Journal: Frontiers in Cellular and Infection Microbiology

Article Title: A Dual Role of Complement Activation in the Development of Fulminant Hepatic Failure Induced by Murine-Beta-Coronavirus Infection

doi: 10.3389/fcimb.2022.880915

Figure Lengend Snippet: Involvement of complement after MHV-3 infection. WT mice were euthanized 0, 6, 12, 24, and 48 hrs after MHV-3 challenge. (A) Immunohistochemical staining for the deposition of C3, C5b-9, and for the expression of C3aR and C5aR in liver tissue samples (CV, central vein; cross, inflammatory infiltrates or necrotic areas). (B, C) Levels of C3aR mRNA and C5aR mRNA, as determined by relative quantitative real-time RT-qPCR in liver tissues at the indicated times. (D, E) Serum concentrations of C3a and C5a at the indicated times. These results are representative of three independent experiments with similar results (n = 4–5 per group). * p <0.05, ** p <0.01, *** p <0.001 compared with 0 time. C3 original magnification, ×200; C5b-9, C3aR, and C5aR original magnification, ×400.

Article Snippet: The monoclonal Ab (mAb) against mouse C5aR (HM1076, clone20/70) and the isotype Rat IgG2b (HI4041, clone RTK4530), were purchased from Hycult Biotech, The Netherlands.

Techniques: Infection, Immunohistochemical staining, Staining, Expressing, Quantitative RT-PCR

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: A Dual Role of Complement Activation in the Development of Fulminant Hepatic Failure Induced by Murine-Beta-Coronavirus Infection

doi: 10.3389/fcimb.2022.880915

Figure Lengend Snippet: Attenuated liver damage in mice treated with anti-C5aR antibody after MHV-3 infection. Mice were administered anti-C5aR antibody or the isotype antibody 12 hrs after MHV-3 infection and euthanized, and liver tissue and serum samples were collected. (A) Histological examination of liver tissue, and immunohistochemical staining for viral antigen and neutrophil infiltration in mouse livers 48 h after virus infection (cross, multifocal damage area). (B, C) Semiquantitative assessment of viral antigen and neutrophil infiltration in livers 48 hrs after virus infection. (D) Serum ALT concentrations 48 hrs after virus infection. (E–G) Serum concentrations of proinflammatory cytokines 24 and 48 hrs after MHV-3 challenge. These results are representative of three independent experiments with similar results (n = 3-4 per group). * p <0.05, ** p <0.01 compared with the isotype control. Original magnification, × 200.

Article Snippet: The monoclonal Ab (mAb) against mouse C5aR (HM1076, clone20/70) and the isotype Rat IgG2b (HI4041, clone RTK4530), were purchased from Hycult Biotech, The Netherlands.

Techniques: Infection, Immunohistochemical staining, Staining

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Gene Set Enrichment Analysis (GSEA) identified GLUT1 and C5aR1 as two top hits related to selective serotonin reuptake inhibitor (SSRI)-induced gene changes. ( B ) GSEA of hepatocellular carcinoma (HCC) RNA-seq data (TCGA cohort) with the SSRI-related gene signature. Sample grouping was made based on the median expression of C5aR1. ( C ) Representative immunohistochemical images showed the expression pattern and cellular distribution of C5aR1 in human HCC tissues. Scale bar, 50 μm. ( D ) Single-cell RNA sequencing analysis showed the expression pattern of C5aR1 with the immune microenvironment of HCC. ( E ) Co-immunofluorescence of C5aR1 (green) with CD163 (red) in HCC samples. Scale bar, 10 μm. ( F ) The drug affinity responsive target stability (DARTS) assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence and absence of 100 μM citalopram treatment. ( G ) The DARTS assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence of different concentrations of citalopram treatment. ( H ) The overall conformation of citalopram binding to C5aR1. ( I ) Representative models of citalopram in pose-1 (left), pose-2 (middle), and allosteric site (right). Several polar interactions were indicated by black dashed lines. ( J ) HEK293T cells were transfected with either WT or mutant C5aR1 expression plasmids for 48 hr, followed by DARTS assay with immunoblotting analysis of C5aR1 protein levels. In all panels, *p < 0.05, **p < 0.01. Values as mean ± SD and compared by the Student’s t test ( F ) or one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups ( G, J ). Figure 2—source data 1. Original western blots for , indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: RNA Sequencing, Expressing, Immunohistochemical staining, Single Cell, Immunofluorescence, Western Blot, Binding Assay, Transfection, Mutagenesis

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Uniform Manifold Approximation and Projection (UMAP) of SMART-seq2-based single CD45 + cells. The tSNE (by cluster) was acquired from http://cancer-pku.cn:3838/HCC/ . ( B ) The UMAP showing C5AR1 expression in HCC immune cell clusters. ( C ) Violin plot showing C5AR1 expression in different immune cell clusters.

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) The drug affinity responsive target stability (DARTS) assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence of different concentrations of selective serotonin reuptake inhibitors (SSRIs) treatment (0, 1, 10, 50, and 100 μM). ( B ) For C5aR1, the predicted binding energy distribution of the clusters with poses more than 50. ( C ) Sequencing analysis showed the successful generation of six C5aR1 mutants. ( D ) The best-scored complex models of C5aR1 with other four different SSRIs. ( E ) HEK293T cells were transfected with either WT or mutant C5aR1 (D282A) expression plasmids for 48 hr, followed by DARTS assay with immunoblotting analysis of C5aR1 protein levels. In all panels, *p < 0.05, **p < 0.01. Values are presented as mean ± SD and compared by one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups ( E ). Figure 2—figure supplement 2—source data 1. Original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Western Blot, Binding Assay, Sequencing, Transfection, Mutagenesis, Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Western blotting showed the knockdown efficiency of GLUT1 in mouse Hepa1-6 cells. ( B ) GLUT1 KD Hepa1-6 cells were subcutaneously injected into the Rag1 −/− or immunocompetent C57BL/6 mice, and mice were treated with 5 mg/kg citalopram when bore visible tumors; 3 weeks later, tumor burden was examined ( n = 6–7 per group). ( C ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host ( n = 7). ( D ) Immunofluorescence analysis of C5a deposition in GLUT1 KD Hepa1-6 tumors from C5ar1 +/− and C5ar1 −/− C57BL/6 host. Scale bar, 50 μm. ( E ) Experimental design of bone marrow transfer experiments. ( F, G, I ) GLUT1 KD Hepa1-6 cells were subcutaneously implanted into syngeneic recipient (r) mice that had been reconstituted with bone marrow cells from either C5ar1 +/− or C5ar1 −/− donor mice. The therapeutic effect of citalopram ( F ), C5a deposition ( G ), and macrophage phagocytosis ( I ) in this model was analyzed. Scale bar, 50 μm. ( H ) The phagocytic capacity of macrophages isolated from GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host. Flow cytometry showed the infiltration of CD45 + CD11b + F4/80 + macrophages ( J ), CD206 + TAMs and CD11b + TAMs ( K ), tumor-infiltrating lymphocytes ( L ) in tumor tissues from orthotopic xenograft model, which was generated in immunocompetent C57BL/6 mice with Hepa1-6 cells ( n = 5 per group). ( M, N ) Measurement of CD8 + T cell function in tumor tissues from the groups mentioned in C and F . ( O ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host upon CD8 + T cell depletion ( n = 7). ( P ) Correlation analysis of C5aR1 expression and immune checkpoint molecules, gene signatures of TAMs, exhausted T cells, and effector Tregs in the TCGA cohort ( n = 371). In all panels, *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant. Values are presented as mean ± SD and compared by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons ( B, C, F, O ), Student’s t test ( H–M ), one-way ANOVA multiple comparisons with Tukey’s method ( B, N ), and the Spearman’s rank correlation methods ( P ). Figure 3—source data 1. Original western blots for , indicating the relevant bands. Figure 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Western Blot, Knockdown, Injection, Immunofluorescence, Isolation, Flow Cytometry, Generated, Cell Function Assay, Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Gene Set Enrichment Analysis (GSEA) plot of phagocytosis pathway in macrophages derived from C5ar1 −/− mice and C5ar1 +/− mice. ( B ) Western blotting and immunofluorescence analysis showed C5aR1 protein levels in Cas9-sgControl, -sg C5ar1 THP-1 subclones. ( C ) Effects of C5aR1 deficiency on the macrophage phagocytosis of HCC-LM3 in the presence or absence of C5a stimulation. ( D ) Effects of different selective serotonin reuptake inhibitors (SSRIs) on the macrophage phagocytosis of HCC-LM3 in the presence of C5a stimulation. ( E ) Reconstituted expression of WT and D282A mutant C5aR1 in C5aR1 KO THP-1 cells. ( F ) The effects of citalopram on macrophage phagocytosis in the absence of C5aR1 with reconstituted expression of C5aR1 WT or C5aR1 D282A . In all panels, *p < 0.05, **p < 0.01, ***p < 0.001. Values are presented as mean ± SD and compared by one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups. Data are representative of three independent experiments ( C, D, F ). Figure 3—figure supplement 2—source data 1. Original western blots for , indicating the relevant bands. Figure 3—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Derivative Assay, Western Blot, Immunofluorescence, Expressing, Mutagenesis

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Conformations of orthosteric binding sites in human (light blue) and mouse (orange) C5aR1. The conformation of human C5aR1 was obtained from the crystal structure (PDB id: 6c1q). The structure of mouse C5aR1 was predicted using the ColabFold (AlphaFold2) software. ( B ) The predicted binding modes of citalopram to human (light blue) and mouse (orange) C5aR1. The conformations of citalopram were shown in pink (binding mode 1) or deep green (binding mode 2) sticks. For mouse C5aR1, green sticks indicate residues set to flexible in the molecular docking process.

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Binding Assay, Software

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: Model depicting the molecular mechanism by which citalopram inhibits the Warburg effect and promotes an anti-tumor response in hepatocellular carcinoma (HCC). In the primary HCC microenvironment (left panel), C5aR1-expressing tumor-associated macrophages (TAMs) exhibit reduced phagocytic capacity and an anti-inflammatory state, which correlates with diminished CD8 + T cell anti-tumor immunity and HCC progression. Upon treatment with citalopram (right panel), the drug not only inhibits the glycolytic metabolism of cancer cells by targeting GLUT1 but also acts on C5aR1 expressed by TAMs, thereby enhancing macrophage-driven anti-tumor immunity. Additionally, citalopram induces a systemic immunostimulatory effect on CD8 + T cell functions through yet-to-be-identified serotonergic mechanisms. The dotted line indicates a causal relationship that has not been fully established through direct evidence.

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Expressing

Journal: European Journal of Immunology

Article Title: TLR activation enhances C5a-induced pro-inflammatory responses by negatively modulating the second C5a receptor, C5L2

doi: 10.1002/eji.201041350

Figure Lengend Snippet: Effect of a pre-exposure to LPS on cell sensitivity to C5a and C5aR expression. (A) Fold increase in the levels of IL-8 – determined as described for – in culture supernatants of PBMCs (1.5×10 5 /well) pre-exposed or not to LPS (100 pg/mL) for the indicated times (starting from 30 min), and subsequently activated (14 h) with C5a (10 nM). (B) C5aR cell-surface expression levels on gated monocytes or neutrophils (inset) at different times following 1×10 6 PBMC (monocytes) or 100 μL whole blood (neutrophils) pre-exposure (30 min) to 100 pg/mL (PBMCs) or 500 pg/mL (whole blood) LPS or a mock-pre-exposure (no LPS), and subsequent activation or not with C5a (10 nM) for the indicated times. (C) C5aR mRNA levels determined by RT-qPCR in RNA samples extracted from PBMC aliquots taken from the experiment described in (B) at the end of the culture (12 h). Results are from one experiment (A and C, ±SD) representative of three. * p <0.05, *** p <0.005 (LPS-pre-exposed versus not pre-exposed, paired Student's t -test).

Article Snippet: At each time point, cell aliquots were tested for C5aR cell-surface expression on gated monocytes or neutrophils – identified by their CD14 + staining and forward and side scatter profiles – by flow cytometry using a human C5aR-specific mAb (S5/1, Hycult), as described .

Techniques: Expressing, Activation Assay, Quantitative RT-PCR

Journal: Journal of Innate Immunity

Article Title: Single-Cell RNA Sequencing in Pediatric Sepsis: γδ T Cell Exhibits a Differentiation to γδT17 Subtype along with Significantly Enhanced Cell Communication with Neutrophils

doi: 10.1159/000547934

Figure Lengend Snippet: Comparison of activation and immune factor expression of the γδ T cells between the pediatric sepsis (sepsis) group and HC group by flow cytometry. a The gating strategy. b Comparison of the proportion of CD69-positive γδ T cells ( n = 30). c Comparison of the proportion of IFN-γ, TNF-α, and IL-17A-positive γδ T cells ( n = 30). d Comparison of the proportion of perforin, GZMB, and GNLY-positive γδ T cells ( n = 20 or 30). e Comparison of the proportion of NKG2A and NKG2D-positive γδ T cell ( n = 30) (ns, p > 0.05; * p < 0.05; ** p < 0.01).

Article Snippet: For surface staining, PBMCs were labeled with the following monoclonal antibodies: anti-human CD45 (HI30), anti-human CD3 (HIT3a), anti-human CD69 (FN50), anti-human NKG2D (1D11) (all from BioLegend, San Diego, CA, USA); anti-human NKG2A (Miltenyi Biotec, Bergisch Gladbach, Germany); mouse anti-human TCR γδ (B1) (BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Comparison, Activation Assay, Expressing, Flow Cytometry