key: cord-0029858-7ei6s9nz
authors: Fu, Li; Wasiak, Sylwia; Tsujikawa, Laura M.; Rakai, Brooke D.; Stotz, Stephanie C.; Wong, Norman C. W.; Johansson, Jan O.; Sweeney, Michael; Mohan, Connie M.; Khan, Aneal; Kulikowski, Ewelina
title: Inhibition of epigenetic reader proteins by apabetalone counters inflammation in activated innate immune cells from Fabry disease patients receiving enzyme replacement therapy
date: 2022-04-13
journal: Pharmacol Res Perspect
DOI: 10.1002/prp2.949
sha: 851910eddf36bc7a9c138b4d6c27b205942559da
doc_id: 29858
cord_uid: 7ei6s9nz

Fabry disease (FD) is a rare X‐linked disorder of lipid metabolism, characterized by the accumulation of globotriaosylceramide (Gb3) due to defective the lysosomal enzyme, α‐galactosidase. Gb3 deposits activate immune‐mediated systemic inflammation, ultimately leading to life‐threatening consequences in multiple organs such as the heart and kidneys. Enzyme replacement therapy (ERT), the standard of care, is less effective with advanced tissue injury and inflammation in patients with FD. Here, we showed that MCP‐1 and TNF‐α cytokine levels were almost doubled in plasma from ERT‐treated FD patients. Chemokine receptor CCR2 surface expression was increased by twofold on monocytes from patients with low eGFR. We also observed an increase in IL12B transcripts in unstimulated peripheral blood mononuclear cells (PBMCs) over a 2‐year period of continuous ERT. Apabetalone is a clinical‐stage oral bromodomain and extra terminal protein inhibitor (BETi), which has beneficial effects on cardiovascular and kidney disease related pathways including inflammation. Here, we demonstrate that apabetalone, a BD2‐selective BETi, dose dependently reduced the production of MCP‐1 and IL‐12 in stimulated PBMCs through transcriptional regulation of their encoding genes. Reactive oxygen species production was diminished by up to 80% in stimulated neutrophils following apabetalone treatment, corresponding with inhibition of NOX2 transcription. This study elucidates that inhibition of BET proteins by BD2‐selective apabetalone alleviates inflammatory processes and oxidative stress in innate immune cells in general and in FD. These results suggest potential benefit of BD2‐selective apabetalone in controlling inflammation and oxidative stress in FD, which will be further investigated in clinical trials.

less effective with advanced tissue injury and inflammation in patients with FD. Here, we showed that MCP-1 and TNFα cytokine levels were almost doubled in plasma from ERT-treated FD patients. Chemokine receptor CCR2 surface expression was increased by twofold on monocytes from patients with low eGFR. We also observed an increase in IL12B transcripts in unstimulated peripheral blood mononuclear cells (PBMCs) over a 2-year period of continuous ERT. Apabetalone is a clinical-stage oral bromodomain and extra terminal protein inhibitor (BETi), which has beneficial effects on cardiovascular and kidney disease related pathways including inflammation. Here, we demonstrate that apabetalone, a BD2-selective BETi, dose dependently reduced the production of MCP-1 and IL-12 in stimulated PBMCs through transcriptional regulation of their encoding genes. Reactive oxygen species production was diminished by up to 80% in stimulated neutrophils following apabetalone treatment, corresponding with inhibition of NOX2 transcription. This study elucidates that inhibition of BET proteins by BD2-selective apabetalone alleviates inflammatory processes and oxidative stress in innate immune cells in general and in FD. These results suggest potential benefit of BD2-selective apabetalone in controlling inflammation and oxidative stress in FD, which will be further investigated in clinical trials.

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding the lysosomal enzyme α-galactosidase A (α-GAL). 1 These mutations result in deficient α-GLA activity and accumulation of globotriaosyceramide (also known as Gb3) and globotriaosylsphingosine (lyso-Gb3) in various tissues and cell types. Gb3 deposits activate pathogenic mechanisms such as harmful pro-inflammatory responses and oxidative stress, 2,3 subsequently leading to tissue injury in organs throughout the body including the heart and kidneys. Cardiovascular and renal dysfunction, which commonly manifest as cardiac hypertrophy and a decline in eGFR respectively, remain the leading causes of death in FD. 4, 5 Enzyme replacement therapy (ERT), the standard of care for nearly 20 years, 6, 7 reduces Gb3 levels and improves clinical outcomes in the short term. 8, 9 However, ERT is less effective when tissue injury has developed and abnormal immune responses persist. 2 Activation of the innate component of the immune system by Gb3 through Toll-like receptor 4 (TLR4) triggers pro-inflammatory responses in FD. 10 Subsequently, activated innate immune cells (e.g., monocytes and neutrophils) produce deleterious inflammatory cytokines and chemokines, driving FD progression. Elevated levels of interleukin-6 (IL-6) and tumor necrosis factorα (TNFα) have been detected in FD patients' peripheral blood mononuclear cells (PBMCs) compared with healthy controls. 10 Increased plasma IL-6 and TNFα levels also positively correlate with incidence of left ventricular hypertrophy in patients with chronic kidney disease (CKD) and cardiovascular disease (CVD). 11, 12 Furthermore, the chemokine monocyte chemoattractant protein-1 (MCP-1), involved in monocyte movement, is associated with CVD progression over 1 year of ERT, 12 while interleukin-12 (IL-12), a monocytederived T helper 1 (Th1)-type cytokine that promotes T cell responses, has been proposed to mediate abnormal T cell activity in patients with FD. 13 T cell appearance in damaged myocardium during long-term ERT 14 implies aberrant T cell infiltration which can result in further tissue damage.

Oxidative stress, associated with excessive production of reactive oxygen species (ROS) is another driver of FD progression. 15 Although ERT has been reported to alleviate oxidative stress in Gb3 stimulated human vascular endothelial cells, 16 recent in vivo studies reveal limitations of ERT in modulating oxidative stress in treated FD patients. [17] [18] [19] Pro-oxidant conditions and oxidative damage correlate with elevated plasma levels of pro-inflammatory cytokines following continuous ERT (~2 years) 19 demonstrating a potential link between abnormal immune responses and ROS production. These findings suggest that novel therapeutic approaches targeting pathological inflammation and/or oxidative stress are necessary to complement the standard of care in FD to further optimize patient outcomes.

Bromodomain and extra-terminal (BET) proteins, termed epigenetic "readers", 20 have been identified as therapeutic targets for disease prevention due to their pivotal role in regulating the transcription of inflammatory genes. [21] [22] [23] BET proteins bind acetylated lysine residues on histone tails and other nuclear proteins 24 through their conserved N-terminal bromodomains (BD1 and BD2) 25, 26 to recruit and/or facilitate assembly of factors needed for regulation of gene expression.

Notably, BET proteins have been shown to cooperate with nuclear fac- Apabetalone binds preferentially to the second bromodomain (BD2) of BET family members BRD2, BRD3 and BRD4 with >20-fold selectivity over BD1. 32, 33 This BD2 dominant binding blocks BET interactions with acetylated lysines on chromatin and transcription factors at latent enhancers and promoters, minimizing maladaptive transcription of disease-driving genes. 26 Preclinical studies have shown that apabetalone reduces the expression of a variety of markers of CVD, CKD, and inflammation in various cell types and disease models. 27, 28, [33] [34] [35] [36] In clinical trials, apabetalone improved cardiac (major adverse cardiovascular events [MACE] ) and renal (serum alkaline phosphatase and eGFR) parameters in CVD patients. 30, 33, [37] [38] [39] These data suggest that treatment with apabetalone may alleviate cardiac, renal, and inflammatory complications in FD patients.

In this study, we first examined the inflammatory status of plasma and PBMCs collected from FD patients. We also tracked immune activation in unstimulated PBMCs over 2 years of continuous ERT.

The effects of ex vivo apabetalone treatment on inflammation burden were examined by assessing pro-inflammatory marker levels in stimulated PBMCs isolated from ERT treated FD patients. Lastly, we investigated apabetalone effects on oxidative stress in stimulated neutrophils, in which the role of BET proteins is currently unknown.

Blood samples were collected from eight FD patients receiving ERT therapy at M.A.G.I.C clinic, Calgary, Alberta, Canada. Patients met the following inclusion criteria: existing diagnosis of FD, according to The Canadian Fabry Association Standard and age ≥18 years.

Inclusion criteria were limited due to the small population from which to draw participants based on the rarity of the disease and 

Blood samples were collected between 7 and 14 days following ERT treatment, which was administered at 14-day intervals for each pa- 

Isolated PBMCs or neutrophils were stained with antibodies to assess purity or immune activation status. The antibodies used in this study were V450 anti-human CD14, APC anti-human CCR2, PE-CF594 anti-human TLR4 (BD Biosciences). Data were acquired with BD FACSCelesta (BD Biosciences) and quantified with FlowJo V10 software (BD Biosciences). 1 μg/ml LPS or 10 ng/ml IFNγ with BETi co-treatments for 4 h at 37°C.

Relative gene expression was determined by real-time PCR as previously described. 27, 34 Briefly, mRNA was isolated using Catcher PLUS kits per manufacturer instructions (ThermoFisher Scientific). Realtime PCR was performed with Taqman primer probes (ThermoFisher Scientific) to determine the abundance of transcripts relative to the endogenous control cyclophilin. The data were analyzed as 2^ (C T cyclophilin -C T tested marker) and then normalized to DMSO treated samples.

Cytokine profiles in plasma or undiluted supernatants from stimu- 

PBMCs were pre-treated with apabetalone, MZ1 or 0.025%

Cells were lysed and sonicated as previously described. 27 20 μg of total protein was added for protein separation. BET proteins were detected with anti-BRD2, anti-BRD3 and anti-BRD4 mAbs (Bethyl) followed by goat anti-rabbit IgG H&L chain specific peroxidase (Calbiochem). Actin was stained by antiβ-actin conjugated to peroxidase (Sigma).

Intracellular ROS levels in LPS-stimulated neutrophils were detected using CellROX ® Green flow cytometry kits (ThermoFisher Scientific)

as per manufacturer instruction. Samples were immediately analyzed using BD FACSCelesta Cell Analyzer. The amount of intracellular ROS was quantified as the percentage of cells having positive green fluorescent signal above background in proportion to total live cells using the Flowjo ® software (BD biosciences).

Statistical significance was calculated using GraphPad Prism 8.0

with Mann Whitney test, one-way ANOVA followed by Dunnett's Multiple Comparison Test where appropriate. p < .05 was considered statistically significant.

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy.

org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, 47 and are permanently archived in the Concise

Guide to PHARMACOLOGY 2019/20. 48

In this study, we recruited eight FD patients receiving ERT therapy (44-74 years old) from a single clinical site. Baseline demographic and biochemical data are shown in Table 1 . Clinical presentations at the time of enrollment are in Table S1 . All patients showed clinical indicators of CVD. Three patients had eGFR below the range of 60 ml/ min/1.73 m 2 (p = .04), which is indicative of reduced kidney function and vulnerability to rapid deterioration. 1, 49 Patients also received standard of care medications for management of other complications (such as CVD or CKD) (Table S1 ). Due to limited blood volume and variation in volume of cells obtained from FD patients, sample number varied in the following analyses.

Pro-inflammatory cytokines are positively associated with FD progression despite ERT treatment. [10] [11] [12] We examined cytokine levels in plasma from ERT-treated FD patients and commercially available plasma from control donors using multianalyte cytokine profiling.

In agreement with previously reported results, 10,12 plasma MCP-1 and TNFα concentrations were greater in FD samples compared with normal controls (p = 0.0062 and p = .0246, respectively) ( Figure 1A , Table S2 ). However, plasma cytokine abundance did not differ by eGFR levels (Table S3) . We also tracked cytokine expression in PBMCs from four FD patients with eGFR >60 (normal eGFR) on continuous ERT therapy before and after 2 years of treatment. PBMCs showed an ~20-fold increase in IL12B transcript levels (p = .03) in the most recent visit compared with 2 years prior ( Figure 1B) . Furthermore, activation markers on monocyte populations ( Figure 1C) were assessed. As shown in Figure 1D , the proportion of monocytes expressing CCR2 was greater in patients with low eGFR (eGFR <60, p = .03), implying that innate immune dysregulation may be associated with renal dysfunction. However, the percentage of TLR4 expressing cells did not differ by eGFR levels.

Overall, these results suggest the persistence of immune dysregulation during continuous ERT in unstimulated PBMCs. .04

ALT, U/L 21 [19] 22 [14] .99

LD, U/L 206 [56] 288 [37] .04

Total bilirubin, µmol/L 11 [7] 7 [3] .39

Gb3, µg/ml 5 [4] 3 .38

Lyso-Gb3, nmol/L 11 [15] 19 [25] . 

To evaluate apabetalone's effects on protein production, we exam- but had no effect on TNFα, IL-6 and IL-8 production ( Figure 3A ).

Furthermore, using multianalyte immunoprofiling, we identified three important pro-inflammatory cytokines, IL-12p40, GM-CSF and MCP-3, that were significantly reduced by apabetalone ( Figure 3B , Table S4 ). Secretion of these proteins was robustly induced by LPS stimulation (up to 400-fold) except for MCP-3 whose induction appeared less extensive, likely due to large variance in basal levels (2 pg/ml-605 pg/ml) ( Figure 3B ). Nevertheless, 1µM apabetalone Figure 3B ). However, BETi treatments had no effect on LPS-induced IL-1β ( Figure 3B ) despite the significant reduction occurred at gene level ( Figure 2 ).

IFNγ plays a well-known role in priming monocytes/macrophages to a pro-inflammatory phenotype during inflammation. 50 IFNγ contributes to pro-inflammatory signaling in FD patients 13 and its production is also a known downstream effect of TLR4 activation. 51 Here, exposure of FD patients' PBMCs (normal eGFR) to IFNγ resulted in the induction of CCL2, TNF, and IL6 by fivefold, ninefold, and sixfold, respectively, but to a lesser extent than with LPS stimulation (Figure 4) . Apabetalone lowered the induced ex- 

In FD, Gb3 accumulation stimulates excessive ROS production causing intracellular oxidative damage, and exacerbating inflammation. [17] [18] [19] Here we investigated ROS levels in stimulated FD neutrophils with BETi co-treatment. Apabetalone attenuated LPSinduced ROS production ( Figure S4A and B, an average of 33-fold) in a dose-dependent manner ( Figure 7A ). Pan-BETi apabetalone (apa 20 μM) countered the induced ROS production to a level comparable with JQ1 ( Figure 7A ), confirming a BET-dependent mechanism.

IFNγ did not alter ROS production from baseline, and apabetalone had no effect on basal ROS levels ( Figure 7B ). ROS production is generated by cellular enzymes such as nicotinamide adenine dinucleotidephosphate-oxidase (NADPH) 52 ; NOX2 and NOX4 genes encode two major subunits of the NADPH complex. NOX2 expression was reduced by BD2-selective BETi treatments (by 29% with apa 1 μM, by 61% with apa 5 μM) and pan-BETi (by 84% with apa 20 μM, by 93% with JQ1) in all examined patient cells ( Figure 7C ), though NOX4 was undetectable. This is the first demonstration that neutrophil-mediated ROS production occurs through a BETdependent process at the transcription level.

Immune cell-mediated inflammation is a major factor driving FD 

In FD, monocytes use TLR4 to sense Gb3 accumulation, triggering pro-inflammatory responses including the production of cytokines and chemokines. 10 The chemokine MCP-1 interacts with its receptor CCR2, promoting monocyte infiltration into injured tissues. 53, 54 In FD, elevated plasma MCP-1 correlates with CVD following 1 year of ERT, 12 indicating ongoing immune dysfunction during treatment. We also detect increased plasma MCP-1 in FD patients undergoing longterm ERT relative to controls ( Figure 1A and Table S2 ). Increased (Table S1 ), suggest persistent inflammation and immune dysfunction despite continuous ERT.

TLR4-activated inflammation in FD has been studied in cultured cell models with LPS or concurrent stimulation with Gb3+ DGJ. 10 In THP1 cells, we demonstrate induced transcription of CCL2 by

Gb3+DGJ, albeit to a lesser extent than with LPS ( Figure 6 ). BET inhibition using BD2-selective BETi (apa 1 μM) counters induced CCL2 expression back to basal levels, indicating sensitivity of CCL2

to BETi. Previously, we have documented that pan-BETi (apa 20 μM)

abrogates transcription of CCL2 in line with reduced production of the encoded protein, MCP-1, in cultured cytokine-stimulated monocytes from diabetic CVD patients. 28 We substantiate this finding, showing that BD2-selective BETi (apa 5 μM) had comparable inhibitory effect on CCL2 transcription relative to pan-BETi (apa 20 μM) (97.8% vs. 99.9%) in LPS or IFNγ stimulated PBMCs from ERT-treated FD patients with clinical indicators of CVD (Table S1 ; Figures 2 and 4) . Results identify a role of BET protein-BD2 domains in CCL2 regulation, suggesting potential for BD2-selective apabetalone in reducing MCP-1 levels in FD. Other chemokines are also involved in monocyte migration/activation. MCP-3 is important for CCR2-mediated monocyte recruitment, 55 and GM-CSF promotes inflammatory activation of monocytes/macrophages. 56 We report increased GM-CSF in plasma from ERT-treated FD patients compared with controls (Table S2 ) and reduced production of both GM-CSF and MCP-3 with BD2-selective apabetalone (1 μM or 5 μM; Figure 3B ). Thus, apabetalone may control monocyte migration by lowering cytokine production in FD patients receiving ERT.

TNFα and IL-6 levels are elevated in ERT-treated FD patient plasma relative to healthy controls. 11, 12 We also observe increased plasma TNFα (twofold) in ERT-treated FD patients versus controls ( Figure 1A , Table S2 ). BET inhibition opposes induced transcription of these two genes in stimulated cell models. 27, 28 We further IL-12, a monocyte-derived cytokine, bridges innate and adaptive immunity to promote Th1 cell-mediated pro-inflammatory responses, 57 which aggravate tissue damage. 58, 59 In FD, IL-12 has been speculated to link abnormal innate and adaptive immune responses, 13 

Excessive ROS production drives oxidative stress and exacerbates inflammation 3,15 in FD cell models. [17] [18] [19] Using cells from ERT-treated FD patients, we demonstrate neutrophil-mediated ROS activity and associated aberrant gene expression with LPS or IFNγ stimulation (Figure 7, S4A) . BETi, particularly BD2-selective apabetalone (1 μM and 5 μM), reduced ROS production in stimulated FD neutrophils ( Figure 7A and B) , and regulated transcription of ROS-related genes such as NOX2 ( Figure 7C ). Furthermore, our data suggest that neutrophil ROS activity and NOX2 expression may be greater in patients with low eGFR than those with normal eGFR ( Figure S4B and D) , implying a connection between renal dysfunction and abnormal neutrophil responses during FD progression. These observations suggest that apabetalone can modulate detrimental BET protein activity in FD neutrophils, at least in vitro. To date, work on BET protein involvement in ROS production has focused on fibrosis and cancer. [60] [61] [62] Our findings indicate that BD2-selective BETi prevent neutrophil-mediated ROS production in the context of FD.

This study has several limitations. and sex-matched untreated or pre-ERT FD controls, due to limited numbers of patients with varying degrees of FD at a single clinic and that increased age is associated with disease progression requiring ERT. Therefore, generalized conclusions cannot be drawn without a larger study. Despite these limitations, we showed elevated inflammatory signaling in immune cells from FD patients over time, without clinical presentation or diagnostic signs of renal complications, such as abnormal eGFR. This persistent inflammation in FD patients regardless of ERT indeed warrants further validation in a greater number of patients. Furthermore, we were able to demonstrate the potential for apabetalone in countering inflammatory processes in activated immune cells in FD patients. Despite limitations, this study demonstrates that apabetalone could be a valuable therapeutic for improving FD patient care.

In summary, our results show that BD2-selective apabetalone counters inflammation and oxidative stress in stimulated innate immune cells from FD patients undergoing continuous ERT. Potential therapeutic effects of BD2-selective apabetalone on FD will be further evaluated in the clinical setting.

The authors would like to thank all the participants who made the 

LF is the principal researcher of the project. LF, SW, and LMT con- 

The study protocol was reviewed and approved by the HREBA

Committee.

Patients declaring interest in study participation provided written informed consent prior to enrollment.

The data sets generated and/or analyzed during the current study are not publicly available. Reasonable requests for data will be considered. All results discussed are provided.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Li Fu https://orcid.org/0000-0003-4983-4578

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