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: bioRxiv

Article Title: Social isolation of aged mice drives dramatic release of inflammatory lipoxygenase-derived oxylipins

doi: 10.64898/2025.12.15.694286

Figure Lengend Snippet: (a) Body weight of adult (5 months) and aged (20 months) mice after being kept in group-housing (Ad-GH and O-GH, respectively) or repeatedly isolated (O-ISO). (b,c) Concentration of the cytokines IL-1β and IL-1ra in pg per 50 mg organ. (d) Principal component analysis of organ-specific oxylipin profiles. Hexagons indicate the mean PCA score of all screened replicates within respective experimental groups. (e) Log 2 -fold changes for the organ-specific concentrations of individual oxylipins for the comparison of aged (O-GH) and adult (Ad-GH) mice kept in group-housing (left panel), and the comparison of aged, repeatedly isolated (O-ISO) and aged, group-housed (O-GH) mice (right panel). Fold-changes that could not be calculated due to missing values (e.g., below limit of detection = nd) are depicted in grey. Statistics : Data are shown as ( a - c ) mean ± SEM. The number of biological replicates is n = 5 for Ad-GH and O-ISO, n = 6 for O-GH. Unpaired, two-tailed Student’s t -tests with or without Welch-correction were performed for indicated comparisons. #, comparison of O-GH versus Ad-GH.

Article Snippet: Quantification of cytokines in organ homogenates was performed using commercially available ELISA kits for murine IL-1β (DY401-05) and IL-1ra (DY406-15), manufactured by R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Concentration Assay, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Social isolation of aged mice drives dramatic release of inflammatory lipoxygenase-derived oxylipins

doi: 10.64898/2025.12.15.694286

Figure Lengend Snippet: (a) Body weight of aged (20 month) mice after being repeatedly isolated into single cages with (O-EX) or without running wheels (O-ISO) for 3 separate nights per week over a period of 8 weeks. (b) Total running performance of O-EX mice is summarised in km over the experimental period of eight weeks. (a,b) Numbers indicate the individual animals in the O-EX cohort. (c,d) Concentration of IL-1β and IL-1ra in pg per 50 mg organ from O-ISO and O-EX mice. (e) Principal component analysis of organ-specific oxylipin profiles from O-ISO and O-EX mice. Hexagons indicate the mean PCA score of all screened replicates within respective experimental groups. (f) Log 2 -fold changes for the organ-specific levels of individual oxylipins comparing the levels of O-EX versus O-ISO mice. Fold-changes that could not be calculated due to missing values (e.g., below limit of detection = nd) are depicted in grey. (g,h) Total amounts of grouped oxylipin species derived from the COX or LOX pathways in (g) fat and (h) liver. Oxylipins were grouped as indicated in . Values are given as ng per 50 mg organ. Limit of detection (LOD) of oxylipins is indicated, if applicable. Statistics : Data are shown as ( a-d , g-h ) mean ± SEM. The number of biological replicates is n = 5 for O-ISO and n = 5-6 for O-EX. Unpaired, two-tailed Student’s t -tests with or without Welch-correction were performed for indicated comparisons.

Article Snippet: Quantification of cytokines in organ homogenates was performed using commercially available ELISA kits for murine IL-1β (DY401-05) and IL-1ra (DY406-15), manufactured by R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Concentration Assay, Derivative Assay, Two Tailed Test

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

Article Title: Distinct roles of hepatocyte- and myeloid cell-derived IL-1 receptor antagonist during endotoxemia and sterile inflammation in mice.

doi: 10.4049/jimmunol.1000872

Figure Lengend Snippet: FIGURE 1. Characterization of floxed il-1rn mice. IL-1Ra levels were measured by ELISA in the sera (A) and in spleen (B), liver (C), lung (D), and skin (E) extracts, 4 h after LPS (2 mg/kg) or saline solution (NaCl) injection, in control mice (WT, n = 3) and heterozygous (IL-1Raflx/+, n = 3) and homozygous (IL-1Raflx/flx, n = 3) floxed il-1rn mice. The results represent the mean 6 SEM of IL-1Ra levels. pp , 0.05; ppp , 0.01; pppp , 0.001, versus NaCl-injected mice by ANOVA, two-tailed t test.

Article Snippet: Endogenous peroxidase was blocked using 0.6% H2O2 for 10 min, and tissue sections were boiled in citrate-based Ag-unmasking solution (10 mM [pH 6]) for 3 min. To evaluate local expression of IL-1Ra, tissue sections were incubated with 14.4 mg/ml (for liver sections) or 7.2 mg/ml (for spleen sections) polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of mouse IL-1Ra Duoset ELISA kit; R&D Systems) for 2 h. The sections were rinsed and incubated for 1 h with 1.6 mg/ml donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, Heidelberg, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Saline, Injection, Control, Two Tailed Test

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

Article Title: Distinct roles of hepatocyte- and myeloid cell-derived IL-1 receptor antagonist during endotoxemia and sterile inflammation in mice.

doi: 10.4049/jimmunol.1000872

Figure Lengend Snippet: FIGURE 2. Characterization of conditional hepatocyte-specific IL-1Ra– deficient mice. A, Alb-Cre–mediated deletion of il-1rn exon 2. Exons are depicted as boxes and numbered. The vector-derived sequences and the mouse il-1rn genomic sequences are represented by bold and thin lines, respectively. LoxP sites are shown as black triangles, and FRT sites are shown as double gray triangles. Diagram is not depicted to scale. B, Il-1rn exon 2 copies were quantified in mouse primary hepatocytes and skin by quantitative PCR on genomic DNA. The number of copies of il-1rn exon 2 was normalized to that of il-1rn exon 3. Location of PCR primers are indicated in A (arrows a and b for exon 2, arrows c and d around exon 3). Results represent exon2/exon3 ratio 3 100. Data are mean 6 SEM for hepatocyte-specific IL-1Ra–deficient mice (IL-1RaDH; n = 3) and control mice (WT; n = 3). ppp p , 0.001, by Student t test. C, Production of IL-1Ra by mouse primary hepatocytes. Hepatocytes were cultured in the presence of LPS (1 mg/ml) or with a combination of IL-1b (1 ng/ml) and IL-6 (10 ng/ml). After 48 h, supernatants were collected, and IL-1Ra levels were measured by ELISA. Values represent the mean 6 SEM of IL-1Ra production by cells isolated from hepatocyte-specific IL-1Ra–deficient mice (IL-1RaDH, n = 3) and control mice (WT, n = 3). pppp , 0.001, versus cells isolated from WT mice with similar stimulation assessed by ANOVA, two-tailed t test. ic, intracellular; s, secreted.

Article Snippet: Endogenous peroxidase was blocked using 0.6% H2O2 for 10 min, and tissue sections were boiled in citrate-based Ag-unmasking solution (10 mM [pH 6]) for 3 min. To evaluate local expression of IL-1Ra, tissue sections were incubated with 14.4 mg/ml (for liver sections) or 7.2 mg/ml (for spleen sections) polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of mouse IL-1Ra Duoset ELISA kit; R&D Systems) for 2 h. The sections were rinsed and incubated for 1 h with 1.6 mg/ml donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, Heidelberg, Germany).

Techniques: Plasmid Preparation, Derivative Assay, Genomic Sequencing, Real-time Polymerase Chain Reaction, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Two Tailed Test

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

Article Title: Distinct roles of hepatocyte- and myeloid cell-derived IL-1 receptor antagonist during endotoxemia and sterile inflammation in mice.

doi: 10.4049/jimmunol.1000872

Figure Lengend Snippet: FIGURE 3. Contribution of hepatocytes and myeloid cells as sources of circulating IL-1Ra after systemic challenge with LPS. IL-1Ra levels were quantified by ELISA in sera of control mice (WT), IL-1RaDH mice, IL-1RaDM mice, and IL-1RaDH+M mice 4 h (A) or 18 h (B) after 10 mg/kg LPS injection. A and B are two independent experiments. Results are shown as individual values for each mouse (symbols) and mean values (lines). pp , 0.05; ppp , 0.01; pppp , 0.001, versus control mice by ANOVA, two-tailed t test.

Article Snippet: Endogenous peroxidase was blocked using 0.6% H2O2 for 10 min, and tissue sections were boiled in citrate-based Ag-unmasking solution (10 mM [pH 6]) for 3 min. To evaluate local expression of IL-1Ra, tissue sections were incubated with 14.4 mg/ml (for liver sections) or 7.2 mg/ml (for spleen sections) polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of mouse IL-1Ra Duoset ELISA kit; R&D Systems) for 2 h. The sections were rinsed and incubated for 1 h with 1.6 mg/ml donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, Heidelberg, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Injection, Two Tailed Test

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

Article Title: Distinct roles of hepatocyte- and myeloid cell-derived IL-1 receptor antagonist during endotoxemia and sterile inflammation in mice.

doi: 10.4049/jimmunol.1000872

Figure Lengend Snippet: FIGURE 4. Contribution of hepatocytes and myeloid cells as sources of IL-1Ra in the liver and spleen in response to LPS. A and C, Quan- tification of IL-1Ra levels in liver and spleen after LPS challenge. IL-1Ra levels were deter- mined by ELISA in liver (A) or spleen (C) extracts of control mice (WT), IL-1RaDH

Article Snippet: Endogenous peroxidase was blocked using 0.6% H2O2 for 10 min, and tissue sections were boiled in citrate-based Ag-unmasking solution (10 mM [pH 6]) for 3 min. To evaluate local expression of IL-1Ra, tissue sections were incubated with 14.4 mg/ml (for liver sections) or 7.2 mg/ml (for spleen sections) polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of mouse IL-1Ra Duoset ELISA kit; R&D Systems) for 2 h. The sections were rinsed and incubated for 1 h with 1.6 mg/ml donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, Heidelberg, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Control

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

Article Title: Distinct roles of hepatocyte- and myeloid cell-derived IL-1 receptor antagonist during endotoxemia and sterile inflammation in mice.

doi: 10.4049/jimmunol.1000872

Figure Lengend Snippet: FIGURE 5. Functional role of hepatocyte- and/or myeloid cell-derived IL-1Ra in the control of LPS-induced lethality. Control mice (WT; n = 19), IL-1RaDH mice (n = 16), IL-1RaDM mice (n = 10), IL-1RaDH+M mice (n = 9), and IL-1Ra–deficient mice (n = 12) were injected i.p. with 10 mg/kg LPS. Survival was scored for 6 d. pp , 0.05 versus WT mice; &p , 0.05 versus IL-1RaDH mice, Kaplan–Meier statistic.

Article Snippet: Endogenous peroxidase was blocked using 0.6% H2O2 for 10 min, and tissue sections were boiled in citrate-based Ag-unmasking solution (10 mM [pH 6]) for 3 min. To evaluate local expression of IL-1Ra, tissue sections were incubated with 14.4 mg/ml (for liver sections) or 7.2 mg/ml (for spleen sections) polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of mouse IL-1Ra Duoset ELISA kit; R&D Systems) for 2 h. The sections were rinsed and incubated for 1 h with 1.6 mg/ml donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, Heidelberg, Germany).

Techniques: Functional Assay, Derivative Assay, Control, Injection

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

Article Title: Distinct roles of hepatocyte- and myeloid cell-derived IL-1 receptor antagonist during endotoxemia and sterile inflammation in mice.

doi: 10.4049/jimmunol.1000872

Figure Lengend Snippet: FIGURE 6. Contribution of hepatocytes and mye- loid cells as sources of circulating IL-1Ra after sys- temic challenge with IL-1b. IL-1Ra (A), IL-6 (B), CXCL1 (C), and CXCL2 (D) levels were quantified by ELISA in sera of control mice (WT), IL-1RaDH

Article Snippet: Endogenous peroxidase was blocked using 0.6% H2O2 for 10 min, and tissue sections were boiled in citrate-based Ag-unmasking solution (10 mM [pH 6]) for 3 min. To evaluate local expression of IL-1Ra, tissue sections were incubated with 14.4 mg/ml (for liver sections) or 7.2 mg/ml (for spleen sections) polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of mouse IL-1Ra Duoset ELISA kit; R&D Systems) for 2 h. The sections were rinsed and incubated for 1 h with 1.6 mg/ml donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, Heidelberg, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Control