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Abstract and Figures

Type 1 diabetes (T1D) is a metabolic disease caused by the autoimmune destruction of insulin-producing β-cells. With its incidence increasing worldwide, to find a safe approach to permanently cease autoimmunity and allow β-cell recovery has become vital. Relying on the inherent ability of apoptotic cells to induce immunological tolerance, we demonstrated that liposomes mimicking apoptotic β-cells arrested autoimmunity to β-cells and prevented experimental T1D through tolerogenic dendritic cell (DC) generation. These liposomes contained phosphatidylserine (PS)—the main signal of the apoptotic cell membrane—and β-cell autoantigens. To move toward a clinical application, PS-liposomes with optimum size and composition for phagocytosis were loaded with human insulin peptides and tested on DCs from patients with T1D and control age-related subjects. PS accelerated phagocytosis of liposomes with a dynamic typical of apoptotic cell clearance, preserving DCs viability. After PS-liposomes phagocytosis, the expression pattern of molecules involved in efferocytosis, antigen presentation, immunoregulation, and activation in DCs concurred with a tolerogenic functionality, both in patients and control subjects. Furthermore, DCs exposed to PS-liposomes displayed decreased ability to stimulate autologous T cell proliferation. Moreover, transcriptional changes in DCs from patients with T1D after PS-liposomes phagocytosis pointed to an immunoregulatory prolife. Bioinformatics analysis showed 233 differentially expressed genes. Genes involved in antigen presentation were downregulated, whereas genes pertaining to tolerogenic/anti-inflammatory pathways were mostly upregulated. In conclusion, PS-liposomes phagocytosis mimics efferocytosis and leads to phenotypic and functional changes in human DCs, which are accountable for tolerance induction. The herein reported results reinforce the potential of this novel immunotherapy to re-establish immunological tolerance, opening the door to new therapeutic approaches in the field of autoimmunity.
| Liposomes are efficiently phagocyted by dendritic cells (DCs) and preserve a high viability. (a) Uptake of liposomes fluorescently labeled with lipid-conjugated fluorescent dye Oregon Green 488 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine by DCs. Upper panel: time course of the capture of fluorescently-labeled PS-liposomes by DCs obtained from control subjects (white circles, n = 5) and patients with type 1 diabetes (T1D) (black circles, n = 10) at 37°C (continuous line) and at 4°C (discontinuous line). Results are mean ± SEM. Comparisons between phagocytosis by control subjects DCs at 37 and 4°C showed significant differences [ ++++ p < 0.0001, two-way analysis of variance (ANOVA)]; also, significant differences were found when comparing phagocytosis in patients with T1D at 37 and 4°C (***p < 0.001, ****p < 0.0001, Two-way ANOVA). No differences were found when comparing PS-liposomes uptake by DCs from control subjects and patients with T1D (Two-way ANOVA). Lower panel: time course of the capture of fluorescently-labeled PC-liposomes by DCs obtained from control subjects (white squares, n = 6) and patients with T1D (black squares, n = 9) at 37°C (continuous line) and at 4°C (discontinuous line). Results are mean ± SEM. Comparisons between phagocytosis by control subjects DCs at 37 and 4°C showed significant differences ( ++++ p < 0.0001, Two-way ANOVA); also, significant differences were found when comparing phagocytosis in patients with T1D at 37 and 4°C (**p < 0.01, ****p < 0.0001, Two-way ANOVA). No differences were found when comparing PC-liposomes uptake by DCs from control subjects and patients with T1D (Two-way ANOVA). (b) Viability of DCs from control subjects (upper panel, white symbols, n ≥ 6) and patients with T1D (lower panel, black symbols, n ≥ 6) assessed by annexin V and 7aad staining. Triangles represent immature DCs (iDCs), circles represent iDCs cultured with PSA-liposomes and PSB-liposomes (PSAB-DCs), squares represent mature PSAB-DCs (mPSAB-DCs) and upside-down triangles represent mature DCs (mDCs). MDCs were induced by culture with cytokine cocktail.
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February 2018 | Volume 9 | Article 2531
ORIGINAL RESEARCH
published: 14 February 2018
doi: 10.3389/mmu.2018.00253
Frontiers in Immunology | www.frontiersin.org
Edited by:
John Isaacs,
Newcastle University,
United Kingdom
Reviewed by:
Bert A. ’T Hart,
Biomedical Primate Research
Center, Netherlands
Elisabetta Padovan,
University Hospital of Basel,
Switzerland
*Correspondence:
Marta Vives-Pi
mvives@igtp.cat
Specialty section:
This article was submitted
to Immunological Tolerance
and Regulation,
a section of the journal
Frontiers in Immunology
Received: 26September2017
Accepted: 29January2018
Published: 14February2018
Citation:
Rodriguez-FernandezS, Pujol-
AutonellI, BriansoF, Perna-BarrullD,
Cano-SarabiaM, Garcia-JimenoS,
VillalbaA, SanchezA, AguileraE,
VazquezF, VerdaguerJ, MaspochD
and Vives-PiM (2018)
Phosphatidylserine-Liposomes
Promote Tolerogenic Features on
Dendritic Cells in Human Type 1
Diabetes by Apoptotic Mimicry.
Front. Immunol. 9:253.
doi: 10.3389/mmu.2018.00253
Phosphatidylserine-Liposomes
Promote Tolerogenic Features on
Dendritic Cells in Human Type 1
Diabetes by Apoptotic Mimicry
Silvia Rodriguez-Fernandez1, Irma Pujol-Autonell1, Ferran Brianso2,3, David Perna-Barrull1,
Mary Cano-Sarabia 4, Sonia Garcia-Jimeno4, Adrian Villalba1, Alex Sanchez2,3,
Eva Aguilera5, Federico Vazquez5, Joan Verdaguer6,7, Daniel Maspoch4,8
and Marta Vives-Pi1,7*
1 Immunology Section, Germans Trias i Pujol Research Institute, Autonomous University of Barcelona, Badalona, Spain,
2 Statistics and Bioinformatics Unit, Vall d’Hebron Research Institute, Barcelona, Spain, 3 Department of Genetics,
Microbiology and Statistics, University of Barcelona, Barcelona, Spain, 4 Catalan Institute of Nanoscience and
Nanotechnology, CSIC and The Barcelona Institute of Science and Technology, Bellaterra, Spain, 5 Endocrinology Section,
Germans Trias i Pujol University Hospital, Badalona, Spain, 6 Department of Experimental Medicine, University of Lleida &
IRBLleida, Lleida, Spain, 7 CIBERDEM, ISCiii, Madrid, Spain, 8 ICREA, Barcelona, Spain
Type 1 diabetes (T1D) is a metabolic disease caused by the autoimmune destruction of
insulin-producing β-cells. With its incidence increasing worldwide, to nd a safe approach
to permanently cease autoimmunity and allow β-cell recovery has become vital. Relying
on the inherent ability of apoptotic cells to induce immunological tolerance, we demon-
strated that liposomes mimicking apoptotic β-cells arrested autoimmunity to β-cells and
prevented experimental T1D through tolerogenic dendritic cell (DC) generation. These
liposomes contained phosphatidylserine (PS)—the main signal of the apoptotic cell mem-
brane—and β-cell autoantigens. To move toward a clinical application, PS-liposomes
with optimum size and composition for phagocytosis were loaded with human insulin
peptides and tested on DCs from patients with T1D and control age-related subjects.
PS accelerated phagocytosis of liposomes with a dynamic typical of apoptotic cell
clearance, preserving DCs viability. After PS-liposomes phagocytosis, the expression
pattern of molecules involved in efferocytosis, antigen presentation, immunoregulation,
and activation in DCs concurred with a tolerogenic functionality, both in patients and
control subjects. Furthermore, DCs exposed to PS-liposomes displayed decreased ability
to stimulate autologous Tcell proliferation. Moreover, transcriptional changes in DCs from
patients with T1D after PS-liposomes phagocytosis pointed to an immunoregulatory pro-
life. Bioinformatics analysis showed 233 differentially expressed genes. Genes involved
in antigen presentation were downregulated, whereas genes pertaining to tolerogenic/
anti-inammatory pathways were mostly upregulated. In conclusion, PS-liposomes
phagocytosis mimics efferocytosis and leads to phenotypic and functional changes in
human DCs, which are accountable for tolerance induction. The herein reported results
reinforce the potential of this novel immunotherapy to re-establish immunological toler-
ance, opening the door to new therapeutic approaches in the eld of autoimmunity.
Keywords: immunotherapy, autoimmunity, human type 1 diabetes, liposomes, tolerance, dendritic cells
Abbreviations: DC, dendritic cell; PS, phosphatidylserine; T1D, type 1 diabetes.
2
Rodriguez-Fernandez et al. Liposomes for Human Autoimmune Diabetes
Frontiers in Immunology | www.frontiersin.org February 2018 | Volume 9 | Article 253
INTRODUCTION
Type 1 diabetes (T1D) mellitus is a metabolic disease caused by
loss of tolerance to self and consequent autoimmune destruction
of insulin-producing pancreatic β-cells (1). When β-cell mass
decreases signicantly, the individual’s endogenous production
of insulin is no longer able to meet metabolic demands, leading to
overt hyperglycemia. Upon diagnosis, patients with T1D require
exogenous insulin administration, and although this treatment
has allowed them to survive, long-term complications due to gly-
cemic imbalances are bound to arise (2, 3). T1D usually appears
during childhood or adolescence, and its incidence is increasing
an average of 4% per year (4). Despite knowing that both genetic
and environmental factors contribute to its development, trig-
gering events remain elusive. e autoimmune attack against
β-cells is led by a mild leukocytic inltrate—insulitis—consisting
of dendritic cells (DCs), macrophages, T and B lymphocytes, and
natural killer cells, which gradually advance through the islets
(5). e rst islet-inltrating cells are DCs (6), which orchestrate
the loss of tolerance to β-cell autoantigens, insulin being a key
autoantigen in human T1D (7).
e rst step to revert T1D would be to arrest the pathologi-
cal recognition of β-cell autoantigens. Many immunotherapies
have prevented and even reverted T1D in animal models (8), but
clinical trials have corroborated how challenging T1D preven-
tion and reversion is, and most of them have been unsuccessful
(9). In this scenario, the development of new therapies to halt
autoimmunity in T1D has become an urgent biomedical matter.
An ideal immunotherapy should restore tolerance to β-cells,
avoiding systemic side eects, and allow islet regeneration. One
of the most ecient physiological mechanisms for inducing
tolerance is apoptosis, a form of programmed cell death lack-
ing inammation. e uptake of apoptotic cells by professional
phagocytes such as macrophages and immature DCs (iDCs) is
named eerocytosis (10). e exposure of “eat-me” signals on
the apoptotic cell surface is what promotes their specic recogni-
tion and phagocytosis. Phosphatidylserine (PS), a phospholipid
usually kept in the inner leaet of the plasma membrane, is a
relevant signal for eerocytosis. is molecule is recognized by
multiple distinct receptors on antigen presenting cells, including
members of the TIM family, Stabilin-2, integrins, CD36, CD68,
among others, as well as by soluble receptors that in turn bind to
membrane receptors (11). Aer the capture, the apoptotic cell
is processed, thus prompting the release of anti-inammatory
signals and presentation of autoantigens in a tolerogenic manner
by DCs (12). Failure of this mechanism, owing to an increase of
apoptotic β-cells or defects in eerocytosis, contributes to the loss
of tolerance to self in the context of T1D (13).
Our group demonstrated that DCs acquired a tolerogenic phe-
notype and functionality aer engulfment of apoptotic β-cells,
and that they prevented T1D when transferred to non-obese dia-
betic (NOD) mice (14, 15). However, since nding a substantial
source of autologous apoptotic β-cells for T1D immunotherapy
would be impossible, we conceived an immunotherapy based
on biomimicry that consisted of liposomes—phospholipid
bilayer vesicles—displaying PS in their surface and containing
autoantigenic peptides, thus resembling apoptotic cells. Indeed,
apoptotic mimicry performed by PS-liposomes successfully
restored tolerance to β-cells in experimental autoimmune diabe-
tes, preventing disease development and decreasing the severity
of insulitis (16). Moreover, by only replacing the autoantigenic
peptide encapsulated within PS-liposomes, we conrmed the
potential of this immunotherapy to prevent and ameliorate
experimental autoimmune encephalomyelitis, the experimental
model of multiple sclerosis (17). In both cases, we demonstrated
that phagocytosis of autoantigen-loaded PS-liposomes induced
a tolerogenic phenotype and functionality in DCs, expansion of
regulatory Tcells and release of anti-inammatory mediators
that are responsible for arresting the autoimmune attack to
target cells. erefore, PS-liposomes could constitute a platform
serving as a physiological and safe strategy to restore peripheral
tolerance in antigen-specic autoimmune diseases. Liposomes,
already used clinically as drugs deliverers for antitumor drugs
and as vaccines (18), have the advantage of being safe and
biocompatible, customizable, easily large-scale produced, and
standardizable.
Aiming for the clinical potential of this strategy, we have
encapsulated human insulin peptides to assess the eect of
PS-liposomes in human DCs from patients with T1D and
control subjects invitro. We herein report that PS-liposomes are
eciently captured by human DCs, thus eliciting transcriptomic,
phenotypic, and functional changes that point to tolerogenic
potential. is immunotherapy constitutes a promising strategy
to arrest autoimmune aggression in human T1D, beneting from
the co-delivery of tolerogenic signals and β-cell autoantigens.
MATERIALS AND METHODS
Patients
Patients with T1D (n=34) and control subjects (n=24) were
included in this study. All patients with diabetes fullled the clas-
sication criteria for T1D. Inclusion criteria were 18–55years of
age, a body mass index (BMI) between 18.5 and 30kg/m2 and,
for patients with T1D, an evolution of the disease longer than
6months. Exclusion criteria were: being under immunosuppres-
sive or anti-inammatory treatment, or undergoing pregnancy or
breastfeeding. For the RNA-sequencing (RNA-seq) experiment,
we selected 8 patients of the 34 that participated in the study,
but BMI was limited to a maximum of 24.9kg/m2, duration of
the disease was restricted to a maximum of 5years (in order to
minimize the eect that long-term hyperglycemia could have
on genetic and/or epigenetic proles) and the presence of other
chronic diseases became an exclusion criterion. All study partici-
pants gave informed consent, and the study was approved by the
Committee on the Ethics of Research of the Germans Trias i Pujol
Research Institute and Hospital.
Cell Separation and Generation of DCs
Peripheral blood mononuclear cells (PBMCs) were obtained
from 50ml blood samples of control subjects and patients with
T1D by means of Ficoll Paque (GE Healthcare, Marlborough,
MA, USA) density gradient centrifugation. Monocytes were
further magnetically isolated using the EasySep Human CD14
3
Rodriguez-Fernandez et al. Liposomes for Human Autoimmune Diabetes
Frontiers in Immunology | www.frontiersin.org February 2018 | Volume 9 | Article 253
Positive Selection Kit (STEMCELL Technologies, Vancouver, BC,
Canada) following the manufacturer’s instructions. Once CD14
purity in the positively selected fraction was >70%, monocytes
were cultured at a concentration of 106 cells/ml in X-VIVO
15 media (Lonza, Basel, Switzerland), supplemented with 2%
male AB human serum (Biowest, Nuaillé, France), 100 IU/ml
penicillin (Normon SA, Madrid, Spain), 100µg/ml streptomycin
(Laboratorio Reig Jofré, Sant Joan Despí, Spain), and 1,000IU/
ml IL-4 and 1,000 IU/ml GM-CSF (Prospec, Rehovot, Israel)
to obtain monocyte-derived DCs. Aer 6 days of culture, DC
dierentiation yield was assessed by CD11c-APC staining
(Immunotools, Friesoythe, Germany) and cell viability was
determined by annexin V-PE (Immunotools) and 7aad staining
(BD Biosciences, San Jose, CA, USA) using ow cytometry (FACS
Canto II, BD Biosciences). e negatively selected fraction of
PBMCs was cryopreserved in Fetal Bovine Serum (ermoFisher
Scientic, Waltham, MA, USA) with 10% dimethylsulfoxide
(Sigma-Aldrich, Saint Louis, MO, USA) and stored for later use.
Peptide Selection and Preparation of
Liposomes
inking in a future clinical application of liposomes, the two
chains of insulin were selected to be encapsulated separately in
order to avoid possible biological eects of insulin. A and B chains
of insulin contain well-known β-cell specic target epitopes in
human T1D (19). Peptide A corresponds to the whole human
insulin A chain (21 aa, N-start-GIVEQCCTSICSLYQLENYCN-
C-end), and peptide B is the whole human insulin B chain (30 aa,
N-start-FVNQHLCGSHLVEALYLVCGERGFFYTPKT-C-end)
(Genosphere Biotechnologies, Paris, France). Peptides were >95%
pure and triuoroacetic acid was removed. Liposomes consisted
of 1,2-dioleoyl-sn-glycero-3-phospho--serine (sodium salt)
(Lipoid, Steinhausen, Switzerland), 1,2-didodecanoyl-sn-glycero-
3-phosphocholine (Lipoid), and cholesterol (Sigma-Aldrich).
Liposomes were prepared using the thin lm hydration method
from a lipid mixture of 1,2-dioleoyl-sn-glycero-3-phospho-
-serine, 1,2-didodecanoyl-sn-glycero-3-phosphocholine and
cholesterol at 1:1:1.33 molar ratio, respectively, as described
(20). Liposomes without PS were generated as controls with
1,2-didodecanoyl-sn-glycero-3-phosphocholine and cholesterol
at 1:1 molar ratio. All liposomes were produced under sterile
conditions and at a nal concentration of 30mM. Lipids were
dissolved in chloroform and the solvent was removed by evapora-
tion under vacuum and nitrogen. e lipids were hydrated with
the appropriate buer (phosphate buered saline or 0.5mg/ml
solution of peptide A or peptide B) and the liposomes obtained
were homogenized to 1 µm by means of an extruder (Lipex
Biomembranes Inc., Vancouver, BC, Canada). Peptide encapsula-
tion eciencies were calculated according to the equation: encap-
sulation eciency (%)=[(Cpeptide,total-Cpeptide,out)/Cpeptide,total] ×100,
where Cpeptide,total is the initial peptide A or peptide B concentration
and Cpeptide,out is the concentration of non-encapsulated peptide.
To measure the Cpeptide,out, liposome suspensions were centrifuged
at 110,000g at 10°C for 30min. e concentration of non-encap-
sulated peptide was assessed in supernatants by PIERCE BCA
protein assay kit (ermoFisher Scientic). In addition to PS-rich
liposomes loaded with insulin peptides [PSA-liposomes (n=3)
and PSB-liposomes (n=3) encapsulating peptide A or peptide B,
respectively], uorescent-labeled liposomes with PS (empty uo-
rescent PS-liposomes, n=4) and without PS (empty uorescent
PC-liposomes, n=4) were also prepared using lipid-conjugated
uorescent dye Oregon Green 488 1,2-dihexadecanoyl-sn-
glycero-3-phosphoethanolamine (Invitrogen, Carlsbad, CA,
USA) and following the aforementioned methodology. Particle
size distributions and stability—expressed as zeta potential
(ζ)—were measured by dynamic light scattering using Malvern
Zetasizer (Malvern Instruments, Malvern, UK) in undiluted
samples. Liposome morphology and lamellarity were examined
by cryogenic transmission electron microscopy (cryo-TEM) in a
JEOL-JEM 1400 microscope (Jeol Ltd., Tokyo, Japan).
Phagocytosis Assay
To assess whether DCs were able to phagocyte liposomes, DCs
were co-cultured with 100µM of empty uorescent PS-liposomes
(n=5 for control subjects and n= 10 for patients with T1D)
or empty uorescent PC-liposomes (n=6 for control subjects
and n=9 for patients with T1D) at 37°C from 5min to 24h.
As control, the same assay was performed at 4°C to conrm that
liposomes were captured by an active mechanism of phagocytosis.
Cells were extensively washed in cold phosphate buered saline
to remove all liposomes attached to the cell membrane. Liposome
uptake was determined by ow cytometry (FACSCanto II, BD
Biosciences).
Assessment of DCs Phenotype after
Liposome Uptake
Although insulin chains were encapsulated separately, DCs
were stimulated by a mixture of PSA-liposomes (50%) and PSB-
liposomes (50%), in order to assess the eect of the whole insulin
molecule as autoantigen. us, DCs from control subjects (n5)
and patients with T1D (n8) were co-cultured with 1mM of
liposomes (PSAB-DCs) for 24h in the presence of 20µg/ml human
insulin (Sigma-Aldrich), and their viability and phenotype were
analyzed by ow cytometry (FACSCanto II, BD Biosciences). e
sample number stated (n) is referred to the minimum number
of control subjects and patients included in each experiment. As
controls, DCs were either cultured with 20µg/ml human insulin
(Sigma-Aldrich) to obtain iDCs or adding a cytokine cocktail
(CC) consisting of tumor necrosis factor (TNF)α (1,000 IU/
ml, Immunotools), IL-1β (2,000 IU/ml, Immunotools) and
Prostaglandin E2 (PGE2, 1µM, Cayman Chemical, Ann Arbor,
MI, USA) for 24 h to obtain mature DCs (mDC). Moreover,
PSAB-DCs were cultured aer phagocytosis with CC for 24h in
order to assess the response in front a pro-inammatory stimulus
(mPSAB-DCs). Phenotyping was performed as follows: DCs were
stained with 7aad (BD Biosciences) and monoclonal antibodies
to CD11c-APC, CD25-PE, CD86-FITC, HLA class I-FITC,
HLA class II-FITC, CD14-PE and CD40-APC (Immunotools),
CD36-APCCy7, TIM4-APC, αvβ5 integrin-PE, CD54-PECy7,
TLR2-FITC, CXCR4-APCCy7, CCR2-APC, DC-SIGN-APC
(Biolegend, San Diego, CA, USA) and CCR7-PECy7 (BD
Biosciences). Corresponding uorescence minus one staining
was used as control. Data were analyzed using FlowJo soware
(Tree Star Inc., Ashland, OR, USA).
4
Rodriguez-Fernandez et al. Liposomes for Human Autoimmune Diabetes
Frontiers in Immunology | www.frontiersin.org February 2018 | Volume 9 | Article 253
T Cell Proliferation Assays
Autologous Tlymphocyte proliferation (n=12 for control sub-
jects and n12 for patients with T1D) was assessed by exposing
PBMCs to the dierent conditions of DCs used in this study.
Briey, PBMCs from the same donor were thawed and stained
with 0.31 µM CellTrace Violet (21) (ermoFisher Scientic)
according to manufacturer’s instructions. e PBMCs were then
co-cultured with iDCs, PSAB-DCs, mPSAB-DCs or mDCs at
a 10:1 ratio (105 PBMCs:104 DCs). For each donor, 105 PBMCs
were cultured in basal conditions as a negative control or with
Phorbol 12-Myristate 13-Acetate (50ng/ml, Sigma-Aldrich) and
Ionomycin (500 ng/ml, Sigma-Aldrich) as a positive control.
Aer 6days of co-culture, proliferation was assessed in the dif-
ferent Tcell subsets with CD3-PE, CD4-APC and CD8-FITC
staining (Immunotools) by ow cytometry (FACS LSR Fortessa,
BD Biosciences). Data were analyzed using FlowJo soware (Tree
Star Inc.).
Cytokine Production
e Human 1/2/17 kit (CBA system; BD Biosciences)
was used to assess cytokine production. Culture supernatants
from DCs and from Tcell proliferation assays (n3 for control
subjects and n3 for patients with T1D) were collected and fro-
zen at 80°C until use. IL-2, IL-4, IL-6, IFN-γ, TNF, IL-17A, and
IL-10 were measured. Data were analyzed using CBA soware.
e production of Human TGF-β1 by DCs aer PSAB-liposome
uptake was determined by ELISA (eBioscience, San Diego, CA,
USA).
RNA-Seq of DCs before and after
Liposome Phagocytosis
Dendritic cells obtained from patients with T1D (n=8) were
cultured in basal conditions (iDCs) or with 1 mM of PSA-
liposomes and PSB-liposomes (PSAB-DCs) at 37°C for 4h. Cells
were then harvested from culture wells using Accutase (eBiosci-
ence), and viability and DC purity were assessed with 7aad (BD
Biosciences), annexin V-PE and CD11c-APC (Immunotools)
staining by ow cytometry (FACS Canto II, BD Biosciences).
Liposome capture control assays were performed for every sample
(see above Phagocytosis Assay section) to conrm phagocytosis.
Supernatant was removed and cell pellets were stored at 80°C
until use. RNA was extracted using RNeasy Micro Kit (QIAGEN,
Hilden, Germany) and following manufacturer’s instructions.
RNA purity, integrity and concentration were determined
by NanoDrop (ND-1000 Spectrophotometer, ermoFisher
Scientic) and 2100 Bioanalyzer (Agilent Technologies Inc.,
Santa Clara, CA, USA). Aerward, 1µg of total RNA was used to
prepare RNA libraries following the instructions of the NebNext
Ultra Directional RNA Library Prep Kit (New England Biolabs,
Ipswich, MA, USA). Library quality controls were assessed using
a TapeStation 2200 (Agilent High Sensitivity Screen Tape) and a
narrow distribution with a peak size of approximately 300bp was
observed in all cases. Libraries were quantied by qPCR using a
QC KAPA kit (Homan-LaRoche, Basel, Switzerland) sequenced
in a NextSeq 500 genetic analyzer (SBS-based sequencing
technology, Illumina, San Diego, CA, USA) in a run of 2× 75
cycles and a high output sequencing mode. Twenty million reads
were obtained and analyzed for each sample. Fastq les coming
from Illumina platform were merged and basic quality controls
were performed with FASTQC and PRINSEQ tools. Paired-end
(forward-reverse) sample merging was carried out with soware
CLCBio Genomics Workbench® version 8.5 (22), following the
RNA-seq analysis pipeline found in CLCBio manuals. Read align-
ment and mapping steps to only gene regions were performed
using CLCBio soware against the human genome (Homo sapi-
ens GRCh38 assembly, at both gene- and transcript-level tracks).
e same soware, with default options, was used to normalize
counts by applying standard “Reads Per Kilobase of transcript
per Million reads mapped” method. e remaining steps of the
analysis were carried out with scripts and pipelines implemented
with R soware (23). e selection of dierentially expressed
genes (DEGs) was performed using the linear model approach
implemented in the limma Bioconductor package (24), with
previous log2-transformation of the normalized data. Adjusted
p values of 0.125, taking into account multiple testing with
the False Discovery Rate method, were considered signicant.
erefore, genes with a p value <0.0013 and Log2 of fold change
>0.05 were considered upregulated, whereas those with Log2 of
fold change <0.05 were considered downregulated. Experimental
data have been uploaded into European Nucleotide Archive (EBI,
https://www.ebi.ac.uk/ena; accession number: PRJEB22240).
DEGs were categorized using Ingenuity Pathway Analysis
Soware (QIAGEN), Protein Analysis rough Evolutionary
Relationships Classication System (25), REACTOME Pathway
database (26) and Gene Ontology Biological Process database
(27). Furthermore, R soware (23) was used to generate a gene
heatmap of DEGs.
Quantitative RT-PCR
To validate transcriptome results, DCs obtained from patients
with T1D (n4) and control subjects (n3) were cultured and
pelleted in three conditions: iDCs, PSAB-DCs and mDCs. RNA
was isolated using RNeasy Micro Kit (QIAGEN), and was reverse-
transcribed with a High Capacity cDNA Reverse Transcription
Kit (ermoFisher Scientic). cDNA synthesis reactions were
carried out using random hexamers (0.5mg/ml, BioTools, Valle
de Tobalina, Madrid, Spain) and reverse transcriptase Moloney-
murine-Leukemia-virus (200 U/ml, Promega, Madison, WI,
USA). Quantitative RT-PCR assays were performed with TaqMan
universal assay (ermoFisher Scientic) on a LightCycler® 480
(Roche, Mannheim, Germany) using the following TaqMan
assays: CYTH4 (Hs01047905_m1), GIMAP4 (Hs01032964_m1),
HPGD (Hs00960590_m1), NFKB inhibitor alpha (NFKBIA)
(Hs00153283_m1), PL AU R (Hs00958880_m1), TNFAIP3
(Hs00234713_m1), tumor necrosis factor superfamily member 14
(TNFSF14) (Hs00542477_m1), and VEGFA (Hs00900055_m1).
Relative quantication was performed by normalizing the expres-
sion for each gene of interest to that of the housekeeping gene
GAPDH (Hs02758991_g1), as described in the 2ΔCt method (28).
Statistical Analysis
e statistical analysis was performed using Prism 7.0 soware
(GraphPad soware Inc., San Diego, CA, USA). Analysis of
TABLE 1 | Data from the control subjects and patients with T1D recruited for the
study.
Control Subjects Patients with T1D p Value
N24 34 —
Gender 11/24 (45.8%) Female 17/34 (50%) Female
13/24 (54.2%) Male 17/34 (50%) Male
Age (years) 30.46±8.18 32.54±8.96 0.4301
BMI (kg/m2) 23.90±2.87 23.80±3.11 0.9075
Age at T1D diagnosis
(years)
NA 20.79±9.90 —
Duration of T1D (years) NA 11.75±9.70 —
HbA1c (%) NA 7.66±1.26 —
Data presented as mean±SD; p value calculated from Mann–Whitney test.
T1D: type 1 diabetes; BMI, body mass index; NA, not applicable.
TABLE 2 | Data from the patients with T1D included in the RNA-seq experiment.
Patient number Gender Age (years) BMI (kg/m2) Age at T1D diagnosis (years) Duration of T1D (years) HbA1c (%)
1 Male 23 21.2 19 4 6.7
2 Female 28 24.4 23 5 6.5
3 Male 28 23.0 25 3 7.4
4 Female 33 21.4 33 0.5 5.9
5 Female 35 24.4 34 1 6.4
6 Female 32 18.6 31 0.5 5.9
7 Male 38 24.2 36 1 12.2
8 Male 21 23.0 16 5 7.9
Mean±SD 29.75±5.85 22.50±2.00 27.13±7.43 2.50±1.98 7.36±2.07
Data presented as mean±SD.
T1D, type 1 diabetes; BMI, body mass index.
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variance (ANOVA) was used for comparisons with several
factors. For comparisons of unpaired data, a non-parametric
Mann-Whitney test was used; for paired comparisons, a non-
parametric Wilcoxon test was used. A p value0.05 was con-
sidered signicant.
RESULTS
Patients with T1D and Control Subjects
Display Similar Features
irty-four patients with T1D (50% female, 50% male) from the
Germans Trias i Pujol Hospital and 24 control subjects (45.8%
female, 54.2% male) met the inclusion and exclusion criteria
and were included in the study (Table1). Age of control subjects
was 30.46±8.18years (mean±SD), while that of patients with
T1D was 32.54±8.96 years; BMI was 23.90 ±2.87kg/m2 and
23.80 ± 3.11 kg/m2, respectively. Patients with T1D had been
diagnosed at 20.79 ± 9.90 years, had a duration of disease of
11.75±9.70years, and a hemoglobin A1c level of 7.66±1.26%.
Control subjects did not signicantly dier from patients with
T1D in terms of age or BMI. Within the 34 patients, we selected
8 for the RNA-seq analysis, 50% female and 50% male, with a
more stringent inclusion and exclusion criteria. eir age was
29.75±5.85years and their BMI was 22.50±2.00kg/m2. ey
had been diagnosed with T1D at 27.13 ± 7.43 years, had a
duration of the disease of 2.50± 1.98 years and a hemoglobin
A1c level of 7.36±2.07%. Specic information on each subject
can be found in Table2.
DC Differentiation Efciency Is Similar in
Patients with T1D and Control Subjects
Monocytes were isolated magnetically from PBMCs. e yield
of monocyte isolation—calculated as the percentage of the
absolute number of CD14+ cells in the positively isolated frac-
tion related to the absolute number of CD14+ cells in PBMCs—
was 54.95 ± 24.97% (mean ± SD) for control subjects and
56.62±18.12% for patients with T1D. e percentage of purity
of CD14+ cells in the isolated fraction was 80.59±10.18% for
control subjects and 79.33±7.56% for patients, and viability was
95.06±4.14 and 94.92± 3.60%, respectively. e eciency of
dierentiation to DCs at day 6 was 87.96 ± 6.61% for control
subjects and 86.92± 6.86% for patients. No statistically signi-
cant dierences were found when comparing these parameters
between both groups. Data are detailed in Table3.
PS-Liposomes Show Multivesicular
Vesicle Morphology and Encapsulate
Insulin Peptides
Liposomes were characterized in terms of diameter, polydis-
persity index (PdI), surface charge (ζ-potential) and eciency
of peptide encapsulation (Tab le 4). All liposomes had a nal
lipid concentration of 30 mM. All liposomes were large to
guarantee ecient phagocytosis, displaying a diameter supe-
rior to 690nm. e presence of PS molecules in liposomes was
conrmed by the negative charge measured at the liposome
surface by ζ-potential (38 mV). Regarding specic features
of PSA-liposomes (n=3), the mean diameter was 690±29nm
(mean±SD), the PdI was 0.40±0.28 and the ζ-potential was
38.57±6.76 mV. e mean of peptide A (human insulin A
chain) encapsulation eciency was 39.74±22.10%. As for PSB-
liposomes (n=3), they had a mean diameter of 788±264nm,
the PdI was 0.52±0.42 and the ζ-potential was 37.50±7.16
mV, and the mean of peptide B (human insulin B chain) encap-
sulation eciency was 93.19±0.92%. Dierences in peptide
encapsulation eciency (PSA vs. PSB) are due to amino acid
composition and dierent solubility of insulin chains A (21
FIGURE 1 | PS-liposomes display multivesicular and multilamellar morphology. Cryogenic transmission electron microscopy images of (A) PSA-liposomes (left) and
(B) PSB-liposomes (right). Bar=0.2μm.
TABLE 4 | Features of the liposomes used in the study.
Liposome type Diameter (nm) Polydispersity index ζ-potential (mV) Encapsulation efciency (%)
PSA-liposomes 690±29 0.40±0.28 38.57±6.76 39.74±22.10
PSB-liposomes 788±264 0.52±0.42 37.50±7.16 93.19±0.92
Fluorescent PS-liposomes 836±217 0.32±0.06 38.90±2.52 (empty)
Fluorescent PC-liposomes 1665±488 0.32±0.09 7.60±2.68 (empty)
Data presented as mean±SD.
TABLE 3 | Data from monocyte’s isolation and dendritic cell differentiation.
Yield
(day 1, %)
Purity
(day 1, %)
Viability
(day 1, %)
Differentiation
efciency
(day 6, %)
Control
subjects
54.95±24.97 80.59±10.18 95.06±4.14 87.96±6.61
Patients 56.62±18.12 79.33±7.56 94.92±3.60 86.92±6.86
p Value 0.3789 0.2286 0.3664 0.5766
Yield: % of the absolute number of CD14+ cells in the positively isolated fraction related
to the absolute number of CD14+ cells in PBMCs. Purity: % of CD14+ cells in the isolated
fraction. Viability: % of Annexin V7aad cells. Differentiation efciency: % of CD11c+ cells.
Data presented as mean±SD; p value calculated from Mann–Whitney test.
T1D, type 1 diabetes.
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Human DCs Display Optimal Kinetics of
PS-Liposomes Phagocytosis without
Affecting Viability
A time course analysis was performed to determine PS-liposomes
uptake kinetics (Figure2A, upper le panel). e capture of empty
uorescent PS-liposomes by DCs was signicantly higher at 37°C
when compared to 4°C (p<0.001), coming from either control
subjects (n=5) or patients (n=10). is result is immunologi-
cally crucial and demonstrates that DCs engulf liposomes by an
active mechanism of phagocytosis. PS-liposomes uptake kinetics
were identical between control subjects and patients with T1D.
To indirectly assess the role of PS in phagocytosis, the
same analysis was performed replacing PS-liposomes with
PC-liposomes (Figure2A, lower le panel). e percentages of
empty PC-liposomes phagocytosis by DCs from control subjects
(n= 6) and patients (n=9) were signicantly higher at 37°C
when compared to 4°C starting at 2h (p<0.0001). e kinetics of
the capture did not dier between control subjects and patients.
When comparing uptake kinetics of PS- and PC-liposomes
(Figure S1 in Supplementary Material), statistically signicant dif-
ferences were found, as expected. e presence of PS signicantly
accelerated phagocytosis in the rst 2h of co-culture (p<0.05)
both in control subjects and patients. In preliminary experiments,
each type of liposome was tested in several sizes (diameter range
505–2,138nm), and similar kinetics of capture were observed,
aa) and B (30 aa) in phosphate buered saline media. B chain
is more positively charged than A chain at neutral pH, result-
ing in a higher encapsulation eciency in negatively charged
liposomes. PSA-liposomes and PSB-liposomes presented
multivesicular vesicle morphology when cryo-TEM analysis
was performed (Figure1).
Fluorescent-labeled PS-liposomes (n= 4) showed a diam-
eter of 836 ± 217 nm, a PdI of 0.32± 0.06 and a ζ-potential
of 38.90 ± 2.52 mV. eir PS-free counterparts, uorescent
PC-liposomes (n=4), had a diameter of 1665±488nm, a PdI
of 0.32±0.09 and a ζ-potential of 7.60±2.68 mV (Table4).
FIGURE 2 | Liposomes are efciently phagocyted by dendritic cells (DCs) and preserve a high viability. (A) Uptake of liposomes uorescently labeled with
lipid-conjugated uorescent dye Oregon Green 488 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine by DCs. Upper panel: time course of the capture of
uorescently-labeled PS-liposomes by DCs obtained from control subjects (white circles, n=5) and patients with type 1 diabetes (T1D) (black circles, n=10) at
37°C (continuous line) and at 4°C (discontinuous line). Results are mean±SEM. Comparisons between phagocytosis by control subjects DCs at 37 and 4°C
showed signicant differences [++++p<0.0001, two-way analysis of variance (ANOVA)]; also, signicant differences were found when comparing phagocytosis in
patients with T1D at 37 and 4°C (***p<0.001, ****p<0.0001, Two-way ANOVA). No differences were found when comparing PS-liposomes uptake by DCs from
control subjects and patients with T1D (Two-way ANOVA). Lower panel: time course of the capture of uorescently-labeled PC-liposomes by DCs obtained from
control subjects (white squares, n=6) and patients with T1D (black squares, n=9) at 37°C (continuous line) and at 4°C (discontinuous line). Results are
mean±SEM. Comparisons between phagocytosis by control subjects DCs at 37 and 4°C showed signicant differences (++++p<0.0001, Two-way ANOVA); also,
signicant differences were found when comparing phagocytosis in patients with T1D at 37 and 4°C (**p<0.01, ****p<0.0001, Two-way ANOVA). No differences
were found when comparing PC-liposomes uptake by DCs from control subjects and patients with T1D (Two-way ANOVA). (B) Viability of DCs from control subjects
(upper panel, white symbols, n6) and patients with T1D (lower panel, black symbols, n6) assessed by annexin V and 7aad staining. Triangles represent
immature DCs (iDCs), circles represent iDCs cultured with PSA-liposomes and PSB-liposomes (PSAB-DCs), squares represent mature PSAB-DCs (mPSAB-DCs)
and upside-down triangles represent mature DCs (mDCs). MDCs were induced by culture with cytokine cocktail.
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independently of liposome size (Figure S2 in Supplementary
Material), thus conrming that PS is the key factor in accelerating
phagocytosis.
e viability of the dierent conditions of DCs (iDCs, PSAB-
DCs, mPSAB-DCs, and mDCs) was assessed to determine
liposome toxicity. e mean viability for each condition was
always >90%, both in DCs obtained from control subjects (n6)
(Figure2B, upper right panel), and patients with T1D (n6)
(Figure2B, lower right panel).
PS-Liposomes Uptake Regulates the
Phenotypic Maturation of Human DCs
Changes in DCs phenotype were determined in control subjects
(n5) and patients with T1D (n8). e membrane molecules
assessed were: PS-receptors (CD36, TIM4, and αvβ5 integrin),
antigen-presentation molecules (HLA-ABC and HLA-DR),
adhesion molecules (CD54), costimulation molecules (CD40
and CD86), activation molecules (CD25), chemokine receptors
(CCR7, CCR2, and CXCR4), and pattern recognition receptors
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(TLR2, CD14, and DC-SIGN). Figure3 shows the relative Median
of Fluorescence Intensity referred to mDCs.
PS-receptors CD36, TIM4 and αvβ5 integrin were expressed
in iDCs. Aer liposome uptake, PSAB-DCs from patients
decreased CD36 expression (p<0.05) and upregulated TIM4
expression (p<0.05) in comparison to iDCs, but PSAB-DCs
presented a higher expression of CD36 and TIM4 than mDCs
from control subjects and patients (p<0.05). Moreover, PSAB-
DCs from patients had increased levels of TIM4 in comparison
to PSAB-DCs from control subjects (p<0.05). e expression of
αvβ5 integrin was higher in PSAB-DCs than in mDCs in patients
(p<0.05). As expected, CD36 and TIM4 were downmodulated in
mDCs (p<0.05), and αvβ5 integrin showed the same tendency.
Regarding HLA molecules, HLA-ABC was expressed similarly
in iDCs and mDCs from both groups, and decreased in PSAB-
DCs from patients aer liposome capture (p<0.05) —and con-
trol subjects displayed the same tendency. Concerning HLA-DR,
iDCs showed a lower expression of this marker when compared
to mDCs (p<0.001). Aer liposome phagocytosis (PSAB-DCs),
the low HLA-DR levels were preserved. As for the expression of
adhesion molecule CD54, it was lower in iDCs from patients in
comparison to mDCs (p<0.05), and control subjects displayed
the same tendency. Aer liposome uptake, no changes in CD54
expression were observed in PSAB-DCs when compared to iDCs,
but mDCs displayed increased levels of CD54 in comparison
to PSAB-DCs (p< 0.05). e expression of CD54 was higher
in mPSAB-DCs exposed to a maturation stimulus despite the
uptake of liposomes (p<0.05).
Expression of costimulatory molecules CD40 and CD86 was
lower in iDCs than in mDCs (p<0.01). Liposome phagocytosis
did not increase the expression of these molecules in PSAB-DCs.
Moreover, PSAB-DCs presented lower levels of these markers
when compared to mDCs (p<0.0001), and even when exposed
to pro-inammatory stimulus (mPSAB-DCs) in comparison to
mDCs (p<0.05). Regarding the expression of activation marker
CD25, it was lower in iDCs when compared to mDCs (p<0.0001).
Upregulation of CD25 was observed aer liposome uptake
in PSAB-DCs from control subjects (p<0.01), but remained
unaltered in patients. PSAB-DCs from both groups presented
CD25 downmodulated when compared to mDCs (p< 0.001).
Furthermore, DCs loaded with liposomes and exposed to pro-
inammatory stimulus (mPSAB-DCs) displayed lower levels of
CD25 than mDCs in patients with T1D (p<0.01).
Chemokine receptors CCR7 and CCR2 were expressed in
iDCs. Aer liposome capture, the expression of both molecules
increased in patients with T1D (p<0.05). CCR7 was upregulated
in PSAB-DCs when compared to mDCs in patients (p<0.05),
and control subjects displayed the same tendency. CXCR4,
overexpressed in mDCs in comparison to iDCs (p<0.05), was
maintained low aer liposome engulfment (PSAB-DCs). e
expression of CXCR4 was higher in DCs exposed to a matura-
tion stimulus despite the uptake of liposomes (mPSAB-DCs)
(p<0.05).
Pattern recognition receptors were assessed in DCs. TLR2
expression was similar in all experimental conditions, despite
showing a tendency to increase aer liposome phagocytosis
(PSAB-DCs) in patients. CD14 was similarly expressed in iDCs
and mDCs, but liposome uptake and maturation stimulus induced
downregulation of this marker (p<0.05). DC-SIGN, expressed in
iDCs, displayed a tendency to be downmodulated aer liposome
capture (PSAB-DCs), especially in controls, which was more
marked aer a pro-inammatory stimulus. Nonetheless, this
marker showed a tendency to remain higher in PSAB-DCs than
in mDCs in patients.
In terms of cytokine secretion by DCs from patients (n3)
and control subjects (n 3) (Figure 4), IL-6 was released in
low amounts aer liposome phagocytosis and, as expected, its
secretion increased aer maturation stimulus. TNF-α was not
increased aer liposome uptake and its secretion increased in
pro-inammatory conditions. Liposome engulfment maintained
a high prole of TGF-β1 secretion both in control subjects and
patients, and tended to decrease in mPSAB-DCs and mDCs,
although non-signicant. Regarding IL-10 production, PSAB-
DCs displayed a tendency to increase the secretion, although
non-statistically signicant, in patients with T1D. IL-2, IL-17A,
and IFN-γ were not detected in any condition of the assay (data
below the detection limit). IL-4 was not considered as it was used
in culture media for DC dierentiation.
PS-Liposomes Uptake Impairs DCs Ability
to Stimulate Autologous T Cell
Proliferation
DCs derived from patients with T1D (n12) and control subjects
(n=12) induced similar levels of autologous Tcell proliferation
(Figure 5). As expected, CD4+ Tcell proliferation induced by
mDCs was higher than proliferation induced by iDCs in both
groups (p<0.01). CD8+ T cell proliferation induced by mDCs
was higher than proliferation induced by iDCs in control sub-
jects (p<0.05), but not in patients. Importantly, the capture of
PSAB-liposomes by iDCs did not increase autologous CD4+ and
CD8+ Tcell proliferation, both in patients and control subjects.
Moreover, a signicant decrease of CD8+ T cell proliferation
induced by PSAB-DCs from patients was observed aer lipo-
some capture, when compared to iDCs (p<0.01). is eect was
reverted aer DCs maturation.
In terms of cytokine production, PBMCs co-cultured with
PSAB-DCs displayed a cytokine prole (IL-6 and IFN-γ) similar
to iDCs (Figure 5). Interestingly, PBMCs showed a tendency
to increase IFN-γ and IL-6 secretion when co-cultured with
mPSAB-DCs or mDCs, respectively, only in patients with T1D.
IL-2, IL-4, IL-10, IL-17A, and TNF-α were not detected in any
condition of the assay (data below the detection limit).
Transcriptional Changes in DCs from
Patients with T1D after PS-Liposomes
Phagocytosis Point to an
Immunoregulatory Prolife
RNA-seq analysis was performed in DCs from 8 patients with
T1D (Ta bl e 2 ) in order to identify transcriptional changes aer
the capture of PS-liposomes. Phagocytosis was veried by ow
cytometry using uorescent liposomes. Aer 4h of co-culture,
73.88±11.57% (mean±SD) of DCs were positive for uorescent
signal.
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FIGURE 3 | Continued
FIGURE 4 | The uptake of PSAB-liposomes by dendritic cells (DCs) does not alter cytokine prole. Concentration (pg/ml) of IL-6, TNF-α, TGF-β1, and IL-10
secreted by DCs obtained from control subjects (white bars, n3) and patients with type 1 diabetes (T1D) (black bars, n3). Bars represent immature DCs (iDCs),
iDCs after the capture of PSA-liposomes and PSB-liposomes (PSAB-DCs), mature PSAB-DCs (mPSAB-DCs), or mature DCs (mDCs). MDCs were induced by
culture with cytokine cocktail. Data presented as mean±SD. Signicant differences were found when comparing the different conditions in the same group of
subjects (*p<0.05, Wilcoxon test), and differences were not found when comparing the same culture conditions between patients with T1D and control subjects
(Mann–Whitney test).
FIGURE 3 | Capture of PSA-liposomes and PSB-liposomes regulates dendritic cell (DCs) phenotype. Relative CD36, TIM4, Integrin αvβ5, HLA-ABC, HLA-DR,
CD54, CD40, CD86, CD25, CCR7, CCR2, CXCR4, TLR2, CD14, and DC-SIGN membrane expression in DCs obtained from control subjects (white bars, n5)
and patients with type 1 diabetes (T1D) (black bars, n8). Bars represent immature DCs (iDCs), iDCs after the capture of PSA-liposomes and PSB-liposomes
(PSAB-DCs), mature PSAB-DCs (mPSAB-DCs), or mature DCs (mDCs), 24h after culture. MDCs were induced by culture with cytokine cocktail. Data presented as
mean±SD of relative Median of Fluorescence Intensity (MFI), this being MFI of each culture condition referred to their respective mDCs control. Signicant
differences were found when comparing culture conditions in the same group of subjects (*p0.05, **p<0.01, ***p<0.001, ****p<0.0001, Wilcoxon test), and
when comparing the same culture conditions between patients with T1D and control subjects (+p<0.05, Mann–Whitney test).
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Integrity of the isolated RNA material was assessed for each
sample, being optimal for RNA-seq experiment: RIN 9.0±0.56
(mean±SD). Bioinformatics analysis of the RNA-seq experiment
revealed that only 233 of 22,711 genes detected were dieren-
tially expressed between iDCs and PSAB-DCs (p value<0.0013,
adjusted p value<0.1254). Of these 233 genes, 203 (87.12%) were
downregulated and the remaining 30 (12.88%) were upregulated,
and 224 corresponded to protein-coding genes. Despite the
heterogeneous basal transcriptomics of DCs from eight patients,
gene expression was clearly altered toward a similar prole
aer PS-liposomes phagocytosis (Figure S3 in Supplementary
Material).
We analyzed several categories and molecules related to DC
function (Table S1 in Supplementary Material). DEGs were mainly
related to metabolism, gene expression, immunoregulation, sig-
nal transduction, molecule transport, post-translational protein
modication, cytokine signaling, cell cycle, vesicle-mediated
processes, DNA replication and repair, antigen processing and
presentation, apoptosis, and cytoskeleton organization (Tab l e 5).
Due to the immunotherapeutic potential of PS-liposomes, DEGs
involved in tolerance were analyzed in detail. DEGs linked to the
immune system were primarily downregulated and involved in
antigen processing and presentation (KBTBD6, BTK, CDC23,
UBE2E3, CD1D), regulation of the immune response (DAPP1,
FIGURE 5 | Capture of PSA-liposomes and PSB-liposomes affects dendritic cells (DCs) functionality. (A) Relative autologous proliferation of CD3+CD4+ and
CD3+CD8+ subsets induced by DCs obtained from control subjects (white bars, n=12) and patients with type 1 diabetes (T1D) (black bars, n12). Autologous
peripheral blood mononuclear cells were stained with CellTrace Violet (CTV) and co-cultured at 1:10 ratio for 6days with each condition of DCs, and proliferation
was measured as the percentage of CTVlow cells. Bars represent immature DCs (iDCs), iDCs after the capture of PSA-liposomes and PSB-liposomes (PSAB-DCs),
mature PSAB-DCs (mPSAB-DC), or mature DCs (mDCs). MDCs were induced by culture with cytokine cocktail. Data presented as mean±SD of relative
proliferation induction, this being the percentage of CTVlow cells in each co-culture condition referred to that of their respective mDCs control. Signicant differences
were found when comparing culture conditions in the same group of subjects (*p0.05, **p<0.01, ***p<0.001, ****p<0.0001, Wilcoxon test), and differences
were not found when comparing the same culture conditions between patients with T1D and control subjects (Mann-Whitney test). (B) IL-6 and IFN-γ secretion (pg/
ml) assessed in supernatants of autologous proliferation co-culture with cells from control subjects (white bars, n3) and patients with T1D (black bars, n3). Bars
represent immature DCs (iDCs), iDCs after the capture of PSA-liposomes and PSB-liposomes (PSAB-DCs), mature PSAB-DCs (mPSAB-DC), or mature DCs
(mDCs). MDCs were induced by culture with cytokine cocktail. Data presented as mean±SD. No differences were found when comparing the different conditions
in the same group of subjects (Wilcoxon test), or when comparing the same culture conditions between patients with T1D and control subjects (Mann–Whitney test).
11
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Frontiers in Immunology | www.frontiersin.org February 2018 | Volume 9 | Article 253
GIMAP4, SLAMF6) and cytokine signaling relevant in the inter-
action between Tcells and DCs (SOCS2, TNFRSF11A). However,
although very few genes were upregulated aer PS-liposomes
phagocytosis, these were related to the prevention of DC matura-
tion [TNFSF14, TNFAIP3, VEGFA, SHB, leukocyte associated
immunoglobulin-like receptor 1 (LAIR1), NFKBIA]. Also, genes
related to apoptosis were downregulated in DCs aer liposome
uptake (BLCAP, PMP22, LMNB1).
Validation by qRT-PCR of the selected gene targets (CYTH4,
GIMAP4, HPGD, NFKBIA, PL AUR, TNFAIP3, TNFSF14,
and VEGFA) conrmed the RNA-seq results, when tested in
DCs from 10 patients with T1D (Figure6). As expected, gene
expression analysis in DCs from 9 control subjects showed the
same pattern. Regarding mDCs—from 4 patients with T1D
and 3 control subjects—we observed a seemingly dierent gene
expression pattern when compared to PSAB-DCs — and with
iDCs. Genes upregulated by PS-liposomes, such as CYTH4 and
TNFSF14, tended to be downregulated in mDCs; other genes
tended to be dierentially expressed in mDCs (NFKBIA, PL AU R,
TNFAIP3, GIMAP4, VEGFA, and HPGD) in comparison to the
other conditions.
DISCUSSION
Apoptosis is a key factor in the maintenance of immunological
tolerance. e uptake of apoptotic cells, through a process called
eerocytosis, results in tolerogenic presentation of autoantigens
inducing specic tolerance rather than autoimmunity (14).
FIGURE 6 | Quantitative RT-PCR validates the RNA-seq results. Relative gene expression of 8 selected targets in immature dendritic cells (iDCs), after phagocytosis
of PSA-liposomes and PSB-liposomes (PSAB-DCs), and in mature dendritic cells (mDCs), analyzed by quantitative RT-PCR. Gene expression signals were
normalized to GAPDH. Bars show the mean±SD of gene expression in control subjects (white bars, n3) and patients with type 1 diabetes (T1D) (black bars,
n4). Statistically signicant differences were found when comparing the different conditions in the same group of subjects (*p<0.05, Wilcoxon test), and
differences were not found when comparing the same culture conditions between patients with T1D and control subjects (Mann–Whitney test).
TABLE 5 | DEGs in dendritic cells (DCs) from patients with type 1 diabetes (T1D) after PSA-liposomes and PSB-liposomes phagocytosis.
Category Number
of DEGs
P value Representative downregulated genes Representative
upregulated genes
Adhesion 3 0.001076 SCYL3, MEGF9 IGSF9
Antigen processing and
presentation
70.001024 KBTBD6, BTK, CDC23, UBE2E3, CD1D, CUL3, KIF11
Apoptosis 6 0.000593 BLCAP, PMP22, LMNB1, CASP3, DCAF7, BCL2L1
Cell cycle 9 0.001162 CSRP2BP, BUB1, MCPH1, CDK13, PCNA, MCM4, SMC2, NCAPG2, AURKA
Cytokine signaling 9 0.001191 TRIM5, SOCS2, STX3, TNFRSF11A, NUP160 TNFSF14, VEGFA, TNF,
IFNLR1
Cytoskeleton organization 6 0.000897 MAPRE2, RMDN1, CKAP2, MDM1, RCSD1, CDC42SE1
DNA replication and repair 8 0.000995 WRNIP1, PAXIP1, MSH2, RAD51C, DCLRE1A, ALKBH1, PARG, MLH1
Gene expression 36 0.001293 ZNF436, MYB, ZFP36L2, MIER3, ZBTB5, HHEX, GTF2B, DYRK2, NFIA, ZBTB39
Immunoregulation 25 0.001146 GIMAP4, SLAMF6, DAPP1, MEF2C, BST1, PROS1, MNDA TNFAIP3, PLAUR, NFKBIA
Metabolism 43 0.001225 C9orf64, HPGD, TIMMDC1, ICK, DDO, DCTD, CDYL2, GLRX, TPK1 MFSD2A
Molecule transport 14 0.001252 ERLIN1, SLC10A7, UNC50, ATP10D, SLC40A1, CLCN3, STIM2 SLC43A3, SLCO4A1,
CLCN6
Post-translational protein
modication
13 0.001056 FBXO36, NSMCE4A, VWA5A, FBXO25, DCAF12, CBX4, RMND5A, LNX2,
BTBD3
PPME1
Signal transduction 18 0.001298 SNN, SKI, PAQR8, UBFD1, N4BP1, FZD5, NET1, ZBED3, FRAT2 SHB
Vesicle-mediated processes 8 0.001208 GOLPH3L, SEC22C, RAB32, EHBP1, KIF20B, SNX18 CYTH4, LDLR
DEGs, differentially expressed genes.
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erefore, the inherent immunomodulatory properties of apop-
totic cells can be useful to design innovative immunotherapies.
Based on this tolerogenic potential of eerocytosis, we generated
liposomes that mimic apoptotic β-cells. At present, liposomes are
clinically used mainly as vehicles for drugs (2931), but they can
be designed to modulate immune responses. is liposome-based
immunotherapy resembles apoptotic cells and acts through the
immunosuppressive signal of PS (32) and tolerogenic autoantigen
presentation. ese large PS-liposomes, aer phagocytosis, are
eective in restoring self-tolerance in experimental autoimmune
diseases (16, 17) by their interaction with DCs and the arrest of
the autoimmune reaction. To explore the clinical potential of this
strategy, we have determined the eect of PS-liposomes loaded
with human insulin peptides in DCs from patients with T1D. is
eect has been assessed in several aspects: phagocytosis, pheno-
typic changes, eect on Tcell proliferation and cytokine prole.
Regarding phagocytosis, lipid membrane composition is
crucial for rapid engulfment by DCs, as demonstrated using PS-
and PC-liposomes. As expected, the presence of PS accelerated
phagocytosis of liposomes both in control subjects and patients
with a dynamic typical of apoptotic cell clearance. PS-liposomes
were more eciently engulfed by DCs than the equivalent ones
without PS in the rst 2h of co-culture, reaching plateau aer
6 h. When encountering PS-liposomes, DCs are deceived into
sensing that they are actual apoptotic cells that need to be rapidly
eerocyted in order to avoid secondary necrosis that could con-
tribute to autoimmunity (33, 34). Moreover, the preservation of
DCs viability proved that PS-liposomes are not toxic, as reported
for other types of liposomes (2932).
e second aspect was the assessment of DCs phenotype.
Aer liposome engulfment, PSAB-DCs maintained high levels of
PS-receptors that mediate this uptake, when compared to mDCs,
pointing to the preservation of phagocytosis ability in tolerogenic
DCs (tolDCs). Interestingly, the upregulation of TIM4 expres-
sion observed in PSAB-DCs from patients might contribute to a
positive feedback of phagocytosis. Upon maturation, PS-receptors
were downmodulated correlating with the phagocytic capacity of
mDCs, as described (35). e expression pattern of molecules
involved in antigen presentation (HLA, costimulatory and
adhesion molecules) in PSAB-DCs concurs with a tolerogenic
function, both in patients and control subjects. e expression
of CD25 activation marker, linked to DCs activation and autoim-
munity (36, 37), conrmed the intermediate activation status of
PSAB-DCs aer phagocytosis. Also, the chemokine receptors
expression pattern supports DCs ability to drive their migra-
tion to secondary lymphoid tissues (38, 39), and moreover, the
high CCR7 expression is associated with induction of tolerance
aer eerocytosis (40). Additionally, the expression of pattern
recognition receptors was not altered by liposomes, as described
for human DCs (32). is phenotype is similar to the previously
observed in mice (16, 17). Of note, RNA-seq analysis reinforces
these results. Furthermore, upon liposome capture, the immu-
nomodulatory cytokine TGFβ-1 was secreted, a reported eect
driven by PS (34) that could suppress DC maturation and dene
the Tcell response aerward. As expected, liposome capture did
not induce IL-6 nor TNF-α secretion by DCs, but maturation did.
Overall, the results point to the tolerogenic eect of these vesicles,
which act on re-establishing self-tolerance. We observed minor
phenotypic dierences between DCs from patients and control
subjects, which could be due to epigenetic changes caused by
autoimmunity and metabolic dysregulation (4143).
e third aspect was the analysis of autologous Tcell prolifera-
tion induced by PSAB-DCs. In agreement with DCs phenotype,
Tcell proliferation induced by PSAB-DCs was similar or even
lower than the induced by iDCs, both in patients and control sub-
jects. Interestingly, in patients with T1D, there was a signicant
reduction in CD8+ Tcell subset proliferation induced by tolDCs
when compared to iDCs. is eect could be related to a reduc-
tion of the Tcell cytotoxic activity, the most important eector
response in human T1D (44, 45). In fact, aer eerocytosis,
DCs present apoptotic cell autoantigens to cognate Tcells in the
absence of costimulation, favoring tolerance to self (12, 13). It
is reasonable to think that liposomes mimicking apoptotic cells
will cause a similar eect, Additional studies using tetramers
would be relevant to determine the antigen-specicity of the
Tcells involved in tolerance induction, even in pro-inammatory
conditions, in which Tcells seem to proliferate more vigorously.
Cytokines produced during the autologous Tcell proliferation
assay induced by PSAB-DCs discard a 1 and 17 prole,
which could be detrimental in the induction of tolerance. In fact,
IFN-γ, which is involved in a 1 response, and IL-6 secretion,
which partially contributes to induce 17 response in T1D (46),
remain poorly secreted in co-cultures of PBMCs with PSAB-
DCs. Interestingly, higher amounts of IL-6 and IFN-γ tend to
be produced by mDCs from patients with T1D when compared
to controls. is feature could reect the ongoing autoimmune
reaction, present in peripheral blood from patients (47).
One of the obstacles of tolerogenic therapies in human disease
is the heterogeneity of the ex vivo-generated tolDCs, which vary
depending on the source, the manufacturing protocols, and the
timespan of the experiment. Work is in progress to dene and
standardize a set of phenotypical and functional characteristics of
tolDCs (48). To date, tolDCs are accepted as maturation-reluctant
cells with low expression of antigen-presenting and costimula-
tory molecules and a tolerogenic-skewed cytokine prole (49).
In this sense, one of the advantages of direct administration of
the liposomes reported herein would be the generation of tolDCs
invivo, avoiding ex vivo cell manipulation. Our previous results
in mice demonstrate this hypothesis (16, 17). However, a global
picture of changes induced by PS-liposomes phagocytosis would
grant a better understanding of tolerogenicity.
To fully characterize the immunomodulatory eects of
liposomes, transcriptomic analysis was performed in DCs. Eight
patients with a short T1D duration were selected in order to
minimize the inuence of long-term hyperglycemia on immune
response, as reported (4143). RNA-seq revealed a set of DEGs
that avoid DCs maturation and contribute to tolerogenic antigen
presentation. One of the most hyperexpressed genes was the
vascular endothelial growth factor (VEGF) A (VEGFA), involved
in cytokine signaling aer eerocytosis (50), iDCs recruitment
and maturation inhibition (51). VEGF increases the expression
of the TNFSF14 gene (52), also upregulated by PS-liposomes
(53). In turn, TNFSF14 upregulates the production of TGF-β by
phagocytes (54), and upon interacting with its ligand in Tcells,
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TNFSF14 regulates T cell proliferation (55), inducing local
immunosuppression (56). Supporting this fact, apoptotic cell
clearance has been described to inhibit inammation via TGF-β
and VEGF production (34). Additionally, VEGFA enhances the
expression of indoleamine 2,3-dioxygenase (IDO) (57), which
in turn codies for an immunomodulatory enzyme expressed
in tolDCs (58). Moreover, the hematopoietically expressed
homeobox (HHEX) gene, a repressor of VEGF signaling (59),
is downregulated aer PS-liposomes phagocytosis, whereas an
inductor of VEGF expression, activating transcription factor 4
(ATF4) (60), is upregulated. Furthermore, the hyperexpressed
SH2 domain containing adaptor protein B (SHB) gene codies
for a protein that regulates VEGF-dependent cellular migration
(61), 2 polarization and Tregulatory cell induction (62). Other
upregulated tolerogenic genes, such as the TNF alpha induced
protein 3 (TNFAIP3) and the LAIR1, can inhibit DC maturation
and their deciency causes autoimmune and autoinammatory
diseases (6366). In the same way, the hyperexpression of the
NFKBIA gene would contribute to inhibit DC maturation and
T cell activation (67, 68). Regarding cytokine signature, our
results agree with those found in phenotypic and functional
experiments. A relevant cytokine for tolerance induction is
TGF-β1, secreted aer eerocytosis (69). Aer PS-liposomes
capture, DCs showed a biological increase of TGF-β1 tran-
scription, although non-signicant, probably due to the short
timespan of the experiment (70). In fact, TGF-β1 was found in
culture supernatants 24h aer PS-liposomes phagocytosis. Also,
the immunoregulatory interferon lambda receptor 1 (IFNLR1)
gene is one of the few overexpressed in DCs aer PS-liposomes
uptake. is receptor induces tolDCs that promote regulatory
Tcell expansion (71). Unexpectedly, the TNF-encoding gene was
upregulated in DCs aer liposome phagocytosis, and the same
tendency was observed in protein secretion. Nevertheless, this
behavior was very dierent to that observed in mDCs, which
secreted higher amounts of TNF. e increase of TNF gene
expression in our RNA-seq agrees with the upregulation of
TNFSF14 and TNFAIP3 genes. Furthermore, a critical role for
TNF has been reported in human tolDCs in the induction of
antigen-specic regulatory Tcells (72). Also, our previous results
showed that murine tolDCs upregulated TNF-gene expression
aer eerocytosis (14). Overall, these results are consistent with
the pleiotropic eects of TNF. Furthermore, in our previous
research, PGE2 was found to be crucial in tolerance induced by
PS-liposomes in mice (16). Strikingly, this pathway does not seem
to be upregulated in human DCs, probably due to divergences
between mice and men. Nevertheless, our data indirectly point
to the involvement of the PGE2 pathway in human DCs: rst,
the downregulation of the hydroxyprostaglandin dehydrogenase
15-(NAD) (HPGD) gene, involved in PGE2 degradation, and
second, a biological upregulation (although non-signicant) of
the peroxisome proliferator activated receptor gamma (PPARG),
a gene induced by prostaglandins which is a negative regulator
of pro-inammatory cytokines (73). Furthermore, PGE2 has
been described to stimulate the synthesis of VEGF (74). In
summary, comparative transcriptome studies identify the whole
molecular features of tolDCs rather than describe a simple state
of maturation or lack thereof in terms of phenotype and function
(75). Further studies are required to nd a common signature
of tolerogenicity, a fact hindered by individual dierences of
human DCs and the heterogeneous results obtained with dif-
ferent agents used to promote tolDCs. Our ndings describe the
specic gene signature of PS-liposomes-induced tolDCs. eir
genomic program, which drives a dierent functionality than
those of iDCs and mDCs, contributes to dissect the complexity
of tolerance regulation.
Perhaps not so peculiarly, most of the alterations found in DCs
aer PS-liposomes capture are also physiopathological strategies
used by tumor cells to escape immune surveillance. Small vesicles
rich in PS are released by tumor cells and act as immunosuppres-
sive agents to inhibit tumor antigen-specic Tcells (76). Tumor
cells can induce immunological tolerance using mechanisms
characteristic of apoptotic cell clearance, and PS-liposomes seem
to make use of the same pathways to achieve similar eects.
e use of PS-liposomes lled with autoantigens is an innova-
tive strategy to arrest autoimmunity by restoring tolerance to self.
As a whole, our results support the tolerogenic behavior of DCs,
induced by the phagocytosis of PS-liposomes, and suggest that,
in the context of autoimmunity, they could act silencing potential
autoreactive T cells. is process could possibly be an active
silencer, and not only a lack of maturation of DCs. In summary,
here we unveil a picture of eerocytosis mimicry that leads to
phenotypic and functional changes in human DCs, accountable
for tolerance induction. e herein reported results reinforce the
potential of this biocompatible immunotherapy to re-establish
immunological tolerance, opening the door to new therapeutic
approaches in the eld of antigen-specic autoimmune disorders.
ETHICS STATEMENT
is study was carried out aer the approval and in strict accord-
ance with the recommendations of the guidelines of Germans
Trias i Pujol Ethical Committee. All subjects gave written
informed consent in accordance with the Declaration of Helsinki.
AUTHOR CONTRIBUTIONS
SR-F, IP-A, MC-S, FV, JV, and MV-P designed the study and inter-
preted the data; SR-F, DM, JV, and MV-P wrote the manuscript;
SR-F, IP-A, DP-B, MC-S, SG-J, and AV performed the experi-
ments; IP-A, SR-F, DM, and MV-P supervised the experiments;
SR-F, EA, FV, and MV-P selected the patients and supervised the
collection of blood samples; and SR-F, IP-A, FB, and AS analyzed
the data. All authors revised the work and gave nal approval of
the version to be published.
ACKNOWLEDGMENTS
We are grateful to the physicians and nurses working at the
Endocrinology Section and the Clinical Investigation Unit
(UPIC) from the Germans Trias i Pujol University Hospital, and
to Dr. J. C arrascal and Ms. B. Quirant-Sanchez, from Immunology
Section, for collecting blood samples and data, respectively.
We are indebted to all blood donors who participated in this
study and made it possible. We would also like to acknowledge
15
Rodriguez-Fernandez et al. Liposomes for Human Autoimmune Diabetes
Frontiers in Immunology | www.frontiersin.org February 2018 | Volume 9 | Article 253
Mr. M. Fernandez for technical assistance with ow cytometry,
Dr. P. Armengol and Ms. A. Oliveira for their support in the
RNA-seq experiment, and Dr. C. Prat for microbiological con-
trols (IGTP). Special thanks to Ms. D. Cullell-Young for English
grammar assistance.
FUNDING
is work has been funded by a grant from the Spanish
Government (FIS PI15/00198) co-nanced with the European
Regional Development funds (FEDER), by Fundació La Marató de
TV3 (28/201632-10), by Catalan AGAUR (project 2014 SGR1365)
and by CERCA Program/Generalitat de Catalunya. CIBER of
Diabetes and Associated Metabolic Diseases (CIBERDEM) is an
initiative from Instituto de Salud Carlos III. ICN2 acknowledges
the support of the Spanish MINECO through the Severo Ochoa
Centers of Excellence Program, under grant SEV-2013-0295.
is work has been supported by positive discussion through A
FACTT network (Cost Action BM1305: www.afactt.eu). COST is
supported by the EU Framework Program Horizon 2020. SR-F
is supported by the Agency for Management of University and
Research Grants (AGAUR) of the Generalitat de Catalunya.
SUPPLEMENTARY MATERIAL
e Supplementary Material for this article can be found online at
https://www.frontiersin.org/articles/10.3389/mmu.2018.00253/
full#supplementary-material.
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CIR-15-0086
Conict of Interest Statement: IP, MC, JV, DM, and MV are inventors in a patent
(WO2015107140) that describes the use of autoantigen-encapsulating liposomes
for the prevention or treatment of autoimmune disorders.
Copyright © 2018 Rodriguez-Fernandez, Pujol-Autonell, Brianso, Perna-Barrull,
Cano-Sarabia, Garcia-Jimeno, Villalba, Sanchez, Aguilera, Vazquez, Verdaguer,
Maspoch and Vives-Pi. is is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). e use, distribution or
reproduction in other forums is permitted, provided the original author(s) and the
copyright owner are credited and that the original publication in this journal is cited,
in accordance with accepted academic practice. No use, distribution or reproduction
is permitted which does not comply with these terms.
... This animal model is mostly induced in mice and rats according to their rapid reproduction and their extensive span of manipulation [60]. The most prevalent rats and mice are Dark Agouti and Lewis rats [61] and C57BL/6 and SJL/J mice, for enduring EAE by subcutaneous injection of myelin-oligodendrocyte glycoprotein (MOG ) and proteolipid protein peptide (PLP [139][140][141][142][143][144][145][146][147][148][149][150][151], along with some adjuvants [62]. The scale of grading the severity of the disease is according to the degree of paralysis, determined by defined scores [63]. ...
... MBP-phosphatidylserine liposomes protect rats against EAE which occurs through an active, antigen-specific suppressor mechanism and has been suggested to take place through tolerance by clonal deletion or clonal inactivation [148]. The most evidenced mechanism of phosphatidylserine is the simulation of apoptotic cells through their presence in liposomes and precipitation of their phagocytosis, leading to alterations in phenotype and function of DCs, amenable to be tolerogen [146,149,150]. Besides, when this phospholipid uses in liposomes that encapsulate the autoantigen of MS, it will have a double-signal of specificity and tolerance to stop the symptoms of this disease synergically. ...
... This mechanism has resulted from the administration of phosphatidylserine liposomes encapsulated MOG [40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55] , an MOG fragment, to EAE mice [151]. Overall, phosphatidylserine liposomes render a biocompatible and safe physiological platform to restore peripheral tolerance in antigen-specific autoimmune disorders such as MS [149]. ...
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Introduction Multiple Sclerosis (MS), as an autoimmune disease, has complicated immunopathology, which makes its management relevant to various factors. Novel pharmaceutical vehicles, especially liposomes, can support efficacious handling of this disease both in early detection and prognosis and also in a therapeutic manner. The most well-known trigger of MS onset is the predominance of cellular to humoral immunity and enhancement of inflammatory cytokines level. The installation of liposomes as nanoparticles to control this disease holds great promise up to now. Areas covered Various types of liposomes with different properties and purposes have been formulated and targeted immune cells with their surface manipulations. They may be encapsulated with anti-inflammatory, MS-related therapeutics, or immunodominant myelin-specific peptides for attaining a higher therapeutic efficacy of the drugs or tolerance induction. Cationic liposomes are also highly applicable for gene delivery of the anti-inflammatory cytokines or silencing the inflammatory cytokines. Liposomes have also been used as biotools for comprehending MS pathomechanisms or as diagnostic agents. Expert opinion The efforts to manage MS through nanomedicine, especially liposomal therapeutics, pave a new avenue to a high-throughput medication of this autoimmune disease and their translation to the clinic in the future for overcoming the challenges that MS patients confront.
... Rights reserved. and phenotypic alterations in human DCs responsible for the induction of tolerance [70]. ...
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... When cells undergo apoptosis, they express the negatively charged phospholipid phosphatidylserine (PS) on their surface (71), which is recognized by receptors on efferocytes, such as stabilin-2 (72), TIM-4 (73), and CD300f (74,75), as shown in Figure 1. The role of PS in apoptosis is extensively reviewed by Birge et al. and several types of nanoparticles containing PS have taken advantage of this pathway to induce tolerance in autoimmune models (76,77). Unfortunately, empty PS liposomes have been shown to induce non-specific immune tolerance which may hamper clinical application (35,78). ...
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... As an autoimmune disorder, type 1 diabetes leads to β-cell destruction which produces insulin. Rodriguez-Fernandez et al. [40] reported that the development of liposomes with phosphatidylserine and β-cell autoantigensmimics apoptotic β-cells which leads to the inhibition of autoimmunity to β-cells, thereby effectively preventing experimental type 1 diabetes through the generation of tolerogenic dendritic cells. ...
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A robust comprehension of phagocytosis is crucial for understanding its importance in innate immunity. A detailed description of the molecular mechanisms that lead to the uptake and clearance of endogenous and exogenous particles has helped elucidate the role of phagocytosis in health and infectious or autoimmune diseases. Furthermore, knowledge about this cellular process is important for the rational design and development of particulate systems for the administration of vaccines or therapeutics. Depending on these specific applications and the required biological responses, particles must be designed to encourage or avoid their phagocytosis and prolong their circulation time. Functionalization with specific polymers or ligands and changes in the size, shape, or surface of particles have important effects on their recognition and internalization by professional and nonprofessional phagocytes and have a major influence on their fate and safety. Here, we review the phagocytosis of particles intended to be used as carrier or delivery systems for vaccines or therapeutics, the cells involved in this process depending on the route of administration, and the strategies employed to obtain the most desirable particles for each application through the manipulation of their physicochemical characteristics. We also offer a view of the challenges and potential opportunities in the field and give some recommendations that we expect will enable the development of improved approaches for the rational design of these systems.
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Dendritic cells (DCs) control adaptive immunity and are therefore attractive for in vivo targeting to either induce immune activation or tolerance, depending on disease. Liposomes, nanoparticles comprised of a lipid bi-layer, provide a nanoplatform for loading disease-relevant antigen, adjuvant and DC-targeting molecules simultaneously. However, it is yet not fully understood how liposomal formulations affect uptake by DCs and DC function. Here, we examined monocyte-derived DC (moDC) and skin DC uptake of six different liposomal formulations, together with their DC-modulating effect. Contrary to literature, we show using imaging flow cytometry that anionic or neutral liposomes are taken up more efficiently than cationic liposomes by moDCs, or by skin DCs after intradermal injection. None of the formulations yielded significant modulation of DC function as determined by the upregulation of maturation markers and cytokine production. These results suggest that anionic liposomes would be more suitable as vaccine carriers for a dermal application.
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Aim: Based on the ability of apoptosis to induce immunological tolerance, liposomes were generated mimicking apoptotic cells, and they arrest autoimmunity in Type 1 diabetes. Our aim was to validate the immunotherapy in other autoimmune disease: multiple sclerosis. Materials & methods: Phosphatidylserine-rich liposomes were loaded with disease-specific autoantigen. Therapeutic capability of liposomes was assessed in vitro and in vivo. Results: Liposomes induced a tolerogenic phenotype in dendritic cells, and arrested autoimmunity, thus decreasing the incidence, delaying the onset and reducing the severity of experimental disease, correlating with an increase in a probably regulatory CD25(+) FoxP3(-) CD4(+) T-cell subset. Conclusion: This is the first work that confirms phosphatidylserine-liposomes as a powerful tool to arrest multiple sclerosis, demonstrating its relevance for clinical application.
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