Journal: Journal of Leukocyte Biology

Article Title: IL-36γ is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components

doi: 10.1189/jlb.4A0315-087R

Figure Lengend Snippet: WT mice were given intranasal purified bacterial flagellin (1 mg). Whole lungs were harvested 2 h later and homogenized and subjected to a targeted RNA microarray analysis. (A) A variety of innate immune response genes were significantly induced in flagellin-treated lungs. Data are means ± sem of 3 mice/group. P < 0.05 for all genes shown, vs. unstimulated controls. (B) Of the IL-36R agonists, only IL-36γ was significantly induced in flagellin-treated lungs (4.2-fold change). No IL-36α or -β mRNA was detected. Data are means ± sem of 3 mice/group. *P < 0.05 vs. controls.

Article Snippet: Generation of rabbit anti-mouse polyclonal IL-36γ-specific Ab An anti-IL-36γ Ab was generated in New Zealand white rabbits with recombinant mouse IL-36γ (carrier free; R&D Systems, Minneapolis, MN, USA) [ 39 ].

Techniques: Purification, Microarray

Journal: Journal of Leukocyte Biology

Article Title: IL-36γ is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components

doi: 10.1189/jlb.4A0315-087R

Figure Lengend Snippet: WT PM were treated with LPS (100 ng), TNFα (100 μg), IL-1β (100 μg), flagellin (1 μg), Poly I:C (1 μg), or PAM3Cys (1 μg). Cell lysates were collected at 4 and 18 h and assessed for IL-36γ mRNA by RT-PCR. Data are means ± sem of 3 wells/group (106 cells/well). *P < 0.05 vs. control for each time point.

Article Snippet: Generation of rabbit anti-mouse polyclonal IL-36γ-specific Ab An anti-IL-36γ Ab was generated in New Zealand white rabbits with recombinant mouse IL-36γ (carrier free; R&D Systems, Minneapolis, MN, USA) [ 39 ].

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Leukocyte Biology

Article Title: IL-36γ is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components

doi: 10.1189/jlb.4A0315-087R

Figure Lengend Snippet: (A) WT PMs were treated with live or HK Sp (MOI 10:1). Cell lysates were collected at 6 and 18 h and assessed for IL-36γ mRNA by RT-PCR. Data are means ± sem of 3 wells/group (106 cells/well). *P < 0.05 vs. control for each time point. (B) WT and MyD88−/− PMs were treated with LPS (100 ng) or with live or HK Kp (MOI 10:1). Cell lysates were collected at 18 h and assessed for IL-36γ mRNA by RT-PCR. *P < 0.05 vs. control. (C) Human AMs were treated with live or HK Kp (MOI 10:1). Cell lysates were collected at 4 and 18 h and assessed for human IL-36γ mRNA by RT-PCR. Data are means ± sem of 4 wells/group (106 cells/well). *P < 0.05 vs. control for each time point.

Article Snippet: Generation of rabbit anti-mouse polyclonal IL-36γ-specific Ab An anti-IL-36γ Ab was generated in New Zealand white rabbits with recombinant mouse IL-36γ (carrier free; R&D Systems, Minneapolis, MN, USA) [ 39 ].

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Leukocyte Biology

Article Title: IL-36γ is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components

doi: 10.1189/jlb.4A0315-087R

Figure Lengend Snippet: (A) WT PMs were treated with LPS (100 ng) or HK Kp (MOI 10:1), with or without the addition of ATP (5 mM), during the last 20 min of incubation. Conditioned medium was collected at 18 h, and IL-36γ protein was assessed by ELISA. Data are means ± sem of 4 wells/group (106 cells/well). *P < 0.05 vs. control. (B) WT PMs were treated with HK Kp (MOI 10:1 for 4 h) and ATP (5 mM for the last 20 min), with or without monensin (25 μM) for the last hour of incubation. Conditioned medium was collected and analyzed for TNFα and IL-36γ by ELISA. Data are means ± sem of 3 samples per group. *P < 0.001 vs. control. (C) WT PMs were treated with or without HK Kp (MOI 10:1). Conditioned medium was collected at 18 h and sonicated (10 s output twice) or not. IL-36γ protein was assessed by ELISA. (D) WT PMs were treated with HK Sp (MOI 10:1), with or without addition of ATP (5 mM) during the last 20 min of incubation. Conditioned medium was collected at 18 h and was sonicated (10s × 2 output) or was not sonicated. IL-36γ protein was assessed by ELISA. (C, D) Data are means ± sem of 3 wells/group (106 cells/well). *P < 0.05 vs. control.

Article Snippet: Generation of rabbit anti-mouse polyclonal IL-36γ-specific Ab An anti-IL-36γ Ab was generated in New Zealand white rabbits with recombinant mouse IL-36γ (carrier free; R&D Systems, Minneapolis, MN, USA) [ 39 ].

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Sonication

Journal: Journal of Leukocyte Biology

Article Title: IL-36γ is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components

doi: 10.1189/jlb.4A0315-087R

Figure Lengend Snippet: (A) WT PMs were treated with HK Sp (MOI 10:1)+ATP (5 mM for 20 min). Conditioned medium was harvested at 6 h and centrifuged at 1500 g for 30 min to remove apoptotic bodies and cell debris. Supernatants were then ultracentrifuged at 25,000 g for 30 min to isolate MPs, assessed by flow cytometry for size relative to submicron calibration beads, and gated to select DAPI-negative particles, corresponding to viable MPs. The MPs were stained for annexin V-PE or a PE-labeled isotype control IgG. Within 95% confidence intervals, as compared to isotype controls, 80% of particles were annexin V positive. (B) WT PMs were treated with HK Sp (MOI 10:1), with or without ATP (5 mM for 20 min). Conditioned medium was collected at 6 h and centrifuged at 1,500 g for 30 min, then ultracentrifuged at 25,000 g for 30 min (MP), followed by 100,000 g for 90 min (exosome). The remaining supernatant was concentrated in a centrifugal concentrator with a 3 kDa cutoff. Pellets from MP and exosome fractions and concentrated conditioned medium were analyzed by Western immunoblot for IL-36γ and IL-1β, with β-actin as the control gene. All lanes were run on a single gel/blot.

Article Snippet: Generation of rabbit anti-mouse polyclonal IL-36γ-specific Ab An anti-IL-36γ Ab was generated in New Zealand white rabbits with recombinant mouse IL-36γ (carrier free; R&D Systems, Minneapolis, MN, USA) [ 39 ].

Techniques: Flow Cytometry, Staining, Labeling, Western Blot

Journal: Cell reports

Article Title: Activation of miR-21-Regulated Pathways in Immune Aging Selects against Signatures Characteristic of Memory T Cells

doi: 10.1016/j.celrep.2018.10.074

Figure Lengend Snippet: (A) Naive CD4 + T cells were transfected with either scrambled control or LNA21. Representative western blots and mean normalized band intensities of PTEN, SPRY1, and b-actin expression after 48 hr are shown (n = 3–4, mean ± SEM). (B and C) Naive CD4 + T cells from young and older individuals were activated with anti-CD3 and anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control RNA or anti-miR-21. (B) Representative histogram of phosphorylated S6 in GFP + cells on day 2 and results from 7 individuals. (C) Representative histograms of phosphorylated S6, AKT, mTOR, and ERK in GFP + cells on day 3 and results of paired samples from 7 individuals. The filled gray histograms represent unstimulated naive CD4 + T cells. (D and E) Naive CD4 + T cells from healthy adults were activated with anti-CD3 and anti-CD28 beads. On day 3, activated cells were treated with combinations of the AKT inhibitor MK-2206 2HCl and the MEK1 and MEK2 inhibitor U0126 for 1.5 hr. (D) Representative histograms show phosphorylated S6, mTOR, and ERK in cells treated with combinations of 40 nM of the AKT inhibitor and 400 nM of MEK1 and MEK2 inhibitor. Numbers in histograms indicate geometric MFI of p-mTOR and p-ERK. (E) Graphs depicting the frequencies of cells with phosphorylated S6 cultured with the indicated concentrations of the AKT inhibitor in the presence or absence of 400 nM of the MEK1 and MEK2 inhibitor (n = 4, mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; all by two-tailed paired t test.

Article Snippet: p-mTOR (S2448; O21-404) , BD Biosciences , Cat# 564242; RRID: AB_2738695.

Techniques: Transfection, Western Blot, Expressing, Transduction, Plasmid Preparation, Cell Culture, Two Tailed Test

Journal: Cell reports

Article Title: Activation of miR-21-Regulated Pathways in Immune Aging Selects against Signatures Characteristic of Memory T Cells

doi: 10.1016/j.celrep.2018.10.074

Figure Lengend Snippet:

Article Snippet: p-mTOR (S2448; O21-404) , BD Biosciences , Cat# 564242; RRID: AB_2738695.

Techniques: Plasmid Preparation, Recombinant, Isolation, SYBR Green Assay, Reporter Assay, Microarray, Luciferase, Software