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Page 24 of 32 Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18

observations may indicate that further immune cell differentiation occurs after their migration to key
pain-relevant nervous tissue regions in response to signals from local tissue-damage.

DISCUSSION
Reports focused on understanding neuroimmune changes when performing a comparative approach
between sexes are rare with most studies applying male rodent models [4,25-27] . In recent years, published
reports provide compelling evidence that activation of spinal microglia play a direct role in generating
[3,4]
pathological pain in males, while in females, the actions of T cells are critically important . Consistent
with prior reports [4,26] , we find that, while the onset, magnitude and spontaneous reversal of allodynia are
similar in males and females [Figure 1], divergent peripheral immune and neuroimmune responses are
present during neuropathy. We demonstrate for the first time that during neuropathy, T cell-associated pro-
and anti-inflammatory responses in males and females are different at discrete anatomical regions critical in
the pain pathway of sciatic neuropathy. During neuropathy, females displayed more profound Th17 specific
responses (IL-17A) than males, both at the injured nerve and in the corresponding LSC [Figure 4]. While
regulatory T cell (Tregs) recruitment (FOXP3 expression) was evident at the injured SCN in both males and
females, only females displayed reliable increases of FOXP3 at the DRGs [Figure 4]. The beneficial role of
blocking the active conformational state of LFA-1 was demonstrated in both sexes [Figure 2] by reducing
immune cell accumulation in damaged SCN [Figure 3]. However, BIRT377 modulated T cell function in a
sex-specific manner [Figure 5]. For example, T cells from females were significantly more responsive to the
anti-inflammatory effects of BIRT377 [Figure 5]. Similarly, BIRT377 treatment elevated T cell-associated anti-
inflammatory factors (FOXP3, IL-10 and TGF-β1) and reduced the proinflammatory T cell cytokine, IL-17A
in the peri-sciatic milieu, predominantly in neuropathic females [Figures 3 and 4]. Interestingly, despite the
fact that there was no additional change in FOXP3 expression in the LSC, the profound reduction of IL-17A in
the LSC in i.v. BIRT377 treated females indicates a limited role exists for spinal Treg actions on pain reversal.
Importantly, a reduction of peri-sciatic IL-17A co-occurs with profound spinal cord suppression of IL-17A,
suggesting the excitatory input from centrally projecting nerve terminals into the lumbar spinal region
ultimately leads to a reduction in proinflammatory factors that includes IL-17A. While these data demonstrate
that a potential role for spinal IL-17A in pro-nociceptive signaling occurs, it remains unclear whether IL-17A
acts in concert with other well-characterized spinal proinflammatory factors, or whether IL-17A is a necessary
factor in pain signaling. Therefore, the results from the current data provide the rationale for performing
future studies to examine whether specifically blocking the spinal actions of IL-17A also suppresses allodynia
from peripheral neuropathy. While these additional studies would aid in understanding the role of IL-17A
in chronic neuropathic pain, the current data are the first documented evidence that a reduction in lumbar
spinal IL-17A expression co-occurs with a reduction in allodynia from CCI in both males and females [Figure 4].
Strikingly, peripheral BIRT377 reduced spinal astrocyte activation, but had little impact on microglial activation
[Figure 8]. Overall, BIRT377 created an anti-inflammatory bias in discrete regions along the pain pathway of
the CCI model in both sexes [Figures 3-7], thereby contributing to pain reversal. A brief summary of immune
changes during CCI-induced neuropathy and BIRT377-mediated effects are listed in Table 1. This initial
comparative analyses of glial/myeloid and T cell-related cytokines and their corresponding transcription
factors that are altered by preventing β2-integrin (LFA-1) signaling, provides insight into possible mechanisms
leading to peripheral sciatic neuropathy between males and females.

Sex differences in peripheral inflammatory reactions to nerve injury
Remarkable sex differences of immune system activity are observed in different disease models [85-89] . Sex
differences in TLR4 responses to pathogen stimulation have been observed, whereby females produce
[85]
similar or less IL-1β and TNF compared to males . Female-derived immune cells are more efficient
[90]
in antigen presentation and initiating adaptive immune responses . In the CCI model, peripheral
inflammatory reactions to nerve injury are mediated by endothelial cells of the blood-nerve barrier and