Low back pain and disc degeneration are decreased following chronic toll-like receptor 4 inhibition in a mouse model  Emerson Krock, Magali Millecamps, J. Brooke Currie, Laura S. Stone, Lisbet Haglund  Osteoarthritis and Cartilage  Volume 26, Issue 9, Pages 1236-1246 (September 2018) DOI: 10.1016/j.joca.2018.06.002 Copyright © 2018 The Authors Terms and Conditions

Fig. 1 Schematic of mouse treatment and behavior schedule. The in vivo mouse study consisted of baseline behavior (green), an acute time course where behavior was assessed 1, 3, 6 and 24 h after TAK-242 treatment (purple), and following a washout period (black), a chronic treatment (blue) with injections Mondays (M), Wednesdays (W), and Fridays (F) for 8 weeks. Pain behavior was assessed as indicated. AEB indicates acetone-evoked behavior, VF indicate von Frey, RR indicates rotarod test and OF indicates open field. Osteoarthritis and Cartilage 2018 26, 1236-1246DOI: (10.1016/j.joca.2018.06.002) Copyright © 2018 The Authors Terms and Conditions

Fig. 2 The effect of TLR agonists on mouse discs. A) Representative image of a Safranin-O stained disc prior to culture showing that the disc was dissected at the growth plate and only cartilaginous endplate remains anteriorly and posteriorly Scale bar indicates 1 mm. B) Discs from 12-week old wild-type male mice were treated with LPS (TLR4 agonist), Pam2CSK4 (TLR2/6 and TLR2 agonist) and Pam3CSK4 (TLR1/2 agonist) for 72 h and conditioned media was analyzed by protein array. The heat map illustrates the log fold difference compared to vehicle-treated discs. C) Changes in select cytokines shown as the mean fold difference ±95% CI compared to vehicle-treated discs. D) IL-1β and E) CXCL5 levels in the disc tissue following TLR ligand treatment were quantified by ELISA. * indicates P < 0.05, *** indicates P < 0.001, n = 5. Data was analyzed by repeated measures one-way ANOVA followed by Tukey's post-hoc test. Osteoarthritis and Cartilage 2018 26, 1236-1246DOI: (10.1016/j.joca.2018.06.002) Copyright © 2018 The Authors Terms and Conditions

Fig. 3 Effect of acute TAK-242 on behavioral signs of axial and radiating pain. A single injection of TAK-242 was given to 7–9-month-old SPARC-null (KO) and wild-type (WT) mice. Frip strength (A), acetone-evoked behavior (a measure of radiating leg pain (B), and sensitivity to von Frey filaments (C) were evaluated 1, 3, 6 and 24 h following injection. Veh indicates vehicle and TLR4-inh indicates TAK-242. Data is presented as mean ± 95% CI and was analyzed by repeated measures two-way ANOVA followed by Tukey's post-hoc test, n = 4–5 wild-type, 9 SPARC-null (KO) per group. Osteoarthritis and Cartilage 2018 26, 1236-1246DOI: (10.1016/j.joca.2018.06.002) Copyright © 2018 The Authors Terms and Conditions

Fig. 4 Behavioral signs of axial and radiating pain are attenuated by chronic TLR4 inhibition. The TLR4 inhibitor TAK-242 was injected 3 times/week for 8 weeks in 7–9-month-old SPARC-null (KO Veh and KO TLR4-Inh) and wild-type (WT Veh and WT TLR-Inh) mice. All behavior was evaluated on non-injection days, approximately 24 h after TAK-242 delivery. Grip strength (axial pain, A), acetone-evoked behavior (cold sensitivity; an index of radiating leg pain B) and mechanical sensitivity to von Frey filaments (C) were assessed on non-injection days during weeks 1, 2, 4, 6 and 8. Baseline to week 4 and week 4 to week 8 area under the curve was calculated using the trapezoid method ((T2 − T1) × ((B1 + B2)/2), where T is time and B is behavioral score) for grip strength (D, E), acetone-evoked behavior (F, G) and von Frey sensitivity (H, I). Data is presented as mean ± 95% CI and analyzed by repeated measures two-way ANOVA (A, B, C) or one-way ANOVA followed by Tukey's post-hoc test (D–I). # indicates P < 0.01, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, n = 5–6 wild-type, 9–10 SPARC-null per group. Osteoarthritis and Cartilage 2018 26, 1236-1246DOI: (10.1016/j.joca.2018.06.002) Copyright © 2018 The Authors Terms and Conditions

Fig. 5 Effect of chronic TLR4 inhibition on locomotion, weight and disc height. To examine the effect of chronic administration of the TLR inhibitor TAK-242 on locomotion, (A) the distance traveled was assessed in a five-minute open field test during weeks 3 and 7 of treatment. (B) Weight was assessed weekly. (C) Following 8 weeks of chronic TLR4 inhibition the lumbar spines of mice were imaged with X-ray and disc height index was determined. KO indicates SPARC-null, WT indicates wild-type, Veh indicates vehicle and TLR4-inh indicates TAK-242. Data is presented as mean ± 95% CI. * indicates P < 0.05, ** indicates P < 0.01. n = 5–6 wild-type, 9–10 SPARC-null per group. Data was assessed by repeated measures two-way ANOVA (A and B) or one-way ANOVA followed by Tukey's post-hoc test (C). Osteoarthritis and Cartilage 2018 26, 1236-1246DOI: (10.1016/j.joca.2018.06.002) Copyright © 2018 The Authors Terms and Conditions

Fig. 6 Pain-related neuroplastic changes in the spinal cord dorsal horn. Following 8-weeks of chronic TLR4 inhibition and behavioral assessment the dorsal horn of the spinal cord was assessed for pain-related neuroplastic changes. (A) CGRP (a pain-related neuropeptide), GFAP (an astrocyte marker) and CD11b (a microglial marker) immunoreactivity were assessed by immunofluorescence in the dorsal horn. Scale bars (white) represent 100 μm % area of immunoreactivity was calculated for CGRP (B), GFAP (C) and CD11b (D) by comparing the area stained above a threshold to the total area of the dorsal horn. KO indicates SPARC-null, WT indicates wild-type, Veh indicates vehicle and TLR4-inh indicates TAK-242. n = 4–5 wild-type, 9 SPARC-null per group. The average of three separate images was calculated for each animal and used to calculate the mean of the treatment group. Data is presented as mean ± 95% CI and was analyzed by one-way ANOVA followed by Tukey's post-hoc text, * indicates P < 0.05, ** indicates P < 0.01. Osteoarthritis and Cartilage 2018 26, 1236-1246DOI: (10.1016/j.joca.2018.06.002) Copyright © 2018 The Authors Terms and Conditions

Fig. 7 Disc secretion of inflammatory mediators is attenuated by chronic TLR4 inhibition. Following 8-weeks of chronic TLR4 inhibition and behavioral assessment discs were extracted and cultured for 48 h. Conditioned culture media was analyzed with protein arrays. Heat map data is presented as the log (fold difference) of (A) SPARC-null vehicle (KO Veh) compared to wild-type vehicle (WT Veh) mice. (B) Select cytokines are shown as mean fold difference ± 95% CI for SPARC-null vehicle compared to wild-type vehicle. Heat map data is presented as the log (fold difference) of (C) SPARC-null TAK-242-treated mice (KO TLR4-Inh) compared to SPARC-null vehicle and select cytokines (D) comparing SPARC-null TAK-242-treated mice to SPARC-null vehicle are shown as mean fold-difference ± 95% CI. # indicated P < 0.1, * indicates P < 0.05, ** indicates P < 0.001. Data was analyzed by two-tail T-tests, n = 5–6 wild-type, 7 SPARC-null (KO) per group. Osteoarthritis and Cartilage 2018 26, 1236-1246DOI: (10.1016/j.joca.2018.06.002) Copyright © 2018 The Authors Terms and Conditions