Temporal Contribution of Myeloid-Lineage TLR4 to the Transition to Chronic Pain: A Focus on Sex Differences

Abstract

Complex regional pain syndrome (CRPS) is a chronic pain disorder with a clear acute-to-chronic transition. Preclinical studies demonstrate that toll-like receptor 4 (TLR4), expressed by myeloid-lineage cells, astrocytes, and neurons, mediates a sexdependent transition to chronic pain; however, evidence is lacking on which exact TLR4-expressing cells are responsible. We used complementary pharmacologic and transgenic approaches in mice to more specifically manipulate myeloid-lineage TLR4 and outline its contribution to the transition from acute-to-chronic CRPS based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We demonstrate that systemic TLR4 antagonism is more effective at improving chronic allodynia trajectory when administered at the time of injury (early) in the tibial fracture model of CRPS in both sexes. In order to clarify the contribution of myeloid-lineage cells peripherally (macrophages) or centrally (microglia), we rigorously characterize a novel spatiotemporal transgenic mouse line, Cx3CR1-Cre ERT2-eYFP ;TLR4 fl/fl (TLR4 cKO) to specifically knock out TLR4 only in microglia and no other myeloid-lineage cells. Using this transgenic mouse, we find that early TLR4 cKO results in profound improvement in chronic, but not acute, allodynia in males, with a significant but less robust effect in females. In contrast, late TLR4 cKO results in partial improvement in allodynia in both sexes, suggesting that downstream cellular or molecular TLR4-independent events may have already been triggered. Overall, we find that the contribution of TLR4 is time-and microglia-dependent in both sexes; however, females also rely on peripheral myeloid-lineage (or other TLR4 expressing) cells to trigger chronic pain.

Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain

“…The copyright holder for this preprint this version posted June 16, 2021. ; https://doi.org/10.1101/2021.06. 15.447407 doi: bioRxiv preprint Following slicing, tissue for biochemical analysis was either immediately flash frozen (CFAtreated tissue) using histo-freeze (Fisher Super Friendly Freeze'It) or was treated with ex vivo BDNF or saline (see below) and was subsequently flash frozen.…”

“…Moreover, sexual convergence onto shared phenotypic behavioural endpoints, such as pain sensitivity, may also be masking sex differences in underlying molecular and cellular mechanisms 5-7 .Recent female-inclusive studies have revealed that pathological neuroimmune signalling in pain processing networks of the spinal dorsal horn diverges between males and females 11 . For example, the microglial TLR4/P2X4 signalling pathway that mediates spinal hyperexcitability and pain hypersensitivity in male rodent models does not have equivalent roles in female models of pathological pain [12][13][14][15] . Instead, hyperexcitability in female rodents is proposed to be driven by T-lymphocyte activation and yet-to-be-identified extracellular signals 11,13,16 .…”

The Role of Microglia in Neuroinflammation of the Spinal Cord after Peripheral Nerve Injury

“…To target TLR4 in specific cell types, two recent studies analyzed cell-specific TLR4 deletions in either microglia (using the CX3CR1 promoter) in a pain model of orthopedic trauma (tibial fracture) or in C-fiber nociceptors (using Nav1.8 promoter) to study mechanical allodynia after PNI. Both studies showed that TLR4 in either microglia or nociceptors influences pain but while microglia TLR4 was more significant in males, the opposite was true for C-fiber nociceptor TLR4 [117,118].…”

The Role of Neuro-Immune Interactions in Chronic Pain: Implications for Clinical Practice

“… 173 Sexual dimorphism in pain is a fascinating area of research, and current preclinical studies span beyond T cell and microglia function and implicate other neuroimmune players, such as myeloid cells and even neurons themselves. 51 , 174 …”

“…Notably, TLRs are found in immune cells, astrocytes and neurons; thus, the specific contribution of microglial TLRs to global knockout genetic models remains unclear. [48][49][50][51] ATP binds to numerous purinergic receptors expressed on microglia and the various binding interactions have contributing roles in neuropathic pain. 52 In particular, P2X purinoceptor 4 (P2XR4) is exclusively upregulated on spinal cord microglia in response to C-C motif chemokine ligand 21 (CCL21) secreted from injured DRG neurons.…”