Fig 1: IL-18 is a bona fide regulator of Paneth cells during AIEC host defence.a Quantitative real-time PCR and ELISA analyses of ileum crypts or tissues for IL-18 in uninfected or AIEC-infected mice. b Quantitative real-time PCR analysis of IL-18-stimulated ileum organoids, derived from the indicated mice, for anti-microbial peptides. c Flow cytometry analysis of IL-18-stimulated ileum organoids, derived from the indicated mice, for CD24+ Lysozyme+ Paneth cells. d Flow cytometry analysis of ileum crypts, derived from IL-22 or IL-18-injected mice, for phospho-Stat3 (pStat3) in the indicated epithelial subsets. Mean fluorescence intensity (MFI) of pStat3 is indicated. e Fresh crypts were stimulated with IL-22, IL-18, or both, to induce the release of anti-microbial peptides (AMP). The AMP-containing supernatants were then incubated with live AIEC and the percentage of bacterial killing was determined by the CFU assay after normalized to unstimulated crypts. f Immunofluorescence analysis of ileum crypts (outlined with a dashed line) for Lysozyme+ Paneth cells at the crypt base in the indicated mice. g Flow cytometry analysis of ileum crypts, derived from the indicated mice, for CD24+ Lysozyme+ Paneth cells. h Colony-forming unit (CFU) of AIEC was analyzed in feces and intestinal tissues isolated from the indicated mice at d6 post infection. i Flow cytometry analysis of PMA/Ionomycin (P/I)-stimulated ileum lamina propria cells for intracellular IFN? in CD4+ or CD8+ T cells in AIEC-infected mice. j CFU of AIEC was analyzed in feces and intestinal tissues isolated from the indicated PBS or IL-18-injected mice at d6 post infection. k Immunofluorescence analysis of ileum crypts for Lysozyme+ Paneth cells at the crypt base in the indicated IL-18-injected mice at d6 post AIEC infection. l Flow cytometry analysis of ileum crypts, derived from the indicated IL-18-injected mice, for CD24+ Lysozyme+ Paneth cells. m Quantitative real-time PCR analysis of ileum crypts from the indicated IL-18-injected mice, for Paneth cell marker Lysozyme and Cryptdin. Each symbol in bar graphs represents an ileum crypt sample (a, d, e, g, l, m), tissue sample (a, h, i, j), or organoid culture (b, c), derived from one mouse. Data shown are representative (f, i, k) or combined (a–e, g, h, j, l, m) results from two independent reproducible experiments. Figures 2f and 4c were performd in the same experiment. Statistical significance is indicated using unpaired two-tailed t test (g, h), One-way ANOVA with Sidak’s multiple comparisons test (a, d, e), or Two-way ANOVA with Tukey’s multiple comparisons test (b, c, i, j, l, m). Data are presented as mean ± SD. Source data are provided as a Source Data file.
Fig 2: Differential role of IL-22 and IL-18 in organoid culture.a Flow cytometry analysis of IL-22 or IL-18-stimulated ileum organoids, derived from the indicated mice, for Ki67+ proliferating cells. Mean fluorescence intensity (MFI) of Ki67+ organoids is shown. b Immunofluorescence analysis of ileum crypts for Ki67+ proliferating cells in the indicated naïve or AIEC-infected mice. Crypt base columnar (CBC, outlined with a solid line) stem cell and the above transit-amplifying (TA, outlined with a dashed line) compartments are indicated. c Flow cytometry analysis of ileum crypts for CD24-/low Ki67+ proliferating cells in the indicated AIEC-infected mice. d Quantitative real-time PCR analysis of IL-22 or IL-18-stimulated ileum organoids, derived from the indicated mice, for stem cell marker Lgr5, Ascl2, and Olfm4. e Quantification of the number of buds per organoid and size in IL-22 or IL-18-stimulated ileum organoids, derived from the indicated mice. “n” indicates the number of images taken from organoids derived from four mice in each group. f Quantification of the number of buds in IL-18-stimulated wild-type ileum organoids. “n” indicates the number of images taken from organoids derived from three mice per group. g Quantitative real-time PCR analysis of IL-22-stimulated ileum organoids, derived from the indicated mice, for stem cell marker Lgr5, Ascl2, and Olfm4. h Quantification of the number of buds per organoid and size in IL-22-stimulated ileum organoids, derived from the indicated mice. “n” indicates the number of images taken from organoids derived from four mice per group. Each symbol in bar graphs represents an ileum crypt sample (c) or organoid culture (a, d, g), derived from one mouse. Data shown are representative (b) or combined (a, c–h) results from two independent reproducible experiments. Statistical significance is indicated using unpaired two-tailed t test (c), One-way ANOVA with Sidak’s multiple comparisons test (f), or Two-way ANOVA with Tukey’s multiple comparisons test (a, d, e, g, h). Data are presented as mean ± SD. Source data are provided as a Source Data file.
Fig 3: IL-22 links IL-18 for IFN? and Paneth cell responses during AIEC infection.a Colony-forming unit (CFU) of AIEC was analyzed in feces and intestinal tissues isolated from the indicated PBS or IL-18-injected mice at day-6 (d6) post infection. b Flow cytometry analysis of PMA/Ionomycin-stimulated ileum lamina propria cells for intracellular IFN? in CD4+ or CD8+ T cells in the indicated PBS or IL-18-injected mice. c Immunofluorescence analysis of ileum crypts for Lysozyme+ Paneth cells at the crypt base in the indicated mice at d6 post infection. d CFU of AIEC was analyzed in feces and intestinal tissues isolated from the indicated PBS or IL-22-injected mice at d6 post infection. e Flow cytometry analysis of PMA/Ionomycin-stimulated ileum lamina propria cells for intracellular IFN? in CD4+ or CD8+ T cells in the indicated PBS or IL-22-injected mice. f Immunofluorescence analysis of ileum crypts for Lysozyme+ Paneth cells at the crypt base in the indicated mice at d6 post infection. Each symbol in bar graphs represents an intestinal tissue sample (a, b, d, e) isolated from one mouse. Data shown are representative (c–f) or combined (a, b) results from two independent reproducible experiments. Statistical significance is indicated using Two-way ANOVA with Tukey’s multiple comparisons test (a, b, d, e). Data are presented as mean ± SD. Source data are provided as a Source Data file.
Fig 4: Cardiomyopcyte‐derived IL‐18 by β‐AR activation contributed to Cx43 up‐regulation in fibroblasts in a paracrine fashion. Quantification of IL‐18 expression in cardiomyocytes by Western blotting analysis (A) (n = 6 mice/group), or concentrations by ELISA (B) in myocardium tissue lysate (n = 6 mice/group) and (C) in plasma (n = 6 mice/group) in 5‐month‐old NTG and β2‐TG mice. D, Immunofluorescence staining of WGA (green), IL‐18 (yellow), α‐SMA (red) and DAPI (blue) in LV myocardium from 5‐month‐old NTG and β2‐TG mice. Scale bar: 20 µm. Concentration of IL‐18 (E) in myocardium tissue lysate and (F) in plasma from control and 7‐day after ISO treatment measured by ELISA (n = 5‐7 mice/group). Concentration of IL‐18 (G) in primary cardiomyocytes in culture or (H) culture media harvested from cardiomyocytes with ISO treatment (1 μmol/L, 48 h) by ELISA (n = 4‐6 independent isolation/2 hearts). I, Western blotting images and quantification of band intensity for Cx43 expression in adult mouse cardiac fibroblasts treated with IL‐18 (10 ng/mL) for 48 h (n = 5 independent isolation/5 hearts). J, Representative images of Cx43 IHC staining in LV sections and (K) quantificational analysis for Cx43 positive area and the ratio of lateral to ID localized Cx43 area in LV sections of mice treated daily with IL‐18 nAb or IgG for 7 d commencing from ISO infusion (n = 7 mice/group). Scale bar: 50 µm. Arrows indicate lateralized Cx43 and arrowheads for ID Cx43 localization. Data were expressed as mean ± SEM. *P < .05, **P < .01 and ***P < .001 vs NTG or control. Statistical significance was determined by two‐tailed unpaired Student's t test
Fig 5: Pro-inflammatory cytokines are increased in COVID-19, and co-treatment of TNF-a and IFN-? induces cell death(A) Heatmap depicting the levels of pro-inflammatory cytokines in serum of patients with COVID-19 and healthy people (Lucas et al., 2020). (B) Pro-inflammatory cytokines released from PBMCs infected with SARS-CoV-2. (C) Percent of BMDMs that are dead 48 h after cytokine treatment using the IncuCyte imaging system and propidium iodide (PI) staining. “Cocktail-1” contained all 8 cytokines (IL-6, IL-18, IFN-?, IL-15, TNF-a, IL-1a, IL-1ß, and IL-2). (D) Percent of BMDMs that are dead 48 h after treatment with the indicated combination of cytokines. (E) Real-time analysis of cell death in BMDMs treated with the indicated cytokines. (F, G) Representative images of cell death in BMDMs (F) and THP-1 cells (G) after 48 h of the indicated treatments. Scale bar, 50 µm. Data are representative of at least three independent experiments. **P < 0.01; ****P < 0.0001. Analysis was performed using the one-way ANOVA (B–D) or the two-way ANOVA (E). Significance asterisks in C and D indicate the comparison to the media-treated control. Data are shown as mean ± SEM (B–E). See also Figure S1.
Supplier Page from BioLegend for Recombinant Mouse IL-18 (carrier-free)