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. 2020 Jan 10:10:2956.
doi: 10.3389/fimmu.2019.02956. eCollection 2019.

Profile of Histone H3 Lysine 4 Trimethylation and the Effect of Lipopolysaccharide/Immune Complex-Activated Macrophages on Endotoxemia

Vichaya Ruenjaiman  1 , Patcharavadee Butta  2 , Yu-Wei Leu  3 , Monnat Pongpanich  4 , Asada Leelahavanichkul  1 , Patipark Kueanjinda  5 , Tanapat Palaga  1   2
Affiliations

Profile of Histone H3 Lysine 4 Trimethylation and the Effect of Lipopolysaccharide/Immune Complex-Activated Macrophages on Endotoxemia

Vichaya Ruenjaiman et al. Front Immunol. .

Abstract

Macrophage plasticity is a process that allows macrophages to switch between two opposing phenotypes based on differential stimuli. Interferon γ (IFNγ)-primed macrophages stimulated with lipopolysaccharide (LPS) [M(IFNγ+LPS)] produce high levels of pro-inflammatory cytokines such as IL-12, TNFα, and IL-6 and low levels of the anti-inflammatory cytokine IL-10, while those stimulated with LPS in the presence of the immune complex (IC) [M(IFNγ+LPS+IC)] produce high levels of IL-10 and low levels of IL-12. In this study, we investigated the plasticity between M(IFNγ+LPS) and M(IFNγ+LPS+IC) in vitro and compared one of the active histone marks [histone H3 lysine 4 trimethylation (H3K4me3)] between M(IFNγ+LPS) and M(IFNγ+LPS+IC) using murine bone marrow-derived macrophages. We found that in an in vitro system, macrophages exhibited functional plasticity from M(LPS) to M(LPS+IC) upon repolarization after 2 days of washout period while IFNγ priming before LPS stimulation prevented this repolarization. Phosphorylation of p38, SAPK/JNK, and NF-κB p65 in M(LPS+IC) repolarized from M(LPS) was similar to that in M(LPS+IC) polarized from resting macrophages. To obtain the epigenetic profiles of M(IFNγ+LPS) and M(IFNγ+LPS+IC), the global enrichment of H3K4me3 was evaluated. M(IFNγ+LPS) and M(IFNγ+LPS+IC) displayed marked differences in genome-wide enrichment of H3K4me3. M(IFNγ+LPS+IC) showed increased global enrichment of H3K4me3, whereas M(IFNγ+LPS) showed decreased enrichment when compared to unstimulated macrophages. Furthermore, M(IFNγ+LPS+IC) exhibited high levels of H3K4me3 enrichment in all cis-regulatory elements. At the individual gene level, the results showed increased H3K4me3 enrichment in the promoters of known genes associated with M(IFNγ+LPS+IC), including Il10, Cxcl1, Csf3, and Il33, when compared with those of M(IFNγ+LPS). Finally, we investigated the impact of M(IFNγ+LPS+IC) on the systemic immune response by adoptive transfer of M(IFNγ+LPS+IC) in an LPS-induced endotoxemia model. The cytokine profile revealed that mice with adoptively transferred M(IFNγ+LPS+IC) had acutely reduced serum levels of the inflammatory cytokines IL-1β and IL-p12p70. This study highlights the importance of epigenetics in regulating macrophage activation and the functions of M(IFNγ+LPS+IC) that may influence macrophage plasticity and the potential therapeutic use of macrophage transfer in vivo.

Keywords: H3K4me3; LPS; endotoxemia; epigenetics; immune complex; macrophage.

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Figures

Figure 1
Figure 1
Repolarization of M(IFNγ+LPS) to M(LPS+IC). (A,B) The protocol used for stimulation of IFNγ-primed macrophages with LPS and LPS with IC is shown with the washout period of 2 or 48 h. IL-10 and IL-12p70 in the culture supernatant harvested from cells treated as described were analyzed by ELISA. The results indicate the means ± SD of triplicates determined from three independent experiments. *A significant difference at p < 0.05.
Figure 2
Figure 2
Repolarization of M(LPS) to M(LPS+IC) and signaling downstream of TLR4. (A) The protocol used for macrophage stimulation with LPS and LPS with IC is shown. (B) IL-10 and IL-12p70 in the culture supernatant harvested from cells treated as described in (A) were analyzed by ELISA. The results indicate the means ± SD of triplicates determined from three independent experiments. *A significant difference at p < 0.05. (C) BMDMs were polarized to M(LPS) and allowed to rest for 2 or 48 h. After re-stimulation with LPS and IC, phosphorylation of MAPKs, NF-κB p65 and Akt was detected in cell lysates by Western blot. β-actin was used as a control. Representative data from one of three independent experiments are shown.
Figure 3
Figure 3
Global enrichment of H3K4me3 in unstimulated macrophages, M(IFNγ+LPS) and M(IFNγ+LPS+IC). BMDMs were primed with IFNγ before stimulation with LPS with or without IC for 4 h. Cells were harvested for ChIP using an anti-H3K4me3 antibody. The ChIP samples were subjected to DNA sequencing. (A) Circos plot showing genome-wide H3K4me3 enrichment in unstimulated macrophages, M(IFNγ+LPS) and M(IFNγ+LPS+IC). The positions of log-transformed H3K4me3 enrichment in unstimulated macrophages (green circle), M(IFNγ+LPS) (red circle) and M(IFNγ+LPS+IC) (blue circle) were aligned according to chromosome position in the outer ring. (B) The total peaks after identification of H3K4me3 enrichment with MACS 1.4 were used to compare the overlap of H3K4me3 enrichment peaks, and the results are presented in a Venn diagram. (C) The cis-regulatory element annotation system (CEAS) showed the distribution pattern of H3K4me3 enrichment between M(IFNγ+LPS) and M(IFNγ+LPS+IC). All ChIP-seq results were analyzed from combined RAW files of two independent experiments.
Figure 4
Figure 4
The epigenomic correlation between M(IFNγ+LPS) and M(IFNγ+LPS+IC). (A) A scatter plot from EpiMINE showing the epigenomic correlation between M(IFNγ+LPS) and M(IFNγ+LPS+IC) in the cis-regulatory elements and other regions. (B) The PCA plots show the differences in the two top principal components between M(IFNγ+LPS) and M(IFNγ+LPS+IC).
Figure 5
Figure 5
Enrichment of H3K4me3 in cis-regulatory regions. (A) ChIP-seq density heatmap of H3K4me3 enrichment at the TSSs and the 1 kb region nearby. (B) ChIP-seq profiling of H3K4me3 enrichment over a 5 kb window around TSSs. (C) Quantification of H3K4me3 enrichment within the promoter regions between M(IFNγ+LPS) and M(IFNγ+LPS+IC).
Figure 6
Figure 6
Enrichment of H3K4me3 in the target loci of M(IFNγ+LPS+IC). IGV was used to compare M(IFNγ+LPS+IC)-related loci of uniquely upregulated genes (A) and uniquely downregulated genes (B) with H3K4me3 enrichment. (C) Quantification of H3K4me3 enrichment in the promoter regions of the target genes between M(IFNγ+LPS) and M(IFNγ+LPS+IC).
Figure 7
Figure 7
KEGG pathway analysis of the differentially H3K4me3-enriched genes in M(IFNγ+LPS+IC). Gene Ontology analysis using KEGG pathway analysis displayed the significant pathways with differential H3K4me3 enrichment genes in M(IFNγ+LPS+IC). The bar plot showed the ranking of pathways correlated to Immune system, Signal transduction and Signaling molecule (dashed line represents adjusted p-value cut-off at 0.05) in M(IFNγ+LPS+IC).
Figure 8
Figure 8
The therapeutic application of M(IFNγ+LPS+IC) in a mouse endotoxemia model. (A) The protocol used for the adoptive transfer of M(IFNγ+LPS+IC) in the endotoxemia model. (B) Blood sera at 1 and 6 h after LPS challenge from mice with adoptive transfer of unstimulated macrophages or M(IFNγ+LPS+IC) were subjected to Bioplex cytokine assays for IL-6, IL-12p70, IL-1β, TNF-α, IL-17, IL-10, and IL-4. *Statistical significance at p < 0.05. The results represent the mean ± SEM of each group (n = 4).
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