Online supplement. Phenotypic, functional and plasticity features of classical and alternatively activated

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1 Online supplement Phenotypic, functional and plasticity features of classical and alternatively activated human macrophages Abdullah Al Tarique*, Jayden Logan *, Emma Thomas *, Patrick G Holt *, Peter D Sly *, Emmanuelle Fantino* * Queensland Children s Medical Research Institute (QCMRI), Royal Children s Hospital, Brisbane, The University of Queensland, Australia, Telethon Kids Institute, The University of Western Australia.

2 Table S1: List of monoclonal antibodies, clones and manufacturers CD antigen Clone Fluorochrome IgG class Source CD1a HI149 BV421 M IgG1, k BioLegend CD1b SN13 PE M IgG1, k BioLegend CD11b ICRF44 APC M IgG1, k BD CD14 HCD14 PE Cy 7 M IgG1, k BioLegend CD23 EBVCS-5 PerCP Cy5.5 M IgG1, k BioLegend CD PE Cy7 M IgG1, k BD CD68 Y1/82A AF488 M IgG2b, k BioLegend CD68 Y1/82A AF647 M IgG2b, k BD CD80 2D10 BV421 M IgG1, k BioLegend CD163 GHI/61 PE-CF594 M IgG1, k BD CD200R OX108 FITC M IgG, k AbDSerotec CD PE Cy 5 M IgG1, k BD CD209 DCN46 BV450 M IgG2b, k BD CD226 11A8 APC M IgG2b, k BioLegend 7-AAD BD Table S2: Antibody panels for phenotyping Panel 1 Panel 2 Panel 3 CD68 AF488 CD64 PE Cy7 CD80 V421 CD206 PE Cy5 CD68 AF488 CD1a V421 CD1b PE CD163 PE-CF594 CD226 APC CD11b APC CD209 V450 CD200R FITC CD23 PerCP Cy5.5

3 Legend for Supplementary Figures Suppl Figure S1: Morphology and CD68 expression of GM-CSF and M-CSF derived macrophages. CD14+ Monocytes from healthy donors were cultured with GM-CSF or M-CSF containing medium for 6 days to differentiate into uncommitted macrophages (M0). (A) Morphology was observed under microscope. (B) Intracellular expression of CD68, a macrophage marker in human, was analysed by flow cytometry. M1 macrophages were then generated by stimulating with LPS alone, IFN-γ alone and LPS plus IFN-γ. M2 polarization was performed with IL-4 alone, IL-13 alone and IL-4+IL-13 for 2-days. Population frequencies of M0, M1 an M2 conditioned cells were assessed by CD1a and CD1b marker combination (C). Bar graph represents the mean and SD of population frequencies of CD64/CD80 and CD11b/CD209 respectively. Data were representative of at least 5 different individual subjects. Statistical significance was calculated using nonparametric one-way ANOVA. Suppl Figure S2: Phenotypic characterization of M-CSF derived human M1 and M2 macrophages. Monocytes from healthy donors were cultured with M-CSF containing medium for 6 days to obtain uncommitted macrophages (M0). M1 macrophages were then generated by stimulating with LPS alone, IFN-γ alone and LPS plus IFN-γ. M2 polarization was performed with IL-4 alone, IL- 13 alone and IL-4+IL-13 for 2-days. Population frequencies of M0, M1 an M2 conditioned cells were assess for two different panels; A: CD64 and CD80, and B: CD11b and CD209. Bar graphs (C & D) represent the mean and SD of population frequencies of CD64/CD80 and CD11b/CD209 population respectively. Data were representative of 3 different individual donors. Statistical significance was calculated using nonparametric one-way ANOVA. Suppl Figure S3: Inflammatory and functional profile of M-CSF derived human M1 and M2 macrophages. M-CSF derived uncommitted macrophages (M0) were induced with

4 LPS alone, IFN-γ alone or LPS+ IFN-γ. M2 polarization was performed with IL-4 alone, IL- 13 alone and IL-4+IL-13 for 2-days. (A) A panel of cytokines and chemokines were analysed in the supernatants of M0, M1 and M2 macrophages. Each symbol represents an independent experiment. Error bars represent the SEM for n=3. Statistical significance was calculated using non-parametric one-way ANOVA. (B) M0, M1 and M2 polarized macrophages were treated with either FITC-labelled E. coli bacteria for phagocytosis and AF-647 labelled dextran (10KD) for endocytosis for 90 mins at 37C. Data reflect 3 independent experiments from 3 independent donors. Error bars represent the SEM. Significance was determined using nonparametric one-way ANOVA among M0, M1 and M2 groups. Suppl Figure S4: Phenotypic and gene expression reversibility of reprogrammed M1 and M2 macrophages. Previously IFN-γ treated M1 and IL-13 treated M2 cells were left in cytokine free medium till 12 days after initial polarization. IL-13 stimulus was then given to M1 cells and IFN-γ was given to M2 cells for next 2 days. (A) Population frequencies of switched M1 (M2 M1) and switched M2 (M1 M2) cells were assessed using two previously described markers; A: CD64 and CD80, and B: CD11b and CD209. (B) Gene expression profile of switched M1 (M2 M1) and switched M2 (M1 M2) cells were analysed by qpcr. Data were representative of 5 individual donors. Mean and SEM are shown. Statistical significance was calculated using nonparametric one-way ANOVA.

5 A GM-CSF differentiated Macrophages M-CSF differentiated Macrophages B CD68 C M0 Isotype Control IFN- LPS LPS+IFN- CD1a 54.7% IL-4 IL-13 IL-4+IL-13 CD1b Suppl Figure S1: Morphology and CD68 expression of GM-CSF and M-CSF derived macrophages. CD14+ Monocytes from healthy donors were cultured with GM-CSF or M-CSF containing medium for 6 days to differentiate into uncommitted macrophages (M0). (A) Morphology was observed under microscope. (B) Intracellular expression of CD68, a macrophage marker in human, was analysed by flow cytometry. M1 macrophages were then generated by stimulating with LPS alone, IFN- alone and LPS plus IFN-. M2 polarization was performed with IL-4 alone, IL-13 alone and IL-4+IL-13 for 2-days. Population frequencies of M0, M1 an M2 conditioned cells were assessed by CD1a and CD1b marker combination (C). Bar graph represents the mean and SD of population frequencies of CD64/CD80 and CD11b/CD209 respectively. Data were representative of at least 5 different individual subjects. Statistical significance was calculated using nonparamatric one-way ANOVA.

6 CD80 A M0 Isotype Control IFN- LPS LPS+IFN- IL-4 CD64 B CD209 M0 IFN- IL-4 LPS IL-13 Isotype Control LPS+IFN- Suppl Figure S2: Phenotypic characterization of M-CSF derived human M1 and M2 macrophages. Monocytes from healthy donors were cultured with M-CSF containing medium for 6 days to get differentiated and uncommitted macrophages (M0). M1 macrophages were then generated by stimulating with LPS alone, IFN- alone and LPS plus IFN-. M2 polarization was performed with IL-4 alone, IL-13 alone and IL- 4+IL-13 for 2-days. Population frequencies of M0, M1 an M2 conditioned cells were assess for two different panels; A: CD64 and CD80, and B: CD11b and CD209. Bar graphs (C & D) represent the mean and SD of population frequencies of CD64/CD80 and CD11b/CD209 population respectively. Data were representative of 3 different individual donors. Statistical significance was calculated using nonparamatric one-way ANOVA. CD11b

7 A B Suppl Figure S3: Inflammatory and functional profile of M-CSF derived human M1 and M2 macrophages. M-CSF derived uncommitted macrophages (M0) were induced with LPS alone, IFN- alone or LPS+ IFN-. M2 polarization was performed with IL-4 alone, IL-13 alone and IL-4+IL-13 for 2-days. (A) A panel of cytokines and chemokines were analysed in the supernatants of M0, M1 and M2 macrophages. Each symbol represents an independent experiment. Error bars represent the SEM for n=3. Statistical significance was calculated using non-parametric one-way ANOVA. (B) M0, M1 and M2 polarized macrophages were treated with either FITC-labelled E. coli bacteria for phagocytosis and AF- 647 labelled dextran (10KD) for endocytosis for 90 mins at 37C. Data reflect 3 independent experiments from 3 independent donors. Error bars represent the SEM. Significance was determined using nonparamatric one-way ANOVA among M0, M1 and M2 groups.

8 A Polarized phenotype CD80 IFN- -M1 IL-13-M2 CD209 Day 6 after initial polarization CD64 CD11b IL-13 IFN- Switching to opposite polarized state CD209 M1 M2 M2 M1 CD80 CD11b Switched M2 (M1 M2) CD64 Switched M1 (M2 M1) B Suppl Figure S4: Phenotypic and gene expression reversibility of reprogrammed M1 and M2 macrophages. Previously IFN- treated M1 and IL-13 treated M2 cells were left in cytokine free medium till 12 days after initial polarization. IL-13 stimulus was then given to M1 cells and IFN- was given to M2 cells for next 2 days. (A) Population frequencies of switched M1 (M2 M1) and switched M2 (M1 M2) cells were assessed using two previously described markers; A: CD64 and CD80, and B: CD11b and CD209. (B) Gene expression profile of switched M1 (M2 M1) and switched M2 (M1 M2) cells were analysed by qpcr. Data were representative of 5 individual donors. Mean and SEM are shown. Statistical significance was calculated using nonparamatric one-way ANOVA.

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