Inhibition of myeloid HDAC2 upregulates glutaredoxin 1 expression, improves protein thiol redox state and protects against high-calorie diet-induced monocyte dysfunction and atherosclerosis
Graphical abstract
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.
References (78)
96 - monocytic glutaredoxin 1 protects mice against obesity, hyperglycemia and atherosclerosis
Free Radic. Biol. Med.
(2016)Macrophage polarization in response to epigenetic modifiers during infection and inflammation
Drug Discov. Today
(2017)Epigenetic regulation of the alternatively activated macrophage phenotype
Blood
(2009)In vivo chromatin remodeling events leading to inflammatory gene transcription under diabetic conditions
J. Biol. Chem.
(2004)- et al.
Mitogen-activated protein kinase phosphatase 1 (MKP-1) in macrophage biology and cardiovascular disease. A redox-regulated master controller of monocyte function and macrophage phenotype
Free Radical Biol. Med.
(2017) Histone deacetylases and atherosclerosis
Atherosclerosis
(2015)- et al.
Histone deacetylase inhibitors: new drugs for the treatment of inflammatory diseases?
Drug Discov. Today
(2005) Potential epigenetic therapeutics for atherosclerosis treatment
Atherosclerosis
(2019)HDAC inhibition helps post-MI healing by modulating macrophage polarization
J. Mol. Cell. Cardiol.
(2018)Valproate attenuates accelerated atherosclerosis in hyperglycemic apoE-deficient mice: evidence in support of a role for endoplasmic reticulum stress and glycogen synthase kinase-3 in lesion development and hepatic steatosis
Am. J. Pathol.
(2009)