Fig 2: Neuronal IKKß promotes astrocyte and microglia responses and inflammatory mediators in CPZ demyelination. (Ai) Iba1 immunostaining of microglia and (Di) GFAP immunostaining of astrocytes in serial brain coronal paraffin sections from nIKKßKO and IKKßff control naïve (CPZ0) or CPZ-fed CPZ3 and CPZ5 mice. Scale bars: 100 µM. Quantitative representation of areas covered by (Aii) Iba1 immunoreactivity and (Dii) GFAP immunoreactivity in the medial corpus callosum in groups of nIKKßKO and IKKßff control mice represented in the photographs. (B, C, and E) Differential expression of the inflammatory marker genes Tnf, Ccl2, and Cxcl16 relative to GusB in total mRNA isolates taken from nIKKßKO and IKKßff control brains from CPZ0 or CPZ-fed CPZ2, CPZ3, CPZ5, CPZ6+0.5, CPZ6+1, and CPZ6+4 mice. Results are means of 2 (nIKKßKO CPZ6+0.5, CPZ6+1, nIKKßKO CPZ6+4) or 3–5 mice (for all other groups and time points) from one representative of two independent experiments. The most relevant statistically significant differences after comparisons between groups are shown by Student’s t-test (Aii and Dii) and by two-way ANOVA with Bonferroni’s test (B, C, and E). *p = 0.05, **p = 0.005, ***p = 0.001

Fig 3: Schematic representation of the different roles of neuronal TNFR in CNS pathology described in the present study. (A) Neuronal TNFR1 and IKKß promote neuroinflammation, demyelination, and axonal damage in the context of demyelination (1) signaled by EAE or CPZ toxicity. solTNF present in the microenvironment of the inflammatory lesion (2) engages TNFR1 present on the surface of neurons (as well as other cell types) and, either directly through a neuronal stress response (3), or indirectly by increasing OLG damage, enhances microglia responses and further promotes solTNF secretion and inflammation (4). (B) Neuronal TNFR2 is necessary for preconditioning neuroprotection against glutamate excitotoxicity. Preconditioning of neurons by challenge with low-dose KA (in vivo, 1a), preincubation with solTNF (in vitro, 1b), or preincubation with a TNFR2 agonist (in vitro, 1c) protects them against subsequent glutamate excitotoxicity (2) induced by high dose KA (in vivo) or NMDA (in vitro). In vitro experiments using TNF and TNFR mutant cells revealed that astrocytes can mediate the preconditioning effect of solTNF, via TNFR1 and expression of tmTNF, which in turn is necessary for interaction with neuronal TNFR2 (3) and the induction of neuroprotection mechanisms (4). Ligation of neuronal TNFR2 by the TNFR2 agonist (1c) is sufficient to reproduce this effect in vitro

Fig 4: Astrocytes stimulation examplemRNA expression determined by qPCR of proinflammatory genes after different stimuli including TNF and IFNg cytokines. (unpaired t test, compared to corresponding condition between WT and MAVS-/- astrocytes, error bars represent SEM).Reprinted with permission from Chao et al. (2019).