LPS injection induces hyperalgesia and peripheral neuroinflammation in mice
To generate systemic inflammation, male C57BL/6 mice were intraperitoneally injected with LPS (2.5 mg/kg) on the first day of experiment (designated as day 0). We monitored weight loss and sensitivities to mechanical stimulation in animals after injection with either vehicle control (saline) or LPS within 8 days. Intraperitoneal injection with LPS caused significant weight loss from day 1 to day 7, compared to saline-injected group (Fig. 1a). In the von Frey tests, no differences in the mechanical withdrawal threshold (MWT) of hind paws were observed between saline- and LPS-injected mice at day 0. From day 1 to day 8, the MWT in LPS-injected group was significantly decreased compared to saline-injected control, verifying that a single intraperitoneal injection of LPS elicited mechanical hyperalgesia in mice (Fig. 1b) [6].
Systemic administration of LPS is known to cause immune responses [40]. To ascertain LPS induced inflammatory response in DRGs, we examined the changes of signaling pathways involved in LPS-triggered inflammatory response. The activations of ERK, p38-MAPK, and NF-κB were more profound in LPS-treated DRGs compared to saline-treated controls (day 7, Fig. 1c). Moreover, the protein levels of certain known molecules involved in inflammation, including TLR4, Hsp70 and COX2, were increased in LPS-treated DRGs compared to saline-treated controls (Fig. 1c, d, and Additional file 1: Figure S2). Furthermore, we analyzed the expressions of inflammatory cytokines in DRGs dissected from the mice injected with either saline or LPS using quantitative real-time PCR (qRT-PCR) an ELISA assays to exclude the serum cytokines induced by systemic LPS administration. Indeed, the expressions of IL-1β and TNF-α were markedly increased both at mRNA levels (Fig. 1e, f) and protein levels (Fig. 1g, h) after LPS injection for 7 days. These results demonstrate that LPS administration induces neuroinflammation in mice.
Mrgprd knockout attenuates LPS-induced inflammatory hyperalgesia
As a member of Mrgpr family, MrgprD is specifically expressed in small-diameter sensory neurons of DRG and modulates pain/itch sensation. To investigate the potential involvement of MrgprD in neuroinflammation, we first examined the MrgprD expression levels after LPS challenge. As illustrated by Western blot analyses, MrgprD protein levels were upregulated from day 5 to day 7 in LPS-treated DRGs compared with saline-treated controls (Fig. 2a, b). Moreover, MrgprD-expressing neurons were enumerated on the sections of lumbar 4 (L4) DRGs derived from Mrgprd−/− mice where the coding region of Mrgprd was replaced with an EGFP gene (Mrgprd∆EGFP) as previously described [37]. Taking advantage of EGFP, the DRG neurons specifically expressing MrgprD can be visualized by imaging GFP fluorescence as shown in Fig. 2c. The percentages of EGFP-positive neurons were significantly increased in LPS-challenged DRGs in comparison with saline-treated DRGs (Fig. 2d, 34 ± 0.81% vs. 28 ± 0.95%, at least 1500 neurons were counted on sections of L4 DRGs using Image J, n = 3 mice/group). These observations strongly suggest an involvement of MrgprD in the LPS-triggered neuroinflammation.
In order to explore the impact of MrgprD on the LPS-induced inflammatory pain, we compared the behaviors of nociceptive responses between wild type (WT) and Mrgprd−/− mice by animal behavioral tests. 7 days after saline injection, Mrgprd−/− mice did not exhibit any differences in the nociceptive responses to mechanical and thermal stimulations compared with their WT littermates (Fig. 2e-2 g). By contrast, 7 days after LPS injection, deletion of Mrgprd resulted in the significant increase in the MWT values (Fig. 2e) and the significant decrease of numbers of paw lifts (Fig. 2f), but without altering the withdrawal latency of hind paw to heat stimulus when compared with saline-injected groups (Fig. 2g). These data indicate that MrgprD is required for maintaining the mechanical and cold allodynia, but not heat hyperalgesia, in a mouse model of LPS-induced inflammatory pain.
Mrgprd knockout attenuates LPS-triggered neuroinflammation in DRG
We next assessed the activations of LPS-triggered signaling pathways in DRGs derived from WT or Mrgprd−/− mice. In saline-treated DRGs, the protein levels of TLR4 and the phosphorylation levels of IKKα/β, ERK, and p38 MAPK were comparable between WT and Mrgprd−/− mice (Fig. 3a). However, in LPS-treated DRGs, deletion of Mrgprd substantially suppressed the up-regulation of TLR4 and the phosphorylation of IKKα/β, without affecting the phosphorylation of ERK and p38 MAPK (Fig. 3b). We further compared the expression levels of pro-inflammatory cytokines in DRGs between LPS-injected WT and Mrgprd−/− mice. Indeed, the LPS injection-induced expressions of IL-1β and TNF-α were significantly reduced in DRGs of Mrgprd−/− mice compared with those of WT counterparts (Fig. 3c, d). During the inflammation, chemokines such as MCP-1 have been implicated in peripheral neuroinflammation and chronic pain sensitization [41]. We then compared the expression levels of MCP-1 in DRGs from WT and Mrgprd−/− mice after saline or LPS administration. As expected, LPS administration caused the up-regulation of MCP-1 in DRGs, and the deletion of Mrgprd substantially attenuated MCP-1 up-regulation in LPS-treated DRG (Fig. 3e). Consistently, the protein levels of IL-1β, TNF-α, and MCP-1 were significantly reduced in DRGs from LPS-injected Mrgprd−/− mice, compared to those from LPS-injected WT mice (Fig. 3f-h). These results indicate a pivotal role of MrgprD in LPS-induced neuroinflammation in DRG.
MrgprD Overexpression Promotes LPS-triggered Activation Of NF-κB
After establishing a correlation between MrgprD and neuroinflammation, we further investigated the underlying mechanism. LPS is well known to stimulate pro-inflammatory gene expression by engaging TLR4 complex that activates bifurcating signaling pathway leading to the activations of MAPK and NF-κB signaling cascades [17, 42]. Of note, TLR4 expression has been recently illustrated in sensory neurons rather than in satellite glial cells in DRG [43]. We therefore first examined whether MrgprD is co-localized with TLR4 in DRG neurons using immunohistochemistry. In the DRG of Mrgprd−/− mice, the expression of EGFP was clearly distributed in a subset of DRG neurons and the nerve fibers, while the immunostaining of TLR4 was readily detected in the cell bodies of DRG neurons (Fig. 4a). As expected, MrgprD-positive neurons were identified with TLR4 expression. Moreover, using Western blotting, the expression of TLR4 and the activation of NF-κB signaling upon LPS stimulation were confirmed in the primary cultured DRG neurons (Additional file 1: Figure S3).
Next, we utilized a HEK-Blue mTLR4 cell model, in which mouse TLR4 and MD2/CD14 were stably overexpressed [44], to assess the impact of MrgprD on LPS-triggered signaling. HEK-Blue mTLR4 cells were transiently transfected with MrgprD expression construct and empty vector, respectively. Confocal microscopy was used to verify the localization of ectopically expressed MrgprD in cell membrane (Additional file 1: Figure S4). At 36 h post transfection, the cells were stimulated with LPS for 30 min and subjected to Western blot analyses. We found that MrgprD overexpression facilitated the LPS-induced activation of NF-κB, but not ERK, JNK or p38 MAPK in HEK-Blue mTLR4 cells (Fig. 4b-d). Moreover, the ectopic expression of MrgprD substantially enhanced the LPS-triggered nuclear translocation of NF-κB p65 subunit (Fig. 4e, f), demonstrating that MrgprD promotes the LPS-triggered signaling that leads to NF-κB activation.
MrgprD Overexpression Enhances Nf-κb P65 Transactivation Activity
NF-κB transcription factors are expressed throughout the peripheral and central nervous systems [20]. The activation of NF-κB is well known to transactivate a variety of genes, especially those encoding pro-inflammatory cytokines and chemokines [45]. Our observation that deletion of Mrgprd attenuated the expressions of IL-1β, TNF-α, and MCP-1 in LPS-challenged DRGs led us to examine whether MrgprD is able to enhance the NF-κB binding capability to target promoter DNA. To this end, HEK293-Blue mTLR4 cells were transiently transfected with MrgprD expression plasmid or empty vector. After 36 h, the cells were stimulated with LPS for 30 min and subjected to chromatin immunoprecipitation (ChIP) to analyze the recruitment of NF-κB p65 to the promoter region of known NF-κB target gene, TNF-α. Indeed, overexpression of MrgprD enhanced the LPS-induced enrichment of p65 to the κB region of TNF-α promoter (Additional file 1: Figure S5a). The p65 enrichment was specific, as there was negligible recruitment of p65 to the β-Actin promoter that does not contain any κB sites (Additional file 1: Figure S5b). Consistently, the amount of the LPS-induced production of TNF-α in the culture medium was significantly increased in MrgprD overexpression group (Additional file 1: Figure S5c). Hence, our results demonstrate that ectopic expression of MrgprD substantially augments the LPS-induced NF-κB transactivation of certain pro-inflammatory cytokine genes, in line with the enhanced NF-κB activation signaling cascade (Fig. 4).
MrgprD mediates NF-κB signaling through interaction with TAK1 and IKKs
We sought to elucidate the mechanism through which MrgprD promotes the cellular signaling that leads to NF-κB activation in DRG. The observation that deletion of Mrgprd markedly suppressed LPS-induced phosphorylation of IKKα/β (Fig. 3a) led us to investigate the interaction of MrgprD with the components in NF-κB signaling pathway upstream of the IKK complex. By co-immunoprecipitation using MrgprD antibody, we found that MrgprD physically associated with TAK1, but not MyD88, TRAF6, or TLR4, in mouse DRGs (Fig. 5a). Such interaction was confirmed in Flag-MrgprD-overexpressing HEK293T cells by immunoprecipitation using anti-Flag (MrgprD) and anti-TAK1 antibodies (Fig. 5b).
We then assessed the impact of MrgprD on the phosphorylation of TAK1 at Thr-187, which is highly correlated with the TAK1 kinase activity [46], in DRGs isolated from WT and Mrgprd−/− mice injected with either saline or LPS. We found that Mrgprd knockout did not alter the LPS-triggered phosphorylation of TAK1 in DRGs (Fig. 5c). In HEK-Blue mTLR4 cells, the stimulation with LPS induced the phosphorylation of TAK1 (Fig. 5d), while MrgprD overexpression did not affect the LPS-induced phosphorylation of TAK1 and p38 MAPK (Fig. 5e). Since it has been proposed that TAK1 can phosphorylate IKKβ and increase its enzyme activity [47], we thus determined whether MrgprD forms a complex with TAK1 together with IKKβ. In DRGs, TAK1 and the IKK complex (IKKα, IKKβ, and IKKγ) were both immunoprecipitated with MrgprD (Fig. 5f). By contrast, p38, ERK, and JNK did not co-precipitated with MrgprD-TAK1 complex (Fig. 5g). These data suggest that MrgprD is involved in TAK1-mediated phosphorylation of IKKs in LPS-triggered NF-κB activation, via forming a complex with TAK1 and IKKs.
Activation Of MrgprD Elicits The Activation Of NF-κB Signaling
MrgprD is activated by β-alanine [30] and has been demonstrated to mediate β-alanine-evoked itch [32]. MrgprD has also been identified as the receptor for alamandine, a new component of renin-angiotensin system (RAS) on the regulation of cardiovascular homeostasis [33, 48]. Recently, we illustrated the expression of MrgprD in mouse peripheral peritoneal macrophages and macrophage-like RAW 264.7 cells [34]. We, therefore, determined the signaling pathways downstream of MrgprD upon the stimulation with β-alanine or alamandine in RAW 264.7 cells. The treatment with β-alanine alone elicited the activation of NF-κB signaling, and the addition of β-alanine reinforced the LPS-induced activation of NF-κB signaling (Fig. 6a, b). By contrast, β-alanine treatments did not alter the phosphorylation levels of ERK, p38 MAPK and JNK in the absence or presence of LPS (Fig. 6a, c). Consistently, the activation of MrgprD by alamandine also induced the activation of NF-κB signaling in the absence of LPS, whilst it had no effect on the phosphorylation of ERK, p38 MAPK and JNK (Additional file 1: Figure S6).
Co-immunoprecipitation assays further confirmed the binding of MrgprD to TAK1 and IKK complex in RAW 264.7 cells (Fig. 6d). We also observed that MrgprD interacted with TAK1 and IKK complex in the absence of LPS. Further, to determine whether LPS stimulation could alter phosphorylated TAK1 and phosphorylated IKKα/β present in MrgprD protein complexes, we immunoprecipitated MrgprD from either resting or LPS stimulated cells. Immunoblotting with phosphorylation-specific antibodies showed that the comparable amounts of phosphorylated IKKα/β and phosphorylated TAK1 interacted with MrgprD between LPS stimulated and unstimulated groups (Fig. 6e-g). Taken together, we speculate that the MrgprD-mediated activation of NF-κB signaling pathway is independent of LPS-TLR4 pathway in RAW 264.7 cells.