Journal: Frontiers in Immunology

Article Title: Targeting immunosuppressive Ly6C+ classical monocytes reverses anti-PD-1/CTLA-4 immunotherapy resistance

doi: 10.3389/fimmu.2023.1161869

Figure Lengend Snippet: Anti-PD-1/CTLA-4/Ly6C treatment reduces tumor growth and increases CD8 T cells/dendritic cells. (A) 344SQ tumor bearing 129/Sv mice were weekly treated with anti-PD-1_CTLA-4 (200 µg of anti-PD-1 plus 200 µg of anti-CTLA-4 per mouse), anti-Ly6C alone (200 µg of anti-Ly6C per mouse), anti-PD-1_CTLA-4_Ly6C (200 µg of anti-PD-1 plus 200 µg of anti-CTLA-4 plus 200 µg of anti-Ly6C per mouse), or their IgG control mixture (IgG control) beginning on week 2 after a subcutaneous cancer cell injection (1 x 10 6 cells per mouse; n = 5) for 4 weeks. Mice received total of 4 treatments starting at week 2 post tumor cell implantation. (B) tSNE CD45 plots from 344SQ tumors treated with IgG control, anti-PD-1_CTLA-4, or anti-PD-1_CTLA-4_Ly6C from (A) . Tumors from (A) were harvested to prepare single cell suspensions for FACS analysis. (C) Percentages of CD8 T cells (left), Effector/memory CD8 T cells (middle), and dendritic cells (right). (D) Percentage of Ly6C- (left) and Ly6C+ (right) CD14+CD115+ monocytes. Kras LSL-G12D /p53 fl/fl mice generated through intratracheal administration of adenovirus expressing Cre recombinase were treated with either anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C for 8 weeks (E) Micro-CT images shown at week 0 (baseline) and week 8 (endpoint) for anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C. Dashed yellow circles indicate lung tumors. H indicates of heart. (F) Percentage change of tumor area was calculated taking into account prior time point and normalized to the baseline measurement. (G) H&E stained lung sections at week 8 from anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C treated mice from (E) . bar= 5 mm. (H) Number of lung tumors on H&E sections from (G) treated with anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C weekly for 8 weeks. (I) IHC of CD8 stained lung tumors from (F) week 8. Percentage of CD8 T cells found in the lung tumors from Kras LSL-G12D /p53 fl/fl mice treated for 8 weeks with anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C. (J) The dendritic cells from CD11c-DTR mice were transferred into C57BL/6 mice to generate the chimerical mice, after 8-week stabilization, the mice were treated with diphtheria toxin twice a week to maintain the depletion of dendritic cells. The B16 melanoma-bearing mice were treated weekly with anti-PD-1_CTLA-4_Ly6C for 3 weeks staring on week 1 after tumor cells inoculation. (K) 344SQ tumor bearing 129/Sv mice were treated weekly with blockade of PD-1, CTLA-4, and Ly6C (200 µg of anti-PD-1 plus 200 µg of anti-CTLA-4 plus 200 µg of anti-Ly6C per mouse) or their IgG control mixture (IgG control) beginning on day 7 after a subcutaneous cancer cell injection (0.1 x 10 6 cells per mouse; n = 5) for 4 weeks. For blocking B7 signal, antibodies (anti-B7: 300 μg of anti-CD80 and 300 μg of anti-CD86 per mouse) were intraperitoneally administered 1 day before the first dose of therapy, and then once a week to maintain the blockade. ANOVA test was used to analyze the data. n.s., not significance; *p < 0.05; **p < 0.01; ***p< 0.001; ****p < 0.0001.

Article Snippet: Anti-mouse PD-1 (clone RMP1-14), anti-mouse CTLA-4 (clone 9D9), anti-mouse CD80 (clone 16-10A1), anti-mouse CD86 (clone GL-1), anti-mouse Ly6C (clone Monts 1), anti-mouse CCL2 (clone 2H5), and the isotype-matched IgG controls were purchased from BioXCell.

Techniques: Control, Injection, Generated, Expressing, Micro-CT, Staining, Blocking Assay

Journal: Advanced Science

Article Title: Mucous Permeable Nanoparticle for Inducing Cuproptosis‐Like Death In Broad‐Spectrum Bacteria for Nebulized Treatment of Acute Pneumonia

doi: 10.1002/advs.202408580

Figure Lengend Snippet: Activation of macrophage response by Cu 2 O‐BSO NPs in vitro. a) Typical scatter plots of MH‐S surface markers CD86 (M1 macrophage marker) and CD206 (M2 macrophage marker) detected by flow cytometry. b) Typical images of MH‐S engulfing a bacterium. The green fluorescence represents GFP‐MRSA, the red fluorescence represents MH‐S, and the blue fluorescence represents the nucleus. The stronger the green fluorescence, the stronger the phagocytosis of MH‐S. c,d) Representative SPM photographs and CFU count of MRSA engulfed by MH‐S. e–g) ELISA results indicating levels of cytokines (IL‐1β, IL‐6, and TNF‐α) secreted by MH‐S in different groups.

Article Snippet: Each group of cells was centrifuged, washed, and resuspended in 500 μL of PBS, which contained an anti‐mouse CD86 antibody coupled with FITC (Elabscience) and an anti‐mouse CD206 antibody coupled with PE (Elabscience).

Techniques: Activation Assay, In Vitro, Marker, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Huanshaodan regulates microglial glucose metabolism reprogramming to alleviate neuroinflammation in AD mice through mTOR/HIF-1α signaling pathway

doi: 10.3389/fphar.2024.1434568

Figure Lengend Snippet: Expression levels of CD86, Arg1 and IL-1β protein in hippocampus and cortex of SAMP8 mice detected by Western-blot, the expression levels of IL-6 and TNF-α in hippocampus and cortex of SAMP8 mice by ELISA. (A,E) Blotting of CD86, Arg1 and IL-1β protein in hippocampus and cortex of mice in each group; (B–D) Expression of CD86, Arg1 and IL-1β protein in hippocampus of mice in each group; (F–H) Expression of CD86, Arg1 and IL-1β protein in cortex of mice in each group; (I–L) Expression of IL-6 and TNF-α protein in hippocampus and cortex of mice in each group. Model group compared with the control group * p < 0.05, ** p < 0.01, *** p < 0.001, Donepezil group compared with the model group # p < 0.05, ## p < 0.01, ### p < 0.001, HSD group compared with the model group $ p < 0.05, $$ p < 0.01, $$$ p < 0.001.

Article Snippet: The main materials that were used in this study were LPS (L8880, Solarbio), MHY1485 (MHY) (M9050-5MG, AbMole), RIPA buffer (high) (R0010, Solarbio), primary antibodies β-Actin and CD86 (42kDa, BM0627; 70kDa, BM4121, BOSTER), Arg1 and mTOR (40kDa, GB11285-100; 289kDa, GB111839-100, Servicebio), p-mTOR and HIF-1α (289kDa, 5536T; 120kDa, 48085S, Cell Signaling Technology), IL-1β (31kDa, 16806-1-AP, Proteintech), Horseradish peroxidase (HRP)-linked secondary antibodies against rabbit and mouse IgG of the primary antibodies (ZB-2301; ZB-2305, Beijing Zhong Shan-Golden Bridge Biological Technology), Glucose (GLU) Assay Kit, Lactic Acid (LD) assay kit, ATP assay kit, Lactate dehydrogenase (LDH) assay kit (A154-1-1; A019-2-1; A095-1-1; A020-2-2, Nanjing Jiancheng Bioengineering Institute), Pyruvate Dehydrogenase (PDH) Activity Assay Kit (E-BC-K650-M, Elabscience), BCA Protein Assay Kit (PC0020, Solarbio), ELISA Kit for Hexokinase 2 (HK2) activity based on enzyme standard of HK2, ELISA Kit for Tumor Necrosis Factor alpha (TNF-α) (MM-45418M1; MM-0132M1, MEIMIAN), ELISA Kit for Interleukin 6 (IL-6) (E-MSEL-M0001, Elabscience) and ELISA Kit for Pyruvate kinase isozymes M2 (PKM2) (SEA588Mu, Cloud Clone Corp), Cell Staining Buffer, PE Anti-Mouse F4/80 Antibody [CI:A3-1], PE/Cyanine7 Anti-Mouse CD86 Antibody [GL-1], Intracellular Fixation/Permeabilization Buffer Kit and APC Anti-Mouse CD206 Antibody (E-CK-A107; E-AB-F0995D; E-AB-F0994H; E-CK-A109; E-AB-F1135E, Elabscience).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Huanshaodan regulates microglial glucose metabolism reprogramming to alleviate neuroinflammation in AD mice through mTOR/HIF-1α signaling pathway

doi: 10.3389/fphar.2024.1434568

Figure Lengend Snippet: The expression levels of p-mTOR/mTOR ratio, HIF-1α, and IL-1β in BV2 cells tested by western blot, the levels of GLU, LD, ATP, LDH and PDH quantified by biochemical test kit and the levels of polarization biomarkers of CD86 and CD206 detected by flow cytometry, the expression levels of IL-6 and TNF-α in BV2 cells tested by ELISA. (A) Blotting of p-mTOR, mTOR and HIF-1α protein in BV2 cells in each group; (B, C) Expression levels of p-mTOR/mTOR ratio and HIF-1α in BV2 cells in each group. (D) GLU content analysis chart; (E) LD content analysis chart; (F) ATP content analysis chart; (G) . LDH activity analysis graph; (H) PDH activity analysis graph. (I) Flow cytometry analysis charts; (J) Flow cytograms of BV2 cells in each group; (K) Blotting of IL-1β protein in BV2 cells in each group; (L) Expression level of IL-1β in BV2 cells in each group. (M, N) Expression level of IL-6 and TNF-α in BV2 cells in each group. Model group compared with control group * p < 0.05, ** p < 0.01, *** p < 0.001, HSD-containing serum-treated group compared with the model group $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, Reverse validation group compared with the HSD-containing serum-treated group & p < 0.05, && p < 0.01, &&& p < 0.001.

Article Snippet: The main materials that were used in this study were LPS (L8880, Solarbio), MHY1485 (MHY) (M9050-5MG, AbMole), RIPA buffer (high) (R0010, Solarbio), primary antibodies β-Actin and CD86 (42kDa, BM0627; 70kDa, BM4121, BOSTER), Arg1 and mTOR (40kDa, GB11285-100; 289kDa, GB111839-100, Servicebio), p-mTOR and HIF-1α (289kDa, 5536T; 120kDa, 48085S, Cell Signaling Technology), IL-1β (31kDa, 16806-1-AP, Proteintech), Horseradish peroxidase (HRP)-linked secondary antibodies against rabbit and mouse IgG of the primary antibodies (ZB-2301; ZB-2305, Beijing Zhong Shan-Golden Bridge Biological Technology), Glucose (GLU) Assay Kit, Lactic Acid (LD) assay kit, ATP assay kit, Lactate dehydrogenase (LDH) assay kit (A154-1-1; A019-2-1; A095-1-1; A020-2-2, Nanjing Jiancheng Bioengineering Institute), Pyruvate Dehydrogenase (PDH) Activity Assay Kit (E-BC-K650-M, Elabscience), BCA Protein Assay Kit (PC0020, Solarbio), ELISA Kit for Hexokinase 2 (HK2) activity based on enzyme standard of HK2, ELISA Kit for Tumor Necrosis Factor alpha (TNF-α) (MM-45418M1; MM-0132M1, MEIMIAN), ELISA Kit for Interleukin 6 (IL-6) (E-MSEL-M0001, Elabscience) and ELISA Kit for Pyruvate kinase isozymes M2 (PKM2) (SEA588Mu, Cloud Clone Corp), Cell Staining Buffer, PE Anti-Mouse F4/80 Antibody [CI:A3-1], PE/Cyanine7 Anti-Mouse CD86 Antibody [GL-1], Intracellular Fixation/Permeabilization Buffer Kit and APC Anti-Mouse CD206 Antibody (E-CK-A107; E-AB-F0995D; E-AB-F0994H; E-CK-A109; E-AB-F1135E, Elabscience).

Techniques: Expressing, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Control, Biomarker Discovery

Journal: BMC Microbiology

Article Title: The activation impact of lactobacillus-derived extracellular vesicles on lipopolysaccharide-induced microglial cell

doi: 10.1186/s12866-024-03217-4

Figure Lengend Snippet: Lac-EVs inhibit M1 macrophage polarization after LPS challenge ( A-B ) The expression of iNOS and Arg1 was measured via RT-qPCR. ( C-D ) Analyses were performed using flow cytometry. The markers of M1 and M2 macrophages were CD86 and CD206, respectively. The ratio of M1 to M2 macrophages is shown in Fig. ( E-F ). The data are shown as the means ± SDs. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001)

Article Snippet: For cell surface staining, 3 μL of PE-conjugated anti-mouse CD86 (E-AB-F0994B Elabscience) was added to each group, and the samples were incubated on ice for 30 min in the dark, followed by fixation and intracellular staining.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: International journal of pharmaceutics

Article Title: Surface conjugation of EP67 to biodegradable nanoparticles increases the generation of long-lived mucosal and systemic memory T-cells by encapsulated protein vaccine after respiratory immunization and subsequent T-cell-mediated protection against respiratory infection

doi: 10.1016/j.ijpharm.2019.05.012

Figure Lengend Snippet: Conjugating EP67 to the surface of biodegradable nanoparticles increases the expression of surface activation markers on immature murine BMDCs after treatment. Immature bone marrow-derived dendritic cells (BMDCs, Day 8 GM-CF/IL-4, male C57BL/6) were incubated with media alone [black bars, Media] or media containing 7.8 μg LPS-free OVA encapsulated at 7.8 wt% in unmodified PLGA 50:50 nanoparticles [dark grey bars, NP(OVA)], PLGA 50:50 nanoparticles surface-modified with inactive scEP67 [grey bars, scEP67-NP(OVA)], or PLGA 50:50 nanoparticle surface-modified with EP67 [white bars, EP67-NP(OVA)] at 37°C for 24 h then analyzed by flow cytometry. Average median fluorescence intensities (MFI) ± SD (n=2 independent treatments) from (A) CD80, (B) CD86, or (C) MHC-II staining of live CD11c+ BMDCs were compared between treatment groups using one-way ANOVA with Tukey’s post-test where **P < 0.01, ***P < 0.001, and ****P ≤ 0.0001. Data are representative of at least three independent experiments.

Article Snippet: Cell surfaces were stained for activation markers by resuspending the cells in FACS Stain Buffer [50 μL] containing half the manufacturer’s suggested amount of PE-Cy5 Anti-Mouse CD11c (Clone N418, eBioscience), PE Anti-Mouse MHC-II (Clone M5/114.15.2, Miltenyi), FITC Anti-Mouse CD80 (Clone 16–10A1, Miltenyi), APC Anti-Mouse CD86 [Clone PO3.3] (Miltenyi), then incubating on ice in the dark for 30 min. FACS Stain Buffer [0.15 mL] was then added and cells were pelleted [500 RCF, 4°C, 5 min.], resuspended in Fixation Buffer (BioLegend) [0.1 mL/well], and incubated on ice [20 min].

Techniques: Expressing, Activation Assay, Derivative Assay, Incubation, Modification, Flow Cytometry, Fluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: The C-type Lectin Receptor CLECSF8 (CLEC4D) Is Expressed by Myeloid Cells and Triggers Cellular Activation through Syk Kinase *

doi: 10.1074/jbc.M112.384164

Figure Lengend Snippet: CLECSF8 expression on human peripheral blood leukocytes. A , PCR of hCLECSF8 and hG3DPH (loading control) on commercial cDNA preparations isolated from various human tissues and cells, as indicated. B , flow cytometric analysis of peripheral blood showing the gating, based on side scatter ( SSC ) and forward scatter ( FSC ) profiles, of granulocytes, monocytes, and lymphocytes and staining with specific cell markers and anti-hCLECSF8, as indicated. See also supplemental Fig. 1 . C , analysis of hCLECSF8 expression on monocyte-derived macrophages and DC. HLA-DR, CD14, DC-SIGN, and CD86 were included as markers of macrophage and DC maturation as described in the text. DCs were matured for 2 days using 1 μg/ml LPS as described under “Experimental Procedures.” The data are representative of at least five independent donors. D , analysis of hCLECSF8 expression on various DC subpopulations in isolated PBMCs. The filled gray histograms represent the isotype controls, and the black histograms represent specific antibody staining, as indicated. The data are representative of at least three independent donors. E , freshly isolated peripheral blood monocytes ( open bars ) and neutrophils ( gray bars ) were treated with various cytokines and TLR agonists for 6 h, and hCLECSF8 expression was analyzed by flow cytometry. The data were normalized to the untreated control value and shown as the mean ± S.E. of pooled data from at least five independent donors. *, p < 0.05. Flag , flagellin.

Article Snippet: The following antibodies were used in these assays: anti-HA (clone 16B12, Covance); anti-hCLECSF8 (MAB2806) and anti-hCLECSF8-APC (FAB2806A, R&D Systems); CD14-PE, CD16-FITC, CD3-FITC, CD4-FITC, CD8-FITC, and CD86-PE (all from Serotec); CD19-PE, CD56-PE, HLA-DR-FITC, and DC-SIGN-FITC (all from BD Biosciences); BDCA-2, CD1c, and BDCA-3 (all from Miltenyi Biotech); as well as irrelevant PE- or FITC-labeled or unlabeled mouse IgG 1 (BD Biosciences), IgG 2b , and IgG 3 (Serotec) control antibodies.

Techniques: Expressing, Control, Isolation, Staining, Derivative Assay, Flow Cytometry