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Page 26 of 32 Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18

Interestingly in DRGs, IL-1β levels were greater in neuropathic females. It is possible that the combination
of T cell-mediated responses, along with myeloid-driven proinflammatory actions culminate in greater
nociceptive factors that induce further hyperexcitability relayed to the spinal cord in females. The fact that
reliable induction of FOXP3 was observed only in female DRGs may reflect the anti-inflammatory rebound
in response to inflammatory signals. Recruitment of Treg cells could function to control bystander injury-
related proinflammatory cytokines.

Sex convergent and sex divergent aberrant spinal immune responses underlying chronic pain
[26]
Despite evidence of microglial activation in neuropathic females , microglial TLR4 signaling is only
necessary for the development of neuropathy in males, whereas astrocytic signaling under neuropathic
conditions is observed in both males and females [3,25,26] . Supporting prior observations, we detected
[4]
astrocytic and microglial activity in both sexes during neuropathy [Figure 8] . However, we noticed that
induction of ipsilateral CCL2 in conjunction with IL-1β was greater in females [Figure 7]. The reduction of
basal IL-10 levels, a finding that our group has previously observed in chronic neuropathic male rats [93,94] ,
appeared more pronounced in neuropathic female than male mice. Note that along with greater astrocyte
(as assessed by increased GFAP) and microglial activation (TMEM119) and increased CCL2 in the
contralateral side, a simultaneous decrease in IL-10 was measured, indicating that contralateral spinal cord
IL-10 expression in females may reflect a greater impact of this cytokine in controlling proinflammatory
contralateral glial activation. It is noteworthy that, other than CCL2 in females, changes in injury-related
contralateral spinal IL-1β or TGF-β1 were not detectable in males or females, despite ongoing contralateral
allodynia. Therefore, the reduction of the basal levels of spinal IL-10, rather than the presence of these
specific proinflammatory cytokines, is likely a better indicator of ongoing allodynia.

We speculate that T cell-mediated proinflammatory cytokines (e.g., IL-17A) at the injury site may
consequently drive sciatic “damage” signals, leading to the release of factors from nerve terminals in the
spinal cord that communicate to pain projection neurons. Astrocytes and microglia local to the dorsal
horn of the spinal cord respond to these damage signals from SCN terminals. Though activated astrocytes
are capable of producing IL-17A [95,96] , contralateral IL-17A was not detected despite astrocyte activation,
suggesting that contralateral IL-17A is not a key factor in contralateral glial activation. In fact, the absence
of contralateral IL-17 may reinforce the possibility that ipsilateral immune-related signaling may drive
contralateral spinal cord pain neuron excitability via astrocyte-specific gap junctional communication [73,80] .
Interestingly, supraspinal mechanisms such as activation of cortical areas important in pain processing,
and descending facilitation from key brainstem areas may contribute in contralateral allodynia as well [97,98] .
Proinflammatory cytokines in pain related brain regions are capable of impairing descending inhibitory
[99]
pain pathways . Whether, differential immune mechanisms following nerve injury influence the
descending pathways involved in manifesting mirror image pain in different sexes would be an interesting
avenue for future exploration.

Though astrocytic activation during neuropathy is common in both sexes, it is possible that microglia in
males and infiltrating Th17 cells in females are the predominant cell types responsible for driving chronic
excitation of astrocytic-neuronal interaction. In support of this possibility, the current report demonstrated
a robust upregulation of IL-17A in the spinal cord of neuropathic females. Our prior data indicates the
presence of activated T cells (T-bet and RORγt mRNA transcripts, which are critical transcription factors
for Th1 and Th17 respectively) in the ipsilateral LSC in neuropathic female rats . Therefore, Th17 cells
[100]
likely infiltrate the ipsilateral spinal cord and interact with astrocytes where ongoing pathology is present,
inducing a feed-forward astrocyte-proinflammatory chemokine (e.g., CCL2) and cytokine production [101,102] ,
as observed in this study. However, re-programming of differentiated T cells and their functional responses
can occur in response to the local cytokine milieu in the CNS [102-104] . Therefore, the absence of contralateral
IL-17A does not prove a lack of T cell recruitment or their actions in contralateral neuropathy.