Lactoylglutathione promotes inflammatory signaling in macrophages through histone lactoylation

Abstract

Chronic, systemic inflammation is a pathophysiological manifestation of metabolic disorders. Inflammatory signaling leads to elevated glycolytic flux and a metabolic shift towards aerobic glycolysis and lactate generation. This rise in lactate corresponds with increased generation of lactoylLys modifications on histones, mediating transcriptional responses to inflammatory stimuli. Lactoylation is also generated through a non-enzymatic S-to-N acyltransfer from the glyoxalase cycle intermediate, lactoylglutathione (LGSH). Here, we report a regulatory role for LGSH in mediating histone lactoylation and inflammatory signaling. In the absence of the primary LGSH hydrolase, glyoxalase 2 (GLO2), RAW264.7 macrophages display significant elevations in LGSH and histone lactoylation with a corresponding potentiation of the inflammatory response when exposed to lipopolysaccharides. An analysis of chromatin accessibility shows that lactoylation is associated with more compacted chromatin than acetylation in an unstimulated state; upon stimulation, however, regions of the genome associated with lactoylation become markedly more accessible. Lastly, we demonstrate a spontaneous S-to-S acyltransfer of lactate from LGSH to CoA, yielding lactoyl-CoA. This represents the first known mechanism for the generation of this metabolite. Collectively, these data suggest that LGSH, and not intracellular lactate, is the primary driving factor facilitating histone lactoylation and a major contributor to inflammatory signaling.

Keywords: Glyoxalase; Inflammation; Lactate; Metabolism; Post-translational modification.

Figure 1

GLO2 ablation exacerbates the inflammatory response. A. The glyoxalase cycle detoxifies the glycolytic by-product, MGO, in a two-step process that cycles GSH. LGSH, the primary substrate for GLO2, serves as an acyl donor to free Lys residues yielding lactoylLys. B. KLA stimulation results in increased COX-2 and iNOS expression, which is exacerbated in KO cells. C-E. PGE₂, PGF_2⍺ and nitrite levels increase in KO cells following KLA treatment. F-H. Pro-inflammatory cytokines TNF⍺ and IL-6 are elevated in KO cells following KLA treatment while IL-10 (anti-inflammatory) is decreased. N = 6 +/− SD. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01 by two-way ANOVA. Relative change: normalized to WT vehicle. Abbreviations: (L)GSH, (lactoyl)glutathione; MGO, methylglyoxal; HTA, hemithioacetal; GLO1, glyoxalase 1; GLO2, glyoxalase 2. KLA, Kdo₂-Lipid A; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; ARG1, arginase 1; STAT3, signal transducer and activator of transcription 3; PG(E₂, F_2⍺), prostaglandin (E₂, F_2⍺); TNF⍺, tumor necrosis factor ⍺; IL(-6,10), interleukin(-6,10). See also Figure S1.

Figure 2

LGSH is a potential metabolic source for lactoyl-CoA. A. Lactoyl-CoA generation is proposed to be derived from lacate and CoA. B-D. Stable isotope tracing of WT and KO cells treated with KLA reveals a significant increase in triose phosphates with no significant alterations in flux to pyruvate or lactate. E. The proposed mechanistic route for the generation of lactoyl-CoA from LGSH. F. Thioester migration is more efficient with lactate than acetate, indicating LGSH is a likely metabolic source for lactoyl-CoA. G. Measurement of acylLys on histone H3.1 H. acyl-CoA and acylGSH incubations with recombinant histone H3.1 demonsrate non-enzymatic acylation in vitro.I. MGO is significantly increased in KLA treated cells, agreeing with B. J. LGSH is significantly increased in KO cells and this effect is potentiated in response to KLA. K. Lactoyl-CoA, and not L. acetyl-CoA, is increased in KO cells. N = 3–6 +/− SD. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05 by two-way ANOVA (B-D, i-l) or one-way ANOVA (H). Relative change: normalized to WT vehicle. Abbreviations: CoA, Coenzyme A.

Figure 3

Histone lactoylation is elevated in a site-specific manner. A. Basal histone lactoylation is higher in KO cells, observed via immunoblotting. Representative blot, N = 3. B. Specific histone sites have quantitative differences in lactoylation status in WT and GLO2 KO cells after inflammatory stimuli. C-K. Acetylation and lactoylation are dynamically regulated at specific loci, normalized to WT vehicle. L. Lactoylation is a low abundance mark compared to other canonical modifications. N = 6 +/− SEM. ∗∗∗^/###P < 0.001, ∗∗^/##P < 0.01 by two-way ANOVA. ∗: WT to WT compared, ^#: WT to KO compared. Abbreviations: ac, acetyl; lac, lactoyl; me, methyl. See also Figure S2, Table S1.

Figure 4

Differences in chromatin accessibility are attributed to increases in site specific elevations of histone lactoylation. A. The number of peaks identified as either unique or common between WT and KO vehicle or between WT and KO KLA samples are shown. The heatmap visualizes the proportion of ATAC peaks overlapping with each ChIP-seq dataset. B,C. PCA analysis (B) and correlation heatmap (C) of ATAC-seq data where N = 3. The The distance matrix in correlation heatmap was calculated as 1 - Pearson correlation coefficient. D. GLO2 KO-driven changes in chromatin accessibility are highly enriched in H3K18lac loci. Shown here are the number of differentially accessible peaks either gaining (Up) or losing accessibility (Down) in KO samples relative to the appropriate WT condition. Also indicated are the hypergeometric test enrichments for overlap of differentially accessible peaks identified in each contrast (column) with ChIP-seq datasets targeting H3K18ac and H3K18lac modifications in vehicle or LPS-treated macrophages (row, GEO accession: GSE115354). Only the tests having adjusted p-values lower than 0.05 are shown. E. Schematic model of chromatin dynamics driven by lactoylation and KLA stimulation. F,G. KEGG pathway enrichment in KLA-induced peaks either commonly (F) or uniquely (G) identified in WT and GLO2 KO samples. In panel (F), the top 20 enriched pathways with an adjusted p-value equal to zero are shown for common changes between genotypes in response to stimulation. The color code denotes the fold enrichment and dot size represents the number of regions observed in each pathway. The top 10 significant pathways that were uniquely identified in each contrast are shown in panel (G). The color represents the -log10-transformed adjusted p-value derived from the binomial tests. The dot size denotes the number of observed regions in each test. See also Table S2-S10. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 5

Enzymatic and non-enzymatic lactoylation are indistinguishable. A. Schematic representation of the inhibitor strategy used. B,C. PGs are significantly elevated in KO cells where p300 inhibition results in a significant increase in the inflammatory response. BET inhibition blunts this response regardless of the metabolic source of lactoylation. Lastly, HDAC inhibition results in a significant increase in PG production, indicating that both enzymatic and non-enzymatic marks are erased by HDACs on chromatin. D. Lactoylation of histones occurs in a site-specific manner, even after inhibition of the enzymatic writer, p300. HDAC inhibition elevates global histone lactoylation. Lactoylation is sensitive to JQ-1 treatment. Samples normalized to WT KLA-treated. Only KLA treated samples are shown. N = 5–6 +/− SD. ∗∗∗∗^/####P < 0.0001, ∗∗∗P < 0.001, ∗∗^/##P < 0.01, ∗P < 0.05 by two-way ANOVA. ∗ indicates comparisons made to WT KLA; ^# indicates comparisons made between WT to KO of the same treatment. Abbreviations: HDAC, histone deacetylase; BET, bromodomain and extra-terminal motif; TSA, trichostatin A. See also Figure S3.