anti human ereg neutralizing antibody  (R&D Systems)

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Sankey diagram of enriched receptor-ligand pairs in SSc skin and at least two lung scRNA-Seq datasets. Ribbon width is proportional to 1/rank of the skin SSc data. (B) Plot of the CellphoneDB ranks (adjusted p-values) of the interaction of EREG with EGFR in our skin scRNA-Seq data as well as our analysis of available data from SSc skin (15) and lung (33, 41, 42). Dotted line shows rank = 0.05. (C) Ereg relative expression during a time course of tissue digestion of healthy mouse skin, n=3 per time point. (D) Expression of EREG in our UMAP embedded scRNA-Seq data. (E) Heatmap of co-expressed genes by SSc EREG-expressing APC (EREG+) compared to healthy EREG+ APC and EREG− APC groups. For clarity, the raw gene list was filtered to genes primarily expressed by immune cells. (F) Expression of FCN1 in our UMAP embedded scRNA-Seq data. (G) Immunofluorescence images of EREG and FCN1 in SSc skin. (H, I) Analysis of EREG expression in SSc compared to healthy controls (H) and compared to modified Rodnan Skin Score (mRSS) (I) using data from (49). (J) Low and high magnification photomicrographs of skin and lung from SSc and healthy subject samples stained with EREG antibody. Dashed boxes delineate region shown in higher magnification image. Arrowheads label positive cells. (K) Enumeration of EREG+ cells in SSc skin dermis and lung (n=3 slides each, skin samples from patients SSc1, 3, and 4, 10 high power fields (hpf) per slide). Slides were imaged with a Keyence BZ-X800 microscope. Low power images are at 10x magnification and stitched together. High power images are 40x magnification. Data are means ± SD (***P<0.001, ****P<0.0001) analyzed with one-way analysis of variance (ANOVA) with Tukey multiple-comparisons test (C) and unpaired two-tailed Student t test (K).

Article Snippet: For EGFR inhibition, sub-confluent HFF were incubated in media alone or with anti-human EREG neutralizing antibody (R&D Biosciences AF1195) 5 μg/mL for 24 hours prior to RNA isolation and qPCR analysis.

Techniques: Expressing, Immunofluorescence, Modification, Staining, Microscopy, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Expression fold change of EREG when THP-1 monocytes were incubated with each indicated cytokine. (B) EREG protein quantification from supernatant of THP-1 incubated with IFNa2 for 4 hours, n=4 per group, data is representative from 2 independent experiments. (C-E) EREG expression fold change from freshly isolated peripheral blood CD14+ monocytes (C) and CD1c+ dendritic cell precursors (D) or cultured human BMDC (E) after incubation with IFNα2. (F) Expression fold change of NOTCH ligands, receptors, and target genes by HFFs incubated with recombinant human EREG (n=5). (G) HES1 expression fold change in SSc fibroblasts after incubation with EREG for 4 hours, n=5 per group. (H) EREG relative expression by BMDC primed with IFNα2 prior to exposure to NOTCH ligand DLL4 (n=3–4 per time point in each group). Statistics compare each group ± DLL4. (I) Relative expression of EGFR ligands by HFF (n=3). Genes with fewer than three points were below detectable level. (J) Changes in ECM gene expression when HFF were incubated with media alone (NT) or EREG neutralizing antibody (Ereg Ab). FNEDA refers to the extra domain A-containing isoform of fibronectin (n=5 per group). (K) Model of EREG-NOTCH circuit between monocyte-derived DC3 and fibroblasts. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001) analyzed with unpaired two-tailed Student t test (A-H, J) and one-way ANOVA with Tukey multiple-comparisons test (I).

Article Snippet: For EGFR inhibition, sub-confluent HFF were incubated in media alone or with anti-human EREG neutralizing antibody (R&D Biosciences AF1195) 5 μg/mL for 24 hours prior to RNA isolation and qPCR analysis.

Techniques: Expressing, Incubation, Isolation, Cell Culture, Recombinant, Derivative Assay, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Experimental diagram depicting adjacent punch biopsies obtained from the forearm of a patient with diffuse cutaneous SSc, which were cultured for 9 days in media alone (NT) or with addition of EREG neutralizing antibody (Ereg Ab). (B) Histology of cultured skin explants, with inset showing higher magnification of dermal collagen. (C, D) Skin explant media was analyzed for pro-COL1A1 N-terminal peptide (PINP) and TNC. (E) Percent reduction of protein by Ereg Ab treatment compared to NT control. (F) LDH activity of skin explant supernatants from patient SSc7. (G-N) Fresh explanted lung tissue from a deceased patient donor with familial idiopathic pulmonary fibrosis was processed for histologic staining, which showed fibroblastic foci formation and hyperplasia of alveolar type II epithelial cells, indicated by arrows (left panel H&E, right panel trichrome). The same tissue was cut into cubes and cultured for 10 days in the presence of the multikinase inhibitor nintedanib (Nin), the Alk5 inhibitor A-1544033 (IN-1130) (Alk5i), EREG antibody (Ereg Ab) or non-treated vehicle control (NT). (H-K) Relative expression of indicated genes, n=4 per group. (L-N) Protein secretion of indicated genes measured by ELISA, n=8 per group. ELISA samples with poor signal and qPCR outliers identified by Grubbs’s test with alpha = 0.05 were excluded. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ****P < 0.0001) analyzed with paired two-tailed Student t test (C-E) comparing NT and Ereg Ab treated samples. In (H-N), comparison of each inhibitor to NT control was analyzed by one-way ANOVA with Dunnett’s multiple-comparisons test whereas Ereg Ab was individually compared to Nin and Alk5i by unpaired two-tailed Student t test.

Article Snippet: For EGFR inhibition, sub-confluent HFF were incubated in media alone or with anti-human EREG neutralizing antibody (R&D Biosciences AF1195) 5 μg/mL for 24 hours prior to RNA isolation and qPCR analysis.

Techniques: Cell Culture, Activity Assay, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A-C) B6 mice were injected subcutaneously with 0.2 mg bleomycin (BLM) and 3 weeks later skin was stained for hematoxylin and eosin (A) and trichrome (B). Epidermis (epi), dermis (dermis) and dermal white adipose tissue (DWAT) are highlighted on histology. (C, D) Immunofluorescence images of PBS and BLM-treated skin 3 weeks post-injection. (E) Hydroxyproline content of the skin at different time points after subcutaneous bleomycin injection (n=3 per group). (F) Heatmap of mean log2(expression fold change) of ECM genes and EGFR ligands at different time points after subcutaneous bleomycin injection compared to the mean of each group and PBS controls, n=3 per time point. (G) Bulk RNA sequencing of dendritic cells isolated from fibrotic skin of Mgl2DTReGFPpANeo mice 3 weeks after subcutaneous bleomycin injection compared to PBS controls (n=3 per group). (H) Relative expression of Ereg at different time points after intratracheal bleomycin administration to B6 mice. (I) B6 mice were injected with bleomycin subcutaneously, then at 2 weeks injected intraperitoneally with Ifnar1-blocking antibody (Ifnar1 Ab), isotype control antibody (iso) or not treated (NT). No significant differences were found between NT and isotype Ab control groups, so they were combined for clarity. At 3 weeks, skin was analyzed for histology (J), dermal skin thickness (K), hydroxyproline (L), and gene expression (M, N). Data from E and F, G, and H are single independent experiments. Data in J-N are aggregated from two separate experiments. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001) analyzed with one-way ANOVA with Tukey multiple-comparisons test (E, F, H, K) and unpaired two-tailed Student t test (L-N).

Article Snippet: For EGFR inhibition, sub-confluent HFF were incubated in media alone or with anti-human EREG neutralizing antibody (R&D Biosciences AF1195) 5 μg/mL for 24 hours prior to RNA isolation and qPCR analysis.

Techniques: Injection, Staining, Immunofluorescence, Expressing, RNA Sequencing Assay, Isolation, Blocking Assay, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Sankey diagram of enriched receptor-ligand pairs in SSc skin and at least two lung scRNA-Seq datasets. Ribbon width is proportional to 1/rank of the skin SSc data. (B) Plot of the CellphoneDB ranks (adjusted p-values) of the interaction of EREG with EGFR in our skin scRNA-Seq data as well as our analysis of available data from SSc skin (15) and lung (33, 41, 42). Dotted line shows rank = 0.05. (C) Ereg relative expression during a time course of tissue digestion of healthy mouse skin, n=3 per time point. (D) Expression of EREG in our UMAP embedded scRNA-Seq data. (E) Heatmap of co-expressed genes by SSc EREG-expressing APC (EREG+) compared to healthy EREG+ APC and EREG− APC groups. For clarity, the raw gene list was filtered to genes primarily expressed by immune cells. (F) Expression of FCN1 in our UMAP embedded scRNA-Seq data. (G) Immunofluorescence images of EREG and FCN1 in SSc skin. (H, I) Analysis of EREG expression in SSc compared to healthy controls (H) and compared to modified Rodnan Skin Score (mRSS) (I) using data from (49). (J) Low and high magnification photomicrographs of skin and lung from SSc and healthy subject samples stained with EREG antibody. Dashed boxes delineate region shown in higher magnification image. Arrowheads label positive cells. (K) Enumeration of EREG+ cells in SSc skin dermis and lung (n=3 slides each, skin samples from patients SSc1, 3, and 4, 10 high power fields (hpf) per slide). Slides were imaged with a Keyence BZ-X800 microscope. Low power images are at 10x magnification and stitched together. High power images are 40x magnification. Data are means ± SD (***P<0.001, ****P<0.0001) analyzed with one-way analysis of variance (ANOVA) with Tukey multiple-comparisons test (C) and unpaired two-tailed Student t test (K).

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Expressing, Immunofluorescence, Modification, Staining, Microscopy, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Expression fold change of EREG when THP-1 monocytes were incubated with each indicated cytokine. (B) EREG protein quantification from supernatant of THP-1 incubated with IFNa2 for 4 hours, n=4 per group, data is representative from 2 independent experiments. (C-E) EREG expression fold change from freshly isolated peripheral blood CD14+ monocytes (C) and CD1c+ dendritic cell precursors (D) or cultured human BMDC (E) after incubation with IFNα2. (F) Expression fold change of NOTCH ligands, receptors, and target genes by HFFs incubated with recombinant human EREG (n=5). (G) HES1 expression fold change in SSc fibroblasts after incubation with EREG for 4 hours, n=5 per group. (H) EREG relative expression by BMDC primed with IFNα2 prior to exposure to NOTCH ligand DLL4 (n=3–4 per time point in each group). Statistics compare each group ± DLL4. (I) Relative expression of EGFR ligands by HFF (n=3). Genes with fewer than three points were below detectable level. (J) Changes in ECM gene expression when HFF were incubated with media alone (NT) or EREG neutralizing antibody (Ereg Ab). FNEDA refers to the extra domain A-containing isoform of fibronectin (n=5 per group). (K) Model of EREG-NOTCH circuit between monocyte-derived DC3 and fibroblasts. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001) analyzed with unpaired two-tailed Student t test (A-H, J) and one-way ANOVA with Tukey multiple-comparisons test (I).

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Expressing, Incubation, Isolation, Cell Culture, Recombinant, Gene Expression, Derivative Assay, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Experimental diagram depicting adjacent punch biopsies obtained from the forearm of a patient with diffuse cutaneous SSc, which were cultured for 9 days in media alone (NT) or with addition of EREG neutralizing antibody (Ereg Ab). (B) Histology of cultured skin explants, with inset showing higher magnification of dermal collagen. (C, D) Skin explant media was analyzed for pro-COL1A1 N-terminal peptide (PINP) and TNC. (E) Percent reduction of protein by Ereg Ab treatment compared to NT control. (F) LDH activity of skin explant supernatants from patient SSc7. (G-N) Fresh explanted lung tissue from a deceased patient donor with familial idiopathic pulmonary fibrosis was processed for histologic staining, which showed fibroblastic foci formation and hyperplasia of alveolar type II epithelial cells, indicated by arrows (left panel H&E, right panel trichrome). The same tissue was cut into cubes and cultured for 10 days in the presence of the multikinase inhibitor nintedanib (Nin), the Alk5 inhibitor A-1544033 (IN-1130) (Alk5i), EREG antibody (Ereg Ab) or non-treated vehicle control (NT). (H-K) Relative expression of indicated genes, n=4 per group. (L-N) Protein secretion of indicated genes measured by ELISA, n=8 per group. ELISA samples with poor signal and qPCR outliers identified by Grubbs’s test with alpha = 0.05 were excluded. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ****P < 0.0001) analyzed with paired two-tailed Student t test (C-E) comparing NT and Ereg Ab treated samples. In (H-N), comparison of each inhibitor to NT control was analyzed by one-way ANOVA with Dunnett’s multiple-comparisons test whereas Ereg Ab was individually compared to Nin and Alk5i by unpaired two-tailed Student t test.

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Cell Culture, Control, Activity Assay, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A-C) B6 mice were injected subcutaneously with 0.2 mg bleomycin (BLM) and 3 weeks later skin was stained for hematoxylin and eosin (A) and trichrome (B). Epidermis (epi), dermis (dermis) and dermal white adipose tissue (DWAT) are highlighted on histology. (C, D) Immunofluorescence images of PBS and BLM-treated skin 3 weeks post-injection. (E) Hydroxyproline content of the skin at different time points after subcutaneous bleomycin injection (n=3 per group). (F) Heatmap of mean log2(expression fold change) of ECM genes and EGFR ligands at different time points after subcutaneous bleomycin injection compared to the mean of each group and PBS controls, n=3 per time point. (G) Bulk RNA sequencing of dendritic cells isolated from fibrotic skin of Mgl2DTReGFPpANeo mice 3 weeks after subcutaneous bleomycin injection compared to PBS controls (n=3 per group). (H) Relative expression of Ereg at different time points after intratracheal bleomycin administration to B6 mice. (I) B6 mice were injected with bleomycin subcutaneously, then at 2 weeks injected intraperitoneally with Ifnar1-blocking antibody (Ifnar1 Ab), isotype control antibody (iso) or not treated (NT). No significant differences were found between NT and isotype Ab control groups, so they were combined for clarity. At 3 weeks, skin was analyzed for histology (J), dermal skin thickness (K), hydroxyproline (L), and gene expression (M, N). Data from E and F, G, and H are single independent experiments. Data in J-N are aggregated from two separate experiments. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001) analyzed with one-way ANOVA with Tukey multiple-comparisons test (E, F, H, K) and unpaired two-tailed Student t test (L-N).

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Injection, Staining, Immunofluorescence, Expressing, RNA Sequencing, Isolation, Blocking Assay, Control, Gene Expression, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A-C) Diagramed in (A), cohorts of B6 and Ereg−/− mice were injected with bleomycin subcutaneously and 35 days later analyzed for skin thickness (B) and histology (C). (D-I) Diagramed in (D), 21 days after bleomycin injection mice began treatment with Ereg antibody (Ereg Ab) compared to controls treated with PBS (NT) for two weeks. Skin was analyzed for dermal thickness (E), hydroxyproline (F), gene expression (G, H) and histology (I), with H&E staining on the top row, trichrome in the middle row, and pEGFR immunohistochemistry (IHC) on the bottom row, n=8 (PBS), 11 (BLM), and 12 (Ereg Ab). (J) B6 and Ereg−/− mice were injected with bleomycin or PBS and 3 weeks later B6 mice were treated for 1 week with Ereg Ab or isotype control Ab, n=3 (PBS), 5 (isotype Ab), 5 (Ereg Ab), and 5 NT Ereg−/−. (K-L) As diagramed in (K), 10 days after intratracheal bleomycin mice were treated with Ereg Ab for two weeks. Lungs were analyzed for histology (L), modified Ashcroft score (M), hydroxyproline (N) and Ereg gene expression (O), n=6 (PBS), 11 (BLM), and 7 (Ereg Ab). Histology images of skin and lung are 10x and 20x magnification, respectively. IHC images are 40x magnification. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P<0.001, ****P<0.0001) analyzed with unpaired two-tailed Student t test (F-H, N, O) and one-way ANOVA with Tukey multiple-comparisons test (B, E, J, M). Data for J is single experiment and data for A-C, D-I, and K-O are aggregated from two independent experiments.

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Injection, Gene Expression, Staining, Immunohistochemistry, Control, Modification, Two Tailed Test

Journal: Bone

Article Title: The reduced osteogenic potential of Nf1 -deficient osteoprogenitors is EGFR-independent

doi: 10.1016/j.bone.2017.10.012

Figure Lengend Snippet: A: Ereg expression in WT and Nf1-deficient mBMSCs (qPCR, n=3). B: Epiregulin protein expression in WT and Nf1-deficient mBMSCs (Western blot, n=3, Right graph: densitometric analysis). C: Egfr expression in WT and Nf1-deficient mBMSCs (qPCR, n=3). D: EGFR protein expression in WT and Nf1 deficient mBMSCs (Western blot, n=3, Right graph: densitometric analysis). E: Level of phosphorylated EGFR (p-EGFR), EGFR and β-actin in A431 cells treated with the conditioned medium (CM) from WT (grey bar) and Nf1-deficient (KO, black bar) mBMSCs in the presence of IgG control or an epiregulin neutralizing antibody (Western blot, n=3, Right graph: densitometric analysis). * and #: p<0.05 between genotypes and treatments, respectively. qPCR gene expression is normalized by Hprt expression.

Article Snippet: Cells were then treated with the conditioned media plus normal goat IgG control (AB-108-C, R&D Systems) or Epiregulin neutralizing antibody (AF1068-SP, R&D Systems) at the final concentration of 0.4 μg/ml.

Techniques: Expressing, Western Blot, Control, Gene Expression

Journal: Bone

Article Title: The reduced osteogenic potential of Nf1 -deficient osteoprogenitors is EGFR-independent

doi: 10.1016/j.bone.2017.10.012

Figure Lengend Snippet: A–B, D–E and G, H: Expression of early osteoblast marker genes (Alpl, Ibsp) in response to EGFR or Epiregulin inhibition during osteogenic differentiation (A–B: AG-1478, D–E: Poziotinib and G, H: epiregulin-neutralizing antibody) in WT and Nf1-deficient (KO) mBMSCs (qPCR, n=3, * and #: p<0.05 between genotypes and treatments, respectively). C, F and I: ALP activity in response to AG-1478, Poziotinib and Anti-Ereg neutralizing antibodies, respectively (n=3, * and #: p<0.05 between genotypes and treatments, respectively). qPCR gene expression is normalized by Hprt expression.

Article Snippet: Cells were then treated with the conditioned media plus normal goat IgG control (AB-108-C, R&D Systems) or Epiregulin neutralizing antibody (AF1068-SP, R&D Systems) at the final concentration of 0.4 μg/ml.

Techniques: Expressing, Marker, Inhibition, Activity Assay, Gene Expression