Modular immune-homeostatic microparticles promote immune tolerance in mouse autoimmune models

Abstract

The therapeutic goal for autoimmune diseases is disease antigen-specific immune tolerance without nonspecific immune suppression. However, it is a challenge to induce antigen-specific immune tolerance in a dysregulated immune system. In this study, we developed immune-homeostatic microparticles (IHMs) that treat multiple mouse models of autoimmunity via induction of apoptosis in activated T cells and reestablishment of regulatory T cells. Specifically, in an experimental model of colitis, IHMs rapidly released monocyte chemotactic protein–1 after intravenous administration, which recruited activated T cells and then induced their apoptosis by conjugated Fas ligand on the IHM surface. This triggered professional macrophages to ingest apoptotic T cells and produce high quantities of transforming growth factor–β, which drove regulatory T cell differentiation. Furthermore, the modular design of IHMs allowed IHMs to be engineered with the autoantigen peptides that can reduce disease in an experimental autoimmune encephalomyelitis mouse model and a nonobese diabetic mouse model. This was accomplished by sustained release of the autoantigens after induction of T cell apoptosis and transforming growth factor–β production by macrophages, which promoted to establish an immune tolerant environment. Thus, IHMs may be an efficient therapeutic strategy for autoimmune diseases through induction of apoptosis and reestablishment of tolerant immune responses.

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Chen et al., Sci. Transl. Med. 13, eaaw9668 (2021) 10 March 2021
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AUTOIMMUNITY
Modular immune-homeostatic microparticles promote
immune tolerance in mouse autoimmune models
Xin Chen1*†, Xiaoshan Yang2*, Pingyun Yuan1*, Ronghua Jin1, Lili Bao2, Xinyu Qiu2, Siying Liu2,
Tao Liu1, John Justin Gooding3, WanJun Chen4, Guozhen Liu5, Yongkang Bai1, Shiyu Liu2*†, Yan Jin2†
The therapeutic goal for autoimmune diseases is disease antigen-specific immune tolerance without nonspecific
immune suppression. However, it is a challenge to induce antigen-specific immune tolerance in a dysregulated
immune system. In this study, we developed immune-homeostatic microparticles (IHMs) that treat multiple
mouse models of autoimmunity via induction of apoptosis in activated T cells and reestablishment of regulatory
T cells. Specifically, in an experimental model of colitis, IHMs rapidly released monocyte chemotactic protein–1
after intravenous administration, which recruited activated T cells and then induced their apoptosis by conjugated
Fas ligand on the IHM surface. This triggered professional macrophages to ingest apoptotic T cells and produce
high quantities of transforming growth factor–, which drove regulatory T cell differentiation. Furthermore, the
modular design of IHMs allowed IHMs to be engineered with the autoantigen peptides that can reduce disease in
an experimental autoimmune encephalomyelitis mouse model and a nonobese diabetic mouse model. This was
accomplished by sustained release of the autoantigens after induction of T cell apoptosis and transforming
growth factor– production by macrophages, which promoted to establish an immune tolerant environment.
Thus, IHMs may be an efficient therapeutic strategy for autoimmune diseases through induction of apoptosis and
reestablishment of tolerant immune responses.
INTRODUCTION
Failure in immune regulation can lead to autoimmune diseases
(1–3). Regulatory T cells (Tregs) have been recognized to be critical
in ensuring immune homeostasis through their ability to induce
and maintain immune tolerance (4). However, experimental and
clinical efforts to use nonspecific bulk Tregs for therapy of auto-
immune diseases have the potential to increase the risk of systemic
immune suppression in patients (5). To overcome these hurdles,
approaches have been reported that focus on the generation of
antigen-specific Tregs that suppress inflammatory pathogenesis and
autoimmunity without compromising the overall immune responses,
including by administering autoantigens during T cell priming
(6,7). Although these approaches have shown efficacy in experi-
mental animal models of autoimmunity and chronic inflammation
(8,9), marked therapeutic effects on human diseases have yet to be
reported (10). This may be attributed to the fact that, in clinical
translation, it is quite difficult to generate long-term immune toler-
ance in the dysregulated immune system of patients with established
autoimmune diseases (11). Some clinical studies have revealed that
it is necessary to disrupt the dysregulated immune response before
resetting the immune system in patients (12). It has been suggested
that, in an established autoimmune disease state, Tregs may lose
their immunosuppressive function and be converted to proinflam-
matory T effector cells, such as T helper 17 cells (TH17). Conse-
quently, patients may show disease relapse (13,14). Thus, disrupting
the inflammatory immune response driving autoimmunity before
reestablishment of a balanced immune system represents a reason-
able strategy for developing antigen-specific immunotherapy.
The strategies used thus far to disrupt a dysregulated immune
response (15), such as radiation (11) and antibodies (16), may also
increase the risk of extensive off-target effects on the immune sys-
tem, leading to immune deficiency. Moreover, effective induction
of antigen-specific immune tolerance requires precise coordination
of T cell programming, which is difficult to achieve. Recently, bio-
materials functionalized with various specific molecules have been
explored for precisely adjusting cell behaviors, such as apoptosis,
differentiation, and migration (17,18). Such biomaterials can be used
as immunomodulators (19), and researchers have reported the tol-
erizing effects of autoimmune disease–relevant peptides bearing
microparticles or nanoparticles applied in mice with experimental
autoimmune encephalomyelitis (EAE) (20,21). This strategy provides
a strategy to perform sequential deletion of the dysfunctional immune
system followed by induction of antigen-specific immune tolerance.
Here, we demonstrate the potential of modular immune-homeostatic
microparticles (IHMs) for promoting immune tolerance in multiple
mouse models of autoimmunity.
RESULTS
Construction and characterization of IHMs
To treat autoimmune diseases, we synthesized IHMs consisting of
modified mesoporous silica nanoparticles (MSNs) and polymer
microspheres (fig. S1). Polymer microspheres were formed using
1School of Chemical Engineering and Technology, Shaanxi Key Laboratory of Energy
Chemical Process Intensification, Institute of Polymer Science in Chemical Engi-
neering, Xi’an Jiao Tong University, Xi’an, Shaanxi 710049, China. 2State Key Labo-
ratory of Military Stomatology and National Clinical Research Center for Oral
Diseases and Shaanxi International Joint Research Center for Oral Diseases, Center
for Tissue Engineering, School of Stomatology, Fourth Military Medical University,
Xi’an, Shaanxi 710032, China. 3School of Chemistry, Australian Centre for Nano-
Medicine and ARC Australian Centre of Excellence in Convergent Bio-Nano Science
and Technology, University of New South Wales, Sydney 2052, Australia. 4Mucosal
Immunology Section, National Institute of Dental and Craniofacial Research, Na-
tional Institutes of Health, Bethesda, MD 20892, USA. 5Graduate School of Biomedical
Engineering, ARC Centre of Excellence for Nanoscale BioPhotonics and Australian
Centre for NanoMedicine, Faculty of Engineering, University of New South Wales,
Sydney 2052, Australia.
*These authors contributed equally to this work.
†Corresponding author. Email: liushiyu@vip.163.com (Shiyu Liu); yanjin@fmmu.edu.cn
(Y.J.); chenx2015@xjtu.edu.cn (X.C.)
Copyright © 2021
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim
to original U.S.
Government Works
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polyacrylic acid–polyhydroxyethyl methacrylate–polylactic acid
(PAA-PHEMA-PLLA) and poly(lactic-co-glycolic acid) (PLGA) by
double emulsion method. The microspheres were then covered by
amino and thiol cofunctionalized MSNs (NH2-MSNs-SH) via an
amidation reaction. Further stepwise modifications with double
epoxy functionalized polyethylene glycol (EO-PEG-EO) and poly-
ethylene glycol dimethacrylate (PEGMEMA) were achieved through
ring opening reactions (between NH2 and EO) and click reactions
(between SH and C═C) to introduce polyethylene glycol (PEG; seg-
ments to against nonspecific protein absorption) and epoxy groups
to generate PLLA/PLGA microspheres–MSNs–PEG/PEGEO (PMPC).
The successful synthesis of the PAA-PHEMA-PLLA copolymer was
confirmed using 1H nuclear magnetic resonance (1H NMR) spectros-
copy (Fig.1A) and Fourier transform infrared (FTIR) spectroscopy
(fig. S2). The polymer microspheres are about 2 m in diameter and
have a smooth surface (Fig.1B). PMPC had rougher surface and a
larger size as compared with the unmodified polymer microspheres
due to covering with the MSNs (Fig.1C and fig. S3). The composi-
tion of PMPC was investigated by energy-dispersive x-ray (EDX)
spectroscopy analysis (Fig. 1D) and thermogravimetric analysis
(TGA; Fig.1E). EDX gave direct evidence for the immobilization of
the MSNs, as determined from the Si signal (characteristic peak
Fig. 1. Construction and characterization of IHMs. (A) 1H NMR spectra of HEMA-PLLA and PAA-PHEMA-PLLA. [, chemical shift. The unit of  is ppm (parts per million)].
The boxes pointed out the major difference of proton signals between HEMA-PLLA and PAA-PHEMA-PLLA regarding their chemical structures, which represents the
conversion of double bonds of monomers into carbon-carbon single bonds and simultaneous formation of the chains of PAA-PHEMA-PLLA after copolymerization.
(B) Scanning electron microscopy image of the polymer microsphere prepared by PAA-PHEMA-PLLA and PLGA with a mass ratio of 1:1 (scale bar, 1 m). (C) Scanning
electron microscopy image and structure diagram of PMPC (scale bar, 1 m). (D) EDX spectroscopy analysis of PMPC containing the mapping analysis of Si (top figures;
scale bars, 1 m) and the full spectrum of all elements (bottom figure) as well as the quantitative analysis from the full spectrum. (E) TGA of the PMPC and polymer micro-
spheres. (F) FTIR spectroscopy of the MSNs, NH2-MSN-SH, EO-PEG-MSN-SH (PMPC without the PEGMEMA modification after removing the polymer microsphere with
dichloromethane treatment), and EO-PEG-MSN-PEGMEMA (PMPC after removing the polymer microsphere with dichloromethane treatment). The insets represent mag-
nified image of characteristic peaks. a.u., arbitrary units. (G) TGA of PMPC before and after the immobilization of FasL and MCP-1. The loading percentages of FasL and
MCP-1 were calculated from the TGA data. (H) Ability of the FasL and MCP-1 coloaded PMPC (IHMs) with and without the PEG segment against the nonspecific absorption
of FITC-labeled serum albumin. (I) Release profiles of autoantigen and MCP-1 from IHMs in vitro in PBS buffer. n = 3 biological replicates per group (G to I). These experi-
ments were repeated at least twice independently to confirm the results. Data are presented by means ± SD.
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located at 1.75 keV). TGA enabled the quantification of MSNs in
the system [53weight % (wt %), where each gram of PMPC con-
tains 0.53 g of MSNs]. Further details of the MSNs were provided by
transmission electron microscope (TEM; fig. S4) and FTIR (Fig.1F),
indicating the successful modification of the MSNs by EO-PEG-EO
and PEGMEMA.
The aim of the MSNs is to manipulate the immune cells via re-
lease of functional biomolecules and direct interaction in a sequential
fashion. To deplete activated T cells in the context of autoimmunity,
we encapsulated monocyte chemoattractant protein–1 (MCP-1), a
cytokine that induces T cell migration to the sites of inflammation
(22), in IHMs. In addition, because the cell death receptor Fas is
increasingly expressed in activated T cells (fig. S5) (23), we immobi-
lized Fas ligand (FasL) onto the surface of IHMs through ring open-
ing reactions between NH2 in FasL and EO on IHMs. Stimulation of
Fas by FasL triggers apoptosis in activated T cells. The immobiliza-
tion of FasL and encapsulation of MCP-1 were investigated by TGA
(Fig.1G). The data indicated that up to 24 wt % of FasL could be
modified on the surface of IHMs and about 15 wt % of MCP-1
could be loaded in the MSNs in maximum relative to control IHMs
not loaded with MCP-1 (IHMMCP-1 null). The loading of FasL and
MCP-1 was also monitored by pore volume analysis through Brunauer-
Emmett-Teller (BET) method (fig. S6), indicating the successful
immobilization of FasL and encapsulation of MCP-1. Because
IHMs were designed to work systemically invivo, the prevention of
nonspecific protein adsorption was important. As shown in Fig.1H,
the amount of nonspecific protein absorption was markedly re-
duced after modification with the PEG layer.
In addition, we investigated whether autoantigens [glutamic
acid decarboxylase524–543 (GAD524–543) or myelin oligodendrocyte
glycoprotein35–55 (MOG35–55)] could be loaded on IHMs and re-
leased over time due to slow biodegradation of the polymer micro-
spheres, where the aqueous solution containing different autoantigens
was used as the aqueous phase for double emulsion to prepare auto-
antigens and MCP-1–coloaded IHMs (IHMMOG or IHMGAD). We
measured the release curves of autoantigens (GAD524–543) and
MCP-1in phosphate-buffered saline (PBS; pH 7.4). As shown in
Fig.1I, most of the MCP-1 was rapidly released from IHMs in
5 days. In contrast, the release of the autoantigen required 30 days
to be completed. The invivo release characteristics of MCP-1 and
autoantigen were also detected after intravenous injection in mice
(fig. S7). The concentration of MCP-1in the blood reached the
highest point 5 hours after injection of IHMGAD (IHMs carrying
GAD524–543) and then decreased rapidly, reaching almost undetectable
concentrations after 24 hours (fig. S7A). In comparison, the amount
of GAD524–543 released in the blood was relatively stable, with a slow
decrease from days 1 to 6, and reached a low point after 12 days (fig.
S7B). The size and surface charge of IHMs were also characterized
(fig. S8). These results collectively indicate that IHMs were success-
fully constructed.
IHMs induce migration and killing of activated T cells in vitro
Our IHMs were designed to recruit activated T cells via MCP-1
release. To verify the chemotactic response of activated T cells to
IHMs, we used a transwell coculture system with exvivo activated
T cells (prestimulated with anti-CD3 and anti-CD28 antibodies) in
Fig. 2. In vitro recruitment and induction of apoptosis of activated T cells
by IHMs. (A) Migration of activated T cells (red) to IHMMCP-1 null or IHMs
(0.1 g/l; green) in a transwell coculture system. Pure MCP-1 was used as a
positive control to induce the migration of activated T cells (scale bar, 1 m).
(B) Western blot analysis and quantification of the expression of apoptosis-
related proteins. The gray values of the blots were measured, and quantifica-
tion was presented as a ratio of Bax/Bcl-2. (C) Activated T cells were treated
with IHMs (0.1 g/l) for 6 hours, and T cell apoptosis was detected by flow
cytometry. n = 3 biological replicates per group (A to C). These experiments
were repeated at least twice independently to confirm the results. Data are
presented by means ± SD; ns, not significant; *P < 0.05 and **P < 0.01 by one-
way ANOVA (A to C). PI, propidium iodide.
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the upper chamber and IHMs in the lower chamber of the plate.
The number of activated T cells that migrated through the transwell
was counted after a 24-hour incubation. The results showed that the
number of activated T cells surrounding IHMs gradually increased
with the incubation time, and the number of T cells recruited by
IHMs was comparable to the number of T cells recruited by pure
MCP-1 at 24hours (Fig.2A). However, the group of IHMMCP-1 null
(IHM without loading of MCP-1) showed negligible T cell migra-
tion, indicating the capacity of MCP-1–loaded IHMs to specifically
promote migration of activated T cells.
Next, we examined the ability of IHMs to induce apoptosis of
activated T cells. We first measured expression of proteins associated
with T cell apoptosis, including cleaved caspase-3, B cell lymphoma-2
(Bcl-2), Bcl-2–associated X protein (Bax), and Bcl-2 homology–
interacting domain death agonist (BID), in activated T cells before
and after treatment with PMPC, IHMMCP-1 null, or IHMs. Compared
to the control groups (nontreated and PMPC-treated activated
T cells), the IHMMCP-1 null and IHM group showed enhanced ex-
pression of cleaved caspase-3 (the active form of caspase-3), Bax,
and tBID (the truncated form of BID that promotes apoptosis),
whereas the amount of Bcl-2 was lower (Fig.2B), suggesting that
T cell apoptosis is initiated by IHMs. Moreover, using an apoptosis
assay by flow cytometry (Fig.2C), we showed that both IHM and
IHMMCP-1 null treatment increased T cell apoptosis as measured by
annexin V staining and propidium iodide staining. IHMs showed
a greater effect than IHMMCP-1 null. These data suggested that MCP-1
promoted the migration of activated T cells to IHMs, which mark-
edly enhanced the ability of IHMs to induce T cell apoptosis.
Fig. 3. IHMs induced immune tolerance in inflammatory colitis. (A) Schematic showing
DSS-induced colitis mouse model and treatment. Mice were treated with PBS, PMPC, IHMMCP-1 null,
or IHMs (20 mg/kg) on day 3. (B) Body weight and (C) DAI of mice in different groups. (D) Per-
centage of CD3+ T cells in colitis mice assessed by flow cytometry. (E) Percentage of apoptotic
CD3+ T cells assessed by flow cytometry. (F) Frequencies of CD4+/IL17+ T cells isolated from individual mice at indicated time points. (G) Frequencies of CD4+/CD25+/
Foxp3+ Tregs. (H) Serum concentrations of cytokines detected by ELISA. (I) Histological structure of the colon tissue assessed by hematoxylin and eosin (H&E) staining and
the calculated histological activity index (scale bar, 500 m). n = 7 animals per group (B to I). These experiments were repeated at least twice independently to confirm the
results. Data are presented as means ± SD; *P < 0.05 and **P < 0.01 compared with IHM group by one-way ANOVA (B and D to I) and Kruskal-Wallis test (C).
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PMPC, which lack FasL, had no effect on the frequency of T cell
apoptosis (Fig.2C).
We also performed migration and apoptosis induction experi-
ments invitro using human peripheral blood–derived T cells. We
showed that IHMs also recruit human T cells (fig. S9A). In addition,
IHMs induced cleavage of caspase-3 (fig. S9B) and apoptosis of
human T cells (fig. S9C). These data collectively indicated that IHMs
effectively promoted migration of activated T cells through MCP-1
and induced their apoptosis by the FasL/Fas pathway invitro.
Biodistribution of IHMs in vivo
Before invivo therapy, we investigated the biodistribution of IHMs
in mice using an exvivo imaging system. We found that the fluorescently
labeled IHMs were soon distributed in the liver and spleen after 72 hours
Fig. 4. Antigen-specific immune tolerance induced by IHMMOG for EAE suppression. (A) Female C57BL/6 mice were immunized with MOG35–55 in complete Freund’s
adjuvant (day 0) via subcutaneous injections to establish EAE and treated with PBS, IHMOVA, or IHMMOG (20 mg/kg) via tail vein 14 days after immunization. (B) EAE mean
clinical scores were recorded. (C) Frequencies of annexin V+PI+CD3+ T cells in the blood were measured 6 hours after injection. (D) H&E staining of the spinal cord sections
(yellow arrows indicate immune cell infiltration; scale bar, 200 m). (E) Luxol fast blue (LFB) staining of the spinal cord sections (yellow arrows indicate demyelination;
scale bar, 200 m). (F) Splenocytes obtained from each group at the end of the experiment were stimulated with MOG35–55 (10 g/ml) for 72 hours. T cell proliferation
was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining. The extent of stimulated proliferation was reported as proliferation index. (G) The
concentrations of cytokines secreted in response to MOG35–55 stimulation in the supernatants of the same splenocytes as in (F) were measured by ELISA. (H) Brain- and
spinal cord–infiltrating cells were isolated at day 41 and pooled for each group. The cells were stained with CD3, CD4, and tetramers recognizing MOG35–55-specific T cells
together with Foxp3 antibody and analyzed by flow cytometry. Total T cells were first determined by CD3+ cell counts, and CD4+/Foxp3+ Tregs were further gated within
CD3+ T cells. MOG35–55-specific Tregs were analyzed in MOG/Foxp3+ double-positive cells. n = 7 (B to G) and 5 (H) animals per group. These experiments were
repeate d at least twice independently to confirm the results. Data are presented as means ± SD; **P < 0.01 by one-way ANOVA (C and F to H) and Kruskal-Wallis test
(B, D, and E).
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of injection, no matter in EAE or nonobese diabetic (NOD) mice (fig.
S10A). We performed immunofluorescence staining to detect which
type of cells takes up IHMs. The results showed that IHMs were
taken up by Marco-positive macrophages (fig. S10B, top), but not
CCR2-positive macrophages (fig. S10B, bottom), in liver and spleen.
IHMs increased the proportion of Tregs and induced immune
tolerance in inflammatory colitis
Previous studies have shown that apoptotic T cells can trigger pro-
fessional phagocytes, such as macrophages, to produce transform-
ing growth factor– (TGF-), which then induces expansion of
tolerance-promoting Tregs (16,23). We hypothesized that IHMs
would induce activated T cell apoptosis invivo and consequently
promote expansion of tolerance-inducing Tregs. To test this hypoth-
esis, we investigated the immunoregulatory function of IHMs in a
well-described dextran sulfate sodium (DSS)–induced experimen-
tal colitis mouse model (24).
DSS was administered orally for 3 days. At day 3, mice were
intravenously injected with either PBS, PMPC, IHMMCP-1 null, or
IHMs (Fig.3A). Ten days after DSS administration, body weight
loss (Fig.3B) and disease activity index (DAI) were measured
(Fig.3C). We found that injection of IHMs could partially inhibit
loss of body weight and decrease DAI score compared to PBS- or
PMPC-treated mice. Although both IHM and IHMMCP-1 null–treated
mice showed lower number of CD3+ T cells (Fig.3D) and higher
number of apoptotic CD3+ T cells than PBS-treated groups (Fig.3E),
IHM-treated mice had the highest number of apoptotic CD3+ T cells
among all the groups, consistent with invitro data. As expected,
IHM injection markedly reduced the proportion of TH17 cells
(Fig.3F) and increased the proportion of Tregs (Fig.3G).
IHMs also elevated TGF- concentrations in serum (Fig.3H).
The circulating amounts of interleukin-17 (IL-17) and IL-6in the
IHM-treated mice also showed reduction compared to other colitis
groups (Fig.3H). Although the amount of tumor necrosis factor–
(TNF-) was not different from that of the IHMMCP-1 null group, it
was lower than that of the other colitis groups (Fig.3H). Histologi-
cal analysis of colon tissue revealed that IHM treatment prevented
tissue damage and infiltration of inflammatory cells into colon tis-
sues compared with all other colitis groups as measured by a histo-
logical activity index (Fig.3I). These data collectively indicate that
IHMs induce immunoregulation and decrease inflammation in the
murine DSS-induced colitis model.
IHMs loaded with autoantigen induced antigen-specific
immunosuppression in mice with EAE
To study the generality of IHM-mediated immunoregulation, we
next treated EAE mice with IHMs that had been loaded with the
autoantigenic peptide MOG35–55 (25). MOG35–55 was chosen because
it has been reported that autoantigenic peptides may direct naïve
CD4+ T cells to differentiate toward antigen-specific Tregs under the
TGF-–rich microenvironment created by phagocytes taking up
apoptotic cells (11,26). We hypothesized that the sustained release
of MOG35–55 by IHMs after induction of T cell apoptosis could in-
duce antigen-specific immune tolerance to suppress EAE. For this,
mice were immunized by MOG35–55 plus complete Freund’s adju-
vant to induce EAE. Mice systemically received PBS, MOG35–55-
loaded IHMs (IHMMOG), or control ovalbumin (OVA) peptide–loaded
IHMs (IHMOVA) at 14 days after immunization (Fig.4A). As ex-
pected, the PBS-treated EAE mice displayed severe clinical symp-
toms. However, IHMMOG-treated mice, but not IHMOVA-treated
mice, displayed obvious suppression of disease symptoms, as deter-
mined by mean clinical score (Fig.4B). To evaluate T cell depletion,
apoptosis of CD3+ T cells in peripheral blood mononuclear cells
(PBMCs) was analyzed 6 hours after IHM treatment. Consistent
with the results from our invitro studies and from the colitis model,
the apoptotic rate of CD3+ T cells markedly increased in the IHMOVA
and IHMMOG groups compared with the PBS group (Fig.4C). In
addition, the proportions of both CD4+ and CD8+ T cells were
decreased at 6 hours, whereas there was no obvious change in the
proportion of B cells or dendritic cells (DCs) (fig. S11). Spinal cord
tissues were collected and processed for histological analysis to
determine inflammatory cell infiltration and demyelination in the
central nervous system. The results showed that IHMMOG-treated
mice exhibited a reduction of infiltrating inflammatory cells as indicated
by yellow arrows (Fig.4D and demyelination (Fig.4E). In contrast,
IHMOVA-treated mice showed no improvement in terms of the pro-
portion of infiltrating cells or demyelination.
T cell activity plays a key role in the development of EAE (27).
To investigate whether the decreased development of EAE in the
IHMMOG-treated mice was associated with a lower response to anti-
gen stimulation, splenocytes were restimulated with MOG35–55 pep-
tide invitro. We found that MOG35–55-specific T cell proliferation
(Fig.4F) and secretion of inflammatory cytokines (Fig.4G) were
markedly decreased in splenocytes isolated from IHMMOG-treated
mice compared to IHMOVA-treated mice (Fig.4,FandG). Further-
more, brain- and spinal cord–infiltrating cells were isolated for the
detection of MOG35–55-specific Tregs. The results showed that treat-
ment with IHMMOG increased the frequency of MOG35–55-specific
Tregs in the brain and spinal cords (Fig.4H). Together, these results
indicated that IHMMOG diminished the inflammatory response and
suppressed EAE in an antigen-specific manner.
Table 1. Composition of microparticles used in this study. PMPC,
PLLA/PLGA microspheres–MSNs–PEG/PEGEO; FasL, Fas ligand; MCP-1,
monocyte chemoattractant protein–1; IHMMCP-1 null, IHM without loading
of MCP-1; IHMs, MCP-1 and FasL coloaded immune-homeostatic mic ropar ticle s;
IHMMOG, MOG35–55-loaded IHMs; IHMGAD, GAD524–543-loaded IHMs; IHMMOG/
MCP-1 null, IHMMOG without MCP-1; IHMMOG/FasL null, IHMMOG without FasL;
IHMGAD/MCP-1 null, IHMGAD without MCP-1; IHMGAD/FasL null, IHMGAD without FasL.
Abbreviation Construction Conjugated biomolecules
FasL or MCP-1 Autoantigen
PMPC
PLLA/PLGA
microspheres–
MSNs–PEG/PEGEO
None None
IHMMCP-1 null PMPC FasL None
IHMs PMPC FasL and
MCP-1 None
IHMMOG PMPC FasL and
MCP-1 MOG35–55
IHMMOG/MCP-1 null PMPC FasL MOG35–55
IHMMOG/FasL null PMPC MCP-1 MOG35–55
IHMGAD PMPC FasL and
MCP-1 GAD524–543
IHMGAD/MCP-1 null PMPC FasL GAD524–543
IHMGAD/FasL null PMPC MCP-1 GAD524–543
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To further explore the role of MCP-1 and FasL in induction of
apoptosis in T cells and subsequent control of EAE-induced pathol-
ogy, we synthesized IHMs with different components (Table1). We
showed that IHMMOG without MCP-1 (IHMMOG/MCP-1 null) or FasL
(IHMMOG/FasL null) was unable to induce T cell apoptosis (Fig.5A).
As a result, there was no obvious improvement in the inflammatory
infiltration of spinal cord (Fig.5B) as well as the induction of
MOG35–55-specific Tregs in IHMMOG/MCP-1 null– or IHMMOG/FasL null–
treated mice (Fig.5C). In addition to MCP-1 and FasL, we next
examined whether host TGF- release plays a role in promoting
Treg development and IHM-based therapies. TGF- was blocked by
intravenous injection of anti–TGF- neutralizing antibody (TGF-),
and an isotype antibody was used as control (contAb) (Fig.5,DandE).
As expected, IHMMOG with TGF- neutralizing antibody injection
(IHMMOG+TGF-) failed to reduce pathology in the spinal cord
of mice after induction of EAE (Fig.5D). TGF- neutralizing anti-
body injection also resulted in a reduction in the proportion of
MOG35–55-specific Tregs (Fig.5E). These data indicated that the
antigen-specific therapeutic effects generated by IHMMOG require
MCP-1–mediated T cell recruitment, FasL-induced T cell apoptosis,
and TGF-.
IHMs loaded with GAD524–543 induced antigen-specific
immune tolerance in diabetic mice
We hypothesized that IHMs have the capacity to induce antigen-
specific immune tolerance in multiple autoimmune diseases by
changing the autoantigens encapsulated in the central polymer
module. To verify this, we next determined the immunoregulatory
effects of IHMs loaded with a diabetes-associated autoantigen,
GAD524–543, in the treatment of experimental diabetes in NOD/
ShiLtJ mice. Diabetes develops from 12 weeks of age in female
NOD/ShiLtJ mice and is characterized by insulitis and a leukocytic
infiltrate in the pancreatic islets (28). GAD524–543 has been identi-
fied as one of the autoantigens in NOD mice and in patients with
type 1 diabetes, the recognition of which can mediate autoimmune
destruction of pancreatic  cells and impair insulin release (29).
Therefore, we treated hyperglycemic NOD mice with glucose val-
ues >200 mg/dl with IHMGAD or IHMOVA.
The results showed that a single injection of IHMGAD blocked
the development of diabetes in NOD/ShiLtJ mice, where mice are
considered diabetic when their blood glucose values rise to >250 mg/dl.
In mice treated with IHMGAD, blood glucose remained below
300 mg/dl, on average, whereas injection of PBS or IHMOVA exhibited
Fig. 5. MCP-1, FasL, and TGF- are indispensable for the induction of antigen-specific immune tolerance by IHMMOG in EAE mice. EAE was induced in mice, and the
mice were treated with PBS, IHMMOG/MCP-1 null, IHMMOG/FasL null, IHMMOG + TGF-, IHMMOG + contAb, or IHMMOG (20 mg/kg) 14 days after immunization. (A) Frequencies of
apoptotic CD3+ T cells in the blood 6 hours after injection. (B) H&E staining of the spinal cord sections (yellow arrows, immune cell infiltration) (scale bar, 200 m).
(C) Brain- and spinal cord–infiltrating lymphocytes were isolated at day 41 and pooled for each group. The cells were then stained with tetramers recognizing
MOG35–55-specific T cells together with Foxp3 antibody and analyzed by flow cytometry. (D) H&E staining of the spinal cord sections (yellow arrows, immune cell infiltration)
(scale bar, 200 m). (E) Brain- and spinal cord–infiltrating lymphocytes were isolated at day 41 and pooled for each group. The cells were then stained with tetramers
recognizing MOG35–55-specific T cells together with Foxp3 antibody and analyzed by flow cytometry. n = 6 (A, B, and D) and 5 (C and E) animals per group. These experiments
were repeated at least twice independently to confirm the results. Data are presented as means ± SD; **P < 0.01 by one-way ANOVA (A, B, and D).
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no suppression of blood glucose (Fig.6A). Like that of EAE mice,
we also detected relevant cell subsets in NOD mice 6 hours after
treatment. The frequencies of both CD4+ and CD8+ T cells were
decreased, whereas no obvious change in the proportion of B cells
or DCs was observed (fig. S12). Histological analysis of the pancre-
ases revealed that the IHMGAD-treated mice preserved islets in
the pancreas, whereas PBS- or IHMOVA-treated mice showed obvi-
ous leukocytic infiltrated in the pancreatic islets and fewer pancre-
atic islets relative to IHMGAD-treated mice (Fig.6B). Analysis of the
pancreatic draining lymph node revealed higher frequencies of Tregs
(Fig.6C) and lower frequencies of CD4+/IFN-+ (interferon-–
positive) TH1 cells (Fig.6D) than in the PBS- or IHMOVA-treated
mice. Moreover, to investigate whether the suppression of diabetes
in IHMGAD-treated mice was associated with the establishment of
GAD524–543-specific immune tolerance, we isolated total CD4+ (in-
cluding CD4+CD25+ and CD4+CD25−) T cells and CD4+CD25− subset
from the spleens of NOD mice at day 41 and then examined
GAD524–543-specific T cell proliferation and cytokine production by
culturing the splenocytes with GAD524–543. If GAD524–543-specific
immune tolerance was restored after treatment, we would expect
to see decreased CD4+ T cell response to GAD524–543 in these mice;
removal of CD4+CD25+ Tregs from the same CD4+ T cells would
reverse the suppression of GAD524–543-specific response in the
CD4+ T cells (11). As controls, the same T cell subsets were also re-
stimulated with anti-CD3 antibody. The results showed that, in
comparison to IHMOVA-treated mice, the proliferation and cyto-
kine production (IFN- and TNF-) of CD4+ T cells from IHMGAD-
treated mice were decreased in response to GAD524–543 stimulation
invitro. By contrast, no obvious change was observed in the prolifera-
tion and cytokine production in CD4+CD25− T cells in response to
GAD524–543 peptide restimulation in the absence of Tregs (Fig.6,EandF).
There was no change of total CD4+ or CD4+CD25− cell prolifera-
tion and cytokine production to anti-CD3 antibody stimulation
among all groups, suggesting that the overall T cell response to
Fig. 6. Antigen-specific immune tolerance induced by IHMGAD in hyperglycemic NOD
mice. (A) Blood glucose was measured to monitor diabetes progression in the NOD mice
treated with PBS or IHMs, which were considered diabetic when the blood glucose value was
higher than 250 mg/dl (blue dashed line). (B) H&E staining of pancreas sections (yellow ar-
rows, immune cell infiltration) (scale bar, 200 m). Three sections were taken from each tissue
for H&E staining, and the average number of islets on each slide was calculated. (C) Represent-
ative flow cytometry data and frequencies of CD25+/Foxp3+ Tregs in pancreas–draining
lymph nodes (DLN). (D) Representative flow cytometry data and frequencies of CD4+/IFN-+
cells in pancreas–draining lymph nodes. (E) The concentrations of cytokines in the supernatant of splenocytes isolated from PBS- or IHM-treated NOD mice in response
to GAD524–543 or CD3 stimulation in the supernatants were measured by ELISA. (F) T cell proliferation was measured by CFSE dilution. The extent of stimulated prolifer-
ation was reported as proliferation index. n = 7 animals per group (A to F). These experiments were repeated at least twice independently to confirm the results. Data are
presented as means ± SD; **P < 0.01 by one-way ANOVA (B to F).
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pan–T cell receptor stimulation in the IHMGAD-treated mice was not
compromised.
We then validated a role for MCP-1 and FasL of IHMs in IHM-
mediated treatment in the NOD mouse model of diabetes. Consistent
with the results in EAE model, IHMGAD without MCP-1 (IHMGAD/MCP-1 null)
or FasL (IHMGAD/FasL null) failed to induce apoptosis of T cells invivo
(Fig.7A) and did not protect NOD mice against islet loss (Fig.7B).
To further confirm that MCP-1 and FasL were necessary for the
induction of Tregs, we isolated total CD4+ T cells and CD4+CD25−
subsets from the spleens of NOD mice after various IHM treat-
ments and then examined GAD524–543-specific T cell proliferation
and cytokine. Results showed that, compared with IHMGAD/MCP-1 null–
or IHMGAD/FasL null–treated mice, the proliferation and cytokine pro-
duction of CD4+ T cells from IHMGAD- treated mice were decreased
in response to GAD524–543 stimulation in cultures. By contrast, no
obvious change was observed in the proliferation and cytokine
production in CD4+CD25− T cells to GAD524–543 peptide restimula-
tion (Fig.7,CandD), suggesting that more Tregs are generated in
the IHMGAD-treated NOD mice but not in the IHMGAD/MCP-1 null–
or IHMGAD/FasL null–treated mice. As expected, IHMGAD with TGF-
neutralizing antibody injection (IHMGAD+TGF-) failed to control
inflammatory cell infiltration of islet tissue in NOD mice (Fig.7E) and
lost the ability to induce Tregs (Fig.7,FandG). Together, these results
provide compelling evidence that the chemotaxis of MCP-1, FasL/
Fas-induced T cell apoptosis, and the release of TGF- were indis-
pensable for IHMMOG and IHMGAD to generate antigen-specific im-
mune tolerance and ameliorate EAE and NOD models, respectively.
DISCUSSION
Autoimmune diseases, including type 1 diabetes, multiple sclerosis,
and lupus, occur when the immune system attacks an individual’s
Fig. 7. MCP-1, FasL, and TGF- are indispensable for the induction of antigen-specific immune tolerance by IHMGAD in hyperglycemic NOD mice. (A) Frequencies
of apoptotic CD3+ T cells in the blood 6 hours after injection of PBS or IHMs. (B) H&E staining of pancreas sections (yellow arrows, immune cell infiltration) (scale bar, 200 m).
(C) T cell proliferation was measured by CFSE staining. (D) The concentrations of cytokines in the supernatant of splenocytes isolated from PBS- or IHM-treated NOD mice
in response to GAD524–543 or CD3 stimulation were measured by ELISA. (E) H&E staining of pancreas sections (yellow arrows, immune cell infiltration) (scale bar, 200 m).
(F) T cell proliferation was measured by CFSE staining. (G) The concentrations of cytokines in the supernatant of splenocytes isolated from PBS- or IHM-treated NOD mice
in response to GAD524–543 or CD3 stimulation were measured by ELISA. n = 6 animals per group (A to G). These experiments were repeated at least twice independently
to confirm the results. Data are presented as means ± SD; *P < 0.05 and **P < 0.01 by one-way ANOVA.
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own tissue or organs (3,25,28, 29). Induction of antigen-specific
immune tolerance holds considerable promise for the treatment of
autoimmune diseases without systemic immune suppression (6,7).
Although multiple strategies for antigen administration have been
performed in animal models, the therapeutic effects of these treat-
ments have not been observed in human clinical trials (10). This
was thought to be due to the fact that autoantigen administration to
modulate T cell receptor signaling alone is not sufficient for treatment
(30). Additional strategies, including the design of vehicles that target
key antigen-presenting cells and the delivery of immunomodulator-
autoantigen combinations, have been used (31). However, the effi-
cacy and potential of these strategies still needs to be fully evaluated.
It is known that the host immune microenvironment plays a key
role in immune tolerance induction, because it is extremely difficult
to generate Tregs in the inflammatory, dysregulated immune system
of patients with established autoimmune diseases (11). To date,
the detailed mechanisms underlying most autoimmune diseases
are yet to be fully established (32,33). Furthermore, no effective
screening methods have been established to detect risk for autoim-
mune diseases before they are established, which makes it difficult
to prevent the autoimmune diseases (10). Therefore, patients with
autoimmune diseases usually have established disease progression
and heavily dysregulated immune responses in which induction of
antigen-specific immune tolerance appears to be difficult (12,34).
In consideration of these challenges, modulation of the immune
system through depletion of activated T cells in advance could en-
hance the efficacy of antigen-specific immune therapy.
In this study, we used FasL to induce apoptosis of activated T cells,
because FasL/Fas signaling is required for depletion of autoreactive
T cells and maintaining immune homeostasis in physiological con-
ditions (35). Previous studies showed that systemically infused
mesenchymal stem cell–derived FasL could ameliorate disease symp-
toms of a systemic sclerosis model by inducing apoptosis of activated
T cells (23). Because IHMs could release MCP-1 to recruit activated
T cells and Fas is increasingly expressed in activated T cells relative
to naïve T cells (23), the system we established should not result in
extensive off-target effects on the immune system. Because IHMs
deplete the dysfunctional immune response, the system developed
in this study expands the potential applications of biomaterials in
immune disease therapy, demonstrating that the biomaterials not
only can be used as modulators as demonstrated previously but also
could be used for reconstruction of immune homeostasis (fig. S13).
Beyond their ability to establish immune homeostasis by recruit-
ment and apoptosis of activated T cells, IHMs can also induce antigen-
specific Tregs for therapy of certain autoimmune disease due to their
controllable release of autoantigenic peptides. Compared with pre-
viously reported biomaterial-based platforms for the induction of
antigen-specific immune tolerance (9,36,37), IHMs depleted the
dysfunctional T cells before autoantigenic peptide administration,
which we hypothesize facilitates immune tolerance induction
(15,38). In this study, although we have not identified the exact lo-
cation where IHMs’ protective effects occur, our data suggest that
this process can occur in the periphery, because we can detect in-
creased apoptosis of T cells in the peripheral blood and increased
Tregs in spleen. In addition, the generation of Tregs may also occur in
the target tissues such as brain/spinal cords and pancreas in the
EAE and NOD mice, respectively. However, it is also possible that
the Tregs generated in the periphery could migrate into the inflamed
tissue. Thus, IHMs induce activated T cell apoptosis in the
periphery and then induce Tregs, which controls inflammation in the
affected tissue.
In this study, we demonstrate that IHMs represent a modular
strategy to treat various autoimmune diseases by simply replacing
the autoantigen loaded into the microparticle. The method we cur-
rently used to detect antigen-specific immune tolerance in NOD
mouse model may not directly represent the number of GAD65-
specific Tregs, whereas the use of GAD65 tetramers would provide
additional direct evidence. Besides, further studies are needed to
optimize IHMs by modifying IHM with other T cell chemoattractant
cytokines and apoptosis-related ligands. Furthermore, therapeutic
effects of IHMs on other autoimmune diseases with specific auto-
antigen need further study. In conclusion, IHMs demonstrated
an effective therapeutic strategy for three distinct models of auto-
immunity by inducing T cell apoptosis and promoting Treg differ-
entiation. This study provides a promising therapeutic strategy to
induce antigen-specific immune tolerance invivo.
MATERIALS AND METHODS
Study design
The experiments in this study were designed to develop efficient
biomaterial-based IHMs for treatment of autoimmune diseases.
Mouse leukocytes and human PBMCs were used to explore the role
of IHMs in T lymphocyte migration and apoptosis invitro. Human
blood samples were collected from healthy donors. The use of
human samples was approved by the ethical review committee of
the Third Affiliated Hospital of Fourth Military Medical University.
All participants were well informed and signed an informed con-
sent form. The whole blood was collected from participants by
nurse practitioners via ulnar vein puncture and was then used for
PBMC isolation. We investigated the invivo role of IHMs using
three mouse models: the inflammatory colitis, EAE, and diabetes
models. All procedures that involved animals were approved by the
Animal Use and Care Committee of the Fourth Military Medical
University. In all experiments, mice of similar age and size were
used across all groups. Mice were randomly assigned to each group,
but the experimenter was not blinded to group identity. The details
of study design, sample sizes (determined according to previous
experimental experience and publications), experimental replicates,
and statistics are provided in the corresponding figures, figure leg-
ends, and data files. Primary data are reported in data file S1.
Mice
Female C57BL/6 mice were purchased from the Animal Center of
Fourth Military Medical University. Female NOD/ShiLtJ mice were
purchased from Nanjing Biomedical Research Institute of Nanjing
University (N000235). Diabetes develops from 12 weeks of age in
female NOD/ShiLtJ mice. Diabetes is characterized by insulitis, leu-
kocytic infiltrate of the pancreatic islets, and marked decreases in
islet number and pancreatic insulin content.
Antibodies
Anti-CD3–allophycocyanin (APC) (0.4 g per test; BioLegend,
100312, lot: B209683), anti–IL-17A–phycoerythrin (PE) (0.125 g per
test; eBioscience, 12-7177-81, lot: 4306419), anti-CD4–PE/cyanine7
(PE/Cy7) (0.25 g per test; BioLegend, 100528, lot: B281900), anti-
CD25–APC (0.25 g per test; BioLegend, 102012, lot: B212677),
anti-Foxp3–PE (1 g per test; eBioscience, 126404, lot: B271499),
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anti–IFN-–PE (5 l per test; BioLegend, 507806, lot: B147494),
anti- CD8–fluorescein isothiocyanate (FITC) (0.5 g per test;
eBioscience, 11-0081, lot: E00115-1631), anti-CD19–PE (0.125 g per
test; eBioscience, 12-0193-82, lot: 2051662), anti-CD11b–peridinin
chlorophyll protein/cyanine5.5 (PerCP/Cy5.5) (0.25 g per test;
BioLegend, 101227, lot: B276003), and anti-CD11c–Alexa Fluor
488 (0.25 g per test; eBioscience, 53-0114-82, lot: 1984152) were
used for flow cytometry. Anti–TGF- neutralizing antibody (Bio X
Cell, BE0057) was used for the blockage of TGF-. Primary anti-
bodies, including caspase-3 antibody (dilute at 1:1000; Cell Signal-
ing Technology, 9662, lot:19), cleaved caspase-3 antibody (dilute at
1:1000; Cell Signaling Technology, 9661, lot: 45), Bax antibody
(dilute at 1:1000; Cell Signaling Technology, 2772, lot: 11), Bcl-2
antibody (dilute at 1:1000; Cell Signaling Technology, 2870), and BID
antibody (dilute at 1:1000; R&D Systems, AF860, lot: CDK031909A),
were used for Western blotting.
T cell culture
For lymphocyte cultures, mice were euthanized, and spleens were
isolated. Cell suspensions were produced by crushing isolated spleens.
Red cell lysis buffer (Beyotime Biotechnology, C3702) was used to
lyse red blood cells. The cell suspension was cultured in RPMI 1640
medium (Gibco) at 37°C in 5% CO2, supplemented with 10% fetal bo-
vine serum (Gibco), 50 mM 2-mercaptoethanol (Sigma-Aldrich),
2 mMl-glutamine (Sigma-Aldrich), and 1% penicillin/streptomycin.
Isolation of PBMCs
Mouse blood samples were obtained from the tail vein, and human
blood samples were obtained from healthy human donors. All
blood samples were collected into heparinized tubes and diluted
1:1in PBS. PBMCs were isolated via density gradient centrifugation
using Lymphocyte Separation Medium [1.0810±0.0005 g/ml for
mouse (Dakewe, 7211011) and 1.077±0.001 g/ml for human
(Dakewe, 7111011)] according to the manufacturer’s instructions.
Cell migration (chemotaxis) assay
The chemotaxis assay was performed in a transwell plate (Millipore).
After 24 to 48 hours of incubation, T cells were activated with anti-
CD3 (2 g/ml; BioLegend, 100314, lot: B316718) and anti-CD28
(2 g/ml; eBioscience, 16-0281-85, lot: 2178222) antibodies. Cells
were then labeled with PKH26 (Sigma-Aldrich) and seeded into the
upper chamber of the transwell plate. Green fluorescent protein–
labeled IHMMCP-1 null or IHMs were added to the lower chamber
and cocultured for 24hours, and then the plate was observed under
a fluorescence microscope. The PKH26-labeled T cells were quanti-
fied by calculating the average number of cells in three microscopic
fields from one well.
Cell isolation and proliferation
To examine the function of antigen-specific Tregs in the spleen,
CD4+, CD4+CD25−, and CD4+CD25+ T cells were magnetic activated
cell sorting (MACS)–sorted using the CD4+CD25+ Regulatory T Cell
Isolation Kit (Miltenyi, 130-091-041) following the manufacturer’s
protocol. For the proliferation assay, splenocytes were labeled with
2.5 M carboxyfluorescein diacetate succinimidyl ester (CFSE; Bio-
Legend, 423801) for 5min according to the manufacturer’s instruc-
tions before culture. CFSE-labeled splenocytes (with Tregs included
in CD4+ T cells and not included in CD4+CD25− T cells) were cul-
tured for 72 hours with either soluble CD3-specific antibody
(anti-CD3) or MOG35–55 or GAD524–543 peptides as indicated. The
extent of stimulated proliferation was analyzed by flow cytometry
and reported as proliferation index analyzed by ModFit software.
Enzyme-linked immunosorbent assay
Peripheral blood samples were collected from mice and centrifuged
at 1000g for 10min to isolate serum. Supernatants of T cells were
collected after stimulation with MOG35–55 or GAD524–543 for 72hours.
Mouse TGF-, IL-17, IL-6, TNF-, and IFN- were detected using
an enzyme-linked immunosorbent assay (ELISA) kit (Neobioscience)
according to the manufacturer’s instructions.
Flow cytometry analysis
For cell surface markers, single-cell suspension was stained with the
following fluorochrome-conjugated antibodies for 30min on ice
under dark conditions. Antibodies for cell surface markers are anti-
mouse CD3-APC, CD4-PE/Cy7, CD8-FITC, CD19-PE, CD11b-PerCP/
Cy5.5, CD11c–Alexa Fluor 488, and CD25-APC. Apoptotic T cells
were detected by staining with CD3-APC antibody for 30min on
ice under dark conditions, followed by Annexin-V-FLUOS Staining
Kit (Roche). Foxp3 expression was examined using the True-Nuclear
Transcription Factor Buffer Set (BioLegend, 424401). For intracel-
lular cytokine measurement, cells were incubated with Cell
Activation Cocktail (BioLegend, 423303) at 37°C for 6 hours to de-
termine the intracellular expression of IL-17 and IFN-. Samples
were analyzed with a flow cytometer (Cytomics FC 500; Beckman
Coulter) equipped with CXP 2.1 software.
Western blotting analysis
Total protein was extracted using radioimmunoprecipitation assay
buffer supplemented with protease inhibitor cocktail (Cell Signaling
Technology). Equal amount of total proteins was separated by 10 or
12% SDS–polyacrylamide gel electrophoresis and transferred elec-
trophoretically onto polyvinylidene difluoride membranes (Millipore,
Germany). The membranes were blocked in 5% bovine serum albu-
min for 2hours at room temperature and incubated with specific
primary antibodies [caspase-3, Bax, Bcl-2, BID, Fas, and glyceraldehyde-
3-phosphate dehydrogenase (GAPDH)] at 4°C overnight. After
three washes in TBST (tris-buffered saline containing 0.1% Tween 20),
the membranes were incubated with horseradish peroxidase–
conjugated secondary antibodies in TBST for 2hours at room
temperature. Blots were captured with the Western Light Chemilu-
minescent Detection System (Tanon). The gray value of the blots in
the pictures was measured with ImageJ software and normalized to
that of GAPDH before comparison. The uncropped pictures of the
Western blotting bands are presented in fig. S14.
Establishment of an experimental colitis model
and treatment by IHM injection
To induce colitis, 8-week-old female C57BL/6 mice were given
3% (w/v) DSS (Mw: 36,000 to 50,000 Da; MP Biomedicals) through
drinking water for 10 days (39). At day 3, PMPC, IHMMCP-1 null, or
IHMs were injected into the disease model mice intravenously. In
the control group, the mice received PBS. Body weight loss, fecal
bleeding, and stool consistency were measured daily. The DAI was
scored according to the following criteria: (i) body weight loss,0 (no
change or increase), 1 (1 to 5%), 2 (6 to 10%), 3 (11 to 20%), and
4 (>21%); (ii) stool consistency or diarrhea, 0 (normal), 1 (slightly
soft), 2 (loose), 3 (unformed/mild diarrhea), and 4 (severe watery
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diarrhea); and (iii) fecal blood,0 (negative fecal occult blood),
1 (faint positive fecal occult blood), 2 (certain positive fecal occult
blood), 3 (moderate rectal bleeding), and 4 (severe rectal bleeding).
The DAI is the sum of the scores for body weight loss, stool consist ency,
and fecal bleeding. All mice were harvested at day 10 and analyzed.
The induced colitis was evaluated as previously described (40).
Peptides
MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) and GAD524–543
(SRLSKVAPVlKARMMEYGTT) were purchased from Ontores.
EAE induction and in vitro cell culture
EAE was induced in 8-week-old female C57BL/6 mice as previously
reported (41). Mice received four subcutaneous injections (two in the
flanks and two at the base of the tail) of 50 l of MOG35–55 (200 g per
mouse), incomplete Freund’s adjuvant, and heat-killed Mycobacterium
tuberculosis (4 mg/ml) (Chondrex). A total of 200ng of Pertussis
toxin (Merck) was injected intravenously on the day of the immuni-
zation and again after 48hours. EAE clinical scores (0,healthy;
1,limp tail; 2,ataxia or paresis of hind limbs; 3, paralysis of hind
limbs or paresis of forelimbs; 4,tetraparalysis; 5,moribund or death)
were recorded daily (33).
For cell cultures, splenocytes were cultured at 37°C in 5% CO2
for 72hours with MOG35–55 (10 g/ml). Cell proliferation was de-
tected by CFSE staining. Cytokines in the supernatants of the cell
cultures were analyzed by ELISA. To examine the MOG35–55- tetramer+
Foxp3+ Tregs in the spinal cord, cells were isolated from brain and
spinal cord as previously reported (16). The isolated T cells were
incubated with a 1:5 dilution of peptide/major histocompatibility
complex (MHC) II tetramers (APC-labeled mouse MHC class II
MOG35–55/I-Ab-tetramers; MBL, TS-M704) for 1 hour at room
temperature protected from light. The cells were then washed and
stained for cell surface markers described above. Samples were ana-
lyzed by flow cytometry.
Histological analysis
Colon tissues were fixed in 4% formaldehyde, embedded in paraffin,
and sectioned into 5-m sections (Leica RM2235 manual rotary
microtome). The sections were prepared for hematoxylin and eosin
staining and evaluated by histological activity index. The histological
activity index was scored according to the following criteria: (i)
severity of inflammation,0 (none), 1 (mild), 2 (moderate), and
3 (severe); (ii) extent of inflammation,0 (none), 1 (mucosa), 2
(mucosa and submucosa), and 3 (transmural); and (iii) crypt damage,
0 (none), 1 (one-third damaged), 2 (two-thirds damaged), 3 (crypt
lost and surface epithelium present), and 4 (crypt and surface epi-
thelium lost). The histological activity index was the sum of the
three parameters. The evaluators were blinded to group identity.
For neuropathological analysis, the spinal cord was obtained and
fixed with 4% paraformaldehyde. Paraffin-embedded 5-m trans-
verse sections were cut from the midlumbar spinal cord and stained
with hematoxylin and eosin and Luxol fast blue to detect inflamma-
tory infiltrates and demyelination, respectively. Inflammation and
demyelination scores were calculated with reference to previously
reported methods (42). Briefly, for inflammation: 0,none; 1,a few
inflammatory cells; 2,organization of perivascular infiltrates; 3,in-
creasing severity of perivascular cuffing; and 4,increasing severity
of perivascular cuffing with extension into the adjacent tissue. For
demyelination: 0,none; 1,rare foci; 2,a few areas of demyelination;
3,many areas of demyelination; and 4,large (confluent) areas of
demyelination.
Statistical analysis
Statistical analysis was performed using SPSS software version 25.0.
All datasets were measured for normality and variance. Statistical
significance of multiple groups was calculated by one-way analysis
of variance (ANOVA). Tukey’s post hoc test was used for post hoc
multiple comparisons to evaluate the significance between groups
after one-way ANOVA. The Kruskal-Wallis test was used if data
did not follow a normal distribution. All data were expressed as
means±SD. P values less than 0.05 were considered significant.
SUPPLEMENTARY MATERIALS
stm.sciencemag.org/cgi/content/full/13/584/eaaw9668/DC1
Materials and Methods
Fig. S1. Synthesis of IHMs.
Fig. S2. The stepwise synthesis of HEMA-PLLA and PAA-PHEMA-PLLA investigated by FTIR
spectroscopy.
Fig. S3. Size distribution of polymer microspheres with and without MSNs.
Fig. S4. TEM images of MSNs before and after modification.
Fig. S5. Expression of Fas protein in naïve T cells and activated T cells.
Fig. S6. The modification and loading procedure of IHMs monitored by Brunauer-Emmett-Teller
measurements, which were presented by the pore size change of IHMs before and after each
step of modification and loading.
Fig. S7. Normalized plasma concentration of MCP-1 and autoantigens released from
GAD524–543-containing IHMs.
Fig. S8. Size and surface charge of microparticles used in the study.
Fig. S9. In vitro chemoattraction and killing of human T cells by IHMs.
Fig. S10. In vivo accumulation and uptake of IHMs by macrophages.
Fig. S11. Peripheral blood cell subsets in EAE mice 6 hours after treatment with IHMMOG.
Fig. S12. Peripheral blood cell subsets in NOD mice 6 hours after treatment with IHMGAD.
Fig. S13. Schematic illustration of IHMs induced antigen-specific immune tolerance via an
apoptosis and reestablishment strategy.
Fig. S14. Uncropped pictures of the Western blot bands presented in this study.
Data file S1. Primary data.
Reference (43)
View/request a protocol for this paper from Bio-protocol.
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Funding: This work was supported by the National Key Research and Development Program
of China (no. 2016YFC1101400 to Y.J.); the National Natural Science Foundation of China
(81601606 to X.C., 31800817 to Siying Liu, and 31670995 to Shiyu Liu); the Young Elite
Scientist Sponsorship Program by CAST (2017QNRC001 to Shiyu Liu); the Fundamental
Research Funds for the Central Universities, open fund of the State Key Laboratory of Military
Stomatology (2017KA02 to X.C.); and the Knowledge Innovation Program of Shenzhen
(JCYJ20170816100941258 to X.C.). W.C. was supported by the Intramural Research Program of
NIH, NIDCR. Funding from the ARC Centre of Excellence in Convergent Bio-Nano Science and
Technology (CE140100036) and an ARC Laureate Fellowship program (FL150100060) is
acknowledged by J.J.G. Author contributions: X.C., X.Y., Shiyu Liu, and P.Y. contributed to the
study design, data acquisition, and interpretation. P.Y. and R.J. characterized properties of
microparticles and polymer microsphere. L.B., X.Q., Siying Liu, and T.L. performed the animal
experiments. G.L., Y.B., J.J.G., and W.C. contributed to data analysis and interpretation. Shiyu Liu,
Y.J., and X.C. developed the concept and supervised experiments. All the authors contributed
to the writing of the manuscript. Competing interests: The authors declare that they have no
competing interests. Data and materials availability: All data associated with this study are
present in the paper or the Supplementary Materials.
Submitted 15 February 2019
Resubmitted 14 March 2020
Accepted 26 January 2021
Published 10 March 2021
10.1126/scitranslmed.aaw9668
Citation: X. Chen, X. Yang, P. Yuan, R. Jin, L. Bao, X. Qiu, S. Liu, T. Liu, J. J. Gooding, W. Chen,
G. Liu, Y. Bai, S. Liu, Y. Jin, Modular immune-homeostatic microparticles promote immune
tolerance in mouse autoimmune models. Sci. Transl. Med. 13, eaaw9668 (2021).
at Xi'an Jiaotong University on July 11, 2021http://stm.sciencemag.org/Downloaded from

autoimmune models
Modular immune-homeostatic microparticles promote immune tolerance in mouse
WanJun Chen, Guozhen Liu, Yongkang Bai, Shiyu Liu and Yan Jin
Xin Chen, Xiaoshan Yang, Pingyun Yuan, Ronghua Jin, Lili Bao, Xinyu Qiu, Siying Liu, Tao Liu, John Justin Gooding,
DOI: 10.1126/scitranslmed.aaw9668
, eaaw9668.13Sci Transl Med
feasible approach for treating autoimmunity.
neuroinflammation. Together, these findings suggest that specific targeting of pathogenic immune cells is a
regulatory T cells. These microparticles reduced disease burden in mouse models of colitis, diabetes, and
. generated microparticles that induced pathogenic T cell death while expanding autoantigen-specificet al
pathogenic, disease-inducing immune cells while leaving the rest of the immune system intact. In this study, Chen
individuals with autoimmunity at increased risk of infection. Thus, there is a need for strategies to deplete only
Current treatment options for many autoimmune conditions rely on systemic immune suppression, putting
Augmenting autoimmunity
ARTICLE TOOLS http://stm.sciencemag.org/content/13/584/eaaw9668
MATERIALS
SUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2021/03/08/13.584.eaaw9668.DC1
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