Yuan et al. Journal of Neuroinflammation(2024) 21:136
https://doi.org/10.1186/s12974-024-03134-3

Journal of Neuroinflammation

Open Access

RESEARCH

CCR5-overexpressing mesenchymal stem cells
protect against experimental autoimmune
uveitis: insights from single-cell transcriptome
analysis
Fa Yuan1, Rong Zhang1, Jiani Li1, Qiannan Lei1, Shuyi Wang1, Fanying Jiang1, Yanan Guo1 and Mengqing Xiang1,2*
Abstract
Autoimmune uveitis is a leading cause of severe vision loss, and animal models provide unique opportunities forstudying its pathogenesis and therapeutic strategies. Here we employ scRNA-seq, RNA-seq and various molecularand cellular approaches to characterize mouse models of classical experimental autoimmune uveitis (EAU),
revealing that EAU causes broad retinal neuron degeneration and marker downregulation, and that Müller glia
may act as antigen-presenting cells. Moreover, EAU immune response is primarily driven by Th1 cells, and resultsin dramatic upregulation of CC chemokines, especially CCL5, in the EAU retina. Accordingly, overexpression of
CCR5, a CCL5 receptor, in mesenchymal stem cells (MSCs) enhances their homing capacity and improves theirimmunomodulatory outcomes in preventing EAU, by reducing infiltrating T cells and activated microglia andsuppressing Nlrp3 inflammasome activation. Taken together, our data not only provide valuable insights into themolecular characteristics of EAU but also open an avenue for innovative MSC-based therapy.
Keywords Experimental autoimmune uveitis, scRNA-seq, CCL5/CCR5, Mesenchymal stem cells

Introduction
Uveitis is a major sight-threatening ocular inflammatory disease worldwide, whose sequelae include cataracts, glaucoma, vitreous opacities, retinal detachment,
and retinal vascular abnormalities, etc [1–5]. Autoimmune uveitis, one kind of uveitis, is estimated to be the*Corresponding author:*Correspondence:
Mengqing Xiang
xiangmq3@mail.sysu.edu.cn
1State Key Laboratory of Ophthalmology, Guangdong Provincial
Key Laboratory of Ophthalmology and Visual Science, Zhongshan
Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China
2
Guangdong Provincial Key Laboratory of Brain Function and Disease,
Zhongshan School of Medicine, Sun Yat-sen University,
Guangzhou 510080, China

fourth leading cause of severe vision loss in the industrialized world [4] and occurs in a variety of diseases[6]. Experimental autoimmune uveitis (EAU) inducedby the interphotoreceptor retinoid-binding protein(IRBP), which shares many pathological characteristics
with those of human uveitis, is widely used to help illuminate the genetic influences on, resolve the pathogenic
mechanisms of, and test potential therapeutic paradigms
for human uveitis since 1988 [7–9]. However, no single
animal model such as EAU can reproduce the full spectrum of features of the human disease. To better understand the pathogenesis of the disease, in addition to the
induced model of autoimmune uveitis, two spontaneous
uveitis models, R161H and Aire−/− mice, were also established subsequently [10, 11]. The classical EAU inducedby IRBP is Th17- or Th1-dependent [12–15], while the© 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
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Yuan et al. Journal of Neuroinflammation(2024) 21:136

two spontaneous uveitis models, which rely on genetic
backgrounds, have been reported to be IFNγ- and Th1dependent, respectively [12, 16].
Previous study suggests that EAU is accompanied byblood-retinal barrier (BRB) breakdown [17]. Meanwhile,
Crane et al. reported that CCR5, a chemokine receptor, plays an important migratory role for cells entering the BRB and retina [18]. It has been suggested thatEAU triggers the expression and release of chemokines,
which in turn facilitate the trans-endothelial migrationof immune cells and mediate the BRB disruption. Nevertheless, CCR5 deletion failed to prevent the development
of EAU, although it did reduce T-cell infiltration into theeye [19]. It has also been speculated that the chemokine
ligands mediate the trafficking of mesenchymal stem
cells (MSCs) and their infiltration of the injured tissue[20]. However, isolated MSCs were found to graduallylose their homing capacity to the targeted lesions during continuous passage [21–23]. Therefore, strategies
for enhancing the homing ability of infused MSCs to theimpaired retinas may benefit therapeutic treatments.
Although single-cell RNA sequencing (scRNA-seq)
analysis has been used to characterize the cell types andgene expression patterns in Aire−/− mouse retinas [16],the classical EAU model induced by IRBP has not yet
been characterized using this powerful and unbiased
method. To investigate the pathogenesis and therapeuticstrategy of uveitis, we characterized the neural retinas of
EAU mice on a C57BL/6 background using both scRNAseq and bulk RNA-seq analyses in the present study. We
found that EAU caused a decrease in marker expression
levels and cell number of all retinal neuron types, andthat Müller glia might act as antigen-presenting cells(APCs) during this process. Moreover, the classical EAU
induced by IRBP peptide 651–670 was Th1-dependent,
and caused dramatic upregulation of the chemokine
CCL5 in the EAU retinas. Based on these observations,
we speculated that MSCs overexpressing human CCR5,
a CCL5 receptor, would have enhanced homing capacityand better attenuate EAU through their immunomodulatory capacity than regular MSCs. And indeed, we wereable to show that CCR5-overexpressing MSCs exhibiteda better therapeutic effect on EAU than regular MSCs,
hence providing a novel MSC-based therapy option.

Results
Diverse immune cells infiltrate into the retina of EAU mice

As a first step to characterize retinal changes in singlecell transcriptome caused by EAU, we established EAU
mouse models by immunization with a human IRBP
peptide 651–670 as described [24]. To confirm thesemouse models, we subsequently performed fundus
imaging, fluorescein fundus angiography (FFA), andoptical coherence tomography (OCT) on mice every 7

Page 2 of 24days postimmunization (d.p.i.) to grade disease severity according to the published clinical grading scale [24,25] (Table S1). The incidence rate was 89.4% ± 7.9% at 21
d.p.i. Figure 1A, B shows representative fundus images
and fluorescein angiograms for the different clinicalgrades (grades 0–4). In general, in the eyes of EAU micefrom low to high grades, there was progressive vascularleakage (Fig. 1A, B), retinal disorganization (Fig. 1C, D),
and increase in the number of infiltrating CD45-positive
leukocytes and CD11b-positive myeloid cells (Fig. 1C-E),
consistent with previous observations [9, 24–27].
Establishing the cell atlas for EAU retinas based on singlecell RNA-Seq

To elucidate changes in the composition and number ofretinal cells when EAU arose, we used mice at grade 2(14 d.p.i.) and their wild-type littermates for scRNA-seq
analysis (Fig. S1). We performed scRNA-seq profilingusing the 10X Genomics platform to obtain 20,448 single
cells from 8 retinas: 2 grade 2 mice (10,369 cells) and 2
wild-type controls (10,079 cells). A total of 147 presumed
doublet cells were excluded from subsequent analysis.
Combining Seurat [28, 29] clustering analysis withknown molecular markers for various cell types (Fig. 2B;
Tables S2, S3), we segregated the single cells into 9 major
cell clusters based on cell types: immune cells [Microglia,
Monocyte/Macrophage, and T/NK (T cell/natural killercell)], retinal cells [AC/HC/RGC (amacrine cell/horizontal cell/retinal ganglion cell), BC (bipolar cell), Cone, MG(Müller glia)/Astrocyte, and Rod], and vascular endothelial cells (VEC) (Fig. 2A). Comparison of control and
EAU cell clusters revealed a decrease in the number ofmultiple neuronal types in EAU retinas, especially in the
BC cluster (20.34–5.02%), Cone cluster (9.49–2.94%), and
AC/HC/RGC cluster (3.76–1.28%) (Fig. 2C). In agreement, bulk RNA-seq analysis showed that in EAU retinas, there was downregulation of marker genes for all 6
retinal neuron types including rods, cones, BCs, ACs,
HCs, and RGCs (Fig. 2D; S2A, B). Immunostaining analysis further validated these results. In EAU retinas, there
was obvious degeneration of rhodopsin+ rod inner andouter segments (Fig. 2E), and in general, progressive lossof Arrestin+ cones, Chx10+ BCs, Tfap2a+ ACs, Rbpms+
RGCs, and Calbindin+ HCs (Fig. 2F-J, L-P). By contrast,
EAU did not appear to significantly alter Sox9+ Müller
glia although resulting in an upregulation of their marker
genes (Fig. 2K, Q). Therefore, EAU may cause not only
progressive degeneration of all retinal neuron types but
also a switch in the expression profile of Müller glia.
The Müller glia may function as antigen-presenting cells

Müller glia are the major glial component of the retina,
which are specified from the retinal progenitor cells [30].
Their processes act as the scaffold of the neural retina,

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Fig. 1 Characterization of EAU retinas. (A) Representative fundus photographs (14 d.p.i.) showing the range of disease severity and corresponding
grades. The asterisks indicate subretinal hemorrhages. (B) Fundus photographs and corresponding fluorescein angiograms (21 d.p.i.) of EAU eyes. The
arrows indicate areas of fluorescein leakage. (C) Fundus photographs (left) and OCT images (right) (14 d.p.i.) showing inflammatory infiltrating cells (arrows), vasculitis (arrowhead), and retinal folds and detachment (asterisks) in EAU eyes. (D) Hematoxylin-eosin staining (14 d.p.i.) revealed inflammatory
infiltrating cells (arrows), vasculitis (arrowheads), and retinal folds and detachment (asterisks) in EAU eyes. (E) EAU increased leukocytes immunoreactive
for CD45 and myeloid cells immunoreactive for CD11b in the retinas (14 d.p.i.). All retinal sections were counterstained with nuclear DAPI. GCL: ganglion
cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. Scale bar: (D, E) 20 μm

ramifying throughout the whole neural retina between
the nerve fiber layer and the outer limiting membrane.
Furthermore, Müller glia are part of the blood-retinal
barrier and are thought to contribute to ocular immune
privilege by inhibiting the proliferation and activation oflymphocytes [31]. Thus, immune cells infiltrating into theretina must necessarily pass and come into contact with
Müller glia.
To investigate the effect of EAU on Müller glia, were-analyzed the MG/Astrocyte cell cluster by UMAP

clustering, which yielded 7 Müller glia clusters (MG_1–7)
when astrocytes were removed (Figs. 2A and 3A). These
Müller glia clusters exhibited distinct expression patternsof Müller cell marker genes (Fig. 3B; S3A), and of all 7
Müller glia clusters, MG_5, which was present mainly in
EAU retinas (Fig. 3A), expressed Gfap (Fig. 3B), suggesting that most Müller glia acquired a reactive state following EAU. Intriguingly, the reactive Müller glia in MG_5
upregulated the major histocompatibility complex class II(MHC II) genes including H2-Ab1 and H2-Eb1 (Fig. 3C),

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Fig. 2 Single-cell transcriptome atlas and neuron loss in EAU retinas. (A) UMAP plot showing different cell-type clusters in a merged dataset from duplicate EAU and control wild-type retinas. (B) Stacked violin plots showing expression patterns of representative cell type marker genes in different cellclusters. (C) UMAP plots and pie charts comparing the cell types in control and EAU retinas. (D) Heatmap of expression levels of a set of marker genes
for cone, rod, bipolar, amacrine, horizontal, retinal ganglion, or Müller glial cells in control and EAU retinas, determined by bulk RNA-seq analysis. (E-K)
Immunostaining of retinal sections for the indicated cell type-specific protein markers showed obvert reduction of retinal neurons after EAU onset. All
retinal sections were also counterstained with nuclear DAPI or hematoxylin (HT). (L-Q) Quantification of cells immunoreactive for the indicated protein
markers in control and EAU retinas. Data are presented as mean ± SD (n = 4–11 retinas per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, nosignificance. AC: amacrine cell; BC: bipolar cell; GCL: ganglion cell layer; HC: horizontal cell; INL: inner nuclear layer; MG: Müller glia; NK: natural killer cell;
ONL: outer nuclear layer; RGC: retinal ganglion cell; S: inner and outer segments; T: T cell; VEC: vascular endothelial cell. Scale bar: (E-K) 20 μm

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Fig. 3 Upregulation of MHC II genes in Müller glia of EAU retinas. (A) UMAP plots of Müller glia clusters in control and EAU retinas. (B) Stacked violin plots
showing expression patterns of known Müller glia and astrocyte marker genes in different Müller cell clusters. (C) Feature plots comparing the expressionof MHC II genes in control and EAU retinas. (D) Immunostaining of control and EAU retinal sections for MHC II proteins I-A/I-E with DAPI counter-labeling.
(E) Triple-immunostaining of control and EAU retinal sections for GS (glutamine synthetase, red), MHC II proteins (I-A/I-E) (green), and Sox9 (magenta) withDAPI counter-labeling (blue). Arrowheads indicate representative GS+Sox9+MHC II+ triple-positive Müller glia. GCL: ganglion cell layer; INL: inner nuclear
layer; ONL: outer nuclear layer. Scale bars: (D, E) 20 μm

Yuan et al. Journal of Neuroinflammation(2024) 21:136

which are mostly expressed by professional APCs [32,33]. In agreement, increasing immunoreactivity for MHC
II I-A/E alloantigens was detected in EAU retinas withincreasing disease grades (Fig. 3D). Moreover, the MHC
class II proteins were found to be enriched in Müller
glial cell bodies (Fig. 3E), suggesting that Müller glia may
function as non-professional APCs in EAU retinas.
Further analysis of our single-cell sequencing data
showed only little or no expression of MHC II-related
genes in amacrine cells, horizontal cells, RGCs, bipolar cells, rods, and cones of EAU retinas. However, theirexpression was detected in the microglia, MG/astrocyte,monocyte/macrophage, and VEC clusters (Fig. S3B).
In agreement, immunostaining of EAU retinal sections
revealed that some Cd31+ VECs, Pax2+ astrocytes andSox9+ Müller glia expressed the MHC II proteins while
there was no MHC II protein expression in Tfap2a+ amacrine cells, Rbpms+ RGCs, and Chx10+ bipolar cells (Fig.
S3C-H).
T cell diversity in the EAU retina

To determine the effect of EAU on immune cells, were-analyzed all immune cell-related clusters (Microglia,
Monocyte/Macrophage and T/NK) by UMAP clustering(Figs. 2A and 4A). By classifying these cells according to
the known immune cell markers (Table S3), we identified 5 subtypes of T cells: Cd8a T cells, Th1 cells, Th17
cells, regulatory T (Treg) cells, and undifferentiated T
cells (Cd4+). Th1 cells were the predominant class of T
helper cells in EAU retinas, accounting for 19.83% of all
immune cells (Fig. 4A). Immunostaining of EAU retinas
verified the abundant presence of Cd8a+ T cells, Tbx21+
Th1 cells, Foxp3+ Treg cells, and Cd4+ undifferentiated T
cells, in addition to Iba1+ microglia and NK1.1+ NK cells,
which were absent or few in control retinas (Fig. 4C-H).
Th1 and Th17 cells are known to mediate interferongamma (IFN-II or IFNγ) signaling and IL17A signaling,respectively [34, 35]. Further examination of the scRNAseq data using cell-cell communication analysis (CellChat[36]) revealed an increase of IFN-II signaling but not
IL17A signaling in EAU retinas (Fig. 4B), consistent withTh1 cells as the predominant class of T helper cells present in EAU retinas.

Page 6 of 24cell-cell adhesion and migration, inflammasome complex, and so on (Fig. 5A), indicating increased immune
response in the EAU retina. Moreover, KEGG enrichment analysis of the upregulated DEGs revealed that themost enriched pathway was cytokine-cytokine receptor
interaction (Fig. 5B). And cell-cell communication analysis of the scRNA-seq data also revealed that a number ofcytokine signaling pathways were enriched in EAU retinas compared to controls, such as IFN-II, IL1, IL2, IL6,
CC chemokine, and CXC chemokine pathways (Figs. 4B,
5C and E-G and 6A and B). Consistent with this, GSEA
for KEGG and Reactome enrichment analyses of thebulk RNA-seq data yielded enriched gene sets associatedwith chemokine signaling pathways and cytokine-cytokine receptor interaction (Fig. 5D; S2C), suggesting elevated chemokine signaling as one of the strong immune
responses elicited by EAU.
Chemokines, a subclass of cytokines, are known to
direct the migration of T cells [37]. As mentioned above,
multiple subtypes of T cells infiltrated EAU retinas, so
conceivably, chemokines may play a crucial role in thisprocess. Cell-cell communication analysis of the scRNAseq data showed that CCL signaling displayed a much
more dramatic strength alteration than CXCL, CX3C or
XC signaling between EAU and control retinas in Monocyte/Macrophage, T/NK, Microglia, and MG/Astrocyte
cell clusters (Figure S4). Therefore, we further examined
and compared the expression levels of CC chemokine
family members in control and EAU retinas by analyzing the scRNA-seq and bulk RNA-seq datasets. Thisrevealed significant upregulation in expression of about adozen CC chemokine family members including Ccl1-9,
Ccl11, Ccl12, Ccl17, Ccl22, and Ccl24 (Fig. 6C, D). This
result was subsequently validated by qRT-PCR assay(Fig. 6E). In particular, among all these chemokine genes,
the expression level of Ccl5 exhibited the most dramatic
upregulation postimmunization, reaching over 2500-foldwhen compared to the control retinas (Fig. 6C-E). This
may explain why Th1 cells were the most abundant T
helper cells infiltrating into EAU retinas (Fig. 4A), given
the fact that Th1 cells predominantly express CCR5, a
receptor of CCL5 [38–41].

Increased expression of CC chemokines in EAU retinas

CCR5-overexpressing MSCs increase migrative capacity
towards CCL5 both in vitro and in vivo

To further ascertain the molecular changes during EAU,
we performed GO enrichment analysis of the significantly upregulated differentially expressed genes (DEGs)
in EAU retinas, which were identified by bulk RNA-seq
analysis (Fig. S2A, B). The enriched GO terms included
MHC protein complex binding, MHC class II protein
complex, chemokine activity, cytokine receptor activity,
immune receptor activity, regulation of immune effector process, mononuclear cell differentiation, leukocyte

Since MSCs have multiple regulatory effects on immunity[42–44] including EAU [45–47] and the chemokine-chemokine receptor axis is indispensable for MSC migration[23, 48], we sought to generate CCR5-overexpressing
MSCs to enhance the homing capacity and therapeutic
effects of MSCs (Fig. S5). First, we induced highly homogeneous MSCs from hiPSCs, using an efficient and chemically defined method [49] (Fig. S5C). Flow cytometricanalysis showed that the hiPSC-derived MSCs expressedYuan et al. Journal of Neuroinflammation(2024) 21:136

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Fig. 4 The diversity of immune cells in EAU retinas. (A) UMAP plots and pie charts of immune cell clusters in control and EAU retinas. The left pie chartshows there are few immune cells in control retinas while the right pie chart shows that Th1 cell is the predominant class of T helper cells in EAU retinas.
(B) Chord diagrams comparing the IFNγ (IFN II) signaling pathway in control and EAU retinas. Edge color denotes the signaling source. Segments withlarge arrows represent signaling targets and the inner bar represents signaling source in which the colors indicate signaling targets. (C-H) Immunostaining of control and EAU retinal sections with antibodies against the indicated immune cell markers with DAPI counter-labeling. GCL: ganglion cell layer;
INL: inner nuclear layer; ONL: outer nuclear layer. Scale bar: (C-H) 20 μm

several typical MSC markers: CD29, CD44, CD73, andCD166, but lacked the expression of hematopoietic cellmarkers CD34 and CD45 (Fig. S5E). To test the multilineage differentiation potential of MSCs, we subjected

them to differentiation procedures in adipogenic, osteogenic or chondrogenic differentiation media. Subsequent
staining by alizarin red S, oil red O and toluidine blue
confirmed that the MSCs had osteogenic, adipogenic andYuan et al. Journal of Neuroinflammation

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Fig. 5 Enrichment of cytokine signaling pathways in EAU retinas. (A) GO enrichment analysis of the upregulated DEGs between EAU and control retinas,
which were identified by bulk RNA-seq analysis. Shown are 15 top enriched GO terms each for molecular function, cellular component and biologicalprocess. (B) KEGG enrichment analysis of the upregulated DEGs between EAU and control retinas. (C) Barplot showing the indicated signaling pathways
ranked based on their differences in overall information flow within the inferred networks. The top signaling pathways (shown in red) were more enrichedin control retinas, whereas the bottom ones (shown in green) were more enriched in EAU retinas. (D) GSEA enrichment analysis of the bulk RNA-seq
data identified enriched gene sets associated with the chemokine signaling pathway or cytokine-cytokine receptor interaction. (E-G) Chord diagrams
comparing IL1 (E), IL2 (F) and IL6 (G) signaling pathways in control and EAU retinas. Edge color denotes the signaling source. Segments with large arrows
represent signaling targets and inner bars represent signaling sources in which the colors indicate signaling targets

chondrogenic capacity, respectively (Fig. S5D). We theninfected MSCs with lentiviruses expressing both CCR5
and tdTomato (referred to as MSCCCR5) or tdTomato
only (referred to as MSCtdTomato), followed by purification of tdTomato-positive MSCCCR5 or MSCtdTomato cellsby fluorescence-activated cell sorting (FACS) (Fig. S5AC, F). FACS-enriched MSCCCR5 cells were further shownto have similar multipotency as regular MSCs (Fig. S5G).
To determine the homing capacity of CCR5-overexpressing MSCs, we first examined the migration of
MSCCCR5 cells towards recombinant human CCL5(hCCL5) through wound healing test (two-dimension)and transwell migration assay (three-dimension). Boththe wound healing test and transwell assay showed that
MSCCCR5 cells responded robustly to hCCL5 stimulation,
while only slight changes occurred for MSCtdTomato cells(Fig. 7A-D). In addition, MSCtdTomato and MSCCCR5 cells
displayed relatively low migration without exogenous
CCL5 treatment (Fig. 7B, D).
Given the dramatic upregulation of Ccl5 in EAU retinas(Fig. 6C-E), we investigated whether CCR5-overexpression in MSCCCR5 cells would potentiate their chemotaxis
towards EAU retinas. MSCtdTomato and MSCCCR5 cells
were transplanted into mice by caudal vein injection at
1 d.p.i. and the retinas were collected from transplanted
EAU animals at 14 d.p.i. for tdTomato immunolabeling analysis (Fig. 7E). We found that tdTomato-positive
cells were increased by more than 2.5-fold in retinas from
MSCCCR5-transplanted EAU mice when compared withthose from MSCtdTomato-transplanted animals (Fig. 7F, G).
Therefore, these results suggest that CCR5 overexpression is able to enhance the CC chemokine-mediated chemotaxis of MSCs both in vitro and in vivo.
Transplantation of MSCCCR5 cells ameliorates EAUGiven that MSCCCR5 cells gain enhanced homing ability
and that MSCs are known to ameliorate EAU [45, 46, 50,51], we asked whether MSCCCR5 cells were better thanregular MSCs in improving the clinical manifestations of
EAU. At 14 and 21 d.p.i., by fundus imaging and OCT,
we found that there were less structural damage andinflammatory infiltrates in the eyes of MSC-transplanted
EAU mice compared to PBS-administered animals, andthat MSCCCR5-transplanted EAU mice appeared to display even milder EAU symptoms than MSCtdTomatotransplanted ones (Fig. 8A, B; S6). Consistent with theseobservations, clinical grade scores at 14 and 21 d.p.i.
were markedly lower for MSC-transplanted EAU micecompared with PBS-treated control animals (Fig. 8C).
Moreover, MSCCCR5-transplanted EAU mice exhibited
significantly lower clinical grade scores than those transplanted with MSCtdTomato cells (Fig. 8C), indicating that
CCR5-overexpressing MSCs have not only a stronger
homing capacity but also better alleviating effect on EAU
than regular MSCs.
MSCCCR5 cells confer protection against microglia
activation and T cell infiltration

To explore the cellular basis of MSC-mediated alleviation of EAU, we examined infiltrating immune cells in
MSCtdTomato- and MSCCCR5-transplanted EAU retinas.
Immunostaining showed that compared to control EAU
retinas, MSCtdTomato-transplanted retinas contained significantly fewer pro-inflammatory M1 microglia positive for Cd16/32, and that the number of M1 microglia
was reduced even more in MSCCCR5-transplanted retinas (Fig. 9A). Similarly, there were obviously fewer infiltrating F4/80+ or Cd206+ macrophages, Cd4+ T cells,
Tbx21+ Th1 cells, and Foxp3+ Treg cells in MSCtdTomatotransplanted retinas, and their numbers were further
decreased in MSCCCR5-transplanted retinas (Fig. 9B-F).
These results together thus suggest that transplantationof CCR5-overexpressing MSCs at the time of immunization may block the progressive development of EAU, perhaps in part by preventing microglia activation and T cell
infiltration.
MSCCCR5 cells suppress activation of the Nlrp3
inflammasome

Previous studies have shown that the primary function ofretinal microglia in autoimmune uveitis is to initiate thedisease, and without microglia, no local inflammation
would develop; moreover, microglia produce cytokines
such as IL-1β and IL-18 by the activation of inflammasomes [52–57]. Additionally, the activation of the
Nlrp3 inflammasome and the maturation of IL-1β havebeen reported in the autoinflammatory uveitis, such as
Behçet’s disease [58]. We therefore investigated whether
EAU would activate inflammasomes in the retina. By
GSEA enrichment analysis of the bulk RNA-seq data, wefound that the DEGs were indeed significantly enriched
for genes associated with the inflammasomes and Nlrp3

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Fig. 6 Chemokine signaling pathways were enriched in EAU retinas. (A, B) Chord diagrams comparing CC chemokine signaling pathway (A) and CXC
chemokine signaling pathway (B) in control and EAU retinas. Edge color denotes the signaling source. Segments with large arrows represent signaling
targets and inner bars represent signaling sources in which the colors indicate signaling targets. (C) Stacked violin plots showing the expression patternsof CC chemokine genes in control and EAU retinas based on scRNA-seq analysis. (D) Dot plot showing the expression of CC chemokine genes in control
and EAU retinas based on bulk RNA-seq analysis. (E) qRT-PCR analysis showing the relative expression levels of the indicated CC chemokine genes incontrol and EAU retinas at 14 d.p.i. Data are presented as mean ± SD (n = 3 mice per group). *p < 0.05, **p < 0.01, ns, no significance. (F) Immunostaining ofcontrol and EAU retinal sections with an anti-CCL5 antibody with DAPI counter-labeling. Scale bar: (F) 20 μm

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Fig. 7 CCR5-overexpressing MSCs possess increased migrative capacity towards CCL5 in vitro and to EAU retinas in vivo. (A) Two-dimensional woundhealing test was performed to detect the migration of MSCtdTomato and MSCCCR5 cells toward hCCL5. Shown are phase-contrast images of the cell culture
at the indicated timepoints following the removal of culture-insert. (B) Transwell migration assay was performed to detect the migration of MSCtdTomato
and MSCCCR5 cells toward hCCL5. MSCs migrated to the lower chamber surface were stained with crystal violet. (C) Quantification of gap closure in thewound healing test showed a significant increase in wound closure rate for MSCCCR5 cells compared to MSCtdTomato cells. Data are presented as mean ± SD
(n = 3 individual samples per group). *p < 0.01. (D) Quantification of migrated cells in the transwell migration assay showed a significant increase for
MSCCCR5 cells compared to MSCtdTomato cells. Data are presented as mean ± SD (n = 3 individual samples per group). *p < 0.05, **p < 0.01, ***p < 0.001, ns,
no significance. (E) Schematic diagram of EAU induction and MSC transplantation into EAU mice. (F) Confocal images of transplanted tdTomato-positive
MSCs located in the retina at 13 days post-injection. (G) Quantification of tdTomato-positive cells in retinas from mice transplanted with MSCCCR5 or
MSCtdTomato cells. Data are presented as mean ± SD (n = 7–10 retinas per group). *p < 0.05. d.p.i.: day(s) postimmunization; GCL: ganglion cell layer; INL:
inner nuclear layer; IRBP: interphotoreceptor retinoid-binding protein; ONL: outer nuclear layer; PTX: pertussis toxin. Scale bar: (A, B) 100 μm, (F), 20 μm

inflammasome pathway (Fig. 10A). And in the scRNAseq data, the Nlrp3 inflammasome genes were also seen
to be upregulated mainly in microglia, monocytes/macrophages, and T/NK cells in EAU retinas (Figure S7A).
At 14 d.p.i., qRT-PCR assay confirmed that the genes
involved in the Nlrp3 inflammasome pathway (e.g. Nlrp3,
IL-1β, Caspase1, and Gsdmd) were significantly upregulated in EAU retinas compared to controls (Fig. 10B),
indicating that EAU results in Nlrp3 inflammasome
activation.
Given the relieving effect of MSCs on EAU, we next
tested whether they would also inhibit Nlrp3 inflammasome activation. Western blot analysis showed significant downregulation of inflammasome-related proteins
Nlrp3 and IL-1β in MSC-transplanted retinas compared
to controls, and their expression levels were the lowest in
MSCCCR5-transplanted retinas (Fig. 10C-E). Consistent
with the in vivo results, lipopolysaccharide (LPS)-treated
BV2 microglia co-cultured with MSCs exhibited reduced
expression of Nlrp3 and IL-1β (Fig. 10F-H), suggestingthat MSCs may also ameliorate EAU by inhibiting Nlrp3
inflammasome activation in microglia. Similarly, we wereable to show that CCR5-overexpressing MSCs inhibited
NLRP3 inflammasome activation in THP-1 monocytesderived macrophages (Figure S7B).

Discussion
In this study, we applied scRNA-seq and bulk RNA-seq
to characterize the classical EAU induced by IRBP peptide 651–670 in C57BL/6 mice. Based on the patternsof gene expression in resident retinal cells and infiltrating immune cells, and the results of immunostaining and
qRT-PCR analyses, we observed/inferred that: (1) EAU
caused a decrease in expression levels of a set of retinal
neuron marker genes and progressive degeneration of all
retinal neuron types; (2) Müller glia might be activatedand function as non-professional APCs in EAU retinas;(3) Th1 cells were the predominant class of T helper cells
in the retinas of mice with IRBP peptide 651-670-induced
EAU; and (4) Cytokines were widely involved in EAU andCCL5 was the most expressed CC chemokine ligand in
EAU retinas. Based on these findings, we proposed andtested a strategy to ameliorate EAU by transplantationof MSCCCR5 cells overexpressing human CCR5. Compared to regular MSCs, MSCCCR5 cells increased targeted
migration toward CCL5 both in vitro and in vivo, better
protected against microglia activation and T cell infiltration, and exhibited a better therapeutic effect on EAU.
Therefore, CCR5-overexpression in MSCs enhances theirhoming capacity and improves therapeutic outcomes,
thereby providing an appealing MSC-based therapy
option.
When autoimmune uveitis encompassed the retina, it
was often associated with autoreactivity to retinal antigens such as the S-antigen (Arrestin) and IRBP, and thiswas referred to as uveoretinitis [16, 24, 59–73]. S-antigen
was highly efficient in the induction of EAU in many different animal species including rats, rabbits, guinea pigs,
and primates [74], and autoimmunity to Arrestin was
crucial for the pathology of uveitis [75]. Our experimental results of scRNA-seq, bulk RNA-seq, and immunolabeling showed that in the IRBP-induced EAU retinas, theexpression of molecular markers of all 6 neuron types
was downregulated and these neuron types underwent
progressive degeneration (Fig. 2). As expected, the number of cones decreased significantly with EAU diseaseprogression, but scRNA-seq analysis revealed that EAU
appeared to affect the number of rods less than cones
although IRBP mRNA was detected in both cones androds [76, 77] (Fig. 2). This phenomenon may be explainedby a theory called “epitope spreading”: autoimmunity
starts with an immune response to a single antigen, andsubsequently extends to other proteins in the same tissue, or to different amino acid sequences within the same
molecule (epitope) [75, 78]. Based on this theory, we have
hypothesized that when EAU is induced by IRBP peptide
651–670, the immune response may be first directed to
IRBP and then extended to Arrestin so that the cones areattacked more by immune cells than rods.
Apart from the impact on retinal neurons, EAU also
caused changes in the state of Müller glia, the major glial
component of the retina. Under physiological conditions,Müller glia are thought to contribute to ocular immune
privilege by inhibiting the proliferation and activation oflymphocytes [31]. They can be induced to generate neurons [79–85], express the MHC class II proteins, andYuan et al. Journal of Neuroinflammation(2024) 21:136

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Fig. 8 Transplantation of MSCs ameliorated EAU in vivo. (A) Representative fundus photographs of the eyes from EAU mice at 21 d.p.i., transplanted with
MSCtdTomato or MSCCCR5 cells, or administered with PBS. (B) Representative fundus photographs (left) and OCT images (right) of the eyes from EAU miceat 21 d.p.i., transplanted with MSCtdTomato or MSCCCR5 cells, or administered with PBS. The arrows point to inflammatory infiltrating cells and the asterisks
indicate retinal folds and detachment in EAU eyes. (C) Clinical grade scores of eyes in the PBS, MSCtdTomato and MSCCCR5 groups at 0, 14 and 21 d.p.i. Data
are presented as mean ± SEM (n = 12–32 eyes per group). *p < 0.05, **p < 0.001, ***p < 0.0001

stimulate the proliferation of T cells as APCs in an MHC
class II-dependent manner [86, 87]. In the present work,
EAU did not appear to affect the number of Müller glia
but upregulated the expression of their marker genes(Fig. 2). Interestingly, Gfap, an indicator of the activatedstate of Müller glia, was upregulated in only one Müller
glia cluster present mainly in EAU retinas (Fig. 3). Moreover, MHC class II proteins were found to be colocalizedwith Müller glial markers Sox9 (in cell bodies) and glutamine synthetase (in radial processes) (Fig. 3). Thus, consistent with a previous study [16], Müller glia may also act
as non-professional APCs in the context of EAU. In addition, our single-cell sequencing data and immunostaining showed that some VECs, astrocytes, microglia andmonocytes/macrophages expressed the MHC II proteins(Figure S3E). Hence, further study is needed to elucidate
the proportion of Müller cells acting as non-professional
APCs compared to other non-professional APCs, and to
determine the relative contribution of Müller glia compared to other APCs in the uveoretinitis disease process.
A reliable method to distinguish astrocytes and Müller

glia deserves further exploration because the expression
profile of Müller glia changed in EAU.
The accumulation of leukocytes in target organs is asign of autoimmunity [88]. The absence or near-absenceof lymphoid cells in wild-type (WT) retinas may be afeature of ocular immune privilege [89]. Activated T
cells specific to retinal antigens mediate EAU in animals, and T cells are also central to the pathogenesis ofhuman uveitis [9, 90, 91]. Our results showed that in WT
control retinas, a small number of microglia and monocyte-lineage cells were present, but T and NK cells were
nearly undetectable; but in EAU retinas, there were many
more nonresident immune cells, and there was a preponderance of T and NK cells and monocyte-lineage cells(Figs. 2 and 4). By scRNA-seq analysis and immunostaining of EAU retinas, we observed aggregates of multiple
types of T cells (Fig. 4). Similarly, a recent study by Peng
et al. showed that multiple types of T cells infiltrated into
the EAU retina, and that the ratio of Th1 cells in the retina was highest on 14 d.p.i. and 21 d.p.i. [92]. Moreover,it has been suggested that Th1 cells are mainly involved

Yuan et al. Journal of Neuroinflammation(2024) 21:136

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Fig. 9 Transplantation of MSCs decreased microglia activation and T cell infiltration. (A-E) Retinal sections from EAU mice transplanted with MSCtdTomato
or MSCCCR5 cells, or administered with PBS were immunostained with antibodies against the indicated protein markers and counter-labeled with DAPI.
Cd16/32 are markers for M1 microglia, F4/80 and Cd206 are markers for macrophages, and Tbx21, Foxp3 and Cd4 are markers for Th1 cells, Treg cells and T
helper cells, respectively. (F) Quantification of Cd4-positive and Foxp3-positive cells in the indicated retinas. Data are presented as mean ± SEM. *p < 0.05,**p < 0.001 (n = 3 retinas per group). Scale bar: (A-E) 20 μm

in the development of acute uveitis, whereas Th17 cells
play a role in the late or chronic phase of uveitis [93]. An
increase in Th17 cells was observed in the retina overtime [92], suggesting that the major pathogenic T cells
may change over the EAU disease process. Future studies targeting later or more severely graded EAU retinas
would help to understand the dynamics throughout thecourse of the disease. The predominance of Th1 cells aseffector T cells in the classical EAU retinas is inconsistent

with previous reports that complete Freund’s adjuvant(CFA) promoted a Th17-dominant response [12, 13].
Conceivably, there are at least two reasons why the differences exist: (1) Chen et al. induced EAU in the B10.RIII
mice instead of C57BL/6 mice [12]; and (2) it was IRBP
peptide 161–180 rather than IRBP peptide 651–670 thatwas used in their study [12]. Previous studies have shownthat autoimmunity to the retina can be either Th17or Th1-driven, depending on the conditions of initialYuan et al. Journal of Neuroinflammation(2024) 21:136

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Fig. 10 Transplantation of MSCs downregulated the expression of genes in the Nlrp3 inflammasome pathway. (A) GSEA enrichment analysis of the bulk
RNA-seq data identified enriched gene sets associated with the inflammasomes and Nlrp3 inflammasome pathway. (B) qRT-PCR assay of the indicatedgenes involved in the Nlrp3 inflammasome pathway in control and EAU retinas at 14 d.p.i. Data are presented as mean ± SD (n = 3–9 retinas per group).
*p < 0.0001. (C) At 21 d.p.i., Western blotting was performed for Nlrp3 and IL-1β p17 expressed in retinas of the PBS, MSCtdTomato and MSCCCR5 groups.
β-tubulin served as the internal protein control. (D, E) Quantification of relative protein expression levels of Nlrp3 and IL-1β p17 in retinas of the PBS,
MSCtdTomato and MSCCCR5 groups. Data are presented as mean ± SEM (n = 7–10 retinas per group). *p < 0.05, **p < 0.01, ***p < 0.001. (F) Western blotting
analysis was performed for Nlrp3 and IL-1β p17 expressed in LPS-treated BV2 microglia cultured in the absence (PBS) or presence of MSCtdTomato or
MSCCCR5 cells. (G, H) Quantification of relative expression levels of Nlrp3 and IL-1β p17 in LPS-treated microglia cultured in the absence (PBS) or presenceof MSCtdTomato or MSCCCR5 cells. Data are presented as mean ± SEM (n = 4–10 individual samples per group). *p < 0.05, ns, no significance

exposure to antigens [13, 89]. And it is known that uveitis is clinically heterogeneous, even though patients may
respond to the same retinal antigen(s) [9]. Moreover, previous observations from EAU and other models of autoimmune diseases have demonstrated that the immune

systems respond to different fragments of a given molecule, and that cell-mediated and humoral responses areinvariably directed against different parts of the molecule[94]. Our data therefore reinforce the notion that noYuan et al. Journal of Neuroinflammation(2024) 21:136

single animal model can reproduce the full spectrum offeatures of the uveoretinitis disease.
Previous studies have revealed the importance of chemokines in the pathogenesis of uveitis [51, 95, 96], andchemokines may be involved in the initiation and/or
amplification steps in the pathogenicity of uveitis [18,96]. In addition, chemokines are powerful chemoattractants for T cells [97]. Our scRNA-seq and bulk RNA-seq
data revealed that a number of cytokines were enriched
in EAU retinas and that the CC chemokine family exhibited a more significant change between EAU and control
retinas than other chemokine families. We examined theexpression of about a dozen of CC chemokines in EAU
retinas by qRT-PCR assay and found that Ccl5 displayed
the most significant change postimmunization. It has
been reported that CCR5, a receptor of CCL5, plays animportant role in the differentiation of Th1 cells [38–41,
98]. In this context, the dramatic upregulation of CCL5
may explain why Th1 cells are the predominant T helper
cells in EAU retinas. Nevertheless, CCR5-deficient micedevelop EAU with a degree of severity comparable tothat in wild-type mice because the reduced T-cell infiltration caused by CCR5 deletion was compensated byaugmented granulocyte infiltration [19]. CCR5 knockout
may cause a significant shift in the gene expression profile of immune cells that ultimately results in changes intheir chemotaxis. As such, CCR5 inactivation may not be
an appropriate strategy for treating EAU. In addition, it
remains unclear whether CCL5 plays a role in the initiation or amplification of EAU, due to the fact that we did
not detect Ccl5 mRNA at different time points during the
progression of EAU. Related to this, the relative contribution of Th1 and Th17 cells at different stages of EAU
needs to be further explored because a previous study
suggested that Th17 cells were responsible for ocularinflammation in the early phase of EAU, whereas Th1 cell
activation was correlated with the late stage and regression of inflammation [99].
In recent years, MSC transplantation has become apromising therapy option for the treatment of immunological and inflammatory disorders, including graft-versus-host disease [100–102], spinal cord injury [103–105],
stroke [23, 106, 107], and of course EAU [45, 46, 50, 51,108]. In MSC therapy, it would be ideal that most of theinfused cells are recruited to the target lesions or tissues[107, 109, 110]. However, it is widely believed that exogenously administered cells are rapidly cleared by the hostand do not engraft for long periods [43, 111]. Although
in some preclinical and clinical trials, MSCs are directly
injected into the target lesions or tissue [112–114],
local administration may cause potential invasive damage. Thus, it is imperative to develop new approaches
to enhance infused MSCs administered by a relatively
safe route, such as intravenous injection, with improved

Page 16 of 24

homing capacity to the target lesions. Additionally,amelioration of EAU has been achieved by inhibition of
migration and recruitment of inflammatory cells into theeye by targeting adhesion molecules and/or chemokinesand chemokine receptors [9, 18, 115]. Considering that:(1) the expression level of CCL5 was the highest among
CC chemokines in EAU retinas (Fig. 6); (2) the numberof CD4+ T cells and Th1 cells was increased by EAU
immunization (Fig. 4); and (3) isolated MSCs were foundto gradually lose their homing capacity and exhibit functional heterogeneity [21–23, 49], we generated functionaland homogeneous hiPSC-derived MSCs and overexpressed hCCR5 in MSCs via lentiviruses. As expected, anincreased number of MSCCCR5 cells was observed in EAU
retinas, exerting superior protection compared to regular
MSCs (Figs. 7, 8, 9 and 10).
Previous work has demonstrated that microglia initiate
neuroinflammation in ocular autoimmunity and mediate
the entry of autoreactive immune cells including T cells
into the retina [53]. We found that after MSCCCR5 treatment, the structure of the retina was more intact (Fig. 8)
and the number of Cd16/32+ M1 microglia, infiltrating
F4/80+ or Cd206+ macrophages, Cd4+ T cells, Tbx21+
Th1 cells, and Foxp3+ Treg cells in the EAU retina wasdrastically reduced (Fig. 9). Nevertheless, Treg cells were
believed to suppress Th1 immune responses in uveitis[116], but they obviously decreased in EAU retinas after
MSC transplantation. A possible explanation for thisis that cell-based therapies have the potential to restore
ocular immune privileges, preventing all types of lymphocytes from infiltrating into the retina.
In EAU retinas, the inflammasome pathway wasenriched and Nlrp3 inflammasome-related genes including Nlrp3, Caspase1, Gsdmd, and Il1b (IL-1β) wereupregulated at RNA levels (Fig. 10). MSCCCR5 treatment
significantly downregulated the expression of Nlrp3 andIL-1β. Similarly, their downregulation was confirmed in
LPS-treated BV2 microglia after co-culture with MSCs
(Fig. 10). Taken together, we have revealed a possible
mechanism underlying the beneficial effects of MSCCCR5
therapy, which may rely on inhibiting the activation of
microglia and inflammasomes, leading to diminishedrelease of inflammatory factors and thereby reducing theinfiltration of autoreactive immune cells such as T cells
into the retina.

Conclusions
In this extensive study employing scRNA-seq and bulk
RNA-seq, our investigation delved into the complexities of EAU induced by the IRBP peptide 651–670 in
C57BL/6 mice, and uncovered a multifaceted impact
of EAU on retinal components, revealing a decrease in
the expression of retinal neuron marker genes and progressive degeneration of various retinal neuron types.

Yuan et al. Journal of Neuroinflammation(2024) 21:136

Page 17 of 24

Additionally, the study shed light on the intriguing roleof Müller glia, which, under EAU conditions, displayed
activation and potential function as non-professional
APCs. The immune cell landscape in EAU retinas
unveiled a prevalence of Th1 cells, contrary to previous
reports, underscoring the heterogeneity of uveitis. Cytokine involvement, particularly the pronounced expression of CCL5, hinted at its potential role in orchestrating
the immune response. We extended this study by proposing and testing a therapeutic strategy involving thetransplantation of CCR5-overexpressing MSCs, whichshowed enhanced homing capacity and promising outcomes in preventing EAU. These specialized cells provided improved protection against microglia activation
and T cell infiltration. Notably, MSCCCR5 treatment
downregulated Nlrp3 inflammasome-related genes, suggesting a mechanism for reducing inflammation and preventing autoreactive immune cell infiltration. This work
not only contributes valuable insights into the molecularcharacteristics of EAU but also opens avenues for innovative MSC-based therapies. Nonetheless, there is still along way to go for clinical application. More details about
MSC transplantation, such as the methods for modifying
MSCs, the time point of transplantation, the number oftransplanted cells, and the frequency of transplantation,
should be evaluated comprehensively.

immunization, 1 µg of pertussis toxin (PTX, List Biological, 180) was injected intraperitoneally (recorded as day0), followed by another injection of 1 µg PTX at 2 d.p.i.
Every 7 days postimmunization, the eyes of these animals
were inspected and graded for disease induction basedon the criteria described previously (Table S1) [24, 25].
Retinas were collected at 14 d.p.i. or 21 d.p.i. for desired
experiments.

Materials and methods

Single-cell RNA sequencing

Animals

scRNA-seq analysis was carried out as previously
described with modification [52, 117]. In brief, retinas
were quickly dissected in Dulbecco’s phosphate-bufferedsaline (DPBS, Hyclone, SH30028.02) and digested using
papain (Worthington Biochemicals, LS003126) with
DNase I (Roche, 10,104,159,001) at 37 °C for 5 min. Then
isometric amount of DPBS containing 10% fetal bovine
serum (FBS, Genial, G11-70500) was added and retinas
were triturated by soft pipetting. The dissociated cells
were filtered using a 40 μm cell strainer (Falcon, 352,340).
Filtered cells were centrifuged and resuspended in 0.4%
bovine serum albumin (BSA, Sigma, B2064) in DPBS.
Cell viability was determined by Cellometer (Nexcelom)
and only cell suspension with a viability of more than 85%
was subjected to scRNA-seq analysis. Single-cell libraries
were constructed from the resuspended cells according
to the manufacturer’s instructions using the Chromium
Single Cell 3′ Library & Gel Bead Kit v3.1 (10X Genomics, 1,000,121), and sequenced on the Illumina 10X platform. Quality checks were performed using the FastQC
software (https://www.bioinformatics.babraham.ac.uk/
projects/fastqc/). scRNA-seq data were pre-processedusing the Cell Ranger pipeline (v6.0.1; 10X Genomics)
with default settings and mm10 as the reference genome.
Further analysis was performed using Python and R packages: Scrublet [118], Seurat [28, 29], and CellChat [36].

All experiments on mice were performed according to
the IACUC (Institutional Animal Care and Use Committee) standards and approved by Sun Yat-sen University and Zhongshan Ophthalmic Center. The C57BL/6
mice were purchased from the Vital River Laboratories(Beijing). Mice were maintained in pathogen-free facilities under standard housing conditions with continuous
access to food and water. All experiments were carriedout in adult mice from 8 to 11 weeks of age. The contribution of gender was not considered in this paper. To
control for sex effects, only female mice were studied.
Experimental autoimmune uveitis induction andassessment

EAU was induced in mice using a protocol described
previously [24] with minor modifications. Briefly, anesthetized 8-week-old C57BL/6 mice (50 mg/kg sodium
pentobarbital, Tocris, 4579) were immunized with 200 µg
human IRBP peptide 651–670 (LAQGAYRTAVDLESLASQLT, Shanghai Hanhong, CSP0725) which wasemulsified in Complete Freund’s Adjuvant (1:1 vol/vol,
CFA, Sigma, F58881) containing an additional 3.5 mg/
mL Mycobacterium tuberculosis H37Ra (BD, 231,141),by injection with a 30G needle into the two thighs(50 µg each) and the tail base (100 µg). Concurrent with

Fundus imaging, fluorescein fundus angiography (FFA),
and optical coherence tomography (OCT) imaging

For all clinical examinations [Fundus imaging, FFA, and
OCT], mice were anesthetized with sodium pentobarbital (50 mg/kg, Tocris, 4579). Local ocular surface anesthesia was achieved by 0.5% dicaine hydrochloride eye
drops (Zhongshan Ophthalmic Center). The pupils weredilated using 0.5% tropicamide and 0.5% phenylephrinehydrochloride (Shenyang Sinqi Pharmaceutical Co., Ltd.).
Artificial tears (Zhongshan Ophthalmic Center) or 1%
hypromellose eye drops (Zhongshan Ophthalmic Center)
were used to maintain corneal moisture and clarity. The
fundus was imaged using the Phoenix Micron IV in vivo
retinal imaging microscope. For FFA, 150 mg/kg fluorescein sodium (Alcon) was injected and the fundus images
were captured in 5 min. OCT images were obtainedusing the Heidelberg Engineering Spectralis HRA + OCT.

Yuan et al. Journal of Neuroinflammation(2024) 21:136

scRNA-seq data reported in this paper were deposited in
the NCBI Sequence Read Archive (SRA) database underaccession numbers SRR25528276 and SRR25528277.
Bulk RNA sequencing

Bulk RNA-seq analysis was performed as previously
described [119] with modification. Total RNA wasextracted using NucleoZOL reagent (MACHEREYNAGEL, 740404.200) following the manufacturer’s
procedure. The quantity and purity of total RNA wereanalyzed with Bioanalyzer 2100 and RNA 6000 Nano
LabChip Kit (Agilent, 5067 − 1511), and only high-quality RNA samples with RIN number > 7.0 were used toconstruct sequencing libraries. The average insert sizefor the final cDNA libraries was 300 ± 50 bp. We performed the 2 × 150 bp paired-end sequencing (PE150)
on an Illumina Novaseq™ 6000 (LC-Bio Technology CO.,Ltd.) following the vendor’s recommended protocol.
We aligned reads of all samples to the mouse reference
genome (mm10) using the HISAT2 software (https://
daehwankimLab.github.io/hisat2/). Hierarchical clusterand scatter plot analyses of gene expression levels were
performed using the R software (http://cran.r-project.
org). Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and GeneSet Enrichment Analysis (GSEA) were carried out using
the clusterProfiler [120] package with p value < 0.05. P
value < 0.05 and log2FoldChange ≥ 1.5 were set as the significant threshold in the DEG (differentially expressedgene) analysis with the DESeq2 package [121]. |NES| > 1
and p.adjust < 0.05 were set as the significant threshold in
GSEA. RNA-seq data reported in this paper were deposited in the NCBI SRA database under accession numbers
SRR25528270-SRR25528275.
Quantitative real-time PCR (qRT-PCR)

Retinas of WT control and EAU mice were dissected
out of the eyeball and harvested. Total RNA was isolated using the NucleoZOL reagent. RNA (1 µg) fromeach sample of different groups was converted to cDNA
using the HiScript II Q RT SuperMix for qPCR (Vazyme
Biotech, R223-01). qRT-PCR was performed using the
Kapa SYBR fast qPCR master mix (Kapa, KK4601) andqTOWER3 G Real-Time PCR system (Analytikjena). The
data were analyzed using the 2−ΔΔct calculation method.
All data were tested for significance using the two-sample
Student’s t-test. The primer sequences used for qRT-PCR
are listed in Table S4.
iPSC culture and MSC induction

We utilized one human induced pluripotent stem cell(hiPSC) line that was previously established in our lab[122]. The cells were cultured on Matrigel (Corning,
354,277)-coated plates in mTeSR1 medium (Stemcell

Page 18 of 24

Technologies, 05445). The medium was changed daily
and the cells were passaged every 4–5 days using
Accutase (Life Technologies, AT-104).
The hiPSCs were differentiated into MSCs as previously
described [49]. First, to differentiate hiPSCs into neuromesodermal progenitors (NMPs), these cells (1–2 × 105
cells/cm2) were seeded on Matrigel-coated plates andcultured in mTeSR1 medium containing 10 µM Y27632(Sigma, Y0503) for 24 h. Neuromesoderm differentiation was initiated by culturing cells in E6 medium (Stemcell Technologies, 05946) supplemented with 20 ng/mL
bFGF (Peprotech, 100-18B), 3 ng/mL TGFβ1 (Peprotech,
100 − 21), and 10 µM Chir99021 (TargetMol, T2310) for 5
days. Next, for MSC differentiation, the NMPs were cultured for another 3 weeks in animal component-free andserum-free medium (MesenCult™-ACF Plus Medium;Stemcell Technologies, 05448). The resulting cells weremaintained in MesenCult™-ACF Plus medium (Fig. S5).
The phenotype and multipotency of hiPSC-derived
MSCs were assessed by flow cytometry analysis and by
their ability to differentiate into mesenchymal-lineage
cells (osteoblasts, adipocytes, and chondrocytes). For
osteogenic differentiation, cells were fixed and incubated with Alizarin Red S (Sigma, A5533) for 30 min forthe detection of calcium deposits. For adipogenic differentiation, cells were fixed and incubated with Oil Red
O (Sigma, O0625) for 20 min for the detection of lipid
droplets. For chondrogenic differentiation, cells were
fixed and incubated with 0.1% toluidine blue (Sigma,
89,640) for 10–15 min. Labeled cells were captured witha microscope (Fig. S5).
Flow cytometry

MSCs were dissociated into single-cell suspension using
Accutase, washed twice in 0.2% cold BSA, and precipitated by centrifugation (500 g at 4 °C). The cell pellet was
resuspended in 0.2% cold BSA, and incubated for 30 min
with the appropriate antibodies (Table S5) on ice protected from light. The cells were then washed twice andprecipitated to remove unbound antibodies. The cellpellet was resuspended in 300 µL 0.2% BSA in a 5-mL
round bottom tube (Falcon, 352,235) for flow cytometricanalysis (Fig. S5). All flow cytometric analysis was conducted with the BD flow cytometer (LSRFortessa™ X-20)
and the data were analyzed using the BD FlowJo software(FlowJo, Ashland, OR, USA).
Construction of viral plasmids and lentivirus preparationand infection

The lentiviral expression vectors used in the present
study were designated as pLV/puro-EF1α-CCR5-T2AtdTomato and pLV/puro-EF1α-tdTomato, which wereconstructed using the gateway method (Fig. S5).

Yuan et al. Journal of Neuroinflammation(2024) 21:136

Lentivirus particles were harvested from 293T cells as
described [52]. In brief, the viral constructs and package plasmids Pspax2 (Addgene #12,260) and PMD2.G(Addgene #12,259) were transfected into HEK293T cells
using the Hieff Trans® Liposomal Transfection Reagent(Yeasen, 40802ES08) according to the manufacturer’s
instruction. After 48 h, the culture supernatants were
harvested and filtered through a 0.45 μm sterile filter (Millipore, SLHV033RB), then ultra-centrifuged at
20,000 g (rotor SW32Ti, Beckman) for 1.5 h at 4℃ toprecipitate the lentiviruses.
MSCs, at 70 ~ 80% confluence in a well of a 6-well plate(Corning, 3516), were infected with the pLV/puro-EF1aCCR5-T2A-tdTomato or pLV/puro-EF1a-tdTomato
viruses in 2 mL fresh MesenCult™-ACF Plus medium inthe presence of 10 µg/mL polybrene (Sigma, TR-1003-G).
After 16 h, the virus and medium mixture was replacedwith fresh MesenCult™-ACF Plus medium.
Fluorescence activated cell sorting (FACS)

Three days after lentivirus infection, MSCs were dissociated using Accutase. After enzyme inactivation, thecell suspension was centrifuged at 186 g for 5 min. The
precipitated cells were resuspended in DPBS containing
2% FBS and 1 mM EDTA, and passed through a 40 μm
cell strainer (Falcon, 352,340) into a 5 mL round bottom tube (Falcon, 352,003). MSCCCR5 and MSCtdTomato
cells were purified by FACS (BD, FACSAriaIII), gatedfor a high level of tdToamto expression. The sorted
MSCCCR5 and MSCtdTomato cells (Fig. S5F) were used forthe wound healing test, transwell assay, transplantation,and co-culture.
HEK293T cell, BV2 microglia and THP-1 monocyte culture

The HEK293T (293T) cells (ATCC, CRL-3216) and
THP-1 monocytes (ATCC, TIB-202) were purchasedfrom ATCC, and the BV2 microglia were from iCell Bioscience (iCell-m011). These cells were tested for mycoplasma contamination before experiments and they
were all negative. The 293T cells and BV2 microglia wereexpanded in the Dulbecco’s modified Eagle’s medium(DMEM, Hyclone, SH30243.01B) supplemented with
10% FBS (Gibco, 10270-106), 1X Penicillin-Streptomycin (Gibco, 15,140,122), and 1X MEM non-essential
amino acids (Gibco, 11,140,050). The THP-1 monocytes
were cultured in RPMI 1640 medium (ThermoFisher,
11,875,093) supplemented with 10% FBS, 1X PenicillinStreptomycin, and 0.05 mM β-Mercaptoethanol (Sigma,
M3148). The cell incubator provided an environment of
5% CO2 and 37℃.
Wound healing test and transwell assay

The migrative capacity of MSCs was evaluated using a
3-well silicone insert (for 2-dimensional migration, Ibidi,

Page 19 of 24

80,369) and an 8 μm-pore transwell chamber system (for3-dimensional migration, Corning, 3422).
In wound healing test, MSCs were seeded into the
3-well culture-insert and cultured until an optically confluent cell monolayer was formed. Cell proliferation wasthen suppressed by treating the cells with the proliferation inhibitor, mitomycin C (1 µg/mL, Selleck, S8146)
for 1 h. The culture-insert was removed to create gaps,
followed by replacement of the old medium with fresh
one with or without 100 ng/mL human CCL5 (R&D system, 278-RN). Gap closure was monitored by taking pictures at different time points (0, 3, 6, 12, 24 h) under aninverted optical microscope.
In the transwell assay, MSCs were seeded onto theupper chamber (2.0 × 105 per well) placed in a 24-wellplate (Corning, 3524), while the lower chamber was
loaded with recombinant human CCL5 (100 ng/mL).
After 4 h of co-culture, we swabbed the MSCs remaining on the upper surface of the membrane and stained
the filter with 0.1% crystal violet (Solarbio, G1064). MSCs
that migrated to the lower surface of the membrane werecounted under an inverted optical microscope.
Cell transplantation

Our experimental design consisted of 3 groups: Group 1,
PBS alone (0.1 mL without MSC administration); Group2, MSCtdTomato transplantation group (2.0 × 106 cells/mouse); and Group 3, MSCCCR5 transplantation group(2.0 × 106 cells/mouse). Cells were suspended in 0.1 mL of
the DPBS and all injections were performed at 1 d.p.i. via
the caudal vein (Fig. 7E). After 13 days (14 d.p.i.) or 20
days (21 d.p.i.) of transplantation, mice were euthanized
and retinas were collected for subsequent experiments.
Hematoxylin-eosin (HE) staining

Eyeballs were fixed overnight in the FAS Eyeball Fixator(Servicebio, D60708) and sectioned in paraffin at 15 μm,
followed by HE staining. Sectioning and staining werecarried out by Servicebio.
Co-culture and LPS treatment

The BV2 microglia were indirectly co-cultured with
MSCs using the 0.4 μm-pore transwell chamber system (Corning, 3412) as previously described [123]. They
were seeded in a 6-well plate. Another 6-well plate wasprepared, in which the transwell inserts were carefully
placed. 500 µL MSCCCR5 or MSCtdTomato cell suspension(1 × 106 cells/mL) was seeded onto the transwell insert.
Isometric amount of DPBS was added into the insert as
the control group. The cells were cultured at 37℃ and
5% CO2 for ~ 24 h until BV2 microglia reached about
95% confluence. Subsequently, the culture medium was
replaced with the medium containing lipopolysaccharide (LPS, 100 ng/mL, Sigma, L4391). Meanwhile, theYuan et al. Journal of Neuroinflammation(2024) 21:136

MSC medium was also replaced with the BV2 microglia
medium containing LPS (100 ng/mL). The inserts werethen placed into the wells of the 6-well plate seeded with
BV2 microglia. After 4 h of culture, the BV2 microglia
were washed twice with DPBS and processed for Western
blotting analysis.
The THP-1 monocytes (5 × 105 cells/well) were seededon 6-well plates and treated with 150 nM phorbol
12-myristate 13-acetate (PMA, Sigma, P1585) for 24 h forthem to transform into adherent macrophages. Meanwhile, the MSCCCR5 and MSCtdTomato cells were prepared
as described above. They were then indirectly co-cultured
with the THP-1 monocytes-derived macrophages andthe culture medium was replaced with the fresh medium
containing 100 ng/mL LPS. After 4 h of co-culture, themacrophages were collected for qPCR analysis.
Western blotting

Total protein was isolated from retinas at 21 d.p.i. or
BV2 microglia using the RIPA buffer (Beyotime, P0013B)
supplemented with PMSF (phenylmethanesulfonyl)(Beyotime, ST505) and quantified using the BCA kit(Beyotime, P0010). Samples were loaded on gradient gels
(GenScript, M00657) for protein separation. The expression levels of target proteins were quantified by ImageJand normalized to β-tubulin in the same sample. Primaryand secondary antibodies for Western blotting are listed
in Tables S6 and S7, respectively.

Immunostaining
Immunofluorescence staining of retinal sections was carried out as previously described [80, 124]. Briefly, for section labeling, retinas were fixed in 4% paraformaldehyde
in PBS for 30 min at 4 °C and sectioned at 15 μm. Sample
sections were washed 3 times with 0.4% Triton X-100
in PBS (PBST) for 5 min each before being incubated in
5% normal donkey serum in PBST for 1 h at room temperature (RT). The primary antibodies (Table S6) in 1%
normal donkey serum in PBST were added for overnightincubation at 4 °C. After washing with PBST, the sections
were incubated with secondary antibodies (Table S7) and
DAPI in PBST for 1 h at RT.
Immunohistochemical staining was performed as previously described [125, 126]. Briefly, eyeballs were fixed
overnight in FAS Eyeball Fixator and sectioned in paraffin
at 15 μm. Then, sections were deparaffinized, subjected
to antigen retrieval, incubated in 3% hydrogen peroxide
to eliminate endogenous peroxidase activity, blocked insolution containing 5% normal donkey serum, 3% BSA,
and 0.2% Triton X-100, incubated with the primary antibodies (Table S6) overnight at 4 °C and the secondary
antibodies (Table S7) for 1 h at RT. Finally, sections weresequentially stained with the AEC substrate (Solarbio,
A2010) and hematoxylin (Servicebio, G1004).

Page 20 of 24

Images were captured by a laser scanning confocal
microscope (Carl Zeiss, LSM700).
Statistical analysis

For quantification of marker-positive cells in retinal sections, optic field represents the area acquired by laser
confocal microscopy at the same magnification. All
experiments have at least three biological replicates. At
least one retina was counted per mouse and three sections were counted per retina. Two optic fields werecounted per section. Statistical analysis was performed
using GraphPad Prism 8.0, R, and Python. The results are
expressed as mean ± SD or mean ± SEM for experiments
conducted at least in triplicates. Student’s t test was used
to assess differences between two groups, and one-way
ANOVA with Bonferroni’s correction was used to assess
differences among three groups. A value of p < 0.05 is
considered statistically significant.
Abbreviations
AC	amacrine cell
APCs	antigen presenting cellsBC	bipolar cell
BRB	blood-retinal barrier
BSA	bovine serum albumin
CFA	complete Freund’s adjuvant
d.p.i.	days postimmunization
DEGs	differentially expressed genes
DMEM	Dulbecco’s modified Eagle’s medium
DPBS	Dulbecco’s phosphate-buffered saline
EAU	experimental autoimmune uveitis
FACS	fluorescence-activated cell sorting
FBS	fetal bovine serum
FFA	fluorescein fundus angiography
GCL	ganglion cell layerGO	Gene OntologyGS	glutamine synthetase
GSEA	Gene Set Enrichment Analysis
HC	horizontal cell
INL	inner nuclear layer
iPSC	induced pluripotent stem cell
IRBP	interphotoreceptor retinoid-binding protein
KEGG	Kyoto Encyclopedia of Genes and Genomes
MG	Müller glia
MHC	major histocompatibility complex
MSCs	mesenchymal stem cellsNK	natural killer cell
NMP	neuromesodermal progenitor
OCT	optical coherence tomography
ONL	outer nuclear layer
PBST	Triton X-100 in PBS
PTX	pertussis toxin
PMA	phorbol 12-myristate 13-acetate
RGC	retinal ganglion cell
RNA-seq	RNA sequencingRT	room temperatureS	inner and outer segment
scRNA-seq	single-cell RNA sequencing
VEC	vascular endothelial cell

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03134-3.

Yuan et al. Journal of Neuroinflammation(2024) 21:136

Supplementary Material 1
Supplementary Material 2
Acknowledgements
We thank Mr. Yunzhao Fu and Ms. Wei Xue for their assistance with EAU
induction; Mr. Mingwei Wang, Dr. Ren Liu, Mr. Yi Tian, and Ms. Yiwen Hong fortheir kind assistance and advice; and the staff of the Laboratory Animal Centerand Core Facilities of the State Key Laboratory of Ophthalmology, Zhongshan
Ophthalmic Center for technical support.

Page 21 of 24

9.
10.

11.
12.

Author contributions
M. X. and F. Y. conceived and designed the research. F. Y., R. Z., J. L., Q. L., S. W., F.
J., Y. G., and M. X. performed the experiments and analyzed the data. M. X. and
F. Y. interpreted the data and wrote the manuscript. All authors contributed to
critical reading of the manuscript.

13.

Funding
This work was supported in part by the National Natural Science Foundationof China (81970794, 81721003), “Technology Innovation 2030-Major Projects”
on Brain Science and Brain-Like Computing of the Ministry of Science andTechnology of China (2021ZD0202603), The Science and Technology Planning
Project of Guangdong Province (2023B1212060018), Science and Technology
Planning Projects of Guangzhou City, Local Innovative and Research Teams
Project of Guangdong Pearl River Talents Program, and the Fundamental
Research Funds of the State Key Laboratory of Ophthalmology, Sun Yat-sen
University.

15.

Data availability
The data are available from the corresponding author on reasonable
request. The scRNA-seq data are deposited in the NCBI SRA database underaccession numbers SRR25528276 and SRR25528277 and bulk RNA-seq data
are also deposited in the NCBI SRA database under accession numbers
SRR25528270-SRR25528275.

Declarations

14.

16.

17.
18.

19.

20.
21.

Competing interests
The authors declare no competing interests.
Ethics approval
The Institutional Animal Care and Use Committee of the Zhongshan
Ophthalmic Center approved this study (approval number: 2020-019).

22.

Received: 9 January 2024 / Accepted: 17 May 2024

23.

24.
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