Sex- and cell-dependent contribution of peripheral high mobility group box 1 and TLR4 in arthritis-induced pain (1)
High mobility group box 1 protein (HMGB1) is a nonhistone nuclear protein that can serve as an alarmin and plays key roles not only in inflammation but also in pain processing. The actions of extracellular HMGB1 are dependent on its redox modifications. During inflammation, all-thiol HMGB1 may be partly oxidized to the disulfide form, which acts on TLR4 and induces cytokine production. Increasing evidence indicates a sexually dimorphic role of TLR4 in pain. Despite both male and female mice expressing spinal TLR4, intrathecal injection of the TLR4 ligand, lipopolysaccharide (LPS) induces hypersensitivity only in male mice. The collagen antibody-induced arthritis (CAIA) model was use for investigating if blockade of peripheral HMGB1 attenuates pain-like behavior in a sex-dependent fashion.
Any differences in disease severity or mechanical hypersensitivity were shown in between the sexes in mice that developed joint inflammation. Mice that had arthritis scores less than 12 on day 12 were excluded from the study and, given these criteria, 35% males and 6% females were excluded. Thus, males showed a lower incidence of CAIA.
Although subcutaneous injection of 2G7, an HMGB1 neutralizing antibody (100 μg/mouse) once a day for 6 days starting from day 12, had no effect on the arthritis score of male or female mice, it significantly reversed CAIA-induced mechanical hypersensitivity at 3, 75, and 120 hours postinjection in male but not in female mice.
Disulfide or all-thiol HMGB1 (1 μg/mouse) was injected into the ankle joint of naive male and female mice and withdrawal thresholds were assessed over 24 hours. Intra-articular disulfide HMGB1 evoked mechanical hypersensitivity in male and female C57BL/6 and BALB/c mice. The mechanical thresholds were still significantly lower in the disulfide HMGB1 male and female groups. All-thiol HMGB1 did not evoke mechanical hypersensitivity in either male or female mice after intra-articular injection.
No differences in mRNA levels were detected for Tlr2 and Rage between male and female mice. However, Tlr4 expression was significantly higher in females compared to male mice. All-thiol HMGB1 did not elevate mRNA for any of the factors assessed in either male or female ankle joints. After intra-articular disulfide HMGB1 injection, female mice showed robust mRNA induction of Tnf, Il1b, Il6, Ccl2, Cxcl1, and Cxcl2 at 2-hour timepoint with Il1b and Cxcl1 levels being higher compared to male mice at this time point. The levels of Tnf, Il1b, Il6, and Ccl2 mRNA were significantly higher in males than females at 6-hour time point.
It was investigated that disulfide HMGB1 induces the production of proinflammatory factors in primary macrophage culture and the levels of these factors differ in cultures prepared from both male and female mice. A significant increase of TNF, IL-6, and CXCL1 levels in culture supernatant of macrophages derived from male mice after stimulation with disulfide HMGB1.
