Silencing miR-21-5p in sensory neurons reverses neuropathic allodynia via activation of TGF-β–related pathway in macrophages
Lynda Zeboudj, … , David Chambers, Marzia Malcangio
Lynda Zeboudj, … , David Chambers, Marzia Malcangio
Published April 18, 2023
Citation Information: J Clin Invest. 2023;133(11):e164472. https://doi.org/10.1172/JCI164472.
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Research Article Immunology Neuroscience

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

Abstract

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.

Authors

Lynda Zeboudj, George Sideris-Lampretsas, Rita Silva, Sabeha Al-Mudaris, Francesca Picco, Sarah Fox, David Chambers, Marzia Malcangio

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Figure 4

miR-21 silencing in sensory neurons prevents neuropathic hypersensitivity and regulates TGF-βR2 expression.

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miR-21 silencing in sensory neurons prevents neuropathic hypersensitivit...
(A) RT-qPCR of miR-21-5p in DRG cultures of WT and miR-21–cKO mice, n = 6–7 cultures per group. (B) RT-qPCR of miR-21-5p in DRGs of WT and miR-21–cKO mice on day 7 after SNI (n = 6–9). (C) ImageStream analyses of exosomes (extracellular vesicles, EVs) isolated from culture media of WT and miR-21–cKO DRG neurons incubated with vehicle or capsaicin (CAPS, 1 μM) for 3 hours (n = 4). (D) Attenuated allodynia in miR-21–cKO mice up to day 7 after SNI in males and females. Data are presented as 50% paw withdrawal thresholds (PWT). +P < 0.05, +++P < 0.001 compared with miR-21–cKO contralateral thresholds; ***P < 0.001 compared with WT contralateral thresholds; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with WT ipsilateral thresholds; by 2-way ANOVA followed by Tukey’s multiple-comparison test (n = 10–14 per group). (E) Intrathecal injection of TGF-βR1 inhibitor SB431542 (100 pmol/mouse) abolished the anti-allodynic effect in miR-21 cKO. Arrow indicates the time of injection given on day 7 after SNI, n = 6. (F) Representative scatterplots of DRG CD11b+F4/80+ cells stained for TGF-βR2 on day 7 after SNI. Bar graphs represent TGF-βR2+ cell number (n = 4). (G) Representative histograms of TGF-βR2 expression in DRG CD11b+F4/80+ cells on day 7 after SNI (MFI), n = 4. (H) RT-qPCR of Tgfbr1, Tgfb1, Tgfbr2, and Tgfbr3 in F4/80+ cells isolated from DRGs of WT and miR-21–cKO mice on day 7 after SNI, n = 5 independent experiments from 4–6 pooled animals in each. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired, 2-tailed Student’s t test (A) or 1-way ANOVA followed by Tukey’s multiple-comparison test (B, C, and E–H).