Fig. 2

Effects of cerebrospinal fluid (CSF) samples from patients with MS on human neuronal cultures. Cortical neurons derived from induced pluripotent stem cells were treated with undiluted human CSF from patients with MS or from patients without neuroinflammatory diseases for 3 days. a Neuron cultures maintained in CSF from control patients verified to have low levels of granzyme B and interleukin-1β had no evidence of injury or toxicity. b Neuron cultures exposed to CSF from patients with MS were fewer in number and had morphological evidence of damage (i.e., shorter axons, simplified arborization). c The toxicity induced by CSF from patients with MS was directly related to the CSF’s granzyme B content. Conversely, neuronal viability diminished with increasing concentration of granzyme B within a given sample. Data are shown as the mean from duplicate experiments. d The neurotoxicity of CSF from patients with MS was significantly greater at 3 days than CSF from control patients and could be abolished by the cell-permeable granzyme B inhibitor Z-Ala-Ala-Asp-chloromethylketone (25 μM) and partially prevented by inhibition of the interleukin-1 receptor using the antagonist AF 12198 (1 μM). A mixture of granzyme B inhibitor and AF 12198 was no more effective than the granzyme B inhibitor alone at preventing cumulative neurotoxicity. ****p < 0.0001 relative to CSF from control patients, ####p < 0.001 relative to CSF from patients with MS, one-way ANOVA with post hoc Tukey’s test, n = 7 per condition. Data are shown as the mean ± standard error of the mean from duplicate experiments