human il 1β neutralizing antibody  (InvivoGen)

Journal: Cellular and Molecular Immunology

Article Title: Macrophages reprogrammed by lung cancer microparticles promote tumor development via release of IL-1β

doi: 10.1038/s41423-019-0313-2

Figure Lengend Snippet: L-MPs induced macrophages to upregulate IL-1β expression. a Human PBMC-derived macrophages were treated with HCC827-MPs at a ratio of 1:20 (macrophages: MPs). After 12 h, the cells were collected, and RNA was extracted for real-time PCR analysis of IL-10 , arginase 1 ( Arg1 ), VEGF and IL-1β . b , c Human PBMC-derived macrophages were treated with HCC827-MPs at different ratios (cell:MPs, 1:1, 1:5, 1:10, 1:20). RNA, protein and cultured medium were collected after 12, 24, or 72 h of treatment, respectively. Then, the IL-1β expression was analyzed by real-time PCR, western blots ( b ) or ELISAs ( c ). d IL-1β mRNA or pro-IL-1β expression of HCC827-MPs, H460-MPs, A549-MPs and Lewis-MPs was analyzed by RT-PCR (left) or western blot (right) analyses. Human PBMC-derived macrophages treated with HCC827-MPs were used as positive controls for A549, HCC827, and H460-MPs. Mouse BMDMs treated with Lewis-MPs were used as a positive control for Lewis-MPs. e Human PBMC-derived macrophages were treated with H460-MPs or A549-MPs for 12 h (left). Mouse BMDMs were treated with Lewis-MPs for 12 h (right). Then, the IL-1β mRNA level was analyzed by real-time PCR. f Human PBMC-derived macrophages were treated with healthy human blood cell-derived MPs at a ratio of 1:20 (cell:MPs). IL-1β mRNA levels were analyzed by real-time PCR (left). BMDMs were treated with wild-type mouse (C57BL/6) blood cell-derived MPs at a ratio of 1:20, and then, the IL-1β mRNA level was analyzed by real-time PCR (right). Error bars indicate the mean ± SEM; n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For antibody treatment, the mice were i.p. injected with 3 mg/kg of purified anti-human IL-1β neutralizing antibody (Thermo Fisher Scientific) 8 weeks after HSC transplantation twice weekly.

Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Positive Control

Journal: Cellular and Molecular Immunology

Article Title: Macrophages reprogrammed by lung cancer microparticles promote tumor development via release of IL-1β

doi: 10.1038/s41423-019-0313-2

Figure Lengend Snippet: TLR3 activation is required for IL-1β induction by L-MPs. a Mouse BMDMs were treated with PKH26-stained Lewis-MPs with or without cytochalasin D (1 μg/ml) for 30 min. Then, the PKH26 fluorescence intensity of macrophages was measured by flow cytometry at different times (left), and the IL-1β expression level was analyzed by real-time PCR (middle) and western blot analyses (right). b Mouse BMDMs were pretreated with or without 2-DG (5 mM) for 30 min. Then, the BMDMs were treated with Lewis-MPs. IL-1β mRNA levels were analyzed by real-time PCR, and mature IL-1β in the supernatant was detected by ELISA after 12 and 72 h. c Mouse BMDMs were incubated with PKH26-labeled Lewis-MPs. After 12 h, the BMDMs were stained with live cell molecular probes, including mitochondria, ER, Golgi, endosome or lysosome trackers. Then, the MP location was observed under a two-photon confocal microscope. Scale bar, 20 µm. d Mouse BMDMs were treated with DNA (1 μg) or RNA (2.5 μg) extracted from Lewis cells or Lewis-MPs for 12 h, and then, the IL-1β mRNA level was analyzed by real-time PCR (left). e Mouse BMDMs were treated with Lewis-MP-derived RNA (10, 5, 2.5, or 1 μg) only or Lewis-MP-derived RNA pretreated with NaOH (0.5 M) for 12 h, and then, the IL-1β mRNA level was analyzed by real-time PCR (right). f Mouse BMDMs or human PBMC-derived macrophages were transfected with TLR3 siRNAs and then treated with Lewis-MPs or HCC827-MPs. After 12 h, the IL-1β mRNA level was analyzed by real-time PCR. g Mouse BMDMs (left) or human PBMC-derived macrophages (right) were treated with Lewis-MPs or HCC827-MPs, respectively. Then, the cells were collected, and the phosphorylation of NF-κB, IKK-α/β, IκBα, p38, JNK, and ERK was detected by western blot analyses at different times. h BMDMs were treated with Lewis-MPs in the presence or absence of NF-κB or MAPK inhibitors, including BAY 11-7085 (BAY), SB203580 (SB), U0126, or SP600125 (SP). After 12 h, the IL-1β mRNA level was analyzed by real-time PCR. Error bars indicate the mean ± SEM; n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For antibody treatment, the mice were i.p. injected with 3 mg/kg of purified anti-human IL-1β neutralizing antibody (Thermo Fisher Scientific) 8 weeks after HSC transplantation twice weekly.

Techniques: Activation Assay, Staining, Fluorescence, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Labeling, Microscopy, Derivative Assay, Transfection, Phospho-proteomics

Journal: Cellular and Molecular Immunology

Article Title: Macrophages reprogrammed by lung cancer microparticles promote tumor development via release of IL-1β

doi: 10.1038/s41423-019-0313-2

Figure Lengend Snippet: L-MP-induced mitochondrial ROS activate NLRP3 for IL-1β cleavage. a Human PBMC-derived macrophages (upper) or mouse BMDMs (bottom) were treated with HCC827-MPs or Lewis-MPs, respectively, at different doses. Then, the cells were collected, and the expression of active caspase-1 (p10) was detected by western blots. b Mouse BMDMs were transfected with or without caspase-1 siRNAs and then treated with Lewis-MPs. RNA and cultured medium were collected after 12 or 72 h of treatment, respectively. Then, IL-1β expression was analyzed by real-time PCR (left) and ELISAs (right). c BMDMs were transfected with or without NLRP3, NLRP1b or AIM2 siRNAs and then treated with Lewis-MPs for 24 h. Cells were collected, and the active caspase-1 levels were detected by western blots. d Mouse BMDMs were treated with Lewis-MPs at different times (upper) or doses (bottom). Then, the BMDMs were stained with CellROX and observed under a two-photon confocal microscope. Scale bar, 20 µm. e , f Mouse BMDMs were treated with Lewis-MPs in the presence or absence of NAC or DPI. The flt1 ROS fluorescence intensity of macrophages was measured via flow cytometry after 24 h. The active caspase-1 levels were detected by western blots ( f , left) after 24 h, and the IL-1β expression was analyzed by ELISAs ( f , right) after 72 h. g Mouse BMDMs were treated with Lewis-MPs at different times (left) and doses (right). Then, the BMDMs were stained with MitoSOX and measured by flow cytometry. Error bars indicate the mean ± SEM; n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For antibody treatment, the mice were i.p. injected with 3 mg/kg of purified anti-human IL-1β neutralizing antibody (Thermo Fisher Scientific) 8 weeks after HSC transplantation twice weekly.

Techniques: Derivative Assay, Expressing, Western Blot, Transfection, Cell Culture, Real-time Polymerase Chain Reaction, Staining, Microscopy, Fluorescence, Flow Cytometry

Journal: Cellular and Molecular Immunology

Article Title: Macrophages reprogrammed by lung cancer microparticles promote tumor development via release of IL-1β

doi: 10.1038/s41423-019-0313-2

Figure Lengend Snippet: Lysosomal calcium release by L-MPs causes mitochondrial ROS production. a Mouse BMDMs treated with or without Lewis-MPs were loaded with the fluorescent Ca 2+ indicator Fluo-4/AM. The Fluo-4 fluorescence intensity of the macrophages was measured via flow cytometry. Cells were stimulated with ionomycin (100 μM, 30 s). b, c Mouse BMDMs were treated with Lewis-MPs with or without BAPTA for 24 h, and then, the Ca 2+ ( b , left), ROS ( b , middle), mitochondrial ROS ( b , right) and active caspase-1 ( c ) levels of the macrophages were measured with flow cytometry or western blots. d Mouse BMDMs were treated with Lewis-MPs with or without ryanodine (Rya). After 24 h, the Ca 2+ ( upper ) and mitochondrial ROS ( bottom ) levels of macrophages were measured via flow cytometry. e , f Mouse BMDMs were treated with or without Lewis-MPs. After 2 h, the cells were collected, and RNA was extracted for mRNA analysis of mucolipin 1 , mucolipin 2 , TPC1 and TPC2 by real-time PCR ( e , left). The BMDMs were transfected with mucolipin 2 siRNAs, and the silencing efficiency of the siRNAs was detected by real-time PCR ( e , middle). The BMDMs transfected with mucolipin 2 siRNAs were treated with Lewis-MPs. After 24 h, the Ca 2+ ( e , right) and ROS ( f , left) levels of the macrophages were measured via flow cytometry. After 72 h, the culture medium was collected, and IL-1β expression was analyzed by ELISAs ( f , right). g Mouse BMDMs were incubated with PKH26-labeled Lewis-MPs at different doses. After 12 h, the BMDMs were stained with LysoSensor. Then, the cells were observed under a two-photon confocal microscope. Scale bar, 20 µm (left). The flt1 LysoSensor fluorescence intensity of the macrophages was measured via flow cytometry (right). h Mouse BMDMs were treated with Lewis-MPs at different times. Then, the expression of V0a2 and V0a3 was detected via western blots. i Mouse BMDMs were treated with Lewis-MPs for 24 h. Immunofluorescence of V0a2 (green, upper), V0a3 (green, bottom) and LAMP1 (red) in the control and Lewis-MPs groups was assessed with two-photon confocal microscopy. Scale bar, 20 µm. j Mouse BMDMs were transfected with V0a2 or V0a3 siRNAs and then treated with Lewis-MPs. After 12 h, the cells were stained with LysoSensor, and the flt1 LysoSensor fluorescence intensity of the macrophages was measured by flow cytometry. k Mouse BMDMs were treated with Lewis-MPs with or without concanamycin B (ConcaB). After 24 h, the cells were stained with Fluo-4 (left) or MitoSox (middle). Then, the Fluo-4 and MitoSox fluorescence intensity of the macrophages was measured by flow cytometry. The active caspase-1 levels were detected by western blots (right). Error bars indicate the mean ± SEM; n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For antibody treatment, the mice were i.p. injected with 3 mg/kg of purified anti-human IL-1β neutralizing antibody (Thermo Fisher Scientific) 8 weeks after HSC transplantation twice weekly.

Techniques: Fluorescence, Flow Cytometry, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Expressing, Incubation, Labeling, Staining, Microscopy, Immunofluorescence, Control, Confocal Microscopy

Journal: Cellular and Molecular Immunology

Article Title: Macrophages reprogrammed by lung cancer microparticles promote tumor development via release of IL-1β

doi: 10.1038/s41423-019-0313-2

Figure Lengend Snippet: L-MP-induced IL-1β generated by macrophages promotes lung tumor development. a–g First, 5 × 10 4 Lewis tumor cells were injected into the right thigh muscle of the mice. After 10 days, 5 × 10 6 Lewis-MPs were injected into the tumor once every 2 days for a total of three times. Two groups of mice ( n = 12) were treated with purified IL-1β antibody (IL-1β-AB, 50 µg) or IgG (50 µg) on days 9 and 12, respectively. On day 15, half of the mice ( n = 6) in each group were sacrificed, and the tumor weight was measured (left). The remaining mice ( n = 6) were used for the long-term survival observation (right). # P < 0.001, MP group compared with Con group. * P < 0.05, MP group compared with MP/IL-1β-AB group. b Tumor sections were labeled with immunofluorescence to indicate the distribution of the macrophages (F4/80, green) and IL-1β (navy blue). The distribution of Lewis-MPs (red) was also recorded. Cell nuclei were stained with DAPI (blue). Scale bar, 30 µm. c Leukocytes in the above tumor tissues were isolated, and then, the ROS levels of tumor-infiltrating macrophages (CD11b + F4/80 + ) were measured by flow cytometry ( n = 6). d The IL-1β expression of tumor tissues was measured by using an ELISA kit. e The active caspase-1 levels of tumor tissues were measured via western blots. f The IL-1β expression of tumor-infiltrating macrophages (CD11b + F4/80 + ) was measured by flow cytometry. g Clodronate (FormuMax Scientific Inc., CA) was i.p. injected into the mice on day 9 (200 µl) and day 12 (100 µl) after tumor inoculation. On day 15, the mice were sacrificed, and the tumor weight was measured (left). The IL-1β expression of the tumor tissues was measured by using an ELISA kit (right). h c-kit and nanog mRNA expression of the tumor tissues from mice in a was measured by real-time PCR. i The isolated tumor cells from mice in a were seeded in soft 3D fibrin gels. The tumor colony (n = 150) size was analyzed. Scale bar, 20 µm. j The isolated tumor cells from mice in g were seeded in soft 3D fibrin gels. The tumor colony ( n = 150) size was analyzed. Scale bar, 20 µm. Error bars indicate the mean ± SEM; n = 3 independent experiments unless otherwise indicated. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For antibody treatment, the mice were i.p. injected with 3 mg/kg of purified anti-human IL-1β neutralizing antibody (Thermo Fisher Scientific) 8 weeks after HSC transplantation twice weekly.

Techniques: Generated, Injection, Purification, Labeling, Immunofluorescence, Staining, Isolation, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Immunology

Article Title: Macrophages reprogrammed by lung cancer microparticles promote tumor development via release of IL-1β

doi: 10.1038/s41423-019-0313-2

Figure Lengend Snippet: hL-MPs facilitate tumor growth in a humanized mouse model. a Human HCC827 lung cancer cells were treated with different concentrations of the mutated EGFR inhibitor icotinib. After 24 h, the cell viability was analyzed with PI and annexin V staining, and the number of HCC827-MPs was calculated by flow cytometry. b Human PBMC-derived macrophages were treated with HCC827-MPs. After 24 h, the Ca 2+ (left) and mitochondrial ROS (middle) levels of the macrophages were measured by flow cytometry. After 72 h, the culture medium was collected, and IL-1β expression was analyzed by ELISAs (right). c Lung cancer patient ( n = 34) and healthy human ( n = 38) peripheral blood samples were collected. Then, the IL-1β serum concentrations were analyzed by ELISAs. d Immunohistochemical staining of IL-1β in patients’ lung cancer tissues and paracancerous normal lung tissues was analyzed. Scale bar, 100 µm. e The association between IL-1β and the survival of patients with lung cancer was analyzed based on a Kaplan-Meier plot ( http://kmplot.com/analysis/ ). f A humanized mouse model ( n = 4) was established as described in the “Methods” section. Nine weeks after CD34 + HSC transplantation, PBMCs, bone marrow cells, and lymphocytes from the spleen, liver and lung were stained with a human CD45 antibody and analyzed by flow cytometry. g First, 5 × 10 6 HCC827 tumor cells were injected into the right femur muscle of 10-week-old humanized mice ( n = 4). Two groups of mice were treated with neutralizing IL-1β-AB (50 µg) or IgG (50 µg) at 8 weeks after HSC transplantation twice weekly. The day after the antibody injection, the mice were injected with 5 × 10 6 HCC827-MPs into the tumor once every 2 days. Twenty three days after the tumor inoculation, the mice were sacrificed, and the tumor weight was measured. Scale bar, 20 mm. h Tumor sections were labeled via immunofluorescence assays to indicate the distribution of the macrophages (CD68 + , green) and IL-1β (navy blue). Cell nuclei were stained with DAPI. Scale bar, 20 µm. i Human tumor cells isolated from tumor-bearing humanized mice were seeded in a 3D culture system, and the colony ( n = 150) size was analyzed. Scale bar, 20 µm. Error bars indicate the mean ± SEM; n = 3 independent experiments unless otherwise indicated. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For antibody treatment, the mice were i.p. injected with 3 mg/kg of purified anti-human IL-1β neutralizing antibody (Thermo Fisher Scientific) 8 weeks after HSC transplantation twice weekly.

Techniques: Staining, Flow Cytometry, Derivative Assay, Expressing, Immunohistochemical staining, Transplantation Assay, Injection, Labeling, Immunofluorescence, Isolation