MFG-E8 attenuates neuro-inflammation in subarachnoid hemorrhage through driving microglial M2 polarization via modulating Integrin β3/SOCS3/STAT3 signaling pathway CURRENT STATUS: POSTED

Background Increasing evidence suggests microglial polarization plays an important role in the pathological processes of neuro-inflammation following subarachnoid hemorrhage (SAH). Previous studies indicated that milk fat globule-EGF factor-8 (MFG-E8) has the potential in anti-apoptosis and anti-inflammation in cerebral ischemia. However, the effects of MFG-E8 on microglial polarization have not been evaluated after SAH. Therefore, the aim of this study was to explore the role of MFG-E8 on anti-inflammation, and its potential mechanism on microglial polarization following SAH. Methods We established the SAH model via prechiasmatic cistern Blood injection in mice. Double-immunofluorescence staining, Western blotting and quantitative real-time polymerase chain reaction (q-PCR) were performed to investigate the expression and cellular distribution of MFG-E8. Two different dosages (1 μg and 5 μg) of recombinant human MFG-E8 (rhMFG-E8) were injected intracerebroventricular (i.c.v.) at 1 h after SAH. Brain water content, neurological scores, beam-walking score, Fluoro-Jade C (FJC) and terminal deoxynucleotidyl transferase dUTP nick endlabeling staining (TUNEL) were measured at 24 h. Intervention of MFG-E8, integrin β3 and phosphorylation of STAT3 were achieved by specific siRNAs (500 pmol/5 µl) and STAT3 inhibitor Stattic (5 µM). The potential signal pathway and microglial polarization were measured by immunofluorescence labeling and Western blotting. polarization integrin β3/SOCS3/STAT3 neuroprotection MFG-E8. rhMFG-E8: recombinant human milk fat globule-EGF factor-8SAH: subarachnoid hemorrhage; EBI: early brain injury; CNS: central nervous system; CSF: Cerebrospinal fluids; DAMP: Damage-associated molecular pattern; IL-1β: Interleukin 1β; IL-6: Interleukin 6; TNF-α: Tumor necrosis factor-α; IL-10: Interleukin 10; iNOS: inducible nitric oxide synthase; TFG-β: transforming growth factor; EGF: epidermal factor; FAK:

composed of epidermal growth factor (EGF)-like sequences and discoidin domain-containing protein 1 (SED-1) [12]. This protein is widely distributed in various tissues of mammalian species including humans. Given its role of a bridging molecule between apoptotic cell and macrophages, MFG-E8 facilitate clearance of pro-inflammatory mediators to prevent secondary injury [13]. Recently, its role attracts more and more attentions by researchers in CNS. Normally, MFGE8 is mainly upregulated in microglia during several pathological process, also small amount of it expressed in astrocytes and neurons [14,15]. MFG-E8 participates in regulating immune responses through inhibiting the release of pro-inflammatory mediators to protect against ischemic cerebral injury [13]. Further, MFG-E8 promote microglia reprograming to shift the microglia phenotype toward alternative (M2) activation [16]. Administration of MFG-E8 could alleviate pathological lesion of Alzheimer's disease (AD) via modulating the alteration of M1/M2 polarization [14]. Recently our laboratory have confirmed that recombinant human MFG-E8 (rhMFG-E8) improve neurological function in an animal model of traumatic brain injury (TBI) by inhibiting neuronal apoptosis [17]. These studies indicated that MFG-E8 could provide neuroprotection in CNS. However, whether MFG-E8 modulate the phenotypes and functions of activated microglia after SAH remains to be reported.
In the present work, we aimed to address the effect of MFG-E8 on microglia polarization, as well as its potential mechanism in a SAH model.

Animals preparation
Adult male C57BL/6J mice weighing 25-28 g were purchased from the Experimental Animal Center of Drum Tower Hospital. All experimental protocols and procedures for this study were approved by the Institutional Animal Care and Use Committee at Drum tower hospital and conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All mice were acclimated in a 12-h light/dark cycle room, and allowed free access to food and water under conditions of controlled humidity and temperature (24 ± 0.5 °C).

Models Of SAH And Experimental Design
Experimental SAH models used in this study were performed as previous study [18]. Briefly, mice Intracerebroventricular Administration Intracerebroventricular (i.c.v) drug administration was performed as previously described [18]. Briefly, mice were placed in a stereotaxic frame after inhalation anesthesia with isoflurane (2% in oxygen gas, 300 ml/min). The needle of a 10-µl Hamilton syringe (Shanghai Gaoge Industry & Trade Co., Ltd., Shanghai, China) was inserted into the left lateral ventricle through a burr hole using the following coordinates: 1.0 mm posterior and 1.5 mm lateral to the bregma, and 3.2 mm below the dural layer.

Quantitative Real-time Polymerase Chain Reaction
Quantitative real-time polymerase chain reaction (qPCR) was performed and analyzed as previously described [20]. Total RNA from brain tissues was extracted using TRIzol Reagents (Invitrogen Life Technologies, USA). RNA quality was insured by gel visualization and spectrophotometric analysis (OD 260/280 ). After reverse transcription, quantitative analysis of the MFG-E8, Integrin β3, IL-1β, IL-6, TNF-α and IL-10 mRNA expression were performed with the real-time PCR method and the primers were synthesized by ShineGene Biotechnology (Shanghai, China) (Additional file Table 1). Test cDNA results were normalized to β-actin. All samples were analyzed in triplicate.  Western blot results were analyzed using Un-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA).
Fluoro-jade C (FJC) Staining FJC staining (Merckmillipore, Germany) was performed according to the operation instructions and to detect degenerating neurons. Briefly, frozen sections were prepared, fixed, and immersed in a basic alcohol solution consisting of 1% sodium hydroxide in 80% ethanol for 5 min, then rinsed for 2 min each in 70% ethanol and distilled water and then incubated in 0.06% potassium permanganate solution for 10 min. Following a 1-2 min water rinse, the slides were transferred for 10 min to a 0.0001% solution of FJC dissolved in 0.1% acetic acid vehicle and then rinsed through three changes of distilled water for 1 min per change. The slides were air-dried, cover slips were applied, and the sections were visualized on an Image J software (Image J 1.4, NIH, USA). Two observer blinded to the experimental group counted the FJC-positive cells in six sections per brain (at 20 × magnification) through the injury's epicenter. The data were presented by the average number of FJC-positive neurons in the fields.

Brain Water Content
Brain water content was measured as previously study [22,23]. Brains were quickly removed at 24 h after SAH. The brainstem was discarded, while the tissue of left hemisphere cortex and right cortex were harvested, and weighted the wet weight of each cortical tissue, then dried for 72 h at 80 °C and the dry weight determined. The percentage of brain water content was calculated as the following formula = [(wet weight − dry weight)/wet weight] × 100%.

Neurologic Evaluation
The neurological deficits were evaluated as previously described modified Garcia scoring (Additional file and the response to vibrissae stimulation. Beam-waling texts were performed and including sevenpoint rating scale. All the tests were evaluated by two independent observer who was blind to the treatment conditions. Higher scores represented better neurological function.

Statistical Analysis
The SPSS 17.0 software package was used for the statistical analysis. All data are expressed as the mean ± Standard Deviation (SD). Comparisons between two groups were performed using Student's t test and multiple comparisons were performed using a one-way ANOVA followed by Tukey's test. A p value < 0.05 was regarded as statistically significant.

General observations and mortality rate
Out of the 217 surgeries in mice that were performed. The mortality rate of SAH group on rhMFG-E8, siRNA and Stattic treatment did not differ significantly from the SAH group and SAH + Vehicle group.
No rats died in Sham group 0% (0/38), and the overall mortality rate of SAH in mice was 17.97% (Additional file Table 3). There was no statistical difference in body weight and body temperature in any of the experimental groups (data not shown).  Treatment with rhMFG-E8 alleviated neuronal damage, brain edema and improved neurobehavioral outcome after SAH To investigate whether MFG-E8 had effect of neuroprotection after SAH, brain edema and neurological score were examined, which revealed that SAH induction aggravated brain water content and neurological impairments compared with sham group at 24 h. While administration two different dosages of rhMFG-E8 (1 µg and 5 µg) via intracerebroventricular (i.c.v.), brain water content was remarkably alleviated and neurological deficits was dramatically recovered compared with SAH + Vehicle group. Moreover, high dosage of rhMFG-E8 performed more effective on neuroprotection ( Fig. 2A-D). To evaluate whether MFG-E8 is relevant to modulate microglial polarization, the protein levels of M1associated marker (CD86) and M2-associated marker (CD206) were measured by Western blots and immunofluorescent staining. The results showed that the proportions of M1 and M2 phenotypes, revealed by Iba-1 + /CD86 + and Iba-1 + /CD206 + staining respectively, were upregulated after SAH at 24 h compared with sham group (Fig. 5A, D). While administration of rhMFG-E8 significantly decreased the number of Iba-1 + /CD86 + cell, and upregulated the ratio of Iba-1 + /CD206 + cell compared with the SAH + Vehicle group (Fig. 5B, E). Similarly, Western blot results showed that SAH induction increased the protein levels of CD86 and CD206 in SAH group and SAH + Vehicle group compared to sham group, while treated with rhMFG-E8 remarkably reduced the expression of CD86, meanwhile further increased the protein level of CD206 (Fig. 5C, F). Considering with these results above, we speculated that the property of MFG-E8 on anti-inflammation might involves microglial polarization, namely, declined the proportion of pro-inflammation M1 phenotype and amplified antiinflammation M2 polarization.

Knockdown MFG-E8 aggravated neuro-inflammation injury and induced M1 microglia activation
To confirm the effects of MFG-E8 on microglia phenotypic conversion process, MFG-E8 siRNA at the concentration of 500 pmol/5 µl were used injecting via i.c.v. 48 h before SAH. As shown in Fig. 6A, the number of Iba-1 + /CD86 + was significant increased after treatment with MFG-E8 siRNA at SAH group compared with SAH + rhMFG-E8 group. Meanwhile, Western blot results showed that MFG-E8 siRNA upregulated the expression of M1 markers (CD86, IL-6) relative to the SAH + rhMFG-E8 group (Fig. 6C-E). While the proportion of Iba-1 + /CD206 + was remarkably reduced (Fig. 6F, G) and the protein levels of M2 markers (CD206, IL-10) greatly attenuated (Fig. 6H-J), when compared with the SAH + rhMFG-E8 group. As expected, there was no statistical difference between the SAH + Vehicle group and SAH + Scramble siRNA group. Taken together, we concluded that rhMFG-E8 was involved in the regulation of microglial polarization process after SAH.
Knockdown integrin β3 and STAT3 inhibition exaggerated M1 microglia polarization and abolished the property of MFG-E8 on anti-inflammation after SAH STAT3, as an important transcription factor, SOCS3/STAT3 signaling pathway was involved in neuroinflammation and microglial polarization. Since phosphorylation STAT3 was mainly expressed in microglia, so we detected its phosphorylation level via double-immunofluorescence staining in microglia. The result showed that SAH induction significantly increased fluorescence intensity of p-STAT3 at SAH + Vehicle group, compared with sham group. Additionally, some of p-STAT3 translocated from cytoplasm to nucleus in Iba-1 + microglia at SAH + Vehicle group, while, it were significantly decreased underwent rhMFG-E8 and STAT3 inhibitor Stattic (5 µM) treatment.
Subsequently, intervention with integrin 3 siRNA enhanced the fluorescence intensity of p-STAT3 and promoted the protein transfer into nucleus of microglia (Fig. 7). This result suggested that MFG-E8 and integrin 3 receptor might be involved in the phosphorylation of STAT3 in microglia after SAH.
Correspondently, intervention of Stattic resulted in remarkably reduction of p-STAT3 (Fig. 8D), meanwhile the expression of M2 markers (IL10, CD206) elevated dramatically and the levels of M1 markers (IL-6, CD86) decreased in comparison with the SAH + rhMFG-E8 group. However, Stattic had no effect on the protein levels of integrin 3 and SOCS3 compared with the SAH + rhMFG-E8 group (Fig. 8B, C). These results suggested that rhMFG-E8 played an important role in M2 polarization, which might be through regulating integrin 3/SOCS3/STAT3 signal pathway.

Discussion
Previous study reported that neuro-inflammatory response was the main mechanism pathological damage of early brain damage following SAH, and regulating inflammatory response can effectively improve the prognosis of SAH patients [27]. In this study, we revealed the neuroprotective effect of rhMFG-E8, mainly through targeting the immunomodulatory functions in animal SAH models. The results showed that the expression of MFG-E8 was time-dependent increased at an early stage of SAH, and which was almost located in microglia and neurons cells. Exogenous rhMFG-E8 effectively attenuated brain edema and improved neurological deficits. rhMFG-E8 up-regulated the expression of we reported that the expressions of pro-apoptosis related proteins and the proportion of apoptotic neurons were attenuated in TBI model after treatment with rhMFG-E8, accompanied by improved neurological performance. This protection was mediated through activation of integrin β3/FAK/PI3K/AKT signal pathways [17]. All this studies indicated that MFG-E8 had potential therapeutic benefits in the brain. In this study, we found that the protein of MFG-E8 was positively coexpressed not only in microglia and but also in neuron following SAH, especially in Iba1 + microglia.
We also found that supplementation of rhMFG-E8 induced a significant reduction of brain edema and preserved neurological function. Correspondingly, down-regulation the expression of MFG-E8 via siRNA aggravated outcomes after SAH. Our results clearly indicated that there has a closely relationship between the level of MFG-E8 and severity of brain damage, and consistent with the other studies to reflect the neuroprotection effects of MFG-E8. . From these evidences, we anticipated that MFG-E8 might regulate the phosphorylation of STAT3 via integrin β3 receptor. Our study also discovered that increased rhMFG-E8 and integrin β3 expression correlate with attenuated inflammation, accompanied by increased the expression of SOCS3. Recent study showed that over-activation of STAT3 might due to the absence of SOCS3 on normal feedback mechanism following pathological stress. Our results were consistent with previous study to underline the possible mechanisms of rhMFG-E8 on anti-inflammation and M2 polarization [14]. In short, we speculated rhMFG-E8 might induce SOCS3 activation through interacting with integrin β3 and interfering with phosphorylation of STAT3 to reduce inflammation after SAH.
As far as we know, we demonstrated for the first time that MFG-E8 confers anti-inflammation and microglia polarization partly depending on the integrin β3/SOCS3/STAT3 signaling pathway following SAH. Additionally, our study mainly focused on the immunomodulation of rhMFG-E8 during the EBI of SAH. However, the long-term effect of rhMFG-E8 on microglia polarization have not been investigation, and should be studied in the future. Moreover, all of the intervention agents we used in our study were siRNAs and Stattic (STAT3 inhibitor). Therefore, the knockout mice need to be used in the further study to keep the data more persuasive.

Conclusions
In conclusion, we demonstrate that MFG-E8 provides neuroprotection via modulation of inflammation after SAH. Treatment with rhMFG-E8 alleviate microglia inflammation response, which relate to mediating M2 microglia shift, and might involve the mechanism of integrin β3/SOCS3/STAT3 signaling pathway. Therefore, MFG-E8 may become a promising candidate to suppress microglia mediated neuro-inflammation and improve the clinical outcomes of SAH patients.        Silencing MFG-E8 aggravated neuro-inflammation and microglia transformed into M1 phenotype. Representative immunofluorescence staining images of M1 phenotype (a) and M2 phenotype (f), showed that silencing MFG-E8 by siRNA abolished the effect of MFG-E8 on M2 phenotype switch compared with the SAH + rhMFG-E8 group (b, g). Meanwhile, Western blotting (c, h) showed that treatment with MFG-E8 siRNA increased the protein levels of CD86 (d) and IL-6 (e), and decreased the expression of CD206 (i) and IL-10 (j) when compared with the SAH + rhMFG-E8 group. The quantitative data are the mean ± SD (n = 5, each; **p 0.01 vs. SAH + Vehicle group; ##p 0.01 vs. SAH + rhMFG-E8 group; &p 0.05 vs. SAH + rhMFG-E8 group).