Journal: PLOS Pathogens

Article Title: C/EBP-β-regulated complement hyperactivation in spleen of SFTSV-infected mice: A clue to targeted complement therapy

doi: 10.1371/journal.ppat.1014144

Figure Lengend Snippet: (A and B) Serum C3a (A) and C5a (B) levels of IFNAR −/− mice infected with SFTSV for 3 days or mock-infected. Data are presented as the mean ± SD. (C) Immunohistochemical staining for C3aR and C5aR in the spleen. The deposition of C3aR and C5aR significantly increased 3 days after SFTSV infection. Scale bars are indicated in the images. (D and E) C3ar1 (D) and C5ar1 (E) mRNA expression levels in the spleen 3 days post-infection. Data are presented as the mean ± SD. (F) Volcano plot indicating DEGs in mock-infected and SFTSV-infected spleens. Upregulated and downregulated genes with FDR < 0.05 and fold change > 2 are colored red and green, respectively. (G) Gene ontology (GO) analysis based on DEGs in SFTSV-infected spleens (FDR < 0.05 and fold change > 2). (H) Gene set enrichment analysis (GSEA) plot for the complement and coagulation cascades pathway. (I) Heatmap showing the expression profiles of genes involved in the complement and coagulation cascades pathway, with complement genes highlighted in red. (J) Circos graph displaying the correlation network between complement genes and the top 25 DEGs associated with the most enriched biological processes in (G) ; the Gapdh gene was added as a reference. Each sector of the circle represents one gene, and the color of each link represents the Spearman correlation coefficient between the expressions of genes. Two-sided p -values, examined by Student’s t-test (A, B, D and E) , are shown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IFNAR −/− C57BL/6 mice (6–8 weeks old) were divided into three groups (n = 5 per group for tissue collection and n = 7 per group for survival rate): mock-infected, SFTSV + vehicle group, and SFTSV + C3aR antagonist (C3aRA, SB290157, TOPSCIENCE) group.

Techniques: Infection, Immunohistochemical staining, Staining, Expressing, Coagulation

Journal: PLOS Pathogens

Article Title: C/EBP-β-regulated complement hyperactivation in spleen of SFTSV-infected mice: A clue to targeted complement therapy

doi: 10.1371/journal.ppat.1014144

Figure Lengend Snippet: C) Intracellular viral RNA (A) and the mRNA expression levels of cytokines IL-1β (B) and IL-6 (C) in SFTSV-infected FRCs treated with or without the C3aR antagonist (SB290157). (D) Schematic diagram of 40-kDa FITC-dextran extravasation analysis using a co-culture of FRCs and the endothelial cell line HMEC-1 (left) and the calculated permeability index in co-cultures treated with SFTSV or SFTSV plus the C3aR antagonist for 24 hpi. Data are presented as the mean ± SD. Two-sided p -values, examined Dunnett’s multiple comparisons test after one-way ANOVA, are shown. ** p < 0.01, *** p < 0.001. (E) A heatmap showing the DEGs of SFTSV-infected FRCs in the presence of the C3aR antagonist or the vehicle. The top 10 upregulated and downregulated genes are highlighted. (F) Significantly enriched pathways analyzed by Metacore using the DEGs of SFTSV-infected FRCs in the presence of the C3aR antagonist or the vehicle.

Article Snippet: IFNAR −/− C57BL/6 mice (6–8 weeks old) were divided into three groups (n = 5 per group for tissue collection and n = 7 per group for survival rate): mock-infected, SFTSV + vehicle group, and SFTSV + C3aR antagonist (C3aRA, SB290157, TOPSCIENCE) group.

Techniques: Expressing, Infection, Co-Culture Assay, Permeability

Journal: PLOS Pathogens

Article Title: C/EBP-β-regulated complement hyperactivation in spleen of SFTSV-infected mice: A clue to targeted complement therapy

doi: 10.1371/journal.ppat.1014144

Figure Lengend Snippet: IFNAR −/− mice were challenged with SFTSV and then administered the C3aR antagonist (C3aRA) or the vehicle control. Viral load in the spleen 3 days after challenge in the SFTSV + vehicle group (n = 5) and the SFTSV + C3aR antagonist group (n = 5). (B) Representative images of the histological analysis of the spleen tissues from mock-infected or SFTSV-infected mice administered the C3aRA or the vehicle 3 days post-infection. Scale bars are indicated in the images. (C-E) The mRNA expression levels of the cytokines IL-6, TNF-α, and IFN-γ in the spleen tissues 3 days post-infection (n = 5 per group). Data are presented as the mean ± SD. Two-sided p-values, examined by Student’s t-test, are shown. * p < 0.05, ** p < 0.01, *** p < 0.001. (F) Kaplan-Meier survival curves of SFTSV-infected IFNAR −/− mice treated with the C3aR antagonist or the vehicle (n = 6 per group). Representative data are shown from two independent experiments. ** p < 0.01 (log-rank test).

Article Snippet: IFNAR −/− C57BL/6 mice (6–8 weeks old) were divided into three groups (n = 5 per group for tissue collection and n = 7 per group for survival rate): mock-infected, SFTSV + vehicle group, and SFTSV + C3aR antagonist (C3aRA, SB290157, TOPSCIENCE) group.

Techniques: Control, Infection, Expressing

Journal: Computational and structural biotechnology journal

Article Title: The complement C3a/C3aR pathway is associated with treatment resistance to gemcitabine-based neoadjuvant therapy in pancreatic cancer.

doi: 10.1016/j.csbj.2024.09.032

Figure Lengend Snippet: Fig. 7. Knockdown of C3aR does not affect the progression of pancreatic cancer cells in vitro. A.A-F Panc-1 cells were infected with C3aR shRNA to knock down the C3aR expression (sh#1, sh#2), or infected with a scrambled shRNA as the control group (sh-Scr). (A) Western blotting was used to detect C3aR expression in control cells and C3aR-deficient cells to evaluate the transfection efficiency. (B) CCK8 assay was used to evaluate the cell proliferation, and the relative optical density of each group at day 0, 1, 2, 3 and 4 was measured. The line chart showed that C3aR knockdown did not change the proliferation capacity of Panc-1 cells. (C) Representative images of EdU assays showed that the knockdown of C3aR did not affect the proliferation capacity of Panc-1 cells. (D) The bar plot showed the percentage of EdU+

Article Snippet: Recombinant human complement C3a protein (MedChemExpress, MCE; HY-P7862), recombinant mouse complement C3a protein (MCE; HY-P7863) and C3aR antagonist, SB290157 (TargetMol) were used according to the manufacturer’s instructions.

Techniques: Knockdown, In Vitro, Infection, shRNA, Expressing, Control, Western Blot, Transfection, CCK-8 Assay

Journal: Computational and structural biotechnology journal

Article Title: The complement C3a/C3aR pathway is associated with treatment resistance to gemcitabine-based neoadjuvant therapy in pancreatic cancer.

doi: 10.1016/j.csbj.2024.09.032

Figure Lengend Snippet: Fig. 8. C3aR antagonist (SB290157) attenuated the effects of C3a activation in pancreatic cancer. (A) Representative images of EdU assays showed that the treatment of SB290157 attenuated C3a-induced proliferation in Panc-1 cells. (B) The bar plot showed the percentage of EdU+ cells per field. (C) Representative images of Transwell assays showed that the treatment of SB290157 attenuated C3a-induced migration in Panc-1 cells. (D) The bar plot showed the percentage of migrated cells per field. (E) CCK-8 assay showed that the treatment of SB290157 attenuated C3a-induced gemcitabine resistance in Panc-1 cells. F-K A mouse subcutaneous tumor model of Panc-1 cells was constructed. The mice were randomly grouped and treated with vehicle, 20 mg/kg gemcitabine, 20 mg/kg SB290157, 20 mg/kg gem- citabine combined with 20 mg/kg SB29015 by intraperitoneal injection once a day. (F) Line charts of volume changes of subcutaneous tumor. (G) On the 14th day of treatment, subcutaneous tumors were separated to show tumor size. Frozen sections were prepared from tumor tissues. IF staining with antibodies to Ki-67 (in red color, (H)) and CC3 (in red color, (J)) was performed to detect proliferation and apoptosis of Panc-1 cells. And the percentage of Ki-67+ cells (I) or CC3+ cells (K) per field was shown by bar plots. * P < 0.05, * * P < 0.01, * ** P < 0.001. GEM, gemcitabine.

Article Snippet: Recombinant human complement C3a protein (MedChemExpress, MCE; HY-P7862), recombinant mouse complement C3a protein (MCE; HY-P7863) and C3aR antagonist, SB290157 (TargetMol) were used according to the manufacturer’s instructions.

Techniques: Activation Assay, Migration, CCK-8 Assay, Construct, Injection, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: Complement dependent TNFα production in neutrophil-like HL60 cells

doi: 10.1016/j.bbrep.2023.101465

Figure Lengend Snippet: Zymosan-treated human serum-induced TNFα production in neutrophil-like HL60 cells. HL60 cells were induced to differentiate into neutrophil-like cells by treatment with 2.5 μM ATRA for 0–5 days. (A) Flow cytometric analysis of the cell surface expression of CR3, C3aR, or C5aR1 in day 0, 1, 3, and 5 ATRA-treated HL60 cells. (B) Day 0–5 ATRA-treated HL60 cells were either untreated (NT) or incubated with 10% human serum or zymosan-treated human serum for 4 h, and the whole cell lysates (Cell) and culture supernatants (Sup.) were then collected. The protein samples were separated in SDS-PAGE, and TNFα and β-actin were detected by immunoblot analysis. RPMI, Human serum, and ZHS in diagonal letters indicate samples of RPMI-1640 medium, RPMI-1640 containing 10% zymosan-treated human serum, or human serum mixed with 1 × SDS-PAGE sample loading buffer, respectively. (C, D) Day 5 ATRA-treated HL60 cells were either untreated (NT) or incubated with 10% human serum or zymosan-treated human serum for indicated time points, and the whole cell lysates (Cell) and culture supernatants (Sup.) were then collected. (E) Day 5 ATRA-treated HL60 cells and PMNs were either untreated (NT) or incubated with 10% human serum (HS) or zymosan-treated human serum (ZHS) for 4 h, and the whole cell lysates (Cell) and culture supernatants (Sup.) were then collected. The protein samples were separated in SDS-PAGE, and TNFα and β-actin were detected by immunoblot analysis. The blot is a representative of 3 independent experiments.

Article Snippet: Anti-C3a Receptor (hC3aRZ8) FITC monoclonal antibody (#130-108-108) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Expressing, Incubation, SDS Page, Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: Complement dependent TNFα production in neutrophil-like HL60 cells

doi: 10.1016/j.bbrep.2023.101465

Figure Lengend Snippet: Schematic diagram of the hypothesis that complement-induced TNFα production in neutrophil-like HL60 cells. The complement activation product C3a binds to C3aR and induces TNFα production in neutrophil-like HL60 cells. The TNFα production is enhanced by intracellular Ca 2+ elevation and is negatively regulated by fibrinogen or fibronectin coating.

Article Snippet: Anti-C3a Receptor (hC3aRZ8) FITC monoclonal antibody (#130-108-108) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Activation Assay

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 1. Cell proliferation and cytokine mRNA and protein expression in cultured human mesangial cells (HMCs). The HMCs were treated with blank medium or medium containing 100 lg/ml immunoglobulin (Ig)A, 100 lg/ml IgA 1 100 lM C3aR antagonist (C3aRA) or 100 lg/ml IgA 1 100 lM C5aR antagonist (C5aRA) for 48 h. The data are expressed as the mean 6 standard deviation (s.d.). (a) HMC proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (n 5 6 for each group). (b) Interleukin (IL)-6 (left row) and monocyte chemotactic protein 1 (MCP-1) (right row) gene expression levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by reverse transcription–quantitative polymerase chain reaction (RT–qPCR). (c) Western blot analysis of IL-6, MCP-1 and GAPDH protein expression. (d) Quantitative analysis for Western blot of IL-6 and MCP-1 protein expression. *P < 005, **P < 001, ***P < 0001 versus negative control.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Expressing, Cell Culture, Standard Deviation, MTT Assay, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 2. Wild-type (WT), C3aR deficient (C3aR–/–) and C5aR–/– mice were immunized with inactivated Sendai virus to induce immunoglobulin (Ig)A nephropathy (IgAN). After 14 weeks of immunization, IgA and C3 deposition in the mesangium and the associated histological changes were analysed. (a,b) Glomerular IgA (upper row) and C3 (low row) deposition was measured by immunofluorescence staining of kidney sections from WT, C3aR–/– and C5aR–/– IgAN mice and negative controls. (c) Mesangial matrix expansion and cell proliferation were demonstrated by periodic acid-Schiff (PAS) staining of renal tissue in the following four groups of mice: negative controls and WT, C3aR–/– and C5aR–/– IgAN mice. Magnification 3400. Scale bars represent 100 lM.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Virus, Immunofluorescence, Staining

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 3. Representative images of immunohistochemical staining for interleukin (IL)-6 and monocyte chemotactic protein 1 (MCP-1) in the kidney sections of wild-type (WT), C3aR deficient (C3aR–/–) and C5aR–/– immunoglobulin (Ig)A nephropathy (IgAN) mice and negative controls. C3aR and C5aR deficiency reduced renal IL-6 and MCP-1 expression compared to WT mice. Scale bars represent 100 lM.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 4. Cytokine and chemokine gene expression in the renal tissues of the following four groups of mice: negative controls and wild-type (WT), C3aR deficient (C3aR–/–) and C5aR–/– immunoglobulin (Ig)A nephropathy (IgAN) mice (n5 7 for each group). Reverse transcription–quantitative polymerase chain reaction (RT–qPCR) was used to quantify the mRNA expression levels of (a) tumour necrosis factor (TNF)-a, (b) transforming growth factor (TGF)-b, (c) interleukin (IL)-1b, (d) interleukin (IL)-6 and (e) monocyte chemotactic protein 1 (MCP-1) relative to glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Data are expressed as the mean 6 standard deviation. *P< 005, **P< 001, ***P< 0001 versus negative control.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Standard Deviation, Negative Control

Journal: bioRxiv

Article Title: Hypoxia-induced Complement Component 3 Promotes Aggressive Tumor Growth in the Glioblastoma Microenvironment

doi: 10.1101/2024.01.28.577617

Figure Lengend Snippet: A-B. Immunofluorescent detection of C3 (red), Nestin (yellow), Nuclei (DAPI, blue), GFAP (green) or Olig2 (cyane) in the perivascular (A) or hypoxic (B) niches of murine RCAS-PDGFB- and RCAS-shp53-induced gliomas. C-D. Hallmark_Hypoxia (C) and Hallmark_Complement (D) signatures mapped on spatially resolved transcriptomics from human GBM samples, displayed with Z-score. E. Spatial correlation between Hallmark_Hypoxia and Hallmark_Complement in one representative tumor (UKF242). P values were corrected for multiple hypothesis testing using the Holm method. F. Distribution of R-values for the correlation between Hallmark_Hypoxia and Hallmark_Complement in a total of 19 human GBM tissue samples with an average correlation score of R=0.54 G-H. Pearson correlation coefficient between Hallmark_Complement and Hallmark_Hypoxia signatures in the TCGA GBMLGG (G) or TCGA GBM (H) dataset. I-K. Expression of CA9, C3, and C3AR1 mRNA in human primary astrocytes (n=3), HMC3 microglia (n=4), or primary human glioma cells U3082MG (n=3), U3065MG (n=3) or U3084MG (n=3) in response to normoxia (21% O2), hypoxia (1% O2) or severe hypoxia (0.1% O2). Error bars represent SEM. *, P < 0.05, **, P < 0.01, or ***, P< 0,001. Statistical tests were one-way ANOVA, with Tukey post-hoc test, or unpaired t-test in case of two sample comparisons.

Article Snippet: The spheres were grown until visible spheres were formed (up to 14 days) with control or treatments with 50 and 250nM C3aR antagonist (Santa Cruz Biotechnology, SB290157).

Techniques: Expressing

Journal: bioRxiv

Article Title: Hypoxia-induced Complement Component 3 Promotes Aggressive Tumor Growth in the Glioblastoma Microenvironment

doi: 10.1101/2024.01.28.577617

Figure Lengend Snippet: A. C3AR1 expression of Pan-Cancer TCGA data of common cancer types (n=33). B. C3AR1 expression in relation to glioma grade as analyzed in TCGA data. C. C3AR1 expression in IDH wildtype glioma compared to IDH mutant with or without 1p/19q codeletion (Tukey post-hoc test) as analyzed in TCGA data. D. C3AR1 expression in GBM compared to non-tumor as analyzed in TCGA data. E. Kaplan-Meier curve showing survival of glioma patients with either high (red) or low (blue) C3AR1 expression based on TCGA data. F . Kaplan-Meier curve showing survival of IDH-wildtype GBM with high (red) or low (blue) C3AR1 expression based on TCGA data. G. UMAP displaying C3AR1 expression in single cell sequencing data from 26 independent datasets compiled in GBmap . H. C3AR1 expression of malignant cells divided into C3AR1-expressing or non-expressing cells. I. Geneset enrichment analysis of the C3AR1-expressing malignant cells. Red colored bars indicate significant Benjamini-Hochberg adjusted P values (padj < 0.05). J. Log-fraction plot of a combination of independent extreme limiting dilution sphere formation assays (n=4) of U3082MG glioma cells treated with SB290157. *, P < 0.05, **, P < 0.01, or ***, P< 0,001. One-way ANOVA, or unpaired T-test (in case of comparison between two groups) with Tukey post-hoc test.

Article Snippet: The spheres were grown until visible spheres were formed (up to 14 days) with control or treatments with 50 and 250nM C3aR antagonist (Santa Cruz Biotechnology, SB290157).

Techniques: Expressing, Mutagenesis, Sequencing, Comparison

Journal: Journal of Neuroinflammation

Article Title: Ceria nanoparticles ameliorate white matter injury after intracerebral hemorrhage: microglia-astrocyte involvement in remyelination

doi: 10.1186/s12974-021-02101-6

Figure Lengend Snippet: A1 astrocytes inhibited microglial phagocytosis of myelin debris via an astrocytic C3-microglial C3aR axis. a Representative images of GFAP and Iba1 double immunostaining in brain sections. Lens: × 100; Scale bar: 200 μm. b Representative TEM image showed microglia phagocytosis of myelin debris. Lens: × 7500, × 15,000; Scale bar: 5 μm (white), 2 μm (yellow). c tSNE plots of brain myeloid cells (Tabula Muris). d Expression of C3aR1 genes in microglia and macrophage (Tabula Muris). e Representative images of C3aR and Iba1 double immunostaining in brain sections. Lens: × 200; Scale bar: 50 μm. f Quantification of fluorescent beads in cultured microglia, * p < 0.01 versus Control, # p < 0.01 versus C3 treatment, ^ p < 0.01 versus A1 astrocyte CM treatment. g Representative images of fluorescent beads uptake in cultured microglia. Lens: × 400; Scale bar: 25 μm

Article Snippet: As shown in Fig. c, d, the data from a single-cell transcriptome database indicated that the C3a receptor (C3aR) is strongly expressed in microglia (Tabula Muris).

Techniques: Double Immunostaining, Expressing, Cell Culture, Control