Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 1. Generation of icIL-1Ra12/2 mice. (A) Targeting strategy used for the introduction of an EGFP–Neo cassette into the icIL-1Ra1–specific first exon (icIl1rn1) of the mouse Il1rn gene. This approach does not interfere with the expression of the other transcript variants. Shown from top to bottom are the WT icIl1rn1 genomic locus (WT allele), the targeting vector and the targeted genomic locus (Mutant allele). Bold lines indicate arms of homology for recombination. Exon 1 is represented as a gray box and numbered. The neomycin resistance gene (Neor) is flanked by two loxP sites (triangles). The primers used to identify recombinant ES cells are indicated as S1 and S2. (B) Southern blot analysis of WT (+/+) and heterozygous (+/2) (one WT allele, one mutant allele) ES cells using a 59 external probe (left panel) or a 39 internal probe (right panel). The WT (6.4-kb [BglII digestion] or 8.9-kb [HindIII digestion] bands) and mutant alleles (7.4-kb [BglII digestion] or 6.2-kb [HindIII digestion] bands) were detected in BglII- or HindIII-digested ES cell genomic DNA, using, respectively, the 59 or 39 probes shown in (A). (C) PCR genotyping to distinguish WT (+/+), heterozygous (+/2) and icIL-1Ra1(2/2) mice. The primers used are indicated as P1, P2, and P3 in (A). The sizes of the WT and icIL-1Ra12/2 PCR products are 300 and 842 bp, respectively. (D) Male (left panel) and female (right panel) WT (male: n = 13/n = 6 [10/17 wk]; female: n = 11/n = 6 [10/17 wk]) and icIL-1Ra12/2 (male: n = 13/n = 13 [10/17 wk]; female: n = 11/n = 9 [10/17 wk]) mice were weighed at 10 and 17 wk of age. Data are shown as the mean 6 SEM. No significant differences in body weight were observed between the genotypes (unpaired two-tailed Student t test). (E) Analysis of IL-1Ra isoforms in lysates of BMDC generated from naive WT and icIL-1Ra12/2 (KO) mice. IL-1Ra was detected using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra isoforms. Protein loading was assessed by the determination of GAPDH expression. One representative sample per genotype out of n = 3 per genotype is shown. Arrows indicate different IL-1Ra isoforms. Theoretical molecular mass of IL-1Ra isoforms are as follows: prosIL-1Ra1, 20 kDa; mature sIL-1Ra1, between 17 and 22 kDa; icIL-1Ra1, 18 kDa; icIL-1Ra3, 16 kDa (13, 69).

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Recombinant, Southern Blot, Two Tailed Test, Generated

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 2. Characterization of BMDC and BMDM from WT and icIL-1Ra12/2 mice. (A) Determination of mRNA levels for icIL-1Ra1 (icIl1rn1) and for sIL-1Ra and icIL-1Ra3 (sIl1rn) in BMDC (left panel) and BMDM (right panel). BMDC and BMDM from naive WT (n = 5) and icIL-1Ra12/2 (n = 5) mice were left untreated (control) or stimulated with 100 ng/ml LPS for 24 h. Total RNA was isolated for qRT-PCR analysis. Results represent icIl1rn1 and sIl1rn mRNA expression levels relative to Mrpl32 mRNA levels. (B) Release of IL-1Ra protein (all isoforms) into the supernatant. BMDC (left panel) and BMDM (right panel) from naive WT (n = 5) and icIL-1Ra12/2 (n = 5) mice were left untreated (control) or stimulated with LPS for 24 h. IL-1Ra in the supernatant was measured using an ELISA that recognizes all isoforms of IL-1Ra. (A and B) Data are shown as the mean 6 SEM of values obtained from three (BMDC) or two (BMDM) independent experiments. The unpaired or paired two-tailed Student t test was used to test statistical significance. All p values ,0.05 were considered to be significant. (C) Analysis of IL-1Ra isoforms in lysates and cell supernatants of BMDC (left panel) and BMDM (right panel) by Western blot. BMDC and BMDM from naive WT and icIL-1Ra12/2 (KO) mice were left untreated (control) or stimulated with LPS for 24 h. IL-1Ra isoforms were analyzed in cell lysates or collected supernatants (30 ml) using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra isoforms. Protein loading was assessed by the determination of GAPDH expression in lysates. One nanogram of rsIL-1Ra served as control. Two rep- resentative samples out of n = 5 per genotype are shown. Arrows indicate different IL-1Ra isoforms. Theoretical molecular mass of IL-1Ra isoforms is as follows: prosIL-1Ra1, 20 kDa; mature sIL-1Ra1, between 17 and 22 kDa; icIL-1Ra1, 18 kDa; icIL-1Ra3, 16 kDa. n.d., not detected. SN, supernatant.

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Control, Isolation, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 3. icIL-1Ra1 deficiency in- creases the clinical severity of Aldara- induced ear thickness and the expression of proinflammatory cytokines. (A) IL- 1Ra isoforms were analyzed in lysates of total dorsal skin from naive WT, hetero- zygous (HET) and icIL-1Ra12/2 (KO) mice using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra iso- forms. Protein loading was assessed by the determination of GAPDH expression. One representative sample out of n = 3 per genotype is shown. Arrow indicates the icIL-1Ra1 isoform. Theoretical mo- lecular mass of icIL-1Ra1: 18 kDa. (B–E) Aldara, or Vaseline as a control, were applied on the right or left ear, respec- tively, of WT (n = 21) and icIL-1Ra12/2

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Expressing, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 4. icIL-1Ra1 is the main IL-1Ra isoform regulating Aldara-induced skin inflammation. (A) Representative immunohistochemical detection of IL-1Ra (brown staining) on ear sections of WT and icIL-1Ra12/2 mice on day 8 after daily application of Vaseline (control) or Aldara. Scale bar, 100 mm. (B) Analysis of IL-1Ra isoforms in lysate of Vaseline-treated (control) (upper panel) and Aldara-treated (lower panel) ears from WT and icIL-1Ra12/2

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Immunohistochemical staining, Staining, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 5. icIL-1Ra1 plays no intracellular role in keratinocytes but is released together with IL-1a upon Aldara treatment. (A) Differential gene expression analysis based on RNA-Seq data of primary keratinocytes from icIL-1Ra12/2 versus WT mice. Primary keratinocytes were isolated from WT (n = 4) and icIL-1Ra12/2 (n = 4) mouse tails and left untreated as a control (left panel) or stimulated with 100 ng/ml rec. m IL-1a (middle panel) or 100 ng/ml rec. m IL-36b (right panel) for 6 h. Total RNA was then isolated from keratinocytes for RNA-Seq analysis. A total of 10,750, 10,709, or 10,727 genes were tested for control, IL-1a–, or IL-36b–treated cells, respectively. The fold-change threshold and Benjamini–Hochberg corrected p value threshold were set to 2 and 0.05, respectively. (B) Ear explants taken from WT (n = 6) and icIL-1Ra12/2 (n = 6) mice at day 8 after daily treatment with Vaseline (control) or Aldara were cultured for 24 h. Levels of IL-1Ra, IL-1a, and IL-1b in the supernatant or the percentage of cytotoxicity were assessed by ELISA or LDH assay, respectively. The IL-1Ra ELISA recognizes all isoforms of IL-1Ra. Data are shown as the mean 6 SEM from one (Figure legend continues)

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Gene Expression, RNA Sequencing, Isolation, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

Journal: Frontiers in Immunology

Article Title: Göttingen Minipigs as a Model to Evaluate Longevity, Functionality, and Memory of Immune Response Induced by Pertussis Vaccines

doi: 10.3389/fimmu.2021.613810

Figure Lengend Snippet: IFN-γ (Th-1), IL-17 (Th-17) and IL-4 (Th-2) secreting cells measured by FluoroSpot assay at M6 and M10 on PBMCs from neonatal minipigs primed with either DTwP or DTaP and boosted with Tdap vaccine, stimulated for 48 h with (A) a pool of pertussis antigens (PTxd, PRN, FIM2/3) and (B) heat-killed B. pertussis (hk Bp ). Data are shown for DTwP ( n = 7) and DTaP ( n = 7) groups as individual and geometric mean titers, after correction for background levels of cytokine secreting cells in the negative control wells (media alone). Data for the control group (TBS, n = 3) are shown as the geometric mean titer for all minipigs at all time-points. * p -value < 0.05 and ** p -value < 0.01 for statistical comparison of DTaP- and DTwP primed groups.

Article Snippet: IFN-γ, IL-4 and IL-17 secreting cells were detected using biotin mouse anti-pig IFN-γ (BD Biosciences #559958), goat anti-porcine IL-4 (R&Dsystems #BAF654) and rabbit anti-swine IL-17A (KingFisher Biotech #PBB0270S-050), respectively, followed by streptavidin-PE (Southern Biotech #7100-09L).

Techniques: Flurospot, Negative Control