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phenotypes.
In vitro
, cultured macrophages exposed to specific cy-
tokine cocktails express a relatively predictable gene expression
profile that has led to a classification system analogous to T-cell
polarization states. Specifically,
in vitro
stimulated macrophages can
be classified within activation states termed M1 and M2 (M2a, M2b,
M2c) depending upon exposure to a particular cytokine(s). Pro-
blematically, this linear classification system has been consistently
used to define microglia/macrophage response following brain
trauma, such that these responses are viewed as ‘either or’ or ‘on/
off’. We have recently shown that TBI induces a milieu of gene
expression responses associated with these defined polarization
states, which vary across time and importantly overlap such that both
M1 and M2 are expressed simultaneously. In the current study we
examined TBI-induced expression of M1 and M2 markers using
immunofluorescent labeling acutely after injury. Specifically, we
show that in the ipsilateral tissue there is simultaneous expression of
antigenic markers associated with both M1 and M2 profiles on the
same cell. Phenotypic antigen markers of M1 and M2 were CD45,
CD68, MARCO, YM1, CD206, and CD36. Co-staining using Iba1
and F4/80 defined these cells as microglia/macrophages. In the
context of trauma, the concept of microglia/macrophage polarization
is semantically and biologically incorrect due to the Janus-faced
nature of these cells.
Keywords: M1, M2, microglia, macrophage, polarization
C7-22
NOX2 REGULATION OF MICROGLIAL ACTIVATION IN
THE TBI BRAIN: A NOVEL MECHANISM FOR NEURO-
PROTECTION AND POST-TRAUMATIC REPAIR
David Loane
, James Barrett, Mariely Alvarez, Bogdan Stoica,
Flaubert Tchantchou, Alan Faden, Alok Kumar
University of Maryland, School of Medicine, Anesthesiology, Balti-
more, USA
Microglia can be polarized towards either an M1-like/classical or
M2-like/alternative activation status in response to injury. These
phenotypes can mediate chronic neuroinflammation or promote tissue
repair, respectively. Activation of NADPH oxidase (NOX2/gp91
phox
)
is an important mechanism involved in pro-inflammatory signaling in
microglia. We reported that NOX2 is chronically expressed in M1-
like microglia in the peri-lesional area 1 year following controlled
cortical impact (CCI) in mice. Here we compared wild-type (WT;
gp91
phox
+
/
+
) and NOX2-deficient (NOX2-KO; gp91
phox-/-
) mice to
investigate the role of NOX2 in posttraumatic microglial polarization.
Three-month old WT or NOX2-KO male mice were subjected to
CCI (6 m/sec, 2 mm depth), and cohorts were followed for 1–28 d
post-injury. M1-/M2-like polarization was analyzed by qPCR, flow
cytometry, Western blot, and immunohistochemistry. Neurogenesis
was assessed using doublecortin (DCX) immunohistochemistry; mo-
tor recovery and histology were assessed using a beam walk test and
stereological methods.
In WT TBI mice, NOX2 was expressed in reactive microglia
(CD68
+
/Clic1
+
) in peri-lesional cortex through 28d; NOX2-KO
significantly reduced CD68/Clic1 expression at 3 and 7d post-injury.
Flow cytometry analysis of isolated microglia/macrophages revealed
that IL-4R
a
and its downstream signaling pathway (STAT6/JAK3)
were significantly increased in CD45
high
microglia/macrophages of
NOX2-KO TBI mice compared to WT controls. M2-like polarization
(Arg1, Ym1, TGF
b
) was increased at 3d post-injury in NOX2-KO,
with effects sustained through 21d. There was significant reduction
M1-like polarized microglia/macrophages (IL-1
b
, TNF
a
, IL-12,
iNOS, CD16/32) in NOX2-KO TBI mice. M1/M2-like changes were
associated with increased numbers of DCX-positive cells in the sub-
ventricular zone, striatum and peri-lesional cortex, indicating in-
creased neurogenesis. NOX2-KO TBI mice also showed significantly
improved motor function and reduced cortical neurodegeneration at
21d.
Thus, after TBI NOX2-KO mice exhibit enhanced IL-4Ra-mediated
signaling, greater M2-like repolarization, and reduced neurodegen-
eration. Moreover, altering the M1-/M2-like balance appears to sup-
port increased neurogenesis. These data indicate that NOX2 drives the
M1-like polarization of microglia/macrophages after TBI, and that
inhibiting this pathway limits tissue damage and may promote tissue
repair.
Keywords: Microglial phenotypes, NOX2, neurodegeneration,
Tissue repair
C7-23
PHOSPHODIESTERASE 4B INHIBITION AS AN ANTI-
INFLAMMATORY TREATMENT FOR TRAUMATIC BRAIN
INJURY
Nicole Wilson
1
, Concepcion Furones
1
, Mark Gurney
2
, Dalton
Dietrich
1
, Coleen Atkins
1
1
University of Miami Miller School of Medicine, Neurosurgery, Mi-
ami, USA
2
Tetra Discovery Partners, Grand Rapids, USA
The anti-inflammatory effects of phosphodiesterase 4 (PDE4) inhibi-
tors are well established in CNS injury models. Knock out studies
have indicated that PDE4B is predominantly responsible for the anti-
inflammatory effects of pan-PDE4 inhibition. In the absence of
PDE4B, neutrophil infiltration and tumor necrosis factor (TNF) are
reduced in models of systemic inflammation. Furthermore, PDE4B2 is
upregulated in the injured cortex after traumatic brain injury (TBI). In
other models, PDE4B2 is expressed in microglia, macrophages and
neutrophils. However, whether PDE4B2 is localized in inflammatory
cells after TBI, and whether PDE4B inhibition reduces inflammation
and improves histopathological outcome after TBI is unknown. To
address this, adult male Sprague Dawley rats received moderate
parasagittal fluid-percussion brain injury (2
–
0.2 atm) or sham sur-
gery. Flow cytometry was used to analyze microglia and infiltrating
myeloid-lineage cells isolated from the injured cortex for PDE4B2
expression at 24 hrs after TBI. PDE4B2 was elevated in microglia and
infiltrating myeloid-lineage cells at 24 hrs TBI. To determine whether
PDE4B inhibition decreases TNF and neutrophil infiltration in the
injured cortex, animals received vehicle (5% DMSO in saline) or a
PDE4B inhibitor, A33 (2-(4-{[2-(5-chlorothiophen-2-yl)-5-ethyl-6-
methylpyrimidin-4-yl]amino}phenyl)acetic acid) at 0.3 mg/kg (i.p.) at
30 min and 5 hrs post-surgery. TNF was measured at 6 hrs post-
surgery using an ELISA. Neutrophil infiltration was assessed at 24 hrs
post-surgery using flow cytometry. PDE4B inhibition significantly
decreased TNF levels and neutrophil infiltration after TBI. To deter-
mine whether PDE4B inhibition reduces pathology after TBI, animals
received vehicle (5% DMSO in saline) or A33 (0.3 mg/kg, i.p.) at
30 min post-surgery and once daily for 3 days. Cortical contusion was
evaluated in serial brain sections stained with hematoxylin and eosin.
PDE4B inhibition significantly reduced cortical contusion volume at 3
days post-injury. These results suggest that inhibiting PDE4B after
TBI may be a viable treatment to reduce inflammation and pathology.
Supported by The Miami Project to Cure Paralysis and NIH/NINDS
NS056072 and NS089351.
Keywords: Phosphodiesterase 4B, PDE4B
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