Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: CD55 is highly and consistently on MDSCs in patients with cancer. ( A–H ) UMAP plots showing MDSCs score, CD55 expression, and colocalization of MDSCs score with CD55 expression in myeloid cells from scRNA-seq datasets of various cancer types, including ( A ) colorectal (n=30), ( B ) gastric (n=55), ( C ) liver (n=66), ( D ) lung (n=54), ( E ) prostate (n=34), ( F ) breast (n=70), ( G ) cervical (n=11), and ( H ) ovarian cancer (n=12). ( I–K ) Flow cytometric analysis of CD55 expression (MFI) and the percentage of CD55-positive cells in ( I ) total MDSCs, ( J ) PMN-MDSCs, and ( K ) M-MDSCs isolated from peripheral blood of healthy donors (n=10) and patients with different cancer types (n=10 per cancer type), as indicated. ( L ) Representative immunofluorescence images showing CD55 (green) and CD33 (red) expression in adjacent normal tissues and tumor tissues from patients with colorectal cancer (n=10). Nuclei were stained with DAPI (blue). Insets show higher magnification of the boxed areas. The quantification of CD33 + CD55 + cells/mm² is presented on the right. Data are presented as mean± SEM. Data of ( I ), ( J ), and ( K ) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. Data of ( L ) was analyzed using Student’s t-test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; MDSCs, myeloid-derived suppressor cells; MFI, mean fluorescence intensity; M-MDSCs, monocytic myeloid-derived suppressor cells; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; scRNA-seq, single-cell RNA sequencing; UMAP, uniform manifold approximation and projection.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Expressing, Isolation, Immunofluorescence, Staining, Derivative Assay, Fluorescence, Single Cell, RNA Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: Global CD55 knockout inhibits tumor progression and alleviates the immunosuppressive tumor microenvironment in MC38-bearing mice. ( A ) Representative images of tumors from WT and Cd55 −/− mice at endpoint. ( B ) Tumor growth curves in WT and Cd55 −/− mice over time. ( C ) Tumor weight at endpoint in WT and Cd55 −/− mice. ( D ) Flow cytometry analysis of immune cell subsets in the spleens of WT and Cd55 −/− tumor-bearing mice, including MDSCs, M-MDSCs, PMN-MDSCs, TAMs, DCs, CD3 + T cells, CD4 + T cells, CD8 + T cells, NK cells, and B cells. ( E ) Flow cytometry analysis of the same immune cell subsets within tumors from WT and Cd55 −/− mice. ( F ) Representative immunofluorescence staining of tumor sections showing CD4 + T cells (yellow), CD8 + T cells (green) and Gr-1 + MDSCs (red) in WT and Cd55 −/− mice. Nuclei were counterstained with DAPI (blue). Quantification of CD4 + T cells, CD8 + T cells and MDSCs per field is shown in the right panels. Data are presented as mean± SEM. All data are presented from n=5 biological replicates per group and were analyzed using Student’s t-test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. DAPI, 4′,6-diamidino-2-phenylindole; DCs, dendritic cells; MDSCs, myeloid-derived suppressor cells; M-MDSCs, monocytic myeloid-derived suppressor cells; NK, natural killer; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; TAMs, tumor-associated macrophages; WT, wild-type.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Knock-Out, Flow Cytometry, Immunofluorescence, Staining, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: MDSC-specific deletion of CD55 contributes to tumor suppression and immunosuppressive tumor microenvironment alleviation in MC38-bearing mice. ( A ) Tumor growth in WT mice that received bone marrow transplantation from either WT or Cd55 − / − donors. Representative tumor images and tumor volume curves are shown. ( B ) Tumor growth in WT and Cd55 − / − mice treated with either isotype control or anti-Gr-1 antibody (MDSCs depletion). Representative tumor images and tumor volume curves are shown. ( C ) Tumor growth in myeloid-specific Cd55 knockout mice ( Cd55 fl/fl Lyz2-cre ) and littermate controls ( Cd55 fl/fl ). Representative tumor images and tumor volume curves are shown. ( D ) Flow cytometric analysis and quantification of MDSCs, PMN-MDSCs, and M-MDSCs in tumors from Cd55 fl/fl and Cd55 fl/fl Lyz2-cre mice. For quantification, the percentages of PMN-MDSCs and M-MDSCs were calculated by multiplying their proportions within the CD11b + gate by the percentage of CD11b + cells within the CD45 + gate. ( E ) Flow cytometric analysis and quantification of T cells, including CD3 + T cells, CD4 + T cells, and CD8 + T cells, in tumors from Cd55 fl/fl and Cd55 fl/fl Lyz2-cre mice. For quantification, the percentages of CD4 + and CD8 + T cells were calculated by multiplying their proportions within the CD3 + gate by the percentage of CD3 + cells within the CD45 + gate. ( F ) Representative immunofluorescence staining of tumor sections from Cd55 fl/fl and Cd55 fl/fl Lyz2-cre mice. CD4 + and CD8 + T cells were stained with anti-CD4 (yellow) and anti-CD8 (green), respectively; MDSCs were stained with anti-Gr-1 (red); nuclei were counterstained with DAPI (blue). Quantification of CD4 + T cells, CD8 + T cells, and MDSCs per field is shown in the right panels. Data are presented as mean±SEM. Data in ( A ), ( C ), ( D ), ( E ), and ( F ) were analyzed using Student’s t-test. Data in ( B ) were analyzed using two-way ANOVA followed by Bonferroni’s post hoc test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; MDSCs, myeloid-derived suppressor cells; M-MDSCs, monocytic myeloid-derived suppressor cells; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; WT, wild-type.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Transplantation Assay, Control, Knock-Out, Immunofluorescence, Staining, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: CD55 deficiency suppresses the formation and immunosuppressive activity of MDSCs. ( A ) Schematic of BM chimera generation: BM cells from CD45.1 (WT) and CD45.2 ( Cd55 − / − ) donor mice were intravenously transplanted into lethally irradiated syngeneic CD45.2 WT recipient mice. 6 weeks post-transplantation, recipient mice were subcutaneously inoculated with the MC38 cell line. Representative flow cytometry plots show the proportion of CD45.1 + and CD45.2 + cells in the peripheral blood. ( B ) Flow cytometric analysis of MDSCs, M-MDSCs, and PMN-MDSCs in the spleen and tumor of chimeric mice gated on CD45.1 (WT donor-derived) and CD45.2 ( Cd55 − / − donor-derived) cells. Quantification of each MDSC subset is shown. ( C ) Flow cytometric analysis of CD55 expression in BM-derived MDSCs from WT and Cd55 − / − mice. MFI is quantified. ( D ) Flow cytometric analysis and quantification of BM-derived MDSC populations (total MDSCs, M-MDSCs, and PMN-MDSCs) from WT and Cd55 − / − mice. For quantification, the percentages of PMN-MDSCs and M-MDSCs were calculated by multiplying their proportions within the CD11b + gate by the percentage of CD11b + cells within the CD45 + gate. ( E ) qPCR analysis of immunosuppressive genes ( Arg1, S100a8, S100a9, Il10, and Tgfb1 ) in freshly isolated BM cells and BM-derived MDSCs from WT and Cd55 − / − mice. ( F ) Proliferation of CFSE-labeled CD8 + T cells co-cultured with BM-derived MDSCs from WT or Cd55 − / − mice at different ratios (2:1, 4:1, 8:1). Proliferation was measured by CFSE dilution; representative histograms and quantification are shown. ( G ) Flow cytometric analysis of IFN-γ and TNF-α production by CD8 + T cells co-cultured with BM-derived MDSCs from WT or Cd55 − / − mice, with or without stimulation. Quantification of cytokine-producing CD8 + T cells is shown. Data are presented as mean±SEM. All groups had n=3 biological replicates. Data in ( B ), ( C ), ( D ), ( F ), and ( G ) were analyzed using Student’s t-test. Data in ( E ) were analyzed using two-way ANOVA followed by Bonferroni’s post hoc test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; ARG-1, arginase-1; BM, bone marrow; CFSE, carboxyfluorescein succinimidyl ester; IFN-γ, interferon-gamma; MDSCs, myeloid-derived suppressor cells; MFI, mean fluorescence intensity; M-MDSCs, monocytic myeloid-derived suppressor cells; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; qPCR, quantitative PCR; TNF-α, tumor necrosis factor-alpha; WT, wild-type.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Activity Assay, Irradiation, Transplantation Assay, Flow Cytometry, Derivative Assay, Expressing, Isolation, Labeling, Cell Culture, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: CD55 interacts with PYGL to promote glycolysis and thereby enhance the immunosuppressive activity of MDSCs. ( A ) Volcano plot showing differentially expressed proteins between CD55-immunoprecipitated (CD55-IP) and IgG control groups in BM-derived MDSCs. ( B ) Volcano plot comparing mRNA profiles of BM-derived MDSCs from Cd55 − / − and WT mice. ( C ) Venn diagram showing overlap of significantly upregulated proteins in the CD55-IP group and mRNA upregulated in WT MDSCs. PYGL was identified as a common target. ( D ) Co-IP analysis demonstrating the interaction between CD55 and PYGL in BM-derived MDSCs. ( E ) Western blot analysis and quantification of CD55, total PYGL, p-PYGL, and the p-PYGL/PYGL ratio in freshly isolated BM cells and BM-derived MDSCs from WT and Cd55 − / − mice. ( F ) Seahorse analysis showing PER and glycolytic function (basal and compensatory glycolysis) in fresh BM and BM-derived MDSCs from WT and Cd55 − / − mice. ( G ) Western blot and quantification of STAT3 and p-STAT3 in fresh BM and BM-derived MDSCs from WT and Cd55 − / − mice. ( H ) Western blot analysis and quantification of CD55, PYGL, p-PYGL and the p-PYGL/PYGL ratio in WT or Cd55 − / − MDSCs transduced with shRNA against Pygl. ( I ) Seahorse analysis showing PER and glycolytic function in shRNA-treated WT and Cd55 − / − MDSCs, with or without Pygl knockdown. ( J ) Western blot and quantification of STAT3 and p-STAT3 in MDSCs from WT and Cd55 − / − mice transduced with control or Pygl-targeting shRNA. ( K ) qPCR analysis of immunosuppressive gene expression ( Arg1, S100a8, S100a9, Il10, and Tgfb1 ) in MDSCs from the indicated groups. Data are presented as mean±SEM. All groups had n=3 biological replicates. Data in ( E ), ( F ), ( G ), ( H ), ( I ), ( J ), and ( K ) were analyzed using two-way ANOVA followed by Bonferroni’s post hoc test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; ARG-1, arginase-1; BM, bone marrow; Co-IP, Co-immunoprecipitation; 2-DG, 2-deoxyglucose; IL, interleukin; IP, immunoprecipitation; MDSCs, myeloid-derived suppressor cells; mRNA, messenger RNA; PER, proton efflux rate; p-STAT3, phosphorylated STAT3; PYGL, glycogen phosphorylase; qPCR, quantitative PCR; Rot/AA, rotenone/antimycin A; shRNA, short hairpin RNA; WT, wild-type.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Activity Assay, Immunoprecipitation, Control, Derivative Assay, Co-Immunoprecipitation Assay, Western Blot, Isolation, Transduction, shRNA, Knockdown, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: Tomatidine inhibits CD55-mediated phosphorylation of PYGL in MDSCs to suppress glycolysis and immunosuppressive. ( A ) Molecular docking model illustrating the predicted interaction interface between CD55 and PYGL. ( B ) SPR sensorgrams showing the binding kinetics between CD55 and PYGL under the potential drug interventions. ( C ) Western blot analysis of CD55, total PYGL, and p-PYGL, as well as the p-PYGL/PYGL ratio, in BM-derived MDSCs treated with increasing concentrations of tomatidine (0–25 μM). Quantification of protein expression levels, including the phosphorylation ratio, is shown below. ( D ) Flow cytometric analysis of MDSC populations (total MDSCs, PMN-MDSCs, and M-MDSCs) in vitro treated with tomatidine. In vitro treatment with tomatidine. A representative gating strategy and quantification of each subset are shown. ( E ) Seahorse analysis was conducted to measure the PER in MDSCs exposed to increasing concentrations of tomatidine. Quantification of both basal and compensatory glycolysis is provided. ( F ) Western blot analysis of total STAT3 and p-STAT3 in BM-derived MDSCs following tomatidine treatment. Protein quantification is shown on the right. ( G ) qPCR analysis of immunosuppressive gene expression ( Arg1, S100a8, S100a9, Il10, and Tgfb1 ) in BM-derived MDSCs treated with tomatidine at indicated concentrations. Data are presented as mean±SEM. All groups had n=3 biological replicates. Data in ( C ), ( D ), ( E ), ( F ), and ( G ) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; ARG-1, arginase-1; BM, bone marrow; 2-DG, 2-deoxyglucose; IL, interleukin; MDSCs, myeloid-derived suppressor cells; M-MDSCs, monocytic myeloid-derived suppressor cells; mRNA, messenger RNA; PER, proton efflux rate; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; p-STAT3, phosphorylated STAT3; PYGL, glycogen phosphorylase; qPCR, quantitative PCR; Rot/AA, rotenone/antimycin A; SPR, surface plasmon resonance.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Phospho-proteomics, Binding Assay, Western Blot, Derivative Assay, Expressing, In Vitro, Gene Expression, Real-time Polymerase Chain Reaction, SPR Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD55-expressing myeloid-derived suppressor cells (MDSCs) drive cancer immunoevasion

doi: 10.1136/jitc-2025-012980

Figure Lengend Snippet: GM-CSF and IL-6 induce CD55 expression in MDSCs via C/EBPβ signaling. ( A ) Flow cytometry analysis showing increased CD55 expression (MFI) on MDSCs after GM-CSF and IL-6 stimulation. ( B ) Schematic of the DNA pulldown assay using a CD55 promoter probe and mass spectrometry analysis. A Venn diagram shows the intersection of identified transcription factors from DNA pulldown, UCSC, Remap, and TF database. ( C ) Luciferase reporter constructs containing WT or mutated CD55 promoter regions at BS1–BS3. ( D ) Relative luciferase activity in BM-derived MDSCs transfected with C/EBPβ and indicated promoter constructs. ( E ) Schematic of the CD55 promoter region with three putative C/EBPβ binding motifs. ( F ) ChIP confirming C/EBPβ binding at CD55 promoter sites. Quantification of enrichment (% of input) shown on the right. ( G ) C/EBPβ expression in BM-derived MDSCs under GM-CSF and IL-6 stimulation, with or without CEBPB knockdown. ( H ) CD55 expression in BM-derived MDSCs under GM-CSF and IL-6 stimulation with or without C/EBPβ knockdown. ( I ) Percentage of BM-derived MDSCs under GM-CSF and IL-6 stimulation among CD45 + cells after C/EBPβ knockdown. Data are presented as mean±SEM. All groups had n=3 biological replicates. Data in ( A ), ( D ), and ( E ) were analyzed using Student’s t-test. Data in ( G ), ( H ), and ( I ) were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and ns for non-significant. ANOVA, analysis of variance; BM, bone marrow; ChIP, chromatin immunoprecipitation; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; LC, liquid chromatography; MDSCs, myeloid-derived suppressor cells; MFI, mean fluorescence intensity; MS, mass spectrometry; WT, wild-type.

Article Snippet: Purified murine CD55 protein (TargetMol) at various concentrations (320, 160, 80, 40, 20, 10, and 5 nM) was preincubated with each test compound (10 μM, dissolved in DMSO, Sigma) and then injected over a sensor chip surface on which murine PYGL protein had been immobilized using standard amine coupling procedures.

Techniques: Expressing, Flow Cytometry, Mass Spectrometry, Luciferase, Construct, Activity Assay, Derivative Assay, Transfection, Binding Assay, Knockdown, Chromatin Immunoprecipitation, Liquid Chromatography, Fluorescence

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: (A) Schematic of yeast-display screen- A library of yeast cells, each displaying a single human protein encoded by a uniquely bar-coded plasmid was pooled and mixed with surface biotinylated Borrelia . The BASEHIT library was scaled to 96-well magnetic separation format for this screen. Magnetic separation using streptavidin microbeads, followed by next-generation sequencing was used to identify yeast displaying proteins that bind to the bacterial cells. Plasmid DNA was isolated and sequenced to identify the proteins (B) Host interactions with B . burgdorferi N40- spirochete from the cultures grown at 33°C or 37°C were surface biotinylated and used for yeast selections, as described above. The data shows average scores of four independent runs of the selection on the 33°C grown sample and one run on the 37°C grown sample. The scores were normalized for diverse microbes (i.e. Escherichia coli , Staphylococcus aureus , Bacillus subtilis , Shigella flexneri , and Bifidobacterium adolescentis ) as background correction for non-specific binding activity. (C) PGLYRP1 interaction with Borrelia species- Samples from 53 Borrelia isolates, grown at two different temperatures (when possible), were screened against the host protein library as described above for B . burgdorferi , as were 370 additional bacterial samples. The score for each gene is defined as the overall enrichment for that gene (relative to the unselected library) multiplied by the percentage of barcodes associated with the gene that enriched (defined as logFC >0). The calculated scores of Borrelia species were compared with those of the other bacteria. The bars represent mean ± SD and p-values were determined using a Mann-Whitney U-test.

Article Snippet: Low passage B . burgdorferi were cultured to a density of ~10 6 −10 7 cells/mL, washed two times with PBS and incubated with either recombinant human PGLYRP1 (with 8X-His tag), recombinant mouse PGLYRP1 (with 6X-His tag) (Sino Biological, #50115-M08H), recombinant human CD55 (with 8X-His tag), or murine CD55 (with 8X-His tag) at room temperature for 1 hour.

Techniques: Plasmid Preparation, Next-Generation Sequencing, Isolation, Selection, Binding Assay, Activity Assay, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: (A) ELISA results show the interaction of human PGLYRP1 with lysate of B . burgdorferi . The lysate was immobilized on microtiter wells and probed with increasing concentrations of either recombinant human PGLYRP1-Fc protein (1–100 ng, red) or Fc control protein (gray). The values plotted represent the mean ± SEM of three replicates from a single experiment. p-value is displayed in the graph and determined using a student t-test. (B and C) Binding of human (B) and mouse (C) PGLYRP1 to B . burgdorferi . The culture was grown to a density of 10 6 CFU/mL and incubated with varying concentrations of recombinant PGLYRP1-His 8 (10 μg/mL in green, 40 μg/ml in red). B . burgdorferi bound to recombinant PGLYRP1-His 8 was measured using a secondary AF488-His 6 monoclonal antibody by flow cytometry. Overlay histograms show protein binding to B . burgdorferi identified by Alexa Fluor 488-His 6 monoclonal antibody. Binding of recombinant human/murine CD55-His 8 (40 μg/ml in blue) to B . burgdorferi was used as control. The background binding of AF488-His 6 antibody alone with B . burgdorferi is shown in gray shaded region. Results from one independent experiment are shown here. For (B) and (C), the Y-axis represents relative cell counts calculated as a percentage of the maximum events ( Borrelia ).

Article Snippet: Low passage B . burgdorferi were cultured to a density of ~10 6 −10 7 cells/mL, washed two times with PBS and incubated with either recombinant human PGLYRP1 (with 8X-His tag), recombinant mouse PGLYRP1 (with 6X-His tag) (Sino Biological, #50115-M08H), recombinant human CD55 (with 8X-His tag), or murine CD55 (with 8X-His tag) at room temperature for 1 hour.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Incubation, Flow Cytometry, Protein Binding

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: (A) ELISA shows the binding of recombinant human PGLYRP1 at two concentrations, 100 and 500 ng/ml to peptidoglycan (PG) sacculi isolated from B . burgdorferi (Bb) as compared to a PBS negative control. The values plotted represent the mean ± SEM of two technical replicates from a single experiment. (B and C) Flow cytometry-based experiment showing binding of human (B) and mouse (C) PGLYRP1 to B . burgdorferi , after pre-incubating the protein in the absence (red) and presence (blue) of B . burgdorferi PG. Recombinant PGLYRP1-His 6 (1 μg/mL) was pre-incubated with Borrelia PG (10 μg/mL) and then added to live B . burgdorferi overlay histograms show PGLYRP1 binding to B . burgdorferi identified by Alexa Fluor 488, His 6 monoclonal antibody. Results from one independent experiment are shown here. For (B) and (C), the Y-axis represents relative cell counts calculated as a percentage of the maximum events ( Borrelia ).

Article Snippet: Low passage B . burgdorferi were cultured to a density of ~10 6 −10 7 cells/mL, washed two times with PBS and incubated with either recombinant human PGLYRP1 (with 8X-His tag), recombinant mouse PGLYRP1 (with 6X-His tag) (Sino Biological, #50115-M08H), recombinant human CD55 (with 8X-His tag), or murine CD55 (with 8X-His tag) at room temperature for 1 hour.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Isolation, Negative Control, Flow Cytometry, Incubation

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: Wild-type BALB/c and PGLYRP1 -/- mice (at least n = 7 in each group) were infected with 1x10 6 spirochetes by subcutaneous injection. (A and B) Skin B . burgdorferi burden was assessed by ear punch biopsies at 14 d (A) and 25 d (B) post infection, by qPCR for Borrelia specific gene ( flaB ) normalized to mouse β-actin . (C and D) B . burgdorferi burden was assessed in hearts (C) and joints (D) at day 25, by qPCR as above. Results from two independent experiments are shown. Each data point represents the value of an individual animal. (E) The extent of splenomegaly was expressed as spleen weights in WT and PGLYRP1 -/- mice infected with B . burgdorferi at day 25. Results from one independent experiment are shown here. Each data point represents the value of an individual animal. The bars represent mean ± SEM and p-values are calculated by student t-test.

Article Snippet: Low passage B . burgdorferi were cultured to a density of ~10 6 −10 7 cells/mL, washed two times with PBS and incubated with either recombinant human PGLYRP1 (with 8X-His tag), recombinant mouse PGLYRP1 (with 6X-His tag) (Sino Biological, #50115-M08H), recombinant human CD55 (with 8X-His tag), or murine CD55 (with 8X-His tag) at room temperature for 1 hour.

Techniques: Infection, Injection

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: (A) Histopathology scores from tibiotarsi for individual mice in infected wild-type mice (WT Inf) and infected PGLYRP1 -/- mice (PGLYRP1 -/- Inf) at 25 days post infection. Tibiotarsi were scored by blinded examination for tenosynovitis on a scale of 0 (negative) to 3 (severe). (B) The severity of cardiac inflammation in the heart of infected WT and PGLYRP1 -/- mice 25 d post infection. Hearts were scored in a blinded fashion for carditis on a scale of 0 (negative) to 5 (severe). Similar to tenosynovitis scores, PGLYRP1 -/- infected mice demonstrated no significant difference in carditis scores compared to WT mice. Results from at least two independent experiments (at least n = 7 in each group) are pooled and shown here. The bars represent mean ± SEM and p-values were calculated by Mann-Whitney U-test.

Article Snippet: Low passage B . burgdorferi were cultured to a density of ~10 6 −10 7 cells/mL, washed two times with PBS and incubated with either recombinant human PGLYRP1 (with 8X-His tag), recombinant mouse PGLYRP1 (with 6X-His tag) (Sino Biological, #50115-M08H), recombinant human CD55 (with 8X-His tag), or murine CD55 (with 8X-His tag) at room temperature for 1 hour.

Techniques: Histopathology, Infection, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: Antibody levels in uninfected wild type BALB/c (WT) and PGLYRP1 -/- mice were compared with those in the infected ones (at least n = 7 in each group). Representative results from one independent experiment are shown. (A) Whole cell lysate of B . burgdorferi was coated on microtiter plate and serum from either uninfected WT, infected WT, uninfected PGLYRP1 -/- or infected PGLYRP1 -/- mice was used at varying dilutions. The binding was measured by secondary goat anti-mouse IgG HRP-conjugated antibody. Statistically significant increase in binding was observed at IgG titers 1:200, 1:2000, 1:20000 in infected WT compared to infected knockout mice. (B-E) Levels of different IgG isotypes (IgG1, B; IgG2a, C; IgG2b, D; IgG3, E) were measured against B . burgdorferi lysate, using mouse serum at varying dilutions. The binding was measured by secondary goat anti-mouse IgG1, IgG2a, IgG2b or IgG3 HRP-conjugated antibody. Statistically significant increase was observed in IgG1, IgG2a, IgG3, and IgG2b at 1:200 and 1:2000 dilution in infected WT compared to infected PGLYRP1 -/- mice. Representative results from one independent experiment are shown. Each data point represents an individual animal in the corresponding group. The bars represent mean ± SEM and p values determined using student t test.

Article Snippet: Low passage B . burgdorferi were cultured to a density of ~10 6 −10 7 cells/mL, washed two times with PBS and incubated with either recombinant human PGLYRP1 (with 8X-His tag), recombinant mouse PGLYRP1 (with 6X-His tag) (Sino Biological, #50115-M08H), recombinant human CD55 (with 8X-His tag), or murine CD55 (with 8X-His tag) at room temperature for 1 hour.

Techniques: Infection, Binding Assay, Knock-Out

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: Serum cytokine profile was assessed in both wild-type and PGLYRP1 -/- mice 25 d post-infection ((at least n = 7 in each group) using a mouse cytokine/chemokine 31-plex (MD-31) array. An increase in pro-inflammatory cytokines IFN-γ (A) and related cytokines- CXCL-9 (B) and CXCL-10 (C) was observed in infected PGLYRP1 -/- mice as compared to parent BALB/c mice. Representative results from one independent experiment are shown. Each data point represents an individual animal in the corresponding group. The bars represent mean ± SEM and p-values determined using student t test.

Article Snippet: Low passage B . burgdorferi were cultured to a density of ~10 6 −10 7 cells/mL, washed two times with PBS and incubated with either recombinant human PGLYRP1 (with 8X-His tag), recombinant mouse PGLYRP1 (with 6X-His tag) (Sino Biological, #50115-M08H), recombinant human CD55 (with 8X-His tag), or murine CD55 (with 8X-His tag) at room temperature for 1 hour.

Techniques: Infection

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: B . burgdorferi is a diderm bacteria where the outer membrane surrounds the peptidoglycan layer and protects it from the external environment. The peptidoglycan meshwork, in turn, surrounds the cytoplasm. Our results suggest PGLYRP1 binds to Borrelia peptidoglycan.

Article Snippet: Low passage B . burgdorferi were cultured to a density of ~10 6 −10 7 cells/mL, washed two times with PBS and incubated with either recombinant human PGLYRP1 (with 8X-His tag), recombinant mouse PGLYRP1 (with 6X-His tag) (Sino Biological, #50115-M08H), recombinant human CD55 (with 8X-His tag), or murine CD55 (with 8X-His tag) at room temperature for 1 hour.

Techniques:

Journal: PLoS Biology

Article Title: Colon stroma mediates an inflammation-driven fibroblastic response controlling matrix remodeling and healing

doi: 10.1371/journal.pbio.3001532

Figure Lengend Snippet: (A) ISH of Adamdec1 in WT or Adamdec1 KO colon at basal state (water-fed mice). Scale bar, 200 μm. n = 2 per cohort. (B) Weight loss in Adamdec1 KO versus WT mice administered 2% DSS for 7 days and followed by H 2 O for 7 days. n = 4 per cohort. Mann–Whitney test, * p < 0.05. (C) Colon length in Adamdec1 KO versus WT mice administered DSS as described above. n = 8 per cohort. Mann–Whitney test, * p < 0.05. For source data for panels B and C, see . (D) HE staining of representative images of colon following DSS in WT and Adamdec1 KO mice. Mice were administered 2% DSS for 7 days, H 2 O for 1 day, and killed on day 8. Scale bar, 600 μm. n = 4 per cohort. (E) IF staining was performed on colons from Adamdec1 KO and WT mice with indicated markers. Mice were administered 2% DSS for 7 days and killed on day 7. αSMA (red), ER-TR7 (blue), DAPI (gray). Indicated ECM component (green). (Top row) Collagen type I (green). Yellow arrow denotes submucosal ECM accumulation. (Middle row) Collagen type VI (green). Yellow dotted line denotes submucosal thickening and edema. (Bottom row) Fibronectin (green). Yellow dotted line denotes hyperplastic response. Yellow arrow denotes muscle thickening. Scale bar, 200 μm. n = 3 per cohort, representative of 2 experiments. DSS, dextran sulfate sodium; ECM, extracellular matrix; HE, hematoxylin–eosin; IF, immunofluorescence; ISH, in situ hybridization; KO, knockout; WT, wild-type.

Article Snippet: The following primary antibodies were used for FACS and IF staining with murine tissues: Ter-119 (TER-119, Biolegend), CD45 (30-F11, Biolegend), EpCAM (G8.8, Santa Cruz Biotechnology), Pdpn (8.1.1, Biolegend), CD31 (390, Biolegend), Procr (eBio1560, Invitrogen), CD90 (53–2.1, Biolegend), CD55 (RIKO-3, Biolegend), αSMA (1A4, Sigma Aldrich), Adamdec1 (Origine #TA323936), Pcolce2 (Proteintech #10607-I-AP), C3 (11H9, Abcam), Sox6 (Abcam #ab30455), ER-TR7 (Abcam #ab51824), Collagen I (Abcam #ab34710), Collagen VI (Abcam #ab6588), and Fibronectin (Abcam #ab2413).

Techniques: MANN-WHITNEY, Staining, Immunofluorescence, In Situ Hybridization, Knock-Out

Journal: PLoS Biology

Article Title: Colon stroma mediates an inflammation-driven fibroblastic response controlling matrix remodeling and healing

doi: 10.1371/journal.pbio.3001532

Figure Lengend Snippet: (A) Single-cell atlas of colon fibroblasts. UMAP of fibroblast (dots) profiles colored by cell type assignment. (B) Expression of canonical and newly characterized markers across fibroblast clusters. Color represents average expression of marker gene within clusters; diameter represents percentage expression of marker gene within cluster. (C) Expression of genes involved in maintaining colon crypt architecture. Color represents average expression of marker gene within clusters; diameter represents percentage expression of marker gene within cluster. (D) GO enrichment of DEGs for each fibroblast cluster at baseline (water-fed mice). (E) IF staining of 3 fibroblasts classes from water-fed mouse colon. Boxes zoom onto fibroblast subsets: green box- fibroblast class 1, orange box- fibroblast class 2, and blue box- fibroblast class 3. Pcolce2 (red), CD55 (white), Procr (green), DAPI (blue). Scale bar, 25 μm. n = 3. (F) IF and FISH of various markers for fibroblast subsets. Fibroblast 1 (orange box): Grem1 (FISH). Fibroblast 2 (green box): Adamdec1 (IF), Agt (FISH), and Sox6 (IF). Fibroblast 3 (blue box): Pi16 (FISH), C3 (IF). Denoted stain (white), DAPI (blue). Scale bar, 50 μm. n = 3. (G) Proposed distribution of 3 fibroblast classes within the colon. BMP, bone morphogenetic protein; DEG, differentially expressed gene; FISH, fluorescence in situ hybridization; GO, gene ontology; IF, immunofluorescence; IstF, interstitial fibroblast; MAF, mucosa-associated fibroblast; UMAP, uniform manifold approximation and projection.

Article Snippet: The following primary antibodies were used for FACS and IF staining with murine tissues: Ter-119 (TER-119, Biolegend), CD45 (30-F11, Biolegend), EpCAM (G8.8, Santa Cruz Biotechnology), Pdpn (8.1.1, Biolegend), CD31 (390, Biolegend), Procr (eBio1560, Invitrogen), CD90 (53–2.1, Biolegend), CD55 (RIKO-3, Biolegend), αSMA (1A4, Sigma Aldrich), Adamdec1 (Origine #TA323936), Pcolce2 (Proteintech #10607-I-AP), C3 (11H9, Abcam), Sox6 (Abcam #ab30455), ER-TR7 (Abcam #ab51824), Collagen I (Abcam #ab34710), Collagen VI (Abcam #ab6588), and Fibronectin (Abcam #ab2413).

Techniques: Expressing, Marker, Staining, Fluorescence, In Situ Hybridization, Immunofluorescence

Journal: PLoS Biology

Article Title: Colon stroma mediates an inflammation-driven fibroblastic response controlling matrix remodeling and healing

doi: 10.1371/journal.pbio.3001532

Figure Lengend Snippet: (A) Genes that are most often differentially expressed in all stromal clusters between water- and DSS-treated samples and whose expression is enriched in gastrointestinal tissues. The frequency of times they are a DEG is plotted on the y-axis. (B) Violin plot of Adamdec1 across fibroblast clusters in water- and DSS-treated samples. Normalized gene expression levels are plotted on the y-axis. Significant DEGs had FDR <0.05, and respective adjusted p -values derived using MAST (see ); * p < 0.05, ** p < 0.001, *** p < 1E-10. (C) IF staining of colon from water- and chronic DSS-treated mice. Adamdec1 (green), DAPI (blue). Scale bar, 100 μm. n = 3. (D) Inferred differentiation trajectory for MAFs into myofibroblast subset populations. Each dot represents a cell, and color represents the estimated pseudotime for each cell. (E) Adamdec1 expression overlaid on top of inferred differentiation trajectory for MAF into myofibroblast subset populations. Each dot represents a cell, and color represents Adamdec1 expression. (F) Dynamics of Adamdec1 expression levels as a function of pseudotime. Each dot represents a cell, and color represents the annotated fibroblast subset. (G) Genes identified in association with MAF-to-myofibroblast inferred differentiation trajectory. Color indicates when gene expression peaks along differentiation trajectory along the x-axis (from left to right). DEG, differentially expressed gene; DSS, dextran sulfate sodium; FDR, false discovery rate; IF, immunofluorescence; MAF, mucosa-associated fibroblast; UMAP, uniform manifold approximation and projection.

Article Snippet: The following primary antibodies were used for FACS and IF staining with murine tissues: Ter-119 (TER-119, Biolegend), CD45 (30-F11, Biolegend), EpCAM (G8.8, Santa Cruz Biotechnology), Pdpn (8.1.1, Biolegend), CD31 (390, Biolegend), Procr (eBio1560, Invitrogen), CD90 (53–2.1, Biolegend), CD55 (RIKO-3, Biolegend), αSMA (1A4, Sigma Aldrich), Adamdec1 (Origine #TA323936), Pcolce2 (Proteintech #10607-I-AP), C3 (11H9, Abcam), Sox6 (Abcam #ab30455), ER-TR7 (Abcam #ab51824), Collagen I (Abcam #ab34710), Collagen VI (Abcam #ab6588), and Fibronectin (Abcam #ab2413).

Techniques: Expressing, Gene Expression, Derivative Assay, Staining, Immunofluorescence

Journal: PLoS Biology

Article Title: Colon stroma mediates an inflammation-driven fibroblastic response controlling matrix remodeling and healing

doi: 10.1371/journal.pbio.3001532

Figure Lengend Snippet: (A) ISH of Adamdec1 in WT or Adamdec1 KO colon at basal state (water-fed mice). Scale bar, 200 μm. n = 2 per cohort. (B) Weight loss in Adamdec1 KO versus WT mice administered 2% DSS for 7 days and followed by H 2 O for 7 days. n = 4 per cohort. Mann–Whitney test, * p < 0.05. (C) Colon length in Adamdec1 KO versus WT mice administered DSS as described above. n = 8 per cohort. Mann–Whitney test, * p < 0.05. For source data for panels B and C, see . (D) HE staining of representative images of colon following DSS in WT and Adamdec1 KO mice. Mice were administered 2% DSS for 7 days, H 2 O for 1 day, and killed on day 8. Scale bar, 600 μm. n = 4 per cohort. (E) IF staining was performed on colons from Adamdec1 KO and WT mice with indicated markers. Mice were administered 2% DSS for 7 days and killed on day 7. αSMA (red), ER-TR7 (blue), DAPI (gray). Indicated ECM component (green). (Top row) Collagen type I (green). Yellow arrow denotes submucosal ECM accumulation. (Middle row) Collagen type VI (green). Yellow dotted line denotes submucosal thickening and edema. (Bottom row) Fibronectin (green). Yellow dotted line denotes hyperplastic response. Yellow arrow denotes muscle thickening. Scale bar, 200 μm. n = 3 per cohort, representative of 2 experiments. DSS, dextran sulfate sodium; ECM, extracellular matrix; HE, hematoxylin–eosin; IF, immunofluorescence; ISH, in situ hybridization; KO, knockout; WT, wild-type.

Article Snippet: The following primary antibodies were used for FACS and IF staining with murine tissues: Ter-119 (TER-119, Biolegend), CD45 (30-F11, Biolegend), EpCAM (G8.8, Santa Cruz Biotechnology), Pdpn (8.1.1, Biolegend), CD31 (390, Biolegend), Procr (eBio1560, Invitrogen), CD90 (53–2.1, Biolegend), CD55 (RIKO-3, Biolegend), αSMA (1A4, Sigma Aldrich), Adamdec1 (Origine #TA323936), Pcolce2 (Proteintech #10607-I-AP), C3 (11H9, Abcam), Sox6 (Abcam #ab30455), ER-TR7 (Abcam #ab51824), Collagen I (Abcam #ab34710), Collagen VI (Abcam #ab6588), and Fibronectin (Abcam #ab2413).

Techniques: MANN-WHITNEY, Staining, Immunofluorescence, In Situ Hybridization, Knock-Out

Journal: Journal of neuroinflammation

Article Title: Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

doi: 10.1186/s12974-023-02920-9

Figure Lengend Snippet: Fig. 2 Localization of CD59 in murine teased nerve and myelinated cultures. A CD59 (green) and ankyrin-G (AnkG, red) staining of WT teased fibers. B CD59 (green) and Caspr (red) staining of WT teased fibers. C CD59 (green) and neurofascin (Nfasc, blue) staining of WT teased fibers. D CD59 (green), myelin basic protein (MBP, red), and neurofascin (Nfasc, blue) staining of WT ICR DRG. CD59 is co-localized with MBP and localized in the internodes and paranodes. There is no CD59 localization in the nodes of Ranvier. A–C Sections taken from a 4-month-old mouse. Immunolabeling procedure handled with methanol and 0.1% triton. Scale bars 20 μm and in D 50 μm

Article Snippet: For murine sciatic nerve and teased cross- and longitudinal sections (including those prepared with the TSA method), we used rabbit polyclonal antibody (pAb) against mouse CD59 (Cardiff University), rat monoclonal antibody (mAb) against mouse CD59 (Cardiff University), chicken pAb against mouse myelin protein zero (P0, AVES-2B Scientific, Oxfordshire, UK), mouse mAb against mouse ankyrinG (AnkG, NeuroMab, Davis, CA, USA), rat mAb against mouse myelin basic protein (MBP) (Millipore, Burlington, MA, USA), rabbit mAb against mouse Caspr (Weizmann Institute [21]), rat mAb against mouse DAF/CD55 (Cardiff University, 2C6), rabbit pAb against mouse Crry (Biorbyt, Cambridge, UK), rabbit pAb against mouse CD46 (Abcam, Cambridge, UK), mouse mAb against mouse beta dystroglycan (Novocastra, Newcastle on Tyre, UK), mouse mAb against mouse myelin-associated glycoprotein (MAG) (Chemicon International, Temecula, CA, USA), chicken pAb against mouse neurofascin (Nfasc, R&D, Minneapolis, MN, USA), rabbit pAb against mouse CD31 (Abcam, Cambridge, UK), and rat mAb against mouse CD31 (Abcam, Cambridge, UK).

Techniques: Staining, Immunolabeling