pcmv6-entry il36rn s113l plasmid  (Addgene inc)

Journal: EMBO Molecular Medicine

Article Title: IL-36 signaling as a drug target in Crohn’s disease patients with IL36RN mutations

doi: 10.1038/s44321-025-00245-z

Figure Lengend Snippet: ( A ) Summary of the clinical history of the IL36RN -mutated patient (IL-36RA patient). ( B ) Sanger sequencing of EDTA blood samples from the IL-36RA patient and the patient’s mother was conducted. The red square indicates the position of the mutation (c.338C) in the IL36RN gene. ( C , D ) HEK 293T cells were transfected with either IL36RN wild-type (WT), IL36RN S113L (S113L), water (H 2 O), or were left untransfected (untrans.). The expression of IL-36RA was analyzed 48 h after transfection ( C ) in the supernatant by ELISA and ( D ) in cell lysates by Western blot analyses. Data represent three independent experiments with n = 6 per condition. The line in the plots indicates the median. ( E ) The activity of NFκB in HEK-Blue IL-36 cells that had been pre-incubated with different concentrations of IL-36RA WT or IL-36RA S113L and subsequently stimulated with IL-36α. The values were normalized to control samples stimulated with IL-36α only. The half maximal inhibitory concentration (IC 50 ) for IL-36RA WT and IL-36RA S113L is indicated in the table below the graph. The data represent four independent experiments. The data are represented as mean ± SD ( n = 8). ( F ) Peripheral blood mononuclear cells (PBMCs) from the IL-36RA patient and one healthy donor (HD) were stimulated in vitro with IL-36α for 7 h or left unstimulated (unstim.). Subsequently, cytokine levels in the supernatant were analyzed. Duplicates represent technical replicates. The line in the plots indicates the median. ( G ) The serum cytokine levels of healthy donors (HDs), Crohn’s disease patients (CD), and the IL-36RA patient at different time points are presented. The line in the plots indicates the median. HD: n = 4, CD: n = 7, IL-36RA patient: n = 4 (same patient, different time points). ( H ) PBMCs of the IL-36RA patient at two different time points, CD patients, and HDs were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin (Iono) or lipopolysaccharide (LPS) for 4 h or with IL-36α for 7 h and subsequently analyzed by mass cytometry. The frequency of NK cells and B cells in unstimulated samples and the frequency of Th17 cells in PMA/Iono-stimulated samples are presented. The line in the plots indicates the median. HD: n = 4, CD: n = 3, IL-36RA patient: n = 2 (same patient, different time points). The statistical significance in ( C , D ) was determined by one-way ANOVA with Tukey’s multiple comparisons test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05. Exact P values for the statistical comparisons are shown in Appendix Table . .

Article Snippet: pCMV6-Entry IL36RN S113L Plasmid , Addgene , 58321.

Techniques: Sequencing, Mutagenesis, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay, Incubation, Control, Concentration Assay, In Vitro, Mass Cytometry

Journal: EMBO Molecular Medicine

Article Title: IL-36 signaling as a drug target in Crohn’s disease patients with IL36RN mutations

doi: 10.1038/s44321-025-00245-z

Figure Lengend Snippet: ( A , B ) Peripheral blood mononuclear cells (PBMCs) of the IL36RN -mutated patient (IL-36RA patient) and one healthy donor (HD) were stimulated in vitro with IL-36α for 7 h or left unstimulated (unstim). Subsequently, cytokine levels in the supernatant were analyzed by cytometric bead array (CBA). Duplicates represent technical replicates. The line in the plots indicates the median. ( B ) Fold change between PBMCs stimulated with IL-36α and those left unstimulated. ( C ) PBMCs of the IL-36RA patient were pre-incubated with 1000 µg/mL spesolimab for 15 min and then stimulated with 100 ng/mL IL-36α for 4 h. Subsequently, the concentration of various cytokines in the supernatant was measured by CBA. Data represent technical replicates. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05. Exact P values for the statistical comparisons are shown in Appendix Table . .

Article Snippet: pCMV6-Entry IL36RN S113L Plasmid , Addgene , 58321.

Techniques: In Vitro, Incubation, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: IL-36 signaling as a drug target in Crohn’s disease patients with IL36RN mutations

doi: 10.1038/s44321-025-00245-z

Figure Lengend Snippet: ( A ) The therapeutic plan of the IL36RN -mutated patient (IL-36RA patient). ( B ) Calprotectin in the stool of IL-36RA patient during spesolimab therapy. ( C ) Endoscopic images showing the luminal inflammation in the ileum of the IL-36RA patient before and during treatment with spesolimab and certolizumab pegol as well as the Simple Endoscopic Score for Crohn’s disease (SES-CD) as assessed during colonoscopy. ( D ) Cytokine levels in the serum of healthy donors (HD), Crohn’s disease patients (CD), and the IL-36RA patient at different time points during spesolimab therapy. The line in the plots indicates the median. HD: n = 5, CD: n = 6, IL-36RA patient: n = 1. ( E–G ) PBMCs from the IL-36RA patient before and during spesolimab therapy and PBMCs of HDs were stimulated in vitro with phorbol 12-myristate 13-acetate (PMA)/ionomycin (Iono) or lipopolysaccharide (LPS) for 4 h or with IL-36α for 7 h. The cells were subsequently analyzed by mass cytometry. HD: n = 3, IL-36RA patient: n = 1. ( E ) Heatmap showing the frequency of the 11 identified clusters in unstimulated PBMCs. ( F ) Frequency of selected clusters in unstimulated PBMCs. ( G ) Frequency of pro-inflammatory cytokine-producing myeloid cells in IL-36α-stimulated samples. The line in the plots indicates the median. .

Article Snippet: pCMV6-Entry IL36RN S113L Plasmid , Addgene , 58321.

Techniques: In Vitro, Mass Cytometry

Journal: EMBO Molecular Medicine

Article Title: IL-36 signaling as a drug target in Crohn’s disease patients with IL36RN mutations

doi: 10.1038/s44321-025-00245-z

Figure Lengend Snippet: ( A ) Frequency of different immune cell populations in lamina propria mononuclear cells (LPMCs) isolated from ileal biopsies of the IL36RN -mutated patient (IL-36RA patient) before and during spesolimab therapy as well as of control Crohn’s disease (CD) patients. CD: n = 5, IL-36RA patient: n = 1. ( B ) Frequency of different immune cell populations in lamina propria mononuclear cells (LPMCs) isolated from colonic biopsies of the IL-36RA patient before and during spesolimab therapy as well as of CD patients. CD: n = 5, IL-36RA patient: n = 1. ( C ) T1 weighted, fat-saturated MRI after i.v. contrast administration. The ischiorectal fossa is shown in an axial plane. The patient had a horseshoe perianal abscess (arrows) in January 2022, which was surgically relieved and subsequently treated with seton stitches. In the further course up to and including January 2023, the abscess healed under the sequential treatment with cyclophosphamide and spesolimab/certolizumab pegol through scarring (arrowheads). .

Article Snippet: pCMV6-Entry IL36RN S113L Plasmid , Addgene , 58321.

Techniques: Isolation, Control

Journal: EMBO Molecular Medicine

Article Title: IL-36 signaling as a drug target in Crohn’s disease patients with IL36RN mutations

doi: 10.1038/s44321-025-00245-z

Figure Lengend Snippet: ( A ) Mutations in IL36RN in a whole-exome sequencing dataset of 45 healthy donors, 86 patients with ulcerative colitis, and 244 patients with Crohn’s disease were identified by searching for mutations predicted by PolyPhen-2 to be damaging. Subsequently, mutations were confirmed by targeted Sanger sequencing. The red square indicates the position of the mutation in the IL36RN gene. ( B, C ) HEK 293T cells were either transfected with IL36RN wild-type (WT), IL36RN P76L (P76L), IL36RN L133I (L133I), water (H 2 O), or left untransfected (untrans.). IL-36RA protein expression was analyzed 48 h after transfection ( B ) in cell lysates by Western blot and ( C ) in the supernatant by ELISA. Data represent three independent experiments with n = 6 per condition. The line in the plots indicates the median. ( D , E ) NFκB activity of HEK-Blue IL-36 cells pre-incubated with different concentrations of IL-36RA WT, IL-36RA P76L, or IL-36RA L133I and subsequently stimulated with IL-36α. Values are normalized to control samples stimulated with IL-36α only. The half maximal inhibitory concentration (IC 50 ) for IL-36RA WT, IL-36RA P76L, and IL-36RA L133I is indicated in the table below the graph. The data represent three independent experiments. Data are represented as mean ± SD ( n = 10). Statistical significance in ( B , C ) was determined by one-way ANOVA with Tukey’s multiple comparisons test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05. Exact P values for the statistical comparisons are shown in Appendix Table . .

Article Snippet: pCMV6-Entry IL36RN S113L Plasmid , Addgene , 58321.

Techniques: Sequencing, Mutagenesis, Transfection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation, Control, Concentration Assay