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. 2018 Aug 21;115(34):E8057-E8066.
doi: 10.1073/pnas.1721815115. Epub 2018 Aug 6.

Macrophage Angiotensin II Type 2 Receptor Triggers Neuropathic Pain

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Free PMC article

Macrophage Angiotensin II Type 2 Receptor Triggers Neuropathic Pain

Andrew J Shepherd et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Peripheral nerve damage initiates a complex series of structural and cellular processes that culminate in chronic neuropathic pain. The recent success of a type 2 angiotensin II (Ang II) receptor (AT2R) antagonist in a phase II clinical trial for the treatment of postherpetic neuralgia suggests angiotensin signaling is involved in neuropathic pain. However, transcriptome analysis indicates a lack of AT2R gene (Agtr2) expression in human and rodent sensory ganglia, raising questions regarding the tissue/cell target underlying the analgesic effect of AT2R antagonism. We show that selective antagonism of AT2R attenuates neuropathic but not inflammatory mechanical and cold pain hypersensitivity behaviors in mice. Agtr2-expressing macrophages (MΦs) constitute the predominant immune cells that infiltrate the site of nerve injury. Interestingly, neuropathic mechanical and cold pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs and AT2R-null hematopoietic cell transplantation. Our study identifies AT2R on peripheral MΦs as a critical trigger for pain sensitization at the site of nerve injury, and therefore proposes a translatable peripheral mechanism underlying chronic neuropathic pain.

Keywords: AT2R; angiotensin; chemogenetics; macrophage; neuropathic pain.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Peripheral AT2R activation mediates neuropathic pain hypersensitivity. (A) Experimental scheme depicting nerve injury-induced neuropathy, pain behavioral assessments, and drug administration timeline in C57BL/6 (B6) mice for BE (data analysis scheme in SI Appendix, Fig. S1A). (B) Systemic administration of PD123319 (10 mg/kg, i.p.) attenuates SNI-induced mechanical hypersensitivity. Mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sham+saline/PD123319 groups; #P < 0.05 and ##P < 0.01 vs. SNI+saline group. (C) Losartan (10 mg/kg, i.p.) has no effects on SNI-induced mechanical hypersensitivity. Mean ± SEM; **P < 0.01 and ***P < 0.001 vs. sham+losartan-ipsilateral (ipsi) group. (D) Intrathecal PD123319 (30 nmol in 10 μL) does not attenuate SNI-induced mechanical hypersensitivity. Mean ± SEM; ***P < 0.001 vs. contralateral (contra) groups; not significant (ns) vs. SNI+saline-ipsi group. (E) Peri-sciatic PD123319 administration (30 nmol in 10 μL) attenuates SNI-induced mechanical hypersensitivity. Mean ± SEM; ***P < 0.001 vs. contra groups; ###P < 0.001 vs. SNI+saline-ipsi group. (F) SNI elevates Ang II levels in injured mouse sciatic nerve, but not in the spinal cord. Mean ± SEM (n = duplicate tissue samples from six mice per group). ***P < 0.001 vs. respective SNI-contralateral groups; not significant (ns) vs. sham/SNI-contralateral or vehicle groups. (G) Experimental scheme depicting cold hypersensitivity assessment, and drug administration timeline in mice subjected to sham/SNI surgery. (H) Systemic administration of PD123319 (10 mg/kg, i.p.) attenuates SNI-induced cold hypersensitivity. Mean ± SEM; *P < 0.05 and ***P < 0.001 vs. sham+PD123319 group; ##P < 0.01 vs. SNI+saline group. Rectangular boxes in BE and H denote postdrug administration time points for behavioral assessment.
Fig. 2.
Fig. 2.
Peripheral AT2R/TRPA1 inhibition attenuates neuropathic pain hypersensitivity. (A) TRPA1 antagonist A967079, but not TRPV1 antagonist AMG9810 (30 mg/kg for each, i.p.), attenuates SNI-induced mechanical hypersensitivity. Rectangular boxes denote postdrug administration behavioral assessment time points. Mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sham-A967079/AMG9810 groups; not significant (ns) vs. sham-A967079 group, n = 8 mice per group. (B) TRPA1 antagonist A967079 attenuates SNI-induced cold hypersensitivity. Rectangular box denotes postdrug administration behavioral assessment time point. Mean ± SEM; ***P < 0.001 vs. sham-A967079 group; ##P < 0.01 and ###P < 0.001 vs. SNI-saline group. (C) Coadministration of the AT2R antagonist PD123319 (3 mg/kg, i.p.) and the TRPA1 antagonist A967079 (10 mg/kg, i.p.) does not additively reverse SNI-induced mechanical (Left) or cold hypersensitivity (Right). Rectangular boxes denote postdrug administration behavioral assessment time points. Mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 vs. respective SNI-contralateral groups; #P < 0.05 vs. 10d-BL time point.
Fig. 3.
Fig. 3.
Absence of AT2R gene expression in DRG sensory neurons and microglia/MΦs without or with nerve injury/neuropathy. (A) The Agtr2 gene (coding for AT2R) is not expressed in neurons and nonneuronal cells in mouse DRG, as verified by lack of GFP signal in DRG sections from Agtr2GFP reporter mice, in which the Agtr2 promoter drives GFP expression. DRG sections are stained with CGRP and NF200 antibodies to mark peptidergic and myelinated sensory neurons. (Scale bars, 50 μm.) (B) A subset of sciatic nerve fibers of Agtr2GFP mice are GFP+ (green). Such fibers are CGRP (red; Upper), and NF200+ (red; Lower) DAPI: blue. (Scale bars, 200 µm.) (C) Seven days following SNI surgery, there is an appreciable increase in Iba1+ cells (red; Center) in ipsilateral vs. contralateral DRG, wherein GFP signal (green; Left) remains negligible. DAPI: blue. (Scale bars, 50 μm.)
Fig. 4.
Fig. 4.
Peripheral MΦ infiltration and AT2R expression therein are associated with nerve injury/neuropathy. (A) Experimental protocol for identification of injury markers in the sciatic nerve of mice subjected to sham or SNI surgery. (BD) Massive MΦ infiltration (Iba1-red, Upper row in B; Iba1-green and F4/80-red in C) and considerable neutrophil infiltration (Ly6g-red, Lower row in B) accompany SNI-induced nerve fiber degeneration (decreased NF200 staining; green) in ipsilateral sciatic nerves, 5 and 15 d after SNI. Sections are costained with nuclear marker (DAPI; blue). (Scale bars, 200 μm.) MΦ density in sciatic nerves is quantified in B. Mean ± SEM; ***P < 0.001 vs. respective sham-ipsilateral groups; not significant (ns) vs. contralateral groups (n = 2 sections per mouse, 4 mice per group). Lower row images in C are magnified (630×) views of the areas marked with white dotted boxes in Upper row images. (D) Enhanced infiltration of MΦs that express GFP (F4/80-red, GFP-green and DAPI-blue) in the ipsilateral sciatic nerves from Agtr2GFP is observed 7 d after SNI, indicating AT2R expression in MΦs under nerve injury/neuropathy conditions. (Scale bars, 200 μm.) Right column images are magnified (630×) views of the areas marked with white dotted boxes on Left column images.
Fig. 5.
Fig. 5.
Peripheral MΦ infiltration is critical for nerve injury/neuropathy-induced mechanical and cold hypersensitivity. (AC) Chemogenetic depletion of peripheral MΦs in MaFIA mice with B/B-HmD administration (2 mg/kg/d for 5 d, starting 6 d after SNI) (A), significantly attenuates SNI-induced ipsilateral hindpaw mechanical (B) and cold hypersensitivity (C), which subsequently returns to predepletion levels 3–4 d after the last B/B-HmD administration. Mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 vs. respective baselines, sham – B/B-HmD-ipsi group or respective SNI – B/B-HmD-contra group; #P < 0.05, ##P < 0.01, ###P < 0.001 and not significant (ns) vs. SNI – vehicle-ipsi group. (D) Histological confirmation of MΦ (Iba1-red) depletion at day 11 after SNI (after fifth B/B-HmD), and repopulation at day 16 after SNI (5 d after final B/B-HmD) in the sciatic nerves (NF200-green) of MaFIA mice, which are quantified in E. (Scale bars, 200 μm.) (E) Mean ± SEM; ***P < 0.001 vs. respective sham – B/B-HmD-ipsi group; ###P < 0.001 and not significant (ns) vs. respective SNI – vehicle-ipsi groups (n = 2 sections per mouse, 4 mice per group). (F and G) Following MΦ depletion, administration of PD123319 on day 10 (red box) has no additional effect on ipsilateral hindpaw mechanical (F) or cold (G) hypersensitivity. Aqua rectangular boxes in B, C, F, and G, and red rectangular boxes in D and E denote postdrug administration behavioral assessment time points. *P < 0.05, and ***P < 0.001 vs. respective SNI-contra group.
Fig. 6.
Fig. 6.
AT2R expression in the hematopoietic lineage is critical for nerve injury/neuropathy-induced mechanical and cold hypersensitivity. (A) Schematic showing generation of Agtr2-WT and Agtr2-KO bone marrow chimeras, and subsequent induction of nerve-injury/neuropathy for pain-related behavioral assessment. (B) SNI induces significant mechanical (Left) and cold hypersensitivity (Right) in Agtr2-WT chimeras, which could be attenuated by systemic administration of the AT2R antagonist PD123319 (10 mg/kg, i.p.). In contrast, Agtr2-KO chimeras show significantly attenuated mechanical (Left) and cold hypersensitivity (Right) upon SNI induction, indicating the critical role of MΦ AT2R in the induction and maintenance of neuropathic pain hypersensitivity. Mean ± SEM; *P < 0.05 and ***P < 0.001 vs. Agtr2-WT or Agtr2-KO sham-ipsi groups; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. Agtr2-WT SNI-ipsi group. Rectangular boxes in B denote postdrug administration behavioral assessment time points. (C) Representative confocal microscopic images of sciatic nerve sections from Agtr2-WT and Agtr2-KO bone marrow chimeras 15 d after SNI. Elevated Iba1 expression (red) is observed within the ipsilateral, but not contralateral nerves of Agtr2-WT (Upper) and Agtr2-KO (Lower) bone marrow chimeras. NF200: green; DAPI: blue. (Scale bars, 200 µm.) (D) Microglial proliferation/density (Iba1: red, DAPI: blue) in spinal cord dorsal horn from Agtr2-WT and Agtr2-KO bone marrow chimera mice, 10 d after sham/SNI surgery. (Scale bars, 50 µm.)

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