Silencing miR-21-5p in sensory neurons reverses neuropathic allodynia via activation of TGF-β–related pathway in macrophages

Neuropathic pain remains poorly managed by current therapies, highlighting the need to improve our knowledge of chronic pain mechanisms. In neuropathic pain models, dorsal root ganglia (DRG) nociceptive neurons transfer miR-21 packaged in extracellular vesicles to macrophages that promote a proinflammatory phenotype and contribute to allodynia. Here we show that miR-21 conditional deletion in DRG neurons was coupled with lack of upregulation of chemokine CCL2 after nerve injury and reduced accumulation of CCR2-expressing macrophages, which showed TGF-β–related pathway activation and acquired an M2-like antinociceptive phenotype. Indeed, neuropathic allodynia was attenuated after conditional knockout of miR-21 and restored by TGF-βR inhibitor (SB431542) administration. Since TGF-βR2 and TGF-β1 are known miR-21 targets, we suggest that miR-21 transfer from injured neurons to macrophages maintains a proinflammatory phenotype via suppression of such an antiinflammatory pathway. These data support miR-21 inhibition as a possible approach to maintain polarization of DRG macrophages at an M2-like state and attenuate neuropathic pain.


Introduction
Peripheral neuropathy is a debilitating condition that often leads to severe and chronic neuropathic pain, for which novel treatment strategies are needed because current drugs have limited efficacy and severe side effects (1,2). In neuropathic pain states, neuronal activity at the site of nerve injury, in dorsal root ganglia (DRGs), and the dorsal horn of the spinal cord provokes immune system responses. Thus, central and peripheral sensitizations are facilitated by microglia, the resident immune cells of the CNS, and monocytes/macrophages in the PNS, respectively (3,4).
Macrophages are a diverse cell population that exhibits remarkable plasticity after injury and disease and expresses specific hallmarks of their tissue of residence (5)(6)(7). Besides a primary function in host defense and inflammatory response, macrophages can release mediators, such as cytokines, that sensitize sensory neurons and contribute to neuropathic pain mechanisms (8). Specifically, after peripheral nerve injury, sensory neuronassociated macrophages (sNAMs) accumulate in lumbar DRGs and at the site of nerve injury (9)(10)(11), and play a mechanistic role, as monocyte/macrophage depletion prevents neuropathic pain-like behavior (12,13). In response to peripheral axon injury, sensory neuron cell bodies upregulate chemokines such as CCL2, which promotes infiltration of monocytes/macrophages in DRGs (14,15) through the satellite cell sheath around the primary sensory neuron in an attempt to clear damaged neurons (16,17). Hence, blood-derived macrophages engraft the pool of sNAMs, rapidly skew to a proinflammatory M1-like phenotype, and facilitate mechanisms of chronic pain (16). Conversely, M2-like macrophages play an antinociceptive role in the resolution of chronic pain, as for instance they can transfer mitochondria contained in vesicles to sensory neurons, which have a high metabolic demand under inflammatory pain conditions (18).
Therefore, the definition of specific modalities that underlie DRG neuron-macrophage communication in initiation, maintenance, and resolution of neuropathic pain holds considerable promise toward the identification of targets for novel antinociceptive strategies.
In this study, we focused on a DRG pathway that includes the upregulation of miR-21 in nociceptive neurons after peripheral injury, and neuron-mediated transfer of exosomes containing miR-21 to macrophages to promote a proinflammatory M1-like phenotype (16). Such a neuron-derived miR-21 contributes to neuropathic pain mechanisms, as both conditional knockout of miR-21 in sensory neurons (miR-21 cKO) and intrathecal delivery of a miR-21 antagomir (antagomir-21) attenuate the development of allodynia (16,(19)(20)(21). Notably, DRG macrophages isolated from miR-21-cKO mice show significant alteration of miR-21 predicted targets 7 days after peripheral nerve injury (spared nerve injury, SNI) (16). Since a single microRNA (miR-NA) can address a multitude of genetic and epigenetic changes, here we identified miR-21 posttranscriptional targets in macrophages following SNI. Our rationale is that the identification of miR-21-mediated mechanisms that regulate macrophage Neuropathic pain remains poorly managed by current therapies, highlighting the need to improve our knowledge of chronic pain mechanisms. In neuropathic pain models, dorsal root ganglia (DRG) nociceptive neurons transfer miR-21 packaged in extracellular vesicles to macrophages that promote a proinflammatory phenotype and contribute to allodynia. Here we show that miR-21 conditional deletion in DRG neurons was coupled with lack of upregulation of chemokine CCL2 after nerve injury and reduced accumulation of CCR2-expressing macrophages, which showed TGF-β-related pathway activation and acquired an M2-like antinociceptive phenotype. Indeed, neuropathic allodynia was attenuated after conditional knockout of miR-21 and restored by TGF-βR inhibitor (SB431542) administration. Since TGF-βR2 and TGF-β1 are known miR-21 targets, we suggest that miR-21 transfer from injured neurons to macrophages maintains a proinflammatory phenotype via suppression of such an antiinflammatory pathway. These data support miR-21 inhibition as a possible approach to maintain polarization of DRG macrophages at an M2-like state and attenuate neuropathic pain.
Silencing miR-21-5p in sensory neurons reverses neuropathic allodynia via activation of TGF-β-related pathway in macrophages Thus, in PMs and BMDMs we manipulated miR-21 expression using mimic-21 and antagomir-21 transfection, and quantified expression of MHCII and CD206 using flow cytometry.
PM transfection with mimic-21 resulted in a 240-fold increase in miR-21-5p, but not miR-155 and miR-706 expression, and a decrease in the miR-21 target Spry2 compared with scramble (N5-control) ( Figure 1, A and B, and Supplemental Figure 2, A and B), indicating high efficiency of transfection. In mimic-21-compared with scramble-transfected macrophages, we found a decrease in Tgfb1 transcripts and a negative Spearman's correlation (r = -0.8, P = 0.0047) between Tgfb1 and miR-21-5p expression (Figure 1, C and D), but Tgfbr1, Tgfbr2, and Tgfbr3 transcripts were not changed ( Figure 1E). We also observed upregulation of Smad7, which encodes an endogenous inhibitory R-SMAD involved in the TGF-β signaling pathway (23) ( Figure 1F), but no change in Smurf2, Bmpr2, Bmp, and Smad5, which are implicated in the TGF-β signaling pathway (23) (Supplemental Figure 2, C-F). Next, flow cytometry analysis of BMDMs transfected with mimic-21 showed lower expression of TGF-βR2 at the single-cell level (mean fluorescence intensity, MFI) and a lower percentage of TGF-βR2 + cells, compared with scramble-transfected BMDMs ( Figure 1G) (gating strategy in Supplemental Figure 2G), but no difference in MHCII + CD206 -(M1-like) and MHCII -CD206 + (M2-like) populations (Supplemental Figure 2H). However, quantification of M1-and M2-like marker gene expression revealed an increase in Tnfa and Il6 ( Figure 1H) but no change in Arg1, Nos2, Rela, and Ym1 ( Figure 1I and Supplemental Figure 2, I and J) in mimic-21compared with scramble-transfected BMDMs. Therefore, a higher level of miR-21 in primary macrophages induces downregulation of antiinflammatory cytokine Tgfb1 and Tgfbr2 and upregulation of proinflammatory cytokine Tnfa and Il6 gene products.
However, using antagomir-21, we obtained only 50% reduction in miR-21 expression in PMs (Supplemental Figure 3A) and a better yield in BMDMs, with 70% reduction in miR-21 expression in 98% of cells (Figure 2A and Supplemental Figure 3B). Thus, we used antagomir-21-transfected BMDMs and found an increase in Spry2 and Tgfb1 (Figure 2, B and C) and significant negative Spearman's correlation (r = -0.63, P = 0.0232) between miR-21 and Tgfb1 expression ( Figure 2D). Remarkably, we found an increase in Tgfbr2 in antagomir-21-compared with scramble-transfected BMDMs, but no changes in Tgfbr1, Tgfbr3, and Smad7 ( Figure  2E). Moreover, Tnfa and Il6 were decreased and Mrc1 and II10 increased (Figure 2, F and G), while other polarization markers, including Ym1 and Arg1, remained unaltered (Supplemental Fig-polarization toward a pronociceptive phenotype can provide elements that can be targeted in macrophages, for instance through DRG-specific delivery of nanoparticles.

miR-21 induces a specific gene expression profile in macrophages.
Further analysis of our genome-wide microarray in WT and miR-21-cKO DRG macrophages (16) revealed dysregulation of genes associated with cell-cell communication, GPCR ligand binding, and TGF-β signaling. Specifically, Tgfbr2 (the gene coding for TGF-β receptor 2, TGF-βR2), was upregulated in cKO macrophages compared with WT (Tgfbr2: P = 0.05, 3.02-fold increase) alongside Tgfbr3 (coreceptor of Tgfbr2), Tgfb1 (ligand for both receptors), and Nfya (encoding nuclear transcription factor Y), which regulates Tgfbr2 transcription (22) (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI164472DS1). Such gene changes in DRG macrophages were strengthened by results obtained in peritoneal macrophages (PMs) transfected with antagomir-21 to downregulate endogenous expression of miR-21, and then exposed to sensory neuron-derived exosomes overexpressing miR-21 to promote transfer of miR-21 from neurons to macrophages. We found 816 differentially expressed genes (DEGs) compared with antagomir-21-transfected PMs, which themselves displayed 4,979 genes that were differentially regulated compared with scramble-transfected PMs (Supplemental Figure 1, B and C). Since macrophages exposed to neuronal exosomes overexpressing miR-21 showed 2,922 DEGs compared with antagomir-21treated PMs (Supplemental Figure 1, B and C), these data suggest that neuron-derived miR-21 modulates the transcriptional profile in macrophages in addition to endogenous miR-21. Macrophage exposure to neuronal exosomes affected pathways related to innate immune system responses and cellular responses to stress and metabolism (Supplemental Figure 1D). Indeed, we observed upregulation of Tnfrsf19 (member of the TNF receptor superfamily; P = 0.029, 1.46-fold increase), together with downregulation of Mrc1 (P = 0.036, 1.69-fold decrease) (Supplemental Figure 1E), all of which are associated with a proinflammatory macrophage phenotype. Furthermore, silencing miR-21 expression in macrophages increased the expression of genes associated with cellular responses to TGF-β. For instance, we observed that Tgfbr1 and Tgfbr2 were upregulated in PMs lacking miR-21 and gene expression returned to basal levels when miR-21-silenced macrophages were incubated with neuron-derived miR-21 (Supplemental Figure 1F). These data suggest that uptake of neuron-derived miR-21 by macrophages results in alteration of the gene expression profile and pathways that are normally under miR-21 control, such as the TGF-β pathway. To validate our bioinformatics analyses, we evaluated the primary macrophage phenotype after transfection with antagomir-21 and a miR-21 mimic (mimic-21).
miR-21 fosters a partial proinflammatory phenotype in macrophages. We began by assessing miR-21 expression in PMs, bone marrow-derived macrophages (BMDMs), and DRG macrophages and found that cultured cells expressed comparable levels that were 28-fold higher than the ex vivo DRG macrophage content (Table 1). Yet, we detected higher levels of macrophages in the ipsilateral compared with contralateral DRG (Table 1). miR-21 RT-qPCR in primary macrophages (BMDMs, PMs) and macrophages isolated from contralateral and ipsilateral WT DRG 7 days after injury. Data are presented as expression relative to internal spike-in miRNAs, n = 4 mice per group.
Overall, these data indicate that miR-21-5p fosters a partial proinflammatory phenotype in macrophages. This suggestion is supported by proinflammatory cytokine upregulation in macrophages overexpressing miR-21 and polarization toward an antiinflammatory phenotype in the absence of miR-21. Moreover, BMDMs behave as a faithful surrogate for DRG macrophages and were used in subsequent selected experiments.   br2, and Tgfbr3 expression was unaltered (Supplemental Figure  4A), suggesting that miR-21 does not regulate the TGF-β pathway in neurons. Yet, in macrophages such TGF-β-related variations occurred concomitant with upregulation of antiinflammatory markers Mrc1 and Il10 (Supplemental Figure 4B) and no change in the proinflammatory markers Il6 and Tnfa (Supplemental Figure  4C). Contralateral DRG macrophages were used for quantification of macrophages under control conditions, since cKO contralateral thresholds were comparable to both WT contralateral and sham thresholds (Supplemental Figure 4D).
Overall, these data suggest that in DRGs the absence of neuron-derived miR-21 results in activation of a TGF-β pathway and M2-like polarization of macrophages. These results correlate with both attenuation of neuropathic allodynia in miR-21-cKO mice and restoration of allodynia following injection of a TGF-βR inhibitor.
Neuronal miR-21 regulates classical CCR2 monocyte/macrophage infiltration in DRGs. Having confirmed our published data (16) showing that F4/80 + cell accumulation is higher in ipsilateral than contralateral WT DRGs after nerve injury, while it is lower in ipsilateral miR-21-cKO DRGs than WT ( Figure 5A), we next evaluated whether neuronal miR-21 affected macrophage proliferation and infiltration on day 7 after SNI. We observed that both WT and cKO mice showed more Ki67 + F4/80 + macrophages in the ipsilateral DRG than contralateral, suggesting that neuron-derived miR-21 does not affect in situ macrophage proliferation ( Figure 5B). Concerning potential effects on infiltration in DRGs after SNI, we monitored expression of 2 chemokine receptors, CCR2 and CCR5, which are implicated in monocyte/macrophage infiltration (26,27). In flow cytometry analysis of CD11b + F4/80 + macrophages of WT DRGs, we found higher numbers of both CCR2 + and CCR5 + cells in ipsilateral compared with contralateral DRGs ( Figure 5, C and D). However, in cKO DRGs, CCR2 + cell number was lower than in WT ( Figure 5C), while CCR5 + cell accumulation was unchanged ( Figure 5D), suggesting that miR-21 affected CCR2 + cell infiltration in DRGs. Notably, these observations were all specific to DRG macrophages, as we did not observe changes in either macrophage infiltration or CCR2 expression at the site of nerve injury (Supplemental Figure 5A). These data in DRG macrophages were further substantiated in transfection experiments, as we observed higher numbers of CCR2 + cells ( Figure 5E) and a trend toward upregulation of Ccr5 mRNA (~40%; Figure 5F) in mimic-21-compared with scramble-transfected BMDMs. Conversely, in antagomir-21-transfected BMDMs we found lower numbers of CCR2 + cells ( Figure 5G) and reduced, yet not significantly, Ccr5 expression ( Figure 5H).
These data suggest that after SNI, miR-21 that is upregulated in neurons can influence CCR2 + monocyte/macrophage infiltration in DRGs. We reasoned that for such a mechanism to be relevant, miR-21 might be involved in upregulation of CCL2 in neurons, which occurs in response to axonal injury (28), albeit through an indirect mechanism, as there is no evidence that this chemokine is a miR-21 target. Indeed, we observed that, in the CD45fraction of the DRG, Ccl2 mRNA was upregulated in WT, but not miR-21-cKO mice ( Figure 6A). In addition, CCL2 protein was increased in WT but not miR-21-cKO ipsilateral compared with contralateral DRG ( Figure 6B) and CCL2 immunostaining was higher in ipsilateral than contralateral and especially in IB4 + miR-21 regulates TGF-β1 release and SMAD activation in macrophages. In antagomir-21-transfected BMDMs, consistent with the Tgfb1 mRNA increase, we found higher TGF-β1 extracellular levels than in scramble-transfected BMDMs ( Figure 3A). Such a release of TGF-β1 was lowered to control levels by incubation with LPS, which itself upregulates miR-21 by 8.57 ± 0.997-fold, (n = 4, P < 0.05) and likely replenished miR-21 in antagomir-21-transfected BMDMs. In addition, TGF-βR2 immunostaining was higher in antagomir-21than scramble-transfected BMDMs ( Figure 3B), along with more SMAD2/3 phosphorylation, and a significant upregulation of SMAD4 that was blocked by the presence of SB431542, a TGF-βR1 inhibitor (Figure 3, C and D). The choice of the TGF-βR1 antagonist was due to TGF-βR1 being the signaling protein required for formation of the active complex with TGF-βR2, which acts as the initial binding protein for TGF-β1 (23). These data suggest that lack of miR-21 in macrophages results in higher basal release of TGF-β1 and activation of TGF-βR2 followed by TGF-βR1-mediated signaling through a SMAD-dependent pathway. This possibility is reinforced by the observation that neither ERK nor p38 phosphorylation was altered by antagomir-21 transfection and involvement of a non-SMAD pathway could be ruled out ( Figure 3E).
Sensory neuron-derived miR-21 regulates TGF-β-related pathway in macrophages. Our next step was to move to an in vivo setting and validate the hypothesis that sensory neuron-derived miR-21 contributes to the development of neuropathic allodynia through downregulation of the TGF-β-mediated pathway in DRG macrophages. For this purpose, we used our miR-21-cKO mice that show significant reduction of miR-21 expression in DRG compared with WT ( Figure 4A), and lack of miR-21 upregulation in DRG ipsilateral to nerve injury ( Figure 4B). Furthermore, since neurons release miR-21 encapsulated in exosomes, we confirmed that exosome release under basal conditions and following activation of nociceptors by capsaicin was not altered in miR-21-cKO mice ( Figure 4C). Then, in behavioral studies, we confirmed that male and female cKO mice developed less severe neuropathic allodynia than WT at 5 to 7 days after SNI ( Figure 4D). Additionally, we observed that single intrathecal administration of SB431542 restored allodynia in miR-21-cKO but not WT mice at 2, 4, and 24 hours after injection ( Figure 4E), suggesting that endogenous TGF-β1 exerted antinociceptive effects in miR-21-cKO mice. Consistent with this possibility, intrathecal infusion of TGF-β1 reduces neuropathic allodynia by about 30% (24), which is comparable to the attenuation of neuropathic allodynia we observed in miR-21 cKO. Moreover, considering that we used SB431542 at a dose of 100 pmol/mouse that completely blocks the TGF-β1-induced anti-allodynic effect in neuropathic mice (25), these results suggest that, in miR-21-cKO mice, the attenuation of allodynia requires TGF-βR1 activation.
In keeping with the possibility that TGF-β pathway-related changes occur in macrophages 7 days after SNI, F4/80 + cells from miR-21-cKO ipsilateral DRGs showed a trend toward higher TGF-βR2 + events and substantially higher TGF-βR2 expression at the cellular level compared with WT (Figure 4, F and G). Moreover, miR-21-cKO isolated F4/80 + cells displayed upregulation of Tgfbr1 and Tgfbr2, and a trend toward Tgfb1 upregulation, but no change in Tgfbr3 compared with WT ( Figure 4H). In contrast, in the non-leukocyte CD45fraction of the DRG, Tgfb1, Tgfbr1, Tgf- neurons as expected (15,29), but not in cKO neurons ( Figure 6C). Consistently with an alteration of the CCL2 levels in miR-21 cKO, we made the following 2 sets of observations in a Transwell plate assay in which cultured DRG neurons were challenged with capsaicin to provide an in vitro model of nociceptive neuron stimulation (schematic in Supplemental Figure 5B). The first one was that capsaicin promoted transmigration of macrophages toward the WT but not cKO neuronal compartment, and this effect was blocked by a CCR2 antagonist ( Figure 6D). The second one was that miR-21-cKO cultured DRG neurons expressed lower intracellular levels of Ccl2 compared with WT DRGs ( Figure 6E) and released lower extracellular CCL2 after incubation with capsaicin ( Figure 6F). Therefore, miR-21 modulation of CCL2 levels in neurons, in synergy with a miR-21-associated increase in CCR2 in macrophages, provides neuroimmune crosstalk by which miR-21 regulates monocyte infiltration in the DRG after nerve injury. In addition, our evidence indicates that in such macrophages, neuron-derived miR-21 inhibits expression of TGF-β1 and TGF-β2 and promotes a proinflammatory phenotype. Taking advantage of such a sharp phenotype, we tested the possibility that antagomir-21-transfected BMDMs exert anti-allodynic effects in neuropathic mice.
Intrathecal delivery of antagomir-21-treated macrophages reverses neuropathic allodynia. Given the contribution of DRG macrophages to both initiation and maintenance of neuropathic hypersensitivity (12), we tested the effect of BMDMs transfected with antagomir-21 on neuropathic allodynia in SNI, and used GFP + BMDMs polarized with IL-4 and TGF-β (M2-like) for comparison (Supplemental Figure 6A).
We then investigated expression of CD206 and MHCII markers in the whole DRG macrophage pool, including injected BMDMs and endogenous cells. We observed an increase in the CD206 + MHCIIpopulation in antagomir-21-BMDM-treated ipsilateral DRGs compared with scramble-BMDMs, with no difference between contralateral DRGs (Figure 8, F and G). However, the MHCII + CD206cell population was not different between antagomir-21-BMDM and scramble-BMDM DRGs (Figure 8, F-H). Therefore, these data show that intrathecal injection of antagomir-21-transfected BMDMs induces reversal of neuropathic allodynia and polarizes DRG macrophages into an M2-like phenotype.
Since we argued that such an M2 polarization shift was unlikely to have occurred within 2 hours of BMDM injection, we further characterized macrophages and found higher numbers of TGF-βR2 + cells in both ipsilateral and contralateral DRGs of antagomir-21-BMDM compared with scramble-BMDM injection ( Figure  8, I and J). These cells were TGF-βR2 + CD206 + (Figure 8K), whereas TGF-βR2 + MHCII + cells were very few and remained unaltered ( Figure 8L).
In additional analyses 48 hours after BMDM injection, when the anti-allodynic effect had faded slightly, ipsilateral DRG CD11b + F4/80 + numbers were comparable between antagomir-21and scramble-BMDM (Supplemental Figure 8A), and similar results were obtained for injected F4/80 + BMDMs (Supplemental Figure 8B). Specifically, these cells had not polarized toward either an M1-like or M2-like phenotype (Supplemental Figure 8C), which indicates adaptability of in vitro-differentiated BMDMs to an in vivo environment. Concerning TGF-βR2, we noticed a trend toward upregulation in F4/80 + cells in ipsilateral and contralateral DRGs in antagomir-21-compared with scramble-BMDM (Supplemental Figure 8D), a trend that was associated with CD206 + but not MHCII + macrophages (Supplemental Figure 8, E and F).
Altogether, these data indicate that intrathecal injection of antagomir-21-transfected BMDMs rapidly alleviates neuropathic allodynia, as macrophages acquire an M2-like phenotype in vivo that is associated with upregulation of TGF-β2R.

Discussion
This study provides evidence for a dual role of miR-21 in neurons and macrophages that promotes pronociceptive mechanisms and pathways in DRGs following peripheral nerve injury. Specifically, in injured sensory neuron cell bodies, miR-21 contributes to CCL2 upregulation that attracts inflammatory macrophages in DRGs. In macrophages, lack of transfer of sensory neuron-derived miR-21 results in upregulation of TGF-βR2, release of TGF-β1, and fosters an antiinflammatory and antinociceptive phenotype. J Clin Invest. 2023;133(11):e164472 https://doi.org/10.1172/JCI164472 and downregulation of Tnfa and Il6. Moreover, antagomir-21 activates the TGF-β canonical pathway via the SMAD family and not the ERK or p38 pathway, suggesting that miR-21 selectively inhibits TGF-β signaling in macrophages.
TGF-β is a pleiotropic and potent antiinflammatory cytokine, and we suggest that neuron-derived miR-21 exerts a pronociceptive action via inhibition of the macrophage TGF-β pathway. This is congruent with evidence that intrathecal administration of TGF-β1 inhibits development of neuropathic allodynia (24) to a similar extent as miR-21 silencing in sensory neurons, and intrathecal injection of bone marrow stromal cells, which release TGF-β1 (25), reduces neuropathic hypersensitivity to a similar extent to BMDMs transfected with antagomir-21. In DRGs, TGF-β1 likely acts via inhibition of proinflammatory cytokine expression (24,33). In this context, our evidence shows that intrathecal delivery of antagomir-21-transfected BMDMs, which express high levels of TGF-βR2 and secrete TGF-β1, exerts prompt anti-allodynic action in neuropathic conditions. We think it is plausible that TGF-β1 would activate TGF receptors in macrophages to maintain an M2-like phenotype via suppression of proinflammatory cytokines. However, we do not exclude a possible effect of TGF-β1 in sensory neurons, which express both TGF-βR1 and TGF-βR2 (34), though the effects of this cytokine in peripheral neurons are both complex and model dependent (35). Nevertheless, TGF-β1 shows unequivocal antinociceptive effects in neuropathic pain models (24), evidence that is indirectly supported by our data that also highlight Spry2 protein regulation (36) rather than TGF-β modulation as a possible miR-21 effect on neuronal regeneration. Macrophages' contribution to the development and maintenance of neuropathic pain is the result of these cells being highly plastic and critical at initiating inflammation and fostering tissue repair, depending on their origin and tissue environments (5). The commonly described M1-and M2-like states oversimplify macrophage heterogeneity and do not fully reflect the vast array of functions that can be adopted during disease and injury. Despite these limitations, the M1-/M2-like phenotype distinction offers valuable insights into investigating macrophage phenotype-dependent processes (37). For instance, intrathecal administration of M2-like macrophages inhibits osteoarthritis-like pain (38,40), while M1-like macrophages exacerbate inflammatory pain (18). Conversely, macrophage depletion reduces neuropathic allodynia (12). Here, we report that antagomir-21-treated macrophages reverse neuropathic allodynia, express higher levels of TGF-βR2, and engraft and shift the pool of DRG macrophages toward a CD206 + MHCII -M2-like phenotype.
Intriguingly, antagomir-21-treated macrophages injected intrathecally accumulate in L3-L4-L5 DRGs and our most plausible explanation is that miR-21 inhibition at least partially promotes macrophage survival. Indeed, tumor suppressors are miR-21 targets, as miR-21 inhibits apoptosis and promotes survival of cancer cells (39,40). However, inconsistencies have emerged since, for example, miR-21 deficiency in macrophages inhibits NLRP3 inflammasome expression, NF-κB activation, and IL-1 secretion, resulting in the reduction of pyroptosis (41). Conversely, overexpression of miR-21 increased cell invasion while decreasing apoptosis of fibroblast-like synoviocytes (42). Such discrepancies can be explained by (a) the type of cells, (b) conditions used for alteration of miR-21 expression, and (c) duration and context of the alteration.
Our data bring what we believe is a novel addition to the concept that neurons instruct immune cells through shuttling of miR-NA in extracellular vesicles to influence the nature of the immune infiltrate in the DRG, which in turn impacts mechanisms underlying noxious signaling. Thus, miR-21 conditional deletion in sensory neurons opposes de novo expression of the chemokine CCL2, which is normally associated with axonal injury (31,32), and results in reduction of CCR2-expressing immune cells in DRGs. Since miR-NAs inhibit their own target expression and there is no evidence that CCL2 is a miR-21 target, this effect on chemokine expression is likely to be indirect. For instance, following nerve injury, in sensory neurons miR-21 binds TLR8 in endosomes/lysosomes, activates ERK, and promotes CCL2 expression, which increases Nav1.8 channel activity and facilitates neuronal excitability (21). Such a miR-21dependent but indirect mechanism could explain the reduction in CCL2 expression that we detected in miR-21-cKO neurons.
Furthermore, lack of miR-21 in neurons is associated with upregulation of TGF-β pathway components in macrophages, which acquire an M2/tissue-repair-like phenotype. Since TGF-βR2 and TGF-β1 are known targets of miR-21, it is feasible that miR-21 transferred from injured neurons to macrophages maintains a proinflammatory phenotype via suppression of such components of this antiinflammatory pathway. Thus, these data indicate that neuronal miR-21 is a key player in the regulation of neuro-immune communication in DRGs and that miR-21 can be inhibited in macrophages to attenuate neuropathic pain.
resident macrophage proliferation since we observe a reduction in CCR2 expression and no alteration of Ki67 + F4/80 + cell numbers.
Here we advanced our understanding of miR-21-specific mechanisms and targets, as we strengthened evidence that silencing miR-21 in sensory neurons attenuates development and maintenance of neuropathic allodynia and show that such an antinociceptive effect is mediated by TGF-β in DRG macrophages.
Of interest, miR-21 silencing in sensory neurons is associated with lower levels of CCL2 and reduced accumulation of monocytes/macrophages expressing CCR2 in DRGs. Whether DRG macrophages derive from circulating monocytes or proliferation of resident macrophages is still a matter of debate (12,43,44). Nevertheless, in our experimental settings, we suggest that miR-21 silencing in neurons reduces circulating monocyte infiltration rather than  Primary macrophage culture. BMDMs were isolated and generated as previously described (50). Briefly, bone marrow cells were obtained by flushing femurs and tibias of adult mice. A single-cell suspension was obtained by passing cells through a 70-μm cell strainer. Cells were then cultured in 10-cm non-tissue culture-treated dishes for 7 days in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS HI; Gibco), 10% (v/v) L929 cell-conditioned medium as a source of macrophage colony stimulating factor (51), and 1% (v/v) penicillin/streptomycin (Gibco/Thermo Fisher Scientific) at 37°C and 5% CO 2 . The macrophage enrichment was validated by flow cytometry using F4/80 and CD11b as markers.
For PMs, mice were euthanized, and lavage of their peritoneal cavity was performed with 10 mL of ice-cold PBS (Sigma-Aldrich)/2 mM EDTA (Invitrogen). Peritoneal cells were centrifuged, resuspend in DMEM, 10% FBS, and 1% (v/v) penicillin/streptomycin and seeded in Petri dishes for a minimum of 2 hours to allow the adhesion of macrophages.
Transmigration assay was performed using cell culture inserts (Transwell plate, Costar) with 8-μm porous polycarbonate filters. DRG neurons (10,000) were cultured in 300 μL F-12/10% FBS in the lower compartment for 24 hours, and then 40,000 WT BMDMs treated with vehicle or CCR2 antagonist (1 μM; Merck) were added to the upper filter. DRG neurons were then stimulated with capsaicin (1 μM) for 3 hours.
Real-time PCR. Total and small RNAs were isolated using a mirVana miRNA Isolation Kit (Invitrogen) according to the manufacturer's instructions and RNA eluted using RNase-free water. Both concentration and purity were measured by a spectrophotometer (NanoDrop ND-100, Labtech). The total RNA samples (100 ng) were reverse transcribed with a Quantitec Reverse Transcription Kit (Qiagen). Real-time PCR was performed using SYBR Green I Master Mix (Roche) and specific primers for Our results support miR-21 inhibition as a strategy to reduce neuropathic pain through maintenance of DRG macrophage polarization in the M2-like state and highlight the important role of the neuro-immune communication that contributes to nociceptive mechanisms.
In conclusion, the findings of this preclinical research may have direct relevance to a substantial clinical problem and provide important evidence for the therapeutic potential of noncoding RNAs as new targets for the treatment of neuropathic pain, perhaps in combination with established therapeutics such as gabapentin, to obtain optimal analgesic efficacy (45). Clinical relevance of noncoding RNAs has been recently highlighted by the first-in-human miRNA-based phase I therapeutic trial in patients with liver cancer (ClinicalTrials.gov NCT01829971), which utilized miRNA mimetics encapsulated in a nanoparticle-based formulation that has shown minimal side effects to date. Our preclinical evidence on the effectiveness of modulating mir-21 levels to mitigate neuropathic nociception may lead to a clinical trial on the analgesic effect of nanoparticles containing antagomir-21 in patients with neuropathic pain.

Methods
Animals. All studies were conducted in C57BL/6 black male and female mice (Charles River). Animals were housed under a 12-hour light/12hour dark cycle with ad libitum access to food and water. miR-21 conditional mutant mice with a null first conditional allele were crossed with Advilin-Cre driver mice for conditional ablation in sensory neurons (16). Adult 8-to 12-week-old miR-21-cKO mice and their control littermates were randomly assigned to groups. Each group contained the same number of age-matched mice of both sexes.
Induction of peripheral neuropathy. Mice were subjected to the SNI model of neuropathic pain, as previously described (46). Briefly, under 2.5% isoflurane anesthesia, a small incision was made in the skin and muscle of the left thigh, and then the sciatic nerve and its 3 terminal branches were exposed. The common peroneal and tibial nerves were located, and the distal nerve stump was removed, leaving the sural nerve intact. Sham operations were performed by exposing the sciatic nerve without excision.
TGF-βR1 inhibitor administration. TGF-βR1 inhibitor (SB431542) was purchased from Selleckchem (S11067). Intrathecal injection was performed on both WT and miR-21-cKO mice on day 5 after SNI under light isoflurane anaesthesia, as previously described (47). Briefly, a spinal cord puncture was made using a 30-gauge needle and Hamilton syringe between L4 and L5 levels to deliver 100 pmol per mouse.
Data availability. All data generated in this study are included in the manuscript. In vivo and in vitro microarray data are deposited into the NCBI Gene Expression Omnibus (GEO) database under accession numbers GSE104270 and GSE227608, respectively.
Statistics. All data are expressed as mean ± SEM. Sample size is stated in the figure legends and was determined according to previous internal data/publications. Data analyses were performed using GraphPad Prism (v.9.0.1) by unpaired Student's t test (2 groups), 1-way ANOVA followed by Tukey's multiple-comparison test (3 or more groups), or 2-way ANOVA followed by Tukey's multiple-comparison test (behavioral data). P values of less than 0.05 were considered significant.
Study approval. All mouse studies were conducted under ethical approval from the local animal guide for care and use of laboratory animals (Biological Services Unit at King's College London), and in accordance with the Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986).

Author contributions
LZ and MM conceptualized the study. LZ, RS, GSL, SAM, FP, SF, and DC developed methodology/experiments. LZ, RS, GSL, and MM carried out the investigation. LZ and MM wrote the original draft of the manuscript. All authors read and approved the final manuscript.