Secukinumab attenuates neuroinflammation and neurobehavior defect via PKCβ/ERK/NF-κB pathway in a rat model of GMH.

AIMS
Germinal matrix hemorrhage (GMH) is a disastrous clinical event for newborns. Neuroinflammation plays an important role in the development of neurological deficits after GMH. The purpose of this study is to investigate the anti-inflammatory role of secukinumab after GMH and its underlying mechanisms involving PKCβ/ERK/NF-κB signaling pathway.


METHODS
A total of 154 Sprague-Dawley P7 rat pups were used. GMH was induced by intraparenchymal injection of bacterial collagenase. Secukinumab was administered intranasally post-GMH. PKCβ activator PMA and p-ERK activator Ceramide C6 were administered intracerebroventricularly at 24 h prior to GMH induction, respectively. Neurobehavioral tests, Western blot and immunohistochemistry were used to evaluate the efficacy of secukinumab in both short-term and long-term studies.


RESULTS
Endogenous IL-17A, IL-17RA, PKCβ and p-ERK were increased after GMH. Secukinumab treatment improved short- and long-term neurological outcomes, reduced the synthesis of MPO and Iba-1 in the perihematoma area, and inhibited the synthesis of proinflammatory factors, such as NF-κB, IL-1β, TNF-α and IL-6. Additionally, PMA and ceramide C6 abolished the beneficial effects of secukinumab.


CONCLUSION
Secukinumab treatment suppressed neuroinflammation and attenuated neurological deficits after GMH, which was mediated through the downregulation of the PKCβ/ERK/NF-κB pathway. Secukinumab treatment may provide a promising therapeutic strategy for GMH patients.


Introduction
Germinal matrix hemorrhage (GMH) is a devastating clinical event for neonates, especially for preterm or low-birthweight infants [1,2]. Its peculiarity of high morbidity and mortality brought heavy burden to the society and survivor's family [3]. It could be predictable that the morbidity would increase as a result of the delivery of higher numbers of premature newborns, due to the improvements of diagnostic tools, reproductive technology and intensive care strategies [4]. Blood components from the ruptured immature vessels are the primary cause of acute and secondary brain injury [1]. Hence, survivors suffer from debilitating consequences, such as neurological de cits, post-hemorrhagic hydrocephalus and developmental delay [5,6]. However, there is no effective management for this severe disease other than prophylactic medications and surgical shunts with signi cant side effects [7]. Therefore, more studies are urgently needed to investigate innovative therapeutic modalities.
The activation of in ammation cascades plays a critical role during the pathological process and aggravates neurobehavioral de cits after GMH [8][9][10]. Meanwhile, some in ammatory markers can be seen elevated in the severely hemorrhagic brain [11]. Inhibition of neuroin ammation is expected to be an effective method to reduce brain injury induced by GMH. Microglia, the main local immune cells, produces in ammatory cytokines and chemokines to elicit neuroin ammation responses [12,13]. Mounting studies uncovered that microglia function as an important in ammatory mediator between secondary brain injuries after hemorrhage [14][15][16].
Secukinumab, a recombinant monoclonal antibody [32], speci cally inhibits IL-17A. It is currently used as a treatment for some chronic immune diseases, such as psoriasis and arthritis [33,34].
Thus, based on the evidence above, we hypothesized that the inhibition of IL-17A, via secukinumab, would potentially reduce neuroin ammation through IL-17RA/PKCβ/ERK/NF-κB pathway in microglia, thereby improving short-term and long-term neurological outcomes after GMH in rats.

Animals and GMH model
All procedures were approved by the Institutional Animal Care and Use Committee at Loma Linda University, and in accordance with United States Public Health Service Policy on Human Care and Use of Laboratory Animals.
A total of one hundred and fty Sprague Dawley (SD) rat pups were purchased from Envigo (Livermore, CA, USA). After arrival, rat pups were housed with their dam and littermates until sacri ce or weaning. GMH surgeries were performed on P7 pups since their brains are comparable to 30-32 weeks of human gestational age. After weaning, they were separated into different cages according to the gender. All animals were housed with controlled appropriate humidity and temperature and a 12h light/dark cycle. All animals had free access to water and food. GMH model was conducted by collagenase as described previously [35]. Brie y, GMH pups were xed on a stereotaxic platform after being anesthetized with 2%-3% iso urane (mixed with oxygen gas and air). A 10µl syringe (Hamilton Co., Reno, NV, USA) was used to inject 0.3 units of collagenase VII-S (Sigma, ST Louis, MO, USA) at the point of 1.6mm right, 1.5mm rostral and 2.7mm deep from the bregma guided by a microinfusing pump (Hamilton Co., Reno, NV, USA). After the injection, the needle remained in place for 5 minutes and was withdrawn over additional 5 minutes to prevent leakage. Then the burr hole was sealed with bone wax and skin was sutured. The respiratory rate, heart-beat, color of skin, myodynamia and body temperature were monitored before recovery from anesthesia. All the pups were placed back to their mother and littermates until sacri ce. Sham pups underwent the same surgical procedures without collagenase infusion.

Experimental design and groups
Both male and female rat pups were randomly divided into the following experimental groups: sham, GMH, GMH + vehicle, GMH + secukinumab, GMH + secukinumab + PMA, GMH + secukinumab + Ceramide C6, and GMH + secukinumab + DMSO. Figure 1 demonstrated the experimental design. Experiment 1. To detect the time course of IL-17A, IL-17RA, PKCβand p-ERK, and to con rm the cellular localization of IL-17RA after GMH. Western blot (WB) was used to analyze the expression of these aforementioned proteins in the whole brain at 6h, 12h, 24h, 3d and 7d after GMH. Immuno uorescence was used to con rm IL-17RA expression on microglia at 24h after GMH. Experiment 2. To investigate the effects of secukinumab on short-term and long-term neurobehavior. Negative geotaxis and righting re ex were employed to test short-term neurobehavior de cits, while Morris water maze, rotarod, and foot fault were employed to assess long-term neurobehavior de cits. Experiment 3. To observe the effects of secukinumab on expression of in ammatory markers.

Neurobehavioral assessment
Negative geotaxis and righting re ex were employed to exam the short-term neurobehavior while water maze, foot fault and rotarod tests were used for long-term neurobehavioral assessment as previously reported from our laboratory [8,37]. All of the tests were executed by two researchers who were double blinded to the experimental groups.

Negative Geotaxis
A rough ramp was adjusted to an angle of 45 degrees. Experimental pups were put on it in a downward orientation and they were expected to adjust to an upward position. The time it took the pups to correct their bodies parallel to the ground plane (90 degrees) and upside down (180 degrees) were recorded. 60 seconds was recorded when pups rolled from ramp or corrected time was over 60 seconds. Each test was repeated three times and the average result of three consecutive days was taken for statistical analysis.

Righting Re ex
Pups were placed supinely on a horizontal plate and the time of automatic rollover was recorded within 20 seconds. Every pup was also examined three times as in negative geotaxis.

Morris Water Maze
Morris water maze was performed to evaluate cognitive function between 22 and 27 days post hemorrhage as previously described [38,39]. Brie y, after the training block, juvenile rats were placed in a round pool (1.10 m in diameter) lled with suitable temperature and opacity water, in which a platform (10cm in diameter) was located at 1cm below the water level. The time and distance it took for the animals to nd platform were recorded over four blocks. Then the platform was removed. A probe trial was performed to measure the percentage of swimming time in the quadrant where the platform was located.

Rotarod and foot fault test
Rotarod and foot fault tests were conducted to detect the motor and proprioception function post hemorrhage as previously described [9,40]. After training, juvenile rats were placed on a rotor, which is 70 mm in diameter, and they must walk forward to maintain their balance. The test started at 5 RPM or 10 RPM and increases by 2 RPM every 5 seconds. The time before the rats fell was recorded by the machine (Columbus Instruments, USA). For the foot fault test, juvenile rats were placed on a shelf with wire grills (0.6×1.5 m, 2.5 cm apart) to move freely and the numbers of missteps were recorded within 1 minute.

Statistical analysis
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 ± SD 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. P values of< 0.05 were considered statistically signi cant. GraphPad Prism 7 (La Jolla) were used for graphing and analyzing all the data. 3.2 IL-17RA is expressed on microglia both in sham and GMH pups. Double immuno uorescence staining was used to determine the cellular localization of IL-17RA on microglia (Iba-1) at 24 hours after GMH. The results indicated that IL-17RA was expressed on microglia in both sham and GMH animals (Figure 3).

Intranasal administration of secukinumab improved
short-term behavioral outcomes at 1h and 6h after GMH surgery.
In a previous study, we con rmed the best dosage (0.6mg/kg) for treatment of GMH[36]. Three different time points (1h, 6h, 12h) were chosen to optimize treatment. Hence, the following animal groups were used for the time-response study: Sham, GMH + vehicle, GMH + secukinumab (0.6mg/kg, 1h), GMH + secukinumab (0.6mg/kg, 6h), and GMH + secukinumab (0.6mg/kg, 12h). Compared to sham, each treatment group had signi cant short-term neurological impairment at 24h after GMH. At 48h and 72h after GMH, the 1h and 6h time-point groups had statistically higher score in both negative geotaxis (90 and 180 degrees) and righting re ex tests ( Figure 4, A-C). Additionally, at 72h after GMH, the 12h timepoint group had statistically higher score in righting re ex as well (Figure 4, C). Based on the experimental evaluation results above, the 1h time point treatment was selected for further research.
IL-1β and MPO were chosen as the indicators of neuroin ammation and immuno uorescence staining was used to count the numbers of microglia. At 24 h after GMH, there were signi cantly increased microglia activation compared to sham animals. Expression of IL-1β and MPO had the same trend as Iba-1 ( Figure 5, A-F). However, immuno uorescence staining showed that secukinumab signi cantly reduced the numbers of Iba-1, IL-1β, and MPO-positive cells in the perihematomal area compared to those in the GMH + vehicle group ( Figure 5, A-F).
3.5 Secukinumab improved long-term behavioral outcomes while PMA or Ceramide C6 abolished its bene cial effects after GMH.
To assess the effects of secukinumab on long-term neurological function (memory and motor skills), neurobehavioral tests were performed between 22nd and 28th days after GMH. For six consecutive days, Morris water maze was performed to evaluate memory function starting at 22 days after GMH. After training, the water maze test was performed every day over a period of four days (Day 1 to Day 4). Animals from different experimental groups began with comparable escape latency and swim distance on Day 1. On Day 2, we observed that all GMH animals, including vehicle and different treatment groups, showed more swim distance (Figure 6, A) and more escape latency (Figure 6, B). However, on Day 3 and Day 4, secukinumab treated group took signi cantly less time and distance to nd the platform compared to the vehicle group ( Figure 6A, B). However, the bene cial effect of treatment was abolished by PMA and Ceramide C6 interventions. There was no statistical difference in the averaged swimming speed of each group (data not shown). A Probe Trial following Day 4 showed that the vehicle-treated group spent signi cantly less time in the target quadrant compared to sham. PMA and ceramide C6 treated animals had similar outcomes compared to the vehicle group, while secukinumab treated animals spent more time in the target quadrant (Figure 6. G). Rotarod and foot fault tests were conducted to evaluate the motor ability on the 28th day after GMH. Vehicle-treated animals had much shorter falling latency on both 5rmp and 10rmp accelerating rods compared to the sham animals. The secukinumabtreated group had shorter latency than the vehicle-treated group (Figure 6, C), and the improvement was abolished by the interventions of PMA and ceramide C6. Meanwhile, the number of total missteps from the vehicle-treated group within 1 minute was more than that of sham. Secukinumab treatment partially normalized the motor function ( Figure 6, E). Yet, the same reversing effect was found with PMA and ceramide C6 treatments. Thus, secukinumab restored the motor function in GMH rats and the activators of PKCβ and p-ERK reversed this effect. There was no statistical difference observed between male and female rats (data not shown) in these tests.

PKCβ activator PMA and p-ERK activator ceramide C6 abolished the anti-in ammatory effects of secukinumab.
Secukinumab inhibition of IL-17A and its downstream factors, such as PKCβ and p-ERK, play a crucial role in expression of NF-κB that leads to suppressed neuroin ammation after GMH. Thus, we evaluated the effects of the treatments on the expression of IL-17RA, PKCβ, p-ERK, NF-κB and in ammation markers, IL-1β, TNF-αand IL-6, at 24 hours post GMH (Figure 7). The expression of IL-17RA, PKCβ, p-ERK, NF-κB, IL-1β, TNF-αand IL-6 increased after GMH. Except for IL-17RA, all these protein expression levels decreased with secukinumab treatment. PMA signi cantly activated the expression of PKCβ at 24 hours after intranasal administration. Western blot outcomes also showed that PMA signi cantly increased phosphorylated ERK and the expression of NF-κB, which was accompanied by increase of in ammation factors, IL-1β, TNF-αand IL-6, at 24 hours after GMH. Furthermore, the results demonstrated that the activator of p-ERK also abolished the effects of secukinumab on the p-ERK and in ammation factors in a similar manner to PMA (Figure 7). Secukinumab did not alter IL-17RA level. Neither did we observe any effect of ceramide C6 on PKCβ.

Discussion
Germinal matrix hemorrhage is devastating disease for neonates with no noninvasive treatments. In ammatory response contributes the brain injury after GMH and microglia play a critic role in mediating the process [9]. In this study, we rst investigated the role of IL-17A inhibitor secukinumab in neuroin ammation after GMH in rat pups and the potential mechanism involving the IL-17RA/PKCβ/ERK/NF-κB signaling pathway. The following outcomes were found. (a) Endogenous protein levels of PKCβ and p-ERK increased, which was accompanied by the increase of IL-17A and its receptor IL-17RA. (b) Intranasal administration of IL-17A antibody secukinumab at 1h after GMH signi cantly improved short-term neurological outcomes; reduced the expression of IL-1β, MPO and Iba-1 near the area of hematoma. (c) Secukinumab attenuated long-term neurobehavioral de cits after GMH. However, the respective activators of PKCβ and p-ERK, PMA and ceramide C6, abolished the treatment effects of secukinumab. (d) Secukinumab suppressed the expression of in ammatory factors after GMH, while PMA and ceramide C6 offset this phenomenon.
The function of IL-17A in the in ammatory cascade has been con rmed by mounting evidence [43,44]. IL-17 is secreted mainly by immune cells, such as Th17 cells and γδT cells [45][46][47]. In the event of brain injuries, these immune cells can be activated through various upstream stimuli [48,49]. Hence, it is deducible that endogenous IL-17A would increase after GMH. Secukinumab, a recombinant monoclonal antibody for IL-17A, is used as an anti-in ammatory treatment for some severe chronic immune diseases, such as psoriasis [33,34]. After GMH, microglia and macrophages recruited from the blood accelerate the release of in ammatory mediators, which contribute to the impairment of neurological behavior.
Consistent with the previous studies, our results showed that secukinumab decreased the expression of pro-in ammatory cytokine IL-1βand MPO in perihematomal areas of GMH, inhibited the activation of microglia, and improved the short-and long-term neurobehavioral outcomes. Also, we observed that secukinumab attenuated the elevated expression of NF-κB, IL-1β, TNF-α and IL-6 in the hemorrhaged brain as shown by Western blot. Interestingly, we noticed that, only early administration of secukinumab (1h) could attenuate the defected of neurological function after GMH. This result suggested that early in ammation response plays a very important role in secondary brain injury after GMH.
In CNS, IL-17RA is expressed on all kinds of resident cells, including microglia, astrocytes, neurons and endothelial cells [43,50,51]. IL-17RA signaling on microglia has been reported by many studies and became the target for drugs [43,52]. There was no intracellular enzymatic activity that has been found on IL-17RA and its complexes [53]. PKCβ, one of the protein kinases, performs its function of controlling other proteins via phosphorylating hydroxyl groups of serine and threonine amino acid residues on these proteins [25]. Under the in uence of PKCβ directly or indirectly, inactive ERK(1/2) changes into active p-ERK [24,25,54], which translocates to the nucleus to trigger the expression of in ammatory activator NF-κB [27,55]. Meanwhile, PKCβ and p-ERK have been reported to be associated with neuroin ammation in microglia [56,57].
In our research, we observed that IL-17A and its receptor increased along with PKCβ and p-ERK in the time course study. Also, the increased expression of PKCβ and p-ERK was attenuated by the treatment of IL-17A inhibitor secukinumab after GMH. To further explore the possible mechanism underlying secukinumab mediated anti-in ammtory response after GMH, PKCβ and p-ERK activator, PMA and ceramide C6, have been chosen. After being administered with secukinumab, both activators abrogated the decreased expression of in ammatory cytokines, IL-1β, TNF-α and IL-6. Meanwhile, consistent with the expression of these proteins, the same effects could be found on the long-term neurological behavior of juvenile rats after GMH as well. However, p-ERK activator Ceramide C6 had no effect on the expression of PKCβ. Therefore, we presumed p-ERK is the downstream protein of PKCβ. Taken together, our data suggested that secukinumab exerted its protective effect at least partially through PKCβ/p-ERK/NF-κB pathway.
Some limitations should not be ignored in this study and more unknown mechanisms need to be explored. First, brain injury has a very complex pathological process after GMH. Except neuroin ammation, other pathological processes also take place, such as the impairment of blood-brain barrier integrity, autophagy and neurogenesis. It is not clear whether they are associated with IL-17A and its pathway. Second, there may be other downstream pathways existing and playing a potential role.
Future studies are needed to fully elucidate the potential mechanisms associated with secukinumab in the setting of experimental GMH. Third, IL-17RA also expresses on other cells, including neurons, and its function need to be further investigated. Last, we have not elucidated whether the change in protein expression was attributed to genetic modi cation. Further studies on a molecular level would be needed.

Conclusion
In this current study, we demonstrated that secukinumab treatment attenuated neurological de cits and inhibited neuroin ammation after GMH in neonatal rat pups. The protective effects were mediated through the activation of PKCβ/ERK/NF-κB signaling pathway. Our study is the rst to demonstrate secukinumab effects on inhibition of neuroin ammation, providing new insight for non-invasive therapeutic strategies for the management of GMH.

Declarations
Authors' contributions SPL and SXD participated in the research design, experimental performance (including animal surgery, Western blotting, and immunohistochemistry, but not the neurobehavioral testing), data analysis, and drafting of the manuscript. YD and JJF provided technical assistance and help with the manuscript preparation. XLZ and XJJ discussed the results and edited parts of the manuscript. XH, JP and GZ performed the intracerebroventricular injection, co-immunoprecipitation, behavioral tests, and data analysis. JJF and JHZ participated in the research design and edited the manuscript. YG and JPT are the corresponding authors; these authors participated in all aspects of the study, including research design, data analysis, and manuscript preparation. The authors read and approved the nal manuscript.

Availability of data and materials
The datasets used and/or analyzed in the current study are available from the corresponding authors on request.