Direct co-culture with human neural stem cells suppresses hemolysate-induced inflammation in RAW 264.7 macrophages through the extracellular signal-regulated kinase pathway

Human neural stem cells for neuroprotective effect

Article information

J Neurocrit Care. 2024;17(2):49-56

Publication date (electronic) : 2024 December 27

doi : https://doi.org/10.18700/jnc.240041

Tae Jung Kim, MD, PhD^,¹^,^*

, Jing Sun, MD, PhD ²^,^*

, Lami Kang, BS ¹, Young-Ju Kim, BS ¹, Sang-Bae Ko, MD, PhD ¹

, Byung-Woo Yoon, MD, PhD ³

¹Department of Neurology and Critical Care Medicine, Seoul National University Hospital, Seoul, Korea

²Department of Neurology, China-Japan Union Hospital, Jilin, China

³Department of Neurology, Uijeongbu Eulji Medical Center, Uijeongbu, Korea

Corresponding Author: Tae Jung Kim, MD, PhD Department of Neurology and Critical Care Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea Tel: +82-2-2072-1810, Fax: +82-2-3672-7553, E-mail: ttae35@gmail.com

The authors contributed equally to this study as co-first authors.

Received 2024 December 9; Revised 2024 December 18; Accepted 2024 December 18.

Abstract

Background

Inflammation following stroke is associated with poor outcomes, and the anti-inflammatory effects of neural stem cells (NSCs) have been reported. However, the direct immunomodulatory effects of NSCs in hemorrhagic stroke remain unclear. In the present study, we investigated the anti-inflammatory mechanism of direct co-culture with NSCs on RAW 264.7 cells stimulated by hemolysate.

Methods

RAW 264.7 cells were stimulated with the hemolysate for 4 hours to induce hemorrhagic inflammation in vitro. Regarding direct co-culture, RAW 264.7 cells were cultured with HB1.F3 cells for 24 hours in normal medium and stimulated with hemolysate for 4 hours. Inflammatory cell signaling molecules, including cycloxygenase-2 (COX-2), interleukin-1β (IL-1β), and extracellular signal-regulated kinase (ERK), as well as tumor necrosis factor-α (TNF-α), were evaluated.

Results

After stimulation with the hemolysate, levels of the inflammatory markers COX-2, IL-1β, and TNF-α were increased in RAW264.7 cells. Inflammatory marker production was reduced in the group subjected to direct co-culture with HB1.F3 in comparison to that in the RAW264.7 group stimulated by the hemolysate. In addition, direct co-culture with HB1.F3 significantly suppressed the phosphorylation of ERK 1/2 in hemolysate-stimulated RAW 264.7 cells. Moreover, treatment of the ERK inhibitor (U0126) suppressed the expression levels of inflammatory markers in hemolysate-stimulated RAW246.7 cells.

Conclusion

These results demonstrate that direct co-culture with HB1.F3 suppresses inflammation by attenuating the ERK pathway. These findings suggest that direct NSC treatment modulates the inflammatory response in hemorrhagic stroke.

Keywords: Neural stem cell; Raw 264.7 cell; Hemorrhagic stroke

INTRODUCTION

Neural stem cells (NSCs) are heterogenous stem cells with multipotency, including functions such as differentiation into multiple cell types, anti-inflammation, and repair, and have been used as therapeutic agents in several neurological disorders [1-3]. Systemic and local immune responses are important in neurological disorders, including cerebrovascular diseases [4-7]. Several studies have suggested that NSCs modulate inflammation and promote angiogenesis in stroke [1-3,8]. In hemorrhagic stroke, such as intracerebral and subarachnoid hemorrhages, inflammatory responses are induced in response to certain blood components, including heme, thrombin, iron, and fibrinogen, which are produced after the rupture of a blood vessel. In addition, these responses are associated with the activation of inflammatory cells [5,9-11]. Macrophages and microglia are early inflammatory cells involved in the innate immune response that play important roles in host defense and early inflammation in response to brain injury after a hemorrhagic stroke. When exposed to inflammatory stimuli, activated macrophages and microglia produce a wide array of proinflammatory mediators and induce tissue damage [6,11,12]. In the present study, we hypothesized that direct co-culture with NSCs can ameliorate hemolysate-induced inflammation in RAW 264.7 macrophages based on the ability of NSCs to modulate the acute inflammatory response. Therefore, we investigated the mechanisms underlying the direct interaction between hemolysate-activated macrophages and NSCs in a hemorrhagic stroke model.

METHODS

Cell culture

RAW 264.7 cells, a murine macrophage-like cell line, were purchased from the American Type Culture Collection (ATCC TIB-71) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; WELGENE Inc.) with 10% fetal bovine serum and 5% antibiotics. The cells were cultured at 37 °C with 5% CO₂ in humidified incubator. Subculture was performed according to the ATCC recommendations. For all experiments, the cells were subjected to no more than 10 passages. An immortal female human NSC line, HB1.F3, was generated from primary fetal human brain cell cultures by infection with the recombinant replication-incompetent retroviral vector pLNX.v-myc [13]. HB1.F3 cells were cultured in the same medium as RAW 264.7 cells at 37 °C with 5% CO₂ in a humidified incubator. The cells were cultured to approximately 80% confluence. The third to fifth passages were used for subsequent experiments. Mycoplasma testing was performed on all cell lines before the experiments.

Preparation of the hemolysate

Hemolysate was prepared as previously described [14]. Briefly, fresh arterial blood from heparinized Sprague-Dawley rats was centrifuged at 2,500×g for 15 minutes at 4 °C and the supernatant was aspirated. The precipitate was mixed with the same amount of cold phosphate-buffered saline and lysed using a freeze-thaw procedure. After centrifugation at 31,000×g for 15 minutes at 4 °C, the supernatant was collected and stored in a freezer. The hemolysate concentration was determined by measuring hemoglobin levels using a spectrophotometer. The detection of two absorbance peaks at 540 and 576 nm confirmed the presence of oxyhemoglobin [15,16]. Cell media with hemolysate by volume was used in this study.

Direct co-culture of RAW 264.7 with HB1.F3 cells

The direct co-culture system of HB1.F3 cells with RAW 264.7 cells was established as follows. RAW 264.7 macrophages were divided into four groups: (1) RAW 264.7 cells cultured in normal medium for 28 hours were labeled R and used as a control group (n=6 in each experiment), (2) RAW 264.7 cells cultured in normal medium for 24 hours and stimulated with hemolysate (10% hemoglobin, g/dL) for 4 hours were labeled RH (n=6 in each experiment), (3) RAW 264.7 cells co-cultured with HB1.F3 cells for 24 hours in normal medium and stimulated with hemolysate for 4 hours were labeled RHF (n=6 in each experiment), and (4) RAW 264.7 cells co-cultured with HB1.F3 cells for 24 hours in normal medium were labeled RF (n=6 in each experiment). For direct co-culture, mixtures of RAW 264.7 cells and HB1.F3 cells at a ratio of 1:4 were prepared. RAW 264.7 cells (1 ×10⁵ cells/well) were co-incubated with HB1.F3 cells (4 ×10⁵ cells/well) in six-well plates at 37 °C in a 5% CO₂ atmosphere to allow cells to adhere [17]. The supernatants were collected at 4 hours after co-culture to assay tumor necrosis factor-α (TNF-α) levels using enzyme-linked immunosorbent assay (ELISA). In addition, the cell lysates were harvested to analyze the expression of cycloxygenase-2 (COX-2), interleukin-1β (IL-1β), and extracellular signal-regulated kinase 1/2 (ERK1/2) pathway-related proteins.

Enzyme-linked immunosorbent assay

TNF-α concentration in the supernatant was measured in each of the four groups after the co-culture of hemolysate-stimulated RAW 264.7 cells with NSCs for 4 hours using ELISA kits (DuoSet ELISA Kit, R&D Systems), according to the manufacturer’s instructions [15-17].

Western blot analyses

Cells from the four groups were lysed with a modified protein extraction reagent for 30 minutes on ice, centrifuged at 14,000×g for 15 minutes, and the supernatant was used as the protein sample. Protein concentration was measured using Bradford reagent (Bio-Rad), according to the manufacturer’s instructions. Equivalent amounts of protein were separated using 8% or 4% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane. After blocking the membrane with 5% skim milk in TBS for 1 hour, the membrane was incubated with primary antibodies [18,19] against COX-2 (Abcam), IL-1β (Abcam), ERK1/2, phosphorylated-ERK 1/2 (p-ERK1/2) (Cell Signaling Technology), and α-tubulin (Sigma-Aldrich) overnight at 4 °C, followed by a horse-radish peroxidase-conjugated anti-rabbit secondary antibody (Cell Signaling Technology) for 1 hour at room temperature. Bands were visualized using an enhanced chemiluminescence detection system (Millipore) and exposed to radiographic film. U0126 (an inhibitor of ERK 1/2) was used to evaluate the correlation between the ERK1/2 pathway and COX-2, IL-1β, and TNF-α production.

Statistical analyses

All data were obtained from three independent experiments and are presented as means and standard deviations, whereas variables that were not normally distributed are presented as the median. Two-tailed unpaired Student t-tests or Mann-Whitney U-test were used to evaluate the differences in mean values between the two groups. Comparisons between groups were evaluated using one-way analysis of variance followed by Tukey’s post-hoc test, if necessary. Statistical significance was set at P<0.05. Statistical analyses were performed using IBM SPSS version 25.0 (IBM Corp.) and GraphPad Prism (version 8.0; GraphPad).

RESULTS

Hemolysate activates COX-2 and IL-1β expression in RAW 264.7 cells

RAW 264.7 cells were stimulated with hemolysate (10% hemoglobin, g/dL) to promote COX-2, IL-1β, and TNF- α expression. COX-2 and IL-1β levels in RAW 264.7 cells increased and reached a peak after 1 h (COX-2) or 4 hours (IL-1β and TNF- α), then decreased gradually after 6 hours (Fig. 1).

Fig. 1.

Hemolysate activates cycloxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) expression in RAW 264.7 cells. (A) Western blot analyses of inflammatory markers. COX-2 levels in RAW 264.7 cells increased and reached a peak level after 1 hour, and IL-1β and TNF-α reached peak levels after 4 hours. The increased inflammatory mediators (COX-2 shown in Fig. 1B, IL1-β in Fig. 1C, and TNF-α in Fig. 1D) decreased gradually. The band intensity was quantified using scanning densitometry and standardized with respect to α-tubulin. Values are presented as mean±standard deviation of three independent experiments.

Direct co-culture with HB1.F3 reduced hemolysate-induced COX-2, IL-1β, and TNF-α expression in RAW 264.7 cells

Hemolysate-stimulated RAW 264.7 cells were directly co-cultured with HB1.F3 cells to evaluate whether HB1.F3 cells modulate COX-2, IL-1β, and TNF-α expression. After hemolysate stimulation, the expression levels of COX-2 and IL-1β in RAW cells were assessed using western blotting and TNF-α levels were examined using ELISA. COX-2, IL-1β, and TNF-α levels were significantly higher after treatment with hemolysate alone for 4 hours than in the control group (Fig. 2). However, direct co-culture with HB1.F3 cells resulted in markedly lower COX-2 and IL-1β levels compared to those of hemolysate-stimulated RAW 264.7 cells alone (Fig. 2B and C). Moreover, HB1.F3 cells markedly suppressed TNF-α production by hemolysate-stimulated RAW 264.7 cells by 44% after 4 hours (Fig. 2D). However, no significant difference was observed between RAW 264.7 cells cocultured with HB1.F3 cells stimulated by hemolysate and RAW 264.7 cells cocultured with HB1.F3 cells.

Fig. 2.

Direct co-culture with HB1.F3 cells reduced the hemolysate-stimulated expression of cycloxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in RAW 264.7 cells. The cells were divided into four groups. One was the control group, which was labeled R. The second group comprised RAW 264.7 cells stimulated with hemolysate for 4 hours and labeled RH. The third group was RAW 264.7 cells co-cultured with HB1.F3 cells and stimulated with hemolysate for 4 hours and labeled RHF. The fourth group was RAW 264.7 cells co-cultured with HB1.F3 cells for 24 hours and labeled RF. (A) Band quantification was performed using scanning densitometry against standardized levels of α-tubulin. (B) Direct co-culture with HB1.F3 cells suppressed COX-2 level compared with the levels in hemolysate-stimulated RAW 264.7 cells alone after 4 hours. (C) IL-1β expression decreased by direct co-culture with HB1.F3 cells after 4 hours compared with the expression in hemolysate-stimulated RAW 264.7 cells alone. (D) Direct co-culture with HB1.F3 cells inhibited TNF-α the hemolysate-stimulated RAW 264.7 cells after 4 hours. These results were obtained using enzyme-linked immunosorbent assay. Values are presented as mean±standard deviation of three independent experiments. NS, not significant. *P<0.05, **P<0.01, and ***P<0.001 compared to the control.

Direct co-culture with HB1.F3 inhibits hemolysate-stimulated ERK1/2 phosphorylation in RAW 264.7 cells

In the direct co-culture system, we investigated the inhibitory effect of HB1.F3 cells on the ERK1/2 pathway using Western blot analysis. The phosphorylation of ERK1/2 increased significantly after hemolysate stimulation for 4 hours, as shown in Fig. 3. However, ERK1/2 pathway activation in hemolysate-induced RAW 264.7 cells was markedly suppressed by direct co-culture with HB1.F3. However, the ERK1/2 pathway in RAW 264.7 cells was not activated by HB1.F3 (Fig. 3B). In addition, the association of the ERK signaling pathway with the hemolysate-activated expression of COX-2, IL-1β, and TNF-α was assessed using an inhibitor of ERK (U0126) in RAW 264.7 cells. RAW 264.7 cells were pretreated with an ERK inhibitor for 1 hour and then stimulated with the hemolysate for 4 hours. After 4 hours, TNF-α levels in the supernatant were examined using ELISA, and COX-2 and IL-1β expression levels in cell lysates were evaluated using western blotting. Treatment with U0126 markedly reduced COX-2, IL-1β, and TNF-α expression in hemolysate-activated RAW 264.7 cells (Fig. 4).

Fig. 3.

Direct co-culture with HB1.F3 cells downregulated the hemolysate-activated phosphorylation of extracellular signal-regulated kinase (ERK) in RAW 264.7 cells. The cells were divided into four groups. One was the control group, which was labeled R. The second group comprised RAW 264.7 cells stimulated with hemolysate for 4 hours and labeled RH. The third group was RAW 264.7 cells co-cultured with HB1.F3 cells and stimulated with hemolysate for 4 hours and labeled RHF. The fourth group was RAW 264.7 cells co-cultured with HB1.F3 cells for 24 hours and labeled RF. (A) Band quantification was performed using scanning densitometry against standardized levels of α-tubulin. (B) ERK phosphorylation increased significantly after hemolysate stimulation for 4 hours. However, ERK pathway activation in hemolysate-induced RAW 264.7 cells was markedly suppressed by direct co-culture with HB1.F3. Moreover, the ERK1/2 pathway in RAW 264.7 cells was not activated by HB1.F3. Values are presented as mean±standard deviation of three independent experiments. p-ERK, phosphorylated ERK; NS, not significant. **P<0.01, ***P<0.001 compared to the control.

Fig. 4.

The phosphorylation of extracellular signal-regulated kinase (ERK) participates in hemolysate-stimulated expression of cycloxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in RAW 264.7 cells. The cells were divided into three groups. One was the control group, which was labelled R. The second group was activated with the hemolysate for 4 hours and labeled RH. The third group was treated with U0126 (an ERK inhibitor) for 1 hour, incubated with hemolysate, and labeled RHU. (A) Band quantification was performed using scanning densitometry against standardized levels of α-tubulin. COX-2 (B), IL-β (C), and TNF-α (D) expressions were significantly suppressed by treatment with U0126 in hemolysate-stimulated RAW 264.7 cells. Values are presented as mean±standard deviation of three independent experiments. *P<0.05, ***P<0.001 compared to the control.

DISCUSSION

Our results showed that direct co-culture of NSCs with RAW 264.7 cells in the same medium reduced inflammation through the ERK1/2 signaling pathway. Moreover, the ERK signaling pathway is involved in the inflammatory response in hemolysate-stimulated RAW 264.7 cells. In hemorrhagic stroke, important pathological mechanisms involved in the process of hemorrhage-induced brain injury include an inflammatory response that correlates with blood vessel rupture. Following the mechanical damage associated with hematoma, secondary damage may be related to extravasated blood components, including erythrocytes and plasma proteins, and these reactions can induce and aggravate brain edema, blood-brain barrier disruption, oxidative stress, and cytotoxic insults [5,10-12,20]. Previous studies have reported that hemolysate, a product of damaged erythrocytes, is associated with brain edema and inflammation after hemorrhagic stroke [10,11,14,15,21]. Consistent with these findings, our results indicated that inflammatory mediators, such as COX-2, IL-1β, and TNF-α, were increased in RAW 264.7 cells stimulated by a hemolysate in an in vitro hemorrhagic stroke model. HB1.F3 is a human NSC with anti-inflammatory and neuroprotective effects studied in several animal models of neurological diseases, including stroke [8,21-26]. Previous studies have demonstrated that HB1.F3 cells modulate the adaptive immune response and have long-term neuroprotective effects based on their trophic effects in ischemic and hemorrhagic stroke [2,21-23,26]. However, innate immune responses associated with microglia and infiltrated inflammatory cells, such as neutrophils and macrophages, are the primary sources of inflammatory reactions that initiate secondary brain injury after hemorrhagic stroke [27,28]. Therefore, direct modulation of the innate immune response in the early acute phase after hemorrhagic stroke is a promising therapeutic approach [27,28]. In our study, direct co-culture with HB1.F3 cells suppressed the inflammatory response by hemolysate stimulation in RAW 264.7 macrophage cells during the acute phase (at 4 hours). Despite the results of in vitro analyses, these reactions could be part of an innate immune reaction after hemorrhagic stroke.

Macrophages are an important innate inflammatory response in hemorrhagic stroke. In addition, ERK pathways are involved in hemolysate-mediated inflammation, and ERK activity can aggravate inflammation and cytotoxic effects by up-regulating COX-2 and inflammatory mediators, such as IL-1β and TNF-α, in stroke [15,29-33]. Therefore, the regulation of these inflammatory mediators, including COX-2, through the ERK pathway, may have anti-inflammatory effects in hemorrhagic stroke. Inhibition of COX-2 activity has been reported to reduce astrogliosis, infiltration of inflammatory cells (including leukocytes and neutrophils), and brain edema, as well as to improve functional outcomes after hemorrhagic stroke [34,35]. Moreover, previous studies have demonstrated that NSCs can suppress the expression of pro-inflammatory cytokines, including IL1-β and TNF-α, and decrease brain edema and inflammation during the acute phase in hemorrhagic stroke [36,37]. Direct co-culture of hemolysate-stimulated RAW 264.7 cells with HB1.F3 cells (that is, NSCs) significantly reduced COX-2 expression and that of pro-inflammatory cytokines, such as IL-1β and TNF-α, through regulation of the ERK pathway. These results indicate that modulation of the ERK1/2 pathway explains the anti-inflammatory effect of direct co-culture with HB1.F3 cells in hemolysate-induced RAW 264.7 cells. However, an unassessed hemolysate-related inflammatory reaction of NSCs to the hemolysate should also be considered.

In conclusion, our in vitro study provided evidence that hemolysate stimulation of RAW 264.7, results in the activation of the ERK and COX-2 signaling pathways. Moreover, HB1.F3 NSCs attenuated the acute inflammatory response in hemolysate-stimulated RAW 264.7 cells by inhibiting the ERK signaling pathway in a direct co-culture system. These results provide evidence that HB1.F3 exerts a direct protective effect against innate inflammatory responses after hemorrhagic stroke. Additional in vivo and clinical studies are required to confirm and elucidate the neuroprotective effects of hemorrhagic stroke on inflammatory processes.

Notes

Ethics statement

An in vitro study was conducted using a cell line; therefore, informed consent was not required. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University Hospital (No. 17-0057-S1A0).

Conflict of interest

Tae Jung Kim is the editor-in-chief, and and Sang-Bae Ko are editorial board members of the journal. However, they were not involved in the peer reviewer selection, evaluation, or decision-making process for this article. No other potential conflicts of interest relevant to this article have been reported.

Author contributions

Conceptualization: TJK, BWY. Methodology: TJK, JS, LK, YJK. Validation: LK, YJK. Formal analysis: TJK, JS. Investigation: TJK, JS, LK, YJK. Resources: TJK, BWY. Data curation: TJK, JS. Visualization: TJK, JS. Supervision: SBK, BWY. Project administration: TJK, SBK. Writing – original draft: TJK, JS. Writing – review & editing: TJK. All authors read and agreed to the published version of the manuscript.

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