Fig 1.
Nlrx1−/− 2D2 mice develop spEAE, which is associated with CNS inflammation and demyelination.
(A) The frequency of spEAE in 2D2 and Nlrx1−/− 2D2 mice. (B) The frequency of spEAE in male and female Nlrx1−/−2D2 mice. (C) Clinical score of Nlrx1−/− 2D2 mice, showing a progressive EAE from the onset (n = 6). (D) A representative immunofluorescent staining of spinal cords from healthy Nlrx1−/−2D2 and spEAE Nlrx1−/−2D2 mice for GFAP, Iba1, and MBP markers. White arrows show astrogliosis (GFAP), microgliosis (Iba1), and focal demyelinating lesions (MBP). (E) The quantification of positive area of stained markers (n = 4 mice per group). (F) The mRNA expression levels of Tnfα, Il-1b, and Ccr5 in spinal cords from healthy Nlrx1−/−2D2 and spEAE Nlrx1−/−2D2 mice, quantified by qPCR (n = 4 or 8 mice per group). (G) A representative HE staining of the spinal cords from Nlrx1−/−2D2 spEAE and healthy Nlrx1−/−2D2 mice. (H) Immunofluorescence analysis of focal lesions in the spEAE spinal cord and the nuclear localization of NF-κB p65 subunit in CD68+ cells (white arrows), 63× magnification. The degree of colocalization was quantified using image J and Pearson correlation coefficient (PCC = 0.6). (I) The percentage of CD45high, CD45low, and CD11b+ MHCII+ cells in the spinal cord of healthy Nlrx1−/−2D2 and spEAE Nlrx1−/−2D2 mice (n = 8 mice per group). (J) Representative flow cytometry plots showing the expression of CD11b+MHCII+ myeloid cells in CD45+ cell population. (K) An immunofluorescent image of CD3+ T cells (shown by white arrows) in the spinal cords of Nlrx1−/−2D2 spEAE mice; magnification 40×; yellow, MBP; blue, DAPI; red, CD3. (L) The percentage of Vβ11+ T cells in the spinal cord and brain of spEAE Nlrx1−/−2D2 (n = 10) compared with healthy Nlrx1−/−2D2 mice (n = 7). (M) Representative flow cytometric analysis of CD45+ Vβ11+ T cells in the brain and spinal cord of spEAE and healthy animals. (N) The expression of T cell–associated transcription factors in the spinal cord (Sc) and lymph nodes (LN) of Nlrx1−/−2D2 spEAE (n = 6) mice compared with Nlrx1−/−2D2 healthy mice (n = 5). All data are presented as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 as determined by the two-tailed Student t test. Underlying data can be found in S1 Data. Ccr5, C-C chemokine receptor type 5; CNS, central nervous system; GFAP, glial fibrillary acidic protein; HE, hematoxylin–eosin; Iba1, ionized calcium binding adaptor molecule 1; Il-1b, interleukin 1 beta; LN, lymph nodes; MBP, myelin basic protein; MHC, major histocompatibility complex; NF-κB, nuclear factor κB; Nlrx1, nucleotide-binding, leucine-rich repeat containing X1; PCC, Pearson correlation coefficient; PCR, quantitative polymerase chain reaction; Rorc, retinoic acid-related orphan nuclear hormone receptor C; Sc, spinal cord; spEAE, spontaneous EAE; Tbet, T-Box transcription factor; Tnfα, tumor necrosis factor alpha.
Fig 2.
CNS inflammation associated with spEAE is more severe in Nlrx1−/−2D2 compared with 2D2 mice.
(A) Representative image of HE staining of lumbar spinal cords from spEAE mice with Nlrx1−/−2D2 or 2D2 genotype. The circles show focal lesions. (B) The number of focal lesions counted in spinal cords from spEAE mice (n = 5, 7 in each group). (C) EAE clinical score in Nlrx1−/− 2D2 (n = 18) or 2D2 (n = 6) mice at the time of euthanization. (D) The percentage of CD45high cells in the spinal cords of Nlrx1−/−2D2 spEAE (n = 6) mice compared with 2D2 spEAE mice (n = 4). (E) The flow cytometry plots of CD45high gate. (F) Similar percentages of Vβ11+ T cells and CD19+ B cells in the spinal cords of spEAE mice (n = 5, 7). (G) Elevated numbers of activated CD11b+MHCII+ monocyte/macrophage in CD45high cell population, quantified in spinal cords from Nlrx1−/−2D2 spEAE (n = 6) or 2D2 spEAE (n = 4) mice by flow cytometry. Data are presented as mean ± SD. *P ≤ 0.05 determined by the two-tailed Student t test. Underlying data can be found in S1 Data. CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; HE, hematoxylin–eosin; MHC, major histocompatibility complex; Nlrx1, nucleotide-binding, leucine-rich repeat containing X1; spEAE, spontaneous EAE.
Fig 3.
Activation of innate immune cells induces CNS inflammation and severe paralysis in Nlrx1−/− Rag−/− mice.
(A) The infiltration of CD45high 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 CD45high cells (myeloid cells) to CD45Low 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.
Fig 4.
Preclinical stages of inflammation in the CNS of Nlrx1−/−2D2 mice.
(A) The percentages of CD45low microglia and CD45high myeloid cells in the spinal cords and brains of Nlrx1−/−2D2 mice compared with 2D2 mice, quantified by flow cytometry, Mann–Whitney U test. (B) Representative plots showing CD45low and CD45high gates in spinal cord and brain samples from Nlrx1−/−2D2 and 2D2 mice, Mann–Whitney U test. (C) The percentage of activated myeloid cells (CD11b+MHCII+) in the spinal cord and brain of Nlrx1−/−2D2 and 2D2 mice (n = 8 mice per group). (D) The percentage of Vβ11+ T cells and CD19+ B cells in the spinal cords and brains of Nlrx1−/−2D2 and 2D2 mice, quantified by flow cytometry (n = 6 mice per group). (E) The mRNA levels of Iba1 and inflammatory mediators in Nlrx1−/−2D2 brains compared with 2D2 brains, quantified by qPCR (n = 7 mice per group). All the data are presented as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 as determined by the Student t test, except when Mann-Whitney U test was specified. Underlying data can be found in S1 Data. CNS, central nervous system; Iba1, ionized calcium binding adaptor molecule 1; MHC, major histocompatibility complex; Nlrx1, nucleotide-binding, leucine-rich repeat containing X1; qPCR, quantitative polymerase chain reaction.
Fig 5.
NLRX1 inhibits the tissue damage and generation of neurotoxic glia in the CNS at the subclinical stage of EAE.
(A) The level of HMGB1 in the spinal cord of asymptomatic Nlrx1−/−2D2 mice compared with 2D2 mice, determined using western blot and quantified by the percentage of HMGB1 to β-tubulin ratio (n = 6 mice per group). (B) The levels of A1- and A2-related gene expression in brain from Nlrx1−/−2D2 mice compared with 2D2 mice (n = 5 mice per group). (C) The expression of A1-related genes in the brains of Nlrx1−/− mice compared with WT mice (n = 5). (D) A heatmap diagram showing the fold change of mRNA levels of pan-, A1- or A2-reactive astrocyte-related genes in Nlrx1−/− glia culture after 24 hours of stimulation with LPS (500 ng/mL) or LPS+IFNγ (10 ng/mL) compared with the corresponding WT controls (n = 4 independent samples in each group). (E) TNFα level in the conditioned medium (CM), collected 24 hours after LPS/IFNγ treatment of glia culture and measured by ELISA (n = 4 independent samples/group). (F) The toxicity of Nlrx1−/− or WT glia CM on MO3.13 and N2A cells after 24 hours, measured by MTT assay (n = 6 independent samples/group). (G) Expression of apoptosis (annexin V) and permeability (propidium iodide [PI]) markers on MO3.13 cells incubated with Nlrx1−/−glia CM compared with WT glia CM for 24 hours (n = 4 independent samples/group). (H) The percentage of annexin V–and PI-positive N2A cells incubated with Nlrx1−/−glia CM compared with WT glia CM for 24 hours (n = 4 independent samples/group). (I) Representative flow cytometry plots used for the quantification of annexin V and PI positivity of MO3.13 and N2A cells. All the data are presented in mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, determined by the two-tailed Student t test. Underlying data can be found in S1 Data. CM, conditioned medium; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; HMGB1, high mobility group box 1; IFNγ, interferon gamma; LPS, lipopolysaccharide; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; NLRX1, nucleotide-binding, leucine-rich repeat containing X1; PI, propidium iodide; TNFα, tumor necrosis factor alpha; WT, wild-type.
Fig 6.
NLRX1: Implications for human MS.
(A) Expression of NLRX1 in PBMC from MS patients (n = 18) compared with healthy donors (n = 31), quantified using qPCR. The data are presented as mean ± SD; *P ≤ 0.05, determined by two-tailed Student t test. (B) The mRNA levels of TNFα expression in PBMC from MS patients (n = 18) and healthy individuals (n = 28), measured by qPCR. (C) Correlation between mRNA levels of NLRX1 and TNFα in the PBMC from MS samples (n = 13), statistically determined using Pearson correlation coefficient, r = 0.604, *P = 0.029. (D) Expression of Nlrx1 in the spleen and brain of C57BL/6J mice with classical EAE compared with healthy mice. The WT mice were immunized with MOG/CFA and pertussis. The brain and spleen were collected 3 weeks after immunization; *P ≤ 0.05, determined by two-tailed Student t test. Underlying data can be found in S1 Data. (E) NLRX1 conservation in orthologs. Evolutionarily conserved positions for the identified mutations are highlighted in black. Organism and RefSeq accession numbers are provided. (F) Pedigrees for families identified with NLRX1 mutations. Black filled symbol, MS; gray filled, unaffected obligate carrier. Heterozygote mutation carriers (MT) and WT genotypes are indicated. CFA, Complete Freund's Adjuvant; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; MT, mutation carrier; NLRX1, nucleotide-binding, leucine-rich repeat containing X1; PBMC, peripheral blood mononuclear cell; qPCR, quantitative polymerase chain reaction; TNFα, tumor necrosis factor alpha; WT, wild-type.