Shahror et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03159-8(2024) 21:170

Journal of Neuroinflammation

Open Access

RESEARCH

Deletion of myeloid HDAC3 promotes
efferocytosis to ameliorate retinal ischemic
injuryRami A. Shahror1, Esraa Shosha1,4, Carol Morris1, Melissa Wild1, Shengyu Mu1, Gabor Csanyi3, Marjan Boerma2,
Nancy J. Rusch1 and Abdelrahman Y. Fouda1,4*
Abstract
Ischemia-induced retinopathy is a hallmark finding of common visual disorders including diabetic retinopathy(DR) and central retinal artery and vein occlusions. Treatments for ischemic retinopathies fail to improve clinicaloutcomes and the design of new therapies will depend on understanding the underlying disease mechanisms.
Histone deacetylases (HDACs) are an enzyme class that removes acetyl groups from histone and non-histone
proteins, thereby regulating gene expression and protein function. HDACs have been implicated in retinal
neurovascular injury in preclinical studies in which nonspecific HDAC inhibitors mitigated retinal injury. Histone
deacetylase 3 (HDAC3) is a class I histone deacetylase isoform that plays a central role in the macrophageinflammatory response. We recently reported that myeloid cells upregulate HDAC3 in a mouse model of retinal
ischemia-reperfusion (IR) injury. However, whether this cellular event is an essential contributor to retinal IR injury is
unknown. In this study, we explored the role of myeloid HDAC3 in ischemia-induced retinal neurovascular injury by
subjecting myeloid-specific HDAC3 knockout (M-HDAC3 KO) and floxed control mice to retinal IR. The M-HDAC3 KO
mice were protected from retinal IR injury as shown by the preservation of inner retinal neurons, vascular integrity,
and retinal thickness. Electroretinography confirmed that this neurovascular protection translated to improved
retinal function. The retinas of M-HDAC3 KO mice also showed less proliferation and infiltration of myeloid cells
after injury. Interestingly, myeloid cells lacking HDAC3 more avidly engulfed apoptotic cells in vitro and after retinal
IR injury in vivo compared to wild-type myeloid cells, suggesting that HDAC3 hinders the reparative phagocytosisof dead cells, a process known as efferocytosis. Further mechanistic studies indicated that although HDAC3 KO
macrophages upregulate the reparative enzyme arginase 1 (A1) that enhances efferocytosis, the inhibitory effectof HDAC3 on efferocytosis is not solely dependent on A1. Finally, treatment of wild-type mice with the HDAC3
inhibitor RGFP966 ameliorated the retinal neurodegeneration and thinning caused by IR injury. Collectively, ourdata show that HDAC3 deletion enhances macrophage-mediated efferocytosis and protects against retinal IR injury,
suggesting that inhibiting myeloid HDAC3 holds promise as a novel therapeutic strategy for preserving retinal
integrity after ischemic insult.
Keywords Retinal ischemia-reperfusion injury, Histone deacetylase 3, Microglia, Macrophages, Efferocytosis*Correspondence:
Abdelrahman Y. Fouda
afouda@uams.edu
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
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in this article, unless otherwise stated in a credit line to the data.

Shahror et al. Journal of Neuroinflammation(2024) 21:170

Introduction
Individuals across the lifespan suffer from visual impairment and blindness caused by diseases of retinal ischemia that include retinopathy of prematurity in neonates,
diabetic retinopathy (DR), and central retinal artery orvein occlusion in older adults. These conditions are collectively termed ischemic retinopathy, and the absence ofeffective therapies represents a major clinical challenge
for efforts seeking to preserve vision and quality of life.
The mechanisms underlying the neurovascular injury
that is a feature of ischemic retinopathy are poorly understood and this knowledge gap limits the design of new
and improved therapeutic interventions. The collective
efforts of the scientific community to identify the pathogenic mechanisms that contribute to ischemia-induced
neurovascular injury of the retina often rely on animalmodels including the ischemia-reperfusion (IR) mousemodel of retinopathy [1–4].
Myeloid cells such as microglia and macrophages(MΦ) play a critical role in the progression of retinal IR
injury [5–9]. Activation and proliferation of microglia and resident MΦ coupled with infiltration of bloodborne monocytes can promote either retinal protection
or degeneration depending on the molecular signature
and activation state of the myeloid cells [10–12]. We andothers have shown that myeloid cells can play a protective role by shifting from a pro-inflammatory phenotype
to a reparative phenotype; the latter myeloid cell phenotype contributes to the resolution of retinal injury [6, 7,10]. Our studies using a mouse model of retinal IR injury
showed that systemic MΦ depletion worsens neurodegeneration and increases retinal hemorrhage suggestingthat MΦ confer neurovascular protection [6]. However,the underlying mechanisms by which myeloid cells canpromote neurovascular repair are poorly understood.
Efferocytosis is the engulfment of apoptotic cells andcell debris by myeloid cells after injury. It is recognized
as a reparative event in several pathological conditions
including stroke [13, 14], cancer [15, 16], and atherosclerosis [17]. Conversely, impaired efferocytosis can lead tothe accumulation of apoptotic cells and damage-associated molecular patterns (DAMPs) after injury, which
fuels a feed-forward cycle of inflammation and further
cell death. Reports of efferocytosis in the injured retina
have focused primarily on photoreceptor degenerationand conflicting results have documented both reparativeand deleterious roles of myeloid cell-mediated phagocytosis [18–21]. Microglia/MΦ-mediated phagocytosis
in the inner retina has been documented after retinal IR
injury, optic nerve crush, and other ocular injuries, but
knowledge of its mechanistic basis and impact on injury
progression is scarce [10, 22–24].
Histone deacetylases (HDACs) are a family of enzymes
that remove acetyl groups from histone and non-histone

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proteins and thereby enable chromatin remodeling toregulate gene expression. The HDACs regulate several
cellular processes including cell metabolism, proliferation, and the immune response [25]. HDAC inhibitors
are reported to be neuroprotective, implying that HDACs
perpetrate neuronal injury. For example, general inhibitors of Class I and II histone deacetylases (HDACs) limit
neuronal damage in the murine model of retinal IR injury[26, 27], suggesting that specific HDAC isoforms withinthese two HDAC classes may participate in the underlying pathogenesis. In this regard, recent attention has beendrawn to the HDAC3 isoform belonging to the ClassI HDACs as a potential culprit. Neuronally expressed
HDAC3 has been implicated in ganglion cell degeneration caused by optic nerve crush [28, 29]. HDAC3 also
is reported to mediate the epigenetic modifications that
promote the expression of MΦ inflammatory genes [30,31]. We recently reported an upregulation of HDAC3 inmyeloid cells in a mouse model of retinal IR injury, suggesting a strong presence of this specific HDAC isoform
at the affected site. We additionally discovered that arginase 1 (A1), a reparative enzyme that hydrolyzes arginine
and promotes MΦ-mediated efferocytosis, and resolution of retinal IR injury, suppresses HDAC3 [5, 13, 32,33]. These findings provide indirect evidence to implicate
myeloid HDAC3 in retinal IR injury and emphasize theimportance of defining its precise role in retinal injury
progression and loss of function.
Here, we present data showing for the first time that
deletion of myeloid HDAC3 is neurovascular protective and leads to the resolution of retinal IR injury. The
underlying mechanism may involve enhanced efferocytosis at the site of injury that promotes the removal of deadcells, which otherwise can contribute to inflammation.
Interestingly, the pro-efferocytic condition caused by
HDAC3 deletion appeared to be partially dependent on
A1, implying that HDAC3 could also regulate efferocytosis by other unique pathways.

Materials and methods
Generation of myeloid-specific KO mice

Myeloid-specific HDAC3 KO mice C57BL/6J floxed
mice with LoxP sites on either side of exon 7 of HDAC3(HDAC3f/f, originally developed by Dr. Scott W. Hiebert)
were obtained from Dr. McGee-Lawrence and bred inour colony [34]. These mice were crossed with LysM Cre
mice (Jackson Laboratory Stock No. 004781) to generateMΦ and microglia cell-specific HDAC3 KO (LysM Cre;HDAC3f/f or M-HDAC3−/−) mice. Mice genotyping andcharacterization are shown in Supplementary Fig. S1.
Myeloid-specific A1 KO mice C57BL/6J floxed mice thathave LoxP sites for A1 (A1f/f) were crossed with LysM Cre
mice (Stock No. 004781) to generate myeloid-specific A1

Shahror et al. Journal of Neuroinflammation(2024) 21:170

KO (M-A1−/−) mice, which were used for studies with isolated MΦ.
Mouse retinal ischemia-reperfusion (IR) injury model and
RGFP966 administration

All experiments were approved by the UAMS Institutional Animal Care and Use Committee (IACUC).
M-HDAC3−/−, HDAC3f/f, and wild-type (WT) C57BL/6J
mice (10–12 weeks old) were anesthetized using a ketamine/xylazine mixture and then subjected to retinal
ischemia followed by reperfusion to achieve IR injury as
described by us and others [10, 35–37]. A needle connected to a raised saline bag was inserted into the anterior chamber of the right eye to raise the intraocularpressure to 110 mmHg (calculated based on the heightof the saline bag). Ischemia was induced for 60 min followed by needle removal to allow reperfusion. The left
eye served as a sham control since we did not detect
differences between contralateral eyes and those fromuninjured mice (Supplementary Fig. S2) [10, 37]. Animals with a drop in pressure due to saline leakage out of
the eye during the procedure were excluded. Mice weredeeply anesthetized and sacrificed by transcardial perfusion or cervical dislocation at the various time points
shown in Table 1 and Supplementary Fig. S3 based on
our previous studies and existing literature.
For studies on WT mice, the specific HDAC3 inhibitor
RGFP966 (Sigma, St. Louis, MO) was dissolved in DMSO
and then diluted in 70% polyethylene glycol (PEG) 200,
30% acetate buffer for intraperitoneal (i.p.) injection aspreviously described for a final DMSO concentration of∼ 10% [44]. RGFP966 was administered (10 mg/kg i.p.) at
1 h after IR and every 48 h thereafter based on previous
literature [45].
Optical coherence tomography (OCT)The eyes of mice were dilated with 1% tropicamide(Akron Pharmaceuticals, IL) and retinas were scannedwith an OCT Ophthalmic Imaging System (Bioptigen
Inc., Durham, NC) under ketamine/xylazine anesthesia.
Mice were placed on a mouse holder to fix the animal’s
posture for scanning. The retina scans were acquired in arectangular volume mode (3 frames/scan, 1000 A scan/Bscan × 100 B scan, 1.4 mm × 1.4 mm). Automated analysis of scans to obtain retinal thicknesses was conductedusing InVivoVue software (Bioptigen Inc., Durham, NC).

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Electroretinography

A full scotopic electroretinogram (ERG) was recorded on
day 14 post-IR as previously described with some modification [46]. Briefly, after 16 h of dark adaptation, mice
were anesthetized using a ketamine/xylazine mixture,and the pupils were dilated with 1% tropicamide (Akron
Pharmaceuticals, IL) eye drops. Mice were then placedon the Celeris electroretinogram system (Diagnosys
LLC, Cambridge, UK) with temperature control (37 °C),and dark-adapted ERGs were recorded. Amplitudes andimplicit times of ERG waveforms were measured at aseries of flash intensities (0.01, 0.1, 1 cd.s/m2) based on astandard machine protocol and literature [46].
Retinal vascular permeability

Albumin extravasation Vascular permeability was
examined by measuring the extravasation of albumin to
the retina on day 2 after IR injury following transcardial
perfusion to remove intravascular albumin as we andothers previously described [35, 47]. Briefly, a 20-gauge
perfusion cannula was introduced into the left cardiac
ventricle of the anesthetized mice. Mice were perfusedwith phosphate-buffered saline (PBS) for 5 min to rinseout blood cells and proteins. Drainage of the blood in the
PBS was conducted by opening the right atrium. Retinas
were then lysed, and Western blotting was performed toquantify retinal vascular leakage by measuring extravascular albumin in the whole retina.
Retinal fluorescein angiography Retinal vascular permeability in vivo was assessed using fluorescein angiography as previously described [48, 49]. Briefly, two days
post-IR injury, mice were anesthetized using a ketamine/
xylazine mixture, and pupils were dilated with 1% tropicamide (Akorn Pharmaceuticals, IL) eye drops. The anesthetized mice were mounted on a maneuverable imaging
platform, and their corneas were lubricated with a thinlayer of GenTeal moisturizing eye gel (Alcon, Geneva,
Switzerland) to keep surface moisture during the procedure. Each mouse received a subcutaneous injection of 50µL of AK-FLUOR® fluorescein sodium (10%; Long Grove
Pharmaceuticals, Rosemont, IL). Rapid image acquisition
ensued over a period of approximately 5 min. Fluoresceinleakage was demonstrated by indistinct vascular borders
progressing to diffusely hazy fluorescence.

Table 1 Time points of the main outcome measures in the in vivo IR studies
Outcome measure
Vascular permeability
Retina thickness measurements (OCT)Neurodegeneration
Retina function (ERG)
Vascular degeneration (Trypsin digests)

Time post-IR
2 days7 days7 days14 days
14 days

References[35, 38, 39][40, 41][35, 41, 42][36, 43][35, 42]

Shahror et al. Journal of Neuroinflammation(2024) 21:170

Evans blue leakage In vivo retinal vascular permeability was assessed by imaging of intravascular Evans Bluedye extravasation at day two post-IR injury as previously
described with some modification [50]. Briefly, anesthetized mice received transcardial injections of Evans Blue
(200 µL, 2% in normal saline), euthanized after 5 min while
under anesthesia, and eyes were fixed with 4% paraformaldehyde (PFA) for 24 h. Retinal flat mounts were mounted
and cover-slipped with Vectashield antifade mounting
medium (Vector Laboratories, Newark, CA), and images
were captured using a fluorescence microscope. Images ofthe entire retina flat mount were processed using ImageJ.
The red color (Evans Blue) was thresholded in each image,the background was subtracted, and the fluorescencearea was binarized. Finally, the leakage area, represented
as a binary area in the entire retina, was measured using
ImageJ.

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performed using ZEN software or ImageJ on single-slice
confocal images. Morphological analysis of myeloid cells
utilized Sholl analysis (Fiji Sholl analysis plugin), as previously described [53]. Random Iba1+ cells in the ganglion cell layer (GCL) were chosen from three different
fields of view and subjected to Sholl analysis. Criteria forcell selection included: (i) non-overlapping with otherlabeled cells, ensuring clear distinction of cell body andprocesses from neighboring cells, and (ii) all cellular processes within the field of view. The analysis determinedthe complexity of myeloid (Iba1+) cell processes by analyzing branch intersections at increasing radial distancesfrom the cell body.
Microscopy-based in vivo efferocytosis assay on retinal flat
mountsEyeballs were isolated on day 14 after IR injury and fixed
overnight in 4% PFA. The retinal vasculature was isolatedby trypsin digestion as we and others have reported [6,35, 51]. The vasculature was air-dried on silane-coatedslides and stained with periodic acid-Schiff and hematoxylin. Acellular capillaries were counted in random fieldsof the mid-retina under the microscope. The number ofacellular capillaries was divided by the field area to get
the number of acellular capillaries per 1 mm of the retina[2].

For microscopy-based quantification of in vivo efferocytosis, double immunolabeling of the microglia/MΦ
marker, Iba1, and TUNEL staining of retinal flat mounts
was conducted on day 2 after IR injury using the ClickiT™ Plus TUNEL Assay (Invitrogen™) following the
manufacturer’s instructions. The number of Iba1+ positive cells associated with TUNEL+ apoptotic bodies wascounted in multiple fields of view in Z-stack images taken
at the ganglion cell layer using an LSM 880 Zeiss confocal
microscope (Supplementary Fig. S4A). The efferocytosis
index (% of dead/dying cells engulfed by myeloid cells)
was calculated using the following equation: ([number of
Iba1+TUNEL+ cells ÷ total number of TUNEL+ cells] ×
100).

Fluorescent immunolabeling

Flow cytometry analysis of retinal immune cell populations

Eyeballs were fixed in 4% PFA and then dissected into
retinal flat mounts. The flat mounts were permeabilized
in 0.1% triton X-100 and then blocked in 10% donkey
serum and 3% bovine serum albumin (BSA) for 30 min.
Subsequently, the flat mounts were incubated in primary
antibodies including Iba1 (FUJIFILM Wako, Cat. #01919741) and NeuN (MilliporeSigma, Cat. #BN78MI) overnight at 4 °C followed by washing in PBS and incubationof secondary antibodies at room temperature for 4 h as
described earlier [35].

Following animal euthanasia by transcardial perfusion, eyes were removed by orbital dissection and retinas were isolated. Retinal tissues (2 to 4 retinas pooled
per preparation) were processed for flow cytometry andanalysis as described by us [54]. Briefly, retinas weredigested in a solution containing 5% filtered fetal bovine
serum (FBS, Gibco; Thermo Fisher, NY) 10 mM HEPES,
0.5 mg/mL of liberase (Sigma Aldrich, St. Louis, MO), and 0.1 mg/mL of DNase (Sigma Aldrich, St. Louis,
MO). Cells were strained through a 40 μm cell strainer
and then incubated with a viability dye (Fixable Viability
Dye eFluor™ 450, eBioscience™) for 30 min. After rinsing the viability dye, cell suspensions were blocked with
1 µg/mL of Fc block anti-mouse CD16/32 and 20% normal rat serum for 10 min at room temperature. Subsequently, retinas were incubated with labeled antibodies
that included PerCP-Cy5.5-conjugated rat anti-mouse
CD11b monoclonal antibody (1:100, Clone M1/70, BD
Bioscience, Cat. #BDB550993), APC-Cy7 rat anti-mouse
CD45 monoclonal antibody (1:100, Clone 30-F11, BD
Bioscience, Cat. #BDB557659), PE-conjugated rat antimouse Ly6C monoclonal antibody (1:100, Clone HK1.4,

Retinal vasculature trypsin digestion and counting ofacellular capillaries

Confocal microscopy and image analysis

Confocal imaging was performed using an LSM 880
Airyscan Zeiss Laser inverted microscope equipped with
405 nm, 488 nm, 561 nm, and 640 nm laser lines. Identical laser intensity settings were applied to all samples
and Z-stacks images (resolution: 1024 × 1024 Pixels) weretaken. Neurodegeneration and myeloid cell proliferation
were determined as the NeuN+ and Iba1+ area, respectively [52]. After the acquisition, a maximum intensity
projection of the Z-stack was applied using ZEN Bluesoftware (Zeiss) or ImageJ. Quantitative analysis wasShahror et al. Journal of Neuroinflammation(2024) 21:170

eBioscience, Cat. #50-245-507), and FITC-conjugated
rat anti-mouse Ly6G (Gr-1) monoclonal antibody (1:100,
Clone RB6-8C5, eBioscience, Cat. #50-991-9). Afterincubation with antibodies, retinas were rinsed 3 times
with cold PBS, fixed with 0.4% PFA, and analyzed using
a BD LSRFortessa flow cytometer (BD Biosciences) and
FlowJo software (Tree Star Inc., San Carlos, CA). Retinal
cell populations initially were gated using the common
leukocyte marker CD45 and the myeloid lineage marker
CD11b. Based on the expression of CD45, myeloid cells
were gated as CD11b+/CD45low microglia and CD11b+/
CD45hi myeloid leukocytes. Myeloid leukocytes weregated further based on Ly6C expression and the granulocyte/neutrophil marker Ly6G. Myeloid leukocytes were
subdivided into classical monocytes (CD11b+/CD45hi/
Ly6Chi/Ly6Gneg), intermediary monocytes (CD11b+/
CD45hi/Ly6Clow/Ly6Gneg), and non-classical monocytes(CD11b+/CD45hi/Ly6Cneg/Ly6Gneg).
Flow cytometry-based in vivo efferocytosis assay

In subsequent experiments, mice were treated with
PSVue 550 eyedrops (Molecular Targeting Technologies, Cat. #P-1005) one day before sacrifice to assess efferocytosis by flow cytometry (Supplementary Fig. S4B).
PSVue dye allows in vivo fluorescent labeling of deadcells since it binds to externalized phosphatidylserine(PtdSer, an “eat-me” signal) on apoptotic cells in live mice[55]. Retinas were then incubated with labeled antibodies that included PerCP-Cy5.5-conjugated rat anti-mouse
CD11b monoclonal antibody (1:100, Clone M1/70, BD
Bioscience, Cat. #BDB550993), APC-Cy7 rat anti-mouse
CD45 monoclonal antibody (1:100, Clone 30-F11, BD
Bioscience, Cat. #BDB557659) and eFluor 660 conjugatedCD68 monoclonal antibody (eBioscience, Cat. #50-068182) with the latter used as a marker of phagocytic cells.
Phagocytic microglia and myeloid leukocytes were gatedfrom CD11b+/CD45low and CD11b+/CD45hi populations,
respectively, as CD68+/PSVue+ cells engaged in efferocytosis of apoptotic cells.
Cell isolation, cell lines, and culture

Bone marrow-derived macrophages (BMDMs) Bone marrow cells were isolated and differentiated into MΦ basedon our published protocol [6]. In brief, both femurs andtibias were harvested and flushed with 20 − 25 ml sterile
PBS using a 27-gauge needle. Flushed cells in PBS werespun down and resuspended in differentiation medium(Dulbecco’s modified Eagle’s medium, DMEM, high glucose, Gibco; Thermo Fisher, NY)) containing 20% FBS(Gibco; Thermo Fisher, NY), 20% L929 conditioned
media, and 1% penicillin-streptomycin (Pen-Strep). Cells
were subsequently plated on uncoated 100-mm dishes.
For studies of in vitro efferocytosis, BMDMs were plated
on 35-mm glass bottom dishes (MatTek Corporation,

Page 5 of 21

Ashland, MA) with a 14-mm glass diameter. Media were
replaced with fresh differentiation media on day 4 after
plating.
K562 lymphoblast cells Suspended K562 cells were cultured in RPMI 1640 medium (Gibco; Thermo Fisher, NY)
containing 2 mM L-glutamine supplemented with 10%
FBS (Gibco; Thermo Fisher, NY) and 100 IU/ml Pen-Strep(Gibco; Thermo Fisher, NY), and maintained in a 5% CO2
incubator at 37 °C [56].
R28 retinal neuronal-like cells The R28 cell line (Kerafast, Boston, MA) was maintained and differentiated as
described previously [52]. Cells were cultured in DMEM
supplemented with 10% FBS (Gibco; Thermo Fisher, NY),
100 U/ml Pen-Strep, and maintained under standard culture conditions (37 °C, 5% CO2). The medium was changed
completely every other day and cultures were passaged at
90% confluence. To induce differentiation, cells were passaged onto laminin-coated plates and supplemented with
250 µM 8-(4-Chlorophenylthio)adenosine 3′,5′-cyclic
monophosphate sodium salt (pCPT-cAMP) for overnightincubation.
Induction of apoptosis in K562 and R28 cells in vitro

K562 cells or R28 cells (apoptotic and non-apoptotic)
were labeled with the Vybrant® CFDA SE Cell Tracer Kit(Invitrogen, Carlsbad, CA) for 30 min at 37 °C, washedtwice with complete media, and resuspended at 5 × 105
cells/ml. BMDM were labeled with CM-DiI Dye® (Invitrogen, Carlsbad, CA) in pre-warmed PBS and incubatedfor 15 min at 37 °C followed by another incubation for
30 min at 37 °C with DMEM (supplemented with 100 IU/
ml Pen-Strep) before rinsing three times with PBS.
Apoptosis of K562 or R28 cells was induced by subjecting the cells to UV-B irradiation for 15 min using a UV
crosslinker (Spectrolinker™ XL-1500, Spectronics Corporation, Melville, NY). Afterward, the cells were resuspended in DMEM containing 10% FBS (Gibco; Thermo
Fisher, NY) and incubated at 37 °C for up to two hours
and monitored for apoptosis induction (visible cell blebbing under the microscope). Apoptosis was also confirmed by Annexin V labeling (Supplementary Fig.
S4C) [56]. UV-B was chosen as the apoptosis induction
method due to its rapid action and widespread use [56,57].
Evaluation of macrophage efferocytosis of apoptotic cells
in vitro

Three sets of experiments using the K562 and R28 cells
prepared as described above were conducted to evaluate
in vitro efferocytosis:

Shahror et al. Journal of Neuroinflammation(2024) 21:170

Page 6 of 21

Experiment 1 CM-DiI labeled BMDMs derived from
HDAC3f/f and M-HDAC3−/− mice were plated on 35-mm
dishes and treated with or without the A1 inhibitor, 2(S)amino-6-boronohexanoic acid (ABH) (100 µM). Thencells were incubated with CFDA-labeled K562 cells (apoptotic or non-apoptotic) at a 1:1 ratio for 45 min at 37 °C
followed by PBS washing to remove non-engulfed K562
cells. Subsequently, the dishes either were imaged undera confocal microscope to detect the efferocytic MΦ orwere trypsinized and immediately analyzed by the BD
Accuri™ C6 Plus Flow Cytometer (BD Bioscience, San
Jose, CA). Cells that were double positive for CM-DiI/
CFDA were regarded to represent phagocytic BMDMs
that engulfed apoptotic cells. Efferocytosis was calculated
as a fold change by dividing CM-DiI+ CFDA+ (phagocytic
BMDMs) by the total number of CM-DiI+ BMDMs.
Experiment 2 CM-DiI labeled BMDMs derived from
A1f/f and M-A1−/− mice and plated on 35-mm dishes wereincubated with CFDA-labeled R28 cells (apoptotic or
non-apoptotic) for 45 min at 37 °C followed by PBS washing to remove non-engulfed cells. The dishes were thenprocessed for imaging under the microscope to detect theefferocytic MΦ.
f/f

Experiment 3 CM-DiI labeled BMDMs derived from A1
and M-A1−/− mice that were plated on 100-mm dishes
were incubated with CFDA-labeled K562 cells (apoptotic and non-apoptotic) for 45 min at 37 °C. After washing the non-engulfed cells, the adherent BMDMs either
were imaged by a confocal microscope or trypsinized and
immediately analyzed by flow cytometry.
Real time-PCR to detect A1 expression in BMDMs

For RNA isolation, 4 × 105 BMDMs derived from
HDAC3f/f and M-HDAC3−/− mice were seeded onto
6-well plates and incubated with 7.5 × 105 K562 cells(apoptotic or non-apoptotic) for 45 min at 37 °C. Following treatment, K562 cells were removed by several
PBS washes. BMDMs were then incubated for another 5hours, and total RNA was isolated using TRIzol reagent(Invitrogen, CA) and converted to cDNA using M-MLV
reverse transcriptase (Invitrogen, CA). The reversetranscriptase qPCR assay was performed using a Verso
cDNA Synthesis Kit (Fisher Scientific, NJ). Gene expression was performed using the PowerTrack™ SYBR Green
Master Mix (Applied Biosystems™) and a CFX96 Touch
Real-Time PCR Detection System (BioRad). Data wereanalyzed using the comparative Ct method with GAPDH
as the reference gene. For A1, the following primer
sequences were used: Forward, ​C​A​G​A​A​G​A​A​T​G​G​A​A​G​A​
G​T​C​A​G; Reverse, ​C​A​G​A​T​A​T​G​C​A​G​G​G​A​G​T​C​A​C​C.

Preparation of BMDMs subjected to efferocytosis forWestern blotting

BMDMs derived from HDAC3f/f and M-HDAC3−/−
mice were seeded into 6-well plates and incubated with
7.5 × 105 K562 cells (apoptotic or non-apoptotic) for
45 min at 37 °C. BMDMs were then washed several times
with PBS to rinse the non-engulfed K562 cells and further incubated for 18 h before protein extraction in RIPA
buffer prior to Western blotting.
Western blotting of frozen retinas and cells

Retinas or cells were collected, snap-frozen, and thenstored in a -80oC freezer for further processing. Tissues
or cells were homogenized in RIPA buffer (Thermofisher) and Western blotting was conducted as previously
described [6]. The primary antibodies were as follows:
HDAC3 (BD Bioscience, San Jose, CA, Cat. #611,124),
albumin (Proteintech, Cat. #16475-1-AP), A1 (GeneTex,
Cat. #GTX109242), and β-actin (Sigma, Cat. #A5441).
Secondary antibodies (Invitrogen) were prepared in 5%
milk in a 1:2000 dilution.
Human eye sections and transcriptome

Postmortem human eye paraffin-embedded sections
were obtained from the National Disease ResearchInterchange (NDRI, Philadelphia, PA). Sections frompatients diagnosed with DR and controls were deparaffinized in xylene (Fisher Scientific, NJ), and rehydrated
in graded ethanol baths (100%, 90%, 70%, 50%) followed
by a final incubation in distilled water. Antigen retrieval
was achieved by microwaving the sections in Tris-EDTA
buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20,
pH 8.0). Sections were incubated in 0.2% Triton X-100
in PBS for 15 min followed by blocking with 3% normal
donkey serum (MilliporeSigma, Cat. # 5,058,837) and 3%
BSA for 1 h. Sections were then incubated overnight at
4 °C with diluted primary antibodies in a blocking bufferthat included Iba1 (FUJIFILM Wako, Cat. # 011-27991),
and HDAC3 (Invitrogen, Cat. #PA5-29026). After primary antibody incubation, sections were incubated withthe appropriate secondary antibody, then washed withcold PBS and cover-slipped with Vectashield antifade
mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Newark, CA) to marknuclei.
Information on HDAC3 expression in the human eye
was obtained by searching the Human Eye Transcriptome Atlas, a publicly available database https://www.
eye-transcriptome.com/search_latest.php.
Statistical analysis

Statistics were performed and graphs prepared using
GraphPad Prism 10 software and data were presented
as mean ± standard deviation (SD). A p-value < 0.05 wasShahror et al. Journal of Neuroinflammation

Fig. 1 (See legend on next page.)(2024) 21:170

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Shahror et al. Journal of Neuroinflammation(2024) 21:170

Page 8 of 21(See figure on previous page.)
Fig. 1 Myeloid HDAC3 deletion is neuroprotective and mitigates retinal thinning after IR injury. (A) Immunolabeling of postmortem human retina sections from control and DR patients show colocalization of HDAC3 with Iba1+ myeloid cells. GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer, n = 3. (B) Graph extracted from a searchable transcriptome database for human eye tissue (https://www.eye-transcriptome.com/) shows highHDAC3 expression in retinal microglia and vitreous MΦ. C-E) Immunolabeling at 7 days after injury and quantification show reduced neurodegeneration(marked by the neuronal marker, NeuN) and microglia/MΦ numbers (marked by Iba1) in the M-HDAC3−/− retinas as compared to HDAC3f/f, n = 5–9. F-H)
Optical coherence tomography (OCT) performed on anesthetized mice 7 days after IR shows preserved total retinal thickness (indicated by a dashed line)
and preserved thickness of the inner nuclear layer (indicated by a star) in M-HDAC3−/− retinas, n = 6–9, *p < 0.05, **p < 0.01, ***p < 0.005, ns: not significant

considered statistically significant. All statistical analyses were performed using the Student’s t-test (for twogroup comparisons) or analysis of variance (ANOVA)
with Tukey’s post hoc test (for comparison of multiple
groups).

Results

retinal thickness in the M-HDAC3−/− mice compared tothe injured HDAC3f/f controls (Fig. 1F-H). Collectively,
these data suggest that myeloid HDAC3 contributes toretinal neuronal degeneration following IR injury andthat HDAC3 deletion mitigates IR-induced retinal degeneration and thinning.

Deletion of myeloid HDAC3 protects retinal neurons fromIR injury and preserves retinal thickness

Deletion of myeloid HDAC3 preserves retinal function after
IR injury

We recently reported that HDAC3 is upregulated inmurine retinal microglia/MΦ following IR injury [5].
Here, we confirm this preclinical finding in human retina
sections from patients with DR, a disease characterizedby varying degrees of retinal ischemia. HDAC3 showed
expression in Iba1+ myeloid cells in retina sections fromcontrol subjects and patients with DR with the lattershowing amoeboid myeloid cells with strong expression of HDAC3. (Fig. 1A). Searching a publicly available
eye transcriptome database showed the highest HDAC3
expression in retinal microglia and vitreous MΦ (Fig. 1B).
To investigate the role of myeloid HDAC3 in retinal injury, we generated a myeloid-specific HDAC3 KO
mouse line using a LysM Cre driver (characterized in
Supplementary Fig. S1). This Cre driver deletes the geneof interest in MΦ and ∼ 30% of microglia [58]. We subjected myeloid HDAC3 KO mice (M-HDAC3−/−) andfloxed controls (HDAC3f/f) to 60 min of ischemia followed by reperfusion (IR) injury. A series of assays toevaluate retinal injury outcomes were conducted at different time points based on our previous work (Supplementary Fig. S3 and Table 1 depict the experimental
timeline) [6]. We initially examined neurodegenerationand myeloid cell proliferation at day 7 after IR by immunolabeling retina flat mounts with the neuronal marker
NeuN and the microglia/MΦ marker Iba1 using ourestablished protocol [6]. Retinas from IR-injured floxed
control mice showed neuronal degeneration and myeloid
cell activation as evidenced by decreased NeuN andincreased Iba1 labeling, whereas retinas from mice lacking myeloid HDAC3 showed neuronal preservation after
IR and decreased Iba1 labeling (Fig. 1C-E). The Shollanalysis on day 2 after IR revealed that myeloid cells fromthe IR-injured groups exhibited reduced branching complexity compared to sham controls. Interestingly, there
were no significant differences in branching complexity within either the IR or sham groups (Supplementary
Fig. S5). OCT at day 7 after IR showed preservation of

To determine whether the retinal neuroprotection andstructural preservation conferred by myeloid HDAC3
deletion translates to improved retinal function, we
conducted electroretinographic (ERG) measurementson mice subjected to IR and then allowed to recover for
14 days. Three progressive flash intensities were used
(Fig. 2A-C). Measurements included analysis of a-wave
amplitude (a measure of photoreceptor function) andb-wave amplitude (a measure of ON bipolar cell function)and oscillatory potential amplitudes (OPs, including thethree major wavelets, OP1, OP2, and OP3, and provide
a measure of inner retina 3rd order neurons function).
Uninjured M-HDAC3−/− and HDAC3f/f mice showed nodifference in ERG response (Supplementary Fig. S6). IR
injury of HDAC3f/f retinas resulted in a marked reduction
in a-wave amplitude at 14 days. This loss of function waspartially ameliorated in injured retinas of M-HDAC−/−mice in which a-wave amplitude was higher than ininjured HDAC3f/f; statistical significance was reached ata flash intensity of 0.1 cd.s/m2 (Fig. 2D, F, H). Similarly,
we observed a drastic reduction in b-wave amplitudes
elicited by 3 flash intensities in injured HDAC3f/f retinas
subjected to ERG at 14 days. In comparison to HDAC3f/f
mice, the b-wave amplitude was higher in M-HDAC3−/−
retinas, reaching significance at the flash intensities of
0.01 cd.s/m2 and 1.0 cd.s/m² (Fig. 2E, G, I). M-HDAC3−/−
retinas showed better OPs only at higher flash intensities, 0.1 and 1 cd.s/m² (Fig. 2J-U). Of note, ERG responsetime was not different between the M-HDAC3−/− andHDAC3f/f IR groups (Supplementary Fig. S7). Collectively, the ERG data confirms the retinal neuronal function preservation after IR with myeloid HDAC3 deletion.
Deletion of myeloid HDAC3 attenuates IR-induced retinal
permeability and vascular degeneration

Next, we examined the effect of myeloid HDAC3 deletionon IR-induced disruption of the blood-retinal barrier. An
intact blood-retinal barrier relies on the integrity of tightShahror et al. Journal of Neuroinflammation(2024) 21:170

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Fig. 2 Myeloid HDAC3 deletion improves the retinal ERG after IR. A-C) Representative a- and b-waveforms elicited by 3 flash intensities in retinas of
HDAC3f/f and M-HDAC3−/− mice on day 14 after IR injury. D-I) Quantification and comparison reveal improved waveform amplitudes in M-HDAC3−/−
retinas with statistical significance achieved at flash intensities of 0.1 cd.s/m2 (a-wave) and 0.01 cd.s/m2 and 1.0 cd.s/m² (b-wave). J-L) Representative
waveforms of oscillatory potentials (OPs) in retinas of HDAC3f/f and M-HDAC3−/− mice elicited by 3 flash intensities at day 14 after IR injury. M-U) Quantification reveals a pattern of improved OP with statistical significance achieved at 1 cd.s/m² (OP2) and at 0.1 cd.s/m2 and 1.0 cd.s/m² (OP3), n = 6–8, *p < 0.05,**p < 0.01

Shahror et al. Journal of Neuroinflammation(2024) 21:170

junctions of the retinal vascular endothelium. Changes inbarrier permeability in perfused mouse retinas on day 2
after IR were assessed by evaluating extravasated albumin on Western blot. Injured retinas from M-HDAC3−/−
mice were profoundly protected from blood-retinal
barrier disruption compared to HDAC3fl/fl retinas as evidenced by reduced albumin leakage (Fig. 3A, B). Reduced
retinal permeability in M-HDAC3−/− mice was confirmed using fluorescein angiography and Evans blue
leakage (Fig. 3C, D, and Supplementary Fig. S8). Vascular integrity was further assessed by conducting trypsin
digestion of retinas and counting acellular capillaries atday 14 after injury. Injured M-HDAC3−/− retinas showed
fewer acellular capillary formations compared to injured
HDAC3f/f retinas, confirming the vascular protective
effect of myeloid deletion of HDAC3 (Fig. 3E, F).
HDAC3 deletion ameliorates the myeloid cell response toretinal injury

Flow cytometric analysis of retina myeloid cells was conducted on day 2 after IR according to our established protocol [54]. Retina myeloid cells were gated using CD45
and CD11b markers and myeloid leukocytes were identified as CD11bhigh, CD45high, whereas microglia were identified as CD11bhigh, CD45intermediate [54]. The IR injury led
to a robust myeloid cell response in retinas (Fig. 4A, B)
in agreement with previous work [10, 54]. Quantificationof myeloid leukocyte and microglia showed a ∼ 10-foldincrease in myeloid leukocyte number (Fig. 4C), whereas
there was a trend toward increased microglia numbers(Fig. 4D). Interestingly, the strong increase in myeloid
cells was dampened in the M-HDAC3−/− retinas injured
by IR. Notably, the frequency of Ly6Clow reparative
monocytes, Ly6Cintermediate and Ly6Chigh inflammatory monocytes was not significantly different between
injured M-HDAC3−/− and HDAC3f/f retinas (Fig. 4EG) despite the lower myeloid leukocytes count in the
M-HDAC3−/− retinas. These results indicate that HDAC3
deletion reduces myeloid cell proliferation/infiltration in
the injured retina after IR.
HDAC3 deletion increases myeloid cell efferocytosis
activity

Since HDAC3 deletion did not affect the myeloid cell
inflammatory response as measured by Ly6C expression, we sought to investigate efferocytosis as a potential reparative mechanism employed by myeloid cells.
We employed two distinct assays to evaluate whether
deletion of HDAC3 increases MΦ-mediated reparative
clearance of apoptotic cells by efferocytosis. Confocal
microscopy was performed to detect co-localization of
TUNEL-labeled apoptotic cells (ACs) with Iba1-labeled
myeloid cells. We chose 2 days post-IR injury as the apoptosis peak at this time point based on our preliminary

Page 10 of 21

studies and published literature [59]. The total number of TUNEL+ cells in M-HDAC3f/f and M-HDAC3−/−
retinas was not significantly different at 2 days post-IR
injury. Interestingly, the M-HDAC3−/− retinas showed
increased Iba1+ myeloid cell association with TUNEL+
ACs as an indication of efferocytosis (Fig. 5A-C). Resultsfrom flow cytometry confirmed that M-HDAC3−/− retinas exhibited higher levels of efferocytosis at 2 days after
IR injury. In these studies, ACs were labeled in vivo using
eye drops with PSVue 550 before retinas were removed
and trypsin-digested for flow cytometry, which quantified myeloid leukocytes and microglia positive for thephagocytic marker CD68 that exhibited engulfed PSVue+
ACs (CD68+/PSVue+). M-HDAC3−/− retinas displayedenhanced efferocytosis as measured by higher numbersof CD68+/PSVue+ myeloid leukocytes (Fig. 5D, E) andmicroglia (Fig. 5F, G). Collectively, these findings imply
that HDAC3 inhibits efferocytosis since its deletionencourages this process.
We explored this possibility and underlying mechanisms using in vitro efferocytosis assays. We labeled
HDAC3−/− and HDAC3f/f (control) MΦ with Dil-red
fluorescent dye for identification and incubated themwith CFDA green-labeled ACs, or non-apoptotic K562
lymphoblasts (NAC), or performed no cell treatment(no ttt). K562 cells are easily induced to be apoptotic andare routinely used in suspension cultures because they
are readily washed from adherent MΦ, which enables
accurate counting of the remaining engulfed K562 cells[7]. Flow cytometric analysis of phagocytic MΦ (Dil+/
CFDA+) showed increased efferocytosis of ACs by
HDAC3−/− compared to HDAC3f/f MΦ (Fig. 5H, I). Collectively, these results suggest the reparative effect of
myeloid HDAC3 deletion involves enhanced efferocytic
functions.
The proefferocytic effect of HDAC3 deletion is partiallydependent on arginase 1

We recently demonstrated that deletion of the enzyme
A1 in MΦ increases the expression of HDAC3 [5]. Since
a contribution of the A1 pathway to efferocytosis has
been reported in rodent models of stroke and atherosclerosis [13, 32, 33], we explored the possibility that
HDAC3 downregulates A1 in a negative feedback loop
to limit efferocytosis. Indeed, the addition of apoptotic
cells to HDAC3−/− MΦ in our in vitro efferocytosis assay
resulted in a marked increase in A1 protein (Fig. 6A, B)
and transcript (Fig. 6C) that were significantly higher
than in HDAC3−/− MΦ treated with apoptotic cells. To
determine whether HDAC3 deletion promotes A1-mediated efferocytosis, HDAC3−/− MΦ and HDAC3f/f MΦwere treated with the A1 inhibitor, ABH, and subjected to efferocytosis. As anticipated, HDAC3 deletion
enhanced efferocytosis whereas A1 inhibition attenuatedShahror et al. Journal of Neuroinflammation(2024) 21:170

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Fig. 3 Myeloid HDAC3 deletion is vascular protective in retinal IR injury. A, B) Albumin extravasation at day 2 post-IR was measured in protein lysatesof perfused retinas by western blot. Extravasated albumin was markedly reduced in M-HDAC3−/− retinas compared to HDAC3f/f controls and was nearly
absent in the contralateral retinas. GAPDH was used as a loading control, n = 6–8. C) Representative retinal fluorescein angiography images at day two
post-IR showing increased retinal vascular permeability. Leaked fluorescein demonstrated by diffusely hazy fluorescence (white arrows) was markedly
reduced in M-HDAC3−/− retinas compared to HDAC3f/f controls and was absent in the sham retinas. D) Quantification of Evans blue leakage demonstrates
a significant reduction in the M-HDAC3−/− IR retinas, n = 4–5. Representative images are included in supplementary figure S8. E, F) Vascular digests at 14days after IR injury showed increased numbers of acellular retinal capillaries (empty basement membrane sleeves - red arrows) in injured HDAC3f/f retinas
compared to injured M-HDAC3−/− retinas, n = 6–8, *p < 0.05, **p < 0.01

Shahror et al. Journal of Neuroinflammation(2024) 21:170

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Fig. 4 M-HDAC3 deletion mitigates the IR-induced microglia proliferation and myeloid leukocyte infiltration into the retina. (A) Representative scattergraphs from flow-cytometric analysis showing the initial gating strategy used to quantify the immune cell populations of microglia, myeloid leukocytes(ML), and lymphocytes in HDAC3f/f and M-HDAC3−/− retinas on day two after IR injury. (B) The ML were further gated based on Ly6C/Ly6G expression into
Ly6Chi/Ly6Gneg monocytes, Ly6Cmoderate/Ly6Gneg monocytes, Ly6Cneg/Ly6Gneg monocytes, and Ly6C+/Ly6G+ granulocytes. (C) The number of CD11b+/
CD45hi ML was reduced in injured M-HDAC3−/− retinas compared to injured HDAC3f/f controls indicating reduced myeloid cell infiltration/proliferation.
(D) Similarly, the number of CD11b+/CD45low microglia was reduced in injured M-HDAC3−/− retinas. E-G) ML further gated into Ly6Clow, Ly6Cintermediate,and Ly6Chigh monocytes showed no change in percentage frequency between the injured groups. For each data point, 3 retinas were pooled and n = 4–5
preparations of pooled retinas were analyzed for each group. *p < 0.05, **p < 0.01

Shahror et al. Journal of Neuroinflammation

Fig. 5 (See legend on next page.)(2024) 21:170

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Shahror et al. Journal of Neuroinflammation(2024) 21:170

Page 14 of 21(See figure on previous page.)
Fig. 5 M-HDAC3 deletion improves macrophage clearance of apoptotic cells through efferocytosisin vivo and in vitro.A) Representative confocal imagesof immunolabeled TUNEL+ apoptotic cells (red) and Iba1+ myeloid cells (green) in retinal flat mounts on day 2 after IR injury. Arrows and arrowheads
depict Iba1-associated and free TUNEL+ cells, respectively. B) The total number of apoptotic cells (ACs, TUNEL+) was not significantly different between the
injured groups. C) Quantification of the efferocytosis index (% Iba1+ ACs / total ACs) indicated enhanced phagocytosis in injured retinas of M-HDAC3−/−
compared to HDAC3f/f mice, n = 6–8. D-G) Flow-cytometric analysis of myeloid leukocytes and microglia co-expressing the phagocytic marker CD68+
and the apoptotic cell signal PSVue reveals more CD68+/PSVue+ cells in retinas of M-HDAC3−/− compared to HDAC3f/f mice at day 2 after IR injury, n = 5
per group. H) Representative scatter plots from flow cytometry studies in which non-apoptotic (NAC) or apoptotic (AC) CFDA green-labeled K-562 cells
were co-cultured with CM-DiI red-labeled MΦ. Control MΦ received no treatment (no ttt). I) Quantification by fluorescence-activated cell sorting (FACS)
of MΦ engaged in efferocytosis of ACs (Dil+/CFDA+) as a percent of total MΦ followed by normalization to the NAC group. Efferocytosis was enhanced in
HDAC3−/− MΦ, n = 5 per group, *p < 0.05, ***p < 0.005. No-treatment group was used as a negative control for gating of CFDA- macrophages

efferocytosis in HDAC3f/f and HDAC3−/− MΦ (Fig. 6D,
E). However, two-way ANOVA statistical analysis showed
no interactive effect between HDAC3 and A1, suggestingthat the inhibitory effect of HDAC3 on efferocytosis is
not solely dependent on A1 (Fig. 6D, E). To confirm that
inhibition of A1 impairs efferocytosis, we performed invitro efferocytosis assays using CM-DiI red-labeled MΦ
from myeloid-specific A1 knockout (M-A1−/−) mice andA1f/f control mice. Confocal imaging revealed that A1−/−
MΦ exhibited impaired efferocytosis compared to control A1f/f MΦ in efferocytosis assays in which engulfmentof CFDA green-labeled R28 (Fig. 7A, B) or K562 (Fig. 7C,D) ACs by MΦ indicated efferocytosis events. Complementary flow cytometry studies confirmed that a higher
percentage of CM-Dil red MΦ were positive for CFDA
green-labeled ACs in MΦ populations from A1f/f compared to M-A1−/− mice (Fig. 7E, F).
Inhibition of HDAC3 is neuroprotective in retinal IR injury

Based on our findings implicating HDAC3 in IR injury,
we performed proof-of-principle in vivo studies to evaluate the therapeutic potential of HDAC3 inhibition. In
these studies, WT mice were dosed with 10 mg/kg i.p.
of the specific HDAC3 inhibitor RGFP966 or vehicle at
1 h after IR and every 48 h thereafter until day 7. The i.p.
route of administration was selected because RGFP966
crosses the blood-retina barrier and no systemic toxicity has been reported using this dose [60]. Compared tovehicle (DMSO) treatment, administration of RGP966
conferred significant neuroprotection as revealed by thepreservation of NeuN-positive neurons (Fig. 8A and B).
OCT analysis also showed preserved retinal morphology and thickness in the RGFP966-treated mice (Fig. 8C
and D). Of note, RGFP966 treatment had no effects on
the contralateral retina thickness as compared to vehicle
treatment (Supplementary Fig. S9). Collectively, HDAC3
inhibition showed similar neuroprotection to the
myeloid-specific deletion of HDAC3.

Discussion
Our study provides the first direct evidence to ourknowledge that the HDAC3 isoform in myeloid cells suppresses efferocytosis and worsens ischemic retinopathy,
and that reparative efferocytosis contributes to injury

resolution in the inner retina. Compelling evidence thatmyeloid HDAC3 plays a detrimental role in retinal IR
injury is provided by the observation of robust neurovascular protection in myeloid-specific HDAC3−/− mice.
This protection is mediated, at least in part, by enhanced
efferocytosis in injured retinas of HDAC3−/− mice thatexhibited higher levels of myeloid cell association with
apoptotic cells. Finally, pharmacological inhibition of
HDAC3 protected against retinal IR injury, raising thepossibility that HDAC3 may have value as a therapeutictarget to mitigate retinal degeneration.
Initially, we considered different mediators of retinal
IR injury including the HDAC gene family. Our interestin the HDAC family was based on evidence that HDAC
pan-inhibitors limit neuronal damage after retinal IR
injury [26, 27]. The HDAC3 isoform causes epigenetic modifications that promote the expression of MΦ
inflammatory genes [30, 31]. Subsequently we observeda robust induction of HDAC3 in retinal myeloid cells in
the mouse model of IR injury [5]. In the present study,
we report colocalization of HDAC3 with the myeloid
cell marker Iba1 in human retinas from control and DR,
which is known to involve chronic ischemia. Interestingly, myeloid cells showed an amoeboid phenotype in
the DR retinas denoting activation. Furthermore, publicly available data showed strong HDAC3 expression
in human retinal microglia and vitreal MΦ. Building onthese findings, we leveraged the use of myeloid-specific
HDAC3−/− mice to implicate HDAC3 in the neurovascular injury caused by retinal IR. Mice lacking HDAC3 inmyeloid cells exhibited marked preservation of the innerretinal neurons as measured by NeuN labeling, whichmarks ganglion cells and other neuron cell types such as
displaced amacrine cells [61, 62]. Mice lacking HDAC3
also showed preserved retinal thickness after IR injury.
Retinal cell function evaluated by ERG was preserved in
M-HDAC3−/− mice. The losses of a- and b- wave amplitudes and OPs that are features of retinal injury anddegeneration [1, 63, 64], and predict vision loss were lesspronounced in retinas of M-HDAC3−/− mice recoveredfor 14 days after IR insult. The partial preservation of thea-wave, b-wave, and OPs in the dark-adapted eye confirmed the collective protection of inner retina neurons
as well as photoreceptors in these animals. In addition toShahror et al. Journal of Neuroinflammation(2024) 21:170

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Fig. 6 Arginase 1 is upregulated in M-HDAC3−/−macrophages and its inhibition impairs macrophage-mediated efferocytosis. A, B) Western blot shows
upregulation of Arginase 1 (A1) in M-HDAC3−/− but not HDAC3f/f MΦ after co-incubation with K-562 ACs. Controls included either co-incubation of MΦ
with K-562 NACs or no treatment (no ttt), n = 4. C) The corresponding A1 transcript was increased in M-HDAC3−/− MΦ co-cultured with K-562 ACs as
compared to control preparations. A1 transcript also increased in HDAC3f/f MΦ co-cultured with ACs but significantly less than in AC/M-HDAC3−/− MΦ cocultures, n = 4. D)In vitro efferocytosis assay in which CFDA green-labeled K-562 NACs or ACs (shown in green) were added to DiI red-labeled MΦ (shownin red) after 45 min pre-incubation with the A1 inhibitor ABH (100 µM). Arrowheads indicate efferocytosis of ACs by HDAC3f/f or M-HDAC3−/− MΦ, n = 4. E)
Quantification of MΦ engaged in efferocytosis of ACs (Dil+/CFDA+) as a percent of total MΦ reveals enhanced efferocytosis in M-HDAC3−/− MΦ. Two-way
ANOVA showed that the effect of HDAC3 deletion and ABH treatment were independently statistically significant but there was no interaction between
the two, n = 4, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

its neuroprotective impact, deletion of myeloid HDAC3
conferred a robust retinal vascular protective effect asmeasured by reduced albumin extravasation and further
confirmed using fluorescein angiography and Evans blue
leakage. M-HDAC3−/− mice retinas had fewer acellularretinal capillaries after IR injury as compared to retinas
from HDAC3f/f IR mice. Retinal permeability was measured at 48 h since previous research has shown that
endothelial tight junction complexes exhibit significant
disruption at this time point after IR [10]. Interestingly,
While neurodegeneration occurs within the first week inthis model, the death of endothelial cells does not happen
until later, which is why we measured vascular degeneration at 14 days [6, 10]. This is in line with reports that

retinal neurodegeneration precedes vascular degeneration [65, 66].
Our subsequent studies began to mechanistically clarify
the contribution of HDAC3 to retinal IR injury. The retinal
IR insult is known to induce a strong monocyte/MΦ cell
response that also includes lesser microglial activation [10–12]. Accordingly, we recorded a 10-fold increase in myeloid
leukocytes in the injured retina of floxed mice. In contrast,
the retinas of M-HDAC3−/− mice exhibited fewer microglia and myeloid leukocytes after IR injury as determined byflow cytometric analysis. This was confirmed by the reduced
number of Iba1+ myeloid cells in M-HDAC3−/− retina flat
mounts after IR. However, the frequency of Ly6Chi monocytes/MΦ remained the same, suggesting that HDAC3

Shahror et al. Journal of Neuroinflammation(2024) 21:170

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Fig. 7 Arginase 1 deletion inhibits efferocytosis in macrophages(A) Representative images from an in vitro efferocytosis assay in which CFDA green-labeled R28 ACs were added to CM-DiI red-labeled MΦ isolated from
M-A1−/− or A1f/f mice. (B) Efferocytosis was reduced in MΦ from M-A1−/− mice, n = 3. C, D) A similar efferocytosis assay but using K-562 ACs also showed
reduced efferocytosis by MΦ from M-A1−/− mice compared to A1f/f mice, n = 5 per group. E, F) Scatter plots and quantification from FACS of CM-DiI+/
CFDA+ MΦ confirmed impaired efferocytosis of K-562 AC by M-A1−/− compared to A1f/f MΦ, n = 5 per group, *p < 0.05, **p < 0.01

Shahror et al. Journal of Neuroinflammation(2024) 21:170

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Fig. 8 HDAC3 inhibition is neuroprotective and mitigates retinal thinning after IR injury. A-B) Retinal flat mounts from vehicle-treated WT mice show amarked loss of NeuN-labeled retinal neurons on day 7 after IR injury. After administration of the HDAC3 inhibitor RGFP966 (10 mg/kg i.p.) at 1 h after IR
and on days 2, 4, and 6, retinal neurons were partially preserved at 7 days. RGFP996 did not reduce NeuN-positive cells in the retinas of the sham animals,n = 7–8. C, D) OCT images and quantification show preserved total retinal thickness on day 7 after IR injury in WT mice treated with RGFP966. Orange
arrows indicate retinal detachment in the IR retinas of vehicle treated group that is absent in the RGFP996 treated group, n = 6–8, *p < 0.05

deletion does not affect myeloid cell inflammatory response
in retinal IR.
Our new finding that myeloid cells lacking HDAC3
exhibit enhanced efferocytosis after IR injury implies that
HDAC3 suppresses the process of reparative efferocytosis
in the retina. Two lines of evidence support this concept.
First, Iba1+ myeloid cells lacking HDAC3 were observed
to co-localize significantly more after IR injury withTUNEL+ apoptotic cells in retinal flat mount images. Second, HDAC3 deletion increased the number of phagocytic
CD68+/PSVue+ microglia and myeloid leukocytes detectedby flow cytometry in injured retinas. These collective results
may indicate that despite the attenuation of myeloid cellproliferation in retinas of M-HDAC3−/− mice after IR injury,
the existing myeloid cell population can more efficiently
engulf apoptotic cells to enact reparative efferocytosis

without overactivation of the phagocytosis process. Indeed,
our in vitro efferocytosis assays confirmed that MΦ lacking HDAC3 more efficiently clear apoptotic cells, pointing
to HDAC3 as a culprit molecule that undermines retinal
repair.
Finally, we explored whether HDAC3 deletion promotes
efferocytosis by upregulating the known pro-efferocytic
enzyme A1 [5], and whether an isoform-specific HDAC3
inhibitor confers neurovascular protection from IR injury.
A1 transcript and protein were significantly higher in
myeloid cells lacking HDAC3 and treated with apoptotic
cells in vitro. Pharmacological inhibition of A1 blunted theenhanced efferocytosis conferred by HDAC3 deletion inMΦ. However, A1 inhibition had similar effects on control
HDAC3f/f MΦ. Thus, in addition to A1, there appear to beother unique and unidentified HDAC3 signaling pathways

Shahror et al. Journal of Neuroinflammation(2024) 21:170

that modulate efferocytosis. The study findings are presented in Fig. 9, and ongoing studies in our lab are examining additional underlying mechanisms of HDAC3 inhibitory
effect on efferocytosis using unbiased RNA-sequencing.

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Regardless of the exact molecular pathways of protection,
our final set of studies demonstrating that systemic administration of the HDAC3 inhibitor, RGFP966 to WT mice protects the retinal neurons and preserves retinal morphology

Fig. 9 Schematic of the study findings. Myeloid HDAC3 suppresses efferocytosis and A1 expression. A1 promotes efferocytosis and plays a role in theenhanced efferocytosis of MΦ lacking HDAC3. HDAC3 may additionally impair efferocytosis by a mechanism unrelated to loss of A1

Shahror et al. Journal of Neuroinflammation(2024) 21:170

after IR injury infers that HDAC3 may be a therapeutic target suitable for clinical intervention to preserve vision. Since
RGFP966 readily penetrates the blood-retina barrier [60],systemic administration was selected over intravitreal injection to allow for multiple dosing. The dose and dosing interval were selected based on previous literature that showedmaximal neuroprotection at 2 weeks post optic nerve crushwith 10 mg/kg RGFP966, given i.p. every 3 days [60]. The
same study showed that daily administration of RGFP966 at
10 mg/kg for 2 weeks did not result in abnormal histopathological effects or toxicity [60]. Furthermore, the 10 mg/
Kg dose was shown to specifically inhibit HDAC3 withminimal inhibition of HDAC1 and HDAC2 [67]. While theplasma half-life of RGFP966 was reported to be relatively
short (peaking within 30 min and declining over the next2 h), our study and others show that it is protective in thisshort ‘pulsed’ dosing [68]. For human translation, localized
HDAC3 inhibition via intravitreal administration could offer
a more targeted approach compared to systemic administration, potentially avoiding systemic adverse effects.
In summary, we have identified the HDAC3 isoform
expressed by myeloid cells as an important mediator of
IR-induced retinal injury. HDAC3 appears to inhibit
reparative efferocytosis in part through suppression of A1
expression. Further investigation into the mechanisms bywhich HDAC3 suppresses efferocytosis is warranted tofacilitate the development of clinically effective HDAC3
inhibitors for the treatment of ischemic retinopathies.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03159-8.
Supplementary Material 1
Acknowledgements
Not applicable.
Author contributions
RAS planned and performed experiments and drafted the manuscript. ES,
CAM, and MW performed experiments. SM, GC, and MB provided scientificguidance and support with techniques. NJR critically evaluated and edited themanuscript. AYF conceived and supervised the whole project and drafted themanuscript.
Funding
This work was supported by the National Institute of Health/National Eye
Institute (NIH/NEI) grant (4 R00 EY029373-03), and UAMS Hornick award to AYF.
Data availability
Data is provided within the manuscript or supplementary information files.

Declarations
Ethics approval
Animal experiments were approved by UAMS IACUC committee.
Consent for publication• Not applicable.

Page 19 of 21

Competing interests
The authors declare no competing interests.
Author details
1
Department of Pharmacology and Toxicology College of Medicine,
University of Arkansas for Medical Sciences (UAMS), 4301 West Markham
Street, Slot 611, BIOMED-1, B306, Little Rock, Office, AR 72205, USA
2
Department of Pharmaceutical Sciences, College of Pharmacy, Universityof Arkansas for Medical Sciences, Little Rock, AR, USA
3
Department of Pharmacology and Vascular Biology Center, Augusta
University, Augusta, GA, USA
4
Clinical Pharmacy Department, Cairo University, Cairo, Egypt
Received: 19 March 2024 / Accepted: 24 June 2024

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