T lymphocytes potentiate endogenous
neuroprotective inflammation in a mouse
model of ALS
Isaac M. Chiua,1, Adam Chenb, Yi Zhenga, Bela Kosarasc, Stefanos A. Tsiftsogloua, Timothy K. Vartanianc,
Robert H. Brown Jr.b, and Michael C. Carrolla
aDepartment of Pathology, Immune Disease Institute, andcBeth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115; andbDay
Neuromuscular Research Laboratory, Massachusetts General Hospital, Charlestown, MA 02129
Edited by Jack L. Strominger, Harvard University, Cambridge, MA, and approved September 18, 2008 (received for review May 13, 2008)
Amyotrophic Lateral Sclerosis (ALS) is an adult-onset, progressive,
motor neuron degenerative disease, in which the role of inflam-
mation is not well established. Innate and adaptive immunity were
investigated in the CNS of the Superoxide Dismutase 1 (SOD1)G93A
transgenic mouse model of ALS. CD4?and CD8? T cells infiltrated
SOD1G93Aspinal cords during disease progression. Cell-specific
flow cytometry and gene expression profiling showed significant
phenotypic changes in microglia, including dendritic cell receptor
acquisition, and expression of genes linked to neuroprotection,
cholesterol metabolism and tissue remodeling. Microglia dramat-
ically up-regulated IGF-1 and down-regulated IL-6 expression.
When mutant SOD1 mice were bred onto a TCR? deficient back-
ground, disease progression was significantly accelerated at the
symptomatic stage. In addition, microglia reactivity and IGF-1
levels were reduced in spinal cords of SOD1G93A(TCR??/?) mice.
These results indicate that T cells play an endogenous neuropro-
tective role in ALS by modulating a beneficial inflammatory re-
sponse to neuronal injury.
amyotrophic lateral sclerosis ? microglia ? neuroimmunology ?
neuroinflammation ? T cells
inherited form of Amyotrophic Lateral Sclerosis (ALS) is linked
to mutations in the Cu2?/Zn2?superoxide dismutase (SOD1)
gene (1). Mice overexpressing human mutant SOD1 develop
motor pathology resembling ALS (2).
In human patients and mutant SOD1 transgenic mice, loss of
motor neurons is accompanied by robust microglia and astrocyte
activation (3). Several lines of evidence implicate involvement of
non-neuronal cells in ALS. Blastocyst chimera studies showed
that mutant SOD1 expressed by neighboring cells negatively
affected survival of motor neurons (4). Conditional deletion
experiments demonstrated that mutant SOD1 reduction in
CD11b? myeloid cells, including microglia, lengthened lifespan
(5). Neonatal bone marrow transplants, replacing the microglia
niche with wild-type cells, also ameliorated disease in mice (6).
One mechanism for microglia induced neurotoxicity may be
reactive oxygen species through NADPH oxidase (7). The role
of inflammation in neurodegenerative disease, however, is com-
plex and may effectuate both beneficial and harmful outcomes
on neuronal survival (8).
Activation of innate (e.g., microglia) and adaptive (e.g., T
cells) immunity has been documented in mutant SOD1 mice (9,
10). Prior studies on neuroinflammatory changes resulted from
whole spinal cord expression profiling (11, 12). Mutant SOD1,
however, dysregulates multiple cell types—including astrocytes
(13, 14), motor neurons, and microglia (5). Therefore, a targeted
analysis of immune cell types is necessary to define their
particular roles in ALS.
In this study, we characterized microglia and lymphocytes
directly isolated from the CNS of SOD1G93Atransgenic mice,
LS is characterized by selective degeneration of motor
neurons, leading to paralysis and death. The most common,
and analysis of these cells identified significant changes in
surface receptor profiles and neurotrophic factor expression.
Specific ablation of T cells led to decreased microglia reactivity,
growth factor expression, and accelerated disease progression.
These findings provide evidence for a beneficial role for inflam-
mation, which poses significant ramifications on immune-
targeted therapies in ALS.
CNS Inflammatory Subsets in Transgenic SOD1G93AMice. To under-
stand cellular contributions that characterize local inflammation
in the CNS microenvironment, we examined population dynam-
ics of microglia and specific lymphocyte subsets during disease
progression. SOD1G93A, SOD1WT, and non- Transgenic (Tg)
mice were analyzed at presymptomatic (day 65), early symptom-
atic (day 100), and end-stage (day 135).
In non-Tg mice, negligible numbers of lymphocytes (CD11b-
CD45hi) were present in the spinal cord at all time-points (Fig.
1 A and B). In SOD1G93Amice, an increased lymphocyte
population was present at day 65 (Fig. 1B). This population
showed significant enlargement with disease progression (Fig.
1A, Gate C), increasing 36-fold relative to non-Tg littermates by
day 135 (Fig. 1B). The resident microglia (CD11b? CD45lo)
population expanded 1.65-fold relative to non-Tg mice by end-
stage (Fig. 1A, Gate A, Fig. 1C). The peripheral monocyte
(CD11b?CD45hi) (Fig. 1A, Gate B).
The infiltrating lymphocyte population was further character-
ized to determine respective contributions by B, T, and natural
killer cell subsets. Spinal cords showed significant accumulation
of CD4?and CD8? T cells, but not CD19? B cells (Fig. 1D).
CD4?cells were positive for pan-T cell marker CD3 and
up-regulated activation marker CD69 [supporting information
(SI) Fig. S1]. A significant natural killer cell population
(NK1.1?) also appeared in SOD1G93Aspinal cords (Fig. 1D).
Lymphocyte influx was more pronounced in the spinal cord than
not occur in non-Tg or wild-type SOD1 transgenic (SOD1WT)
mice (Table S1). Therefore, recruitment of peripheral NK and
T cells contributes significantly to neuroinflammatory popula-
tions in ALS Tg mice.
not increase detectably
analyzed data; and I.M.C. and M.C.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0804610105 PNAS ?
November 18, 2008 ?
vol. 105 ?
no. 46 ?
by FACS (Fig. S4). Purified microglia were immediately lysed in TRIzol, RNA
transcribed into cDNA with iScript cDNA synthesis kit (Bio-Rad). Quantitative
and detailed protocols, see SI Text.
ACKNOWLEDGMENTS. We thank Michael Haas, Vijay Kuchroo, and Tammy
Hshieh for helpful, critical discussions; Eugene Ponomarev for CNS cell isola-
tion and adult microglia culture advice; Laura Santambrogio, Jeng-Shin Lee,
and Viraga Haridas for technical help; and Monica Carrasco for help with
image analysis. This work was funded by the Amyotrophic Lateral Sclerosis
Association (M.C.C., I.M.C.); National Institutes of Health Training Grant AI
007306-23 (to I.M.C.); and the ALS Association, National Institute for Neuro-
logical Disease and Stroke, National Institute for Aging, Angel Fund, Project
ALS, Pierre L. de Bourgknect ALS Research Foundation, and Al-Athel Amyo-
trophic Lateral Sclerosis Foundation (R.B.).
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