Journal: Frontiers in Immunology

Article Title: A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment

doi: 10.3389/fimmu.2022.728455

Figure Lengend Snippet: Characterization of Sgpl1 M467K mutation. (A) Venn diagram of the shared genes with homozygous mutation between different mice. Numbers represent the number of shared genes among different mice. Red marked Sgpl1 gene is the only gene which is in all four lymphopenia mice of the same colony (1-756, B1413, 4-1234, 2-987) but absent from the B6 gene list . (B) Mononucleotide mutation was identified and localized on 1400 (Ref. NM_009163), T changed to A. (C) Multiple protein sequence alignment of 6 Sgpl1 proteins from Mus musculus and the homologues, including Mus musculus (NP_033189.2), Homo sapiens (NP_003892.2), Caenorhabditis elegans (NP_505372.1), Drosophila melanogaster (NP_652032.1), Danio rerio (XP_005156777.1), Saccharomyces cerevisiae (NP_010580.1). (D, E) Comparison of serum (D) and cutaneous (E) C18-Ceramide, Sphingosine, S1P concentration of wild type and Sgpl1 M467K mice; five wild-type and five Sgpl1 M467K mice were analyzed, please note S1P concentration of wild type was under detection threshold and was defined as 0 during statistic evaluation; original TIC within . *, p<0.05; ***, p<0.001; ****, p<0.0001; unpaired t-test, error bar represents s.e.m. (F) Western blot analysis of SGPL1 expression in kidney of wild type and Sgpl1 M467K mice; experiment repeated twice; full-length pictures were shown in .

Article Snippet: The primary antibodies were used to detect SGPL1 protein (Cat#A15745, 1:1,000, ABclonal) and GAPDH (Cat#ab9485,1:3,000, Abcam).

Techniques: Mutagenesis, Sequencing, Comparison, Concentration Assay, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment

doi: 10.3389/fimmu.2022.728455

Figure Lengend Snippet: Sgpl1 M467K mice exhibit severe cutaneous pathology after IMQ treatment. (A) Ear images of WT and Sgpl1 M467K mice in steady-state. Left, H&E staining, scale bar, 500µm; right, ear photo. Representative image from ≥3 mice of each genotype. (B) Experiment flow chart for IMQ application; IMQ was applied on the mouse ear from day 0 to day 6 and the thickness, erythema, and scaling score of the treated ear were recorded each day; ear hyperpigmentation was checked after more than 30 days. (C) Ear images of WT and Sgpl1 M467K mice at day 7 of IMQ induction. Top, ear photo; middle and bottom images, H&E stained; bottom images were enlarged from square marked middle regions, middle and bottom images scale bars represent 500µm and 100µm, separately; black triangle, epidermis, and acanthosis; red triangle, the dermis. Representative images were depicted from ≥ 6 ears per genotype. (D) The pathological pattern was determined by ear thickness, erythema, and scaling score of each experimental day (for each category please refer to , details see Materials and Methods ). Experiments were repeated five times with three mice from each genotype, one was chosen as representative. ****, p<0.0001; ***, p<0.001; **, p<0.01; ns, not significant; analyzed with two-way ANOVA. (E, F) Representative ear images, 30 days IMQ-treatment cessation; (E) ear photo. (F) Fontana-Masson stained ear section; bottom images were enlarged from top ones, triangle pointed to epidermal melanin. Representative images from ≥ 6 ears per genotype. (G) Melanin content was assessed from the epidermal region of seven randomly selected independent Fontana-Masson stained sections (see Materials and Methods ). ****,p<0.0001, unpaired t-test, error bar represents s.e.m.

Article Snippet: The primary antibodies were used to detect SGPL1 protein (Cat#A15745, 1:1,000, ABclonal) and GAPDH (Cat#ab9485,1:3,000, Abcam).

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment

doi: 10.3389/fimmu.2022.728455

Figure Lengend Snippet: Sgpl1 M467K mutation impaired αβT cell development and cell count in blood and LN. (A, B) Thymocytes were analyzed with a flow cytometer. (A) Representative flow cytometer figure; the DN, DP, CD4 subsets were defined from all cells; the CD8 + was further defined from CD8 single-positive cells by removing the ISP population. (B) Absolute number of different thymocytes subsets within a thymus. Data were pooled from sixteen mice from four different experiments. (C, D) Periphery blood lymphocytes and T cell count were accessed with Attune Flow cytometry. (C) Representative flow cytometer figure; the same amount of CD45 + cells were acquired using the same flow speed, the different length of time under the same arbitrary unit reflects the CD45 + cell concentration. (D) Blood cell concentration. Experiments repeated more than 10 times, data was from fourteen mice of one representative experiment. (E) Absolute cell counts of LN CD45 + and abT cells. Data pooled from eighteen mice from three independent experiments. (F) Skin αβT count under steady-state. Data pooled from two experiments of thirteen mice. ****, p<0.0001; ***, p<0.001; **, p<0,01; ns, not significant; analyzed with unpaired t-test, error bar represents s.e.m.

Article Snippet: The primary antibodies were used to detect SGPL1 protein (Cat#A15745, 1:1,000, ABclonal) and GAPDH (Cat#ab9485,1:3,000, Abcam).

Techniques: Mutagenesis, Cell Counting, Flow Cytometry, Concentration Assay

Journal: Frontiers in Immunology

Article Title: A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment

doi: 10.3389/fimmu.2022.728455

Figure Lengend Snippet: Sgpl1 M467K skin exhibited elevated VγδT cell count after IMQ treatment. (A) Total white blood cell count within steady-state or inflamed skin (two ears). (B, C) T cell count in steady-state skin determined by flow cytometer. (B) Representative flow cytometer figure, T cells were grouped from the Lin - (not CD11b - or MHCii - ) cells by CD3 + , which was further divided into TCRβ + αβT cells and TCRβ - CD3 medium γδT cells and CD3 hi dendritic epidermal T cells (DETC). (C) γδT cell count of two ears. (D–F) Skin T cell count after IMQ treatment. (D) Experiment flow chart for IMQ application, mouse ears were treated with IMQ from day 0 to day 6. (E) Representative flow cytometer figure, the same as (B) ; (F) absolute T cell count after IMQ stimulation. (G–I) Vγ4 Vγ6 γδT cell subsets within the steady-state or inflamed ear. (G) Representative flow cytometer figure of steady-state skin; left, Vγ4 or Vγ6 subpopulation composition of skin γδT cells; right, blue dots are CD3 hi γδT hi DETC, red dots are γδT - CD3 low αβT, black dots are CD3 medium γδT medium Vγ4 + T cells, green dots are Vγ4 - CD3 bright γδT low Vγ6 T cells. (H) Representative flow cytometer figure of Vγ4, Vγ6 cells within inflamed skin. Representative figure of steady-state skin data were displayed from one of three independent experiments, which analyzed thirteen wild-type mice and eleven mutant mice; inflamed skin data is from two independent experiments, which analyzed six wild-type mice and six mutants; steady-state ear Vγ4 Vγ6 distribution data were pooled from two experiments, which analyzed eight wild-type and six mutant mice; inflamed ear Vγ4, Vγ6 distribution data displayed are from one experiment, which analyzed four wild-type and four mutant mice; ****, p<0.0001; **, p<0.01; *, p<0,05; ns, not significant; unpaired t-test.

Article Snippet: The primary antibodies were used to detect SGPL1 protein (Cat#A15745, 1:1,000, ABclonal) and GAPDH (Cat#ab9485,1:3,000, Abcam).

Techniques: Cell Counting, Flow Cytometry, Mutagenesis

Journal: Frontiers in Immunology

Article Title: A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment

doi: 10.3389/fimmu.2022.728455

Figure Lengend Snippet: Elevated levels of IL17a + cells contributed to IMQ-induced acute pathology in Sgpl1 M467K skin lesions. (A) Cytokine expression within psoriatic lesions was measured with quantitative PCR 7 days after IMQ treatment. The experiment was conducted two times. (B–D) Lesional γδ17T count after 7 days after IMQ induction was determined with flow cytometer; (b) representative flow cytometer figure for γδ7T cell or IL17 + αβT cell population. (C) γδ17T proportion within the γδT population; (D) γδ17T cell counts within the skin lesion. (E–G) Draining LN cell count after 6 days of IMQ treatment determined by flow cytometer. (E) Representative flow cytometer figure; left, total draining LN cells were sub-grouped to αβT cells and γδT cells; right, γδT cells were tested for IFNγ or IL17a expression. (F, G) αβT cell, γδT, and γδ17T cell count within draining LN. For both skin and its draining LN analysis, data were pooled from two experiments, which analyzed five wild-type and six mutants; ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant; unpaired t-test. (H, I) IL17 neutralization postponed pathology progression on IMQ treated Sgpl1 M467K skin; (H) experiment flow-chart, IL17 antibodies or Isotype antibodies were administrated at days -2, 1,3, and 5; IMQ was painted on mouse ears from day 0 to day 6; (I) Pathology score was determined by ear thickness, erythema, and scaling score from day 0 to day 7; the statistical analysis is listed on the left table; similar results were obtained from two repeated experiments; analyzed with two-way ANOVA.

Article Snippet: The primary antibodies were used to detect SGPL1 protein (Cat#A15745, 1:1,000, ABclonal) and GAPDH (Cat#ab9485,1:3,000, Abcam).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cell Counting, Neutralization

Journal: Frontiers in Immunology

Article Title: A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment

doi: 10.3389/fimmu.2022.728455

Figure Lengend Snippet: Sgpl1 M467K mutation partially impairs γδT cell migration. (A–C) Thymic γδT cell count was accessed with flow cytometer. (A) Representative flow cytometer figure; left, γδT cells were grouped from total thymocytes; middle, the γδT cell were subdivided into CD24 + and CD24 - populations, or Vγ1 and Vγ4 populations on the right. (B, C) Absolute cell count of thymic γδT cell population; data pooled from three experiments (B) ; data pooled from three experiments, which analyzed seven wild-type and five mutant mice. (D–G) γδT cells counts within mesenteric LN (mLN) were accessed with a flow cytometer. (D) Representative flow cytometer figure; left, γδT cells were grouped from total mLN cells; the CD44 expression, or IL17a + , IFNγ + , Vγ4 + , Vγ1 + populations were analyzed. (E) γδT and γδ17T cell count. (F) γδT cell CD44 mean fluorescent density (MFI). (G) Vγ4 + or Vγ1 + cell count within mLN. Data were from two experiments, three-four mice were used for each experiment. (H) DETC count from two ears; gating strategy refers to Figure 3B . Data pooled from thirteen wild-type and eleven mutant mice. ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant; unpaired t-test.

Article Snippet: The primary antibodies were used to detect SGPL1 protein (Cat#A15745, 1:1,000, ABclonal) and GAPDH (Cat#ab9485,1:3,000, Abcam).

Techniques: Mutagenesis, Migration, Cell Counting, Flow Cytometry, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Resident Immunity in Tissue Repair and Maintenance: The Zebrafish Model Coming of Age

doi: 10.3389/fcell.2019.00012

Figure Lengend Snippet: Selected list of key transgenic zebrafish lines and antibodies to detect immune cells and/or inflammation markers.

Article Snippet: Rabbit Spi1 spleen focus forming virus (SFFV) (Pu.1) , progenitor myeloid cells , GTX128266-S Genetex , , , In our hands (CMH) we did not achieve clear results in either paraffin or cryosections with or without antigen retrieval.

Techniques: Transgenic Assay, In Situ, Hybridization, Labeling, Staining, Virus, Purification

Journal: PLoS ONE

Article Title: The hemagglutinin-neuramidinase protein of Newcastle disease virus upregulates expression of the TRAIL gene in murine natural killer cells through the activation of Syk and NF-κB

doi: 10.1371/journal.pone.0178746

Figure Lengend Snippet: (A) HN-induced TRAIL expression in NK cells was mediated by spleen tryosine kinases (Syk) and NF-κB pathways. Purified murine splenic IFN-R1 -/- NK cells were pre-cultured with herbimycin A or PDTC, and then incubated with soluble recombinant NDV HN. Cells were stained with a PE-conjugated mAb against TRAIL. PBS-treatment NK cells were used as negative controls. (B) Cellular proteins were extracted and subjected to western blotting with a mAb against TRAIL. Results are representative of three independent experiments. (C) Phosphorylation of Syk and IκBα in HN-induced IFN-R1 -/- NK cells. Purified IFN-R1 -/- NK cells were incubated with recombinant NDV HN for 15, 30, 45, 60, and 90min. Cellular proteins were extracted, and Syk, phosphorylated Syk (pSyk), IκBα, and phosphorylated IκBα (pIκBα) were detected by western blotting. β-actin was used as a loading control. (D) Purified IFN-R1 -/- NK cells were pre-cultured with the sodium salt of N-acetylneuraminic acid, 2,3-dehydro-2-deoxy, or herbimycin A, prior to incubation with HN or a mixture NDV 7793 and neutralizing antibodies against HN. Cellular proteins were extracted, and Syk, pSyk, IκBα, and pIκBα were detected by western blotting. β-actin was used as a loading control. Results are representative of three independent experiments using different mice.

Article Snippet: A neutralizing mAb against NDV HN, a mAb against NKp46 conjugated to PE, a mAb against TRAIL conjugated to PE, rat IgG 2a conjugated to PE, a mAb against spleen tyrosine kinases (Syk), a mAb against IκBα and a mAb against phosphorylated IκBα (pIκBα) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Purification, Cell Culture, Incubation, Recombinant, Staining, Western Blot, Phospho-proteomics, Control