Our results reveal sex differences in the DRG in both the EAE mouse model of MS, and our TNFα-treated culture model. Both male and female EAE DRGs exhibit increased staining for inflammatory markers, but notably, males have greater macrophage/ monocyte staining than females at the onset of EAE. In addition, male macrophages and monocytes have more Dectin-1 staining in male EAE mice, suggestive of greater inflammatory activation. Using an in vitro preparation of DRG neurons stimulated with the inflammatory cytokine TNFα, we observed that males preferentially activated P38 MAPK, and presented with disrupted mitochondrial morphology, elevated mitochondrial superoxide production, and cytochrome c release. This mitochondrial pathology is likely responsible for the increased caspase 3 cleavage we observed in the male DRG both in vitro and in vivo. Females, however, preferentially activated the transcription factor NFκB rather than P38, and while their mitochondrial morphology was also altered, they did not elevate superoxide production, cytochrome c release, or caspase 3 cleavage. Rather, we show that female EAE DRGs have increased levels of the injury and plasticity-associated factors, ATF3 and pCREB in vivo. These observations have significant implications for our understanding of neuropathy and pain in chronic neuroinflammatory disorders such as MS.
First, we found a significant increase in global inflammation and immune cell infiltration indicated by CD45, CD3, and Iba1 staining at the level of the DRG in the EAE model in both sexes. Many studies of neuropathic pain using peripheral nerve injury models have focused on the spinal cord and report that females have a T-cell-mediated pain phenotype [44, 45]. This, however, may not be true for the onset of peripheral sensitization in the EAE DRG, as we saw no sex differences in CD3 T cell infiltration between the sexes. However, the indication that microglia/macrophages are responsible for pain in the male spinal cord [44–47] may be analogous to the EAE DRG, as evidenced by our finding that more cells were stained for Iba1 in males, and that a greater number of these Iba1-positive cells were co-labelled with the inflammatory marker Dectin-1. DRG macrophages have already been identified as important players in peripheral sensitization and neuropathic pain in a multitude of studies [48]. For example, their secretion of pro-inflammatory mediators, including TNFα, can directly increase sensory neuron excitability [49–51]. We found increased TNFα mRNA in both male and female EAE DRGs, and our lab has previously confirmed that TNFα is increased peripherally in EAE [21]. This motivated us to use TNFα as our in vitro stimulus to further elucidate sex differences in intracellular signaling in DRG neurons.
To determine if the inflammatory profile we identified in EAE DRGs has consequences on intracellular neuronal processes, we moved in vitro, where we observed sex differences in the downstream signaling pathways engaged by TNFα stimulation. Our finding that male neurons are biased towards activation of P38 in response to TNFα is supported by studies which have shown that males activate P38 in the spinal cord in mouse models of neuropathic pain [52, 53]. Additionally, we observed that female neurons preferentially activate NFκB with TNFα stimulation. This aligns with reports indicating that females preferentially activate NFκB in endothelial cells and inflamed lung tissue [54, 55].
Given the strong links of both P38 and NFκB to functional changes in the mitochondria [22–25], we went on to conduct a surface-level investigation of the changes that occur in mitochondrial morphology in response to TNFα. We found that female neurons increased their mitochondrial footprint in response to 24 hours of TNFα treatment, and that their mitochondrial morphology responded more quickly to treatment than males. We observed more fragmented mitochondria, and less tubular mitochondria in females after one hour of TNFα treatment compared to control, which may indicate a maladaptive phenotype such as impaired mitochondrial fission. However, at 6 and 24 hours of TNFα treatment, female mitochondrial networks appeared to shift their response to TNFα treatment, as they exhibited less fragmented mitochondria and more tubular mitochondria compared to control. Male neurons also exhibited less fragmentation and more tubular networks at 24 hours of treatment but showed no significant changes at earlier timepoints.
We hypothesize that the decreased fragmentation and increased tubular mitochondrial networks we observed after TNFα treatment may be evidence of an adaptive response to inflammation, which appears be more effective in females than in males. To further investigate this mechanism, we turned to functional readouts of mitochondrial health in our TNFα-stimulated culture, superoxide production and cytochrome C release. We found that male DRG neurons produce more superoxide and release more cytochrome C than females in response to TNFα. This led us to speculate that DRG neurons initiate an adaptive response to inflammation that is more effective in females than in males, potentially due to their differential activation of the TNFα signaling pathways.
The dramatic increase in cytochrome C release from male neurons lead us to investigate caspase 3 cleavage. We found that both in vitro and in vivo, male DRGs exhibit more cleaved caspase 3 staining than females. This finding aligns with TNFα signaling literature as P38 can cause cytochrome C release and eventually, intrinsic apoptosis through B-cell lymphoma (Bcl) protein regulation [40]. In addition, previous work from our lab has investigated sex differences in the mouse EAE spinal cord and found significantly greater increases in the levels of amyloid precursor protein (APP) and non-phosphorylated neurofilament (SMI32) in males, suggesting that male spinal neurons are more sensitive to axonal injury and degeneration in EAE [56]. These results support our interpretation that male EAE DRG neurons are more prone to neuronal injury than females. Neuronal injury in males may also explain previous work from our lab which has shown that while female EAE DRG neurons are hyperexcitable, males are not [21]. We hypothesize that while female pain is driven by hyper-excitability in the periphery, this is not the source of pain in males. Instead, male pain in EAE may be mediated by a central mechanism.
Finally, we examined mouse EAE DRGs for the expression of the injury and plasticity-associated proteins ATF3 and pCREB. Females had significantly more ATF3-positive cells than males, suggesting that females engage an adaptive mechanism in response to inflammation. NFκB is known to induce ATF3 signaling, acting in a negative feedback loop to dampen its own pro-inflammatory signaling, as ATF3 can bind and inhibit the P65 subunit of NFκB [57–59]. There was also significantly more pCREB staining in the female EAE DRG than in males, which provides further evidence of a regenerative phenotype. Much like ATF3, CREB can directly inhibit NFκB and pro-inflammatory signaling [60]. It has also been linked to aberrant plasticity, neuronal hyperexcitability, and pain in spinal cord injury [42, 43]. While activation of these transcription factors in the female EAE DRG may protect neurons from inflammation, mitochondrial pathology, and neuropathy, they may also be responsible for neuronal hyperexcitability and pain via maladaptive plasticity.
Thus, we propose that a regenerative mechanism is induced by preferential NFκB signaling in the female EAE DRG (Fig. 9.1). While this mechanism prevents neuropathy, it may be either insufficient to prevent neuronal hyperexcitability and pain or may even contribute to pain through aberrant plasticity [61, 62]. Males, however, preferentially activate P38, which leads to mitochondrial dysfunction and caspase 3 cleavage (Fig. 9.2) This pathway may cause male DRGs to become more prone to neuropathy. These studies reveal that there are distinct pathological changes that occur at the level of the DRG between the sexes. There is a need for future therapeutic strategies to treat neuropathic pain to be tailored in a sex specific manner. Our findings suggest that targeting processes of axonal degeneration and neuropathy will be more efficacious in males while modulating inflammation and aberrant plasticity will be more beneficial in females.