Page 95 - Read Online

Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18 Page 17 of 32

of IL-17A than neuropathic males (P = 0.002). These data suggest that Th17 cells may play a more
prominent role in females with peripheral neuropathy. This is further supported by BIRT377-mediated
reduction of IL-17A mRNA levels in SCNs of pain-reversed females (P < 0.0001). Spinal IL-17A mRNA
transcripts were absent under non-neuropathic sham-treated conditions in males and females [Figure 4F].
Compared to sham controls, large increases in LSC IL-17A mRNA levels were observed ipsilaterally in
neuropathic females (P < 0.0001), with modest increases in IL-17A in neuropathic males [Figure 4F]. IL-17A
was not reliably detected in the DRGs or contralateral spinal cord samples in any groups. BIRT377 treatment
abolished ipsilateral spinal IL-17A mRNA levels in neuropathic females (P < 0.0001) and, to a lesser extent,
in neuropathic males. These data, along with the data presented in Figure 3G suggest that though there was
no difference in overall content of the T cell (CD3 mRNA) population, the quality and differentiation status
of these T cells varied in neuropathic animals. These data also show that the actions of BIRT377 on these
differentiated T cell subsets is most pronounced in females and may reflect a phenotypic change from
proinflammatory to anti-inflammatory, rather than simply reflecting a suppression of T cell recruitment.

BIRT377 treatment exerts sex-dependent differential effects on T cell differentiation and
functional responses
While BIRT377-mediated reduction of IL-17A is indicative of effects of BIRT377 on CD4 T cell
differentiation and function, these effects may also be due to the indirect effects of a general reduction in
[83]
proinflammatory cytokine production (such as TNF) from monocytes that promote Th17 differentiation .
Therefore, to examine the direct actions of BIRT377 on CD4 T cell differentiation and function, CD4
naïve T cells were given conditioned media to induce the generation of either a Treg or Th17 phenotype,
in the presence of control (media only) or BIRT377. Subsequently, the proportion of T cells positive
+
for RORγt (transcription factor required for the generation of Th17 cells), was analyzed. BIRT377 only
+
reduced the generation of RORγt T cells in females (P = 0.0007), but not in males [Figure 5A]. Additionally,
+
CD4 T cells that are IL-17A , and also produce TNF, were examined as an indication of their functional
+
+
+
proinflammatory capacity. Compared to conditioned media alone, the population of IL-17A TNF CD4
+
T cells was substantially reduced by BIRT377 exposure only in CD4 T cells derived from females (P =
0.001), but not males [Figure 5B]. Furthermore, Treg generation and function in the presence of BIRT377
was examined. Fully differentiated Tregs exert their immune suppressive actions by producing the
[57]
characteristic anti-inflammatory cytokines, IL-10 and TGF-β1 . Therefore, the expressions of IL-10 and
TGF-β1 proteins were examined as direct evidence of the fully differentiated functional Treg cells. Given
that FOXP3 drives Treg generation concurrent with IL-10 and TGF-β1 production, FOXP3 expression
was considered redundant. During Treg differentiation in the presence of BIRT377, a large increase in the
production of the IL-10 (P = 0.0005) and TGF-β1 (P = 0.014) was observed [Figure 5C and D] in female-
derived pooled T cells, while BIRT377-induced changes in these anti-inflammatory cytokines were absent in
male derived T cells. While a trend of increased IL-10 CD4 T cells was also observed in males [Figure 5C],
+
+
these data demonstrate that female T cells are much more responsive to BIRT377-mediated modulation of
pro- and anti-inflammatory T cell-related cytokines. Therefore, BIRT377 regulates one aspect of T cell
differentiation and function more readily in females under pathological conditions, which may provide a
mechanism for the IL-17A reduction reliably detected only in females at the SCN [Figure 4].

BIRT377 modulates the proinflammatory cytokine milieu in the DRGs to favor pain reversal
The most widely examined proinflammatory cytokines known to be critical for pain processing were
examined in the ipsilateral DRGs. As predicted, neuropathic male and female (CCI + Veh) mice revealed
increases in CCL2 (male: P = 0.01; female: P = 0.016), IL-1β (males: P = 0.0005; female: P < 0.0001), TNF (P <
0.0001: both sexes). In support of prior reports, a compensatory elevation in anti-inflammatory IL-10 (P < 0.0001:
both sexes) mRNA levels in the ipsilateral DRGs as compared to Sham + Veh was also measured [Figure 6].
While CCL2 mRNA levels were increased in males and females following CCI, BIRT377 did not alter CCL2
mRNA levels under either condition, and sex differences were not observed. However, a reduction in IL-1β