Journal: Life Science Alliance

Article Title: IL-36 signaling in keratinocytes controls early IL-23 production in psoriasis-like dermatitis

doi: 10.26508/lsa.202000688

Figure Lengend Snippet: (A) Comparison of body weights from 8- to 20-wk-old Il36r fl/fl (FL) and Il36r ΔK (DK) mice. (B) The indicated organs were collected from FL and DK mice, and levels of Il1rl2 mRNA expression (coding for IL-36R) were determined by qRT-PCR. The results represent mRNA expression relative to L32. Results are from one representative experiment of two. (C, E) Keratinocytes were prepared from FL and DK mice and then cultured for 6 h with medium alone, murine IL-1β (100 ng/ml), or murine IL-36β (100 ng/ml). (C) Levels of mRNA expression of the genes coding for the indicated proteins in stimulated keratinocytes were determined by qRT-PCR. The results represent mRNA expression relative to L32 . Results are pooled from five independent experiments. (E) The levels of CXCL1 were measured in the cell supernatants by ELISA. (D, F) BMDCs were prepared from Il36r fl/fl (FL) and Il36r ΔK (DK) mice. BMDCs were cultured for 24 h with medium alone, LPS (10 ng/ml), or murine IL-36β (100 ng/ml). (D) Levels of mRNA expression of the genes coding for the indicated proteins in stimulated BMDCs were determined by qRT-PCR. The results represent mRNA expression relative to L32. Results are pooled from two independent experiments. (F) Levels of IL-6 were measured in the cell supernatants by ELISA. (B, C, D, E, F) Statistical analysis was performed using the Mann–Whitney test (B), RM one-way ANOVA, and Welch t tests (C, D, E, F). Numbers indicated represent the P -values when significance was found. A P -value < 0.05 was considered as significant.

Article Snippet: On day 3, primary murine keratinocytes were stimulated with 100 ng/ml murine IL-36β (generously given by Amgen Inc.) or 100 ng/ml murine IL-1β (#211-11B; Peprotech) in K-SFM medium/P/S for 6 h, and untreated cells were used as a control.

Techniques: Comparison, Expressing, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: IL-36 signaling in keratinocytes controls early IL-23 production in psoriasis-like dermatitis

doi: 10.26508/lsa.202000688

Figure Lengend Snippet: Il36r −/− (KO) and Il36r ΔK (DK) mice as well as their littermate controls, respectively, Il36r +/+ (WT) and Il36r fl/fl (FL) mice, were challenged with the topical application of Aldara on one ear during 3 or 7 d. RNASeq was performed on treated (AL) and untreated (CT) ears. (A, B) Definition of the strategy used to identify IL-36–dependent genes at d3 (A) and d7 (B) of Aldara treatment. The volcano plot (left panel) represents, on the x-axis, the mean value of the expression of the genes in the compared conditions indicated above (in log 2 scale), and the y-axis represents the fold change in the comparison (in log 2 scale). Horizontal dashed blue line represents a fold change of two (up- or down-regulation). Red dots indicate genes with a Benjamini–Hochberg–corrected P -value lower than 0.05. Gene numbers are summarized in the table below the volcano plot. Differentially expressed (DE) genes in WT AL versus WT CT were selected and grouped based on the four comparisons described in the Venn diagram (right panel, non-DE, non-differentially expressed). IL-36–dependent genes were selected as non-differentially expressed in KO AL compared with KO CT ears and DE in KO AL compared with WT AL ears.

Article Snippet: On day 3, primary murine keratinocytes were stimulated with 100 ng/ml murine IL-36β (generously given by Amgen Inc.) or 100 ng/ml murine IL-1β (#211-11B; Peprotech) in K-SFM medium/P/S for 6 h, and untreated cells were used as a control.

Techniques: Expressing, Comparison

Journal: Life Science Alliance

Article Title: IL-36 signaling in keratinocytes controls early IL-23 production in psoriasis-like dermatitis

doi: 10.26508/lsa.202000688

Figure Lengend Snippet: Il36r −/− (KO) and Il36r ΔK (DK) mice as well as their littermate controls, respectively, Il36r +/+ (WT) and Il36r fl/fl (FL) mice, were challenged with the topical application of Aldara on one ear during 3 or 7 d. RNASeq was performed on treated (AL) and untreated (CT) ears. (A, B) Definition of the strategy used to identify genes dependent of IL-36 signaling in keratinocytes (KC-36) at d3 (A) and d7 (B) of Aldara treatment. The volcano plot (left panel) represents, on the x-axis, the mean value of the expression of the gene in the compared conditions indicated above (in log 2 scale), and the y-axis represents the fold change in the comparison (in log 2 scale). Horizontal dashed blue line represents a fold change of two (up- or down-regulation). Red dots indicate genes with a Benjamin–Hochberg–corrected P -value lower than 0.05. Gene numbers are summarized in the table below the volcano plot. Differentially expressed (DE) genes in FL AL versus FL CT were selected. Genes from this group that were also found in IL-36–dependent genes (as defined in , center panel) were further grouped based on the four comparisons described in the Venn diagram (right panel, non-DE, non-differentially expressed). KC-36–dependent genes were selected as non-differentially expressed in DK AL compared with DK CT ears and DE in FL AL compared with DK AL ears.

Article Snippet: On day 3, primary murine keratinocytes were stimulated with 100 ng/ml murine IL-36β (generously given by Amgen Inc.) or 100 ng/ml murine IL-1β (#211-11B; Peprotech) in K-SFM medium/P/S for 6 h, and untreated cells were used as a control.

Techniques: Expressing, Comparison

Journal: Life Science Alliance

Article Title: IL-36 signaling in keratinocytes controls early IL-23 production in psoriasis-like dermatitis

doi: 10.26508/lsa.202000688

Figure Lengend Snippet: Il36r −/− (KO) and Il36r ΔK (DK) mice as well as their littermate controls, respectively, Il36r +/+ (WT) and Il36r fl/fl (FL) mice, were challenged with the topical application of Aldara on one ear during 3 or 7 d. RNASeq was performed on treated and untreated ears. (A, B) These graphs represent the log 2 of the fold changes of gene expression of the 30 most up-regulated and down-regulated genes among IL-36–dependent genes (as defined in ) in Aldara-treated compared with untreated ears at d3 (A) and d7 (B) of treatment. (C, D) This graph represents the log 2 of the fold changes of gene expression of the keratinocyte IL-36R (KC-36)–dependent genes (as defined in ) in Aldara-treated compared with untreated ears at d3 (C) and d7 (D) of treatment. Results are combined from five independent experiments.

Article Snippet: On day 3, primary murine keratinocytes were stimulated with 100 ng/ml murine IL-36β (generously given by Amgen Inc.) or 100 ng/ml murine IL-1β (#211-11B; Peprotech) in K-SFM medium/P/S for 6 h, and untreated cells were used as a control.

Techniques: Gene Expression

Journal: Annals of Translational Medicine

Article Title: IL-36β promotes anti-tumor effects in CD8 + T cells by downregulating micro-RNA let-7c-5p

doi: 10.21037/atm-21-5991

Figure Lengend Snippet: IL-36β in cancer tissues is downregulated. (A) IL-36β mRNA levels in cancer tissue (T) samples were 0.392±0.070, while the levels in paracancerous tissue specimens (NT) were 1.493±0.113, and the difference was statistically significant (P<0.05). (B) Immunohistochemistry was performed for IL-36β protein detection in tissue samples (magnification, 200×). Positive signals in pancreatic cancer tissue samples (a). No expression in pancreatic cancer tissue specimens (b). Positive signals in paracancerous tissue specimens (c). No expression in paracancerous tissue samples (d). *, P<0.05. IL, interleukin.

Article Snippet: Panc02 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) When cells reached approximately 30% confluency, appropriate amounts of IL-36β lentivirus (GeneChem Co., Shanghai) were used to transfect them according to a multiplicity of infection (MOI) of 10.

Techniques: Immunohistochemistry, Expressing

Journal: Annals of Translational Medicine

Article Title: IL-36β promotes anti-tumor effects in CD8 + T cells by downregulating micro-RNA let-7c-5p

doi: 10.21037/atm-21-5991

Figure Lengend Snippet: Positive expression rates for the IL-36β protein in cancerous and paracancerous tissue samples

Article Snippet: Panc02 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) When cells reached approximately 30% confluency, appropriate amounts of IL-36β lentivirus (GeneChem Co., Shanghai) were used to transfect them according to a multiplicity of infection (MOI) of 10.

Techniques: Expressing

Journal: Annals of Translational Medicine

Article Title: IL-36β promotes anti-tumor effects in CD8 + T cells by downregulating micro-RNA let-7c-5p

doi: 10.21037/atm-21-5991

Figure Lengend Snippet: IL-36β overexpression in tumor cells inhibits tumor growth in vivo. In total, 1×106 Panc02-Control, Panc02-NC, and Panc02-IL-36β cells were injected subcutaneously into C57BL/6 mice, and the tumor sizes were monitored every other day. Data (mean ± SEM) are from three independent experiments (n=5/group). Until the end of the study, the tumor diameter was markedly reduced in the Panc02-IL-36β group (9.45±0.36 mm) compared with the Panc02-Control (16.59±0.08 mm) and Panc02-NC (17.01±0.65 mm) groups (P<0.05). IL, interleukin; NC, negative control; SEM, standard error of mean.

Article Snippet: Panc02 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) When cells reached approximately 30% confluency, appropriate amounts of IL-36β lentivirus (GeneChem Co., Shanghai) were used to transfect them according to a multiplicity of infection (MOI) of 10.

Techniques: Over Expression, In Vivo, Control, Injection, Negative Control

Journal: Annals of Translational Medicine

Article Title: IL-36β promotes anti-tumor effects in CD8 + T cells by downregulating micro-RNA let-7c-5p

doi: 10.21037/atm-21-5991

Figure Lengend Snippet: Tumoral IL-36β overexpression enhances type 1 immune responses in the tumor microenvironment. On day 33, tumor samples were obtained to generate single-cell suspensions. (A) Percentages of CD45+ cells in tumor cell suspensions. (B,C) Representative flow-cytograms and CD8+ T or NK cell rates within tumor CD45+ cells. *, P<0.05. IL, interleukin; NK, natural killer.

Article Snippet: Panc02 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) When cells reached approximately 30% confluency, appropriate amounts of IL-36β lentivirus (GeneChem Co., Shanghai) were used to transfect them according to a multiplicity of infection (MOI) of 10.

Techniques: Over Expression

Journal: Annals of Translational Medicine

Article Title: IL-36β promotes anti-tumor effects in CD8 + T cells by downregulating micro-RNA let-7c-5p

doi: 10.21037/atm-21-5991

Figure Lengend Snippet: Adenovirus with IL-36β overexpression inhibits tumor growth after intratumoral injection. In total, 1×106 Panc02 cells were administered by subcutaneous injection into C57BL/6 mice. On the 7th day, the animals were randomized into three groups and injected separately with 1×109 PFU IL-36β overexpression adenovirus, 1×109 PFU adenovirus empty vector, and 100 μL PBS. Tumor sizes were monitored every other day. Data (mean ± SEM) are from three independent experiments (n=5/group). Until the end of the study, the tumor diameter was markedly reduced in mice injected with IL-36β adenovirus (11.82±1.28 mm) compared with the control PBS (16.8±1.57 mm) and adenovirus empty vector (16.31±1.40 mm) groups (P<0.05). IL, interleukin; PFU, plaque-forming units; PBS, phosphate-buffered saline; SEM, standard error of mean.

Article Snippet: Panc02 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) When cells reached approximately 30% confluency, appropriate amounts of IL-36β lentivirus (GeneChem Co., Shanghai) were used to transfect them according to a multiplicity of infection (MOI) of 10.

Techniques: Over Expression, Injection, Plasmid Preparation, Control, Saline

Journal: Annals of Translational Medicine

Article Title: IL-36β promotes anti-tumor effects in CD8 + T cells by downregulating micro-RNA let-7c-5p

doi: 10.21037/atm-21-5991

Figure Lengend Snippet: Adenovirus with IL-36β overexpression induces CD8+T, NK, and γδT cell aggregation. On day 27, tumor resection was performed to generate single-cell suspensions. (A) Percentages of CD45+ cells in tumor cell suspensions. (B,C) Representative flow-cytograms, and CD8+ T, NK1.1+, or γδT cell rates among tumor CD45+ cells. *, P<0.05. IL, interleukin; NK, natural killer.

Article Snippet: Panc02 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) When cells reached approximately 30% confluency, appropriate amounts of IL-36β lentivirus (GeneChem Co., Shanghai) were used to transfect them according to a multiplicity of infection (MOI) of 10.

Techniques: Over Expression

Journal: Annals of Translational Medicine

Article Title: IL-36β promotes anti-tumor effects in CD8 + T cells by downregulating micro-RNA let-7c-5p

doi: 10.21037/atm-21-5991

Figure Lengend Snippet: Heat map of 21 miRNAs generated with the R software (pheatmap package). (A) Human; (B) mouse. Myeloid Differentiation Factor 88 (MYD88, a critical upstream effector of IKK/NF-κB signaling). (C) The results of qRT-PCR indicated that let-7c-5p downregulation was the most consistent. Control group, WT CD8+; IL-36β group, WT CD8+/IL-36β stimulation (***, P<0.0001). KO, knockout; qRT-PCR, quantitative real-time PCR; IKK, IKappaBbeta kinase; NF-κB, nuclear factor-kappaB; IL, interleukin; WT, wild type.

Article Snippet: Panc02 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) When cells reached approximately 30% confluency, appropriate amounts of IL-36β lentivirus (GeneChem Co., Shanghai) were used to transfect them according to a multiplicity of infection (MOI) of 10.

Techniques: Generated, Software, Quantitative RT-PCR, Control, Knock-Out, Real-time Polymerase Chain Reaction

Journal: Annals of Translational Medicine

Article Title: IL-36β promotes anti-tumor effects in CD8 + T cells by downregulating micro-RNA let-7c-5p

doi: 10.21037/atm-21-5991

Figure Lengend Snippet: IL-36β downregulates IFN-γ in let-7c-5p+ CD8+ T cells. Data are mean ± SEM. ***, P<0.001 (two-tailed unpaired Student’s t-test). IL, interleukin; IFN, interferon; SEM, standard error of mean.

Article Snippet: Panc02 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) When cells reached approximately 30% confluency, appropriate amounts of IL-36β lentivirus (GeneChem Co., Shanghai) were used to transfect them according to a multiplicity of infection (MOI) of 10.

Techniques: Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: IL-36 synergized with IL-17A to promote inflammatory cytokines expression in NHDF. (A, B) RNA sequencing was performed for NHDF cultured with human IL-17A (100 ng/ml), IL-36 ligand (IL-36α 30 ng/ml + IL-36β 30 ng/ml + IL-36γ 30 ng/ml), and IL-36 ligand (IL-36α 30 ng/ml + IL-36β 30 ng/ml + IL-36γ 30 ng/ml) plus IL-17A (100 ng/ml) proteins for 24 hours. Differential gene expression profiling (FCH ≥ 2, FDR < 0.05). FCH, fold change; FDR, false discovery rate. (A) Venn diagram displaying the number and overlap of differentially expressed genes compared with those of the control. (B) Hierarchically clustered heatmap of inflammatory related differential gene expression of these groups. (C) The synergy effect of IL-17A and IL-36 ligand in the induction of IL-6 secretion. NHDF cells were cultured with 0.5 ng/ml IL-17A and 15 ng/ml IL-36α, 1 ng/ml IL-36β or 2 ng/ml IL-36γ overnight. (D) Various gene expression was analyzed by RT-PCR in NHDF cell following stimulation by IL-17A, IL-36 or IL-17A&IL-36 combined. Results demonstrated the synergistic effect of IL-17A and IL-36 in inducing inflammatory related gene expression. *P<0.05, **P<0.01, ***P<0.01, ****P<0.0001, ns, no significance.

Article Snippet: IL-36β (85 pM, Novoprotein, China; Cat#CK25) and serial diluted mAb/BsAb were added into plate, then incubate for 24 hours.

Techniques: Expressing, RNA Sequencing, Cell Culture, Gene Expression, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: HB0043 effectively antagonized the production of IL-6 induced by IL-17A and IL-36 in NHDF cells. (A–C) The ability of HB0043 to block IL-36R signaling and IL-17A signaling was measured and compared to that of HB0034 and HB0017, using IL-17A and IL-36R blockade cell-based assay. The secretion of IL-6 was monitored, which was induced by 0.5 ng/ml human IL-17A and 15 ng/ml IL-36α (A) , 1 ng/ml IL-36β (B) or 2 ng/ml IL-36γ (C) in NHDF cells overnight. Data are expressed as means ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.01, ****P < 0.0001, as determined by one-way ANOVA.

Article Snippet: IL-36β (85 pM, Novoprotein, China; Cat#CK25) and serial diluted mAb/BsAb were added into plate, then incubate for 24 hours.

Techniques: Blocking Assay, Cell Based Assay

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: Dual blockade of IL-17A and IL-36R showed stronger inhibition of inflammation relative to mAbs alone in IMQ-induced mouse psoriasis model. (A) Schematic plot showing therapeutic dosing regime of BALB/C mice for experimental data in IMQ-induced psoriasis model. Mice were smeared with 5.0% IMQ (62.5mg) on the shaved back skin from Day1 to Day 7 daily. Antibodies (HB0017, HB0034SA, HB0017+HB0034SA) were administered intraperitoneally into mice at 50 mg/kg twice a week. The control group received PBS intraperitoneal injections. (B) Body weight. (C) PASI score evaluation result, (D, E) Back skin samples were collected for subsequent histopathological evaluation (D) and H&E staining (E) (black arrow: stratum corneum thicken, green arrow: loose structure, red arrow: acanthosis, yellow arrow: trochanterellus lengthen and rodlike, blue arrow: inflammatory cell infiltration, orange arrow: blooding in papillary layer). (F) Relative skin mRNA expression of skin inflammation related cytokines (IL-17A, IL-22, IL-23, IL-36α, IL-36β, IL-36γ). *P<0.05, **P<0.01, ***P<0.01, ****P<0.0001.

Article Snippet: IL-36β (85 pM, Novoprotein, China; Cat#CK25) and serial diluted mAb/BsAb were added into plate, then incubate for 24 hours.

Techniques: Inhibition, Control, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: IL-36 and IL-17 secreted by multiple cell types have overlapping and non-overlapping downstream effects. IL-36 cytokines are expressed by a broad variety of cell types such as epithelial cell, fibroblast, keratinocytes, lymphocytes, monocytes, myeloid dendritic cells (DCs), monocyte-derived DCs, plasmacytoid DCs, and plasma cells. There is a feedback loop between the IL-36 and Th17 cytokines. Indeed, IL-36 cytokines are not only regulated by Th17 cytokines, but also act as activators of Th17 cells which regulate the differentiation and enhance the expression of Th17 cytokines. IL-17 is mainly secreted by IL-23-IL-17 axis, as well as other cell populations, such as CD8+ (Tc17) cells and various subsets of innate lymphocytes including γδT cells, natural killer T (NKT) cells, group 3 innate lymphoid cells (ILC3s), and ‘‘natural’’ Th17 cells. IL-36 and IL-17 signaling promote secretion of inflammatory and chemotactic molecules and immune cell infiltration and immune regulation, which closely related to many inflammatory diseases.

Article Snippet: IL-36β (85 pM, Novoprotein, China; Cat#CK25) and serial diluted mAb/BsAb were added into plate, then incubate for 24 hours.

Techniques: Derivative Assay, Clinical Proteomics, Expressing