Animals

Two-day-old (P2) newborn C57BL/6 mice were provided by ZHBY Biotech Co. Ltd. (Jiangxi, China). All mice were treated in strict accordance with the guidelines of the Ethics Committee of Fujian Medical University, and all animal experiments were approved by the Ethics Committee of Fujian Medical University (IACUC FJMU 2023-Y-0438). Efforts were made to reduce the number of mice used and their suffering.

Primary brain microglia and bone marrow-derived macrophage (BMDM) isolation

Primary brain microglia were isolated from P2 newborn C57BL/6 mice. In brief, the neonatal mice cerebral cortices without blood vessels and meninges were rinsed repeatedly for blood stain removal with cold Hank’s solution brain, followed by digestion with 0.25% trypsin for 10 minutes. The precipitate was incubated in DMEM/F12 with 10% FBS in an incubator at 37°C in 5% CO₂ and 95% air to halt digestion. The filtered mixed glial cells were centrifuged at 1000 r/min for 5 min to obtain sediment, which was afterward resuspended in DMEM/F12 with 20% FBS. Cells were seeded into 75 cm² flasks at a density of 3 × 10⁵ cells/cm² and cultured for 9-11 days. When the cells reached confluence and were maintained for 3 days (the 11^th day), the culture medium was switched to DMEM/F12 with 10% FBS and the culture flasks were placed in a shaking incubator for 12 hours to collect the floating cells, which represented the pure primary microglia.

Mouse bone marrow cells isolated by flushing the marrow space of the femur and tibia with 10% complete culture medium in 5-week-old mice were dispersed into a single-cell suspension. After adding erythrocyte lysate for 10 minutes and discarding the supernatant, the floating cells resuspended by medium were treated with 40 ng/ml GM-CSF for 8 days and cultured in the incubator at 37oC in 5% CO₂.

Transient cell transfection with si-IL-33/si-NC

When the density of microglia reached 70% confluence, the culture medium was replaced with 1 ml of serum-free medium. The lyophilized siRNA powder (si-IL-33 or si-NC, Jiangxi Zhonghong Boyuan Biotechnology Co., Ltd) was reconstituted in DEPC-treated water at a concentration of 125 µl per OD unit.

For transfection, 5 µl of Lipofectamine 3000 (L3000015, Invitrogen) and 12.5 µl of reconstituted siRNA were separately diluted in 125 µl of Opti-MEM medium (Gibco), incubated for 5 minutes at room temperature, then combined and incubated for an additional 15 minutes to allow complex formation. The transfection mixture was then added dropwise to the cells. After 4 hours of transfection, the medium was replaced with complete culture medium containing 20% serum, and cells were incubated for 48 hours. Transfection efficiency was evaluated by Western blot and qRT-PCR.

Microglia exposure to OGD/R injury

For imitating oxygen–glucose deprivation, the si-IL-33 (or not) microglia were cultured with the sugar-free medium in an incubator with 94% N₂, 5% CO₂ and 1% O₂ for 2 hours. To induce reperfusion injury, the medium of cells was switched to the regular medium in an incubator at 37oC in 5% CO₂ and 95% air for another 24 hours.

Macrophage polarization and macrophage – OGD/R microglia co-cultures

The direct co-culture system was applied in this experiment. The treated microglia and M1 (M2) macrophages were mixed in a ratio of 1 : 2 directly and incubated for 24 hours. The supernatant of the co-culture system was collected for ELISA and WB assay, and the remaining cells were used for immunofluorescence assay.

Quantitative real-time RT-PCR (qRT-PCR) assay

The transcriptional level of IL-33 was detected by qRT-PCR assay for verifying transfection efficiency. Total RNA was extracted from cells using an Ultrapure RNA Kit (CW0581M, CWBIO) and Trizon Reagent (CW0580S, CWBIO). Samples were then reverse-transcribed to cDNA from RNA by HiScript II Q RT SuperMix for qPCR (R223-01, Vazyme). cDNA was amplified by ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme) on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories Inc.). All procedures followed the manufacturer’s instructions. β-actin was used as an internal reference for normalization, and the relative expression was calculated according to the 2^-ΔΔCt method. The primers for IL-33 and β-actin, synthesized by General Biosystems (Anhui) Co., are presented in Table 1.

Western blot

Antibodies against IL-33, STAT1, IRF5, IRF4, CEBP-β, Beclin-1, P62 (1 : 500, AF6300/DF6283/DF6198/AF7747/AF5128/AF5384, Affinity Biosciences Group Ltd.), PPAR-γ, STAT6, IRF8, STAT3, ULK1 (1 : 2000, 16643-1-AP/66717-1-Ig/60199-1-lg/20986-1-ap, Proteintech Group, Inc) and the internal control β-actin (1 : 2000, TA-09, Beijing Zhong Shan Golden Bridge Biological Technology Co., Ltd) were applied. The cells were lysed with a Cell Lysis kit (C1053, Applygen Technologies Inc.) before the cells were placed on ice for 20 minutes. The total proteins were extracted by centrifugation at 12,000 r/min for 10 minutes, and the supernatant containing proteins was retained for subsequent assays. The total proteins were reserved at –20oC. A BCA kit (E-BC-K318-M, Elabscience) was used for verifying protein concentration. Equal amounts of extracted protein were loaded in 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) and separated by gel electrophoresis under constant flow at 300 mA for 1.5 hours. The target proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore). The membranes were incubated with primary antibodies at 4°C overnight before incubation with 5% non-fat milk to prevent non-specific binding. After rinsing with TBST, membranes were incubated with diluted horseradish peroxidase (HRP)-conjugated secondary antibodies (1 : 2000, ZB-2305, Beijing Zhong Shan Golden Bridge Biological Technology Co., Ltd) for 2 h the next day. The densities of the binding bands were visualized by the Ultra High Sensitivity Chemiluminescent Imaging System and quantified with the Image J software (National Institutes of Health, Bethesda, MD, USA).

Enzyme-linked immunosorbent assay (ELISA)

The expression of interferon γ (IFN-γ), IL-10, and TGF-β in cell supernatants was measured using ELISA kits (Jiangsu Meimian Industrial Co., Ltd). In accordance with the manufacturer’s instructions, all samples, including standards, were run in triplicate. Linear regression equations for standard curves were calculated with standard concentrations and optical density (OD) 450. The actual sample concentration was obtained by substituting the OD value of the sample into an equation and multiplying it by the dilution factor.

Multiple immunofluorescence assay

All samples were fixed with 4% parapformaldehyde for 30 minutes after rinsing with PBS 3 times. The cells were blocked with 5% BSA at 37°C for 30 minutes. Without washing, cells were incubated overnight at 4°C with primary antibodies against CD16+CD32 (1 : 200, Abcam Trading (Shanghai) Co., Ltd.) and CD206 (1 : 200, Affinity Biosciences Group Ltd.). The next day, cells were washed three times with PBS and incubated with ABflo® 488-conjugated Goat anti-Rabbit IgG (H+L) (1 : 200, AS053, ABclonal) for 30 minutes at 37°C. After three PBS washes, cells were blocked again with 5% BSA at 37°C for 30 minutes. Without washing, cells were incubated with anti- Iba1 primary antibody (1 : 200, Proteintech Group, Inc.) at 37°C for 3 hours, followed by three PBS washes. Cells were then incubated with Cy3-conjugated secondary antibody (1 : 200, AS007, ABclonal) for 30 minutes at 37°C for 30 minutes in the dark. The slides were counterstained with nuclear dye DAPI (Jiangsu KeyGEN BioTECH Co., Ltd.) and observed using a fluorescent microscope.

Statistical analysis

All experiments were performed at least in triplicate. All the data are presented as the mean ± standard error of the mean (SEM). Statistical differences between groups were evaluated using an independent samples t-test, while statistical differences among groups were analyzed by one-way ANOVA. A p-value < 0.05 was considered statistically significant. GraphPad 9.0 software was used for statistical analysis and graphing.

Effect of IL-33 deficiency on macrophage polarization and the inflammatory phenotype of OGD/R microglia

To elucidate the involvement of IL-33 in cerebral I/R, the effect of IL-33 on the relevant cytokines of OGD/R microglia was assessed. The efficiency of the IL-33 interference vector effect was validated by qPCR and WB. To assess changes in M1 and M2 macrophage polarization, intracellular markers of M1 (STAT1, STAT3, IRF5, IRF8) and M2 (STAT6, PPAR-γ, IRF4, CEBP-β) were measured by Western blot (WB) (Fig. 1A). The results from M2/si-IL-33 microglia co-cultures with OGD/R conditions showed that the expression levels of STAT6, PPAR-γ, IRF4, and CEBP-β were significantly increased (p < 0.05), while those of STAT1, IRF8, STAT3, and IRF5 were significantly reduced (p < 0.05). IL-33 knockdown markedly reduced the pro-inflammatory phenotype. The co-expression between M1-type macrophage marker CD16+CD32 and microglial cell marker Iba1 and the co-expression between M2-type macrophage marker CD206 and microglial cell marker Iba1 were detected via immunofluorescence assay (Fig. 1B, C). Compared with M1/si-IL-33 microglia co-cultures, CD206 expression was higher in M2/si-IL-33 microglia co-cultures. ELISA results showed that the M2-type extracellular markers IL-10 and TGF-β were significantly increased (p < 0.05), whereas the M1-type marker IFN-γ was significantly decreased (p < 0.05) in M2/si-IL-33 co-cultures compared with M1/si-IL-33 co-cultures (Fig. 1D).

Fig. 1

Effect of IL-33 deficiency on macrophage polarization and the inflammatory phenotype of OGD/R microglia. A) Expression of M1-type intracellular markers and M2-type intracellular markers was detected by WB. *p < 0.05, **p < 0.01. All experiments were performed in triplicate A) Expression of M1-type intracellular markers and M2-type intracellular markers was detected by WB. *p < 0.05, **p < 0.01, ***p < 0.001 , ****p < 0.0001. All experiments were performed in triplicate B, C) Immunofluorescence detection of M1-type macrophage markers and co-expression of M2-type macrophage markers with microglia markers D) Expression of M1-type extracellular markers and M2-type extracellular markers in cell supernatants by ELISA assay. Compared to the OGD/R + M1 group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All experiments were performed in triplicate

Effect of IL-33 deficiency and macrophage polarization on autophagy of OGD/R microglia

Autophagy, a critical cellular process for the degradation and recycling of damaged organelles and proteins, has been shown to be upregulated in models of oxygen-glucose deprivation (OGD), a common in vitro model for ischemia-reperfusion injury. The expression of autophagy-related proteins such as Beclin-1 and Unc-51-like kinase 1 (ULK1) increases under OGD conditions, while P62, an autophagy adaptor protein, typically shows an inverse relationship with autophagic activity. Elevated Beclin-1 and ULK1 levels indicate enhanced autophagic flux, as Beclin-1 is integral to autophagosome formation, while ULK1 is a key initiator of autophagy signaling pathways. Moreover, P62 is known to accumulate when autophagy is inhibited, serving as a marker for impaired autophagy, while its degradation reflects effective autophagic processes.

In this study, WB analysis was performed to examine the expression of Beclin-1, P62, and ULK1 in the OGD/R model (Fig. 2). Compared to the OGD/R model group, co-culture with M1 macrophages significantly increased the expression of Beclin-1 and ULK1, while reducing P62 expression. The activation of autophagy by M1 macrophages may play a role in facilitating cellular survival under conditions of ischemic stress by removing damaged organelles and proteins [6].

Fig. 2

Effect of IL-33 deficiency and macrophage polarization on autophagy of OGD/R microglia. Expression of autophagy signaling pathway proteins Beclin-1, p62, and ULK1 in cells. Compared with the control group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All experiments were performed in triplicate

Conversely, co-culture with M2 macrophages and IL-33 deficiency both resulted in a reduction in Beclin-1 and ULK1 expression, alongside an increase in P62 levels, suggesting that M2 macrophages may suppress autophagy. Additionally, the absence of IL-33, an alarmin that has been shown to promote autophagy under certain pathological conditions, further attenuates autophagy signaling, indicating its possible role in regulating the balance between inflammation and autophagy. The increase in P62 levels in these conditions suggests that autophagic degradation is impaired, leading to the accumulation of cellular debris and potentially exacerbating ischemia-reperfusion injury.

In the context of ischemic injury, autophagy serves a dual role: while moderate autophagy can protect cells by removing damaged components, excessive or dysregulated autophagy can lead to autophagic cell death, contributing to tissue damage. Thus, the differential modulation of autophagy by M1 and M2 macrophages, as well as the influence of IL-33, highlights the complex interplay between macrophage polarization, inflammatory responses, and autophagic activity in ischemic injury models.