A well-characterized mouse model of PM was used in this study [
29,
30]. Briefly, adult male C57BL/6 mice aged 8–16 weeks were clinically examined and scored. Clinical scoring consisted of a beam balancing test, a postural reflex test, and the presence of piloerection, seizures, or reduced vigilance [
31]. In healthy animals, the score is 0 points; 13 points are attributed to terminally ill animals that have to be euthanized due to ethics within the observation period. Additionally, an open field test (as described previously [
32]) and temperature measurements were carried out. After clinical scoring, meningitis was induced by intracisternal injection of 10
5 colony-forming units of
S.
pneumoniae type 2 (D39 strain) under short-term anesthesia induced by isoflurane. Mice injected with equal volume of PBS served as negative controls (
n = 8). In total, 100 mice were used in this study; two of them had to be euthanized immediately after intracisternal injection due to clinical signs of brain stem injury caused by the injection procedure. At 21 h after infection, all 98 mice achieved disease scores between 4 and 9 and were randomly allocated to the different i.p. treatment groups which were as follows: antibiotic therapy with 100 mg/kg ceftriaxone in combination with 100 μg anti-HMGB1 chicken polyclonal antibody (SHINO-TEST Corporation,
n = 8), 10 mg/kg paquinimod (Active Biotech,
n = 8), anti-HMGB1 + paquinimod (
n = 6), 0.5 mg/kg dexamethasone (every 8 h,
n = 9), anti-HMGB1 + dexamethasone (
n = 8), 12 mg/kg daptomycin (
n = 6), or anti-HMGB1 + daptomycin (
n = 8). If not otherwise stated, treatments were given once per day. Administration of ceftriaxone alone (
n = 12) served as positive control, while adjuvant injection with anti-HMGB1 isotype antibodies (
n = 12) or DMSO (paquinimod vehicle,
n = 9) were used as placebo-treated controls. Adjunctive treatments were injected 5 min before ceftriaxone injection. All treatments were applied immediately after one another. At the time when treatment is started, MRP14 and HMGB1 are conceivably present in the CSF since previous Western blot and ELISA experiments showed that MRP14 and HMGB1 are released into the CSF within the first 18 to 24 h post-infection in murine pneumococcal meningitis [
28,
29]. Moreover, MRP14 and HMGB1 were detected in CSF samples withdrawn from patients with pneumococcal meningitis at the time of admission to the hospital [
28,
29]. Twenty-four hours after therapy, animals were blindly clinically evaluated again. Then, mice were anesthetized with ketamine/xylazine, and a catheter was placed into the cisterna magna. CSF samples were withdrawn for the determination of CSF leukocyte counts and the intracranial pressure was measured. Subsequently, blood samples were obtained by transcardial puncture for the assessment of bacterial titers and total leukocyte counts. After deep anesthesia induced by thiopental, mice were perfused with ice-cold phosphate-buffered saline containing heparin. Brains were removed and frozen immediately. The experiment was performed in batches of 8 mice, with 1–2 mice randomly allocated to one of the different treatment groups, so that for every treatment group, the procedure was repeated at least thrice.