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