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Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18 Page 15 of 32

of changes observed in males. mRNA levels of the anti-inflammatory cytokines, IL-10 (male: P < 0.0001;
female: P = 0.034) and TGF-β1 (male: P = 0.0001; female: P = 0.046) were increased in CCI + Veh mice
compared to sham-operated conditions (Sham + Veh) [Figure 3D and E]. These data reflect the predicted
peri-sciatic anti-inflammatory compensatory response to control ongoing inflammation at the injured
SCN [47,51] . Neuropathic females had lower levels of IL-10 mRNA than their male counterparts (CCI + Veh:
P = 0.001). Interestingly, while BIRT377 treatment in neuropathic males did not induce further increases
in these anti-inflammatory cytokines, notable mRNA increases in both IL-10 (P = 0.002) and TGF-β1 (P =
0.006) were measured in females [Figure 3D and E]. Additionally, because low-level cytokine/chemokine
expression remains unaltered in sham-surgery animals given BIRT377 (Sham + BIRT), these data suggest
a permissive effect of BIRT377’s action in and around activated peripheral immune cells that have already
migrated to the local site of injury in the female SCN microenvironment.

To confirm the presence of the monocytes/macrophages and T cells, which are well-characterized to
produce CCL2, IL-1β, TNF, IL-10 and TGF-β1, mRNA levels for CD11b (pan myeloid cell marker) and
CD3 (pan T cell marker) were evaluated [Figure 3F and G]. While all neuropathic mice (CCI + Veh) reveal
significant increases in peri-sciatic CD11b (male: P < 0.0001; female: P = 0.0001) and CD3 (male: P < 0.0001;
female: P = 0.005) mRNA levels, reflecting that these peripheral immune cells have migrated to the damaged
SCN, the magnitude of increase was greater in males than females for CD11b (P = 0.0002). Sham animals
treated with BIRT377 did not result in alterations of CD11b and CD3 mRNA levels. However, BIRT377 did
significantly reduced mRNA levels of CD11b (P = 0.0002) and CD3 (P = 0.016) in neuropathic males. These
data indicate that by 4 days following i.v. BIRT377 injection, a reduction in both monocyte/macrophage and
T cell recruitment around the injured nerve occurred in males. Importantly, these data demonstrate that
despite similar levels of CD11b and CD3 mRNA in CCI + Veh and CCI + BIRT females, TNF and IL-1β are
reduced, indicating that BIRT377 alters functional responses of immune cells previously recruited around
the SCN in females. That is, BIRT377 may be exerting actions on immune cells beyond simply preventing
leukocyte trafficking. Given the observed elevation in anti-inflammatory IL-10 and TGF-β1 mRNA levels
and reduction in proinflammatory cytokines discussed above, BIRT377 may be dampening the degree of
peripheral “damage” signals relayed from the peripheral nervous system to the central nervous system
(CNS).

Sex differences observed in the effects of BIRT377 on reduced mRNA levels of T cell-specific
pro- and anti-inflammatory responses
Previous reports demonstrate potential differential contribution of T cell-mediated responses in males and
[3]
females . In the current report, the T cell specific factors, FOXP3 (anti-inflammatory-like T cells) and IL-17A
(proinflammatory-like T cells) were analyzed in key anatomical regions of the pain pathway following CCI
[Figure 4]. A potential cellular source of anti-inflammatory cytokines is from a subset of T cells referred to
as Tregs cells [57,82] . The transcription factor responsible for generating Treg cells is FOXP3 [57,59] . Therefore, to
identify a possible source of IL-10 and TGF-β1 (demonstrated in Figure 3D and E), the contribution of Treg
cells was indirectly explored by examining FOXP3 mRNA levels. Compared to sham treatment, elevated
FOXP3 mRNA was observed in SCN of males (P < 0.0001) and females (P = 0.003) given vehicle. FOXP3
mRNA levels were further elevated from CCI + Veh group following BIRT377 treatment, only in females (P <
0.0001) [Figure 4A]. Similarly, DRGs revealed elevated FOXP3 mRNA levels in CCI + Veh females (P = 0.034),
while no such increases were observed in males [Figure 4B]. However, BIRT377 did not alter basal FOXP3
mRNA levels in DRG in sham or CCI groups. These data indicate a modest recruitment of Tregs in female
DRGs. In contrast to effects observed in females of peripheral tissues (SCN and DRG), only males revealed
changes in FOXP3 mRNA levels in the ipsilateral spinal cord, with no FOXP3 mRNA changes observed in the
contralateral spinal cord [Figure 4C and D]. Th17-specific proinflammatory cytokine, IL-17A, increases were
detected in SCN of both neuropathic males (P = 0.04) and females (P < 0.0001), when compared to sham-
surgery groups [Figure 4E]. Neuropathic (CCI + Veh) females displayed about twice as much upregulation