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The importance of being “pure” neutrophils

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1 The importance of being “pure” neutrophils
Federica Calzetti, BS, Nicola Tamassia, PhD, Fabio Arruda-Silva, MS, Sara Gasperini, PhD, Marco A. Cassatella, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 139, Issue 1, Pages e6 (January 2017) DOI: /j.jaci Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Patterns of gene expression and cytokine production by Neu, wbNeuM, and wbNeuE incubated with R848, poly(I:C), or TNF-α. Neu, wbNeuM, and wbNeuE were incubated with 5 μM R848, 50 μg/mL poly(I:C), or 10 ng/mL TNF-α for mRNA expression analysis by RT-quantitative PCR (A) and cytokine production by ELISA (B). Analysis of IL-6 and TNF-α was performed after 4 hours of incubation, whereas that of CXCL8, IL-10, IFIT1, and ISG15 was performed after 20 hours. Panels A and B display representative experiments out of 3 performed with similar results. MNE, Mean normalized units; n.d., not done. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Effects of the removal of various contaminating leukocyte subtypes from wbNeuM and wbNeuE. Percentages of decrease in cytokine production (A) and gene expression (B-D), observed in wbNeuM (Fig 2, A and B) and wbNeuE (Fig 2, A-D) depleted of slan+CD16+-monocytes, alone (Fig 2, A and B) (n = 2-5), or together with B cells, natural killer cells, or pDCs (Fig 2, C and D) (n = 3). Analysis of IL-6 and TNF-α mRNA expression/production was performed after 4 hours, whereas that of IL-10, IFIT1, and ISG15 was performed after 20 hours of incubation (Fig 2, C and D). *P < .05 by 1-way ANOVA followed by Tukey posttest. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 CXCL8 production and gene expression patterns in Neu and NeuF incubated with R848 or poly(I:C). Neu and NeuF were incubated for 20 h with either 5 μM R848 or 10 ng/ml TNFα for CXCL8 release (A), or with either R848 or 50 μg/ml poly(I:C) for IL-6 and TNFα (B), or IFIT1 and ISG15 (C), mRNA expression analysis, by RT-qPCR, performed after 4 and 20 h of incubation, respectively. Panel A reports the mean of 2 experiments, while panels B and C display representative experiments out of 3 performed with similar results. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Assessment of slan+CD16+-monocyte removal from wbNeuM and wbNeuE. After wbNeuM and wbNeuE neutrophils isolation, slan+CD16+-monocytes were removed by separation using αM-DC8-linked microbeads via MACS column. Flow cytometry analysis was then used to assess the levels of contaminant slan+CD16+-monocytes present in neutrophils preparations. Accordingly, contaminating PBMCs were first identified by SSClow/CD45 positivity (left panels, black gate), and then slan+CD16+-monocytes were selected among them, as M-DC8/CD16 double positive cells (right panels, red gate) and overlayed (red dots) on total CD45 cells. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 IL-10 secretion assay. 2 x 106 WbNeuE were treated with or without 5 μM R848 or 10 ng/mL TNFα for 14 h and then assayed for IL-10 secretion. Histograms, displaying 1 representative experiment out of 2 with similar results, show that only slan+CD16+-monocytes (right panel), but not neutrophils (left panel), secrete IL-10. Similar results were obtained using WbNeuM. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 Assessment of slan+CD16+-monocyte, B cell, NK cell and pDC removal from wbNeuE. After isolation, wbNeuE were stained with FITC-conjugated antibodies towards M-DC8 (for slan+CD16+-monocytes) alone or in combination with CD19 (for B cells), CD56 (for NK cells) or CD303 (for pDCs). Panels show that removal of stained cells was efficiently achieved by anti-FITC microbeads via MACS column separation. In fact, contaminating PBMCs were first gated by SSClow/CD45 positivity (upper panels, black gate), and then slan+CD16+-monocytes (red dots), B cells (light blue dots), NK cells (green dots) and pDCs (purple dots) were identified, using specific CD markers overlaid on HLA-DR/CD123 dot plots. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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