Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet: MNP FcγR A:I Ratio Modulates Intestinal Type 17 Immunity (A) Type-17-associated cytokine production by day-14 LPMCs stimulated with Ova and Ova-IC for 16 h (n = 5–9). Paired samples represent cells isolated from the same mouse. Data are pooled from 2 independent experiments. (B) qPCR of type 17 cytokines in WT and Fcgr2b −/− whole colonic tissue after cDSS versus controls (n = 4–9 per group). Data are normalized to uninflamed healthy colon. Medians are indicated. Data are representative of 2 independent experiments. (C) Flow-cytometry plots of colonic IL-17A-expressing T cell subsets in co-housed sex-matched WT and Fcgr2b −/− mice at day 21 after aDSS (n = 6–8 per group) versus controls (day 0) (n = 3–5 per group). (D) Quantification of absolute numbers of colonic IL-17A-producing T cells shown in (C). Medians are indicated. Data are representative of 3 independent experiments. (E) Quantification of absolute numbers of colonic IL-17A-producing T cell subsets in co-housed WT and Fcgr2b −/− mice at day 15 after aDSS and weekly treatment with anti-IL-1R1 IgG-blocking antibody or control IgG (n = 5–7 per group). Medians are indicated. Data are representative of two independent experiments. (F) Colonic IL-17A-expressing T cell subsets in M-TG and N-TG littermate controls at day 21 after aDSS (n = 5 or 6 per group) versus controls (n = 5 or 6 per group). Data are representative of two independent experiments. (G) Quantification of absolute cell counts of colonic IL-17A-producing T cell subsets as shown in (F). Medians are indicated. (H) Weight loss of Fcgr2b −/− mice treated with control or anti-IL-1R1 IgG antibodies after aDSS treatment (n = 5–7 per group). Mean ± SEM are indicated. Data are representative of two independent experiments. (I) Colonic neutrophil infiltration in WT and Fcgr2b −/− mice treated as in (H) (n = 5–7 per group). Medians are indicated. p values were calculated with a ratio paired t test (A), the nonparametric Mann-Whitney U test (B–G and I), or a two-way ANOVA (H). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S6 .

Article Snippet: InVivoMab anti-mouse IL-1R1 antibody , BioXCell , Cat#BE0256; RRID: AB_2661843.

Techniques: Isolation, Flow Cytometry, Expressing, Blocking Assay, Control, MANN-WHITNEY

Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet:

Article Snippet: InVivoMab anti-mouse IL-1R1 antibody , BioXCell , Cat#BE0256; RRID: AB_2661843.

Techniques: Virus, Control, Recombinant, Staining, SYBR Green Assay, Protease Inhibitor, Protein Extraction, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Chromatography, Library Quantification, SNP Genotyping Assay, Gene Expression, Microarray, Infection, Software, Transfection, Transferring

Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet:

Article Snippet: Murine primers: Ccl2 (Mm00441242_m1), Csf2 (Mm01290062_m1), Cxcl1 (Mm04207460_m1), Cxcl2 (Mm00436450_m1), Fcer1g (Mm02343757_m1), Fcgr1 (Mm00438874_m1), Fcgr2b (Mm00438875_m1), Fcgr3 (Mm00438882_m1), Fcgr4 (Mm00519988_m1), Gapdh (Mm99999915_g1), Hprt (Mm03024075_m1), Ifng (Mm01168134_m1), Il1b (Mm00434228_m1), Il6 (Mm00446190_m1), Il10 (Mm01288386_m1), Il17a (Mm00439618_m1), Il22 (Mm01226722_g1), Il23a (Mm00518984_m1), Tnf (Mm00443258_m1), and Tnfsf15 (Mm00770031_m1).

Techniques: Virus, Control, Recombinant, Staining, SYBR Green Assay, Protease Inhibitor, Protein Extraction, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Chromatography, Library Quantification, SNP Genotyping Assay, Gene Expression, Microarray, Infection, Software, Transfection, Transferring

Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet:

Article Snippet: HRP-coupled secondary anti-goat (Santa Cruz, sc-2056), anti-mouse (Dako), anti-rabbit (Thermo Fisher Scientific), and anti-β-actin (Sant Cruz, sc-47778) antibodies were detected by enhanced chemiluminescence (Amersham, GE Healthcare) and X-ray films.

Techniques: Virus, Control, Recombinant, Staining, SYBR Green Assay, Protease Inhibitor, Protein Extraction, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Chromatography, Library Quantification, SNP Genotyping Assay, Gene Expression, Microarray, Infection, Software, Transfection, Transferring

Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet: Mucosal FcγR Expression Correlates with IL-1β and CXCL8 (A) Log2 fold-change comparison of differentially expressed cytokine and chemokine genes across two independent UC cohorts. Data are from GEO: GSE38713 (UC = 15, HC = 13) and GEO: GSE59071 (UC = 74, HC = 11). (B and C) Correlation summary of FCGR2A expression with UC-associated cytokine and chemokine gene transcripts (B) and correlation with IL1B and CXCL8 (C) in n = 85 mucosal biopsies. Data are from GEO: GSE59071 . (D) Hierarchical clustering of cytokine and chemokine and FCGR2A expression in healthy controls and non-inflamed and inflamed UC patients. Data are derived from GEO: GSE38713 . (E and F) Correlation of Fcgr3 expression with Il1b (E) and candidate-gene (F) expression in whole inflamed colonic tissue by qPCR (n = 16). Data are representative of two independent experiments. (G) IL1B and CXCL8 expression in healthy human LPMC stimulated with Ova or Ova-IC for 16 h (n = 5 per condition). Means ± SEM are indicated. Data are representative of two independent experiments. (H) IL-1β, CXCL1, and CXCL2 in supernatants of inflamed murine LPMCs stimulated with Ova or Ova-IC for 16 h (n = 3-9). Paired samples represent LPMCs from a single mouse. Data are pooled from two independent experiments. (I) Flow-cytometry profiling of pro-IL-1β-expressing cells at day 14 after aDSS (n = 6). Medians are indicated. Data are representative of three independent experiments. (J) Pro-IL-1β expression by colonic CX3CR1 + MNP subsets and neutrophils in Rag2 −/− mice treated with control or anti-flagellin-enriched serum IgG and 7-day aDSS or uninflamed H 2 O-treated controls (n = 5 per group). Medians are indicated. (K) Day 7 weight loss for aDSS-treated mice shown in (J) (n = 5 per group). Medians are indicated. p values were calculated via limma with multiple correction using the BH procedure (A), linear regression analysis (B, C, E, and F), Student’s two-tailed t test (G), ratio paired t test (H and I), or the Kruskal-Wallis test with Dunn’s multiple-comparisons test (J). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S3 .

Article Snippet: TaqMan Gene Expression ( Il1b ) , Thermo Fisher Scientific , Mm00434228_m1.

Techniques: Expressing, Comparison, Derivative Assay, Flow Cytometry, Control, Two Tailed Test

Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet: IgG-Induced IL-1β Production by Human Macrophages (A) Venn diagram of significant co-expressed genes between UC and Ova-IC-stimulated colonic macrophages (left) and IC-induced UC-associated cytokines and chemokines (right), as determined by sdef. Human UC data are from GEO: GSE38713 (UC = 15, HC = 13). For macrophage stimulation, n = 4 per condition. (B) GSEA of inflammasome genes (left) and heatmap of selected core enrichment genes (right) in flow-sorted murine intestinal CX3CR1 + Ly6C lo MHC-II hi macrophages stimulated with Ova and Ova-IC for 4 h (n = 4 per condition). (C) IL-1β production by WT and Nlrp3 −/− BMDMs primed with Ova and Ova-IC for 4 h followed by 30 min of ATP stimulation ( ∗∗∗∗ , WT versus Nlrp3 −/− Ova-IC; ####, WT Ova versus Ova-IC) (n = 3 per condition). Means ± SEM are indicated. Data are representative of three independent experiments. ND, not detected. (D) IL-1β ELISA of human MDMs stimulated with plate-coated IgG (c-IgG) and intestinal commensals (Comm.) for 24 h ± anti-FcγRIIA IgG-blocking antibody (n = 3 per condition). Means ± SEM are indicated. Data are representative of two independent experiments. (E) qPCR of IL1B mRNA (left, n = 3 per condition) and western blot of IL-1β in cell lysates and cell-free supernatants (right) from human MDMs as stimulated in (D). For qPCR (left), means ± SEM are indicated. Data are representative of two independent experiments. (F) The effect of NLRP3 inhibition on IgG-induced MDM IL-1β production. Western blot (left) and IL-1β production (left, ELISA) by human MDMs stimulated as in (D) after NLRP3 “Trim-Away” or ± NLRP3 inhibitor MCC950 (right, ELISA) (n = 3 per condition). Means ± SEM are indicated. (G) IL-1β production by human MDMs stimulated as in (D) ± mitochondrial ROS inhibitor mitoTEMPO (n = 3 per condition). Means ± SEM are indicated. Data are representative of 2 independent experiments. (H) Fold change in IL1B expression in Ova-IC-stimulated MDMs, normalized to R/R fold change (n = 6 per group). Data are pooled from 3 independent experiments. Medians are indicated. (I) Schematic of FcγR A:I ratios with FCGR2A SNP (dbSNP: rs1801274) and murine transgenic models. (J) Il1b induction in WT, Fcgr2b −/− , and M-TG BMDMs stimulated with Ova and Ova-IC for 3 h (n = 3 per condition). Means ± SEM are indicated. Data are representative of 3 independent experiments. p values were calculated via the standard DESeq2 method with multiple correction using the BH procedure (A and B), two-way ANOVA with Tukey’s multiple comparisons test (C, F, and J), one-way ANOVA with Tukey’s multiple-comparisons test (D, E, and G), or parametric Student’s t test (H). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S4 .

Article Snippet: TaqMan Gene Expression ( Il1b ) , Thermo Fisher Scientific , Mm00434228_m1.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Western Blot, Inhibition, Expressing, Transgenic Assay

Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet:

Article Snippet: TaqMan Gene Expression ( Il1b ) , Thermo Fisher Scientific , Mm00434228_m1.

Techniques: Virus, Control, Recombinant, Staining, SYBR Green Assay, Protease Inhibitor, Protein Extraction, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Chromatography, Library Quantification, SNP Genotyping Assay, Gene Expression, Microarray, Infection, Software, Transfection, Transferring

Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet:

Article Snippet: TaqMan Gene Expression ( HPRT1 ) , Thermo Fisher Scientific , Hs02800695_m1.

Techniques: Virus, Control, Recombinant, Staining, SYBR Green Assay, Protease Inhibitor, Protein Extraction, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Chromatography, Library Quantification, SNP Genotyping Assay, Gene Expression, Microarray, Infection, Software, Transfection, Transferring

Journal: Genes

Article Title: Development of a 76k Alpaca ( Vicugna pacos ) Single Nucleotide Polymorphisms (SNPs) Microarray

doi: 10.3390/genes12020291

Figure Lengend Snippet: Number of scaffold fragment lengths containing one SNP identified in this study and included in the microarray.

Article Snippet: Thirty (10 from Pasco and 20 from Puno) trios (sire, dam and progeny) were identified based on their pedigrees and were genotyped with the generated alpaca SNP microarray.

Techniques: Microarray

Journal: Genes

Article Title: Development of a 76k Alpaca ( Vicugna pacos ) Single Nucleotide Polymorphisms (SNPs) Microarray

doi: 10.3390/genes12020291

Figure Lengend Snippet: Number of SNPs selected for the microarray.

Article Snippet: Thirty (10 from Pasco and 20 from Puno) trios (sire, dam and progeny) were identified based on their pedigrees and were genotyped with the generated alpaca SNP microarray.

Techniques: Microarray

Journal: Genes

Article Title: Development of a 76k Alpaca ( Vicugna pacos ) Single Nucleotide Polymorphisms (SNPs) Microarray

doi: 10.3390/genes12020291

Figure Lengend Snippet: Coverage of the alpaca genome with SNPs included in the microarray.

Article Snippet: Thirty (10 from Pasco and 20 from Puno) trios (sire, dam and progeny) were identified based on their pedigrees and were genotyped with the generated alpaca SNP microarray.

Techniques: Microarray

Journal: Genes

Article Title: Development of a 76k Alpaca ( Vicugna pacos ) Single Nucleotide Polymorphisms (SNPs) Microarray

doi: 10.3390/genes12020291

Figure Lengend Snippet: Population structure based on microarray genotyping (Pacomarca—red, Quimsachata—green, Racco—blue and Gacocen—black).

Article Snippet: Thirty (10 from Pasco and 20 from Puno) trios (sire, dam and progeny) were identified based on their pedigrees and were genotyped with the generated alpaca SNP microarray.

Techniques: Microarray

Journal: Genes

Article Title: Development of a 76k Alpaca ( Vicugna pacos ) Single Nucleotide Polymorphisms (SNPs) Microarray

doi: 10.3390/genes12020291

Figure Lengend Snippet: Heat map of genomic relationships among animals based on microarray genotyping Gacocen (bottom left), Pacomarca and Quimsachata (center area) and Racco (top right).

Article Snippet: Thirty (10 from Pasco and 20 from Puno) trios (sire, dam and progeny) were identified based on their pedigrees and were genotyped with the generated alpaca SNP microarray.

Techniques: Microarray

Journal: Genes

Article Title: Development of a 76k Alpaca ( Vicugna pacos ) Single Nucleotide Polymorphisms (SNPs) Microarray

doi: 10.3390/genes12020291

Figure Lengend Snippet: Inbreeding coefficient (F) of alpacas genotyped by microarray.

Article Snippet: Thirty (10 from Pasco and 20 from Puno) trios (sire, dam and progeny) were identified based on their pedigrees and were genotyped with the generated alpaca SNP microarray.

Techniques: Microarray

Journal: Genes

Article Title: Development of a 76k Alpaca ( Vicugna pacos ) Single Nucleotide Polymorphisms (SNPs) Microarray

doi: 10.3390/genes12020291

Figure Lengend Snippet: Level of heterozygosity of alpacas genotyped by microarray.

Article Snippet: Thirty (10 from Pasco and 20 from Puno) trios (sire, dam and progeny) were identified based on their pedigrees and were genotyped with the generated alpaca SNP microarray.

Techniques: Microarray

Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet: Mucosal FcγR Expression Correlates with IL-1β and CXCL8 (A) Log2 fold-change comparison of differentially expressed cytokine and chemokine genes across two independent UC cohorts. Data are from GEO: GSE38713 (UC = 15, HC = 13) and GEO: GSE59071 (UC = 74, HC = 11). (B and C) Correlation summary of FCGR2A expression with UC-associated cytokine and chemokine gene transcripts (B) and correlation with IL1B and CXCL8 (C) in n = 85 mucosal biopsies. Data are from GEO: GSE59071 . (D) Hierarchical clustering of cytokine and chemokine and FCGR2A expression in healthy controls and non-inflamed and inflamed UC patients. Data are derived from GEO: GSE38713 . (E and F) Correlation of Fcgr3 expression with Il1b (E) and candidate-gene (F) expression in whole inflamed colonic tissue by qPCR (n = 16). Data are representative of two independent experiments. (G) IL1B and CXCL8 expression in healthy human LPMC stimulated with Ova or Ova-IC for 16 h (n = 5 per condition). Means ± SEM are indicated. Data are representative of two independent experiments. (H) IL-1β, CXCL1, and CXCL2 in supernatants of inflamed murine LPMCs stimulated with Ova or Ova-IC for 16 h (n = 3-9). Paired samples represent LPMCs from a single mouse. Data are pooled from two independent experiments. (I) Flow-cytometry profiling of pro-IL-1β-expressing cells at day 14 after aDSS (n = 6). Medians are indicated. Data are representative of three independent experiments. (J) Pro-IL-1β expression by colonic CX3CR1 + MNP subsets and neutrophils in Rag2 −/− mice treated with control or anti-flagellin-enriched serum IgG and 7-day aDSS or uninflamed H 2 O-treated controls (n = 5 per group). Medians are indicated. (K) Day 7 weight loss for aDSS-treated mice shown in (J) (n = 5 per group). Medians are indicated. p values were calculated via limma with multiple correction using the BH procedure (A), linear regression analysis (B, C, E, and F), Student’s two-tailed t test (G), ratio paired t test (H and I), or the Kruskal-Wallis test with Dunn’s multiple-comparisons test (J). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S3 .

Article Snippet: Human primers: CXCL8 (Hs00174103_m1), FCER1G (Hs00175408_m1), FCGR1B (Hs02341825_m1), FCGR2A (Hs01013401_g1), FCGR2B (Hs01634996_s1), FCGR3A (Hs02388314_m1), FCGR3B (Hs04334165_m1), GAPDH (Hs02786624_g1), HPRT1 (Hs02800695_m1), and IL1B (Hs01555410_m1). qPCR was carried performed on the Viia 7 PCR machine (Life Technologies).

Techniques: Expressing, Comparison, Derivative Assay, Flow Cytometry, Control, Two Tailed Test

Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet:

Article Snippet: Human primers: CXCL8 (Hs00174103_m1), FCER1G (Hs00175408_m1), FCGR1B (Hs02341825_m1), FCGR2A (Hs01013401_g1), FCGR2B (Hs01634996_s1), FCGR3A (Hs02388314_m1), FCGR3B (Hs04334165_m1), GAPDH (Hs02786624_g1), HPRT1 (Hs02800695_m1), and IL1B (Hs01555410_m1). qPCR was carried performed on the Viia 7 PCR machine (Life Technologies).

Techniques: Virus, Control, Recombinant, Staining, SYBR Green Assay, Protease Inhibitor, Protein Extraction, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Chromatography, Library Quantification, SNP Genotyping Assay, Gene Expression, Microarray, Infection, Software, Transfection, Transferring

Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet:

Article Snippet: Human primers: CXCL8 (Hs00174103_m1), FCER1G (Hs00175408_m1), FCGR1B (Hs02341825_m1), FCGR2A (Hs01013401_g1), FCGR2B (Hs01634996_s1), FCGR3A (Hs02388314_m1), FCGR3B (Hs04334165_m1), GAPDH (Hs02786624_g1), HPRT1 (Hs02800695_m1), and IL1B (Hs01555410_m1). qPCR was carried performed on the Viia 7 PCR machine (Life Technologies).

Techniques: Virus, Control, Recombinant, Staining, SYBR Green Assay, Protease Inhibitor, Protein Extraction, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Chromatography, Library Quantification, SNP Genotyping Assay, Gene Expression, Microarray, Infection, Software, Transfection, Transferring