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Thermo Fisher anti-human il-1β neutralizing antibody

L-MPs induced macrophages to <t>upregulate</t> <t>IL-1β</t> 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

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1) Product Images from "Macrophages reprogrammed by lung cancer microparticles promote tumor development via release of IL-1β"

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

Journal: Cellular and Molecular Immunology

doi: 10.1038/s41423-019-0313-2

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

Figure Legend 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

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

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

Figure Legend 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

Techniques Used: 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

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

Figure Legend 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

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

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

Figure Legend 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

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

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

Figure Legend 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

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

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

Figure Legend 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

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

Injection:

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

Purification:

Transplantation Assay:

Expressing:

Derivative Assay:

Real-time Polymerase Chain Reaction:

Cell Culture:

Western Blot:

Reverse Transcription Polymerase Chain Reaction:

Positive Control:

Activation Assay:

Staining:

Fluorescence:

Flow Cytometry:

Enzyme-linked Immunosorbent Assay:

Incubation:

Labeling:

Microscopy:

Transfection:

Phospho-proteomics:

Immunofluorescence:

Control:

Confocal Microscopy:

Generated:

Isolation:

Immunohistochemical staining:

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A, Cytokine array showing altered abundance of chemokines/cytokines in serum-free CM collected from CAF-2 cells stably expressing scramble or IRAK4 shRNAs. B, Quantitative PCR showing fold changes in mRNA levels of the indicated genes in CAF-2 expressing scramble shRNA treated with DMSO, IRAK1/4 inhibitor or AS2444697 overnight, or stably expressing two different shIRAK4s. Data (means ± SEM) represents one of two experiments done in biological duplicates and technical triplicates. C, Consecutive IHC sections showing expression of IL-1β in α-SMA+ CAFs in two representative human PDAC samples from TMA (US Biomax PA 2081. Of all 117 PDAC samples, 96 showed positive, and 21 showed negative IL-1β IHC staining in α-SMA+ CAFs. D, Confocal IF images showing presence of IL-1β in α-SMA+ CAFs in two human surgical PDAC and murine KPC tumors. E, ELISA assay showing abundance of IL-1β protein in CM collected from the indicated cells treated with DMSO or AS2444697 overnight (left panel), or from CAF-2 cells stably expressing scramble shRNA or two difference shIRAK4s (right panel). Experiments were done twice in triplicates. Data presented as means ± SEM (*p<0.05, **p<0.01 by two-tailed t test). F, Western blots showing dose-dependent suppression of p-IRAK4 and p-p65 levels in CAF-2 treated overnight with nIL-1β antibody or Anakinra.

Journal: Cancer research

Article Title: Tumor-Stroma IL-1β-IRAK4 Feedforward Circuitry Drives Tumor Fibrosis, Chemoresistance, And Poor Prognosis In Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-17-1366

Figure Lengend Snippet: A, Cytokine array showing altered abundance of chemokines/cytokines in serum-free CM collected from CAF-2 cells stably expressing scramble or IRAK4 shRNAs. B, Quantitative PCR showing fold changes in mRNA levels of the indicated genes in CAF-2 expressing scramble shRNA treated with DMSO, IRAK1/4 inhibitor or AS2444697 overnight, or stably expressing two different shIRAK4s. Data (means ± SEM) represents one of two experiments done in biological duplicates and technical triplicates. C, Consecutive IHC sections showing expression of IL-1β in α-SMA+ CAFs in two representative human PDAC samples from TMA (US Biomax PA 2081. Of all 117 PDAC samples, 96 showed positive, and 21 showed negative IL-1β IHC staining in α-SMA+ CAFs. D, Confocal IF images showing presence of IL-1β in α-SMA+ CAFs in two human surgical PDAC and murine KPC tumors. E, ELISA assay showing abundance of IL-1β protein in CM collected from the indicated cells treated with DMSO or AS2444697 overnight (left panel), or from CAF-2 cells stably expressing scramble shRNA or two difference shIRAK4s (right panel). Experiments were done twice in triplicates. Data presented as means ± SEM (*p<0.05, **p<0.01 by two-tailed t test). F, Western blots showing dose-dependent suppression of p-IRAK4 and p-p65 levels in CAF-2 treated overnight with nIL-1β antibody or Anakinra.

Article Snippet: Details of inhibitors were: IRAK1/4 inhibitor (Sigma, cat# I5409), AS2444697 (Tocris, cat#5430), IMD-0354 (Tocris, cat#2611), anti-mouse IL-1β neutralizing antibody (Invivogen, clone 7E3), anti-human IL-1β neutralizing antibody (Invivogen, clone 4H5).

Techniques: Stable Transfection, Expressing, Real-time Polymerase Chain Reaction, shRNA, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot

A, Western blots showing changes in p-IRAK4 and p-p65 levels of SC00A5 cells incubated overnight with serum-free DMEM alone or at 1:1 ratio with serum-free CM collected from the indicated PDAC lines. B, IL-1β ELISA of CM collected from the indicated PDAC lines used in Fig. 5A. Experiment was done twice in triplicates and data represented as means ± SEM. C. Quantitative PCR of IL-1β mRNA in the indicated fibroblast lines incubated with or without serum-free MIA Paca-2 CM overnight. D, Venn diagrams of genes expressed at ≥1.5-fold difference FDR <0.1 in DMSO vs. IRAK1/4 inhibitor treatment in Capan-1 and PANC-1 cells. Expression values used were gene-wise z-score normalized. E, Western blots showing changes in p-IRAK4 and p-p65 levels of SC00A5 cells incubated with serum-free DMEM, MIA Paca-2 or Capan-1 CM in the absence or presence of neutralizing anti-human IL-1β antibody. F, Gel contraction assay showing a role of IL-1β in MIA Paca-2 CM in inducing collagen contraction of SC00A5 cells. Experiment was done three time in triplicates and presented as means ± SEM. G, Western blots confirming IL-1R protein knockdown in SC00A5 cells stably expressing two different shIL-1R (left panel). These three SC00A5 lines were then treated with serum-free DMEM or MIA Paca-2 CM overnight, followed by western blots showing changes in p-IRAK4 and p-p65 levels. (*p<0.05, **p<0.01, ***p<0.001).

Journal: Cancer research

Article Title: Tumor-Stroma IL-1β-IRAK4 Feedforward Circuitry Drives Tumor Fibrosis, Chemoresistance, And Poor Prognosis In Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-17-1366

Figure Lengend Snippet: A, Western blots showing changes in p-IRAK4 and p-p65 levels of SC00A5 cells incubated overnight with serum-free DMEM alone or at 1:1 ratio with serum-free CM collected from the indicated PDAC lines. B, IL-1β ELISA of CM collected from the indicated PDAC lines used in Fig. 5A. Experiment was done twice in triplicates and data represented as means ± SEM. C. Quantitative PCR of IL-1β mRNA in the indicated fibroblast lines incubated with or without serum-free MIA Paca-2 CM overnight. D, Venn diagrams of genes expressed at ≥1.5-fold difference FDR <0.1 in DMSO vs. IRAK1/4 inhibitor treatment in Capan-1 and PANC-1 cells. Expression values used were gene-wise z-score normalized. E, Western blots showing changes in p-IRAK4 and p-p65 levels of SC00A5 cells incubated with serum-free DMEM, MIA Paca-2 or Capan-1 CM in the absence or presence of neutralizing anti-human IL-1β antibody. F, Gel contraction assay showing a role of IL-1β in MIA Paca-2 CM in inducing collagen contraction of SC00A5 cells. Experiment was done three time in triplicates and presented as means ± SEM. G, Western blots confirming IL-1R protein knockdown in SC00A5 cells stably expressing two different shIL-1R (left panel). These three SC00A5 lines were then treated with serum-free DMEM or MIA Paca-2 CM overnight, followed by western blots showing changes in p-IRAK4 and p-p65 levels. (*p<0.05, **p<0.01, ***p<0.001).

Techniques: Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Collagen Gel Contraction Assay, Stable Transfection

A, Alamar blue assay showing viability of the indicated PDAC lines cultured in serum-free medium alone or at 1:1 ratio with different CAF CM for 72 hours. B, NF-κB luciferase reporter assay of MIA Paca-2 cells incubated without or with different CAF CM overnight. C, Alamar blue assay showing gemcitabine dose-response inhibition of MIA Paca-2 and CFPAC-1 cells cultured with CM collected from SC00A5, or CAF-2 cells expressing scramble shRNA or two different shIRAK4s for 72 hours. Changes in IC50 of gemcitabine in each condition were shown. D, Caspase 3/7 luciferase reporter activity in MIA Paca-2 cells cultured in the indicated CAF CM, treated with vehicle (PBS) or gemcitabine for 24 hours. E, Alamar blue assay showing viability of two PDAC lines cultured in serum free medium alone or CAF-2 CM for 72 hours in the absence or presence of neutralizing anti-human IL-1β antibody. F, NF-κB luciferase reporter assay of MIA Paca-2 cells treated with anti-IL-1β neutralizing antibody without or with CAF-2 CM overnight. All experiments were done three times in triplicates and data presented as means ± SEM. (*p<0.05, **p<0.01, ***p<0.001).

Journal: Cancer research

Article Title: Tumor-Stroma IL-1β-IRAK4 Feedforward Circuitry Drives Tumor Fibrosis, Chemoresistance, And Poor Prognosis In Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-17-1366

Figure Lengend Snippet: A, Alamar blue assay showing viability of the indicated PDAC lines cultured in serum-free medium alone or at 1:1 ratio with different CAF CM for 72 hours. B, NF-κB luciferase reporter assay of MIA Paca-2 cells incubated without or with different CAF CM overnight. C, Alamar blue assay showing gemcitabine dose-response inhibition of MIA Paca-2 and CFPAC-1 cells cultured with CM collected from SC00A5, or CAF-2 cells expressing scramble shRNA or two different shIRAK4s for 72 hours. Changes in IC50 of gemcitabine in each condition were shown. D, Caspase 3/7 luciferase reporter activity in MIA Paca-2 cells cultured in the indicated CAF CM, treated with vehicle (PBS) or gemcitabine for 24 hours. E, Alamar blue assay showing viability of two PDAC lines cultured in serum free medium alone or CAF-2 CM for 72 hours in the absence or presence of neutralizing anti-human IL-1β antibody. F, NF-κB luciferase reporter assay of MIA Paca-2 cells treated with anti-IL-1β neutralizing antibody without or with CAF-2 CM overnight. All experiments were done three times in triplicates and data presented as means ± SEM. (*p<0.05, **p<0.01, ***p<0.001).

Techniques: Alamar Blue Assay, Cell Culture, Luciferase, Reporter Assay, Incubation, Inhibition, Expressing, shRNA, Activity Assay

A, Final weights and representative pictures of Capan-1 tumors injected subcutaneously alone, or in 1:2 ratio with CAF-2 expressing scramble shRNA, shIRAK4 or shIL-1R. (N=8/group). B, C, Quantification of (B) proliferating (dual pan-cytokeratin+ and Ki-67+ cells) or (C) α-SMA+ cells in the indicated Capan-1 tumors. D, Final weights and representative pictures of KPC2 tumors grown in mice treated with vehicle (V), anti-mouse IL-1β neutralizing antibody (nIL-1β), gemcitabine (GEM), AS2444697, or in dual combinations. (N=10–14/group combined from two independent experiments). E, F, G, Quantification of proliferating (dual pan-cytokeratin+ and Ki-67+ cells), apoptotic cells (cleaved caspase-3+), or degree of fibrosis (Sirius Red+ area) of KPC2 tumors treated as indicated (N=10/group). H, (Left) Representative IHC images showing assigned intensity scores (0=absence/faint, 1=weak, 2=moderate, 3=strong) of stromal IL-1β staining in a PDAC tissue microarray (N=125). (Right) Kaplan-Meier survival analysis and median survival of 125 patients based on low (0–1) or high (2–3) stromal IL-1β IHC score. I, Schematics depicting the proposed model in which PDAC cells and CAFs conspire to establish a feedforward IL-1β–IRAK4 circuitry that leads to tumor fibrosis, chemoresistance and poor prognosis.

Journal: Cancer research

Article Title: Tumor-Stroma IL-1β-IRAK4 Feedforward Circuitry Drives Tumor Fibrosis, Chemoresistance, And Poor Prognosis In Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-17-1366

Figure Lengend Snippet: A, Final weights and representative pictures of Capan-1 tumors injected subcutaneously alone, or in 1:2 ratio with CAF-2 expressing scramble shRNA, shIRAK4 or shIL-1R. (N=8/group). B, C, Quantification of (B) proliferating (dual pan-cytokeratin+ and Ki-67+ cells) or (C) α-SMA+ cells in the indicated Capan-1 tumors. D, Final weights and representative pictures of KPC2 tumors grown in mice treated with vehicle (V), anti-mouse IL-1β neutralizing antibody (nIL-1β), gemcitabine (GEM), AS2444697, or in dual combinations. (N=10–14/group combined from two independent experiments). E, F, G, Quantification of proliferating (dual pan-cytokeratin+ and Ki-67+ cells), apoptotic cells (cleaved caspase-3+), or degree of fibrosis (Sirius Red+ area) of KPC2 tumors treated as indicated (N=10/group). H, (Left) Representative IHC images showing assigned intensity scores (0=absence/faint, 1=weak, 2=moderate, 3=strong) of stromal IL-1β staining in a PDAC tissue microarray (N=125). (Right) Kaplan-Meier survival analysis and median survival of 125 patients based on low (0–1) or high (2–3) stromal IL-1β IHC score. I, Schematics depicting the proposed model in which PDAC cells and CAFs conspire to establish a feedforward IL-1β–IRAK4 circuitry that leads to tumor fibrosis, chemoresistance and poor prognosis.

Techniques: Injection, Expressing, shRNA, Staining, Microarray

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

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

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

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

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

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

Effect of interleukin-1β (IL-1β) on neurokinin-1 receptor (NK-1R) expression in U87 MG cells (A) and primary rat astrocytes (B). Cells were incubated with IL-1β at the indicated concentrations for 3 h, and total RNA was isolated and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis. PCR was performed for NK-1R, as well as for β-actin for U87 MG cells or GAPDH (primary rat astrocytes), as a control to ensure that RNA amounts were equal. The data shown are representative of three experiments.

Journal: Glia

Article Title: Interleukin-1? Upregulates Functional Expression of Neurokinin-1 Receptor (NK-1R) via NF-?B in Astrocytes

doi: 10.1002/glia.20079

Figure Lengend Snippet: Effect of interleukin-1β (IL-1β) on neurokinin-1 receptor (NK-1R) expression in U87 MG cells (A) and primary rat astrocytes (B). Cells were incubated with IL-1β at the indicated concentrations for 3 h, and total RNA was isolated and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis. PCR was performed for NK-1R, as well as for β-actin for U87 MG cells or GAPDH (primary rat astrocytes), as a control to ensure that RNA amounts were equal. The data shown are representative of three experiments.

Article Snippet: Recombinant human and rat IL-1β were purchased from R&D Systems (Minneapolis, MN); rabbit anti-human IL-1β neutralizing antibody (IL-1βAb) from Sigma (St. Louis, MO); caffeic acid phenethyl ester (CAPE) from Calbiochem-Novabiochem (San Diego, CA); and SP from Sigma (St. Louis, MO).

Techniques: Expressing, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction

Time course of interleukin-1β (IL-1β) effect on neurokinin-1 receptor (NK-1R) mRNA expression in U87 MG cells. U87 MG cells were incubated with (+) or without (−) IL-1β (4 ng/ml) for the time points post-treatment as indicated. Total RNA was isolated and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis. PCR was performed for NK-1R as well as for β-actin as a control to ensure that RNA amounts were equal. The data shown are representative of three experiments.

Journal: Glia

Article Title: Interleukin-1? Upregulates Functional Expression of Neurokinin-1 Receptor (NK-1R) via NF-?B in Astrocytes

doi: 10.1002/glia.20079

Figure Lengend Snippet: Time course of interleukin-1β (IL-1β) effect on neurokinin-1 receptor (NK-1R) mRNA expression in U87 MG cells. U87 MG cells were incubated with (+) or without (−) IL-1β (4 ng/ml) for the time points post-treatment as indicated. Total RNA was isolated and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis. PCR was performed for NK-1R as well as for β-actin as a control to ensure that RNA amounts were equal. The data shown are representative of three experiments.

Techniques: Expressing, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction

Effect of interleukin-1β (IL-1β) Ab on IL-1β-induced neurokinin-1 receptor (NK-1R) mRNA expression in U87 MG cells. U87 MG cells were incubated with (+) or without (−) IL-1β (4 ng/ml) and/or rabbit anti-human IL-1βAb (50 μg/ml) or normal rabbit IgG (50 μg/ml) for 3 h. Total RNA was isolated and subjected to reverse transcription-polymerase chain reaction (RT-PCR) and electrophoresis for NK-1R mRNA. PCR was performed for NK-1R as well as for β-actin as a control to ensure that RNA amounts were equal. The data shown are representative of two experiments.

Journal: Glia

Article Title: Interleukin-1? Upregulates Functional Expression of Neurokinin-1 Receptor (NK-1R) via NF-?B in Astrocytes

doi: 10.1002/glia.20079

Figure Lengend Snippet: Effect of interleukin-1β (IL-1β) Ab on IL-1β-induced neurokinin-1 receptor (NK-1R) mRNA expression in U87 MG cells. U87 MG cells were incubated with (+) or without (−) IL-1β (4 ng/ml) and/or rabbit anti-human IL-1βAb (50 μg/ml) or normal rabbit IgG (50 μg/ml) for 3 h. Total RNA was isolated and subjected to reverse transcription-polymerase chain reaction (RT-PCR) and electrophoresis for NK-1R mRNA. PCR was performed for NK-1R as well as for β-actin as a control to ensure that RNA amounts were equal. The data shown are representative of two experiments.

Techniques: Expressing, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Electrophoresis

Induction of neurokinin-1 receptor (NK-1R) protein expression by interleukin-1β (IL-1β). A: Effect of IL-1β on NK-1R protein expression in U87 MG cells. U87 MG cells were incubated with or without (control) IL-1β at 4 ng/ml for 3 h. Cell lysates were quantified with a DC protein assay kit. Equal amounts (5 μg) of protein extracted from treated and untreated U87 MG cells were applied onto a nitro-cellulose membrane for immunoblot assay. The results were recorded on the film (1-min exposure). B: U87 MG cells were incubated with or without IL-1β (4 ng/ml) for 3 h. The cells were then removed from the culture plate and resuspended in 100 μl of phosphate-buffered saline (PBS). After incubation with 20 μl of FITC-conjugated substance P (SP) (1:1,000) for 45 min at 4°C, the cells were washed twice with PBS and fixed with 1% paraformaldehyde in PBS. Fluorescence was analyzed on an EPICS-elite flow cytometry. The data shown are representative of three experiments.

Journal: Glia

Article Title: Interleukin-1? Upregulates Functional Expression of Neurokinin-1 Receptor (NK-1R) via NF-?B in Astrocytes

doi: 10.1002/glia.20079

Figure Lengend Snippet: Induction of neurokinin-1 receptor (NK-1R) protein expression by interleukin-1β (IL-1β). A: Effect of IL-1β on NK-1R protein expression in U87 MG cells. U87 MG cells were incubated with or without (control) IL-1β at 4 ng/ml for 3 h. Cell lysates were quantified with a DC protein assay kit. Equal amounts (5 μg) of protein extracted from treated and untreated U87 MG cells were applied onto a nitro-cellulose membrane for immunoblot assay. The results were recorded on the film (1-min exposure). B: U87 MG cells were incubated with or without IL-1β (4 ng/ml) for 3 h. The cells were then removed from the culture plate and resuspended in 100 μl of phosphate-buffered saline (PBS). After incubation with 20 μl of FITC-conjugated substance P (SP) (1:1,000) for 45 min at 4°C, the cells were washed twice with PBS and fixed with 1% paraformaldehyde in PBS. Fluorescence was analyzed on an EPICS-elite flow cytometry. The data shown are representative of three experiments.

Techniques: Expressing, Incubation, DC Protein Assay, Western Blot, Fluorescence, Flow Cytometry

Effect of nuclear factor-κB (NF-κB) inhibitor caffeic acid phenethyl ester (CAPE) on interleukin-1β (IL-1β)-induced activation of the NF-κB promoter (A) and neurokinin-1 receptor (NK-1R) mRNA expression in U87 MG cells (B) and primary rat astrocytes (C). A: U87-MG cells were transfected with the plasmid containing the NF-κB promoter (pNF-κB-luc) and incubated with or without IL-1β and/or CAPE for 12 h. Data presented as means ± SD of triplicate cultures, a representative of three independent experiments. B, C: U87 MG cells and primary rat astrocytes were pre-incubated with or without CAPE (25 μg/ml) for 2 h, the cells were then treated with IL-1β (4 ng/ml) for 3 h. Total cellular RNA extracted was subjected to reverse transcription-polymerase chain reaction (RT-PCR) for NK-1R mRNA expression. PCR was performed for NK-1R as well as for β-actin U87 MG cells or GAPDH for primary rat astrocytes as a control to ensure that RNA amounts were equal. The data shown are representative of three experiments.

Journal: Glia

Article Title: Interleukin-1? Upregulates Functional Expression of Neurokinin-1 Receptor (NK-1R) via NF-?B in Astrocytes

doi: 10.1002/glia.20079

Figure Lengend Snippet: Effect of nuclear factor-κB (NF-κB) inhibitor caffeic acid phenethyl ester (CAPE) on interleukin-1β (IL-1β)-induced activation of the NF-κB promoter (A) and neurokinin-1 receptor (NK-1R) mRNA expression in U87 MG cells (B) and primary rat astrocytes (C). A: U87-MG cells were transfected with the plasmid containing the NF-κB promoter (pNF-κB-luc) and incubated with or without IL-1β and/or CAPE for 12 h. Data presented as means ± SD of triplicate cultures, a representative of three independent experiments. B, C: U87 MG cells and primary rat astrocytes were pre-incubated with or without CAPE (25 μg/ml) for 2 h, the cells were then treated with IL-1β (4 ng/ml) for 3 h. Total cellular RNA extracted was subjected to reverse transcription-polymerase chain reaction (RT-PCR) for NK-1R mRNA expression. PCR was performed for NK-1R as well as for β-actin U87 MG cells or GAPDH for primary rat astrocytes as a control to ensure that RNA amounts were equal. The data shown are representative of three experiments.

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Incubation, Reverse Transcription Polymerase Chain Reaction

Measurement of cytosolic [Ca2+]i in cultured U87 MG cells and primary rat astrocytes in response to substance P (SP). Cells incubated with or without (control) interleukin-1β (IL-1β) (4 ng/ml) were loaded with 2.5 μM fura-2 AM for 30 min and exposed to SP (10−7 M) and/or (CP-96,345, 10−6 M) and change in [Ca2+]i was recorded from 20–40 cells. The data shown are representative of five experiments.

Journal: Glia

Article Title: Interleukin-1? Upregulates Functional Expression of Neurokinin-1 Receptor (NK-1R) via NF-?B in Astrocytes

doi: 10.1002/glia.20079

Figure Lengend Snippet: Measurement of cytosolic [Ca2+]i in cultured U87 MG cells and primary rat astrocytes in response to substance P (SP). Cells incubated with or without (control) interleukin-1β (IL-1β) (4 ng/ml) were loaded with 2.5 μM fura-2 AM for 30 min and exposed to SP (10−7 M) and/or (CP-96,345, 10−6 M) and change in [Ca2+]i was recorded from 20–40 cells. The data shown are representative of five experiments.

Techniques: Cell Culture, Incubation

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anti-human il-1β neutralizing antibody (Thermo Fisher)

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