Fig. 5: HB-EGF exerts anti-inflammatory and tissue-protective effects on CNS-resident and -infiltrating cell types.

a, RT–qPCR analysis of Il1b, Cd68, Tnf and Lif expression in microglia pre-activated with LPS for 8 h and stimulated with ACM derived from pseudohypoxic (CoCl₂) astrocytes, where astrocyte-derived HB-EGF was blocked by an anti-HB-EGF (αHB-EGF) antibody for 24 h. n = 3/4 per group. b, RT–qPCR analysis of Icam1 expressed by primary mouse BMVECs stimulated with TNF-α, IFN-γ ± HB-EGF or vehicle. n = 3 per group. c, Schematic of i.c.v. injection of TNF-α and IL-1β ± HB-EGF or vehicle, followed by intracellular flow cytometry after 24 h. d, MFI of GM-CSF production by CD45^intCD11b⁺ microglia (left) and CD45^hiCD11b⁺Ly6C⁺ monocytes (right) following i.c.v. injection of TNF-α and IL-1β ± HB-EGF or vehicle. n = 3 per group. e, Quantification of survival and the expression (% of parent) of differentiation markers by primary mouse oligodendrocytes at day 5 of culture in the presence of HB-EGF, T3, PDGF/FGF or vehicle quantified by flow cytometry. n = 11 per group. f, Representative scatter plots depicting PDGFRα and O4 expression by primary mouse oligodendrocytes at day 5 of culture in the presence of HB-EGF, T3, PDGF/FGF or vehicle. g, RT–qPCR analysis of Ptprz1, Pdgfra, Plp and Mbp expression by O4⁺ oligodendrocytes following i.c.v. injection of TNF-α, IL-1β ± HB-EGF. n = 3 per group. h,i, Immunostaining (h) and quantification (i) of Olig2⁺ cells in optic nerves stimulated with IFN-γ ± HB-EGF or vehicle. n = 5–7. Scale bar, 50 µm. j–l, Schematic (j), fluoromyelin staining (k) and quantification (l) of LPC-induced demyelination in the corpus callosum. n = 28. Scale bar, 400 µm. Data are shown as median with the 25th and 75th percentiles. m, Representative scatter plots of neuronal cells stained with Annexin V (A-V) and propidium iodide (PI) following stimulation with TNF-α ± HB-EGF or vehicle. n, Quantification of early apoptotic (A-V⁺PI⁻), late apoptotic (A-V⁺PI⁺) and necrotic (A-V⁻PI⁺) neuronal cells following stimulation with TNF-α ± HB-EGF, or vehicle. n = 4 per group. o,p, Immunostaining (o) and quantification (p) of RBPMS⁺ retinal ganglion cells in retinae stimulated with IFN-γ ± HB-EGF or vehicle. n = 11–19 per group. Scale bar, 50 µm. Data are shown as mean ± s.d. One-way ANOVA with Dunnett’s (tested against control) or Tukey’s multiple comparisons test if not indicated otherwise. Unpaired t-test in l.

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