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: Frontiers in Immunology

Article Title: CAR-T cells and CAR-Tregs targeting conventional type-1 dendritic cell suppress experimental autoimmune encephalomyelitis

doi: 10.3389/fimmu.2023.1235222

Figure Lengend Snippet: XCR1 CAR-T cells engraft and deplete DC1 in immunodeficient mice. RAG2 −/− mice were iv injected with 1 x 10 6 XCR1, control, or mock transduced (A, B, G–J, P) CD8 + , ( C-F, O ) CD3 + (+ 10 μg IL-2), or (K–N, Q) CD4 + T cells on day 0 (n = 3/group). On day 7 or 12, spleen and ILN were collected and analyzed via flow cytometric analysis for CAR-T and DC subsets. Shown (A) are representational dot plots of the following: row 1, donor T cells (TCRβ + CD8 + ) assessed for SSC-A (y-axis) and mCherry (x-axis); row 2, recipient CD45 + cells for MHCII (y-axis) and CD11c (x-axis). Shown (B) are representational dot plots of recipient DCs (MHCII + CD11c + ) for CD11b (y-axis) and XCR1 (x-axis), and DCs for BST2 (y-axis) and XCR1 (x-axis) from the spleen of mice treated with CD8 + CAR-T cells. Graphed are the percent CAR + (mCherry + ) of the (C) TCRβ + (G) , CD8 + , and (K) CD4 + T cells and (D, H, L) the percent DCs (MHCII + CD11c + ) of total CD45 + from the ILN and spleen of mice treated with (C, D) CD3 + (G, H) , CD8 + , and (K, L) CD4 + CAR-T cells. The percent DC1 (XCR1 + CD11b − ), DC2 (XCR1 − CD11b + ), and pDC (XCR1 − BST2 + ) of total DC are shown for the spleen (E, I, M) and the ILN (F, J, N) for mice treated with (E, F) CD3 + , (I, J) CD8 + , and (M, N) CD4 + CAR-T cells. Graphed (O) is the Percent change in body weight from day 0 to the end of the experiment on day 12 of mice treated with CD3 + CAR-T cells. On day 7, a quarter of the total splenocytes were restimulated with 10 μg/mL LPS in T cell media for 24 h, and then ng/mL IL-12 was determined via MSD. Shown (P, Q) are ng/mL of IL-12 from (P) CD8 + or (O) CD4 + CAR-T cell-treated mice. Statistical significance was analyzed by use of one-way ANOVA Tukey’s multiple comparisons test. *p < 0.05. Error bars represent SEM. Data (A–J) are representative of three independent similar experiments.

Article Snippet: Female C57BL/6J “CD45.2” (Stock# 000664), B6.SJL- Ptprc a Pepc b /BoyJ “CD45.1” (Stock# 002014), B6.Cg-Foxp3tm2Tch/J “FIG” (Stock# 006772), and B6.Cg-Tg(TCRaTCRb)425Cbn/J “OTII” (Stock# 004194) were purchased from Jackson Laboratories; C57BL/6NCrl “CD45.2” and B6.SJL- Ptprc a Pepc b /BoyCrCrl “CD45.1” were purchased from Charles River Laboratories; B6.129S6- Rag2 tm1Fwa “RAG2 −/− “ were purchased from Taconic; and XCR1-DTR-cre mice were designed and generated by Horizon Discovery using CRISPR-Cas9 (now Inotiv Co, St. Louis, MO) and colony maintained at Charles River Laboratory.

Techniques: Injection, Control

Journal: Frontiers in Immunology

Article Title: CAR-T cells and CAR-Tregs targeting conventional type-1 dendritic cell suppress experimental autoimmune encephalomyelitis

doi: 10.3389/fimmu.2023.1235222

Figure Lengend Snippet: XCR1 CAR-T cells systemically deplete DC1 when engrafted with encephalitogenic T cells in immunodeficient mice. RAG2 −/− mice (n = 4/group) were iv injected with 1 x 10 6 XCR1, control, or mock transduced CD45.1 + CD8 + CAR-T alongside 1 x 10 6 encephalitogenic CD4 + CD45.2 + T cells and 10 µg of IL-2 IP on day 0. On day 6, ILN, CLN, spleen, lung, and liver were collected and analyzed via flow cytometric analysis for CAR-T cells and DC subsets. Shown (A) the percent CAR + (mCherry + ) CD45.1 + of viable and (B) the percent Ki67 + of donor CD45.1 + cells and (C) the percent DC of total CD45.2 + . Shown (D) are representational dot plots of DC (MHCII + CD11c + ) for XCR1 (y-axis) and CD11b (x-axis) for each organ. Graphed are (E) the percent DC1 (XCR1 + CD11b − ), (F) DC2 (XCR1 − CD11b + ), and (G) pDC (XCR1 − BST2 + ) of total DCs. Statistical significance was analyzed by use of one-way ANOVA Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent SEM.

Article Snippet: Female C57BL/6J “CD45.2” (Stock# 000664), B6.SJL- Ptprc a Pepc b /BoyJ “CD45.1” (Stock# 002014), B6.Cg-Foxp3tm2Tch/J “FIG” (Stock# 006772), and B6.Cg-Tg(TCRaTCRb)425Cbn/J “OTII” (Stock# 004194) were purchased from Jackson Laboratories; C57BL/6NCrl “CD45.2” and B6.SJL- Ptprc a Pepc b /BoyCrCrl “CD45.1” were purchased from Charles River Laboratories; B6.129S6- Rag2 tm1Fwa “RAG2 −/− “ were purchased from Taconic; and XCR1-DTR-cre mice were designed and generated by Horizon Discovery using CRISPR-Cas9 (now Inotiv Co, St. Louis, MO) and colony maintained at Charles River Laboratory.

Techniques: Injection, Control

Journal: Frontiers in Immunology

Article Title: CAR-T cells and CAR-Tregs targeting conventional type-1 dendritic cell suppress experimental autoimmune encephalomyelitis

doi: 10.3389/fimmu.2023.1235222

Figure Lengend Snippet: XCR1 CAR-T cells suppressed the onset of CD4 + T cell-induced passive EAE. (A-G) EAE was induced in RAG2 −/− mice by iv injection with 2 × 10 6 CD45.2 + CD4 + encephalitogenic T cells followed by PTx on days 0 and 2. In addition, mice were treated with 2 × 10 6 XCR1, control, or mock CD45.1 + CD8 + CAR-T (n = 8/group) and 10 µg of IL-2. Shown (A) are the mean clinical EAE scores and (B) the mean percent change in body weight from day 0 through the end of the experiment on day 24 for each group. Shown (C) is the initial day of EAE disease onset from day 0 through day 24 for XCR1 and control CAR-T treatment groups. Spleen, ILN, and CNS were harvested from five mice from each group on day 24 and analyzed via flow cytometry for presence of CAR-T cells and DC subset. Shown (D) are representative dot plots of the following: first row, CD45.1 + donor cells assessed for mCherry; second row, CD45.2 + cells assessed for MHCII (y-axis) and CD11c (x-axis); and third row, DC (CD11c + MHCII + ) assessed for SSC-A (y-axis) and XCR1 (X-axis) from the spleen. The percent (E) CAR + (mCherry + ) T cells, (F) percent DC (CD11c + MHCII + ) of the CD45.2 + cells, and (G) percent DC1 (XCR1 + ) of the total CD45.2 + DC are shown for each organ on day 24. Statistical significance was analyzed by use of (A) Friedman test followed by Dunn’s multiple comparisons test, (B) two-way repeated measures ANOVA followed by Šídák multiple comparison test, (C) Gehan–Breslow–Wilcoxon test, (E–G) one-way ANOVA Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SEM. Data are representative of two independent experiments.

Article Snippet: Female C57BL/6J “CD45.2” (Stock# 000664), B6.SJL- Ptprc a Pepc b /BoyJ “CD45.1” (Stock# 002014), B6.Cg-Foxp3tm2Tch/J “FIG” (Stock# 006772), and B6.Cg-Tg(TCRaTCRb)425Cbn/J “OTII” (Stock# 004194) were purchased from Jackson Laboratories; C57BL/6NCrl “CD45.2” and B6.SJL- Ptprc a Pepc b /BoyCrCrl “CD45.1” were purchased from Charles River Laboratories; B6.129S6- Rag2 tm1Fwa “RAG2 −/− “ were purchased from Taconic; and XCR1-DTR-cre mice were designed and generated by Horizon Discovery using CRISPR-Cas9 (now Inotiv Co, St. Louis, MO) and colony maintained at Charles River Laboratory.

Techniques: IV Injection, Control, Flow Cytometry, Comparison

Journal: Frontiers in Immunology

Article Title: CAR-T cells and CAR-Tregs targeting conventional type-1 dendritic cell suppress experimental autoimmune encephalomyelitis

doi: 10.3389/fimmu.2023.1235222

Figure Lengend Snippet: XCR1 CAR-T cells suppress the onset of CD3 + T cell-induced EAE. RAG2 −/− mice were iv injected with 1 x 10 6 purified CD3 + encephalitogenic T cells, to induce EAE, on day 0. Subsequently, RAG2 −/− mice were injected IP with 100 ng PTx on days 0 and 2. In addition, mice were iv treated with 1 x 10 6 XCR1 or control CD8 + CAR T cells (n = 6/group) on day 0. Shown (A) are the daily mean clinical EAE scores and (B) the mean percent change in body weight and (C) the initial day of EAE onset from day 0 through day 14 for XCR1 and control CAR-T cell-treated mice. The CNS was harvest on day 19 and analyzed via flow cytometry for the presence of CAR-T cells and different DC subsets. Shown (D) are representational dot plots; first column, CAR-T cells (CD45.1 + CD8 + ) assessed for CD8 (y-axis) and mCherry (x-axis); second column, total DCs (MHCII + ) assessed for SSC-A (y-axis) and CD11c (x-axis); third column, DCs (MHCII + CD11c + ) assessed for XCR1 (y-axis) and CD11b (X-axis); fourth column, DCs assessed for BST2 (y-axis) and CD11b (X-axis). The percent CAR + T cells of CD45.1 + donor cells (E) and the percent DCs of total CD45.2 + F4/80 − MHCII + cells (F) for CAR-treated mice. Shown (G) are the percent DC1, DC2, and pDC, of total DCs. Statistical significance was analyzed by use of (A) Wilcoxon matched pairs test, (B) two-way repeated measures ANOVA, (C) Gehan–Breslow–Wilcoxon test and (D-G) unpaired two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SEM.

Article Snippet: Female C57BL/6J “CD45.2” (Stock# 000664), B6.SJL- Ptprc a Pepc b /BoyJ “CD45.1” (Stock# 002014), B6.Cg-Foxp3tm2Tch/J “FIG” (Stock# 006772), and B6.Cg-Tg(TCRaTCRb)425Cbn/J “OTII” (Stock# 004194) were purchased from Jackson Laboratories; C57BL/6NCrl “CD45.2” and B6.SJL- Ptprc a Pepc b /BoyCrCrl “CD45.1” were purchased from Charles River Laboratories; B6.129S6- Rag2 tm1Fwa “RAG2 −/− “ were purchased from Taconic; and XCR1-DTR-cre mice were designed and generated by Horizon Discovery using CRISPR-Cas9 (now Inotiv Co, St. Louis, MO) and colony maintained at Charles River Laboratory.

Techniques: Injection, Purification, Control, Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: CAR-T cells and CAR-Tregs targeting conventional type-1 dendritic cell suppress experimental autoimmune encephalomyelitis

doi: 10.3389/fimmu.2023.1235222

Figure Lengend Snippet: XCR1 and MOG CAR-Tregs are functional and suppress EAE. (A, B) 50,000 XCR1, control, or MOG CD4 + CAR-Treg were cocultured with 5 x 10 5 irradiated RAW, RAW–XCR1, HEK, or HEK–MOG overnight in complete media with 3 ng/mL IL-2. Shown (A) are representational histograms of CD69 (x-axis) expression of XCR1 (top) or MOG (bottom) CAR-Treg cocultured with respective cell lines, mouse T-activator beads “CD3/CD28” at a 10:1 ratio, or alone. Graphed (B) are the percent CD69 + of XCR1 (left), MOG (middle), or control (right) CAR-Treg following cocultured with respective cell lines. (C, D) 25,000 CTV-stained OTII responder T cells were cocultured with 50,000 irradiated (30 Gy) BMDC and 50,000 RAW, RAW–XCR1, or RAW–MOG cells in complete media with 64 nM OVA323-339. XCR1 or MOG CAR-Treg were titrated and added at designated ratios. Shown (C) are representative histograms of CTV dilution of responder OTII T cells under each condition. Graphed (D) is the percent suppression of responder OTII T cells at designated ratios with XCR1 or MOG CAR-Tregs cocultured with RAW–XCR1 (left) and RAW–MOG (right). (E) RAG2 −/− (n = 4–5/group) were iv injected with 1 x 10 6 purified CD4 + encephalitogenic T cells and 5 × 10 5 XCR1, control, MOG, or mock CAR-Treg on day 0 and IP with 120 ng PTx on days 0 and 2. Shown (E) are the daily mean clinical EAE scores from day 0 through day 20. (F) C57BL/6 mice (10/group) were injected with MOG 35–55 in CFA followed by 120 ng PTx IP on days 0 and 2. 5 × 10 5 XCR1, control, MOG CAR-Treg, or PBS were injected on day 1. Shown (F) are the mean clinical EAE scores from day 0 through day 15. (G) C57BL/6 mice (5–10/group) were injected with 2 × 10 6 purified CD4 + encephalitogenic T cells followed by 120 ng PTx IP on days 0 and 2. Then, 2.5 × 10 5 XCR1, control, or mock CAR-Treg were iv injected on day 13. Shown (G) are the mean clinical EAE scores from day 0 through day 40. Statistical significance was analyzed by use of (B, D) unpaired two-tailed t test and (E, G) Friedman test followed by Dunn’s multiple comparisons test for day 8 through end of experiment. *p < 0.05, ****p < 0.0001 Error bars represent SEM. Data (A-D) are representative of three independent experiments.

Article Snippet: Female C57BL/6J “CD45.2” (Stock# 000664), B6.SJL- Ptprc a Pepc b /BoyJ “CD45.1” (Stock# 002014), B6.Cg-Foxp3tm2Tch/J “FIG” (Stock# 006772), and B6.Cg-Tg(TCRaTCRb)425Cbn/J “OTII” (Stock# 004194) were purchased from Jackson Laboratories; C57BL/6NCrl “CD45.2” and B6.SJL- Ptprc a Pepc b /BoyCrCrl “CD45.1” were purchased from Charles River Laboratories; B6.129S6- Rag2 tm1Fwa “RAG2 −/− “ were purchased from Taconic; and XCR1-DTR-cre mice were designed and generated by Horizon Discovery using CRISPR-Cas9 (now Inotiv Co, St. Louis, MO) and colony maintained at Charles River Laboratory.

Techniques: Functional Assay, Control, Irradiation, Expressing, Staining, Injection, Purification, Two Tailed Test