Fig. 4

Stimulation of M2a polarized bone marrow-derived macrophages (M2a BMDMs) from 2-month-old Prg4 gene-trap (Prg4^GT/GT) and Prg4^+/+ mice by Pam3CSK4, a Toll-like receptor 2 (TLR2) agonist, and efficacy of recombinant human proteoglycan-4 (rhPRG4) in reducing the secretion of cytokines and chemokines in Pam3CSK4-stimulated Prg4^GT/GT M2a BMDMs. M2a polarization was performed using interleukin-4 (IL-4)/IL-13. M2a BMDMs were treated with Pam3CSK4 (100ng/mL) ± rhPRG4 (200μg/mL) for 24h followed by gene expression studies of inducible nitric oxide synthase (iNOS), IL-1β, IL-6, IL-10, and TGF-β. Cytokine and chemokine levels in media supernatants were quantified using a murine cytokine array. The proteome profiler cytokine array panel map is presented in Supplementary Figure 3. Data is presented as mean ± standard deviation of three independent experiments, with cells derived from 2–3 animals per experiment. Analyses of cycle threshold (Ct) values and cytokine/chemokine levels were performed using one-way ANOVA, followed by Tukey’s post hoc test. ns non-significant; *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001. A Pam3CSK4 induced higher IL-1β, IL-6, and lower IL-10 expressions in M2a-polarized BMDMs from Prg4^GT/GT animals compared to BMDMs from Prg4^+/+ animals. M0-polarized BMDMs from each genotype were used as controls. B rhPRG4 treatment reduced iNOS, IL-1β, and IL-6 expression in Pam3CSK4-stimulated M2a BMDMs. C Representative cytokine array blots showing attenuated signal intensities with rhPRG4 treatment. D rhPRG4 treatment reduced the secretion of inflammatory cytokines and chemokines by Pam3CSK4-stimulated M2a BMDMs.