Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Real-time PCR probes and details of primers

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Real-time Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Examples of imunohistochemical staining for the IL-1 family. IL-1β (row A), IL-1Ra (row B), and IL-1 receptor, type I (row C) in grade-1 non-degenerate discs (column 1) and grade-12 degenerate discs (column 2), IgG controls (row D) were all negative. Immunopositivity is revealed by brown staining. N.B In non-degenerate discs, no cell clusters were seen and little immunopositivity was observed in the single cells. In degenerate discs, a large number of cell clusters were observed, which were predominately immunopositive. Bars = 570 μm.

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Staining

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Immunopositive staining for the IL-1 family in human intervertebral discs. Numbers of cells with immunopositivity for IL-1α (a) , IL-1β (b) , IL-1 receptor antagonist (c) , IL-1 receptor, type I (d) , and IL-1β-converting enzyme (e) , according to place of origin in the disc and grade of intervertebral disc degeneration ( n = 30). Data are presented as means ± 2 standard errors (as a representative of 95%CI). * P < 0.1,; ** P < 0.05

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Staining

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Effects of IL-1β on gene expression in cells from non- degenerate or degenerate intervertebral discs

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Expressing

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Effect of IL-1 treatment on the IL-1 family gene expression in human intervertebral disc cells. Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene and untreated controls (hence control is graphed at 1 on the log scale) for IL-1α (a) , IL-1β (b) , IL-1 receptor antagonist (IL-1Ra) (c) , and IL-1 receptor, type I (IL-RI) (d) following IL-1β treatment of disc cells from two regions of non-degenerate (non-deg) ( n = 6) and degenerate ( n = 24) discs. ** P < 0.05.

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Expressing, Control

Journal: Nature Communications

Article Title: Inhibition of interleukin-1β reduces myelofibrosis and osteosclerosis in mice with JAK2 -V617F driven myeloproliferative neoplasm

doi: 10.1038/s41467-022-32927-4

Figure Lengend Snippet: a Upper panel: Serum IL-1β (pg/ml) in normal controls (NC; n = 20) and MPN patients ( n = 120); ET ( n = 42), PV ( n = 44), PMF ( n = 34). Correlation ( r ) between % JAK2 -V617F in granulocytes and log transformed serum IL-1β in MPN patients. Limit of detection is shown by dashed green line at y = 0.01 pg/ml. Lower panel: IL-1β mRNA expression relative to β-actin in granulocytes of NC ( n = 12) and MPN patients ( n = 46); ET ( n = 11), PV ( n = 18), PMF ( n = 17). Correlation between log transformed IL1B mRNA expression and % JAK2 -V617F. b Upper panel: Serum IL-1RA (pg/ml) in NC ( n = 20) and MPN patients ( n = 120); ET ( n = 42), PV ( n = 44), PMF ( n = 34). Correlation ( r ) between % JAK2 -V617F and log transformed serum IL-1RA. Lower panel: IL1RN (IL-1RA) mRNA expression relative to β-actin in NC and MPN patients. Correlation between log transformed IL1RN mRNA expression and % JAK2 -V617F. Two-tailed unpaired non-parametric Mann–Whitney t -test was performed in a and b . c Representative histogram showing the expression of interleukin 1 receptor type 1 (IL-1R1) in peripheral blood hematopoietic stem cells (HSCs) from isotype control, NC ( n = 5), ET ( n = 6), PV ( n = 5), and PMF ( n = 7). Bar graph showing the percentages of IL-1R1 + HSC), common myeloid progenitors (CMP), granulocyte macrophage progenitor (GMP), megakaryocyte erythroid progenitor (MEP) and megakaryocyte progenitor (MkP). Graph showing correlation ( r ) between % JAK2 -V617F and percentages of IL-1R1 + HSCs. d Representative histogram showing the expression of interleukin 1 receptor accessory protein (IL-1RAcP) in peripheral blood HSC from NC and MPN patients. Bar graph showing the percentages of IL1RAcP + HSC, CMP, GMP, MEP, and MkP in NC ( n = 5), ET ( n = 6), PV ( n = 5), and PMF ( n = 7). Correlation ( r ) between % JAK2 -V617F and percentages of IL-1RAcP+ HSPCs. Two-tailed unpaired t-test was performed for statistical comparisons in c and d . Spearman correlation ( r ) and two-tailed t -test was performed for correlation analysis in a – d . All data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. See also Supplementary Figs. – . Source data and exact p values are provided as a Source Data file.

Article Snippet: Frozen PBMCs from MPN patients and normal controls were thawed and stained after blocking Fcγ receptors (#564220, BD) with following human antibodies: lineage-FITC (1:20; 348701), CD34-Pacific Blue (1:100; 343512), CD38-APC (1:50; 356606), CD123-BV605 (1:100; 306026), and CD41-PE-Cy5 (1:50; 343512) from BioLegend, CD45RA-BV786, (1:50; 563870; BD biosciences) and IL-1R1-PE (1:20; FAB269P), IL-1RAcP-PE (1:20; FAB676P) or isotype goat IgG-PE antibody (1:20; IC108P) from R&D systems.

Techniques: Transformation Assay, Expressing, Two Tailed Test, MANN-WHITNEY, Control

Journal: The Journal of Neuroscience

Article Title: System x c − Activity and Astrocytes Are Necessary for Interleukin-1β-Mediated Hypoxic Neuronal Injury

doi: 10.1523/JNEUROSCI.2459-07.2007

Figure Lengend Snippet: Comparison of ischemic brain damage between wild-type and IL-1RI null mutant animals. A, Representative TTC staining of coronal brain sections (2 mm) 70.5 h after a 90 min reversible MCAO for wild-type (IL-1RI+/+; left) and IL-1RI-deficient (IL-1RI−/−; right) male mice. The lack of TTC staining indicates an infarcted area. B, Infarct volumes in the hemisphere (hemi), cerebral cortex (cortex), and caudate–putamen (CP) of wild-type (IL-1RI+/+; gray bars) and IL-1RI knock-out (IL-1RI−/−; hatched bars) male mice were measured over five coronal sections. Infarct volumes are reported as the mean ± SEM percentage of non-ischemic hemisphere, corrected for edema. Actual infarct volumes for wild-type versus null mutant animals were as follows: hemisphere, 177.3 ± 18.0 versus 65 ± 16.4 mm3; cortex, 99.8 ± 8.2 versus 29.7 ± 11.3 mm3; caudate–putamen, 31.2 ± 2.0 versus 18.1 ± 2.9 mm3. An asterisk indicates a value significantly different from wild-type animals, as assessed via an unpaired t test (n = 9 animals per group). Significance was assessed at p < 0.05. C, Post-ischemic neurological deficits were scored as described in Materials and Methods at 24 and 72 h after MCAO in wild-type (IL-1RI+/+; squares; gray bars) and IL-1RI null mutant (IL-1RI−/−; circles; hatched bars) male mice. Neurological scores between groups were different at 24 and 72 h after MCAO, as determined by a Mann–Whitney U test (n = 9 animals per group, statistical significance was assessed at p < 0.05).

Article Snippet: In experiments using recombinant mouse IL-1ra (rIL-1ra) (R & D Systems) or monoclonal anti-mouse IL-1RI antibody directed against the extracellular domain of mouse IL-1RI (MAB7711; R & D Systems), the respective solutions were prepared with IL-1β (1.5 ng/ml; 1.5×) and added as described above.

Techniques: Comparison, Mutagenesis, Staining, Knock-Out, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: System x c − Activity and Astrocytes Are Necessary for Interleukin-1β-Mediated Hypoxic Neuronal Injury

doi: 10.1523/JNEUROSCI.2459-07.2007

Figure Lengend Snippet: Role of IL-1RI signaling in the potentiation of hypoxic neuronal injury by IL-1β in vitro. A, B, Mixed cortical cell cultures were treated with 1 ng/ml IL-1β for 20–24 h in the presence or absence of rIL-1ra (10–1000 ng/ml; A) or anti-IL-1RI (0.1–100 μg/ml; B), washed, and then deprived of oxygen (5 h). The percentage of total neuronal cell death was determined 20–24 h later. An asterisk indicates values significantly greater than hypoxia alone, whereas # denotes a significant diminution of the IL-1β-mediated increase in injury (IL-1β) as determined by one-way ANOVA followed by a Student–Newman–Keuls t test. Significance was assessed at p < 0.05 (n = 3–9 cultures pooled from 2–3 different experiments).

Article Snippet: In experiments using recombinant mouse IL-1ra (rIL-1ra) (R & D Systems) or monoclonal anti-mouse IL-1RI antibody directed against the extracellular domain of mouse IL-1RI (MAB7711; R & D Systems), the respective solutions were prepared with IL-1β (1.5 ng/ml; 1.5×) and added as described above.

Techniques: In Vitro

Journal: The Journal of Neuroscience

Article Title: System x c − Activity and Astrocytes Are Necessary for Interleukin-1β-Mediated Hypoxic Neuronal Injury

doi: 10.1523/JNEUROSCI.2459-07.2007

Figure Lengend Snippet: Astrocyte IL-1RI signaling is required for the mediation of IL-1β-induced hypoxic neuronal injury. Chimeric mixed cortical cell cultures were obtained by plating wild-type (IL-1RI+/+) or IL-1RI-deficient (IL-1RI−/−) neurons on either wild-type (IL-1RI+/+) or IL-1RI-null mutant (IL-1RI−/−) astrocytes These cultures were treated with 1 ng/ml IL-1β for 20–24 h (black bars) or vehicle (hatched bars), washed, and then deprived of oxygen. A, The percentage of total neuronal cell death was determined 20–24 h later as described in Materials and Methods (n = 18–24 cultures pooled from 4 different experiments). B, After 60 min, cells were washed with HBSS, and 14C-l-cystine was added for 5 min as described in Materials and Methods. Data are expressed as 14C-l-cystine uptake in picomoles per minute per milligram protein (n = 6–12 cultures pooled from 2–4 different experiments). C, After 120 min of hypoxia, supernatant was collected to measure accumulation of glutamate in the bathing medium via HPLC. Data are expressed as the mean ± SEM glutamate accumulation in micromolar (n = 6 cultures pooled from 3 different experiments). An asterisk indicates a value significantly greater than oxygen deprivation alone (−IL-1β), as determined by two-way ANOVA followed by a Bonferroni's t test for multiple comparisons. Significance was assessed at p < 0.05.

Article Snippet: In experiments using recombinant mouse IL-1ra (rIL-1ra) (R & D Systems) or monoclonal anti-mouse IL-1RI antibody directed against the extracellular domain of mouse IL-1RI (MAB7711; R & D Systems), the respective solutions were prepared with IL-1β (1.5 ng/ml; 1.5×) and added as described above.

Techniques: Mutagenesis

Journal: Nature communications

Article Title: IL1R1 + cancer-associated fibroblasts drive tumor development and immunosuppression in colorectal cancer.

doi: 10.1038/s41467-023-39953-w

Figure Lengend Snippet: Fig. 2 | IL1R1 and IL1B expression in CAF subtypes and their association with survival in CRC patients. a UMAP plot showing CAF subtypes identified in the CLZ, Lee and Qian scRNA-seq datasets. b IL1R1 expression in the three CRC CAF subtypes (iCAF, IL1R1+ iCAF and myCAF). The bar length shows the percentage of IL1R1- expressing cells and the color gradient shows the mean of normalized counts. c IL- 1β scores in the CAF subpopulations from the CLZ, Lee, and Qian datasets. Hor- izontal lines show the median. Holm’s adjusted pairwise two-sided Wilcoxon signed rank test (***p < 0.001). d Heatmap showing the expression of IL1R1+ iCAF-related genes in the three CAF subtypes of the three datasets. The color gradient shows the relative expression (mean of scaled expression values) and the bubble size shows the percentage of expressing cells. Genes appearing in bold define the IL1R1+ iCAF signature. e Correlation between IL1R1 expression with iCAF and IL1R1+ iCAF scores in CMS4 patients (TCGA dataset, n = 143). f Kaplan-Meier curves showing the prognostic value of IL1R1, iCAF and IL1R1+ iCAF scores on the overall survival of CRC patients (GSE39582 dataset). The upper row shows all patients (n = 562) and the lower row shows the subset of CMS4 patients (n = 126). The third quartile expres- sion value was used as a threshold to split patients into low and high expressors (75% low vs 25% high expressing patients). Non-adjusted p-values and hazard ratio

Article Snippet: CT5.3 fibroblasts were dissociated into a single cell suspension and stained for 30minat at 4 °Cwith an anti-IL1R1 antibody (FAB269P, R&D Systems) and DAPI.

Techniques: Expressing

Journal: Nature communications

Article Title: IL1R1 + cancer-associated fibroblasts drive tumor development and immunosuppression in colorectal cancer.

doi: 10.1038/s41467-023-39953-w

Figure Lengend Snippet: Fig. 5 | IL1R1+ iCAFs regulate immune cells in the tumor microenvironment. a Potential LR interactions between IL1R1+ iCAFs and epithelial cells, macrophages and T cells detected by LIANA in the Lee scRNA-seq dataset. b Experimental setup of a CD8+ T cell proliferation assay. Gp33-specific T cells were isolated from spleens and lymph nodes of P14 TCRVα2Vβ8 mice and co-cultured with L cells (mouse fibroblast cell line). c Proliferation of CD8+ T cells after 72 h of co-culture with fibroblasts pretreated with IL-1β (1 ng/ml) alone or in combination with anti-IL-1β (100 ng/ml) as analyzed by flow cytometry. Data from two independent experi- ments (shown as filled and empty circles) are represented. Statistically significant differences were determined using a nested ANOVA followed by Tukey’s post-hoc test. d Chord diagram showing LR interactions (LIANA aggregate score <0.05) between ligands borne by immune cells (macrophages, CD4+ T cells, CD8+ T cells and Tregs) and receptors borne by IL1R1+ iCAFs in the Lee scRNA-seq dataset. Arrow thickness and opacity shows higher ranked LIANA scores. Arrows outlined in red highlight the IL-1β-IL1R1 pair. e CD163 (left) and CD206 (right) expression on PBMC-

Article Snippet: CT5.3 fibroblasts were dissociated into a single cell suspension and stained for 30minat at 4 °Cwith an anti-IL1R1 antibody (FAB269P, R&D Systems) and DAPI.

Techniques: Proliferation Assay, Isolation, Cell Culture, Co-Culture Assay, Cytometry, Expressing

Journal: Nature communications

Article Title: IL1R1 + cancer-associated fibroblasts drive tumor development and immunosuppression in colorectal cancer.

doi: 10.1038/s41467-023-39953-w

Figure Lengend Snippet: Fig. 7 | A summary of the roles played by the IL1R1+ CAFs in the CRC TME. The IL1R1+ subtype of CAFs is linked to lower survival and to higher tumor growth and immune evasion (M2 polarization and T cell exhaustion markers). IL1R1+

Article Snippet: CT5.3 fibroblasts were dissociated into a single cell suspension and stained for 30minat at 4 °Cwith an anti-IL1R1 antibody (FAB269P, R&D Systems) and DAPI.

Techniques:

Journal: PLoS ONE

Article Title: Crucial Role of IL1beta and C3a in the In Vitro -Response of Multipotent Mesenchymal Stromal Cells to Inflammatory Mediators of Polytrauma

doi: 10.1371/journal.pone.0116772

Figure Lengend Snippet: A) MSC on glass slides were stained with an anti-IL1RI antibody (left) or the non-specific IgG isotype control (right). Gene expression levels of B) PTGES, C) TSG6 and F) MMP1 were measured with quantitative real-time PCR and normalized to those of HPRT1 following total RNA isolation 24 h post stimulation. D) PGE 2 and E) TSG6 levels in supernatants were detected with the Biotrend PGE2 Enzyme Immunoassay Kit and TSG6 ELISA. MSC were seeded at 5.2*10 3 cells/cm 2 and stimulated with the PTC or IL1beta after the anti-IL1RI antibody or the non-specific (ns) IgG isotype control had been added. Scatter plots and mean from up to 4 donors are presented.

Article Snippet: In experiments, in which the binding of IL1beta to the IL1 receptor I (IL1RI) was inhibited to suppress IL1beta signaling, 2 μg/ml of the polyclonal goat anti-human IL1RI antibody (AF269, R&D Systems, Wiesbaden, German) was added to the medium prior to addition of IL1beta or the PTC.

Techniques: Staining, Control, Gene Expression, Real-time Polymerase Chain Reaction, Isolation, Enzyme-linked Immunosorbent Assay