IL-20R Activation via rIL-19 Enhances Hematoma Resolution through the IL-20R1/ERK/Nrf2 Pathway in an Experimental GMH Rat Pup Model

Aims Blood clots play the primary role in neurological deficits after germinal matrix hemorrhage (GMH). Previous studies have shown a beneficial effect in blood clot clearance after hemorrhagic stroke. The purpose of this study is to investigate interleukin-19's role in hematoma clearance after GMH and its underlying mechanism of IL-20R1/ERK/Nrf2 signaling pathway. Methods A total of 240 Sprague-Dawley P7 rat pups were used. GMH was induced by intraparenchymal injection of bacterial collagenase. rIL-19 was administered intranasally 1 hour post-GMH. IL-20R1 CRISPR was administered intracerebroventricularly, or Nrf2 antagonist ML385 was administered intraperitoneally 48 hours and 1 hour before GMH induction, respectively. Neurobehavior, Western blot, immunohistochemistry, histology, and hemoglobin assay were used to evaluate treatment regiments in the short- and long-term. Results Endogenous IL-19, IL-20R1, IL-20R2, and scavenger receptor CD163 were increased after GMH. rIL-19 treatment improved neurological deficits, reduced hematoma volume and hemoglobin content, reduced ventriculomegaly, and attenuated cortical thickness loss. Additionally, treatment increased ERK, Nrf2, and CD163 expression, whereas IL-20R1 CRISPR-knockdown plasmid and ML385 inhibited the effects of rIL-19 on CD163 expression. Conclusion rIL-19 treatment improved hematoma clearance and attenuated neurological deficits induced by GMH, which was mediated through the upregulation of the IL-20R1/ERK/Nrf2 pathways. rIL-19 treatment may provide a promising therapeutic strategy for the GMH patient population.


Introduction
Germinal matrix hemorrhage (GMH) is defined by the rupture of thin-walled blood vessels located in the germinal matrix of preterm/low-birthweight infants [1,2]. It is one of the leading causes of morbidity and mortality among preterm infants [3]. Survivors suffer from severe long-term neurological, motorial, and cognitive deficits, which contribute to cerebral palsy, developmental delays, mental retardation, and posthemorrhagic hydrocephalus (PHH) [4,5]. Cur-rently, there are no safe-effective drug therapies to treat the GMH patient population. Thus, a safe and noninvasive strategy is essential in the management for GMH.
After the rupture of blood vessels, blood components permeate into the brain parenchyma [6,7]. Cytotoxins such as heme, peroxiredoxin 2 (PRX-2), and iron are released from metabolized blood cell (RBC) causing secondary brain injury through the increase in neuroinflammation, oxidative stress, neuronal death, and/or apoptosis [8,9]. Additionally, the intracerebroventricular blood clot directly impairs the circulation and absorption of the cerebrospinal fluid (CSF), leading to the development of PHH [10].
Microglia play a pivotal role in the pathological process after injury, through the regulation of the immune response, oxidative stress, and the removal of blood products or tissue debris [11]. Within the neonatal CNS, microglia play a positive role in blood-brain development and postnatal development and were shown to be neuroprotective after germinal matrix hemorrhage [2]. In a neonatal model of GMH, microglia/macrophages participate in the phagocytosis of red blood cells (RBCs) which enhanced hematoma resolution. The quick resolution of hematoma resulted in the reduction of ventricular dilation and improved long-term neurobehavior [12].
Scavenger receptor CD163, a class B glycoprotein, has been shown to induce microglia/macrophage phagocytosis which induced hematoma clearance [11,13]. CD163 is primarily expressed on alternatively activated microglia/macrophages (M2) [14]. The CD163 function in the neonatal CNS is yet to be evaluated.
Because of the aforementioned evidence, we hypothesized that recombinant IL-19 treatment increases hematoma resolution, expression of scavenger receptors on microglia/macrophages, M2 microglia, reduces ventricular dilatation, and improves short-and long-term neurological deficits and that the beneficial effects may be mediated by the activation of the IL-20R1/ERK/Nrf2/CD163 signaling pathway.

Materials and Methods
2.1. Animals and GMH Model. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Loma Linda University, and in accordance with the National Institute of Health guidelines for the treatment of animals. A total of two hundred and forty seven-dayold (P7) female and male Sprague-Dawley (SD) neonatal rats were purchased from Envigo (Livermore, CA, US). P7 pups were used for the GMH model as their brains are comparable to 30-32 weeks of human gestation. We experienced zero mortality of rat pups during these experiments. Upon arrival or after surgeries, pups remained with the mother until euthanasia or the weaning process for long-term experiments. After being weaned, two pups of the same gender were housed per cage. All animals are housed on a 12 : 12 hour light/dark cycle in controlled appropriate humidity and temperatures with plenty of food and water.
The GMH model was induced by stereotactic-guided injection of bacterial collagenase as previously described in [22]. Neonatal rat pups were anesthetized with 5% isoflurane and placed onto a stereotactic head frame with a neonatal adaptor in a prone position. A scalp incision is made along the midline exposing the bregma. A burr hole is drilled on the right side of the skull at coordinates of 1.6 mm (right lateral), 1.5 mm (rostral), and dura of 2.7 mm (depth) from bregma. 0.3 units of collagenase VII-S (VII-S, Sigma; 0.3 U in 0.5 μl of PBS) was infused by a 10 μl syringe (Hamilton Co. US) guided by a microinfusion pump (Harvard Apparatus, Hollison, MA). To minimize the leakage of collagenase from the burr hole, the syringe was kept in place for 5 minutes after injection and was withdrawn at a speed of 0.5 mm/min.
Bone wax was used to seal the burr hole, and a suture was used to close the incision line. Lastly, the pups were placed on a 37°C heated blanket for recovery, and after recovery, the pups were placed back with the dam. During the operation, the skin color, temperature, respiratory rhythm and frequency, and heart rates were closely monitored. The procedure for sham groups were similar to the vehicle groups, yet they were only subjected to needle insertion without collagenase injection.

Experimental Design and Groups.
Both male and female pups were randomized into the following experimental groups: sham, GMH + vehicle, GMH + rIL19, GMH + rIL-19 + IL-20R1 CRISPR (knockdown), GMH + rIL-19 + IL-20R1 CRISPR diluent, MH + rIL-19 + ML385, and MH + rIL-19 + DMSO. Figure 1 shows each animal group and experimental design. Experiment 1. Experiment 1 was performed to detect the endogenous levels of IL-19, IL-20R1, IL-20R2, and CD163. Western blot (WB) was used to analyze the expression of these aforementioned proteins in the whole brain at 12 h, 24 h, 72 h, 5 d, and 7 d after GMH. To confirm if the complex of IL-20 receptor type 1 (IL-20R1 and IL-20R2) is expressed on microglia, the colocalization of IL-20R (IL-20R1, IL-20R2) and CD11b was conducted for immunofluorescence. Experiment 2. Experiment 2 was performed to investigate the effects of rIL-19 treatment on short-and long-term neurobehavior after GMH. Negative geotaxis and righting reflex were used to test short-term neurobehavior deficits. The Morris water maze, rotarod, and foot fault were used to assess long-term neurobehavior deficits. Experiment 3. Experiment 3 was performed to observe the effects of rIL-19 on hematoma resolution after GMH. Hematoma volume, hemoglobin content, ventricular volume, and relative cortical thickness were evaluated.   [25] or 5% dimethyl sulfoxide (DMSO) was intraperitoneally administered 1 h before surgery, and every 24 h for 3 consecutive days.

Neurobehavioral Examination.
Negative geotaxis and righting reflex were employed to examine the short-term neurobehavior and water maze, foot fault, and rotarod test for long-term neurobehavior as previously reported in [22]. All of tests were executed in a blinded manner.
2.4.1. Negative Geotaxis. Neonatal pups were placed on a 45°r ough ramp in a downward orientation, and the time it takes for the pup to recognize that it is on an incline and to correct its positioning by making a 180-degree turn facing upwards within a 60-second duration is recorded. This test was repeated three times for three consecutive days post-GMH, and the average of each day was taken for statistical analysis.

Righting
Reflex. The pups were placed supinely on a horizontal plate, and the time it takes for the pup to flip/rollover was recorded within a 20-second duration. Every pup also was examined in repeats of three as described for negative geotaxis.

Morris Water
Maze. The Morris water maze was performed to evaluate spatial and reference memory at 22 and 27 days postictus as previously described in [26,27]. After the training block trial, rats undergo 4 trial blocks to find the platform. The time and distance it takes the animal to find the platform was recorded. On the last day, the platform is removed to perform the probe trial, where the time spent swimming in the quadrant where the platform is located is measured.

Rotarod and Foot Fault
Test. Rotarod and foot fault tests were conducted to evaluate the motor, locomotor, and balance after injury and treatment as previously described in [28,29]. Rats were placed on an accelerating rod, which is 70 mm in diameter, at starting speeds of 5 RPM or 10 RPM. Falling latency was recorded by the Rotor Rod instrument (SD Instruments, San Diego, CA). During the foot fault test, animals were placed on a grid wire (0:6 × 1:5 m, 2.5 cm apart) where the number of missteps were recorded in a one-minute interval.

Hematoma Volume and Hemoglobin Content Measurement.
Hematoma measurement was adapted from the following manuscript [12]. Rats were anesthetized by 5% isoflurane, followed by transcardiac perfusion with 4°C phosphatebuffered saline (PBS). The whole brain was extracted and cut into 5 coronal sections equidistantly. The ImageJ software (NIH) was used to count the area of hematoma (mm 2 ), and then the hematoma volume was estimated with the Group (short-term, long term): Hemoglobin assay was conducted to measure hemoglobin content as previously reported in [30]. Extracted forebrain tissue was placed into glass tubes with 3 mL of PBS. The brains were homogenized for one minute (Fisher Scientific, USA) followed by ultrasonication for one minute to lyse erythrocyte membranes. After centrifugation for thirty minutes (2°C), the supernatant was extracted from the sample. Drabkin's reagent (Sigma-Aldrich) is combined with supernatant at a 4 : 1 ratio and left to react for 15 minutes at room temperature. Using a spectrophotometer (540 nm; Genesis 10UV; Thermo Fisher Scientific), absorbance was calculated into hematoma volume (μL) as described in [12].

Ventricular Volume and Cortical Thickness Estimation.
At 28 days postictus, animals were euthanized, and the whole brains were extracted and prepared for histology. To be better observed and measured, histological slices were stained by Nissl. Ventricular volume and cortical thickness were evaluated as previously described in [31]. The brains were embedded with Optimal Cutting Temperature (OCT, Fisher Scientific, Waltham, MA) and sectioned into 20 μm thick slices (Leica Microsystems, LM3050S). Samples were stained with Cresyl violet, and optical dissector principles were used to delineate cerebral structure borders as described in [32]. Ventricular volume and cortical thickness were analyzed using the ImageJ software (NIH) as previously described in [33].
2.8. Immunofluorescence. Immunofluorescence staining was performed as previously described in [35]. The animals were sacrificed and transcardially perfused with PBS followed by 4% formalin. 2.9. Rigor and Statistical Analysis. All animals in this study were numbered and randomly sorted into each of the experimental animal groups using Excel. The protocol of randomization by Excel is as follows: (1) sequence the pups vertically in an Excel spreadsheet; (2) use "RAND" function to generate a random number for each pup; (3) rerank the random numbers in an ascending or descending order; and (4) designate a certain number of pups into each experimental group according to the study design. All experimental groups and identifying numbers were unknown to the surgeons and researchers conducting methodology. Animal sample size evaluation was determined using a type I error rate of 0.05 and a power of 0.8 on a 2-sided test by power analysis. Parametric data was described as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's post hoc test. Longitudinal data were analyzed using two-way repeated measure ANOVA with Tukey's post hoc test. The counting data was tested using chi-square test. P values of < 0.05 were considered statistically significant. GraphPad Prism 7 (La Jolla) were used for graphing and analyzing all the data.

Results
3.1. Endogenous Expression of IL-19, IL-20R1, IL-20R2, and CD163 Increased after GMH. The endogenous proteins were examined by WB at 0 (sham), 12, 24, and 72 hours, and 5 and 7 days post-GMH. Endogenous IL-19 expression was significantly increased at 24 hours to 5 days, where peak expression was seen at 72 hours after GMH (Figures 2(a) and 2(b)). Both subunits of IL-20R expression increased after GMH, where

Oxidative Medicine and Cellular Longevity
IL-20R1 increased at 24 hours to 5 days, and peak expression was achieved at 72-hour time point (Figures 2(a) and 2(c)), while IL-20R2 increased at 12 hours to 7 days, and peak expression was seen at 5 days after GMH (Figures 2(a) and 2(d)). The endogenous expression of CD163 decreased at 12 and 24 hours and then increased significantly at 72 hours to 7 days postictus (Figures 2(a) and 2(e)). Based on these results, the 3-day time point was chosen to evaluate the mechanism of action.
Double immunofluorescence staining was used to determine the cellular localization of IL-20R1 and IL-20R2 in the CNS at 72 hours after GMH. Both IL-20R1 and IL-20R2 were found to be colocalized with CD11b at the site of perihematoma (Figures 3(a) and 3(c)). These results indicate that IL-20R type 1 is expressed on microglia/macrophage cells.

Intranasal Administration of Murine Recombinant IL-19
Improved Short-and Long-Term Behavioral Outcomes after GMH. Three different dosages of rIL-19 (3.3 μg/kg, 10 μg/kg, and 30 μg/kg) were administered intranasally to determine the best dose response. Animals were randomly divided into the following groups: Sham, GMH + vehicle, GMH + low dosage rIL-19 (3.3ug/kg), GMH + middle dosage rIL-19 (10ug/kg), and GMH + high dosage rIL-19 (30ug/kg) animal group. When compared to sham, all GMH groups had significantly short-term neurological impairments. At the 24-hour time point after GMH, middle and high dosage of rIL-19 significantly improved motor coordination (negative geotaxis) when compared to the vehicle group (Figures 4(a) and  4(b)), yet righting reflex demonstrated no significant difference. At 48 and 72 hours after GMH, middle and high dosage groups significantly improved short-term behavior when compared to the vehicle group (Figures 4(a)-4(c)). Both middle and high dosages of rIL-19 improved short-term neurological deficits, and the medium dosage (10 μg/kg) was selected as the best dose.
To assess the effects of rIL-19 on long-term neurological function (spatial memory, reference memory, and motor coordination), neurobehavioral tests were performed at 22 to 27 days after GMH. No significant difference was seen in escape latency and swim distance among all animal groups at blocks 1 and 2. The rIL-19-treated group took significantly less time and distance to find the platform when compared to the vehicle group (Figures 5(a)-5(c)). There was no statistical difference in the average swimming speed of each group (data not shown). The Probe Trial demonstrated that the   Oxidative Medicine and Cellular Longevity vehicle-treated group spent significantly less time in the target quadrant when compared to sham animals, whereas rIL-19-treated animals spent more time in the target quadrant compared to the vehicle group ( Figure 5(d)). Thus, rIL-19 improved spatial and reference memory after GMH. Rotarod and foot fault tests were conducted to evaluate motor coordination on the 28th day after GMH. rIL-19treated animals demonstrated shorter falling latency on both 5 RMP and 10 RMP accelerating rods compared to the sham group. rIL-19-treatment group had significantly lower falling latencies than the vehicle-treated group ( Figure 5(f)). Additionally, rIL-19-treated animals had significantly lower number of foot faults when compared to vehicle ( Figure 5(e)). rIL-19 attenuated long-term motor coordination deficits induced by GMH. There was no statistical difference observed between male and female rats (data not shown) during these tests.

rIL-19 Reduced Hematoma Volume and Hemoglobin
Content at 72 Hours Post-GMH. To assess the effect of rIL-19 on hematoma volume, rat pup brains were evaluated at 24 and 72 hours after GMH. At 24 hours, no statistical difference was found between vehicle and rIL-19 treatment groups (Figures 6(a) and 6(c)). At the 72-hour time point, rIL-19   Oxidative Medicine and Cellular Longevity significantly decreased hematoma volume when compared to the vehicle group (Figures 6(b) and 6(c)). The same trend was seen in hemoglobin assay, as rIL-19 treatment significantly decreased hemoglobin content when compared to the vehicle group ( Figure 6(d)).

rIL-19 Enhanced the CD163 Expression on
Microglia/Macrophages at 72 Hours after GMH. Scavenger receptor CD163 was colocalized with CD11b to determine CD163 + microglia/macrophages cells around the site of perihematoma and was quantified as previous described in [12]. 72 hours after GMH, the number of microglia/macrophages was significantly increased in the GMH groups when compared to sham (Figure 7). rIL-19-treated group, significantly increased CD163 + microglia/macrophages positive cells when compared to sham and vehicle (Figure 7).

rIL-19 Reduced Ventricular Volume and Attenuated
Brain Atrophy 28 Days after GMH. Nissl staining was performed to help assess ventricular volume and relative cortical thickness at 28 days after GMH. Compared to sham animals, the vehicle group had significantly increased ventricle dilation and decreased cortical thickness, whereas rIL-19 treatment group significantly reduced ventricular dilation and protected against the loss of cortical thickness (Figure 8).
3.6. The Nrf2 Inhibitor ML385 and IL-20R1 CRISPR Inhibited the Signaling Pathway Induced by rIL-19 after GMH. IL-19 activation of ERK and its downstream protein Nrf2 play a pivotal role in the expression of CD163 and its enhancement of hematoma clearance after GMH. Thus, we evaluated the effects of the rIL-19 on p-ERK, Nrf2, and CD163 at 72 hours postictus. rIL-19 significantly increased p-ERK, Nrf2, and CD163, when compared to sham and vehicle (Figures 9 and  10). Knockdown of IL-20R1 by specific CRISPR plasmid significantly attenuated the expression of IL-20R1, p-ERK, and Nrf2, which was accompanied by a decrease in the expression of CD163 at 72 hours after GMH (Figures 9(a) and 9(b)). Furthermore, the upregulation of Nrf2 by rIL-19 was inhibited by Nrf2 inhibitor ML385, which also demonstrated to

Discussion
GMH is a one of the most devastating stroke subtypes for newborns, where the major neurological complications after hemorrhage are neuroinflammation, cerebral palsy, PHH, and motor and cognitive deficits [36]. Currently, GMH research has primarily focused on neuroinflammation [29,37], reactive astrogliosis [31,35], blood-brain barrier (BBB) destruction [38,39], and hematoma resolution [12,40] which has been closely associated with hematoma. In this study, our focus is on the blood clot which is primary causative factor in GMH-induced secondary brain injury. Here, we are the first to investigate the therapeutic effects of rIL-19 and its role in hematoma clearance by activating IL-20R1 signaling pathway after GMH. The following observations were made: (a) Endogenous protein levels of IL-19 and its receptor IL-20R were significantly expressed postictus, which correlated with an increase in endogenous CD163; (b) IL-20R1 was shown to be expressed on microglia/macrophages cells after GMH; (c) rIL-19 treatment significantly improved short-and long-term neurological function after GMH; (d) rIL-19 treatment significantly reduced GMH-induced ventricular dilation and cortical thickness loss; (e) rIL-19 decreased hematoma volume and hemoglobin content; and (f) IL-20R1 CRISPR-knockdown plasmid and Nrf2 inhibitor ML385 abolished the effects of rIL-19 treatment on the upregulation of the proposed signaling pathway. Resident microglia and peripheral macrophages express the same set of protein markers, making it difficult to distinguish in CNS [41]. We will refer to resident and recruited  [11,42]. Three days post-GMH, we demonstrated an increase in the number of CD11b + cells, which is consistent with previous studies [43]. This basic pathological process provides a prerequisite for further research on hematoma clearance in GMH. Scavenger receptor CD163 is primarily expressed on M2 microglia/macrophages and plays an important role in phagocytosis [14,44]. Increased expression of endogenous CD163 at the 72hour time point of the time-course study may be the result of the change in the M1/M2 ratio. Hemoglobin (Hb), which is released from metabolized erythrocytes, binds to haptoglobin (Hp) forming a Hb-Hp complex [45]. This complex is

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Oxidative Medicine and Cellular Longevity recognized by CD163, which results in the engulfment of the hematoma [46,47]. Thus, targeting CD163 may be beneficial for blood clot clearance after GMH. IL-19 has been previously reported to play an antiinflammatory role in various disease models [48][49][50]. Yet its role in the GMH pathophysiology remains to be elucidated. The IL-19 has been shown to be expressed on various immune cells such as monocytes, B cells, and T cells [51,52]. Interestingly, IL-19 binds with IL-20 receptor type 1, which specifically contains the IL-20R1 subunit [53]. Like IL-19, we demonstrated that IL-20R type 1 was also expressed on microglia/macrophages after GMH. During the time-course experiments, we saw an inconsistent tendency between IL-20R1 and IL-20R2 after GMH, which may be related to the IL-20R type 2. It is reported that IL-19 has a higher affinity to IL-20R type 1, since IL-19 showed higher affinity to bind IL-20R2 subunit than other ligands such as IL-20 [54].
Following the binding of IL-19 with IL-20R1 and IL-20R2, the cytoplasmic tail of IL-20R1 directly initiated the phosphorylation of ERK1/2 [18] or indirectly stimulated the phosphorylation of the molecule through the JAK/STAT signaling pathway [55,56]. Mounting evidence suggest that phosphorylated ERK may stimulate the expression of Nrf2, thereby blocking the Nrf2-binding domain with Keap-1 [57,58]. Nrf2 then enters into the nucleus to increase the transcription activity of CD163 [59,60]. Experiment 4 demonstrated the synchronization IL20R1/ERK/Nrf2/CD163 signaling pathway in sham, vehicle, and rIL-19 treatment groups. To study the rIL-19 mechanism of action on hematoma clearance, we evaluated its effects on the IL-20R1/ERK/Nrf2 signaling axis with the use of inhibitors. IL-20R1 CRISPR-knockdown plasmid ameliorated the therapeutic effects of rIL-19 on hematoma clearance, which effected the expression of CD163 and Nrf2. Nrf2 inhibitor ML385 also  This study demonstrated that rIL-19 promoted the hematoma clearance after GMH through the activation of IL-20R1/ERK/Nfr2 signaling pathway. Although we present compelling evidence, there are some limitations to this study. First, we only tested the expression of proteins and did not measure the gene activity of each target molecule. Second, we did not measure the concentration of rIL-19 in serum or brain tissue. Third, it is possible that there are other proteins involved in hematoma clearance, and the present experimental result may be the part of a larger pathway. Lastly, rIL-19 and ERK have been shown to have anti-inflammatory and proregeneration effects in other models [61]. Because of this, rIL-19 promotion of hematoma clearance cannot be entirely attributed as the sole cause of neuroprotection after GMH.

Conclusion
In this current study, we demonstrated that rIL-19 treatment attenuated neurological deficits and promoted hematoma clearance after GMH in neonatal rat pups. The protective effects were mediated through the activation of IL-20R1/ERK/Nrf2 signaling pathway. Our study is the first to demonstrate rIL-19 effects on hematoma clearance, providing new insight for noninvasive therapeutic strategies for the management of GMH.

Data Availability
The data support the findings of this study and are available from the corresponding author upon reasonable request.

Conflicts of Interest
The authors declare no conflicts of interest.

Authors' Contributions
SPL and JF worked on the experimental design. SPL, GZ, SXD, and JP conducted the experiments and analyzed the data and the manuscript. BL and JF worked on the data interpretations and manuscript preparation. JPT and JHZ participated in the experimental design, data analysis and interpretation, and manuscript preparation. All authors have read and approved the final manuscript.