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Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18 Page 27 of 32

Sex-specific mechanistic differences of BIRT377 pain reversal
BIRT377-mediated effects on myeloid/glial activation
Numerous reports suggest that blocking LFA-1 actions restricts migration of monocytes and T cells to
injured tissues [37,105] . Following BIRT377 treatment, both males and females display decreases in peri-sciatic
IL-1β and TNF, which are generally myeloid-derived. Reduced CD11b levels around the injured nerve are
found only in males. Together, these data suggest BIRT377 may reverse pain in males mainly by blocking
myeloid cell migration and consequent exposure to proinflammatory cytokines. However, in females, the
effect of blocking the active conformation of LFA-1 by BIRT377 appears to directly alter transcriptional
regulation of pro- and anti-inflammatory cytokines of myeloid-derived cells. Previous studies suggest
that a lack of LFA-1 interaction with leukocytes increases IL-10, switching macrophage activation from a
proinflammatory bias to an anti-inflammatory state [38,42] . Therefore, BIRT377-mediated re-programing of
myeloid cell function may also occur, and possible sex differences regarding these observations need to be
further explored.

BIRT377-mediated effects on T cells
Though the exact mechanism(s) are unclear, LFA-1 signaling interacts with T cell activation, and therefore,
modulates adaptive immune responses [44,54] . While sex was not specified, previous studies suggest that
+
blocking LFA-1 actions decreases Th17 differentiation and increases FOXP3 Tregs [39,106] . Consistent with
the in vitro T cell findings demonstrated in the current report, in vivo increases in IL-10 and TGF-β1 were
observed along with increases in FOXP3 and reduced IL-17A levels at the SCN only in females following
BIRT377 treatment. Therefore, BIRT377 treatment is beneficial for pain reversal by affecting both immune
cell migration and modulation of their actions at local sites of inflammation, thereby, indirectly influencing
the spinal-immune milieu during neuropathy. Interestingly, for both sexes, BIRT377 did not change
microglial activation, suggesting that reducing astrocytic activation and increasing IL-10 levels at the spinal
cord is sufficient to reverse allodynia.

We have recently reported that intrathecal (spinal) application of BIRT377 in a rat CCI model leading
to chronic neuropathy, dorsal horn spinal astrocyte activation and IL-1β are reduced, as evidenced by
[107]
immunohistochemical staining and image analysis measures . Though BIRT377-mediated effects on mRNA
levels of IL-17A, IL-10, and TGF-β1 are supportive of the protein levels of these cytokines [Figure 5], direct
quantification of protein levels of all the diverse immune markers would further strengthen the findings in the
current report [30,32] , along with semi-quantitative analysis of expression markers using immunohistochemical
and image analysis methods that can capture within-region specific changes in comparatively sparse T cell
subtypes [33,108] .

In conclusion, this study supports the presence of divergent proinflammatory cytokines in males and
females following peripheral nerve injury, which has important implications when developing pain
therapeutics. Despite the observed cytokine/chemokine and related transcription factor expression
differences in SCN, DRG and LSC, systemic blockade of LFA-1 activation is beneficial for pain reversal in
both sexes. Therefore, BIRT377 may serve as a novel therapeutic for chronic pain and other CNS diseases.

DECLARATIONS
Acknowledgments
The authors thank Dr. Vojo Deretic in the Department of Molecular Genetics and Microbiology at UNM
for providing resources for magnetic separation of CD4 T cells.

Authors’ contributions
Performed behavioral assessments and extracted RNA from DRG tissues: Havard MA
Performed behavioral assessments: Sanchez JE, Harris NW
Experimental design, flow cytometry, real time PCR, data analysis and manuscript preparation: Noor S