HDAC1 dysregulation promotes pro-inflammatory microglial activation and aggravates post-stroke neuroinflammation

Abstract

Background: Histone deacetylase 1 (HDAC1) is a key epigenetic regulator involved in DNA repair and neuronal survival. While HDAC1 downregulation has been implicated in ischemic brain injury, its role in regulating microglial functional shift and neuroinflammation remains unclear. This study aimed to investigate how HDAC1 dysfunction influences microglial activation states and contributes to neuroinflammatory processes in ischemic stroke.

Methods: Using a rat model of endothelin-1-induced focal cerebral ischemia, HDAC1 knockdown was achieved via stereotactic co-injection of HDAC1 siRNA. Immunofluorescence, Western blotting, ELISA, and oxidative stress assays were performed to assess neuroinflammation, microglial polarization, and related signalling pathways. In parallel, HDAC1 was silenced in HMC3 human microglial cells, with or without IFN-γ stimulation, to evaluate transcriptional responses associated with pro-inflammatory activation. Finally, HDAC1 was selectively reactivated in vivo and in vitro using Compound 5104434 to assess behavioural, neuroinflammatory and mechanistic effects after ischaemic or inflammatory injury.

Results: HDAC1 knockdown in vivo led to a pronounced shift towards a pro-inflammatory microglial activation, evidenced by increased CD86 expression, along with elevated levels of IL-1β, IL-6, TNF-α, ROS, LDH and MMP activity. T-cell infiltration was also significantly enhanced. In vitro, HDAC1 deficiency sensitized microglia to IFN-γ, further amplifying the expression of pro-inflammatory genes. Mechanistically, HDAC1 knockdown activated the NF-κB pathway and its downstream effectors MAP3K8, AP-1 and SAT1, while IFN-γ stimulation predominantly drove STAT3 phosphorylation. Notably, pNF-κB was upregulated even in the absence of exogenous stimulation, indicating that HDAC1 intrinsically suppresses pro-inflammatory signalling. HDAC1 enzymatic reactivation by compound 5104434 promotes functional recovery and suppresses NF-κB-driven microglial activation after stroke.

Conclusion: HDAC1 acts as a key repressor of NF-κB-driven pro-inflammatory microglial activation and neuroinflammation in stroke. Its loss exacerbates inflammatory cascades, immune cell infiltration, and neuronal injury, underscoring HDAC1 as a potential therapeutic target for limiting secondary brain damage after ischaemic stroke.

Keywords: HDAC1; inflammation; microglia; stroke.

Figure 2.

Ischaemia insult suppresses anti-inflammatory microglial activation after stroke. (A) Representative immunofluorescence images showing Iba-1 (red), CD206 (green, M2 marker), and DAPI (blue) in Sham, Stroke, and Stroke + HDAC1 KD groups. Scale bar: 150 μm. Amplified images reveal a marked reduction in CD206 expression following stroke; however, no obvious further decrease was observed in the Stroke + HDAC1 KD group compared to the Stroke group. Scale bars: 50 μm. (B) Quantification of CD206⁺/Iba-1⁺ microglia, expressed as a percentage of total Iba-1⁺ cells. CD206 expression is significantly decreased after stroke in the Stroke and Stroke + HDAC1 KD groups (**p < 0.001, one-way ANOVA with Tukey’s post hoc test). N = 8 per group. Data are presented as mean ± SEM.

Figure 3.

HDAC1 knockdown promotes T cell infiltration in the ischaemic brain. (A) Representative immunofluorescence images of brain sections stained for CD3 (green, T cell marker) and DAPI (blue, nuclei) in Sham, Stroke, and Stroke + HDAC1 KD groups. The Stroke group exhibits increased CD3⁺ cell presence compared to Sham controls, which is further enhanced in the Stroke + HDAC1 KD group. Scale bars: 150 μm; Scale bars: 50 μm in amplified views. (B) Quantification of CD3⁺ cell density, expressed as a percentage of total cells per field of view. CD3⁺ cell infiltration is significantly increased in the Stroke group and further elevated following HDAC1 knockdown (*p < 0.05, one-way ANOVA with Tukey’s post hoc test). N = 8 per group. Data are presented as mean ± SEM.

Figure 4.

HDAC1 knockdown exacerbates stroke-induced neuroinflammation, oxidative stress, and neuronal injury. Bar graphs depict quantification of key inflammatory and injury markers in Sham, Stroke, and Stroke + HDAC1 KD groups. (A) MMPs activity measured by gelatinase assay. Stroke significantly increases MMPs activity, which is further enhanced by HDAC1 knockdown. (B and C) Pro-inflammatory cytokines IL-1β and IL-6 levels, showing a marked increase in the Stroke group, with further elevation in the Stroke + HDAC1 KD group. (D) TNF-α levels follow a similar pattern, with the highest expression observed in the Stroke + HDAC1 KD group. (E) LDH levels, indicating neuronal injury, are significantly increased following stroke and further elevated by HDAC1 knockdown. (F) ROS levels measured by H₂O₂ production demonstrate significantly elevated oxidative stress in the Stroke + HDAC1 KD group. N = 8 per group. Statistical significance: * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001 (one-way ANOVA with Tukey’s post hoc test). Data are presented as mean ± SEM.

Figure 1.

HDAC1 knockdown promotes pro-inflammatory microglial activation after stroke. (A) Representative immunofluorescence images of brain sections stained for Iba-1 (red, microglia), CD86 (green, M1 marker), and DAPI (blue, nuclei) in Sham, Stroke, and Stroke + HDAC1 KD groups. Scale bar: 150 μm. Amplified images highlight increased colocalization of CD86 with Iba-1 following stroke, which is further enhanced by HDAC1 knockdown. Scale bar: 50 μm. (B) Quantification of CD86⁺/Iba-1⁺ microglia, expressed as a percentage of total Iba-1⁺ cells. CD86 expression is significantly upregulated in the Stroke group and further increased following HDAC1 knockdown (*p < 0.05, one-way ANOVA with Tukey’s post hoc test). N = 8 per group. Data are presented as mean ± SEM.

Figure 5.

HDAC1 knockdown enhances pro-inflammatory microglial activation in vitro. (A) Representative immunofluorescence images of cultured human macrophage cell line-HMC3 stained for Iba-1 (red, microglial marker), CD86 (green, M1 marker), and DAPI (blue, nuclei) under different conditions: Control, HDAC1 KD, Control + IFN-γ, and HDAC1 KD + IFN-γ. Scale bars: 50 μm. (B) Quantification of M1 (CD86⁺) microglial cell number per field of view, showing a significant increase in CD86 expression with HDAC1 KD, IFN-γ treatment, and HDAC1 + IFN-γ treatment. (C) Percentage of CD86⁺ microglia per total microglial population, demonstrating that HDAC1 KD enhances M1 polarization, particularly in response to IFN-γ stimulation. N = 5 independent experiments. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA with Tukey’s post hoc test). Data are presented as mean ± SEM.

Figure 6.

HDAC1 knockdown suppresses anti-inflammatory microglial activation in vitro. (A) Representative immunofluorescence images of cultured microglia stained for Iba-1 (red, microglial marker), CD206 (green, M2 marker), and DAPI (blue, nuclei) under different conditions: Control, HDAC1 KD, Control + IFN-γ, and HDAC1 KD + IFN-γ. Scale bars: 50 μm. (B) Quantification of M2 (CD206⁺) microglial cell number per field of view, showing a significant decrease in CD206 expression with HDAC1 KD and IFN-γ treatment. (C) Percentage of CD206⁺ microglia per total microglial population, indicating that HDAC1 KD suppresses M2 polarization, particularly in response to IFN-γ stimulation. N = 5 independent experiments. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA with Tukey’s post hoc test). Data are presented as mean ± SEM.

Figure 7.

HDAC1 knockdown enhances pro-inflammatory transcriptional responses. (A) GeneMania network analysis showing predicted interactions of HDAC1 with transcriptional regulators involved in inflammatory signalling, including NF-κBIA, NF-κB1 and NF-κB2. Physical interactions, genetic interactions, co-expression, and pathway connections are represented with different colours. (b) Real-Time PCR for gene expression analysis of NF-κBIA, NF-κB1, NF-κB2, MAP3k8, AP-1, and SAT1 in microglia under Control, HDAC1 KD, Control + IFN-γ, and HDAC1 KD + IFN-γ conditions. Loss of HDAC1 significantly enhances the expression of NF-κB-related genes, as well as AP-1 and SAT1, particularly in response to IFN-γ stimulation. N = 16 independent experiments. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA with Tukey’s post hoc test). Data are presented as mean ± SEM.

Figure 8.

HDAC1 KD modulates NF-κB and STAT signalling pathways under IFN-γ stimulation. (A) Western blot analysis showing the expression levels of HDAC1, pNF-κB, total NF-κB, pStat1, total STAT1, pStat3 and total STAT3 across experimental groups: Control, HDAC1 KD, Control + IFN-γ, and HDAC1 KD + IFN-γ. GAPDH served as the loading control. HDAC1 KD resulted in altered expression and phosphorylation of key signalling proteins, particularly under IFN-γ stimulation. (B) Quantification of Western blot results, presented as normalized expression relative to control levels. HDAC1KD significantly reduced HDAC1 expression. Increased phosphorylation of NF-κB was observed in HDAC1 KD, with the highest levels in HDAC1 KD + IFN-γ. Phosphorylation of STAT1 was significantly upregulated under IFN-γ stimulation. HDAC1 KD increased STAT3 phosphorylation, and IFN-γ stimulation promoted its levels. N = 6 independent experiments. Data are presented as mean ± SEM, and statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Tukey’s post hoc test).

Figure 9.

HDAC1 enzymatic reactivation by Compound 5104434 promotes functional recovery and suppresses NF-κB–driven microglial activation after stroke. (A) Neurological assessments showed mNSS scores (left) at PSD1, PSD3, and PSD7, with Compound 5104434 treatment significantly enhancing recovery compared to vehicle-treated stroke rats on PSD7. Cylinder test performance (right) was also improved on PSD7, indicating better forelimb function. N = 6 per group. (B) Microglial activation in peri-infarct cortex at PSD 7. Quantification of Iba-1⁺ cells (top) and representative immunofluorescence images (bottom; Iba-1, red; DAPI, blue). Scale bar: 100 μm. N = 6 per group. (C) Primary microglial polarization in vitro. Representative confocal images of Iba-1 (red) and CD86 (green) expression in control, IFN-γ, and IFN-γ + Compound 5104434 conditions (top). Quantification of CD86⁺ ratio per field (bottom). Scale bar:100 μm. N = 6 independent experiments per group. (D) Gene expression analysis by qPCR. Fold changes in NFKBIA, NFKB1, NFKB2, MAP3K8, AP-1, and SAT1 following IFN-γ stimulation with or without Compound 5104434. N = 6 independent experiments per group. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (One-way ANOVA with Tukey’s post hoc test).