Sex-dependent role of microglia in disulfide high mobility group box 1 protein-mediated mechanical hypersensitivity

Abstract

High mobility group box 1 protein (HMGB1) is increasingly regarded as an important player in the spinal regulation of chronic pain. Although it has been reported that HMGB1 induces spinal glial activation in a Toll-like receptor (TLR)4-dependent fashion, the aspect of sexual dimorphisms has not been thoroughly addressed. Here, we examined whether the action of TLR4-activating, partially reduced disulfide HMGB1 on microglia induces nociceptive behaviors in a sex-dependent manner. We found disulfide HMGB1 to equally increase microglial Iba1 immunoreactivity in lumbar spinal dorsal horn in male and female mice, but evoke higher cytokine and chemokine expression in primary microglial culture derived from males compared to females. Interestingly, TLR4 ablation in myeloid-derived cells, which include microglia, only protected male mice from developing HMGB1-induced mechanical hypersensitivity. Spinal administration of the glial inhibitor, minocycline, with disulfide HMGB1 also prevented pain-like behavior in male mice. To further explore sex difference, we examined the global spinal protein expression using liquid chromatography-mass spectrometry and found several antinociceptive and anti-inflammatory proteins to be upregulated in only male mice subjected to minocycline. One of the proteins elevated, alpha-1-antitrypsin, partially protected males but not females from developing HMGB1-induced pain. Targeting downstream proteins of alpha-1-antitrypsin failed to produce robust sex differences in pain-like behavior, suggesting that several proteins identified by liquid chromatography-mass spectrometry are required to modulate the effects. Taken together, the current study highlights the importance of mapping sex dimorphisms in pain mechanisms and point to processes potentially involved in the spinal antinociceptive effect of microglial inhibition in male mice.

Figure 1.

Intrathecal injection of disulfide HMGB1 leads to upregulation of Iba1 expression in spinal lumbar dorsal horn. Representative confocal images of Iba1 immunoreactivity in (A) male and (B) female spinal dorsal horns 24 hours postinjection of either vehicle (saline) or disulfide HMGB1. Bar graphs display quantification of Iba1 signal intensity in (C) male and (D) female mice expressed as percentage change to the control saline group. Data are presented as mean ± SEM, n = 5 to 6 mice/group. *P < 0.05 vs control group. dsHMG, disulfide HMGB1, scale bar: 100 μm. HMGB1, high mobility group box 1 protein.

Figure 2.

Disulfide HMGB1 induces cytokine and chemokine expression to higher levels in primary microglial culture generated from male compared to female mice. Bar graphs represent mRNA levels for (A) Tnf, (B) Ccl2, (C) Il1b, and (D) Il6 in primary cultures of microglia 6 hours after stimulation with disulfide HMGB1 (1 μg/mL) or no stimulation. Data are represented as mean ± SEM, n = 4 to 5 experimental replicates/group, *P < 0.05, **P < 0.01 vs unstimulated group. Unstim, unstimulated; dsHMG, disulfide HMGB1; HMGB1, high mobility group box 1 protein.

Figure 3.

Blockade of microglial function elicits antinociceptive effects in disulfide HMGB1-subjected male but not female mice. Withdrawal threshold values after i.t. injection of disulfide HMGB1 (1 μg/mouse) or vehicle (saline) in (A) male and (B) female mice lacking TLR4 in microglia (LysM-TLR4^fl/fl) or TLR4^fl/fl (control mice). Withdrawal response before, and 6 and 24 hours after the first day i.t. injection of a combination of disulfide HMGB1 (1 μg/mouse) and minocycline (30 μg/mouse, microglial inhibitor) or disulfide HMGB1 (1 μg/mouse) and vehicle (PBS), as well as 6 hours after the second day intrathecal injection of minocycline (30 μg/mouse) or vehicle in (C) male and (D) female mice. Data are presented as mean ± SEM, n = 4 to 8 mice/group, *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle groups. ds HMGB1, disulfide high mobility group box 1 protein.

Figure 4.

LC/MS-MS reveals 12 spinal proteins to be differentially expressed in response to sex and minocycline treatment. Venn diagram displays 3 statistically significant contrasts (P < 0.05)—sex difference, treatment effect and the interaction between sex and treatment—analyzed in the 54 proteins identified by LC-MS/MS. LC-MS/MS, liquid chromatography-mass spectrometry.

Figure 5.

Minocycline treatment induces upregulation of anti-inflammatory and antinociceptive factors in the spinal dorsal horns of disulfide HMGB1-subjected male but not female mice. Bar graphs depict the effect size in protein expression of (A) A1AT5, (B) A1AT4, (C) A1AT2, (D) SPA3K, (E) SPA3N, (F) HPT, (G) VTDB, and (H) HEMO between (1) male vs female injected with disulfide HMGB1, (2) males injected with disulfide HMGB1-minocycline compared to disulfide HMGB1-vehicle, and (3) females injected with disulfide HMGB1-minocyline compared to disulfide HMGB1-vehicle. Data are presented as relative levels in log₂ scale, n = 6 mice/group. HMGB1, high mobility group box 1 protein.

Figure 6.

Alpha-1-antitrypsin, but not its downstream target neutrophil elastase, partially reverses disulfide HMGB1-induced pain-like behavior in male but not female mice. Representative Western blot images of (A) alpha-1-antitrypsin (A1AT), neutrophil elastase (ELA2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from protein extracts of lumbar spinal cords of male and female mice injected with disulfide HMGB1 in combination with vehicle (+veh) or minocycline (+mino). Bar graphs depict quantification of (B) A1AT and (C) ELA2 immunopositive bands normalized to GAPDH and presented as percentage change to vehicle control groups. Withdrawal response before, and 3, 6, and 24 hours after the first day i.t. injection of disulfide HMGB1 (1 μg/mouse) in combination with either alpha-1-antitrypsin (15 ng/mouse), sivelestat (0.5 ng, neutrophil elastase inhibitor), or vehicle (PBS), and 3 and 6 hours after the second day i.t. injection of either alpha-1-antitrypsin (30 ng/mouse), sivelestat (1 ng/mouse), or vehicle in (D) male and (E) female mice. Data are presented as mean ± SEM, n = 6 mice/group for Western blot results and n = 5 to 12 mice/group for behavioral results, *P < 0.05, ***P < 0.001 vs control groups. ds HMGB1, disulfide high mobility group box 1 protein.

Figure 7.

proteinase-activated receptor 2 depletion partially protects male and female mice from developing disulfide HMGB1-induced mechanical hypersensitivity. (A) Representative Western blot images and (B) quantification of proteinase-activated receptor 2 (PAR2) immunopositive bands normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and presented as percentage change to vehicle control groups. Withdrawal response after i.t. injection of disulfide HMGB1 (1 μg) or vehicle in (C) male and (D) female wild-type (PAR2^+/+) and whole-body PAR2 knockout (PAR2^−/−) mice. Data are presented as mean ± SEM, n = 6 mice/group for Western blot results and n = 4 to 7 mice/group for behavioral results, *P < 0.05, **P < 0.01, ***P < 0.001 vs control groups. ds HMGB1, disulfide high mobility group box 1 protein.