Secukinumab attenuates reactive astrogliosis via IL‐17RA/(C/EBPβ)/SIRT1 pathway in a rat model of germinal matrix hemorrhage

Abstract Aims Reactive astrogliosis plays a critical role in neurological deficits after germinal matrix hemorrhage (GMH). It has been reported that interleukin‐17A and IL‐17A receptor IL‐17RA/(C/EBPβ)/SIRT1 signaling pathway enhances reactive astrogliosis after brain injuries. We evaluated the effects of secukinumab on reactive astrogliosis in a rat pup model of GMH. Methods A total of 146 Sprague Dawley P7 rat pups were used. GMH was induced by intraparenchymal injection of collagenase. Secukinumab was administered intranasally 1 hour post‐GMH. C/EBPβ CRISPR or SIRT1 antagonist EX527 was administrated intracerebroventricularly (icv) 48 hours and 1 hour before GMH induction, respectively. Neurobehavior, Western blot, histology, and immunohistochemistry were used to assess treatment regiments in the short term and long term. Results The endogenous IL‐17A, IL‐17RA, C/EBPβ, and GFAP and proliferation marker CyclinD1 were increased, while SIRT1 expression was decreased after GMH. Secukinumab treatment improved neurological deficits, reduced ventriculomegaly, and increased cortical thickness. Additionally, treatment increased SIRT1 expression and lowered proliferation proteins PCNA and CyclinD1 as well as GFAP expression. C/EBPβ CRISPR activation plasmid and EX527 reversed the antireactive astrogliosis effects of secukinumab. Conclusion Secukinumab attenuated reactive astrogliosis and reduced neurological deficits after GMH, partly by regulating IL‐17RA/(C/EBPβ)/SIRT1 pathways. Secukinumab may provide a promising therapeutic strategy for GMH patients.


| INTRODUC TI ON
Germinal matrix hemorrhage (GMH) is one of the leading causes of mortality and morbidity in preterm and low-birthweight infants, occurring in approximately 3.5 per 1000 births in the United States. 1,2 The germinal matrix contains various thin-walled blood vessel, which leaves them susceptible to spontaneous rupture due to respiratory and hemodynamic fluctuations seen in preterm infants. [3][4][5] Currently, there is no effective therapeutics in the management of GMH, 6 and new treatment strategies are warranted.
Although previous studies have shown that reactive astrogliosis (also known as astrocyte activation) is necessary for poststroke CNS repair, [7][8][9] recent studies have demonstrated that reactive astrogliosis plays a pivotal role in neurological injury in CNS injury models such as hemorrhagic stroke. [10][11][12][13] Reactive astrogliosis is characterized by astrocyte hypertrophy and astrocyte proliferation, which have been shown to contribute to glial scar formation. 14 Despite glial scarring being necessary to seal the site of injury and protect damaged neural tissue, this repair mechanism inhibited the regrowth of damaged axons which contributed to further neurological deficits brought on by CNS injury. 15,16 Interleukin-17A and its receptor IL-17R play a significant role in inflammation and BBB breakdown after stroke. 17 IL-17 is predominantly produced by various immune cells such as natural Th17 cells, γδT cells, T-helper cells, and innate lymphoid cells. 18 After hemorrhagic stroke, there is an increased level of IL-17A, one of six subtypes of the IL-17 family (IL-17A to IL-17F) within the CNS. 19 IL-17 receptor (IL-17R) has five subtypes which include IL-17RA to IL-17RE, where IL-17A and IL-17F can bind to IL-17RA. IL-17RA is found on various cell types in the CNS such as microglia, neurons, and astrocytes. [20][21][22] The CCAAT/enhancer binding proteins β (C/EBPβ), a transcription factor, has been reported to be activated by IL-17RA/ ACT1/TRAF6 signaling pathway. 17,23 Increased C/EBPβ expression has been shown to suppress the expression and activity of silent information regulator 1 (SIRT1). [24][25][26] Previous studies have demonstrated that SIRT1 plays an essential role in cell proliferation and cell survival. 27 SIRT1 attenuates reactive astrogliosis by targeting nuclear factor ƙB (NF-ƙB) 28,29 and signal transducing activator of transcription 3 (STAT3). 30 Thus, IL-17A activation of the IL-17RA/C/EBPβ signaling pathway may play a role in reactive astrogliosis, whereas SIRT1 signaling pathways attenuate reactive astrogliosis after GMH.
Secukinumab, a recombinant monoclonal antibody, selectively targets IL-17A, is currently used as a treatment for severe chronic immune diseases that are associated with IL-17A. 31,32 In this present study, we hypothesized that the inhibition of IL-17A, via secukinumab, would attenuate C/EBPβ inhibitory effects on SIRT1 resulting in the reactive astrogliosis, thereby improving short-and long-term neurological deficits 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 the National Institute of Health guidelines for the treatment of animals.
Timed pregnant Sprague Dawley (SD) rats were purchased from Envigo (Livermore). The rat pups were housed with dams until sacrificed (short-term time points) or weaned (long-term time points); after the weaning process, each cage housed two rats of the same gender. All rats were housed in a controlled humidity and temperature room with a 12-h light/dark cycle and were raised with free access to water and food. A total of 146 P7 rat pups (brain development comparable to 30-32 weeks of human gestation) were used. GMH was induced by stereotactic-guided injection of bacterial collagenase as previously described. 33 Rat pups were fixed on a stereotaxic plate while anesthetized with 2%-3% isoflurane (mixed with oxygen gas and air). A 10-μL Hamilton syringe (Hamilton Co.) was used to inject 0.3 units of collagenase VII-S (Sigma) at stereotactic coordinates from bregma of 1.6 mm (right lateral), 1.5 mm (rostral) and 2.7 mm (depth) from the dura.
After injection, the needle remained for 5 minutes and then was withdrawn slowly for an additional 5 minutes to minimize potential "back-leakage." The burr hole created for the insertion of the Hamilton syringe was sealed with bone wax and the incision site sutured. Respiration, heartbeat, skin color, myodynamia, and body temperature were monitored before and after recovery from anesthesia. After recovery, rat pups were placed back with the dam.
Sham animal groups were subjected to needle insertion without collagenase injection. 33

| Animal experimental groups and treatments
Male and female SD rat pups were randomized into experimental and sham groups. The animal groups and experimental design are shown in Figure 1. Each animal group had zero mortalities during these experiments.

| Neurobehavioral assessments
Short-term behavior (24, 48 and 72 hours postictus) was evaluated using righting reflex and negative geotaxis (at 90° and 180° inclined surface) as previously described. 33 Morris water maze was used to evaluate spatial learning and memory at 24-28 days postictus, and motor function was assessed by foot fault test and Rotarod at 28 days postictus as previously described. 33 Neurobehavioral function was evaluated in a blinded manner.

| Animal perfusion and tissue extraction
Animals were euthanized using isoflurane (≥5%) which was followed by transcardiac perfusion with ice-cold phosphate-buffered saline (PBS) for Western blot or ice-cold PBS followed by 10% formalin for histology samples.

| Western blotting
Ipsilateral forebrain samples were extracted from experimental rat pups for Western blot (WB) samples as previously described. 34 Samples were homogenized in RIPA lysis buffer (sc-24948, Santa Cruz, USA) followed by centrifugation at 14 000 for 30 minutes; once finished, the supernatant was removed from the remainder of the sample. Protein concentrations for immunoblotting were determined by detergent-compatible protein assay (Bio-Rad). ; and anti-goat (1:5000, Santa Cruz, USA). The membrane was then exposed to radiography films to display the protein bands. Lastly, the density of bands was analyzed for the relative density of the resultant protein immunoblot by ImageJ software (NIH). 35
Immunofluorescence staining was performed as previously described. 34 Sections were stained with primary antibodies of IL-17RA

| Nissl staining
Nissl staining was performed as previously described in Ref. 36 to examine the relative cortical thickness and ventricular volume. 36,37

| Statistical analysis
Data are expressed as mean ± standard deviation and were analyzed by GraphPad Prim 7.0 software (GraphPad Software Inc). One-way ANOVA on ranks was performed to compare the difference among each group and followed by the Tukey multiple comparison post hoc test. P-value < 0.05 was considered statistically significant.

| Time course study: IL-17A, IL-17RA, SIRT1, CyclinD1, and GFAP expression after GMH
The endogenous protein levels were evaluated by Western blot at 0, 12, 24, 72 hours, and 5 and 7 days post-GMH. Endogenous IL-17A expression was significantly increased at 72 hours, and 5 and 7 days after GMH (Figure 2A,B). Endogenous IL-17RA was significantly increased at 24 hours and peaked at 5 days post-GMH ( Figure 2A,C). The endogenous expression of SIRT1 was significantly decreased at 24 hours after GMH (Figure 2A,D), and proliferation marker CyclinD1 expression was significantly increased at  (Figure 2A,E). GFAP significantly increased at 24 hours after GMH and remained elevated (Figure 2A,F). Based on these results, the 5-day time point was chosen to study the mechanism of action.
Immunofluorescence staining was used to determine the cellular localization of IL-17RA in the CNS at 72 hours after GMH. IL-17RA was found to be colocalized with GFAP, NeuN, and Iba1 at the site of perihematoma (Figure 2G-J). These results indicate that IL-17RA is expressed on astrocyte, neuronal, and microglia cells.

| Secukinumab improved short-and long-term neurological function after GMH
Short-term neurological function was examined in the following animal groups for the best dose-response study: sham, GMH + vehicle, GMH + secukinumab 0.3 mg/kg, GMH + secukinumab 0.6 mg/kg, and GMH + secukinumab 0.9 mg/kg animal groups.
GMH rats had significantly short-term neurological deficits compared to shams. At 24 hours, all groups had significant neurological impairment in all neurobehavioral tests when compared to sham ( Figure 3A,B). However, secukinumab at dosages of 0.6 mg and 0.9 mg/kg significantly improved short-term neurological outcomes at the 2 and 3 days after GMH (P < 0.05, Figure 3A,B). Based on the short-term neurological function outcomes, 0.6 mg/kg was chosen as the best dose and is used for the following experiments.
For seven consecutive days, Morris water maze was performed to evaluate spatial learning and memory starting at 24 days after GMH.

| Secukinumab reduced ventricular volume and preserved cortical thickness after GMH
Nissl staining was performed to evaluate ventricular volume and relative cortical thickness at 28 days after GMH. Compared with sham, the vehicle group had significantly enlarged ventricles and decreased cortical thickness, while secukinumab-treated group significantly attenuated ventricular dilation and preserved cortical thickness ( Figure 5).

| Secukinumab reduced astrogliosis around the site perihematoma after GMH
At 72 hours postictus, astrocyte proliferation was accessed by immunofluorescence to determine the effect of the treatment.

| D ISCUSS I ON
In this present study, we first time investigated the therapeutic effects of secukinumab against reactive astrogliosis by inhibiting  lead to glial scar formation. 7,38 Reactive astrogliosis has been shown to be a byproduct of stroke, causing changes to the brain tissue, blood vessel, CNS microenvironment, and disrupting neurotransmissions. 14 During the acute phase of CNS injury, the proliferation of astrocytes occurred which contributed to cell death, neovascular remodeling, multicellular inflammation, loss of axons and synapse in perilesion, and loss of oligodendrocytes. 38 Astrocytes play a primary role in the production of inflammatory cytokines, chemokines, and ROS, 13 leading to the inhibition of oligodendrocyte progenitor differentiation and hydrocephalus, resulting in secondary brain injury. 39 Then newly proliferated and hypertrophic astrocyte cells, fibroblast-linage cells and inflammatory cells consisted of astrogliosis scar formation under modulating by intrinsic signaling transduction such as STAT3 and NF-ƙB pathway. 38,40,41 After the acute phase of brain injury, scar formation attenuated neuronal regeneration, blocked axon prolongation, inhibited synapse remodeling, and reduced the thickness of ipsilateral cortex, 14,15 leading to long-term neurobehavioral deficits.
Additionally, glial scarring blocks the circulation of cerebrospinal fluid (CSF) and contributed to the enlargement of ventricles, which impaired neurological function. 42 Thus, targeting reactive astrogliosis may be beneficial for the treatment of GMH.
IL-17A, secreted by immune cells, 43,44 plays a vital role in the stroke pathophysiology. In addition to its pro-inflammatory effect, IL-17A has been shown to promote hyperproliferation and differentiation of various cell types; the most notable are Th17 cells and γδT cells due to their production of IL-17A. 17,[45][46][47][48] After hemorrhagic stroke, macrophages become activated and secret IL-23, which promotes the activation of γδT cells and Th cells. 18 Due to the blood-brain barrier (BBB) rupture after hemorrhagic stroke, 49 IL-17A enters into the brain tissue through the serum. 50 The expression of IL-17A has been shown to increase in both the serum and brain tissue after hemorrhagic stroke, 50,51 We observed increased IL-17A receptor (IL-17RA) expression on astrocytes, in concurrence to previous reports. 52 IL-17 receptor (IL-17RA) and its complexes have no intracellular enzymatic activity but implement their function by protein-protein signal transmission. 53 The most notable pathway is Act1 (one of two IL-17RA complexes) activation of C/EBPβ. 17,53 Various articles indicate that C/EBPβ is expressed in astrocytes and a plays role in inflammation, cell proliferation, and metabolism. 24,54 After activation, C/EBPβ is rapidly transported to the nucleus and binds to HDAC1.
Consequently, the C/EBPβ-HDAC1 complex was found to negatively regulate SIRT1 expression and activity. 24,25 SIRT1 plays a significant role in the deacetylation of NF-ƙB, 29,55 which resulted in a decrease in proliferation proteins such as CyclinD1 and PCNA. 29,56 Additionally, SIRT1 has been shown to downregulate STAT3 30 and reduced reactive astrogliosis. 57 In this study, we observed a significant reduction in endogenous SIRT1 expression along with an increase in cell proliferation marker CyclinD1 after GMH. The time course of these markers showed a similar trend to that of IL-17A and IL-17AR, suggesting F I G U R E 7 Secukinumab reduced astrocyte scar formation at 7 d after GMH. A, Repetitive images of GFAP staining. B, indicates the photographed areas. C, Quantitative analysis of area density of GFAP staining. Scale bar = 50 µm, * P < 0.05 vs sham, @ P < 0.05 vs GMH+ vehicle, mean ± SD, one-way ANOVA, Tukey's test, n = 6/group. DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; GMH, germinal matrix hemorrhage that IL-17A/IL-17AR/SIRT1 signaling pathway may play a role in the regulation of reactive astrogliosis after GMH. We also observed that secukinumab, which selectively targets IL-17A, 32 significantly improved short-and long-term neurological deficits induced by GMH.
These behavioral benefits were associated with reduced ventricular dilation, decreased astrocyte activation and proliferation, and reduced glial scar tissue formation around the site of perihematoma.
To study the secukinumab mechanism of action, we evaluated its effects on the (C/EBPβ)/SIRT1 axis. C/EBPβ CRISPR activation plasmid ameliorated the therapeutic effects of secukinumab on astrocyte proliferation and activation, which was associated with increased expression of C/EBPβ, CyclinD1, PCAN, and GFAP, and decreased SIRT1 expression. SIRT1 inhibitor EX527 also reversed the effect of secukinumab. This study demonstrated that secukinumab attenuated reactive astrogliosis after GMH through the attenuation of the IL-17RA/(C/EBPβ) signaling pathway, removing the inhibitory effects on SIRT1.
Second, we did not measure the concentration of secukinumab in serum or brain tissue. Third, IL-17RA was also expressed in neurons and microglia. Secukinumab effects on these cell types need further investigation. Lastly, secukinumab and SIRT1 have been shown to be anti-inflammatory in several CNS injury models. 58,59 Because of this, secukinumab's reduction of reactive astrogliosis cannot be entirely attributed as the sole cause of neuroprotection in GMH-treated animals.

| CON CLUS ION
In the current study, we demonstrated that secukinumab treatment attenuated neurological deficits and reactive astrogliosis after GMH in rat pups. The protective effects were mediated by the inhibition of IL-17RA/(C/EBPβ) signaling pathway. Our study is the first to demonstrate secukinumab effects on glial scarring, providing new insight for therapeutic strategies for the management of patients with GMH.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.