Histone lactylation-ROS loop contributes to light exposure-exacerbated neutrophil recruitment in zebrafish

Abstract

Light serves as a crucial external zeitgeber for maintaining and restoring physiological homeostasis in most organisms. Disrupting of light rhythms often leads to abnormal immune function, characterized by excessive inflammatory responses. However, the underlying regulatory mechanisms behind this phenomenon remain unclear. To address this concern, we use in vivo imaging to establish inflammation models in zebrafish, allowing us to investigate the effects and underlying mechanisms of light disruption on neutrophil recruitment. Our findings reveal that under sustained light conditions (LL), neutrophil recruitment in response to caudal fin injury and otic vesicle inflammation is significantly increased. This is accompanied by elevated levels of histone (H3K18) lactylation and reactive oxygen species (ROS) content. Through ChIP-sequencing and ChIP‒qPCR analysis, we discover that H3K18 lactylation regulates the transcriptional activation of the duox gene, leading to ROS production. In turn, ROS further promote H3K18 lactylation, forming a positive feedback loop. This loop, driven by H3K18 lactylation-ROS, ultimately results in the over recruitment of neutrophils to inflammatory sites in LL conditions. Collectively, our study provides evidence of a mutual loop between histone lactylation and ROS, exacerbating neutrophil recruitment in light disorder conditions, emphasizing the significance of maintaining a proper light-dark cycle to optimize immune function.

Fig. 1. Sustained light increases H3K18 lactylation and ROS levels.

a–d Q-PCR analysis showed that the expression levels of the lactylation-related genes ep300a and ep300b were significantly upregulated in juvenile fish reared under LL conditions. e–h Q-PCR analysis showed that the expression levels of the lactate metabolism-related genes ldha and gapdh were significantly upregulated in juvenile fish reared under LL conditions. i The lactate content in the juvenile fish reared under LL conditions was significantly increased. j, k Western blotting results showed that the level of histone lactylation (H3K18) was significantly upregulated in the juvenile fish of the LL group. l The hydrogen peroxide content in the juvenile fish under LL condition was significantly increased. m, n Fluorescent staining results showed that LL treatment significantly increased the levels of reactive oxygen species (ROS) in juvenile fish (n = 15). o–q Q-PCR analysis showed that the expression levels of akt1 and akt3 were upregulated under LL conditions, but akt2 was not statistically significant. Data were statistically analyzed using unpaired t tests. r–t Western blot analysis showed a significant increase in Akt phosphorylation levels in zebrafish under LL condition. All experiments were repeated three times. Bar graphs represent the mean ± standard error of the mean (SEM). (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 2. Evaluation of the physiological toxicity of lactic acid and 2DG on zebrafish larvae.

a The chemical formula of lactic acid. b Experimental procedure for the effects of lactic acid and 2DG on zebrafish growth and development. c Heartbeat of 48 hpf zebrafish larvae (beats per minute, n = 15). d Heartbeat of 72 hpf zebrafish larvae (beats per minute, n = 15). e Survival rate curve of zebrafish larvae from 0 to 96 hpf (% survival, n = 15). f Tail swing frequency of 30 hpf zebrafish larvae (swing per minute, n = 15). g Statistical graph of body length of 96 hpf zebrafish larvae (mm, n = 15). h, i Morphological images of 96 hpf zebrafish larvae observed under a stereo microscope (scale bar: 350 μm). There were no significant differences in morphology between the lactic acid- and 2DG-treated groups compared to the control group. Data were analyzed using one-way ANOVA and log-rank test. All experiments were repeated three times. Bar graphs represent the mean ± standard error of the mean (SEM). (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 3. H3K18 lactylation regulates neutrophil recruitment.

a, b Western blotting results showed that exogenous addition of lactic acid significantly enhanced the lactylation of H3K18. c, d Inhibition of lactate with 2DG resulted in a significant downregulation of H3K18 lactylation. e, f Statistical analysis showed that lactate significantly increased neutrophil recruitment at the site of injury under LD conditions, while 2DG significantly decreased neutrophil recruitment (n = 30). The white rectangles in the images represent the counting areas (scale bar: 200 μm). g Neutrophils migrated to the caudal fin injury site after 3 h of injury after lactic acid and 2DG treatment under LL condition. The white rectangles in the images represent the counting areas (scale bar: 200 μm). h Statistical analysis revealed that lactic acid significantly increased the recruitment of neutrophil to the injury site, while 2DG significantly decreased neutrophil recruitment (n = 30). i, j In larvae that were not subjected to fin injury, there was no significant difference in the neutrophil counts in the counting area between the control group and the treatment group. The white rectangles in the images represent the counting areas (scale bar: 200 μm) (n = 15). k, l Lactic acid increased the recruitment of neutrophils to otic vesicles 3 h after LPS challenge, while 2DG significantly decreased the recruitment (n = 30). The white rectangles in the images represent the counting areas (scale bar: 200 μm) (n = 30). m, n In larvae that were not subjected to microinjection of LPS, there was no significant difference in the neutrophil counts in the counting area between the control group and the treatment group. The white rectangles in the images represent the counting areas (scale bar: 200 μm) (n = 15). Unpaired tests and one-way ANOVA were used to analyze the differences. All experiments were repeated three times. Bar graphs represent the mean ± standard error of the mean (SEM). (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 4. H3K18 lactylation regulates ROS levels and akt expression.

a KEGG enrichment analysis showed the signaling pathways of genes bound by histone lactylation (H3K18). b GO-BP enrichment analysis showed the biological processes of genes bound by histone lactylation (H3K18). c–f ChIP-qPCR results showed that under LD conditions, lactylated H3K18 binds to the transcription region of akt1 and duox, and similar results are observed under LL conditions. g Q-PCR showed that lactic acid significantly upregulated the expression of akt1, while 2DG significantly downregulated the expression of akt1 under LL condition. h–j Western blotting showed that lactic acid and 2DG significantly upregulated and downregulated the expression of Akt protein under LL condition. k Q-PCR showed that lactic acid significantly upregulated the expression of duox, while 2DG significantly downregulated the expression of duox under LL condition. l, m Fluorescent staining results showed that lactic acid upregulated the levels of reactive oxygen species (ROS), while 2DG downregulated the levels of ROS under LL condition. n Q-PCR showed that lactic acid significantly upregulated the expression of duox, while 2DG significantly downregulated the expression of duox under LD condition. o, p Fluorescent staining results showed that lactic acid upregulated the levels of reactive oxygen species (ROS), while 2DG downregulated the levels of ROS under LD condition. Data were statistically analyzed using unpaired t tests and one-way ANOVA. All experiments were repeated three times. Bar graphs represent the mean ± standard error of the mean (SEM). (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 5. ROS promote H3K18 lactylation and lactic acid metabolism.

a, b Fluorescence staining experiments revealed a significant increase in reactive oxygen species (ROS) levels in zebrafish larvae upon the addition of 100 μM hydrogen peroxide (n = 15). c Q-PCR results showed a significant upregulation of the oxidative stress gene duox upon treatment with 100 μM hydrogen peroxide. d, e The addition of 100 μM hydrogen peroxide significantly reduced the expression of the histone delactylation genes hdac3 and hdac12. f The expression of the histone lactylation gene ep300a was significantly upregulated. g, h Treatment with 100 μM hydrogen peroxide significantly increased the level of histone lactylation (H3K18). i, j Fluorescence staining experiments showed a significant inhibition of reactive oxygen species (ROS) levels in zebrafish larvae under LL condition upon treatment with 10 μM DPI. k Treatment with 10 μM DPI significantly reduced the expression level of the oxidative stress gene duox. l, m DPI treatment significantly inhibited the H3K18 lactylation under LL condition. Data were statistically analyzed using unpaired t tests and one-way ANOVA. All experiments were repeated three times. Bar graphs represent the mean ± standard error of the mean (SEM). (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 6. ROS and Akt regulate neutrophil recruitment under LL condition.

a–c Western blotting results showed that MK-2206 significantly inhibited the expression of Akt. d The image showed that neutrophils migrated to the damaged caudal fin 3 h after injury after treatment with DPI. e The statistical graph shows that MK-2206 and DPI significantly inhibited the recruitment of neutrophils to the injured site of the caudal fin under LD condition (n = 20). f The image showed that neutrophils migrated to the damaged caudal fin 3 h after injury after treatment with MK-2206 and DPI. White rectangles represent counting areas (scale bar: 200 μm). g The statistical graph shows that MK-2206 and DPI significantly inhibited the recruitment of neutrophils to the injured site of the caudal fin under LL condition (n = 20). h The image showed that after treatment with MK-2206 and DPI, neutrophils migrated to the otic vesicle inflammation site 3 h after injury. White rectangles represent counting areas (scale: 200 μm). i The statistical graphs show that MK-2206 and DPI significantly inhibited the recruitment of neutrophils to the otic vesicle inflammation under LL condition (n = 20). The data were analyzed using unpaired t tests and one-way ANOVA. All experiments were repeated three times. Bar graphs represent the mean ± standard error of the mean (SEM). (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 7. Illustration of the mechanism underlying the regulation of photoperiodic disruption in neutrophil recruitment.

Photoperiodic interference affects the level of H3K18 lactylation, regulating the transcriptional activation of the duox gene and resulting in an increase in reactive oxygen species (ROS) levels. Additionally, H3K18 lactylation activates Akt expression. The generation of ROS further enhances the level of H3K18 lactylation, forming a positive feedback loop between histone lactylation and ROS. This positive feedback loop leads to excessive recruitment of neutrophil under LL condition.