Fig 2: Neutrophil depletion attenuates mechanical allodynia and accumulation of CatE-positive cells in the DRG during the preclinical phase of EAE. (A) The PWT in MOG35-55-immunized WT mice that were treated with Ly6G-specific antibody (500 µg, i.p.) or isotype control IgG2a (500 µg, i.p.). n = 6 mice per group, two-way ANOVA Tukey test, ***P < 0.001. (B) Immunoblot for ELA2 and MPO in the L5 DRG of neutrophil-depleted mice on day 5 after MOG35-55 immunization. The columns represent the relative values of ELA2 or MPO proteins normalized to ß-actin. n = 3 mice per group, unpaired t test, ***P < 0.001. (C) Immunoblot for CatE in the L5 DRG of neutrophil-depleted mice on day 5 after MOG35-55 immunization. The columns represent the relative values of CatE protein normalized to ß-actin. n = 3 mice per group, unpaired t test, ***P < 0.001. (D) Images show CatE (green) and Nissl (blue) in the L5 DRG of neutrophil-depleted mice on day 5 after MOG35-55 immunization. Arrowheads indicate CatE-positive cells. Bottom image indicates the enlarged image of inset. Scale bars = 100 µm and 10 µm. The columns represent the density of CatE-positive cells in the L5 DRG. n = 5 mice per group, unpaired t test, ***P < 0.001. Data represent the mean ± SEM. ANOVA, analysis of variance; DRG, dorsal root ganglion; EAE, experimental autoimmune encephalomyelitis; PWT, paw withdrawal threshold; WT, wild-type.

Fig 3: MOG35-55 induces CatE in neutrophils. (A) Representative dot plot for flow cytometry of Ly6G+/CD11b+ cells in isolated neutrophils from the bone marrow of WT mice. Gating is based on isotype control for Ly6G and CD11b. (B) Immunoblot for CatE in neutrophils after stimulation with MOG35-55 (25 µg/mL) for 6 hours. The band for CatE is absent in CatE-/- neutrophils. The columns represent the relative values of CatE protein normalized to ß-actin. n = 3 mice per group, unpaired t test, ***P < 0.001. (C) Quantitative PCR analyses of CatE mRNA in MOG35-55-stimulated neutrophils. The columns represent the relative values of CatE mRNA normalized to ß-actin. n = 3 mice per group, unpaired t test, ***P < 0.001. (D) Enzymatic activity for CatE in cell lysate of MOG35-55-stimulated neutrophils. n = 3 mice per group, two-way ANOVA Tukey test, **P < 0.01. (E) Images show CatE (green), ELA2 (red), and Nissl (blue) in MOG35-55-stimulated neutrophils. Broken lines indicate outline of neutrophils. Scale bars = 10 µm. The profile plots fluorescence intensity (values from 0 to 255) of CatE and ELA2 fluorescence at the position along the black line in the image that was cropped from merged image. Data represent the mean ± SEM. ANOVA, analysis of variance; WT, wild-type.

Fig 4: CatE-dependent production of elastase in neutrophils contributes to the induction of mechanical allodynia during the preclinical phase of EAE. (A) The PWT of naive C57BL/6 mice after intravenous injection of MOG35-55 (25 µg/mL)-stimulated WT neutrophils (n = 10), MOG35-55-stimulated CatE-/- neutrophils (n = 10), MOG35-55-stimulated WT monocytes (n = 6), or OVA323-339 (25 µg/mL)-stimulated WT neutrophils (n = 6). Two-way ANOVA Tukey test, vs 0 hours: ***P < 0.001, †P < 0.05; WT neutrophils (MOG35-55) vs CatE-/- neutrophils (MOG35-55): ††P < 0.01, †††P < 0.001; two-way ANOVA Tukey test, vs 0 hours (MOG35-55-stimulated WT monocytes): 2 hours: P > 0.9999; 4 hours: P = 0.9986; 6 hours: P > 0.9999; vs 0 hours (OVA323-339-stimulated WT neutrophils): 2 hours and 4 hours: P > 0.999; 6 hours: P = 0.9998. (B) The PWT of naive C57BL/6 mice after intrathecal injection of saline- or MOG35-55-stimulated WT neutrophils and MOG35-55-CatE-/- neutrophils. n = 10 mice per group, two-way ANOVA Tukey test, vs 0 hours: ***P < 0.001, WT neutrophils (MOG35-55) vs CatE-/- neutrophils (MOG35-55): †††P < 0.001. (C) Systemic (100 mg/kg, n = 10) or intrathecal injection (100 ng, n = 6) of sivelestat attenuated mechanical allodynia caused by intravenous injection of MOG35-55-stimulated neutrophils. Two-way ANOVA Tukey test, vs saline (i.p.), 0 hours: ***P < 0.001, saline (i.t.), 0 hours: ‡‡‡P < 0.001; saline (i.p.) vs sivelestat (i.p.): †††P < 0.001; saline (i.t.) vs sivelestat (i.t.): §P < 0.05, §§P < 0.01, §§§P < 0.001. (D) Neutrophil elastase (NE) activity in the supernatant of WT and CatE-/- neutrophils 6 hours after MOG35-55 (25 µg/mL) stimulation. n = 3 mice per group, two-way ANOVA Tukey test, ***P < 0.001. (E) Whole-cell lysate of DRG from MOG35-55-immunized CatE-/- mice was incubated with recombinant mouse CatE (rmCatE, 1.25 µg) for 6 hours with or without pepstatin A (10 µM), a specific inhibitor of aspartic protease. n = 4 mice per group, one-way ANOVA Tukey test, *P < 0.05, **P < 0.01. (F) Quantitative PCR analyses of Elane mRNA in MOG35-55-stimulated neutrophils. The columns represent the relative values of Elane mRNA normalized to ß-actin. n = 3 mice per group, two-way ANOVA Tukey test, *P < 0.05. (G) The PWT of naive WT or CatE-/- mice after intrathecal injection of recombinant mouse neutrophil elastase protein (rmNE, 1 µg). n = 10 mice per group, two-way ANOVA Tukey test, vs 0 hours (WT, rmNE): ***P < 0.001; vs 0 hours (CatE-/-, rmNE): †††P < 0.001. (H) The PWT of MOG35-55-immunized mice 3 and 24 hours after intrathecal injection of pepstatin A-penetratin (PepA-p). n = 6 mice per group, two-way ANOVA Tukey test, vs day 5 pre: *P < 0.05, ††P < 0.01, †††P < 0.001, ### P < 0.001; PepA-p (300 ng) vs PepA-p (3000 ng): §P < 0.05. (I) Neutrophil elastase activity in the DRG of MOG35-55-immunized mice. The DRG was collected from PepA-p-treated mice after behavioral analyses on day 7. n = 6 mice per group, one-way ANOVA Tukey test, *P < 0.05, ***P < 0.001. Data represent the mean ± SEM. ANOVA, analysis of variance; DRG, dorsal root ganglion; EAE, experimental autoimmune encephalomyelitis; PWT, paw withdrawal threshold; WT, wild-type.

Fig 5: 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.