Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: IFN-φ neutralization abolishes ST-induced interferon production in zebrafish embryos. (A) Representative immunohistochemical staining of IFN-φ (brown DAB precipitate) in 1 dpf wild-type (WT) embryos under four conditions: (a.1) unstimulated control, (a.2) ST stimulation (10 6 cells/ml for 2h), (a.3) anti-IFN-β antibody pretreatment alone, and (a.4) anti-IFN-β pretreatment followed by ST stimulation. Scale bar: 50 μm. (B) Quantification of IFN-φ-positive cells from two slides per group (eight sections/slide, three embryos/section) using Fiji-ImageJ. Data represent mean ± SEM ( n = 3 independent experiments); * p < 0.05 versus unstimulated control WT, # p < 0.05 versus ST-stimulated WT.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Neutralization, Immunohistochemical staining, Staining, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: IFN-I signaling is essential for IL-1β–mediated protection against ST stimulation. (A) Locomotor activity (total distance traveled) assessed 72h after stimulation at 28°C. Mortality count (C) and Caspy2 expression in WT embryos after ST stimulation (10 6 cells/ml, 2h) with or without anti-IFN-β antibody pretreatment. The corresponding bar graph (D) represents the protein expression levels as a percentage of the control. (E) Quantification and (F) representative IHC images of mature IL-1β (mIL-1β)–positive cells (brown DAB precipitate) in: (f.1) ST-stimulated embryos, (f.2) pretreatment with anti-IFN-β alone, and (f.3) pretreatment with anti-IFN-β followed by ST stimulation. Scale bar: 200 μm. Data represent mean ± SEM ( n = 3 independent experiments); * p < 0.05 versus unstimulated control WT, # p < 0.05 versus ST-stimulated WT.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Activity Assay, Expressing, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: Effectiveness of CRISPR/Cas9 depletion of loc795232. Basal expression of loc795232 mRNA transcripts in unstimulated WT or KO embryos ( n = 100/group) at 1 dpf by RT-qPCR (A) or by in situ hybridization (B) . All qPCR data normalized to β-actin and expressed as fold change relative to 0h WT unstimulated control. Data represent mean ± SEM of three biological replicates. Independent groups of 1 dpf embryos previously treated or not with Pam3CSK4 or MCC950 and subjected to ST stimulation for 2h, as well as KO larvae stimulated with ST were processed for Natterin detection by WB (C) using anti-Natterin serum (62 kDa dimeric form) and anti-IgG TrueBlot HRP. The corresponding bar graph represents the protein expression levels as a percentage of the control (D) , and Ponceau S staining is visualized in the first horizontal line.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: CRISPR, Expressing, Quantitative RT-PCR, In Situ Hybridization, Control, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: Natterin is required for Gbp4 induction. Constitutive expression of gbp4 (A) and gbp1 (C) , was analyzed in unstimulated WT embryos ( n = 100/group) at 24h intervals by RT-qPCR. 1 dpf ST-responsive expression of gbp4 (B) and gbp1 (D) was assessed in WT and KO groups. All qPCR data normalized to β-actin and expressed as fold change relative to 0h WT unstimulated control. * p < 0.05 versus unstimulated control WT; # p < 0.05 versus ST-stimulated WT.

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: Natterin is essential for proteolytic activation of Caspy and Caspy2 during ST stimulation. (A) Developmental expression profile of caspy2 mRNA in unstimulated WT embryos ( n = 100/group) from 24 to 120 hpf. (B) One day post-fertilization ST-induced caspy2 expression in WT versus natterin knockout (KO) embryos 2h post-stimulation. All qPCR data normalized to β-actin and expressed as fold change relative to 0h WT unstimulated control. Data represent mean ± SEM; * p < 0.05 versus unstimulated control WT, # p < 0.05 versus ST-stimulated WT. Western blot analysis of mature Caspy ( Supplementary Figure S3 ) and Caspy2 ( Supplementary Figure S4 ) expression in whole embryo lysates at indicated times post-ST stimulation (15 and 30 min, 1 and 2h). Data are normalized to the unstimulated control and presented as percentage values. Quantitative densitometry is shown in bar graphs (C, D) .

Article Snippet: They were then incubated overnight at 4°C with the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IL-1β antibody that recognizes the rainbow trout mature IL-1β peptide with 20 kDa according to (#CLF016HP, Cedarlane, 1/3,000) or the polyclonal rabbit HRPO-labeled IgG anti-rainbow trout IFN-β antibody (#CLF005HP, Cedarlane, 1/3,000) according to .

Techniques: Activation Assay, Expressing, Knock-Out, Control, Western Blot

Journal: Biomolecules

Article Title: Intestinal Barrier in Post- Campylobacter jejuni Irritable Bowel Syndrome

doi: 10.3390/biom13030449

Figure Lengend Snippet: EEA1 and cytokine intensity grade (0–4) at surface cells in the colon mucosa of PI-IBS patients and controls.

Article Snippet: Briefly, 3 µm paraffin sections were deparaffinized followed by retrieval of the antigenic epitopes via treatment in a pressure cooker in citrate buffer pH 6 for 3 min. Unspecific bindings were blocked by a serum-free protein block (DAKO, Glosdrup, Denmark) for 15 min at room temperature and the following polyclonal primary antibodies—rabbit-anti-human-EEA1 (abcam, Cambridge, UK), IL-1β (Bioss, Woburn, MA, USA), IL-22 (abcam), IL-4 (Tebubio, Frankfurt, Germany), IL-10 and IFNγ (both Peprotech, Hamburg, Germany), and goat-anti-human-IL-6 (R&D systems, Minneapolis, MN, USA) and -TNFα (PeproTech)—were applied in dilutions previously titrated in antibody diluent (DAKO) at 4 °C overnight.

Techniques:

Journal: Biomolecules

Article Title: Intestinal Barrier in Post- Campylobacter jejuni Irritable Bowel Syndrome

doi: 10.3390/biom13030449

Figure Lengend Snippet: Immunohistochemistry of Th1 cytokines (red) in the colon mucosa of controls and PI-IBS patients of IL-1β ( a , b ), IL-6 ( c , d ), IL-22 ( e , f ), INFγ ( g , h ) and TNFα ( i , j ), (counterstaining with hematoxylin = violet). Staining for IL-1β and IL-22 intensified in surface enterocytes in PI-IBS. Bar = 100 µm.

Article Snippet: Briefly, 3 µm paraffin sections were deparaffinized followed by retrieval of the antigenic epitopes via treatment in a pressure cooker in citrate buffer pH 6 for 3 min. Unspecific bindings were blocked by a serum-free protein block (DAKO, Glosdrup, Denmark) for 15 min at room temperature and the following polyclonal primary antibodies—rabbit-anti-human-EEA1 (abcam, Cambridge, UK), IL-1β (Bioss, Woburn, MA, USA), IL-22 (abcam), IL-4 (Tebubio, Frankfurt, Germany), IL-10 and IFNγ (both Peprotech, Hamburg, Germany), and goat-anti-human-IL-6 (R&D systems, Minneapolis, MN, USA) and -TNFα (PeproTech)—were applied in dilutions previously titrated in antibody diluent (DAKO) at 4 °C overnight.

Techniques: Immunohistochemistry, Staining

Journal: Veterinary Research

Article Title: Innate immune response in experimentally induced bovine intramammary infection with Staphylococcus simulans and S. epidermidis

doi: 10.1186/1297-9716-42-49

Figure Lengend Snippet: The ELISA kit, anti-cytokine antibodies (ab) and cytokine protein standards used in this study.

Article Snippet: IL-1β , Biotin-conjugated mouse anti-bovine IL-1β (detection ab) , AHP851B AbD Serotech , 2 μg/mL in 1% BSA-PBS.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Recombinant

Journal:

Article Title: The Secretory Route of the Leaderless Protein Interleukin 1? Involves Exocytosis of Endolysosome-related Vesicles

doi:

Figure Lengend Snippet: ProIL-1β is contained in part in vesicles cofractionating with lysosomes. Western blot analysis of PNS (5% of total, lanes 1 and 4), P1 (lanes 2 and 5), and P2 (lanes 3 and 6) or supernatants from 106 cells (lanes 7 and 8) obtained from activated monocytes cultured in the absence (−) or presence (+) of 100 μM glibenclamide (glib). After removal of 5% PNS, PNS were treated with proteinase K (PK) before the ultracentrifugation. Lanes 9 and 10, P1 from undigested PNS was untreated (lane 9) or solubilized (+TX100, lane 10) with Triton X-100 before proteinase K (PK) digestion. Filters were hybridized with rabbit anti-CD (A), melted, and rehybridized with mouse anti-IL-1β (B) antibodies. The migration of the three molecular forms of CD and proIL-1β and IL-1β is indicated.

Article Snippet: In double-labeling experiments, the sections were first incubated with anti-IL-1β followed by 10 nm of goat anti-mouse IgG gold conjugates (British BioCell International, Carditt, UK) and then incubated with anti-CD or anti-Lamp-1 followed by 18 nm of protein-A gold.

Techniques: Western Blot, Cell Culture, Migration

Journal:

Article Title: The Secretory Route of the Leaderless Protein Interleukin 1? Involves Exocytosis of Endolysosome-related Vesicles

doi:

Figure Lengend Snippet: Kinetic analyses of cytosolic and particulated IL-1β. LPS-activated monocytes were pulsed with 1 mCi/ml methionine-cysteine Promix [35S] for 15 min (time of chase, 0) and chased in complete medium for 1, 3, 6, or 15 h in the absence (open symbols) or presence (closed symbols) of 100 μg of pepstatin and leupeptin. At the end of each period of chase subcellular fractionation was carried out as above, P1 and P2 were treated with proteinase K, followed by protease inhibitors and solubilization, and the IL-1β present in the pooled P1 and P2 fractions (A), in the cytosol (B), and in the supernatants (C) was analyzed by immunoprecipitation with anti-IL-1β antiserum followed by SDS-PAGE and autoradiography as described (Rubartelli et al., 1990 ). Data are expressed as densitometric areas (AU, arbitrary units). One representative experiment of three performed is shown.

Article Snippet: In double-labeling experiments, the sections were first incubated with anti-IL-1β followed by 10 nm of goat anti-mouse IgG gold conjugates (British BioCell International, Carditt, UK) and then incubated with anti-CD or anti-Lamp-1 followed by 18 nm of protein-A gold.

Techniques: Fractionation, Immunoprecipitation, SDS Page, Autoradiography

Journal:

Article Title: The Secretory Route of the Leaderless Protein Interleukin 1? Involves Exocytosis of Endolysosome-related Vesicles

doi:

Figure Lengend Snippet: Immunoelectron microscopy analysis of the colocalization of IL-1β and CD (A–C, E, and F) and of IL1β and Lamp-1 (D and G) in P1 fraction. Double immunolabeling with anti-IL-1β (10-nm gold particles) and anti-CD (18-nm gold particles) antibodies reveals the presence of both molecules in organelles (>200 nm in diameter), which display the typical morphology of late endosomes and early lysosomes (A, arrow) or more dense, mature lysosomes (B, arrow). These organelles appear double immunolabeled also for IL-1β (10-nm gold particles) and Lamp-1 (18-nm gold particles) (D, arrow). Mature lysosomes showing positive staining for IL-1β alone are also found (C, arrow). Dense vesicles, <200 nm in diameter, appear either positively immunolabeled for IL-1β only (E, arrowheads), for both IL-1β and CD (F, arrowhead), or for both IL-1β and Lamp-1 (G, arrowhead). Bars, 200 nm.

Article Snippet: In double-labeling experiments, the sections were first incubated with anti-IL-1β followed by 10 nm of goat anti-mouse IgG gold conjugates (British BioCell International, Carditt, UK) and then incubated with anti-CD or anti-Lamp-1 followed by 18 nm of protein-A gold.

Techniques: Immuno-Electron Microscopy, Immunolabeling, Staining

Journal:

Article Title: The Secretory Route of the Leaderless Protein Interleukin 1? Involves Exocytosis of Endolysosome-related Vesicles

doi:

Figure Lengend Snippet: Migration of proIL-1β on Percoll density gradient. P1 and P2 fractions were pooled and further fractionated by centrifugation on a 25% Percoll density gradient under conditions that separate heavy-density lysosomes from lower-density endosomes. Membranes were collected from the individual fractions and tested for β-hexosoaminidase activity or analyzed by Western blot using anti-CD, anti-IL-1β, or anti-Rab7 antibodies. Fraction 1 represents the lowest density; fraction 18 is the highest density. The reactivity of proCD and CD (A), IL-1β (B), and Rab7 (C) was quantified by image digitalization and plotted as arbitrary units (a.u.); β-hexosoaminidase activity (C) is expressed as percent of total. The Western blots hybridized with anti-CD and anti-IL-1β are shown as insets in A and B, respectively. One representative experiment four is shown.

Article Snippet: In double-labeling experiments, the sections were first incubated with anti-IL-1β followed by 10 nm of goat anti-mouse IgG gold conjugates (British BioCell International, Carditt, UK) and then incubated with anti-CD or anti-Lamp-1 followed by 18 nm of protein-A gold.

Techniques: Migration, Centrifugation, Activity Assay, Western Blot

Journal:

Article Title: The Secretory Route of the Leaderless Protein Interleukin 1? Involves Exocytosis of Endolysosome-related Vesicles

doi:

Figure Lengend Snippet: Effects of lysosomotropic drug treatment on secretion and vesicular accumulation of IL-1β. A, Monocytes were stimulated 1 h with LPS without (nil; lanes 1, 4, and 7) or with 50 mM NH4Cl (NH4Cl pre; lanes 2, 5, and 8) and cultured 2.5 h in the absence or presence of the same drug as indicated. Alternatively, monocytes were stimulated 1 h with LPS without NH4Cl, followed by 2.5 h of culture with the drug (NH4Cl chase; lanes 3, 6, and 9). Filters were hybridized with anti-IL-1β (upper panels) or anti-CD (lower panels) antibodies. Cyt, cytosolic fractions (lanes 1–3); P1, P1 pellet (lanes 4–6); sec, supernatants (lanes 7–9). (B) Supernatants from monocytes untreated (nil, lane 1), chased (lanes 2–4), or pretreated (lanes 5–7) as in A with 50 mM NH4Cl (lanes 2 and 5), 50 μM chloroquine (chlor; lanes 3 and 6), or 1 μM BafA1 (lanes 4 and 7). Filters were hybridized with anti-IL-1β antibody.

Article Snippet: In double-labeling experiments, the sections were first incubated with anti-IL-1β followed by 10 nm of goat anti-mouse IgG gold conjugates (British BioCell International, Carditt, UK) and then incubated with anti-CD or anti-Lamp-1 followed by 18 nm of protein-A gold.

Techniques: Cell Culture

Journal:

Article Title: The Secretory Route of the Leaderless Protein Interleukin 1? Involves Exocytosis of Endolysosome-related Vesicles

doi:

Figure Lengend Snippet: Exocytosis of IL-1β–containing vesicles is regulated by extracellular [Mg2+]. (A) Supernatants from monocytes incubated 2.5 h with LPS in medium alone (lanes 1 and 5) or plus 5 mM EDTA (lanes 3 and 7), 5 mM MgCl2 (lanes 2 and 6), or 5 mM EDTA plus 5 mM MgCl2 (lanes 4 and 8). When indicated (+ATP, lanes 5–8), 1 mM ATP was added during the last 30 min. (B) PNS (5%) and P1 from activated monocytes cultured 1 h in the absence (−, lanes 1 and 2) or presence of 5 mM MgCl2 (Mg2+, lanes 3 and 4) or 5 mM EDTA (lanes 5 and 6). Filters were hybridized with anti-IL-1β (upper panels) or anti-CD (lower panels).

Article Snippet: In double-labeling experiments, the sections were first incubated with anti-IL-1β followed by 10 nm of goat anti-mouse IgG gold conjugates (British BioCell International, Carditt, UK) and then incubated with anti-CD or anti-Lamp-1 followed by 18 nm of protein-A gold.

Techniques: Incubation, Cell Culture

Journal:

Article Title: The Secretory Route of the Leaderless Protein Interleukin 1? Involves Exocytosis of Endolysosome-related Vesicles

doi:

Figure Lengend Snippet: Exocytosis of IL-1β–containing vesicles is regulated by extracellular osmotic conditions. (A) Supernatants from monocytes incubated 2.5 h with LPS in isotonic conditions (medium alone, iso, lanes 1 and 4) or plus 0.1 M sucrose (hyper, lanes 2 and 5) or diluted 1:2 with water (hypo, lanes 3 and 6) in the absence (−, lanes 1–3) or presence of 1 mM ATP (+, lanes 4–6) for the last 30 min. Filters were hybridized with anti-IL-1β (upper panels) or anti-CD (lower panels). (B) PNS and P1 from monocytes incubated 2.5 h with LPS in isotonic conditions (medium alone, iso, lanes 1 and 2) or plus 0.1 M sucrose (hyper, lanes 3 and 4) or diluted 1:2 with water (hypo, lanes 5 and 6).

Article Snippet: In double-labeling experiments, the sections were first incubated with anti-IL-1β followed by 10 nm of goat anti-mouse IgG gold conjugates (British BioCell International, Carditt, UK) and then incubated with anti-CD or anti-Lamp-1 followed by 18 nm of protein-A gold.

Techniques: Incubation

Journal:

Article Title: The Secretory Route of the Leaderless Protein Interleukin 1? Involves Exocytosis of Endolysosome-related Vesicles

doi:

Figure Lengend Snippet: Two step model for IL-1β secretion, showing vesicle-mediated transport from the cytosol to the extracellular space. The translocation step is blocked by glibenclamide and when the ΔpH between cytosol and vesicles is abolished; the exocytosis is induced by high extracellular ATP and by hypotonic medium, whereas it is inhibited by low extracellular ATP and by hypertonic conditions.

Article Snippet: In double-labeling experiments, the sections were first incubated with anti-IL-1β followed by 10 nm of goat anti-mouse IgG gold conjugates (British BioCell International, Carditt, UK) and then incubated with anti-CD or anti-Lamp-1 followed by 18 nm of protein-A gold.

Techniques: Translocation Assay