In this study, we confirmed that TGR5 in injured nerves protect against pSNL-induced mechanical allodynia by modulating neuroinflammation. Specifically, the expression of TGR5 increased in injured nerves, and local activation of TGR5 alleviated mechanical allodynia by reversing the increase of pro-inflammatory cytokines/chemokines as well as the increase of monocytes/macrophages induced by pSNL. On the contrary, specific knockdown of TGR5 in myeloid cells aggravates mechanical allodynia by increasing the expression of pro-inflammatory mediators and the proportion of monocytes/macrophages.
TGR5, a GPCR protein, is abundantly expressed in the gallbladder epithelium, and is minimally to moderately expressed in almost all other tissues and cell types (Vassileva et al. 2006). In the nervous system, TGR5 is found in both neurons and non-neuronal cells, including resident and infiltrating immune cells (Lieu et al. 2014). The distinct functions of TGR5 in neurons and non-neuronal cells was previously reported. For neurons, administration of the TGR5 agonist increases the intrinsic excitability of DRG neurons and activates colon-innervating DRG neurons separated from naïve mice (Alemi et al. 2013, Castro et al. 2019). In non-neuronal cells, TGR5 activation mitigates LPS-induced inflammatory responses in both microglia and macrophages (Hogenauer et al. 2014, Yanguas-Casas et al. 2017). Moreover, the known roles of TGR5 expressed in different anatomical region in pain modulation are also inconsistent. In the mouse model of paclitaxel-induced peripheral neuropathic pain, TGR5 expressed in the DRG neurons contribute to the upregulation of CCR5, and then mediates the deoxycholic acid induced mechanical allodynia (Zhong et al. 2023). In the mouse model of spared nerve injury (SNI)-induced neuropathic pain, intrathecal injection of TGR5 agonist alleviates mechanical allodynia by inhibiting the activation of glial cells and modulating the function of GABAA receptors in the spinal cord(Wu et al. 2023). In this study, TGR5 in the injured sciatic nerve was mainly expressed in the macrophages, and pSNL-induced mechanical allodynia was significantly alleviated by peri-sciatic administration of the TGR5-specific agonist INT-777. Underlying this phenotype, the molecular mechanism regulated by TGR5 activation is more consistent with a negative regulation effect on inflammation rather than a positive impact on neuron excitability. The inference was validated by bulk RNA-seq analysis of injured nerve tissues. The sequence data indicated that, among the entire transcriptome changes, the inflammatory response is the primary process engaged by DEGs which were regulated reversely by pSNL and TGR5 activation. In addition, pSNL-induced mechanical allodynia was further exaggerated when TGR5 was specifically knockdown in myeloid cells along with the transcriptomic alterations predominantly related to the inflammatory response. Taken together, although the specific role of TGR5 expressed by neuron fibers in pain modulation was not evaluated in this study, the present data proved that increased TGR5 in the injured sciatic nerve is a molecular target to modulate mechanical allodynia by regulating neuroinflammation.
In the field of neuropathic pain, ample evidence has underscored the substantial role of neuroinflammation in driving both peripheral and central sensitization, thereby enhancing pain hypersensitivity (Grace et al. 2014, Bethea and Fischer 2021). Recent findings indicate that specific subsets of immune cells contribute to the alleviation of neuropathic pain (Fiore et al. 2023). Among them, macrophages are a subset of cells which exhibit remarkable plasticity and adopt functionally distinct phenotypes (Mosser et al. 2020). The inflammatory mediators, which are highly expressed by pro-inflammatory M1 macrophages, such as IL-1β (Binshtok et al. 2008), IL-6 (Liu et al. 2019) and TNF-α (Jin and Gereau 2006) were reported to induce neuronal sensitization and circulating leukocytes recruitment into inflamed tissue (Wang et al. 2022). The antinociceptive properties of the mediators secreted by anti-inflammatory M2 macrophages, such as IL-10 (Kwilasz et al. 2019, Niehaus et al. 2021) and IL-4 (Celik et al. 2020, Labuz et al. 2021) have also been validated. In this study, we confirmed the dynamic changes of macrophages and their close proximity to neuron fibers in injured nerves. In the context of TGR5 manipulation, accompanied by the alterations of mechanical allodynia, pro-inflammatory mediators and the proportion of monocytes/macrophages in the sciatic nerve were significantly regulated, with no significant difference detected in the expression of anti-inflammatory mediators. Activation of microglia in the SDH is an important part of central sensitization and is known to contribute to the neuropathic pain (Inoue and Tsuda 2018). In the present study, manipulations of TGR5 in the sciatic nerve significantly altered the activation level of microglia. In summary, the high relevance between macrophage-mediated neuroinflammation and pSNL-induced mechanical allodynia was further confirmed in this study. Regarding the neuronal mechanisms that mediate the effect of TGR5-modulated neuroinflammation on pain perception, although five genes involved in dendritic spine morphogenesis and negative regulation of excitatory postsynaptic process were detected to be downregulated by TGR5 knockdown, the specific roles of these genes still need to be further investigated.
Since its detection, the modulatory effects of TGR5 on the functions of monocytes and macrophages have been extensively studied. Specifically, the production of pro-inflammatory cytokines in macrophages, such as TNF-α, IL-6 and IL-1β, are reported to be inhibited by TGR5 activation via NF-κB pathway (Pols et al. 2011, Yang et al. 2017), and to be exacerbated by TGR5 deficiency via stabilizing the β-catenin destruction complex (Rao et al. 2020). The production of anti-inflammatory cytokines, such as IL-4 and IL-10, is reported to be upregulated by macrophage TGR5 activation, but the controversy remains regarding whether the presence of TGR5 is indispensable for the polarization of anti-inflammatory M2 macrophages (Perino et al. 2014, Zhao et al. 2022). The expressions of chemokines, such as CCL2, CCL3 and CCL4, are also regulated by TGR5. In the presence of LPS, TRG5 deficiency exaggerates the upregulation of chemokines, while the activation of TGR5 poses the opposite impact on macrophages via the mTOR-C/EBPβ pathway (Perino et al. 2014). In addition, the activation of NLRP3 inflammasome is inhibited by bile acids via TGR5-cAMP-PKA axis (Guo et al. 2016), and the deficiency of TGR5 exaggerates the inflammatory response of macrophages stimulated with palmitic acid by promoting the activation of NLRP3 inflammasome (Shi et al. 2021). In the present study, the increase of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, as well as chemokines CCL3 and CCL2 in sciatic nerves was exacerbated by myeloid-cell specific TGR5 knockdown. These results are consistent with the known effects of TGR5 on macrophages. Regarding the NLRP3 inflammasome, although the expression of NLRP3 was not altered, the expression of the adaptor protein ASC, which connects the inflammasome sensor molecule to caspase-1, as well as the expression of caspase-1, which initiates downstream responses, were significantly upregulated by TGR5 knockdown, indicating the further activation of NLRP3 inflammasome. As for the specific molecular pathways underlying the modulations of TGR5 on inflammatory mediators in macrophages, which located in the injured sciatic nerve, further research is needed to illustrate the intrinsic alterations of these cells in situ rather than the changes induced by mimicked inflammatory response in cell experiments.
The endogenous ligands of TGR5 are primary bile acids synthesized from cholesterol and secondary bile acids metabolized by the gut microbiota (Collins et al. 2023). The majority of bile acids are generated and exist in the enterohepatic system and a minority of them spillover into the circulation (Perino et al. 2021). In the peripheral and central nervous system, the presence of bile acids is confirmed (Xing et al. 2023). In addition to being transported from circulation, bile acids are also synthesized locally in the brain and spinal cord, as evidenced by the detection of rate-limiting enzymes for synthesis (Hurley et al. 2022). In the present study, data of bulk RNA-seq indicated that genes involved in the biosynthetic and metabolic process of cholesterol were significantly regulated in the sciatic nerve after pSNL, implying the potential alterations of endogenous ligands of TGR5. However, whether the protein levels of bile acids in injured sciatic nerves changes and whether these ligands participate in the modulation of pathological processes of neuropathic pain need to be further studied.
The primary limitation of this study is the use of only male mice. Given the well-documented sex differences in neuroinflammatory mechanisms (del Rivero et al. 2018, Gregus et al. 2021), whether TGR5 plays the same regulatory role in neuroinflammation and mechanical allodynia in female mice remains unknown.
In conclusion, this study established that TGR5, expressed by macrophages within injured nerves, is a protective factor against mechanical allodynia. This protective effect involves alleviating neuroinflammation by modulating the pSNL-induced expression of inflammatory mediators. Consequently, TGR5, which operates at the peripheral nerve injury site, has emerged as a promising molecular target for treating neuropathic pain.