Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: Demographic information of Intervertebral disc donors.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques:

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: Disc NLRP3 levels correlate with the pain and disc degeneration level in LDD patients. Disc specimens from 24 participants who had different levels of Thompson classification of the degeneration and scores for pain were analyzed. (A, B) The NLRP3 protein levels in disc tissue were quantified by ELISA in all 24 specimens. Reads of NLRP3 OD values at 450nm were presented. The correlation between NLRP3 protein levels and pain score ( A , r 2 = 0.85, p<0.0001) or Thompson classification of the degeneration level ( B , p=0.003) was assessed.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: NLRP3 is exclusively expressed in disc microglia. Single cell expression profile for mouse spinal cord was obtained from Panglaodb. (A–F) In all analyzed 6 mouse spinal cord samples, NLRP3 (blue rectangle) was exclusively expressed in disc microglia clusters (red rectangle).

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: Generation of microglia-specific NLRP3-KO or NLRP3-overexpressing mice. (A) Illustration of mice with microglia-specific depletion of NLRP3 (Tmem119p-CreERT2; NLRP3 (fx/fx)) and their control NLRP3(fx/fx) mice, as well as mice with microglia-specific persistent expression of NLRP3 (Tmem119p-CreERT2; NLRP3mut) and their control NLRP3mut mice. (B) NLRP3 staining was done in spinal discs from tamoxifen-challenged mice. (C, D) Dissociated cells from spinal discs of the mice were FAC sorted for CD68+Tmem119+ microglia, the NLRP3 levels of which were checked by ELISA. (C) The relative levels to those from NLRP3(fx/fx) (=1) were shown. (D) The presentative flow charts of FACS sorting CD68+Tmem119+ microglia. *p<0.05. ns, non-significant. Scale bars are 100µm.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques: Control, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: NLRP3 depletion in microglia reduces phagocytosis potential and release of pro-inflammatory cytokines. (A) Phagocytosis for zymosan was assessed in sorted disc microglia from mice with microglia-specific alteration in NLRP3 expression. (B) ELISA for IL-1β, TNFα, IFNɣ, ARG1 and CD163 in sorted disc microglia from mice with microglia-specific alteration in NLRP3 expression. The relative levels to those from NLRP3mut (=1) were shown. *p<0.05. ns, non-significant.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: NLRP3 depletion in microglia reduces disc degeneration and associated pain. The effects of altering NLRP3 levels in microglia on disc degeneration and associated pain were examined in a mouse model for LDD. A total of 8 groups of mice were included in this experiment. Group 1, NLRP3 (fx/fx) mice received sham operation (Sham); Group 2: NLRP3 (fx/fx) mice received LDD induction (LDD); Group 3: NLRP3mut mice received sham operation; Group 4: NLRP3mut mice received LDD induction; Group 5, Tmem119p-CreERT2; NLRP3 (fx/fx) mice received sham operation; Group 6: Tmem119p-CreERT2; NLRP3 (fx/fx) mice received LDD induction; Group 7: Tmem119p-CreERT2; NLRP3mut mice received sham operation; Group 8: Tmem119p-CreERT2; NLRP3mut mice received LDD induction. Mice were analyzed 8 weeks after LDD or at age of 23-week-old. (A, B) Surgical induction of LDD and the quantification of disc degeneration were performed, shown by representative images (A) and by quantification for degenerative scores (B) . (C) A Von Frey filament test for pain evaluation, shown by the relative mechanically induced withdrawal threshold and by thermally induced withdrawal latency of the paw (normalized to those from NLRP3mut (=1)). *p<0.05. ns: no significance.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques:

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: Reduction in disc degeneration and associated pain by NLRP3 depletion in microglia may result from an alleviation of neuroinflammation. (A–D) ELISA for NLRP3 (A) , IL-1β (B) , TNFα (C) and IFNɣ (D) levels in disc tissue sham/LDD-treated mice. The relative levels to those from NLRP3mut (=1) were shown. *p<0.05. ns, no significance.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: The role of ethanolamine phosphate phospholyase in regulation of astrocyte lipid homeostasis

doi: 10.1016/j.jbc.2021.100830

Figure Lengend Snippet: Identifying dietary regulation of genes in neurons and astrocytes using translating ribosomal affinity purification (TRAP). A , graphic showing possible metabolic outcomes of phosphoethanolamine (PEtN). B , Etnppl mRNA expression using qRT-PCR of input and pulled-down (IP) neuron (Syn-Cre) and astrocyte (Aldh1l1-Cre) fractions tissue harvested from ribo-tag mice exposed to fed, fasted diet, or ketogenic diet of TRAP screen. Samples sizes [Syn-Cre: fed (n = 5), fasted (n = 7), ketogenic diet (n = 6)]. Aldh1l1-Cre: fed (n = 5), fasted (n = 4), ketogenic diet (n = 8). C , protein expression of Etnppl from fed and overnight fasted adult mice n = 3. D , Etnppl mRNA expression using qRT-PCR across ages (prenatal day 18 (E18) and postnatal day 7 and 35 (P7 and P35) and CNS regions (cortex, spine, and hippocampus)). (n = 3) Data are expressed as mean ± S.D. Represented data analyzed using multiple Student’s two-tailed t-tests. ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.

Article Snippet: Constitutive Etnppl KO mice were acquired from Taconic Biosciences (#10062).

Techniques: Affinity Purification, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The role of ethanolamine phosphate phospholyase in regulation of astrocyte lipid homeostasis

doi: 10.1016/j.jbc.2021.100830

Figure Lengend Snippet: Expression of Etnppl and PEtN-related genes across early development. A , Etnppl protein expression in the whole brain across ages. WT and Etnppl KO fasted cerebellum (CB) tissue used as positive and negative controls for Etnppl protein expression repectively. B , Etnppl protein expression in the liver across ages. WT and Etnppl KO fasted CB and liver tissue used as positive and negative controls for Etnppl protein expression repectively. C , Etnppl and PEtN-related gene mRNA expression in the whole brain using qRT-PCR across ages P3 (n = 3), P4 (n = 4), P14 (n = 3), P21 (n = 4), P28 (n = 4). D , Etnppl and PEtN-related gene mRNA expression in the liver using qRT-PCR across ages P3 (n = 3), P4 (n = 4), P14 (n = 3), P21 (n = 4), P28 (n = 4). Data (as mentioned in text) are expressed as mean ± S.D. Represented data analyzed using ordinary measures two-way analysis of variance with Sidak’s tests for multiple comparisons. Outliers were removed after using Grubb’s outlier test. ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.

Article Snippet: Constitutive Etnppl KO mice were acquired from Taconic Biosciences (#10062).

Techniques: Expressing, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: The role of ethanolamine phosphate phospholyase in regulation of astrocyte lipid homeostasis

doi: 10.1016/j.jbc.2021.100830

Figure Lengend Snippet: Examining regulation of PEtN-related genes by diet and glucocorticoids. A , mRNA expression of Etnppl and other PEtN-related genes in the whole brain from adult chow-diet-fed, fasted, and refed after fasted mice using qRT-PCR. (n = 8). B , mRNA expression of Etnppl and other PEtN-related genes in the liver from adult chow-diet-fed, fasted, and refed after fasted mice using qRT-PCR. (n = 8). C , mRNA expression of PEtN-related genes in wild-type P2 1° astrocytes after a 24- or 72-h exposure to the glucocorticoid agonist dexamethasone. [dexamethasone] = 100 nM. (n = 3). Data in A and B are expressed as mean ± S.E.M. Represented data analyzed using ordinary measures two-way analysis of variance with Sidak’s tests for multiple comparisons. Data in C is expressed as mean ± S.D. Represented data analyzed using Student’s two-tailed t-tests. ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.

Article Snippet: Constitutive Etnppl KO mice were acquired from Taconic Biosciences (#10062).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The role of ethanolamine phosphate phospholyase in regulation of astrocyte lipid homeostasis

doi: 10.1016/j.jbc.2021.100830

Figure Lengend Snippet: Impact of Etnppl loss on gene expression and metabolic substrate use in the brain and liver. A , Etnppl protein expression in cerebellum from 9-week-old, 18-h fasted Etnppl KO and WT mice. B , Etnppl protein expression in liver from 9-week-old, 18-h fasted Etnppl KO and WT mice. C , cortex mRNA expression of Etnppl , genes that are indicators of CNS health, PEtN-related genes, and other metabolically relevant genes from 9-week-old, 18-h fasted Etnppl KO and WT mice. n = 4. D , liver mRNA expression of Etnppl , genes that are indicators of CNS health, PEtN-related genes, and other metabolically relevant genes from 9-week-old, 18-h fasted Etnppl KO and WT mice. n = 4. E , oxidation of 1- 14 C oleic acid to 14 CO 2 in P2 1° cortical astrocytes derived from Etnppl KO and WT mice (n = 6). [etomoxir] = 100 μM. F , incorporation of 1- 14 C ethanolamine into membranes in cultured P2 1° cortical astrocytes derived from Etnppl KO and WT mice (n = 6). [dexamethasone] = 100 nM. Data are expressed as mean ± S.D. Represented data analyzed using Student’s two-tailed t-tests. Outliers were removed after using Grubb’s outlier test. ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.

Article Snippet: Constitutive Etnppl KO mice were acquired from Taconic Biosciences (#10062).

Techniques: Gene Expression, Expressing, Metabolic Labelling, Derivative Assay, Cell Culture, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The role of ethanolamine phosphate phospholyase in regulation of astrocyte lipid homeostasis

doi: 10.1016/j.jbc.2021.100830

Figure Lengend Snippet: Loss of Etnppl does not result in major changes to oxygen consumption or abundance of many hippocampal PEtN-related metabolites. A , seahorse assay mitochondrial stress test measuring oxygen consumption using cultured P2 1° cortical astrocytes derived from Etnppl KO and WT mice after overnight incubation with dexamethasone and ethanolamine (EtN) (n = 6). [dexamethasone] = 100 nM, [EtN] = 5 mM. B , relative abundances of PEtN-associated metabolites in whole hippocampus from 18-h fasted 9-week-old Etnppl KO and WT (n = 6). Data in A are expressed as mean ± S.E.M. Represented data analyzed using multiple Student’s two-tailed t-tests. Statistical significance of represented metabolites in B determined using two-stage false discovery rate (FDR) method of Benjamini, Krieger, and Yekutieli with an FDR (Q) of 10%. Fold changes in green boxes are significantly increased, fold changes in red boxes are significantly decreased, and fold changes in yellow boxes are not significantly affected by genotype. The same data are represented as mean of relative species abundance ± S.D. in adjacent graphs in panels C – E . ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.

Article Snippet: Constitutive Etnppl KO mice were acquired from Taconic Biosciences (#10062).

Techniques: Cell Culture, Derivative Assay, Incubation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The role of ethanolamine phosphate phospholyase in regulation of astrocyte lipid homeostasis

doi: 10.1016/j.jbc.2021.100830

Figure Lengend Snippet: Phospholipid abundance and composition are altered in cortex after loss of Etnppl. A , volcano plot representing PC and PE species fold changes comparing Etnppl KO with WT using 18-h fasted cortex from 9-week-old mice. n = 5. B , volcano plot representing phospholipid species fold changes comparing Etnppl KO with WT using 18-h fasted liver from 9-week-old mice. C , total relative phospholipid abundance in the cortex from 18-h fasted, 9-week-old Etnppl KO and WT mice. n = 5. D , total relative phospholipid abundance in the cortex from 18-h fasted liver from 9-week-old Etnppl KO and WT mice. n = 5. E , relative total AA abundance in PC species in the cortex from 18-h fasted, 9-week-old Etnppl KO and WT mice. n = 5. F , relative total AA abundance in PE species in the cortex from 18-h fasted, 9-week-old Etnppl KO and WT mice. n = 5. G , relative total DHA abundance in PC species in the cortex from 18-h fasted, 9-week-old Etnppl KO and WT mice. n = 5. H , relative total DHA abundance in PE species in the cortex from 18-h fasted, 9-week-old Etnppl KO and WT mice. n = 5. Data are expressed as mean ± S.D. Represented data analyzed using Student’s two-tailed t-tests. ∗α = 0.05; ∗∗α = 0.01; ∗∗∗α = 0.001; ∗∗∗∗α = 0.0001; ns, not significant.

Article Snippet: Constitutive Etnppl KO mice were acquired from Taconic Biosciences (#10062).

Techniques: Two Tailed Test