Elsevier

Atherosclerosis

Volume 328, July 2021, Pages 23-32
Atherosclerosis

Inhibition of myeloid HDAC2 upregulates glutaredoxin 1 expression, improves protein thiol redox state and protects against high-calorie diet-induced monocyte dysfunction and atherosclerosis

https://doi.org/10.1016/j.atherosclerosis.2021.05.002Get rights and content

Highlights

  • Myeloid HDAC2 deficiency in high-calorie diet-fed LDL-R−/− mice reduces atherosclerosis in males by 39% but has no effect in female mice.

  • HDAC2 deficiency increases acetylation on histone 3 and induces Grx1 expression in male but not female blood monocytes.

  • HDAC2 deficiency reduces protein S-glutathionylation and chemotactic activity of male but not female monocytes.

Abstract

Background and aims

The thiol transferase glutaredoxin 1 controls redox signaling and cellular functions by regulating the S-glutathionylation status of critical protein thiols. Here we tested the hypothesis that by derepressing the expression of glutaredoxin 1, inhibition of histone deacetylase 2 prevents nutrient stress-induced protein S-glutathionylation and monocyte dysfunction and protects against atherosclerosis.

Methods

Using both a pharmacological inhibitor and shRNA-mediated knockdown of histone deacetylase 2, we determine the role of this deacetylase on glutaredoxin 1 expression and nutrient stress-induced inactivation of mitogen-activated protein kinase phosphatase 1 activity and monocyte and macrophage dysfunction. To assess whether histone deacetylase 2 inhibition in myeloid cells protects against atherosclerosis, we fed eight-week-old female and male HDAC2−/−MyeloidLDLR−/− mice and age and sex-matched LysMcretg/wtLDLR−/− control mice a high-calorie diet for 12 weeks and assessed monocyte function and atherosclerotic lesion size.

Results

Myeloid histone deacetylase 2 deficiency in high-calorie diet-fed LDLR−/− mice reduced atherosclerosis in males by 39% without affecting plasma lipid and lipoprotein profiles or blood glucose levels but had no effect on atherogenesis in female mice. Macrophage content in plaques of male mice was reduced by 31%. Histone deacetylase 2-deficient blood monocytes from male mice showed increased acetylation on histone 3, and increased Grx1 expression, and was associated with increased MKP-1 activity and reduced recruitment of monocyte-derived macrophages, whereas in females, myeloid HDAC2 deficiency had no effect on Grx1 expression, did not prevent nutrient stress-induced loss of MKP-1 activity in monocytes and was not atheroprotective.

Conclusions

Specific histone deacetylase 2 inhibitors may represent a potential novel therapeutic strategy for the prevention and treatment of atherosclerosis, but any benefits may be sexually dimorphic.

Introduction

Atherosclerosis is a progressive chronic inflammatory disease and the main contributor to cardiovascular diseases (CVD) worldwide [[1], [2], [3]]. The exact mechanisms underlying these diseases are still not fully understood, but increasing evidence suggests that diet-induced monocyte and macrophage dysfunction plays a major causal role in the conversion of acute into chronic inflammation [4,5] and the onset of atherogenesis [5]. Current lipid-lowering therapies have only had a limited effect on CVD mortality [6], particularly in patients unable to tolerate statins (15–20%) [7]. Novel strategies are urgently needed to further lower the incidence of CVD and CVD-related deaths.

The recruitment of monocyte-derived macrophages into plaques is a rate liming step in atherogenesis [2,8,9]. Nutrient stress triggered by a high-calorie diet (HCD) promotes monocyte reprogramming and dysfunction, increases monocyte chemotaxis and accelerates the recruitment of monocyte-derived macrophages into atherosclerotic plaques [10], a process that is mediated by oxidative stress and increased protein S-glutathionylation [11]. Protein S-glutathionylation is the reversible formation of mixed disulfides between protein thiols and the tripeptide glutathione (GSH) in response to oxidative stress [12]. The reduction of these mixed disulfides is catalyzed by thiol transferases called glutaredoxins, the most abundant and cytosolic form of this enzyme being glutaredoxin 1 (Grx1) [13]. We showed that Grx1 protects blood monocytes from nutrient stress-induced priming, dysregulation, and hypersensitization to chemokines [14]. Our most recent data demonstrates that Grx1 deficiency sensitizes monocytes and macrophages to nutrient stress-induced dysfunction and reprogramming, accelerating diet-induced obesity, metabolic syndrome, hyperglycemia, insulin resistance and atherosclerosis [14,15].

Epigenetic modifications in monocytes and macrophages, including histone acetylations, control cellular functions, including monocytes adhesion and macrophage polarization and immune responses, and are associated with altered gene expression profiles [[16], [17], [18], [19], [20], [21]]. Acetylation of histone lysine residues is highly dynamic and mediated by two families of enzymes: histone acetyltransferases (HAT) and histone deacetylases (HDAC) [22,23]. In general, histone acetylation leads to loosening of the chromatin and increased gene expression, albeit not in all cases, whereas deacetylation usually leads to reduced gene expression [24,25]. Both HAT and HDAC are involved in the regulation of macrophage activation, immune response and functional differentiation [26,27].

Acetylation of non-histone proteins in monocytic cells has also been reported and affects protein stability, interactions with other proteins and protein functions [[28], [29], [30]]. For example, HDAC1, 2 and 3 deacetylate MKP-1, decreasing MAPK signaling and dampening innate immune functions [31,32]. HDAC inhibitors (HDACi) promote MKP-1 acetylation and increase binding of MKP-1 to its substrates, resulting in the inhibition of p38 MAPK activity and the downregulation of inflammatory genes, including TNF-α, iNOS and IL-1β [33,34]. Furthermore, in lipopolysaccharide (LPS)-treated cultured murine RAW macrophage-like cells, HDACi and RNAi-mediated HDAC1-3 silencing in a dose-dependent manner suppressed mRNA levels of pro-inflammatory mediators, including iNOS, TNF-α, IL-1β, and IL-6 [35,36].

HDAC regulate the expression of many genes associated with inflammation and CVD [[37], [38], [39], [40], [41]]. It is therefore not surprising that in recent years, HDACi have emerged as potential therapies for the suppression of macrophage-mediated inflammation and the prevention and treatment of atherosclerosis [[42], [43], [44]]. HDACi inhibit cholesterol metabolism, stimulate transcription of proapoptotic genes and increase inflammation resolution [[45], [46], [47], [48]]. It has been reported that the knockdown of HDAC2 in LPS-stimulated macrophages decreases the expression of pro-inflammatory genes, including IL-12, TNF- α and iNOS, and adoptive transfer of HDAC2-deficient macrophages in mice attenuates LPS-triggered inflammatory responses [49]. HDACi have anti-atherogenic effects as they downregulate macrophage pro-inflammatory activation by reducing cytokine expression and stress-induced macrophage apoptosis mediated by Fas–Fas ligand interactions and cytokine withdrawal [[50], [51], [52], [53]]. However, the complex roles HDAC play in atherogenesis are still poorly understood.

Here we report on a novel pathway through which HDAC2 controls monocyte and macrophage signaling and function, and promotes atherosclerosis. Targeting this pathway may present a new therapeutic avenue for the prevention and treatment of atherosclerosis.

Section snippets

Isolation of blood monocytes and bone marrow-derived macrophages

Blood was collected from mice by cardiac puncture. Red blood cells were lysed and monocytes were isolated and purified by negative selection (RoboSepTM Mouse Monocytes Isolation Kit, StemCell Technologies) using a RoboSep automated cell isolation system (StemCell Technologies). To generate bone marrow-derived macrophages (BMDM), myeloid progenitor cells were isolated from the bone marrow of C57BL/6 mice and cultured for 5 days in the presence of 50 ng/ml macrophage colony-stimulating factor

Results

To identify a potential role for HDACs in the regulation of the thiol transferase Grx1 in macrophages, in a proof-of-principle study, we treated BMDM, a model of blood monocyte-derived macrophages, with the class I HDAC inhibitor Scriptaid (1–5 μM) for up to 72 h. We found that in the presence of Scriptaid, Grx1 showed a strong, dose- and time-dependent induction, both at the mRNA and the protein level (Supplementary Fig. 1A and B). Grx1 showed the highest mRNA expression (13-fold increase) at

Discussion

Monocyte and macrophage functions are under the control of epigenetic enzymes and targeting epigenetic enzymes has proven to be an effective tool to dampen inflammatory responses [65,66]. HDACs, which mediate lysine deacetylation on histones, regulate immunometabolism and inflammatory outputs of macrophages [31]. In this study, we demonstrate that HDAC2 inhibition protects monocytes and macrophages against nutrient stress-induced dysfunction and reprogramming. The HDACi Scriptaid induced the

Financial Support

This project was supported by a grant to R.A. from the NIH (AT006885).

Author contributions

L.W. and Y.J.A. performed experiments and contributed significantly in preparing the manuscript. R.A. provided funding, designed the experiments and contributed significantly to the preparation and editing of the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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