Sex- and cell-dependent contribution of peripheral high mobility group box 1 and TLR4 in arthritis-induced pain

Abstract

Spinal high mobility group box 1 protein (HMGB1) plays crucial roles in arthritis-induced pain; however, the involvement of peripheral HMGB1 has not been examined previously. In this study, we addressed the role of peripheral HMGB1 and explored if sex contributes differentially to nociception in arthritis. We found Hmgb1 expression to be elevated in the ankle joints of male and female mice subjected to collagen antibody-induced arthritis. Blocking the action of peripheral HMGB1, however, only reversed collagen antibody-induced arthritis-mediated hypersensitivity in males. Intra-articular injection of the toll-like receptor (TLR)4-activating, partially reduced disulfide, but not the fully reduced all-thiol, HMGB1 evoked mechanical hypersensitivity in both sexes. A sex-dependent temporal profile in expression of inflammatory factors in the ankle joint was observed in response to intra-articular injection of disulfide HMGB1, with male mice showing a delayed, yet longer-lasting increase in mRNA levels for several of the investigated factors. Intra-articular HMGB1 did not induce cellular infiltration in the ankle joint suggesting its action on tissue resident cells. To further explore possible sex differences in cellular involvement, we used the macrophage inhibitor, minocycline, and mice with specific TLR4 depletion in myeloid cells or nociceptors. We found that inhibition of resident macrophages attenuated HMGB1-induced pain-like behavior only in male mice. Interestingly, although the contribution of TLR4 on myeloid cells to nociception was minimal in females compared to males, TLR4 on nociceptors are important for HMGB1-induced pain in both sexes. Collectively, our work highlights sex- and cellular location-dependent roles of HMGB1 and TLR4 in peripheral pain mechanisms.

Figure 1.

Collagen antibody-induced arthritis (CAIA) induces joint inflammation and mechanical hypersensitivity in both male and female mice and increases expression of Hmgb1 mRNA in the ankle joints of the hind legs. (A) Scoring of arthritis assessed by visual inspection of the hind and front paws (score 0-60) and (B) mechanical hypersensitivity assessed by von Frey filaments for 16 days after CAIA induction. (C and D) Hmgb1 mRNA levels in male and female ankle joints collected on day 16. Data are presented as mean ± SEM, n = 11 mice per group for arthritis scores and behavioral data, and n = 10 to 14 for mRNA data, *P < 0.05, **P < 0.01 vs saline group. HMGB1, high mobility group box 1 protein.

Figure 2.

HMGB1 neutralizing antibody (2G7) reverses CAIA-induced mechanical hypersensitivity in male but not in female mice without affecting arthritis scores. (A and C) Arthritis scores and (B and D) mechanical hypersensitivity assessed in the CAIA model after subcutaneous injection of 2G7 (100 μg/mouse) or vehicle once a day from day 12 to day 16 in (A and B) male and (C and D) female mice. Data are presented as mean ± SEM, n = 6 mice/group, *P < 0.05, **P < 0.01 vs CAIA vehicle group. CAIA, collagen antibody-induced arthritis; HMGB1, high mobility group box 1 protein.

Figure 3.

Intra-articular injection of disulfide HMGB1 (dsHMG), but not all-thiol HMGB1 (atHMG), into ankle joint induces mechanical hypersensitivity in male and female mice. Withdrawal threshold responses, assessed by von Frey filaments subsequent to intra-articular injection of dsHMG (1 μg/mouse), and HI indexes calculated for 0 to 6 h for C57BL/6 (A) male and (B) female and BALB/c (C) male and (D) female mice. Mechanical hypersensitivity and HI index (0-6 hours) induced by an intra-articular injection of all-thiol HMGB1 (1 μg) to BALB/c (E) male and (F) female mice. Data are presented as mean ± SEM, n = 4 to 8 mice/group, *P < 0.05, **P < 0.01 vs vehicle control group. HI, hyperalgesic index; HMGB1, high mobility group box 1 protein.

Figure 4.

Intra-articular injection of disulfide HMGB1, but not all-thiol HMGB1, induces mRNA levels of inflammatory factors at different timepoints in ankle joints of male and female mice. (A) mRNA expression for HMGB1 receptors Tlr2, Tlr4, and Rage in noninjected ankle joints of males and female mice. mRNA expression for inflammatory factors (B) Tnf, (C), Il1b, (D) Il6, (E) Ccl2, (F) Cxcl1, (G) Cxcl2, (H) Cox2, and (I) Ngf in male and female mice injected with dsHMG or atHMG. Phosphate buffered saline-injected mice were used as vehicle control group and depicted as dashed lines in the graphs. Data are presented as mean ± SEM, n = 4 to 8 mice/group, *P < 0.05, **P < 0.01 male vs female. dsHMG: dsHMGB1, atHMG: atHMGB1. HMGB1, high mobility group box 1 protein.

Figure 5.

Injection of disulfide HMGB1 into the ankle joints does not induce local cellular infiltration. (A) Representative images of ankle joints stained by hematoxylin and eosin. Tb: tibia, Ta: talus, and Ca: calcaneus. Scale bar: 100 μm. mRNA expression for (B) Cd34, (C) Mpo, and (D) Cd11b in ankle joints of male and female mice injected with dsHMG. Phosphate buffered saline-injected mice were used as control group and depicted as dashed lines in the graphs. Data are presented as mean ± SEM, n = 5 to 6 mice/group. HMGB1, high mobility group box 1 protein.

Figure 6.

Addition of disulfide HMGB1 in culture induces higher levels of proinflammatory factors in male compared to female macrophages. Levels of (A) TNF, (B) IL-6, (C) CCL2, and (D) CXCL1 measured in culture supernatant after 24-hour stimulation of 1 μg/mL disulfide HMGB1 or phosphate buffered saline (vehicle control) of macrophages generated from male and female mice. Data are presented as mean ± SEM, n = 4 mice/group, *P < 0.05, **P < 0.01, ***P < 0.001. HMGB1, high mobility group box 1 protein.

Figure 7.

Inhibition of resident macrophages prevents male but not female mice from developing disulfide HMGB1-induced mechanical hypersensitivity. Withdrawal thresholds and HI indexes (0-6 hour) after intra-articular injection of disulfide HMGB1 (1 ug/mouse) in combination with either vehicle (phosphate buffered saline) or disulfide HMGB1 (1 ug/mouse) in combination with either vehicle or minocycline (30 ug and 100 ug/mouse) in (A and B) male and (C and D) female mice. Data are presented as mean ± SEM, n = 4 to 11 mice/group, *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle group. HI, hyperalgesic index; HMGB1, high mobility group box 1 protein.

Figure 8.

Depletion of TLR4 in nociceptors prevents HMGB1-induced hypersensitivity in male and female mice, whereas TLR4 depletion in myeloid cells protects male but not female mice from HMGB1-induced pain-like behavior. Withdrawal thresholds and HI indexes (0-6 hour) after intra-articular injection of disulfide HMGB1 (1 μg/mouse) or phosphate buffered saline (vehicle) in mice lacking TLR4 in (A and B) Na_v1.8-expressing neurons (Nav 1.8-TLR4^fl/fl) or (C and D) Lys-M-expressing myeloid cells (LysM-TLR4^fl/fl) in (A and C) male and (B and D) female mice. Data are presented as mean ± SEM, n = 5 to 8 mice/group, *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle group. HI, hyperalgesic index; HMGB1, high mobility group box 1 protein.