doi: 10.1093/brain/aww113.
Epub 2016 May 31.
IL-10-dependent Tr1 cells attenuate astrocyte activation and ameliorate chronic central nervous system inflammation
Affiliations
- PMID: 27246324
- PMCID: PMC4939696
- DOI: 10.1093/brain/aww113
Item in Clipboard
IL-10-dependent Tr1 cells attenuate astrocyte activation and ameliorate chronic central nervous system inflammation
Brain.
2016 Jul.
Abstract
SEE WINGER AND ZAMVIL DOI101093/BRAIN/AWW121 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: The innate immune system plays a central role in the chronic central nervous system inflammation that drives neurological disability in progressive forms of multiple sclerosis, for which there are no effective treatments. The mucosal immune system is a unique tolerogenic organ that provides a physiological approach for the induction of regulatory T cells. Here we report that nasal administration of CD3-specific antibody ameliorates disease in a progressive animal model of multiple sclerosis. This effect is IL-10-dependent and is mediated by the induction of regulatory T cells that share a similar transcriptional profile to Tr1 regulatory cells and that suppress the astrocyte inflammatory transcriptional program. Treatment results in an attenuated inflammatory milieu in the central nervous system, decreased microglia activation, reduced recruitment of peripheral monocytes, stabilization of the blood-brain barrier and less neurodegeneration. These findings suggest a new therapeutic approach for the treatment of progressive forms of multiple sclerosis and potentially other types of chronic central nervous system inflammation.
Keywords: T-lymphocytes; astrocyte; interleukin 10; multiple sclerosis; neuroinflammation.
© The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain.
Figures
Nasal anti-CD3 ameliorates disease in a progressive model of EAE.
(
A
and
B
) Clinical scores of EAE in NOD mice treated nasally with CD3 specific or isotype control mAbs, administered daily (1 µg/mouse) from Day 30 after EAE induction (progressive phase) for the duration of the experiment. Representative data of eight independent experiments with
n
= 8 mice/group (mean and SEM), statistical analysis by two-way ANOVA and linear regression. (
C–J
) At the experimental endpoint (Day 70) mice Rotarod performance was evaluated (
G
), and (
l–J
) histopathology analysis of lumbar spinal cord serial sections from EAE NOD mice treated with anti-CD3 or isotype control as in
A
was performed. Sections were stained with haematoxylin and eosin (H&E), Luxol Fast blue stain or Bielschowsky’s silver impregnation for analysis of mononuclear cell infiltration and vacuoles indicating oedema, demyelination or axonal loss (axonal injury with spheroids is identified by red arrows), respectively. Representative data of two independent experiments with
n
= 6 mice/group, statistical analysis by Student’s
t
-test (
E–G
) Quantification of dystrophic axons as determined by amyloid precursor protein (APP) staining and lesions load, respectively. Representative data of two independent experiments with
n
= 6 mice/group, statistical analysis by Student’s
t
-test. (
H–J
) Blood–brain barrier permeability. (
H
) Extravasation of endogenous fibrin and antibodies (IgG), and (
J
) exogenous tracer dye Evans Blue or FITC-conjugated dextran to the spinal cord. Representative data of two independent experiments with
n
= 5 mice/group. (
K
) Clinical scores of EAE in NOD mice treated nasally with CD3 specific or isotype control mAbs from Day 45 after EAE induction. Representative data of two independent experiments with
n
= 8 mice/group (mean and SEM), statistical analysis by two-way ANOVA. Scale bar = 100 µm for low power magnification (
C
and
H
), and 50 µm (
C–E
and
H
) or 20 µm (
J
) for higher magnification. *
P
< 0.05, **
P
< 0.01, ***
P
< 0.001, n.s. = not significant.
Nasal anti-CD3 induces an IL-10+LAP+ T- cell that attenuates progressive EAE in an IL-10 dependent manner.
(
A–I
) NOD mice treated nasally with CD3 specific or isotype control mAbs following EAE induction as in (
Fig. 1
A). IL-10 secretion by splenic T cell in response to MOG
35–55
(20 µg/ml) or anti-CD3 mAbs (0.2 µg/ml) stimulation was determined by enzyme-linked immunosorbent assay (
A
), and IL-10 and LAP expression by splenic CD4
+
T cells was examined by fluorescence-activated cell sorting (FACS) (
B
and
C
). Data are representative of four independent experiments with
n
= 6 mice/group (mean and SEM). Statistical analysis by Student’s
t-
test. (
D
) Proportion of MOG-specific T cells within LAP
+
and LAP
−
splenic T cells in NOD EAE mice treated with nasal anti-CD3. Data are representative of two independent experiments with
n
= 5 mice/group (mean and SEM). Statistical analysis by Student’s
t-
test. Relative and absolute numbers of CNS-infiltrating CD4
+
T cells was determined by FACS (
E
and
F
, respectively). (
G
) Quantitative PCR analysis of the expression of
Il10
,
Foxp3, Tgfb1
,
Gata3
,
and Il4
mRNA of CD4
+
T cells isolated from the CNS; expression is presented relative to
Gapdh.
(
H
) IL-10 expression by CNS-infiltrating CD4
+
T cells was examined by FACS (
I
and
K
) FACS-sorted CD4
+
T cells from nasal anti-CD3 treated NOD EAE mice (Day 60) were used in a standard suppression assay with naive CD4
+
responder T cells at a ratio of 1:1 (
I
). To test the role of IL-10 in
in vitro
suppression, isotype control (IC) or anti-IL-10 R (50 µg/ml) blocking antibodies were added to co-cultures (
K
). Representative data of two independent experiments with
n
= 4 mice/group, statistical analysis by Student’s
t-
test. (
J
) Clinical scores of EAE in NOD mice following adoptive transfer (4 × 10
6
cells/mouse) of splenic CD4
+
, or CD4
+
LAP
neg
T cells sorted from chronic NOD EAE mice (Day 60) that were treated with anti-CD3 as in
Fig. 1
A. Representative data of two independent experiments with
n
= 5 mice/group (
L
) Clinical scores of EAE in NOD mice. At the onset of the chronic phase of EAE mice were treated daily with nasal CD3 specific or isotype control mAbs, and were intraperitoneally injected every fourth day (black arrows) with anti-IL-10 receptor blocking mAbs (αIL-10 R) or appropriate control (IgG) (0.5 mg/mouse). Representative data of two independent experiments with
n
= 8 mice/group. *
P
< 0.05, **
P
< 0.01, ***
P
< 0.001, n.s. = not significant; n.d. = not detected.
Nasal anti-CD3 induces IL-10
+
Tr1-like cells.
(
A–H
) EAE was induced in IL-10:GFP F1 hybrid mice (B6NODF1IL-10:GFP) and mice were treated nasally with CD3 specific or isotype control mAbs as in
Fig. 1
A. (
A
) Nasal anti-CD3 attenuates the of progressive phase of EAE in the B6NODF1IL-10:GFP mice. Data are representative of two independent experiments with
n
= 8 mice/group (means and SEM). (
B
) GFP (IL-10) expression by CD4
+
T cells in the spleen and cervical lymph nodes (cLN) was examined by flow cytometry. (
C
) Heat map depicting the differential mRNA expression profiles in naïve CD4
+
or CD4
+
GFP/IL-10
+
T cells isolated from the spleen or cLNs of B6NODF1IL-10:GFP mice during the progressive phase of EAE following nasal treatment, as detected by microarray data analysis. (
D
) Functional enrichment analysis of the differentially expressed genes. Flow cytometry analysis of IL-10 and Ki-67 expression by CD4+ T cells isolated from anti-CD3 treated NOD mice (
E
), and their IL-10 expression following CFSE-labelling and
ex vivo
restimulation (
F
). Data are representative of two independent experiments with
n
= 5 mice/group. (
G
and
H
) PCA of differentially regulated genes of
in vivo
and
in vitro
sorted regulatory T cell listed in (
Supplementary Tables 1
and
Supplementary Data
); circles indicate grouping. Biplot in
H
displays the top 20 genes with the maximal variances that drive the population functional segregation (PC2). (
I
) Gene set enrichment analysis (GSEA) of the upregulated genes
in vitro
differentiated Tr1 cells, and the
in vivo
induced IL-10
+
T cells. Normalized enrichment score (NES) of 1.389 and FDR q-value (FDR) of 0.0382. (
J
) Clinical scores of EAE in wild-type F1 hybrid mice (B6NODF1) mice. At the onset of the chronic phase mice were treated with anti-CD3 or subjected to adoptive transfer of sorted
in vitro
differentiated Tr1 or Tho cells. Representative data of two independent experiments with
n
= 7 mice/group (means and SEM). Statistical analysis of EAE clinical score in (
A
and
J
) by two-way ANOVA. **
P
< 0.01, ***
P
< 0.001.
Nasal anti-CD3 attenuates adaptive T-cell responses.
Splenic T cell recall response to MOG
35–55
(20 µg/ml) or anti-CD3 stimulation (0.2 µg/ml); proliferation (
A
) and secretion of the cytokines IL17A (
B
). Data are representative of four independent experiments with
n
= 8 mice/group (mean and SEM). Statistical analysis by Student’s
t-
test. (
C
and
D
) EAE NOD mice were treated with CD3 specific or isotype control mAbs during the chronic phase as in
Fig. 1
A. (
C
) IL17A expression by splenic CD4
+
T cells was examined by flow cytometry. (
D
) Quantitative PCR analysis of the expression of
Rorc
,
Il17a
,
Csf2
,
Tbx21
,
and Ifng
mRNA of CD3
+
CD4
+
T cells isolated from the CNS; expression is presented relative to
Gapdh
. Data are representative of three independent experiments with
n
= 6 mice/group (mean and SEM). Statistical analysis by Student’s
t-
test. *
P
< 0.05, **
P
< 0.01, ***
P
< 0.001. n.s. = not significant.
Nasal anti-CD3 suppresses astrocyte activation.
(
A–E
) Naïve or EAE NOD mice treated with CD3-specific or isotype control mAbs during the chronic phase as in (
Fig. 1
A). (
A
) Heat map depicting mRNA expression, as detected by Nanostring nCounter analysis, in astrocytes isolated from naïve or EAE NOD mice treated with CD3 specific or isotype control mAbs.
Upper panels
: Histogram presentation of normalized gene expression in each gene cluster. Representative data of three independent experiments, statistical analysis by one-way ANOVA, followed by Tukey
post hoc
analysis. (
B
) Quantitative PCR analysis of
Ccl2
,
Ccl5
,
Il6
,
Tlr2
, and
Aqp4
expression in astrocytes isolated from naïve and EAE NOD mice treated as in
A
; expression is presented relative to
Gapdh
. Representative data of three independent experiments, statistical analysis by one-way ANOVA, followed by Tukey
post hoc
analysis. (
C
) Immunohistochemistry analysis of spinal cords for aquaporin 4 (AQP4) expression, and the presence of astrocytes (GFAP) on subsequent sections. Scale bar = 50 µm. Data are representative of two independent experiments with
n
= 6 mice/group. (
D
) Relative expression (to NOD naïve group) of genes associated with the control of demyelination in astrocytes isolated from EAE NOD mice treated with anti-CD3 or isotype control. Representative data of three independent experiments. Statistical analysis by Student’s
t-
test. (
E
) Quantitative PCR analysis of
Mmp3
,
Mmp9
and
Vegfa
expression in astrocytes isolated from naïve and EAE NOD mice treated as in
A
; expression is presented relative to
Gapdh
. Representative data of three independent experiments, statistical analysis by one-way ANOVA, followed by Tukey
post hoc
analysis. (
F
) Cultured astrocytes were pre-treated for 1 h with IL-10 (100 ng/ml), or vehicle (PBS), followed by activation with IL1β (20 ng/ml) or left untreated. Quantitative PCR analysis of
Ccl2
,
Ccl5
,
Csf2
,
Nos2
,
Mmp3
, and
Mmp9
; expression is presented as fold change from untreated, relative to
Gapdh
. Data from five independent experiments (mean and SEM). Statistical analysis by two-way ANOVA, followed by Tukey
post hoc
analysis. *
P
< 0.05, **
P
< 0.01.
Astrocytic IL-10 receptor has a pivotal role in mediating the therapeutic effect of nasal anti-CD3.
(
A
) Schematic map of the astrocyte-specific shRNA lentiviral vector. (
B–D
) Intracerebroventricular injection of astrocyte-specific sh
IL10ra
lentivirus ameliorates disease severity. NOD mice were injected intracerebroventricularly (i.c.v.) with 10
7
IU of shControl, or sh
IL10ra
lentivirus (LV, black arrow), at Day 35 after EAE induction (progressive phase).
n
= 10 mice per group. Two weeks after i.c.v. injection the experiment was terminated and (
B
) immunostaining analysis of spinal cord slices from mock- or lentivirus-infected mice identify GFP
+
expression only in GFAP
+
astrocytes. (
C
)
Il10ra
expression levels were determined by quantitative PCR in astrocytes isolated from naïve or EAE NOD mice; expression normalized to
gapdh
and presented relative to that of cells from naïve mice. Representative data of two independent experiments, Statistical analysis by one-way ANOVA, followed by Tukey
post hoc
analysis. (
D
) EAE clinical scores. Representative data of two independent experiments. Statistical analysis by two-way ANOVA. **
P
< 0.01, ***
P
< 0.001.
Nasal anti-CD3 attenuates microglial activation.
(
A–C
) Naïve or EAE NOD mice treated with CD3 specific or isotype control mAbs during the chronic phase as in
Fig. 1
A. (
A
) Heat map depicting mRNA expression, as detected by Nanostring nCounter analysis, in microglial cells isolated from naïve or EAE NOD mice treated with CD3 specific or isotype control mAbs.
Upper panels
: Histogram presentation of normalized gene expression in each gene cluster. Representative data of three independent experiments, statistical analysis by one-way ANOVA, followed by Tukey
post hoc
analysis. (
B
) Quantitative PCR analysis of
Il1b
,
Il6
,
Nos2
,
Cd40
, and
Irf7
expression in microglial cells isolated from naïve and EAE NOD mice treated as in
A
; expression is presented relative to
Gapdh
. Representative data of three independent experiments, statistical analysis by one-way ANOVA, followed by Tukey
post hoc
analysis. (
C
) Mean normalized expression of genes associated with M1 or M2 phenotype in microglia (
Supplementary Table 6
). Statistical analysis by Student’s
t
-test. (
D
) Primary microglia were pretreated for 1 h with IL-10 (100 ng/ml), or vehicle (PBS), followed by activation with GM-CSF (25 ng/ml) or left untreated. Quantitative PCR analysis of
Il1b
,
Il6
,
Il12b
,
Cd40
,
H2aa
, and
TNF
; expression is presented as fold-change from untreated relative to
Gapdh
. Data from three independent experiments (mean and SEM). Statistical analysis by Student’s
t-
test. *
P
< 0.05, **
P
< 0.01, ***
P
< 0.001. (
E–G
) Microglial IL-10 receptor role in mediating the therapeutic effect of nasal anti-CD3. (
E
) Schematic map of the microglia-specific shRNA lentiviral vector. (
F
and
G
) Intracerebroventricular injection of microglia-specific sh
IL10ra
lentivirus ameliorates disease severity. NOD mice were injected i.c.v. with 10
7
IU of shControl, or sh
IL10ra
lentivirus (LV, black arrow), at Day 37 after EAE induction (progressive phase).
n
= 10 mice per group. (
F
)
Il10ra
expression levels were determined by quantitative PCR in microglia isolated from naïve or EAE NOD mice; expression normalized to
Gapdh
and presented relative to that of cells from naïve mice. Representative data of two independent experiments, Statistical analysis by one-way ANOVA, followed by Tukey
post hoc
analysis. (
G
) EAE clinical scores. Representative data of two independent experiments. Statistical analysis by two-way ANOVA. **
P
< 0.05,**
P
< 0.01, ***
P
< 0.001.
Nasal anti-CD3 decreases the recruitment of Ly6Chi monocytes to the CNS and modulates their phenotype.
(
A
) Recruitment of inflammatory monocytes (defined either as of CD11b
+
Ly6C
high
or CD11b
+
CD45
high
cells) to the CNS NOD EAE mice, treated with CD3 specific or isotype control mAbs during the chronic phase as in
Fig. 1
A, was analysed by flow cytometry and presented as cell frequency, and total cell numbers. Representative data of three independent experiments. Statistical analysis by Student’s
t
-test. (
B
) Volcano plot depicting changes in mRNA expression in CD11b
+
Ly6C
high
monocytes isolated from the CNS of NOD EAE mice (as in
A
), as detected by Nanostring nCounter analysis. Changes in gene expression (at least 2-fold) are indicted by colour (Red = increased expression; blue = reduced expression). Representative data of two independent experiments. (
C
) Mean normalized expression of genes associated with M1- or M2-phenotype in microglia (
Supplementary Table 6
). Statistical analysis by Student’s
t
-test. *
P
< 0.05, **
P
< 0.01.
Comment in
-
Your nose knows how to target brain inflammation.Brain. 2016 Jul;139(Pt 7):1866-9. doi: 10.1093/brain/aww121. Brain. 2016. PMID: 27343218 Free PMC article.
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