Fig. 3.

A20^Cx3Cr1-KO mice are hypersensitive to LPS. a Rectal body temperature responses and b survival were analyzed in function of time in control (A20^FL; n = 13) and A20^Cx3Cr1-KO (n = 14) mice after intraperitoneal injection of 3.5 mg/kg LPS. The combined results of three independent experiments are shown. Body temperature data are means ± SEM. Statistical differences were determined by a REML analysis for rectal body temperatures and a Mantel–Cox test for the survival curve (**p < 0.01, ***p < 0.001). c Immunohistochemistry images showing Iba-1⁺ expression in the cerebral cortex, hippocampus and spinal cord of control (A20^FL) and A20^Cx3Cr1-KO mice 10 h post LPS challenge. Scale bars represent 100 µm (cortex and hippocampus) and 200 µm (spinal cord), insert: 10 and 20 µm, respectively. Representative images are displayed. Data are representative of two independent experiments. d Number of Iba-1⁺ ramified parenchymal microglia in brain. Each symbol represents data from one mouse, n = 4 per group. Data are presented as mean ± SEM. Significant differences were determined by a two-way ANOVA with Tukey correction for multiple comparison (*p < 0.05, ****p < 0.0001). e, f Three-dimensional reconstruction (scale bars represent 10 µm, e) and Imaris-based semiautomatic quantification of cell morphology (f) of cortical Iba-1⁺ microglia. Each symbol represents the average of three measured cells per mouse; n = 5 per group. Data are presented as mean ± SEM. Significant differences were determined by two-way ANOVA with Tukey correction for multiple comparison (**p < 0.01, ***p < 0.001, ****p < 0.0001). g Bioplex analysis of cytokine levels in CSF 10 h post LPS. Each symbol represents one mouse. Data are representative of three independent experiments. Significant differences were determined by a one-way ANOVA with Tukey correction for multiple comparison (**p < 0.01, ****p < 0.0001). h RNA prepared from FACS-sorted microglia from control (A20^FL) and A20^Cx3Cr1 mice either or not injected with LPS for 10 h was submitted for RNA sequencing. Heat map of expression values for inflammatory genes that are upregulated in LPS-stimulated A20^Cx3Cr1 microglia compared with LPS-stimulated control microglia. Each column represents microglia data from one individual mouse, with four or five mice per group. Color code presents linear scale