Journal: iScience

Article Title: Microglial transcription profiles in mouse and human are driven by APOE4 and sex.

doi: 10.1016/j.isci.2021.103238

Figure Lengend Snippet: Figure 2. Sex and APOE genotype drive amyloid pathology and microglial gene expression profiles in APOE-FAD mice (A) Representative images of immunohistochemical staining for amyloid-b (red) and the microglial marker IBA1 (green) in male and female APOE3- and APOE4-FAD mice. Scale bar, 50 mm. (B) Samples separate by APOE and sex along PC1, with APOE3-FAD males clustering separately from mice of both sexes and APOE3-FAD females. A large proportion of the genes driving this separation (76.4%) are DAM-APOE genes. APOE3-FAD males are shown in yellow, APOE3-FAD females in gray, APOE4-FAD males in magenta, and APOE4-FAD females in teal. (C) Heatmap of the DAM-APOE genes associated with sample clustering along PC1. Gene expression levels were normalized, scaled, and centered and are displayed as Z scores. (D and E) Levels of the DAM-APOE marker, CD11c, are increased in APOE3-FAD females and in APOE4-FAD mice of both sexes, both at the (D) transcript and (E) protein levels. (D) Gene expression of Itgax in normalized counts; data are presented as mean (GSEM) values; n = 5–7/group. *p < 0.05, **p < 0.01; one-way ANOVA with Tukey’s multiple comparisons test correction. (E) Representative immunofluorescence images of microglia (IBA1, red) and CD11c (green) in the hippocampal subiculum of male and female APOE3- and APOE4-FAD mice. Scale bar, 25 mm.

Article Snippet: For IBA1/CD11c double labeling, antigen retrieval was performed using boiling EDTA and sections were incubated for 2 days at 4 C in primary antibodies directed against IBA1 (1:500) and CD11c (#NB110-97871, 1:500, Novus Biologicals).

Techniques: Gene Expression, Immunohistochemical staining, Staining, Marker

Journal: Nature Communications

Article Title: Co-delivery of sorafenib and an FSP1 inhibitor triggers dual ferroptosis in tumor cells and immunosuppressive macrophages for enhanced immunotherapy in mouse models of hepatocellular carcinoma

doi: 10.1038/s41467-025-65056-9

Figure Lengend Snippet: a Schematic illustration of the induction of M2 TAMs, the 1-6/TAM spheroids formulation, the transwell co-incubation, and their respective treatments. b Western blot analysis of GPX4 and FSP1 expression in M2 TAMs after different treatments. The experiment was repeated three times independently with similar results. Uncropped blots in Source Data. c Quantification of grayscale intensity of GPX4 and FSP1 in ( b ) ( n = 3 independent experiments). Protein expression levels in PBS group were normalized to 1. d Live/dead staining of M2 TAMs after different treatments (Scale bar = 200 μm). Viable cells were stained with calcein-AM (green), and dead cells were stained with PI (red). The experiment was repeated three times independently with similar results. e Quantification of the percentage of viable and dead M2 TAMs in ( d ) ( n = 3 independent experiments). f Flow cytometric analysis of Annexin V-FITC/propidium iodide (PI)-stained M2 TAMs (gated on F4/80 + CD206 + macrophages) after different treatments. g Quantification of the percentage of apoptotic cells in ( f ) ( n = 3 independent replicates). The experiment was repeated twice independently with similar results. h The cell proliferation of M2 TAMs after different treatments measured by CCK8 ( n = 3 independent experiments). The concentrations of SF and vF contained in nanoparticles were both 3 μM. i Fluorescence images of C11 BODIPY 581/591 -stained M2 TAMs after different treatments (Scale bar = 100 μm). The experiment was repeated three times independently with similar results. j Fluorescence images of live/dead stained 1-6/TAM spheroids after different treatments (Scale bar = 500 μm). Viable cells were stained with calcein-AM (green), and dead cells were stained with PI (red). Z-stack scanning of the spheroids was performed with slices distanced by 14.36 μm. The experiment was repeated three times independently with similar results. k Flow cytometric analysis of anti-CRT-stained Hepa1-6 cells after different treatments. l Quantification of the percentage of CRT positive cells in ( k ) ( n = 3 independent replicates). The experiment was repeated twice independently with similar results. m The ATP assay of Hepa1-6 cells after different treatments ( n = 3 independent experiments). n The HMGB1 assay of Hepa1-6 cells after different treatments ( n = 3 independent experiments). o Flow cytometric analysis of anti-CD80/CD86-stained BMDCs (gated on CD11c + DCs) after different treatments. p Quantification of the percentage of mature DCs in ( o ) ( n = 3 independent replicates). The experiment was repeated twice independently with similar results. q The assay of TNF, IL-6, and IL-12 in BMDCs after different treatments ( n = 3 independent experiments). Unless specified otherwise, error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test ( c , g , h , l – n , p , q ) and P -values were indicated. Source data are provided as a Source Data file. The elements in Fig. 5a were created by Adobe Illustrator.

Article Snippet: PerCP/Cyanine5.5 anti-mouse F4/80 (1:200, #E-AB-F0995J), APC anti-mouse CD206 (1:200, #E-AB-F1135E), PE/Cyanine7 anti-mouse CD86 (1:200, #E-AB-F0994H), Fluor Red 780 anti-mouse CD80 (1:200, #E-AB-F0992S), APC anti-mouse CD11c (1:200, #E-AB-F0991E), Fluor Violet 450 anti-mouse CD3 (1:200, #E-AB-F1013Q), Fluor Red 780 anti-mouse CD4 (1:200, #E-AB-F1097S), PerCP/Cyanine5.5 anti-mouse CD8 (1:200, #E-AB-F1104J), FITC anti-human/mouse CD44 (1:200, #E-AB-F1100C), PE anti-mouse Foxp3 (1:200, #E-AB-F1238D), FITC anti-mouse MHC II (1:200, #E-AB-F0990C), APC anti-mouse CD62L (1:200, #E-AB-F1011E), APC anti-mouse PD-L1 (1:200, #E-AB-F1132E), PerCP anti-human CD45 (1:200, #E-AB-F1137F), Fluor647 anti-human CD68 (1:200, #E-AB-F1299M), FITC anti-human CD206 (1:200, #E-AB-F1161C), PE anti-human CD80 (1:200, #E-AB-F1232D), APC anti-human HLA-DR (1:200, #E-AB-F1111E), PE anti-human CD11c (1:200, #E-AB-F1118D), APC anti-human CD3 (1:200, #E-AB-F1001E), and FITC anti-human CD8 (1:200, #E-AB-F1110C) were purchased from Elabscience (Wuhan, China).

Techniques: Formulation, Incubation, Western Blot, Expressing, Staining, Fluorescence, ATP Assay

Journal: Nature Communications

Article Title: Co-delivery of sorafenib and an FSP1 inhibitor triggers dual ferroptosis in tumor cells and immunosuppressive macrophages for enhanced immunotherapy in mouse models of hepatocellular carcinoma

doi: 10.1038/s41467-025-65056-9

Figure Lengend Snippet: a Body weight of mice with intravenous treatments during a 28-day period ( n = 5 independent mice). b The blood analysis for the liver/kidney functions including ALT, AST, BUN, and CRE was determined on day 28 following the treatments ( n = 5 independent mice). c The curve of injected drug concentration (ID %) versus time point was plotted in subcutaneous HCC mice ( n = 6 independent mice). d Fluorescence imaging of the biodistribution in orthotopic HCC mice at 12 h after different treatments (tumors marked with yellow circles; n = 6 independent mice). e Tumor-to-background ratio for different treatments in ( d ) ( n = 6 independent mice). f Quantitative biodistribution analysis of DiR-labeled nanoparticles in major organs and liver tumors in ( d ) ( n = 6 independent mice). g Fluorescence images of Sv@PM-M2p inside HCC cells, M2 TAMs, M1 TAMs, DCs, and T cells in tumors ( n = 6 independent mice; Scale bar = 100 μm). Sv@PM-M2p was labeled with Rhodamine (red), the nuclei and cell markers (HCC, CK8; M2, CD206; M1, CD86; DC, CD11c; T, CD3) were stained with DAPI (blue) and antibody (green), respectively. h Quantification of the percentage of colocated cells in ( g ) ( n = 6 independent mice). Unless specified otherwise, error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test ( c , e , f , h ) and P -values were indicated. Source data are provided as a Source Data file.

Article Snippet: PerCP/Cyanine5.5 anti-mouse F4/80 (1:200, #E-AB-F0995J), APC anti-mouse CD206 (1:200, #E-AB-F1135E), PE/Cyanine7 anti-mouse CD86 (1:200, #E-AB-F0994H), Fluor Red 780 anti-mouse CD80 (1:200, #E-AB-F0992S), APC anti-mouse CD11c (1:200, #E-AB-F0991E), Fluor Violet 450 anti-mouse CD3 (1:200, #E-AB-F1013Q), Fluor Red 780 anti-mouse CD4 (1:200, #E-AB-F1097S), PerCP/Cyanine5.5 anti-mouse CD8 (1:200, #E-AB-F1104J), FITC anti-human/mouse CD44 (1:200, #E-AB-F1100C), PE anti-mouse Foxp3 (1:200, #E-AB-F1238D), FITC anti-mouse MHC II (1:200, #E-AB-F0990C), APC anti-mouse CD62L (1:200, #E-AB-F1011E), APC anti-mouse PD-L1 (1:200, #E-AB-F1132E), PerCP anti-human CD45 (1:200, #E-AB-F1137F), Fluor647 anti-human CD68 (1:200, #E-AB-F1299M), FITC anti-human CD206 (1:200, #E-AB-F1161C), PE anti-human CD80 (1:200, #E-AB-F1232D), APC anti-human HLA-DR (1:200, #E-AB-F1111E), PE anti-human CD11c (1:200, #E-AB-F1118D), APC anti-human CD3 (1:200, #E-AB-F1001E), and FITC anti-human CD8 (1:200, #E-AB-F1110C) were purchased from Elabscience (Wuhan, China).

Techniques: Injection, Concentration Assay, Fluorescence, Imaging, Labeling, Staining

Journal: Nature Communications

Article Title: Co-delivery of sorafenib and an FSP1 inhibitor triggers dual ferroptosis in tumor cells and immunosuppressive macrophages for enhanced immunotherapy in mouse models of hepatocellular carcinoma

doi: 10.1038/s41467-025-65056-9

Figure Lengend Snippet: a Schematic illustration of the research procedure in subcutaneous HCC mouse model. b Relative tumor volume curves of different treatment groups ( n = 5 independent mice). c Animal survival after different treatments ( n = 5 independent mice). d Quantification of fluorescence intensity in Supplementary Fig. f ( n = 5 independent mice). e Immunofluorescence images of tumor tissue slices collected from different groups stained with anti-F4/80/CD206, anti-F4/80/CD86, and anti-CD11c/MHC II after different treatments ( n = 5 independent mice; Scale bar = 100 μm). f Quantification of the percentage of F4/80 + CD206 + M2 TAMs, F4/80 + CD86 + M1 TAMs, and CD11c + MHC II + actDCs in ( e ) ( n = 5 independent mice). g Schematic illustration of the research procedure in orthotopic HCC mouse model. h Relative bioluminescence intensity curves for the various treatment groups ( n = 5 independent mice). i Animal survival after different treatments ( n = 5 independent mice). j Flow cytometric analysis of anti-F4/80/CD206-stained macrophages, anti-F4/80/CD86-stained macrophages, anti-CD11c/MHC II-stained DCs, anti-CD3/CD4-stained T cells, anti-CD3/CD8-stained T cells, and anti-CD4/Foxp3-stained T cells in tumor tissues after different treatments ( n = 5 independent mice). Unless specified otherwise, error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test ( b , d , f , h ) or log-rank test ( c , i ) and P -values were indicated. Source data are provided as a Source Data file. The elements in Fig. 7a, g were created by Adobe Illustrator.

Article Snippet: PerCP/Cyanine5.5 anti-mouse F4/80 (1:200, #E-AB-F0995J), APC anti-mouse CD206 (1:200, #E-AB-F1135E), PE/Cyanine7 anti-mouse CD86 (1:200, #E-AB-F0994H), Fluor Red 780 anti-mouse CD80 (1:200, #E-AB-F0992S), APC anti-mouse CD11c (1:200, #E-AB-F0991E), Fluor Violet 450 anti-mouse CD3 (1:200, #E-AB-F1013Q), Fluor Red 780 anti-mouse CD4 (1:200, #E-AB-F1097S), PerCP/Cyanine5.5 anti-mouse CD8 (1:200, #E-AB-F1104J), FITC anti-human/mouse CD44 (1:200, #E-AB-F1100C), PE anti-mouse Foxp3 (1:200, #E-AB-F1238D), FITC anti-mouse MHC II (1:200, #E-AB-F0990C), APC anti-mouse CD62L (1:200, #E-AB-F1011E), APC anti-mouse PD-L1 (1:200, #E-AB-F1132E), PerCP anti-human CD45 (1:200, #E-AB-F1137F), Fluor647 anti-human CD68 (1:200, #E-AB-F1299M), FITC anti-human CD206 (1:200, #E-AB-F1161C), PE anti-human CD80 (1:200, #E-AB-F1232D), APC anti-human HLA-DR (1:200, #E-AB-F1111E), PE anti-human CD11c (1:200, #E-AB-F1118D), APC anti-human CD3 (1:200, #E-AB-F1001E), and FITC anti-human CD8 (1:200, #E-AB-F1110C) were purchased from Elabscience (Wuhan, China).

Techniques: Fluorescence, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Co-delivery of sorafenib and an FSP1 inhibitor triggers dual ferroptosis in tumor cells and immunosuppressive macrophages for enhanced immunotherapy in mouse models of hepatocellular carcinoma

doi: 10.1038/s41467-025-65056-9

Figure Lengend Snippet: a Schematic illustration of the research procedure in PDX subcutaneous mouse model. b Relative tumor volume curves of different treatment groups ( n = 5 independent mice). c Collected tumor tissues of mice at the end of treatment in different groups. d Tumor weight at the end of treatment in different groups ( n = 5 independent mice). e Immunofluorescence images of tumor tissue slices collected from different groups stained with Liperfluo after different treatments ( n = 5 independent mice; Scale bar = 100 μm). f Quantification of fluorescence intensity in ( e ) ( n = 5 independent mice). g Flow cytometric analysis of anti-CD68/CD206-stained macrophages (gated on CD45 + cells), anti-CD68/CD80-stained macrophages (gated on CD45 + cells), anti-HLA-DR/CD11c-stained DCs (gated on CD45 + Lin-1 – T cells), and anti-CD3/CD8-stained T cells (gated on CD45 + cells) in tumor tissues after different treatments ( n = 5 independent mice). h Quantification of the percentage of CD68 + CD206 + M2 TAMs, CD68 + CD80 + M1 TAMs, HLA-DR + CD11c + actDCs, and CD3 + CD8 + Tc cells in ( g ) ( n = 5 independent mice). Unless specified otherwise, error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test ( b , d , f , h ) and P -values were indicated. Source data are provided as a Source Data file. The elements in Fig. 9a were created by Adobe Illustrator.

Article Snippet: PerCP/Cyanine5.5 anti-mouse F4/80 (1:200, #E-AB-F0995J), APC anti-mouse CD206 (1:200, #E-AB-F1135E), PE/Cyanine7 anti-mouse CD86 (1:200, #E-AB-F0994H), Fluor Red 780 anti-mouse CD80 (1:200, #E-AB-F0992S), APC anti-mouse CD11c (1:200, #E-AB-F0991E), Fluor Violet 450 anti-mouse CD3 (1:200, #E-AB-F1013Q), Fluor Red 780 anti-mouse CD4 (1:200, #E-AB-F1097S), PerCP/Cyanine5.5 anti-mouse CD8 (1:200, #E-AB-F1104J), FITC anti-human/mouse CD44 (1:200, #E-AB-F1100C), PE anti-mouse Foxp3 (1:200, #E-AB-F1238D), FITC anti-mouse MHC II (1:200, #E-AB-F0990C), APC anti-mouse CD62L (1:200, #E-AB-F1011E), APC anti-mouse PD-L1 (1:200, #E-AB-F1132E), PerCP anti-human CD45 (1:200, #E-AB-F1137F), Fluor647 anti-human CD68 (1:200, #E-AB-F1299M), FITC anti-human CD206 (1:200, #E-AB-F1161C), PE anti-human CD80 (1:200, #E-AB-F1232D), APC anti-human HLA-DR (1:200, #E-AB-F1111E), PE anti-human CD11c (1:200, #E-AB-F1118D), APC anti-human CD3 (1:200, #E-AB-F1001E), and FITC anti-human CD8 (1:200, #E-AB-F1110C) were purchased from Elabscience (Wuhan, China).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: Conversion of iMo to an inflammatory phenotype is associated with nuclear translocation of NF-κB and production of IL-1β that requires ADAM17 proteolytic activity. (A) NF-κB cytosolic to nuclear translocation (n = 6/group). (B) Intracellular IL-1β production in brefeldin A–treated iMo at 2 h of shear and treatment with allosteric affinity stabilizing anti-CD11c or IgG control that binds the common epitope of CD11c (n = 6 per group) (two-way ANOVA with Tukey posttest, *p < 0.05 from control within each Ab treatment, #p < 0.05 between CAD and NSTEMI within Ab treatments, $p < 0.05 from the CD11c IgG treatment for each patient cohort). (C) Combined expression of nuclear NF-κB (n = 4; CAD; n = 2, NSTEMI) and (D) cytosolic IL-1β (n = 3 CAD, n = 2 NSTEMI) after treatment with ADAM17 inhibitor TMI-1 or vehicle in the presence of the IgG control to CD11c or the low affinity promoting Ab after 2 h of shear-resistant arrest to VCAM-1 at 2 dynes/cm2. Two-way ANOVA with Tukey posttest, *p < 0.05 from the control treatment compared with that of the ADAM17 inhibitor.

Article Snippet: The membrane was subsequently blocked with 5% milk for 2 h, and primary Ab was used to probe for CD11c (NBP-234491; R&D Systems), ADAM17 (ab57484; Abcam), CD16 (MEM-154; Sigma Aldrich), Syk (MA1-19332; Thermo Fisher Scientific), and p-Syk (44-233G; Thermo Fisher Scientific).

Techniques: Translocation Assay, Activity Assay, Shear, Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: Monocyte recruitment to inflamed endothelium under shear stress is β1- and β2-integrin–dependent on binding to VCAM-1. (A) Monocytes were perfused into the A-Chip onto TNF-α–stimulated HAEC in the presence of blocking Abs to CD11c, CD11b, VLA-4, or a nonspecific isotype IgG (Control) and TEM of adherent cells (white arrows) assessed by phase-contrast imaging after 10 min of shear at 2 dynes/cm2. Images display a time course of monocyte arrest and transmigration (phase dark) over 2 min. Scale bar, 10 μm. Monocyte subsets were determined based upon on-chip analysis of relative fluorescence intensity of Abs to CD14, CD16, and CCR2, which enabled the assessment of the number of arrested per field of view (FOV) at original magnification ×40. (B) Quantification of arrested iMo (filled bars) and the number of those arrested cells that have transmigrated (open bars) over 10 min of constant shear (2 dynes/cm2). Data are represented as mean arrested or transmigrated cells per FOV ± SD (n = 4–5 per group) (two-way ANOVA with Tukey posttest *p < 0.05 between isotype control within each cohort TEM color, #p < 0.05 compared with healthy, $p < 0.05 between CAD and NSTEMI cohorts for each Ab blocking treatment).(C) Monocyte subset arrest frequency on rVCAM-1 on the A-Chip. Data are represented as mean ± SD monocyte subset fraction of total arrested measured on the day of percutaneous coronary intervention or venous blood draw from healthy (n = 6), CAD (n = 8), and NSTEMI (n = 20) analyzed via two-way ANOVA with Tukey posttest. *p < 0.05 between healthy age-matched subject groups.

Article Snippet: The membrane was subsequently blocked with 5% milk for 2 h, and primary Ab was used to probe for CD11c (NBP-234491; R&D Systems), ADAM17 (ab57484; Abcam), CD16 (MEM-154; Sigma Aldrich), Syk (MA1-19332; Thermo Fisher Scientific), and p-Syk (44-233G; Thermo Fisher Scientific).

Techniques: Shear, Binding Assay, Blocking Assay, Control, Imaging, Transmigration Assay, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: Recruitment of iMo on VCAM-1 is regulated by the expression level and affinity state of CD11c, which discriminates cardiac patients from healthy controls. (A) Integrin dependence of iMo arrest following treatment with a pan–anti-CD11c mAb to a nonblocking common epitope (IgG), an allosteric anti-CD11c low affinity–inducing mAb, and anti–VLA-4 functional blocking mAb (healthy, n = 5; CAD, n = 5; NSTEMI, n = 21). Data are represented as mean ± SD iMo arrest number (two-way ANOVA with Tukey posttest, *p < 0.05 between patient groups, #p < 0.05 from the anti-CD11c IgG control). Arrest fraction of iMo/10,000 monocytes infused into the A-Chip plotted against an individual patient’s mean receptor number for (B) CD11c (n = 6, healthy; n = 8, CAD; n = 20, NSTEMI), (C) VLA-4 (n = 6, healthy; n = 10, CAD; n = 7, NSTEMI), (D) CD11b (n = 6, healthy; n = 10, CAD; n = 7, NSTEMI), and (E) CX3CR1 (n = 6, healthy; n = 8, CAD; n = 7, NSTEMI) receptor expression measured on whole blood monocytes via FACS analysis with the resulting Pearson correlation. (F) Principle component plot of integrin and chemokine receptor expression from six random individuals from each of the healthy, CAD, and NSTEMI groups, in which each data point represents a unique individual. The plot represents a two-dimensional space that contains 80.7% of the original expression data, in which the x-axis or dimension 1 (Dim1) represents 52.6% of the original expression data, and the y-axis given by dimension 2 (Dim2) represents 28.1% of the original input of expression data. (G) Heatmap of the integrin and chemokine receptor parameters used in the principal component clustering analysis. Relative weighting of the correlations depicted by a gradient in which blue is the minimum, white (0) is the median, and red indicates the maximum.

Article Snippet: The membrane was subsequently blocked with 5% milk for 2 h, and primary Ab was used to probe for CD11c (NBP-234491; R&D Systems), ADAM17 (ab57484; Abcam), CD16 (MEM-154; Sigma Aldrich), Syk (MA1-19332; Thermo Fisher Scientific), and p-Syk (44-233G; Thermo Fisher Scientific).

Techniques: Expressing, Functional Assay, Blocking Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: iMo form focal adhesions through activated CD11c and diffusion of VLA-4 that precedes conversion to a CD16− phenotype postarrest on VCAM-1. (A) TIRF images of iMo CD11c (green) and VLA-4 (red) expression at time of attachment to human rVCAM-1 (T = 0) and at 60 min postarrest at 2 dynes/cm2 shear stress for healthy, CAD, and NSTEMI (scale bar, 10 μm). Quantification of healthy (black, n = 7), CAD (blue, n = 7), and NSTEMI (orange, n = 5) iMo (B) CD11c and (C) CD49d (VLA-4) membrane receptor MFI per cell area at time of arrest to establish baseline fluorescence/cell area (bar graphs) and dynamically over time using live TIRF. Data are represented as percentage change from baseline fluorescence at the time of attachment for CD11c or VLA-4 receptors (one-way ANOVA with Tukey posttest, *p < 0.05 comparing the maximal percentage change to the baseline at T = 0. Scale bar, 10 μm). (D) Representative images of iMo from healthy, CAD, and NSTEMI patients depict CD16 expression (red) and CD14 (green) at t = 0 min (time of arrest) and 60 min postarrest. (E) CD16 receptor expression on adherent iMo assessed from the time of capture to 60 min postadhesion from healthy (n = 6), CAD (n = 7), and NSTEMI (n = 5) patients under constant flow or under static conditions (translucent points and lines). iMo conversion was classified as expressing fewer than 10,000 receptors, which was equivalent to background nonspecific fluorescence intensity. Data are represented as mean CD16 receptor number at time of arrest to VCAM-1 (one-way ANOVA with Tukey posttest, *p < 0.05 comparing the peak increase/decrease the baseline at T = 0. Scale bar, 10 μm). (F) Fraction of iMo that have converted relative to baseline CD16 receptor expression from healthy (n = 6), CAD (n = 7), and NSTEMI (n = 7) patients at 75 min under constant shear and treatment with CD11c allosteric or a pan-CD11c Ab control. Data are represented as mean CD16 expression/time ± SD (two-way ANOVA with Tukey posttest, #p < 0.05 compared with healthy controls, *p < 0.05 compared with the CD11c IgG treatment for each healthy age-matched subject cohort, $p < 0.05 between CAD and NSTEMI). Scale bar, 10 μm.

Article Snippet: The membrane was subsequently blocked with 5% milk for 2 h, and primary Ab was used to probe for CD11c (NBP-234491; R&D Systems), ADAM17 (ab57484; Abcam), CD16 (MEM-154; Sigma Aldrich), Syk (MA1-19332; Thermo Fisher Scientific), and p-Syk (44-233G; Thermo Fisher Scientific).

Techniques: Diffusion-based Assay, Expressing, Shear, Membrane, Fluorescence, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: An allosteric shift in CD11c affinity following iMo arrest on VCAM-1 induces membrane coalescence with CD16 and subsequent cleavage by ADAM17. (A) Schematic depicting the coalescence and (B) immunofluorescent images of CD11c (green), ADAM17 (purple), and CD16 (red) within focal adhesive contact on VCAM-1 at the time point of capture (scale bar, 10 μm). Associated Pearson correlation of the coalescence of membrane (C) CD11c–CD16 and (D) CD11c–ADAM17 receptors under static conditions from healthy, CAD, and NSTEMI iMo (n = 4–5). (E) Schematic representation of high affinity CD11c coclustered with CD16. This results in outside-in signaling associated with phosphorylation of Syk and association with ADAM17. (F) Western blot of coimmunoprecipitation of CD11c with ADAM17 and CD16 and associated signaling component p-Syk following 15 min of shear stress or static conditions on arrested monocytes from a healthy donor using allosteric affinity modulating mAbs to CD11c or a pan anti-CD11c control IgG (n = 7). (G) CD16, (H) p-Syk/Total Syk, and (I) ADAM17 band density normalized to CD11c protein expression quantified by densitometry (n = 7). Data are represented as mean band intensity normalized to CD11c ± SD (Student t test, *p < 0.05 between static and shear conditions) (n = 7). (J) Schematic representation of phenotypic conversion dependent upon ADAM17-mediated cleavage of CD16 and an allosteric shift to low affinity CD11c at focal sites of VLA-4–mediated adhesion. (K) ADAM17-dependent cleavage of CD16 on iMo from CAD and NSTEMI when treated with ADAM17 inhibitor (TMI-1 at 12 nM) (translucent lines) or DMSO vehicle control (filled lines) over 75 min at 2 dynes/cm2 postarrest to VCAM-1. * represents a significant difference between vehicle and TMI-1 at the given time point that is sustained after 15 min of shear. One-way ANOVA with Tukey posttest, *p < 0.05. (L) iMo conversion frequency after 75 min of shear postarrest to VCAM-1 in the presence of ADAM17 inhibitor or vehicle control. Data are represented as mean conversion ± SD (Student t test. *p < 0.05 between treated and vehicle control).

Article Snippet: The membrane was subsequently blocked with 5% milk for 2 h, and primary Ab was used to probe for CD11c (NBP-234491; R&D Systems), ADAM17 (ab57484; Abcam), CD16 (MEM-154; Sigma Aldrich), Syk (MA1-19332; Thermo Fisher Scientific), and p-Syk (44-233G; Thermo Fisher Scientific).

Techniques: Membrane, Adhesive, Phospho-proteomics, Western Blot, Shear, Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: Integrin expression and iMo arrest as a function of measures of myocardial injury and incidence of MI in cardiac patients. Bivariate plots of iMo average integrin receptor expression (CD11c [black]; CD11b [blue]; VLA-4 [red]) from each NSTEMI patient versus corresponding serum measures of (A) CK-MB, (B) LVEF%, (C) and peak troponin T (CD11c: n = 33; CD11b: n = 30; VLA-4: n = 31), with resulting Pearson correlation coefficient. (D) Number of iMo arrested under shear stress in the A-Chip from patients experiencing a primary NSTEMI (n = 9) versus those returning to the clinic for a repeat MI within 3 y (n = 4). Student t test ***p < 0.001.

Article Snippet: The membrane was subsequently blocked with 5% milk for 2 h, and primary Ab was used to probe for CD11c (NBP-234491; R&D Systems), ADAM17 (ab57484; Abcam), CD16 (MEM-154; Sigma Aldrich), Syk (MA1-19332; Thermo Fisher Scientific), and p-Syk (44-233G; Thermo Fisher Scientific).

Techniques: Expressing, Shear