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: PLOS Biology

Article Title: Staphylococcal toxin PVL ruptures model membranes under acidic conditions through interactions with cardiolipin and phosphatidic acid

doi: 10.1371/journal.pbio.3003080

Figure Lengend Snippet: (A) 25 most enriched genes identified in a CRISPR screen in human macrophages treated with PVL. (B) Relative abundance levels of total sphingomyelin (SM) and phosphatidylcholine (PC) in wild type and SGMS1 KO #1 and #2 THP-1 macrophages. Mean ± SEM from five independent experiments, P values (one-way ANOVA followed by Dunnett’s multiple Comparison test) are shown. (C) Cell death (Draq7-positive) of wild type and two independent SGMS1 KO THP-1 macrophages treated with PVL (62.5 ng/ml) or LukAB (15.6 ng/ml) overtime. Mean ± SEM from three independent experiments, P values (one-way ANOVA) are shown. (D) Flow cytometry of cell-surface levels of C5aR1 and LukS-PV in WT THP-1 macrophages treated with methyl-beta-cyclodextrin (MβCD) or control PBS. Blue line indicates isotype control, red line antigen-specific antibodies. Data representative of three independent experiments. (E) Mean fluorescence intensity (MFI) of C5aR1 and LukS-PV in PBS- or MβCD-treated macrophages. Mean ± SEM from three independent experiments. ns = not significant; unpaired t test. (F) Cell death (Draq7-positive) of human THP-1 macrophages treated with PVL (62.5 ng/ml) or LukAB (15.6 ng/ml) with PBS or MβCD (2.5 or 5 mM) overtime. Mean ± SEM from three independent experiments, P values (one-way ANOVA) are shown. (G) Sulforhodamine B encapsulated liposome of POPC/sphingomyelin/cholesterol (w/w 4:1:1) were treated with purified PVL (1 µg/ml), LukF-PV, LukS-PV, saponin, or control PBS and fluorescence determined relative to Triton X-100 treatment. Mean ± SEM from three independent experiments. The data underlying this Figure can be found in S1 Data.

Article Snippet: Membranes were blocked with TBS containing 0.2% Tween 20 (TBS-T) containing 5% skim milk for 1 h and were then probed with primary antibodies to LukS-PV (made inhouse), human C5aR1 (clone B-6, #sc-271949, Santa Cruz Biotechnology) at 4°C overnight.

Techniques: CRISPR, Comparison, Flow Cytometry, Control, Fluorescence, Purification

Journal: PLOS Biology

Article Title: Staphylococcal toxin PVL ruptures model membranes under acidic conditions through interactions with cardiolipin and phosphatidic acid

doi: 10.1371/journal.pbio.3003080

Figure Lengend Snippet: (A) Cell-surface levels of C5aR1 and LukS-PV in WT Cas9 THP-1 macrophages, SGMS1 knockout, and C5aR1 knockout clones using flow cytometry. Blue line represents isotype control treatment, red line antigen-specific staining. Data representative of four independent experiments. (B) Flow cytometric analysis and mean fluorescence intensity (MFI) of C5aR1 and LukS-PV from panel A. Mean ± SEM from four independent experiments. ns = not significant; P values are shown by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Localization of LukS-PV (red) in WT and SGMS1 KO macrophages treated with PVL (62.5 ng/ml) or LukS-PV (62.5 ng/ml) for 5 or 15 min. Plasma membrane was stained with DeepRed Cytopainter (green). White arrows indicate internalized LukS-PV signal. Scale bar is 20 µm. (D) WT and C5aR1-deficient human induced pluripotent stem cell (iPSC)-derived macrophages were treated with PVL (62.5 ng/ml; 5 or 15 min) and probed with anit-LukS-PV (red) and C5aR1 (green) antibodies, and nuclei stained with DAPI (blue). Scale bar is 20 µm. (E) Human iPSC-derived macrophages were treated with MβCD for 30 min prior to PVL exposure for 15 min. Cells were probed with anti-LukS-PV (green) and C5aR1 (red) antibodies. Scale bar is 20 µm. (F) Colony forming unit (CFU) of S. aureus in WT, SGMS1 KO, and C5aR1 KO human THP-1 macrophages, infected at an MOI of 10, at 1 and 4 h post infection (hpi). Mean ± SEM from six independent experiments. One-way ANOVA showed no statistical significance (n.s.). (G) Cell death (Draq7-positive) of WT, SGMS1 KO, and C5aR1 KO THP-1 macrophages after infection with S. aureus . Mean and ± SEM from three independent experiments. *** indicate P -value <0.01 (one-way ANOVA between WT and SGMS1 KO). The data underlying this Figure can be found in S1 Data.

Article Snippet: Membranes were blocked with TBS containing 0.2% Tween 20 (TBS-T) containing 5% skim milk for 1 h and were then probed with primary antibodies to LukS-PV (made inhouse), human C5aR1 (clone B-6, #sc-271949, Santa Cruz Biotechnology) at 4°C overnight.

Techniques: Knock-Out, Clone Assay, Flow Cytometry, Control, Staining, Fluorescence, Comparison, Clinical Proteomics, Membrane, Derivative Assay, Infection