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Meningeal Mast Cell Show No Sex differences in EAE C57BL/6 Mice for
ILC2 Promoting factors: Answers into the Autoimmune Sex Disparity
Kevin Cao, Abigail Short (Russi), Mark Ebel, Julianne Hatfield, Melissa A. Brown
Adlai E. Stevenson High School SPARK Program and Feinberg School of Medicine, Department of Immunology
Main Question: Do mast cells from male and female C57mice have ILC2 promoting factor
differences, mirroring the absence of differences in EAE severity?
Abstract
It has been long known that females are more susceptible to a number of autoimmune
diseases such as systemic lupus erythematous, rheumatoid arthritis, and myasthenia gravis.
One disease that shows this prominent sex disparity is multiple sclerosis (MS).
→ Multiple sclerosis has several subtypes of disease that vary in severity and are driven by
different immune responses. As a result, the cellular mechanisms that drive these
different responses are of great interest in learning more about autoimmunity.
→ In this study, the meninges (mast cell rich tissues that surround the brain and spinal cord)
from mice with the induced mouse model of MS (called EAE) were examined for factors
that promote type 2 innate lymphoid cells (ILC2). ILC2s may be protective in EAE by
mediating a less severe autoimmune T cell reaction (Th2).
Mast
Cell (From
Female)
ILC
2
ILC2
Mast cells can produce factors that activate innate lymphoid cells. In the SJL
model, bone marrow derived mast cells from male and female mice produce
strikingly different mediators. Mediators produced by mast cells derived from
male mice favor ILC2- and Th2-promoting factors. However, mast cells derived
from female mice produce more pro-inflammatory mediators.
Th17
pathogenic
Th2
Protective
ILC
(Innate
Lymphoid
Cell)
Method of Meningeal RNA extraction:
Disease Variants of multiple sclerosis: Promoted by specific Innate Lymphoid Cell types
Different T cell responses direct differing auto-inflammatory responses in EAE/MS.
→The Th2 (known as T-helper type 2 cell) response has been known to produce a limited and less
severe disease response. In human and mouse disease, Th2 responses correlate with reduced
disease severity. Type 2 innate lymphoid cells skew T-helper cells towards the Th2 variant, thus
promoting a Th2 response and protection from EAE.
*Th2 responses dominate the auto-reactive T cell response in male mice with EAE.
→ The Th1 and Th17 responses are both known to cause a more severe disease pathology in mice
and humans. The Th1 and Th17 responses of EAE may be mediated by type 1 innate lymphoid cells
(ILC1) and type 3 innate lymphoid cells (ILC3), respectively.
*Th1/Th17 responses dominate the auto-reactive T cell response in female mice with EAE.
Mast Cell activation of ILC1, ILC2, or ILC3 in mouse EAE disease
Although epithelial cells are the primary activators of innate lymphoid cells, mast cells also produce
many of the factors that promote ILC function. Thus, mast cells, through their activation of ILC2s
and modulation of subsequent T cell responses, may represent a starting point in the sex
differences of disease.
Sex Differences shown with SJL Mouse Model
SJL Mouse Strain: Relapse and Remitting disease model
→ EAE induced on male mice of the SJL background exhibit far less severe disease,
mirroring the sex dimorphism observed in human disease. Male SJL mice with
EAE have an increased proportion of ILC2s, a dominate Th2 response, and
therefore a less severe disease course. (Short, Brown, 2015)
→ Similar to human disease, female SJL EAE mice exhibit a more severe disease
course. In female SJL mice, the main immune response that drives the
demyelination is the Th1/Th17 response. Fittingly, the female SJL mice with
EAE/MS show a reduced amount of ILC2 cells.
1) 4 female and 4 male mice were immunized for EAE.
2) Both cohorts were then sacrificed 6 days post immunization. The
meninges were dissected away from the mouse skull. Meningeal tissue
from each group was pooled and placed in RNA stabilization buffer at
-80C.
3) The pooled meningeal samples were homogenized and meningeal
RNA was extracted and isolated.
Mast cell gene expression of ILC2 promoting factors:
1) cDNA was synthesized from the isolated RNA using reverse
transcriptase.
2) Meningeal gene expression was determined by real-time PCR. Genespecific primers were used. Expression was compared to expression of
Hprt, a housekeeping gene, to determine relative expression. A
housekeeping gene is a gene common and standard in all strain of SJL
and C57BL/6 mice.
Relative expression/HPRT
Meningeal TNFa Gene Expression
0.4
3
Mast
Cell (From
Male)
Mast
Cell (From
Male)
Relative expression/HPRT
Th1/Th17
Mast
Cell (From
Female)
SJL Model
Innate lymphoid cells (ILCs) are a
heterogeneous group of hematopoietic
cells that serve as a mediator between
the innate and adaptive immune
systems. ILCs respond to a wide range
of stimuli.
Future Directions and Other Experiments:
→ Look for any post-translational factors that may alter the production of
ILC2-promoting or Pro-Inflammatory genes at protein level - Perform
ELISA (Enzyme Linked Immunosorbent Assay) to detect
cytokine levels in serum, CSF, and other tissue.
Find confounders that may alter Th1/Th17 activating process
→ Look for ILC1 and ILC3 promoting factors to recapitulate the
promotion of different ILCs during inflammation.
→ Explore other ILC2 promoting factors from other cells such as
epithelial cells and work to establish association between the disease.
ILCs are generally classified into 3
groups: ILC1: Generally associated with
a Th1 response. ILC2: Associated with a
Th2 response. See Figure 4 for further
description of the cytokines that will
promote these cells. ILC3s are
commonly associated with Th17
responses.
Figure 5
15
6
4
10
2
5
0
0
0.2
Meningeal TSLP Gene Expression
0.8
0.6
0.4
Female SJL Day6
15
10
5
0.2
0.0
Female SJL Day6
Male SJL Day6
Meningeal CRTH2 Gene Expression
0.10
Meningeal TNFa Gene Expression
Meningeal IL33 Gene Expression
Meningeal TSLP Gene Expression
0.3
Male SJL Day6
Male SJL Day6
Female SJL Day6
Meningeal HPDGS Gene Expression
Male SJL Day6
Meningeal IL1b Gene Expression
2.0
0.8
1.5
0.6
1.0
0.4
0.5
0.2
0.0
0
Female SJL Day6

C57BL/6 EAE
mouse model
0.0
Female SJL Day6
Male SJL Day6
Female SJL Day6
Male SJL Day6
Figure 5: Expression in SJL Mice
Meninges of SJL male and female mice were
harvested on day 6 of the disease course.
A student’s T-test was performed to differentiate a
statistical significant between the SJL expression and
that of the C57BL/6 that saw differences p-value <.05.
Test determined that both male and female C57BL/6
meninges showed lower expression than the SJL by a
10 fold average. SJL females and both C57BL/6 sexes
show similar disease severity, and both SJL females
and C57BL/6 mice appear to have a lower gene
expression of ILC2 promoting factors.
0.05
1
0.0
Female B6 D6
Male B6 D6
0.08
Female B6 D6
Male B6 D6
0.00
Female B6 D6
0.06
0.06
1.0
0.04
0.04
0.5
0.02
0.02
Female B6 D6
Male B6 D6
0.0
0.00
Female B6 D6
Figure 7
Male B6 D6
ILC1/ILC2
?
Th2
response
?
Th1/Th17
Future question:
What drives mast cell differences in gene expression ILC2 promoting
factors between SJL and C57BL/6 strain?
Bone marrow derived mast cells corroborate low expression in
meninges for ILC2 promoting factors:
0.08
1.5
ILC2
Male B6 D6
Meningeal IL1b Gene Expression
Meningeal HPGDS Gene Expression
Piecing together the
cellular mechanism
2
0.1
0.00
ILC2
Innate Lymphoid Cells:
Immunological Significance in an
EAE context
Meningeal expression of ILC2 promoting factors in C57BL/6 mice is
reduced compared to SJL mice:
0.15
2
0
Th2
The meninges are a group of layered
tissue that provide selective protection
and nutrient delivery to the brain and
spinal cord. The meninges are sites of
robust inflammatory immune response
during the EAE/MS.
The meninges house a large number of
mast cells and thus can be utilized as a
window into in vivo mast cell function.
Bone Marrow Mast
cell cultures were
later used to corroborate evidence of
meningeal expression
with pure mast cell
populations.
cDNA from the same Male and Female mice samples were used in two trials measuring the
expression of genes : Il33, Tnfa, Tslp, Crth2, Il1b, Hpgds. Shown if figure 4
Student’s T-test was used measuring for a significant statistical difference between the male
and female for the first and second respectively samples yielded p-values >0.5 for all except
TNFa (P<.05)
Figure 4
Meningeal IL33 Gene Expression
***No Overt Sex differences in the C57BL/6 disease model***:
C57BL/6 Mouse Strain: Progressive disease model
→ There are no sex differences in regards to the severity of the disease in the
C57BL/6 mice when immunized for EAE.
→ The cellular mechanism explaining why there are no sex differences in B6 mice
is currently unknown, but it is known that the disease for both sexes is driven
by Th1/Th17 responses.
Significance of the meninges for
EAE:
Meninges show no sex differences in ILC2 promoting factors in
gene expression:
Meningeal CRTH2 Gene Expression
C57BL/6
Model
Strain specific
Mast
Cell (From
Female)
Female B6 D6
Male B6 D6
1
Figure 7
Flow cytometry run under
dyes FITC-A and APC. Cells
were gated under FCɛ and ckit receptors. Flow
cytometry indicated 98.6%
mast cell population in male
C57BL/6 culture and 99.1 in
female C57BL/6 culture.
4
Mast cells were then separated in 2 groups, one being
stimulated with IgE antibody (a common activator of
the cell) and the other having no stimulation. Mast cell
cultures were incubated for 26 days, then Mast cells
were preloaded with IgE-DNP (2μg/ml) overnight.
The mast cells were then lysed in cell buffer and then
exacted for RNA. The subsequent RNA of the cultured
mast cells were then synthesized into cDNA and run
similarly under a real time PCR, looking for the same
gene expression as the meninges.
turn into pictures … put into methods
Break up graph, 4 graphs .
*Marrow derived culture provides for a more mast cell
rich sample, thus limiting the possibility for other
innate immune or epithelial RNA to be found. As the
more “pure” sample shows, mast cell expression of
ILC2 is in line with the meningeal expression.
Are ILC2’s reduced in meninges during EAE? Do ILC1 and ILC3
make up the majority during the disease course?
Figure 6(below): Bone marrow Mast
cell culture gene expression:
Figure 6
Fold induction (IgE/NS)
Multiple sclerosis is a debilitating autoimmune disease of the central nervous system (CNS)
affecting more than 3 million people worldwide with 400,000 patients in the United States alone.
(Hauser SL et al. Neuron. 2006. and Noseworthy JH et al. NEJM. 2000.)
The disease is characterized by significant loss of motor and sensory function due to the destruction
of the myelin coating the nerve axons by inflammatory CD4+ T cells
Mast
Cell (From
Female)
Meninges and mast cells display no differences in ILC2
promoting factors between sexes:
Tnfa: Encodes tumor necrosis factor, a proinflammatory cytokine secreted by a variety of
lymphoid cells, including mast cells. TNFa recruits
activated neutrophils.
Il1b: Encodes IL-1b, which is produced by mast cells.
Il-1b drives Th17 responses from T cells.
Fold induction (IgE/NS)
Outcomes
What is multiple sclerosis (MS) and what is the disease pathophysiology of it?
ILC2
Crth2: Encodes the chemokine receptor for
prostaglandin D2, expressed primarily on Th2 cells and
ILC2s.
Tslp: Encodes thymic stromal lymphopoietin, which is
produced by mast cells and expands ILC2s.
Il33: Encodes the cytokine IL-33, which is secreted by
mast cells and induces proliferation of ILC2s.
Ptgds: Encodes the enzyme PGD Synthase, which
synthesizes prostaglandin D2, a strong
chemoattractant for ILC2s. Mast cells are a primary
source of PGD2s.
pathogenic
Promoter of variants
?
“Pathogenic”
Th1
BACKGROUND
Mast
Cell (From
Male)
“Protective”
Relative expression/HPRT
Intrinsic promoter
The meninges (discussed more below) are mast cell rich tissues and thus can
be used as a window into the in vivo mast cell responses. RNA was isolated
from meningeal samples from male and female immunized C57BL/6 mice (6
days post immunization). Complementary cDNA was synthesized and the
relative gene expression of certain genes (see right) was analyzed via
quantitative real time PCR.
Inflammatory Promoting Cytokine
Genes
Relative expression/HPRT
Mast
Cell
(From
Male)
Gene Expression of ILC2 Promoting Factors in Mast cells
in C57BL/6 Male and Female Mice:
ILC2 Promoting Cytokine
Genes
During EAE in both the male and female mice, the
Th1/Th17 immune response dominates. Given that the
gene expression of ILC2 promoting factors is both
relatively low and similar between the sexes, it
appears that ILC2 activation does not contribute to the
sex difference or cellular mechanism of EAE in
C57BL/6 mice.
15
Bone Marrow Mast Cell IL33
Fold Induction
50
Bone Marrow Mast Cell TNFa
Fold Induction
40
10
30
20
5
10
0
1.0
Female B6 Day6
Male B6 Day6
Bone Marrow Mast Cell TSLP
Fold Induction
0.8
8
Female B6 Day6
Male B6 Day6
Wild Type
Bone Marrow Mast Cell IL1b
Fold Induction
6
0.6
4
0.4
2
0.2
0.0
0
Female B6 Day6
Male B6 Day6
0
Female B6 Day6
Male B6 Day6
3
W/Wv C-Kit
mutation