Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet: CD163 + macrophages accumulate in the lung in severe COVID-19 (A) Overview of study design and analyses. CT, computed tomography; BAL, bronchoalveolar lavage; scRNA-seq, single-cell RNA sequencing; snRNA-seq, single-nucleus RNA sequencing; IHC, immunohistochemistry; IF, immunofluorescence microscopy; MELC, multi-epitope ligand cartography; EM, electron microscopy; VCin, inspiratory vital capacity; PBMC, peripheral blood mononuclear cells; IAV, Influenza A virus. (B) Postmortem analysis of consecutive histological sections of non-COVID-19 (left) and COVID-19 autopsy lung samples (right) by hematoxylin and eosin (H&E; top) and CD68 IHC (bottom). Scale bar, 100 μm. (C) IF of CD68 (green) and CD163 (red) in lung tissue autopsy samples of COVID-19 patients and non-COVID-19 controls. Arrows indicate CD68 + CD163 – macrophages, and arrowheads indicate CD68 + CD163 + macrophages. Scale bar, 20 μm. (D) Quantification of CD68 + macrophage density (left) and the proportion of CD163 + macrophages (right) in lung autopsy samples from fifteen donors (as in C). Mann-Whitney test; ∗ p < 0.05. (E) Representative images of consecutive histological sections of lung autopsy samples. H&E (left), CD68 IHC (middle), and SARS-CoV-2 RNA-FISH (right). Arrowheads indicate SARS-CoV-2 RNA-positive macrophages. Scale bars, 50 μm, 25 μm. RNA-FISH, RNA-fluorescence in situ hybridization. (F) Lung autopsy samples of 9 COVID-19 patients were analyzed by MELC with a panel of 22 markers on 19 fields of view (FOVs). Two-dimensional embedding computed by UMAP on 9,684 computationally identified CD45 positive cells (T cells, CD3 + ; B cells, CD20 + ; NK cells, CD56 + ; neutrophils, MRP14 + /CD66b + ; monocytes, MRP14 + /CCR2 + ; macrophages, MRP14 + /HLA-DR + ). (G) Relative proportion (of total CD45 + cells) of cell types in all 19 FOVs (left), and average cell numbers (summary, right).

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Computed Tomography, RNA Sequencing Assay, Immunohistochemistry, Immunofluorescence, Microscopy, Electron Microscopy, MANN-WHITNEY, Fluorescence, In Situ Hybridization

Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet:

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Immunohistochemistry, Plasmid Preparation, Recombinant, Staining, Lysis, Protease Inhibitor, Mass Spectrometry, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Journal of Neuroinflammation

Article Title: Expansion and activation of distinct central memory T lymphocyte subsets in complex regional pain syndrome

doi: 10.1186/s12974-019-1449-9

Figure Lengend Snippet: A table showing the mass cytometry panel used to immunophenotype the blood of CRPS and healthy control participants. The columns represent the rare-earth metal isotype used for conjugation, species reactivity, target antigen, clone, manufacturer and staining concentration. The antibody from Fluidigm was purchased metal labelled, and antibodies from all other manufacturers were purchased, metal-conjugated and validated in-house by the Ramaciotti Facility for Human Systems Biology

Article Snippet: 152 Gd , Human , CD66b , 80H3 , Fluidigm , 3 (μl/ml) , FACS.

Techniques: Mass Cytometry, Control, Conjugation Assay, Staining, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Neutrophils aggravate inflammatory lesions in intestinal organoids from necrotizing enterocolitis

doi: 10.3389/fimmu.2025.1582526

Figure Lengend Snippet: Assessment of neutrophil survival and activation using analysis by flow cytometry. Flow cytometry was performed to assess (1) neutrophil survival using propidium iodide (PI) and Annexin-V staining, and (2) activation using anti-CD66b and anti-CD11b antibodies, in the following conditions (1): Organoid differentiation medium (ODM) (2), Roswell Park Memorial Institute 1640 medium (RPMI) (3), ODM + lipopolysaccharide (LPS), and (4) RPMI + LPS. (A) Neutrophil survival was assessed at during multiple timepoints up until 72 (h) Survival rates decreased steadily until 24 h in all groups with no significant differences between groups. After 24 h, neutrophils in RPMI only showed significantly lower survival rates, suggesting a detrimental effect of RPMI alone on neutrophil viability. At 72 h, increased survival of neutrophils in LPS was observed. (B, D) Neutrophil activation was analyzed by CD66b (B) and CD11b (D) expression for 72 h at multiple timepoints. Activation markers (CD66b and CD11b) increased across all conditions at 24 h, with neutrophils in RPMI only showing similar activation levels to other conditions after 48 (h). This suggests a delayed activation in RPMI compared to other groups. (C, E) CD66b (C) and CD11b (E) signal intensity were measured at multiple time points up to 72 (h) CD66b signal intensity rose in (1) RPMI (2), ODM, and (3) ODM+LPS, while the signal intensity in RPMI+LPS solely rose within the first 12 h, upon which a decrease was seen (C) . CD11b intensity increased over time and plateaued after 12 h, with RPMI showing a less pronounced increase, indicating delayed activation (E) .

Article Snippet: Next, neutrophils were either stained with (1) PI (Becton Dickinson and Company, Cat. 51-66211E, dilution: 1:20 in Annexin Binding Buffer (10X Annexin Binding Buffer, Becton Dickinson and Company, Cat. 5166121E) diluted 1:10 in Aqua dest.) and Annexin (Cat. 556419, dilution: 1:50 in Annexin Binding Buffer) for detection of apoptosis and necrosis, or (2) antibodies against CD66b (APC anti-human CD66b, ReafinityTM, mAb, Clone: REA306, human IgG1, Milenyi Biotec, Bergisch Gladbach, Germany, Cat. 130-117-692, dilution: 1:50 in autoMACS Rinsing Solution (Milenyi Biotec, Cat. 130-091-222) with 0,5% BSA (MACS Staining Buffer) and CD11b (Vioblue anti-human CD11b, ReafinityTM, mAb, Clone: REA713, human IgG1, Milenyi Biotec, Cat. 130-110-554, dilution: 1:50 in MACS Staining Buffer) for detection of neutrophil activation.

Techniques: Activation Assay, Flow Cytometry, Staining, Expressing

Journal: bioRxiv

Article Title: Tumor-specific neutrophils originating from meninges promote glioblastoma

doi: 10.1101/2023.05.23.542010

Figure Lengend Snippet: (A to G) Identification of tumor-specific neutrophils (TSNs) in human glioblastomas (GBM). (A) Diagram of profiling myeloid cells in human GBM tumors. (B) Major cell types identified in human GBM tumors. Baso, basophils; Ma_MRC1, Ma_BNIP3, Ma_LYZ, Ma_MKI67, macrophages with corresponding markers; Mi, microglia; N, neurons; Neu, neutrophils; NK, natural killer cells; Ol, oligodendrocytes; TSN, tumor-specific neutrophils; Tu, tumor cells. (C) Neutrophil subtypes are defined in human GBM tumors and patients’ blood. Neu_CXCR2, Neu_ISG15, Neu_S100A12, neutrophils with corresponding markers; TSN, tumor-specific neutrophils. (D) Top expressing genes in neutrophil subtypes as defined in (C) . (E) Violin plot of the expression of neutrophil marker genes ( CXCR2 and S100A8 ), immunosuppressive genes ( CD274 and IDO1 ), and myeloid recruitment-related genes ( CXCL8 , CCL3 , CCL4 , and IL1B ) in neutrophil subtypes. (F and G) Representative images of CD66b/CD274 immunofluorescence staining (F) or CD274 immunohistochemistry (G) of human GBM tumors. White arrows, CD66b + CD274 + cells; black arrow, CD274 + neutrophil with the lobulated nucleus. (H to L) TSNs are correlated with glioma grades and poor prognosis. (H) TSN scores are positively correlated with glioma grades in the TCGA dataset (Student’s t -test). (I) Correlation between TSN scores and CD274 expression levels in the TCGA dataset. The correlation coefficient (R) and p -value were calculated by Pearson correlation. (J) TSN scores are enriched in the mesenchymal subtype than in other molecular subtypes of human GBM (Student’s t -test). (K and L) Overall survival of TCGA GBM cases (K) or TCGA glioma cases (L) stratified by TSN scores (log-rank test).

Article Snippet: For the immunofluorescence staining, 5 μm-thick paraffin sections were deparaffinized and stained with anti-human CD66b (1:100, GTX19779, GeneTex), anti-human CD274 (1:100, #13684, Cell Signaling Technology), anti-mouse Ly6G (1:300, GB11229, ServiceBio), or anti-mouse Cd274 (15μg/mL, MAB1561, R&D Systems).

Techniques: Expressing, Marker, Immunofluorescence, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: Tumor-specific neutrophils originating from meninges promote glioblastoma

doi: 10.1101/2023.05.23.542010

Figure Lengend Snippet: (A) Dot plot of major cell types identified by profiling myeloid cells in human GBM tumors. Three top marker genes for each cell type are shown. Baso, basophils; Ma_MRC1, Ma_BNIP3, Ma_LYZ, Ma_MKI67, macrophages with corresponding markers; Mi, microglia; N, neurons; Neu, neutrophils; NK, natural killer cells; Ol, oligodendrocytes; TSN, tumor-specific neutrophils; Tu, tumor cells. (B) Expression of the neutrophil marker gene ( CXCR2 ) and immunosuppressive genes ( CD274 , IDO1, IDO2 , and CEACAM1 ) in major cell types identified in (A) . (C) Frequency of TSNs and other subtypes in the total neutrophils of GBM patients’ blood and tumors defined by scRNA-seq. (D) Representative image of CD66b/CD274 immunofluorescence staining of human low-grade glioma tumor. White arrows, CD66b + CD274 + cells.

Article Snippet: For the immunofluorescence staining, 5 μm-thick paraffin sections were deparaffinized and stained with anti-human CD66b (1:100, GTX19779, GeneTex), anti-human CD274 (1:100, #13684, Cell Signaling Technology), anti-mouse Ly6G (1:300, GB11229, ServiceBio), or anti-mouse Cd274 (15μg/mL, MAB1561, R&D Systems).

Techniques: Marker, Expressing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Tumor-specific neutrophils originating from meninges promote glioblastoma

doi: 10.1101/2023.05.23.542010

Figure Lengend Snippet: (A to C) Presence of TSNs in the meninges of mouse glioma models. C57BL/6 wild-type mice were intracranially implanted with glioma cells. (A) TSNs and other neutrophil subtypes identified in mouse meninges and glioma tumors. Neu_Camp, Neu_Cxcr2, Neu_Isg15, Neu_Mki67, Neu_S100a6, Neu_Slpi, neutrophils with corresponding markers; TSN, tumor-specific neutrophils. The black arrow denotes TSNs in the mouse meninges of tumor-bearing condition. (B) TSN frequency in the total neutrophils of mouse spleens and meninges defined by scRNA-seq. (C) The percentage of TSNs (Cd45 + Cd274 + Cd11b + Ly6G + ) in neutrophils (Cd45 + Cd11b + Ly6G + ) of the spleens, meninges, and gliomas of tumor-bearing mice was quantified by FACS analyses (Student’s t -test). ( D to G ) Tumor-induced myelopoiesis in the meninges. C57BL/6 wild-type mice were intracranially implanted with glioma cells. (D) The percentage of neutrophils (Cd45 + Cd11b + Ly6G + ) in total immune cells (Cd45 + ) of tumors at different time points post-implantation. (E) The percentage of granulocyte-monocyte progenitors (GMP, Lin - Sca-1 - c-Kit + Cd34 + Cd16/32 + ; Lin: B220, Cd2, Cd3, Cd5, Cd8, Gr-1, and Ter119) in total Lin - cells of the bone marrow, meninges, spleens, and gliomas of tumor-bearing mice were determined at day 5 post-implantation by FACS analyses (Student’s t -test). (F) Neutrophil proliferation occurs in the meninges. Ki67-RFP reporter mice were intracranially implanted with glioma cells. The percentage of Ki67-RFP + cells in neutrophils (Cd45 + Cd11b + Ly6G + ) of the spleens, meninges, and tumors were assessed by FACS analyses (Student’s t -test). ( G ) Cxcr2 deletion in host mice retains neutrophils in the meninges. Cxcr2 -/- or control Cxcr2 +/+ mice were intracranially implanted with glioma cells. The percentage of neutrophils (Cd45 + Cd11b + Ly6G + ) in total immune cells (Cd45 + ) of the meninges was quantified by FACS analyses (Student’s t -test). (H) Peripheral circulating immune cells were not sufficient to count for TSNs in mouse gliomas. A parabiosis between C57BL/6 wild-type mice and H11-tdTomato reporter mice was established. C57BL/6 mice in parabiotic pairs were then intracranially implanted with glioma cells. The percentage of tdTomato + cells in neutrophils (Cd45 + Cd11b + Ly6G + ) of the bone marrow, meninges, spleens, and tumors of C57BL/6 mice was determined by FACS analyses (Student’s t -test). ( I to K ) Presence of TSNs in the meninges of human GBM patients. ( I ) Major cell types identified in GBM patients’ meninges. B, B cells; DC, dendritic cells; En, endothelial cells; Flc, fibroblast-like cells; Ma, macrophages; Mast, mast cells; Mo, monocytes; N, neurons; Neu, neutrophils; SM, smooth muscle cells; T/NK, T cells / natural killer cells. (J) TSN frequency in the total neutrophils of GBM patients’ blood and meninges defined by scRNA-seq. (K) The percentage of neutrophils (CD45 + CD11b + CD14 - CD15 + HLA-DR - CD33 low CD66b + ) in total immune cells (CD45 + ) of the meninges of GBM or control patients was examined by FACS analyses (Student’s t -test). (L and M) Regulation of human TSN development by distinct signaling pathways. (L) KEGG enrichment diagram of human TSN marker genes. (M) Heat map of SCENIC binary regulon activities in TSNs and other neutrophil subtypes. Components of the NF-κB pathway are highlighted in orange. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the immunofluorescence staining, 5 μm-thick paraffin sections were deparaffinized and stained with anti-human CD66b (1:100, GTX19779, GeneTex), anti-human CD274 (1:100, #13684, Cell Signaling Technology), anti-mouse Ly6G (1:300, GB11229, ServiceBio), or anti-mouse Cd274 (15μg/mL, MAB1561, R&D Systems).

Techniques: Control, Protein-Protein interactions, Marker

Journal: International Journal of Molecular Sciences

Article Title: Low-Density Neutrophils Contribute to Subclinical Inflammation in Patients with Type 2 Diabetes

doi: 10.3390/ijms25031674

Figure Lengend Snippet: Clinical characteristics of the study population.

Article Snippet: Non-sorted HDNs or PBMCs (10 6 cells/mL) were incubated in PBS for 20 min at room temperature with various cell surface markers of neutrophils (Alexa Fluor 647 mouse anti-human CD66b, clone G10F5; Sony Biotechnology, San Jose, CA, USA), monocytes (Brilliant Violet 421 mouse anti-human CD14, clone M5E2; Sony Biotechnology, San Jose, CA, USA), NET components (rabbit anti-human citrullinated H3 (H3Cit); Abcam, Toronto, ON, Canada), and a neutrophil activation marker (PE mouse anti-human MPO, clone 5B8, BD Biosciences, San Jose, CA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Low-Density Neutrophils Contribute to Subclinical Inflammation in Patients with Type 2 Diabetes

doi: 10.3390/ijms25031674

Figure Lengend Snippet: Circulating neutrophils, HDNs, LDNs, and their respective ratios to lymphocytes. Isolated ( A ) LDNs, ( B ) HDNs, and ( C ) total neutrophil counts were determined by flow cytometry, and the values obtained were divided by the HV and T2D lymphocyte counts, respectively ( D – F ). The data are expressed as the absolute number of cells per liter of blood ( A – C ) or as a ratio ( D – F ). All values are presented as mean ± SEM. p < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01 vs. HV).

Article Snippet: Non-sorted HDNs or PBMCs (10 6 cells/mL) were incubated in PBS for 20 min at room temperature with various cell surface markers of neutrophils (Alexa Fluor 647 mouse anti-human CD66b, clone G10F5; Sony Biotechnology, San Jose, CA, USA), monocytes (Brilliant Violet 421 mouse anti-human CD14, clone M5E2; Sony Biotechnology, San Jose, CA, USA), NET components (rabbit anti-human citrullinated H3 (H3Cit); Abcam, Toronto, ON, Canada), and a neutrophil activation marker (PE mouse anti-human MPO, clone 5B8, BD Biosciences, San Jose, CA, USA).

Techniques: Isolation, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Low-Density Neutrophils Contribute to Subclinical Inflammation in Patients with Type 2 Diabetes

doi: 10.3390/ijms25031674

Figure Lengend Snippet: In Vitro isolated HDN and LDN adhesion to hECM. Isolated HDNs and HDNs were pre-treated with anti-human ß2 integrin/CD18 Ab and/or DNase I for 30 min prior to incubation with PBS, PMA (25 nM), and CRP (5 mg/L) for an additional 7.5 min on hECM-coated 48-plates. Adhered neutrophils were counted in four fields of view per well by optical microscopy using a digital camera. Adhesion of ( A ) basal (PBS) HDNs and LDNs from HVs and patients with T2D, agonist-induced ( B ) HDNs, and ( C ) LDNs from HVs and patients with T2D was expressed by the average number of adhered neutrophils/field from the four FOVs of each well. All values are presented as mean ± SEM. p < 0.05 was considered statistically significant. (aa p < 0.01, vs. corresponding HV; ** p < 0.01, *** p < 0.001 vs. PBS; † p < 0.05, †† p < 0.01, ††† p < 0.001 vs. corresponding PBS pre-treatment; ‡ p < 0.05 and ‡‡ p < 0.001 vs. DNase I).

Article Snippet: Non-sorted HDNs or PBMCs (10 6 cells/mL) were incubated in PBS for 20 min at room temperature with various cell surface markers of neutrophils (Alexa Fluor 647 mouse anti-human CD66b, clone G10F5; Sony Biotechnology, San Jose, CA, USA), monocytes (Brilliant Violet 421 mouse anti-human CD14, clone M5E2; Sony Biotechnology, San Jose, CA, USA), NET components (rabbit anti-human citrullinated H3 (H3Cit); Abcam, Toronto, ON, Canada), and a neutrophil activation marker (PE mouse anti-human MPO, clone 5B8, BD Biosciences, San Jose, CA, USA).

Techniques: In Vitro, Isolation, Incubation, Microscopy

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

Article Title: Mitocryptides from Human Mitochondrial DNA-Encoded Proteins Activate Neutrophil Formyl Peptide Receptors: Receptor Preference and Signaling Properties.

doi: 10.4049/jimmunol.1701719

Figure Lengend Snippet: FIGURE 6. MCTs trigger L-selectin shedding and granule mobilization in human neutrophils. Neutrophil exposure to MCT-ND4 (10 and 100 nM, FPR2), MCT-ND6 (100 nM, FPR1), or MCT-COX1 (100 nM, dual agonist) for 10 min at 37˚C induces CD62L shedding (A), upregulation of cell surface CD11b (B), and upregulation of cell surface CD66b (C) in a manner similar to the FPR1 agonist fMLF (10 and 100 nM). Data are presented as percentage of cleavage/upregulation compared with neutrophils incubated with buffer for 10 min at 37˚C (mean + SD, n = 3). Dashed lines represent the unchanged control levels, depicted as 100%.

Article Snippet: Allophycocyaninin- and PE-conjugated Abs against CD62L and CD11b were from Becton Dickinson (San Jose, CA), and FITC-conjugated Ab against CD66b was from AbD Serotec/Bio-Rad (Sundbyberg, Sweden). rTNF-a was from R&D Systems Europe (Abingdon, Oxon, U.K.), and HRP was from Boehringer Mannheim (Mannheim, Germany).

Techniques: Incubation, Control