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Page 2 of 32 Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18

onset, magnitude and duration of allodynia. Hindpaw sensitivity before and after CCI and application of intravenous
BIRT377 was assessed. Peripheral and spinal tissues were analyzed for protein (multiplex electrochemiluminescence
technology) and mRNA expression (quantitative real-time PCR). The phenotype of peripheral T cells was determined
using flow cytometry.

Results: Sex differences in proinflammatory CCL2 and IL-1β and the anti-inflammatory IL-10 were observed from
a set of cytokines analyzed. A profound proinflammatory T cell (Th17) response in the periphery and spinal cord
was also observed in neuropathic females. BIRT377 reversed pain, reduced IL-1β and TNF, and increased IL-10 and
transforming growth factor (TGF)-β1, also an anti-inflammatory cytokine, in both sexes. However, female-derived
T cell cytokines are transcriptionally regulated by BIRT377, as demonstrated by reducing proinflammatory IL-17A
production with concurrent increases in IL-10, TGF-β1 and the anti-inflammatory regulatory T cell-related factor,
FOXP3.

Conclusion: This study supports that divergent peripheral immune and neuroimmune responses during neuropathy
exists between males and females. Moreover, the modulatory actions of BIRT377 on T cells during neuropathy are
predominantly specific to females. These data highlight the necessity of including both sexes for studying drug
efficacy and mechanisms of action in preclinical studies and clinical trials.

Keywords: Neuropathic pain, glia, neuroimmune, peripheral immune, T cells

INTRODUCTION
While male and female rodent models of peripheral neuropathic pain generate similar clinical features
such as pathological sensitivity to light touch referred to as allodynia, emerging evidence suggests that the
biochemical and cellular aspects underlying allodynia are different between the sexes. Clinical evidence
[1,2]
strongly implicates sex differences in pain sensitivity , and preclinical data supports these clinical
findings by demonstrating that peripheral immune and glial cells exhibit sex differences in response
[3-6]
to peripheral nerve injury leading to neuropathy . Understanding sex divergent components of pain
pathophysiology has drawn significant attention and is of paramount importance for identifying effective
pain therapeutics in males and females.

Chronic neuropathic pain following peripheral nerve injury involves dynamic neuroimmune interactions
between peripheral immune cells that traffic to the injured nerve, the dorsal root ganglia (DRG), and
the spinal cord, and the actions of glial cells within the spinal cord . Peripheral nerve injury leads
[7-9]
to alterations in proinflammatory cytokines including interleukin (IL)-1β, IL-6, and TNF, and anti-
inflammatory cytokines such as IL-10 and transforming growth factor-β (TGF-β1) at anatomical sites of
the pain pathway [10-16] . Spinal glia and DRG satellite glia contribute to persistent allodynia by responding
to and releasing these proinflammatory cytokines and by reducing IL-10 expression [12,14,17-19] . Additionally,
the chemokine CCL2 is elevated in DRG of injured nerves and facilitates leukocyte migration to DRG and
spinal cord [19,20] . However, most of these reports were studied male models of neuropathy or the sex was
unspecified, with few studies utilizing females [21-24] , or compared sex differences underlying pathological
pain [3-5,25-28] .

A substantial role of the adaptive immune response is now recognized as underlying aberrant
neuroimmune actions following nerve injury. Activated CD4 T cells, specifically proinflammatory Th1 and
Th17 cells, infiltrate the injured peripheral nerve and the lumbar spinal cord (LSC) [29-33] , and are thought
[34]
to contribute to glial activation . Conversely, anti-inflammatory T regulatory (Treg) cells control spinal
[35]
glial-immune proinflammatory activation and are protective against neuropathy . A few studies have