Journal: eLife

Article Title: Microglial SIRPα regulates the emergence of CD11c+ microglia and demyelination damage in white matter

doi: 10.7554/elife.42025

Figure Lengend Snippet: Figure 4. Activation of microglia in the brain white matter of CD47 KO mice. (A) Immunofluorescence staining of coronal brain sections prepared from control (WT) or CD47 KO mice at 19 wks of age with antibodies to Iba1 (red) and CD11c (green). Merged images are shown. The boxed areas in the upper panels are shown at higher magnification in the lower panels. fi, fimbria. Scale bars: 100 mm (upper panels), 50 mm (lower panels). (B) Figure 4 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.42025 19 of 29 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody PE conjugated rat mAb to mouse CD172a (SIRPa) (clone P84) eBioscience (Cat# 12-1721-80) RRID:AB_11149864 FCM (1:100) Antibody PE conjugated rat mAbs to CD68 (clone FA-11) BioLegend (Cat# 137013) RRID:AB_10613469 FCM (1:100) Antibody PE conjugated rat mAb to mouse CD14 (clone Sa14-2) BioLegend (Cat# 123309) RRID:AB_940582 FCM (1:100) Antibody PE conjugated recombinant antibody (Ab) to Dectin-1 (REA154) Miltenyi Biotec (Cat# 130-102-987) RRID:AB_2651541 FCM (1:5) Antibody PE conjugated recombinant Ab to CD47 (REA170) Miltenyi Biotec (Cat# 130-103-108) RRID:AB_2659745 FCM (1:10) Antibody Rat mAb to myelin basic protein (MBP) (clone 12) Merck (Cat# MAB386) RRID:AB_94975 IHC (1:500) Antibody Rabbit pAb to Olig2 Immuno-Biological Laboratories (Gunma, Japan) (Cat# 18953) RRID:AB_1630817 IHC (1:400) Antibody Alexa Fluor 488 goat anti-rabbit IgG Molecular Probes (Cat# A11034) RRID:AB_2576217 IHC (1:200) Antibody Cy3-conjugated AffiniPure Goat anti-rabbit IgG Jackson Immuno Research (Cat# 111-165-144) RRID:AB_2338006 IHC (1:400) Antibody Cy3-conjugated AffiniPure Goat anti-rat IgG Jackson Immuno Research (Cat# 112-165-167) RRID:AB_2338251 IHC (1:200) Antibody Cy3-conjugated AffiniPure Goat anti-mouse IgG Jackson Immuno Research (Cat# 115-165-166) RRID:AB_2338692 IHC (1:400) Antibody Streptavidin, Alexa Fluor 488 conjugate Molecular Probes (Cat# S11223) RRID:AB_2336881 IHC (1:400) Commercial assay or kit Black-Gold II myelin staining kit Merck Cat# AG105 Commercial assay or kit Tyramide Signal Amplification (TSA) Biotin System kit Perkin Elmer Cat# NEL700A001KT Commercial assay or kit RNeasy Mini kit Qiagen Cat# 74106 Commercial assay or kit QuantiTect Reverse Transcription kit Qiagen Cat# 205313 Commercial assay or kit QuantiTect SYBR Green PCR kit Qiagen Cat# 204143 or 24163 Commercial assay or kit GeneChip 3’IVT Express Kit Affymetrix Cat# 901228 or 901229 Commercial assay or kit Ovation Pico WTA system V2 NuGEN Cat# 3302–12/ 60/–A01 Commercial assay or kit Encore Biotin Module NuGEN Cat# 4200–12/ 60/–A01 Chemical compound, drug Tamoxifen Toronto Research Chemicals Inc. Cat# T006000 Chemical compound, drug 4’,6-Diamidino-2phenylindole Nacalai Tesque (Kyoto, Japan) Cat# 11034–56 Continued on next page Sato-Hashimoto et al. eLife 2019;8:e42025.

Techniques: Activation Assay, Immunofluorescence, Staining, Control

Journal: eLife

Article Title: Microglial SIRPα regulates the emergence of CD11c+ microglia and demyelination damage in white matter

doi: 10.7554/elife.42025

Figure Lengend Snippet: Figure 5. Microarray transcriptome analyses of the white matter and the brain mononuclear cells of CD47 KO mice. (A,B) The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with DAVID. Statistically significant (p-value <0.01) KEGG enrichment pathways of up- (A) or downregulated (B) genes in the white matter (optic nerve and optic tract) (upper panels) or in the brain mononuclear cells (lower panels) of CD47 KO mice. Enrichment score is expressed as –Log (p-value). ARVC, Arrhythmogenic right ventricular cardiomyopathy; HCM, Hypertrophic Figure 5 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.42025 19 of 29 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody PE conjugated rat mAb to mouse CD172a (SIRPa) (clone P84) eBioscience (Cat# 12-1721-80) RRID:AB_11149864 FCM (1:100) Antibody PE conjugated rat mAbs to CD68 (clone FA-11) BioLegend (Cat# 137013) RRID:AB_10613469 FCM (1:100) Antibody PE conjugated rat mAb to mouse CD14 (clone Sa14-2) BioLegend (Cat# 123309) RRID:AB_940582 FCM (1:100) Antibody PE conjugated recombinant antibody (Ab) to Dectin-1 (REA154) Miltenyi Biotec (Cat# 130-102-987) RRID:AB_2651541 FCM (1:5) Antibody PE conjugated recombinant Ab to CD47 (REA170) Miltenyi Biotec (Cat# 130-103-108) RRID:AB_2659745 FCM (1:10) Antibody Rat mAb to myelin basic protein (MBP) (clone 12) Merck (Cat# MAB386) RRID:AB_94975 IHC (1:500) Antibody Rabbit pAb to Olig2 Immuno-Biological Laboratories (Gunma, Japan) (Cat# 18953) RRID:AB_1630817 IHC (1:400) Antibody Alexa Fluor 488 goat anti-rabbit IgG Molecular Probes (Cat# A11034) RRID:AB_2576217 IHC (1:200) Antibody Cy3-conjugated AffiniPure Goat anti-rabbit IgG Jackson Immuno Research (Cat# 111-165-144) RRID:AB_2338006 IHC (1:400) Antibody Cy3-conjugated AffiniPure Goat anti-rat IgG Jackson Immuno Research (Cat# 112-165-167) RRID:AB_2338251 IHC (1:200) Antibody Cy3-conjugated AffiniPure Goat anti-mouse IgG Jackson Immuno Research (Cat# 115-165-166) RRID:AB_2338692 IHC (1:400) Antibody Streptavidin, Alexa Fluor 488 conjugate Molecular Probes (Cat# S11223) RRID:AB_2336881 IHC (1:400) Commercial assay or kit Black-Gold II myelin staining kit Merck Cat# AG105 Commercial assay or kit Tyramide Signal Amplification (TSA) Biotin System kit Perkin Elmer Cat# NEL700A001KT Commercial assay or kit RNeasy Mini kit Qiagen Cat# 74106 Commercial assay or kit QuantiTect Reverse Transcription kit Qiagen Cat# 205313 Commercial assay or kit QuantiTect SYBR Green PCR kit Qiagen Cat# 204143 or 24163 Commercial assay or kit GeneChip 3’IVT Express Kit Affymetrix Cat# 901228 or 901229 Commercial assay or kit Ovation Pico WTA system V2 NuGEN Cat# 3302–12/ 60/–A01 Commercial assay or kit Encore Biotin Module NuGEN Cat# 4200–12/ 60/–A01 Chemical compound, drug Tamoxifen Toronto Research Chemicals Inc. Cat# T006000 Chemical compound, drug 4’,6-Diamidino-2phenylindole Nacalai Tesque (Kyoto, Japan) Cat# 11034–56 Continued on next page Sato-Hashimoto et al. eLife 2019;8:e42025.

Techniques: Microarray

Journal: bioRxiv

Article Title: Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

doi: 10.1101/2020.03.18.997080

Figure Lengend Snippet: ( a ) Outline of our “-omics” approach in human fibrotic lung integrating proteomics, secretomics, and genomics technology platforms to study the contribution of leukocytes and pathologic fibroblasts and to identify new therapeutic targets. ( b ) Single-cell, force-directed layout of fibrotic lung tissues. Shaded regions indicate the location of manually gated cell populations: green-shaded area represents leukocytes (CD45+), pink area represents epithelial cells (CK7+), blue area represents endothelial cells (CD31+) and grey dotted circle highlights the fibroblasts (CD45-CK7-CD31-). ( c ) Frequencies of cell populations in the lung detected by mass cytometry (CyTOF). Data are displayed as mean ± SD of 11 fibrotic and 3 normal control lung samples. ( d ) Principal component analysis (PCA) computed on mass cytometry data sets from fibroblast clusters from 11 individual pulmonary fibrosis patients (PF) and 3 normal donors (NC) demonstrating that fibrotic and normal fibroblasts were distinct from each other. ( e ) ViSNE maps of fibroblast mass cytometry data demonstrating that the abundance of fibroblasts differed; in normal controls the lung fibroblasts appeared heterogeneous while the fibroblasts clustered tightly together in fibrotic lungs (Blue: highlighted by the black dotted circle). The data demonstrate a representative example per group and each point in the viSNE map represents an individual cell. ( f ) ViSNE analysis of mass cytometry data of fibrotic lung (blue dots), normal lung (orange dots) and normal peripheral blood mononuclear cells (PBMCs, green dots) revealed increased activation of the JUN and AKT pathways in fibrotic lung fibroblasts. Schematic diagram of the location of the indicated cell types on the viSNE map are based on the expression of lineage-specific markers: epithelial cells (Epi), natural killer cells (NK), plasmacytoid dendritic cells (pDC), endothelial cells (EC), and macrophages (Mac). Red indicates high and blue low protein expression. ( g ) Representative mass cytometry plots of the pro-fibrotic fibroblast population in fibrotic lung compared with normal lung. ( h ) Immune fluorescent stains confirmed increased CD47 and PD-L1 co-expression in lung fibroblasts from fibrotic lungs compared to normal controls (activated fibroblasts expressing FSP1+ Collagen1+ and SMA+). The arrow indicates the blood vessel. (Scale bars, 100 μm). ( i ) RNA expression analysis of JUN , PD-L1 and CD47 in fibrotic and normal lung fibroblasts as detected by QPCR. Data are expressed as mean ± SD of 5 fibrotic fibroblasts and 3 normal fibroblasts and are representative of at least three experiments. Data were analyzed by twotailed unpaired t -test, * P < 0.05. See Supplementary Table 4 for statistical details.

Article Snippet: For CD47 antibody blockade experiments, mice were injected intraperitoneally (IP) with a dose of 500 μg CD47 antibody (Clone MIAP410, Bioxcell) diluted in 100 μl of PBS on day 4.

Techniques: Biomarker Discovery, Mass Cytometry, Control, Activation Assay, Expressing, RNA Expression

Journal: bioRxiv

Article Title: Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

doi: 10.1101/2020.03.18.997080

Figure Lengend Snippet: ( a ) ViSNE map of concatenated fibroblasts (CD45-CD31-CK7-population) from fibrotic lung (black dotted circle) and normal lung demonstrating increased expression of PDGFRa, podoplanin, CD47 and PD-L2 but not calreticulin in subsets of fibroblasts in fibrotic lungs. ( b ) Representative CyTOF plots of PD-L2 and calreticulin protein in fibroblasts from fibrotic and normal lungs indicating increased PD-L2 but no difference in calreticulin expression. ( c ) Quantitation of CD47 and PD-L1 immune stains in fibrotic and normal lung biopsies. Data are expressed as mean ± SD and analyzed by two-tailed unpaired t -test, ** P < 0.01; **** P < 0.0001. The immune stains were evaluated by a blinded pathologist, in addition to image J software. ( d ) A representative haematoxylin and eosin staining of fibrotic and normal lung tissue. The inserted black frames highlight the fibrotic and normal areas. Scale bar, 100 μm. ( e ) Multiplexed ion beam imaging (MIBI) and relevant quantitation demonstrated the co-expression of JUN and FOS with CD47 in fibroblasts in fibrotic plaques in lungs of idiopathic pulmonary fibrosis patients. Representative MIBI analysis of lung biopsy sections from 5 patients with idiopathic pulmonary fibrosis were stained with metal-conjugated antibodies. In total, 10 different markers (JUN, JUNB, JUND, FRA1, FRA2, FOS, FOSB, COLLAGEN1, CD47 and Hematoxylin) were analyzed. Eight fields of view were acquired with ten repeat scans over a single area. Experiments were run multiple times, representative examples and related analyses are shown as mean ± SD. Scale bar, 100 μm. ( f ) ELISA detected increased levels of secreted PD-L1 in fibrotic lung BAL compared to normal lungs. Data are expressed as mean ± SD of 5 fibrotic and 3 normal samples. Data were analyzed by two-tailed unpaired t -test, * P < 0.05. See Supplementary Table 4 for statistical details.

Article Snippet: For CD47 antibody blockade experiments, mice were injected intraperitoneally (IP) with a dose of 500 μg CD47 antibody (Clone MIAP410, Bioxcell) diluted in 100 μl of PBS on day 4.

Techniques: Expressing, Quantitation Assay, Two Tailed Test, Software, Staining, Imaging, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

doi: 10.1101/2020.03.18.997080

Figure Lengend Snippet: ( a ) We analyzed dendritic cells in fibrotic and normal lungs with mass cytometry and found increased percentages of myeloid dendritic cells (mDC: CD45+ nonB nonT nonNK nonmacrophage CD11c+CD123-) in fibrotic lung but no difference for plasmacytoid dendritic cells (pDC: CD45+ nonB nonT nonNK nonmacrophage CD11c-CD123+). ( b ) IDO protein expression in macrophages from fibrotic lungs is decreased compared to macrophages from normal control lungs. Raw values of means of CyTOF data are displayed on a per-patient basis with mean ± SD of 11 fibrotic and 3 normal samples and analyzed by two-tailed unpaired t -test, * P < 0.05. ( c ) The viSNE maps colored by intensity of expression (red is high, and blue is low) demonstrate the expression of IDO, ARG1, CD47, CD16, CD163 and CD11c in macrophages derived from fibrotic lungs which clustered spatially within the black circled area. (d) Representative histogram of mass cytometry data demonstrates decreased alveolar macrophages (AM) but increased interstitial macrophages (IM) in human fibrotic lungs. ( e ) Individual viSNE analysis of AM and IM from fibrotic lung (blue) and normal lungs (orange) suggested the immunophenotypes of AM and IM in the fibrotic tissues are clearly different from those in the normal lungs. Macrophages derived from fibrotic lungs are highlighted by the dotted black circles. ( f ) Quantitation of PD-1+ expression on macrophages (CD68+) in fibrotic and normal lung biopsies. Data are expressed as mean ± SD and analyzed by unpaired t test with Welch’s correction (Two-tailed), ** P < 0.01. The immune stains were evaluated by a blinded pathologist, in addition to image J software. See Supplementary Table 4 for statistical details.

Article Snippet: For CD47 antibody blockade experiments, mice were injected intraperitoneally (IP) with a dose of 500 μg CD47 antibody (Clone MIAP410, Bioxcell) diluted in 100 μl of PBS on day 4.

Techniques: Mass Cytometry, Expressing, Control, Two Tailed Test, Derivative Assay, Quantitation Assay, Software

Journal: bioRxiv

Article Title: Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

doi: 10.1101/2020.03.18.997080

Figure Lengend Snippet: ( a ) Heatmap demonstrating dynamic chromatin changes in fibrotic lung fibroblasts with ( JUN -KO) or without (Control) JUN deletion and normal lung fibroblasts with (TetO- JUN Dox+) or without (TetO- JUN Dox-) JUN activation. ( b ) Representative genome browser tracks comparing ATAC-seq signal in fibrotic lung fibroblasts (with ( JUN -KO) or without (Control) JUN -knockout) and also ChIP-seq signal in normal lung fibroblasts (with (TetO- JUN Dox+) or without (TetO- JUN Dox-) JUN overexpression) with A549, MCF7, h1-hESC, HepG2 and K562 from published data at JUN , CD47 and CD274 loci. The red boxes highlight ATAC-seq and ChIP-seq peaks in the promoter sites of JUN , CD47 and CD274 (and enhancer is shown in green). We also compared our peaks with H3K4me3 or H3K27Ac (=histone mark for open chromatin), H3K9me3 or H3K27me3 (=histone mark for closed chromatin), ChIP-seq data generated from normal human lung fibroblasts is from published data, which highlighted the same areas respectively. ( c ) Gene expression changes in primary lung fibroblasts from JUN knockout (KO) compared to overexpression (OE). QPCR values were normalized to the value in JUN KO. Four experimental repeats. Ratio paired t test, ** P < 0.01; **** P < 0.0001. ( d ) Representative flow cytometry histograms showing reduced expression of pJUN, PD-L1 and CD47 after JUN overexpression (OE) or KO. Yellow plot: JUN overexpression; Black plot: JUN knockout. ( e ) Vector maps of the control and CD47 enhancer constructs used to engineer reporter cell lines. ( f, g ) CD47 enhancer reporter assays demonstrating doxycycline-induced JUN expression initiated CD47 enhancer expression which disappeared when JUN expression was turned off ( f ) or JUN was knocked out ( g ). Data are expressed as mean ± SD, Ordinary one-way ANOVA (Tukey’s multiple comparisons test), n.s., non-significant; * P < 0.05; *** P < 0.001; **** P < 0.0001. See Supplementary Table 4 for statistical details.

Article Snippet: For CD47 antibody blockade experiments, mice were injected intraperitoneally (IP) with a dose of 500 μg CD47 antibody (Clone MIAP410, Bioxcell) diluted in 100 μl of PBS on day 4.

Techniques: Control, Activation Assay, Knock-Out, ChIP-sequencing, Over Expression, Generated, Gene Expression, Flow Cytometry, Expressing, Plasmid Preparation, Construct

Journal: bioRxiv

Article Title: Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

doi: 10.1101/2020.03.18.997080

Figure Lengend Snippet: ( a, b ) Quantitative comparative analysis of ATAC-seq peaks obtained from fibrotic lung fibroblasts with and without JUN deletion as well as normal lung fibroblasts with or without JUN overexpression. The top ten significant pathways which were associated with down regulation (labeled as Promoter Down in red) or up regulation (labeled as Promoter Up in blue) of the promoters were shown. ( c ) Venn Diagram generated by comparing downregulated promoters in fibrotic lung fibroblasts after JUN deletion with published RNA-seq data of bulk fibrotic lung samples demonstrating that 1.6% or 70 of the genes which overlapped between these two distinct data sets encoded profibrotic pathways (red) and pathways which encoded T-cell exhaustion (green). ( d ) Reporter assays for the CD47 enhancer demonstrating continuously increasing activation of the CD47 enhancer (E7TK) reflected by increased EGFP expression with increased JUN expression ( JUN -OE) while the CD47 enhancer activity decreased with doxycycline removal (turns JUN off) in a timely dependent manner and JUN deletion with CRISPR-Cas9 knock-out ( JUN -KO) abolished the enhancer activity. Meanwhile the control TK vector showing no differences with JUN modification. Scale bar, 100 μm. See Supplementary Table 4 for statistical details.

Article Snippet: For CD47 antibody blockade experiments, mice were injected intraperitoneally (IP) with a dose of 500 μg CD47 antibody (Clone MIAP410, Bioxcell) diluted in 100 μl of PBS on day 4.

Techniques: Over Expression, Labeling, Generated, RNA Sequencing, Activation Assay, Expressing, Activity Assay, CRISPR, Knock-Out, Control, Plasmid Preparation, Modification

Journal: bioRxiv

Article Title: Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

doi: 10.1101/2020.03.18.997080

Figure Lengend Snippet: ( a ) The secreted proteins in the lung bronchoalveolar lavage (BAL) of fibrotic lung patients were quantified by Luminex assay, showing IL-6 as the highest expressed cytokine across all fibrotic patient BAL samples. Data were normalized by protein levels of the BAL of normal lung and presented as mean ± SD. ( b ) Cytokines and chemokines in the fibrotic mouse BAL after Jun induction were quantified by Luminex assay. IL-6 was consistently among the most highly expressed cytokines in Jun -induced mouse fibrotic lungs indicative of IL-6-JAK-STAT pathway activation. Data were normalized by normal lung expression and presented as mean ± SD. ( c ) The cytokines/chemokines released from Jun -induced, lung-fibrotic, mouse-derived whole bone marrow, fibroblasts and monocytes/macrophages in the medium after 48h of Dox-initiated Jun induction were quantified by Luminex assay, demonstrating that whole bone marrow and fibroblasts are secreting increased IL-6 in response to Jun. Data are presented as mean ± SD. ( d ) Increased IL-6 expression levels were detected by QPCR and flow cytometry in primary lung fibroblasts with JUN knock-out (KO) or overexpression (OE). Four experimental repeats. Ratio paired t test, *** P < 0.001. ( e, f ) IL-6 increased CD47 enhancer activity at concentrations as low as 1 ng/ml ( e ) and protein expression at 10 ng/ml ( f ) in a dose-dependent fashion. Data are expressed as mean ± SD, Ordinary one-way ANOVA with multiple comparisons test, n.s., non-significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. See Supplementary Table 4 for statistical details.

Article Snippet: For CD47 antibody blockade experiments, mice were injected intraperitoneally (IP) with a dose of 500 μg CD47 antibody (Clone MIAP410, Bioxcell) diluted in 100 μl of PBS on day 4.

Techniques: Luminex, Activation Assay, Expressing, Derivative Assay, Flow Cytometry, Knock-Out, Over Expression, Activity Assay

Journal: bioRxiv

Article Title: Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

doi: 10.1101/2020.03.18.997080

Figure Lengend Snippet: ( a ) Schematic maps showing that the promoter sites (highlighted in red) for IL-6, IL-6R, and IL-6ST depended on JUN expression in normal lung fibroblasts with (TetO- JUN Dox+) or without (TetO- JUN Dox-) JUN overexpression and fibrotic lung fibroblasts with ( JUN -KO) or without (Control) JUN knockout with CRISPR-Cas9 but not in other cell lines like A549, MCF7, h1-hESC, HepG2 and K562. We also compared our data to publicly available H3K4me3 or H3K27Ac (=histone mark for open chromatin), H3K9me3 or H3K27me3 (=histone mark for closed chromatin) ChIP-seq data generated from normal human lung fibroblast from published data to confirm the regions of open chromatin for the IL-6 family members. ( b ) IL-6 expression in the bronchoalveolar lavages (BAL) of fibrotic and normal lungs were measured by ELISA showing dramatically increased secreted IL-6 protein. Data are expressed as min to max of 5 fibrotic and 3 normal samples. Data were analyzed by unpaired t test with Welch’s correction (Two-tailed), *** P < 0.001. ( c ) CD47 constituent enhancer-driven EGFP reporter (E7TK) expression was activated and increased in lung fibroblast cells treated with IL-6 in a dose dependent manner. Control cells were transduced with the lentiviral cassette containing the thymidine kinase (TK) minimal promoter only. Scale bar, 100 μm. See Supplementary Table 4 for statistical details.

Article Snippet: For CD47 antibody blockade experiments, mice were injected intraperitoneally (IP) with a dose of 500 μg CD47 antibody (Clone MIAP410, Bioxcell) diluted in 100 μl of PBS on day 4.

Techniques: Expressing, Over Expression, Control, Knock-Out, CRISPR, ChIP-sequencing, Generated, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Transduction

Journal: bioRxiv

Article Title: Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

doi: 10.1101/2020.03.18.997080

Figure Lengend Snippet: ( a ) Wholelung scaffold map for bleomycin-induced lung fibrosis in mice. Each node represents unsupervised cell clusters. ( b ) Representative mass cytometry plot demonstrating increased expression of immune checkpoint proteins CD47 and PD-L1 in fibroblasts and an expansion of CD11b+F4/80+ macrophages, regulator T cells (CD3+CD4+CD25+FOXP3+) and exhausted T cells (CD3+CD8+PD-1+TIM3+) in mouse model after fibrosis induction with bleomycin for 2 weeks. ( c, d ) Representative images of Micro CT scans of wildtype and B6.129S2- Il6 tm1Kopf /J (IL-6KO) mice highlighting increased fibrosis in the lung after fibrosis induction (wildtype and IL-6KO mice) and much improved fibrosis after treatment with HAC (anti-PD-L1) alone or combined with a blocking antibody against CD47 or/and IL-6. Data are expressed as mean ± SD of 5 animals and analyzed by using one-way ANOVA for multiple comparisons test. n.s., non-significant; * P < 0.05; **** P < 0.0001. ( e ) Trichrome of lung sections of control mice, mice after fibrosis induction with bleomycin (wildtype and IL-6KO mice) and mice after treatment with blocking antibodies against IL-6 and CD47 and HAC (the blocking reagent against PD-L1) demonstrating dramatically improved fibrosis (significantly decreased blue stained areas on Masson’s trichrome stain which correspond to cross-linked collagen) and diminished PD-L1 expression in FSP1+ fibroblasts after treatment. Scale bar, 100 μm. See Supplementary Table 4 for statistical details.

Article Snippet: For CD47 antibody blockade experiments, mice were injected intraperitoneally (IP) with a dose of 500 μg CD47 antibody (Clone MIAP410, Bioxcell) diluted in 100 μl of PBS on day 4.

Techniques: Mass Cytometry, Expressing, Micro-CT, Blocking Assay, Control, Staining

Journal: bioRxiv

Article Title: Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

doi: 10.1101/2020.03.18.997080

Figure Lengend Snippet: ( a ) Histogram plots of mass cytometry data of phosphor p-JUN expression in lung fibroblasts comparing two different mouse models of lung fibrosis—the bleomycin-induced lung fibrosis model abundantly used by many labs and the JUN-induced lung fibrosis model—both demonstrated increased activation and phosphorylation of JUN after initiation of lung fibrosis in mice. ( b ) The time course of bleomycin induction in mice and in vivo treatment with blocking antibodies. ( c, d ) Morphological and molecular markers of representative histologic sections of wildtype and B6.129S2- I16 tm1Kopf /J ( IL-6KO ) mice lung tissues after fibrosis induction and treatment with blocking antibodies against immune checkpoint inhibitors and IL-6. Hematoxylin-Eosin (H.E.) stains and CD47, FSP1 counterstained with DAPI ( c ), Masson’s Trichrome stains and PD-L1 and FSP1 with DAPI ( d ) demonstrating improved fibrosis along with decreased CD47 and PD-L1 immune checkpoint protein expression in fibroblasts (FSP1+). Scale bar, 100 μm. ( e ) Quantitation of PD-L1 and CD47 expression in fibroblasts and collagen fibrosis of 10 high power fields (40x) of trichrome-stained sections. Data are expressed as mean ± SD, ordinary one-way ANOVA (Dunnett’s multiple comparisons test), n.s., non-significant; **** P < 0.0001. The immune stains were evaluated by a blinded pathologist, in addition to image J software. ( f ) In vivo analysis of human fibrotic fibroblasts in kidney capsule adoptive transfer assay in NSG mice to study efficacy of PD-1/PD-L1 blockade with HAC protein. Representative bioluminescence imaging (BLI) image and quantification of luminescence intensity, trichrome and anti-GFP staining of kidney area with the xenograft demonstrate that PD-1/PD-L1 blockade with HAC increased fibrotic fibroblast clearance compared to placebo (PBS). Data are expressed as mean ± SD and analyzed by using two-way ANOVA followed by Tukey’s multiple comparisons test. ** P < 0.01. Scale bar, 100 μm. See Supplementary Table 4 for statistical details.

Article Snippet: For CD47 antibody blockade experiments, mice were injected intraperitoneally (IP) with a dose of 500 μg CD47 antibody (Clone MIAP410, Bioxcell) diluted in 100 μl of PBS on day 4.

Techniques: Mass Cytometry, Expressing, Activation Assay, Phospho-proteomics, In Vivo, Blocking Assay, Quantitation Assay, Staining, Software, Adoptive Transfer Assay, Imaging

Journal: bioRxiv

Article Title: Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

doi: 10.1101/2020.03.18.997080

Figure Lengend Snippet: Left: In fibrotic lung, we find persistent myofibroblast activation in fibrotic plaques and JUN upregulation. JUN expression in fibrosis-associated fibroblasts (FAFs) appears to directly control the promoters and enhancers of CD47 and CD274 (PD-L1). The direct consequence is increased expression of these immune checkpoint proteins in fibroblasts and dormant macrophages which do not phagocytose, but continue to release chronic inflammatory cytokines. JUN also directly regulates IL-6 at the chromatin level. The increased expression and secretion of this potent cytokine leads to a suppressive adaptive immune response—chiefly T cell exhaustion and upregulation of regulatory T cells. Right: Disrupting the suppression of the innate and adaptive immunity with CD47 and PD-L1 inhibitors as well as the proinflammatory IL-6 cytokine pathway stimulated phagocytic removal of profibrotic fibroblasts and T-cell activation leading to clearance of the fibrosis in the lung.

Article Snippet: For CD47 antibody blockade experiments, mice were injected intraperitoneally (IP) with a dose of 500 μg CD47 antibody (Clone MIAP410, Bioxcell) diluted in 100 μl of PBS on day 4.

Techniques: Activation Assay, Expressing, Control

Journal: Frontiers in Immunology

Article Title: IL-27 Derived From Macrophages Facilitates IL-15 Production and T Cell Maintenance Following Allergic Hypersensitivity Responses

doi: 10.3389/fimmu.2021.713304

Figure Lengend Snippet: CD3, CD47, and CD172a expression in human ACD clusters. (A–C) Immunofluorescence staining of CD3 (green), CD47 (red), CD172a (purple), CD14 [green, a serial slide section with the staining of CD172a (purple)], and Hoechst (blue) in human donor-matched patch-test negative control and patch-test (+) ACD skin. White dashed lines mark the epidermal-dermal junction. Data are representative of 3 patient samples per tested condition. (A) Scale bars are 200 µm (left) and 100 µm (right). (B, C) Scale bars are 20 µm.

Article Snippet: Mouse IgG1 isotype control (MOPC-21) (Tonbo Biosciences), Goat IgG isotype control (R&D Systems), Sheep IgG isotype control (R&D Systems), Rabbit isotype control (Southern Biotech, Birmingham, AL), anti-human CD14 (61D3, Tonbo Biosciences), anti-human iNOS (polyclonal, Thermo Fisher Scientific), anti-human CD8 (MCD8, Santa Cruz Biotechnology, Dallas, TX), and IL27R (polyclonal, R&D Systems), anti-human IL-27 (polyclonal, R&D Systems), anti-human CD86 (IT2.2, Biolegend), anti-human CD3 (SP7, Abcam, Cambridge, England), anti-human CD47 (polyclonal, R&D Systems), anti-human SIRP alpha (CD172a) (OTI7B3, Origene), anti-human IL-15 (polyclonal, R&D systems), anti-human BCL2 (clone 100, BioLegend), anti-mouse CD3 (17A2, Tonbo Biosciences), and anti-mouse CD8 (YTS 105.18, Novus Biologicals, Littleton, CO) followed by reaction with Cy3, Alexa Fluor 555, Alexa Fluor 647, Alexa Fluor 488, or FITC-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Expressing, Immunofluorescence, Staining, Negative Control

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: SARS-CoV-2 infection is associated with increased CD47 levels. A) TF protein abundance in uninfected (control) and SARS-CoV-2-infected (virus) Caco-2 cells (data derived from . P-values were determined by two-sided Student’s t-test. B) CD47 and SARS-CoV-2 N protein levels and virus titres (genomic RNA determined by PCR) in SARS-CoV-2 strain FFM7 (MOI 1)-infected air-liquid interface cultures of primary human bronchial epithelial (HBE) cells and SARS-CoV-2 strain FFM7 (MOI 0.1)-infected Calu-3 cells. Uncropped blots are provided in Suppl. Figure 1. C) CD47 mRNA levels in post mortem samples from COVID-19 patients (data derived from ). P-values were determined by two-sided Student’s t-test.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Infection, Quantitative Proteomics, Control, Virus, Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 aging” (A) and “CD47 hypertension” (B). C) Overview figure of the data derived from the literature searches. Age-related increased CD47 levels may contribute to pathogenic conditions associated with severe COVID-19.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 diabetes” (A). B) Overview figure of the data derived from the literature search. Hyperglycaemia- and diabetes-induced increased CD47 levels may contribute to immune escape of SARS-CoV-2-infected cells.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay, Infection