During peripheral infection, excessive production of pro-inflammatory cytokines in the aged brain from primed microglia induces exaggerated behavioral pathologies. While the pro-inflammatory cytokine IL-6 increases in the brain with age, its role in microglia priming is not known. This study examined the functional role of IL-6 signaling on microglia priming. Our hypothesis is that IL-6 signaling mediates primed states of microglia in the aged. An initial study assessed age-related alteration in IL-6 signaling molecules; sIL-6R and sgp130 were measured in cerebrospinal fluid of young and aged wild-type animals. Subsequent studies of isolated microglia from C57BL6/J (IL-6) and IL-6 knock-out (IL-6) mice showed significantly less MHC-II expression in aged IL-6 compared to IL-6 counterparts. Additionally, adult and aged IL-6 and IL-6 animals were administered lipopolysaccharide (LPS) to simulate a peripheral infection; sickness behaviors and hippocampal cytokine gene expression were measured over a 24 h period. Aged IL-6 animals were resilient to LPS-induced sickness behaviors and recovered more quickly than IL-6 animals. The age-associated baseline increase of IL-1β gene expression was ablated in aged IL-6 mice, suggesting IL-6 is a key driver of cytokine activity from primed microglia in the aged brain. We employed in vitro studies to understand molecular mechanisms in priming factors. MHC-II and pro-inflammatory gene expression (IL-1β, IL-10, IL-6) were measured after treating BV.2 microglia with sIL-6R and IL-6 or IL-6 alone. sIL-6R enhanced expression of both pro-inflammatory genes and MHC-II. Taken together, these data suggest IL-6 expression throughout life is involved in microglia priming and increased amounts of IL-6 following peripheral LPS challenge are involved in exaggerated sickness behaviors in the aged.

The inclusion of females in preclinical pain studies has become more commonplace in the last decade, as the National Institutes of Health (NIH) “Sex as a Biological Variable” (SABV) mandate was released. Presumably, basic researchers have not had a comprehensive understanding about neuroimmune interactions in half of the population and how hormones play a role in this. To date, we have learned that sex hormones contribute to sexual differentiation of the nervous system and sex differences in behavior throughout the lifespan; however, the cycling of sex hormones does not always explain these differences. Here, we highlight recent advances in our understanding of sex differences and how hormones and immune interactions influence sensory neuron activity to contribute to physiology and pain. Neuroimmune mechanisms may be mediated by different cell types in each sex, as the actions of immune cells are sexually dimorphic. Unfortunately, the majority of studies assessing neuronal contributions to immune function have been limited to males, so it is unclear if the mechanisms are similar in females. Finally, pathways that control cellular metabolism, like nuclear receptors have been shown to play a regulatory role in both pain and inflammation. Overall, communication between the neuroimmune and endocrine systems modulate pain signaling in a sex-dependent manner, but more research is needed to reveal nuances of these mechanisms.

Pain disorders induce metabolic stress in peripheral sensory neurons by reducing mitochondrial output, shifting cellular metabolism, and altering energy use. These processes implicate neuronal metabolism as an avenue for creating novel therapeutics. Liver kinase B1 (LKB1) mediates the cellular response to metabolic stress by inducing the AMPK pathway. The LKB1-AMPK pathway increases energy producing processes, including mitochondrial output. These processes inhibit pain by directly or indirectly restoring energetic balance within a cell. Although the LKB1-AMPK pathway has been linked to pain relief, it is not yet known which cell is responsible for this property, as well any direct ties to cellular metabolism. To elucidate this, we developed a genetic mouse model where LKB1 is selectively removed from Nav1.8-pain sensory neurons and metabolically stressed them by fasting for 24 hours. We found females, but not males, had neuron-specific, LKB1-dependent restoration of metabolic stress-induced mitochondrial metabolism. This was reflected in mechanical hypersensitivity, where the absence of LKB1 led to hypersensitivity in female, but not male, animals. This discrepancy suggests a sex- and cell-specific contribution to LKB1-depdendent fasting-induced mechanical hypersensitivity. While our data represent a potential role for LKB1 in anti-pain pathways in a metabolic-specific manner, more must be done to investigate these sex differences.

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Background Pain induces metabolic stress in pain sensing neurons (nociceptors); alleviating this stress represents an avenue for treating pain. Liver kinase B1 (LKB1) is involved in maintaining energy homeostasis and is a key mediator of the cellular response to metabolic stress. If cellular energy modulation and pain relief is LKB1‐dependent, then its removal could lead to metabolic imbalance and innocuous stimuli becoming painful (allodynia). Hypothesis In a novel mouse transgenic model where LKB1 is removed from Nav1.8+ nociceptors (Nav1.8LKB1‐), we predict nociceptors will have reduced abilities to cope with metabolic stress, leading to energy imbalance and subsequent allodynia. Methods After baselining for mechanical allodynia, animals were fasted for 24hr to induce metabolic stress. A subset of animals had their primary dorsal root ganglia nociceptors cultured for 6 hours for the Seahorse MitoStress assay. The remainder of animals were tested for mechanical allodynia once a day for 5 days after food was returned. Results As expected, a 24hr fast induced mechanical allodynia in a genotype dependent manner, with Nav1.8LKB1‐ animals responding to the fast in a sexually dimorphic manner. While fasting initially caused allodynia in males of both genotypes, Nav1.8LKB1‐ animals took longer to recover. However, in females, only Nav1.8LKB1‐animals exhibited allodynia after fasting. A sexually dimorphic and genotype dependent effect was also seen in neuronal mitochondrial function. In control females, fasting increased neuronal oxidative metabolism, suggesting the LKB1 pathway is necessary to mitigate fasting‐induced energy depletion. Neither fasting nor genotype changed neuronal mitochondrial function in males, suggesting an alternate mechanism was responsible for their initial allodynia. Conclusions Although Nav1.8LKB1‐ animals of both sexes developed genotype‐dependent allodynia in response to a metabolic stressor (24hr fast), only females exhibited a difference in mitochondrial function in response to this fast. These data support existing literature that suggests neuronal processes are responsible for the development of allodynia in females but not males and suggests that these differences are metabolic in nature. However, additional studies are needed to determine how other sex‐ and cell‐specific metabolic processes can contribute to pain development. This includes how the metabolism of immune cells, such as microglia and macrophages, which have been shown contribute to pain in males but not females, can be affected in an LKB1‐dependent manner to reduce a painful phenotype. Acknowledgements: We would like to thank Luz Barron and Han Jeong for their contributions.