Fig 3. Activation of innate immune cells induces CNS inflammation and severe paralysis in Nlrx1^−/− Rag^−/− mice.

(A) The infiltration of CD45^high leukocytes to the brain of Nlrx1^−/−Rag^−/− mice (n = 3) compared with Rag^−/− mice (n = 5) 14 days after immunization with MOG-CFA emulsion plus PTX, quantified by flow cytometry as shown in representative plots, ANOVA test. (B) The percentage of activated CD11b⁺MHCII⁺ microglia/macrophages in CD45⁺ cells, quantified by flow cytometry as shown in representative plots, Mann–Whitney U test. (C) Adoptive transfer of 2D2 T cells followed by MOG-CFA/PTX immunization caused hind limb paralysis in Nlrx1^−/−Rag^−/− mice, which was not observed in Rag^−/−. (D) The clinical score of mice 3 weeks after adoptive transfer and immunization (n = 4 mice per group). (E) The ratio of CD45^high cells (myeloid cells) to CD45^Low cells (microglia) in the spinal cords of Nlrx1^−/−Rag^−/− mice compared with Rag^−/− mice following adoptive T-cell transfer and MOG-CFA/PTX immunization (n = 4 mice per group). (F) The HE staining of spinal cords from Nlrx1^−/−Rag^−/− mice and Rag^−/− mice, black arrows show the infiltration of mononuclear cells, magnification 40×. (G) The expression of GFAP, Iba1, and MBP in the spinal cords of Nlrx1^−/−Rag^−/− mice compared with Rag^−/− mice, magnification 40×. (H) Quantification of stained markers (Iba1, microglia; GFAP, astrocyte; MBP, myelin basic protein) using Image J software (n = 3 mice per group). (I) The percentage of Vβ11⁺ T cells in the spinal cord, brain, and spleen of Nlrx1^−/−Rag^−/− mice compared with Rag^−/− mice (n = 4 mice per group). (J) The percentage of activated CD11b⁺MHCII⁺ microglia/macrophage in the spinal cords, brains, and spleens of Nlrx1^−/−Rag^−/− mice compared with Rag^−/− mice (n = 4 mice per group). All the data are presented as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 determined by the two-tailed Student t test, except for the data that mentioned one-way ANOVA with Tukey post hoc or Mann–Whitney U test. Underlying data can be found in S1 Data. CFA, Complete Freund's Adjuvant; CNS, central nervous system; GFAP, glial fibrillary acidic protein; HE, hematoxylin–eosin; Iba1, ionized calcium binding adaptor molecule 1; MBP, myelin basic protein; MHC, major histocompatibility complex; MOG, myelin oligodendrocyte glycoprotein; Nlrx1, nucleotide-binding, leucine-rich repeat containing X1; PTX, pertussis toxin; Rag, recombination-activating gene.