory effector through the IL-10/STAT3 signaling pathway in myocardial ischemia/reperfusion.

Discussion
In the current work, we showed that NIL10, a novel anti-inflammatory nanoparticle, induces cardiac protection after acute myocardial infarction following cardiac ischemia/reperfusion. Administration of NIL10 24 h after IR attenuated interstitial fibrosis and improved LVEF in mice and pigs by days 3 and 7 after IR. The same pattern applied to IL-10 knockout mice, while in IL-10-receptor-deficient mice, NIL10 did not have an effect on cardiac recovery. As shown by confocal microscopy, NIL10 binds to IL-10R, inducing accumulation of M2-like macrophage populations after 3 days of IR, in which anti-inflammatory cytokines IL-4, IL-7, IL-10, IL-13, IL-16, and IL-27 were increased, at least through STAT-3-dependent inhibition of NF-κB nuclear translocation.
IL-10 is an anti-inflammatory cytokine that leads to cardiovascular protection in human atherosclerosis [31], acute coronary syndrome [32], unstable angina [33], and heart failure [34]. IL-10 plays an important role in the final outcome of myocardial infarction. A positive correlation between serum IL-10 levels with LVEF in patients after myocardial infarction suggests the implication of IL-10 in predicting acute and chronic heart failure [35]. This is shown in patients with metabolic syndrome, in which higher IL-10 serum levels are associated with a lower incidence of severe coronary artery disease [32,35]. Here, we provide a novel compound based on the anti-inflammatory properties of IL-10, in which, both in mice and pigs, it has been shown to prevent cardiac necrosis after myocardial ischemia/reperfusion.
The described benefits of using small molecules, including oligopeptides, include acting as an agonist of several proteins. We previously described the cardioprotective effect of intravenous administration of NAP9. The nanoparticles carry a small peptide, which specifically binds to EMMPRIN, a protein involved in the extracellular matrix degradation during myocardial infarction in mice and pigs subjected to myocardial IR [36]. Others have found that the synthetic IL-10 analogue peptide, IT9302, can interact with IL-10 receptor and mimic its anti-inflammatory effects both in vitro in human melanoma cells [37] (in monocyte differentiation to TGF-β dendritic cells [26]) and in rabbits subjected to acute pancreatitis [38] and acute lung injury [39]. However, the potential benefits of IT9302 in other inflammatory conditions are poorly understood.
The use of several types of peptides in rodent models of myocardial infarction has been widely described. Examples include the use of Tat-DAXXp, a fusion peptide with Tat-cell-penetrating peptide and the death-associated protein peptide [40], a synthetic apolipoprotein A-I mimetic peptide 4F [41], the E-domain region of the human MGF protein [42], or UM206, a peptide derived from regions of high homology between Wnt proteins [43]. Each example is a promising strategy for preventing progression of disease in rodents, although no studies have yet been performed in large animal models prior to validating the strategy in patients suffering a myocardial infarction. In the current work, we provided the efficacy of using a new IT9302 conjugated nanoparticle, both in mice and in pigs, with similar results.
Currently, the efficacy of specific treatments after myocardial infarction is limited for reasons that include lack of specific biodistribution, reduced half-life, or even toxicity issues. Given their small size, instead of using antibodies, many therapeutic strategies have used nanoparticles conjugated with the therapeutic principle, enabling them to freely travel throughout the vasculature and reach the target faster. They are also biocompatible, with very little chance of rejection [44]. NIL10 is coated with PEGs (see methods for details), conferring a feature that implies a significant drug delivery (peptide IT9302 binding to IL10 receptor) to target cells and tissues with respect to empty peptide administration and decrease immunogenicity. Most importantly, PEG-coated NIL10 shields the surface from aggregation, phagocytosis, and degradation, thus prolonging systemic circulation time with respect to other molecules, including chemicals, peptides, or antibodies [45]. In addition, Z-potential and size ensured nanoparticle stability and tissular accessibility after IV administration.
The use of nanoparticles is preferred over antibodies for the treatment of inflammatory diseases, in which eliciting immunostimulation is undesirable and should be minimized [46]. NIL10 activates the IL10 downstream signaling pathway by interacting with the IL10 receptor in vivo, while the use of anit-IL10 antibodies has the opposite effect, as shown when promoting collateral growth in mice subjected to hind limb ischemia. In this case, administration of recombinant IL10 induced reperfusion recovery, while incubation with anti-IL10 prevented the effect [20]. The use of anti-IL10 antibodies was also assayed in other inflammatory diseases, including psoriasis lesions; anti-IL10 antibody treatment upregulates IL-17A, which plays a central role in psoriasis pathogenesis [47]. Furthermore, nanoparticles are extremely versatile, as additional components can be incorporated into their composition to enable the acquisition of new functionalities. Such is the case of NAP9 [27,36] and NIL10-both are nanoparticles based on lipid micelles and incorporate a fluorochrome in their composition to gain visualization by fluorescence, conferring specificity.
Theranostics is defined as the combination of diagnostic and therapeutic capability on a single agent and, depending on their composition, nanoparticles are unique compounds suitable to be used as theranostic agents. Chemicals, antibodies, and peptides have been used to induce and/or inhibit several signaling pathways associated with the onset and progression of disease. However, very few compounds, including specific engineered nanoparticles, may have the ability to have a theranostic effect. As mentioned above, we engineered gadolinium containing NAP9 nanoparticles, revealing a theranostic effect against cardiac ischemia/reperfusion in mice and in pigs. NAP9 nanoparticles incorporate a small peptide, AP9, which specifically binds to EMMPRIN, an inflammatory molecule that induces MMP9 expression and activity in response to cardiac ischemia. NAP9 significantly improved cardiac function after IR and, thanks to the presence of gadolinium, NAP9 was visualized by MRI, correlating the extension of cardiac necrosis and healing overtime [27,36].
IL-10 triggers downstream signaling through binding to IL-10 receptors, which phosphorylate JAK1 and further activate the intracellular domain of IL-10R-a, required for the recruitment and phosphorylation of STAT3, which, at the end, regulates gene expression of anti-inflammatory genes [19]. Interestingly, others found that the IL-10 functional domain homolog IT9302 induced monocyte differentiation to TGF-β tolerogenic dendritic cells by an independent JNK/STAT3 signaling pathway [37]. However, our results point to STAT3 activation as a mechanism induced by NIL10 in cardiac protection against myocardial infarction. This apparent contradiction may lie in the mechanism of IT9302 administration, which, in the case of NIL10, specifically binds to IL-10 receptor, as shown by confocal microscopy when compared to its negative control, NIL10SC ( Figure 1). Indeed, in IL-10 receptor null mice subjected to myocardial IR, administration of NIL10 had no effect, while in IL10 knockout animals, NIL10, but not NIL10SC, recovered cardiac protection, as detected by an improvement of LVEF in these mice (Figure 3).
The anti-inflammatory effect of IL-10 is in addition driven by STAT3-mediated induction of several mediators, including certain transcription factors, such as ETV3 and the Strawberry notch homologue-2, which inhibit NF-kB-mediated transcription in LPSstimulated macrophages [30]. Here, we also found that NIL10 induced STAT3-NF-κB inhibition in LPS-stimulated RAW 264.7 macrophages, as confirmed by the reversion of NIL10-mediated inhibition of iNOS expression in LPS-stimulated cells by pharmacological inhibition of STAT-3 in this context.

Conclusions
In conclusion, we provided a feasible delivery of a novel compound to target the inflammatory response after acute myocardial infarction and reperfusion, representing a reliable strategy to preserve heart contractility, as a mechanism to prevent the progression of myocardial necrosis. In addition to NIL10, our approach supports the use of additional peptides against specific targets susceptible to inhibition in the acute phase of heart failure postinfarction. Further studies to test the long-term effects of NIL10 administration in pigs will be a key step to initiating validation in the clinical setting.