Journal: Hepatobiliary Surgery and Nutrition

Article Title: EREG-secreting THBS1 + tissue monocytes are recruited by C5a to promote rapid liver regeneration in patients and mice during the ALPPS procedure

doi: 10.21037/hbsn-24-391

Figure Lengend Snippet: The potential role of C5a in the recruitment of THBS1 + tissue monocytes. (A) The results of a GO analysis of bulk FLR tissue. (B) Bubble plots of THBS1 + tissue monocyte receptor interactions (left) and hepatocyte ligand interactions (right) generated according to the CellphoneDB. The color is coded by the mean intensity of the interaction, and the size of the bubble is determined by the −log 10 P. (C) t-SNE plot showing clusters generated by scRNA-seq data. The color is coded by the expression level of C5a. (D) t-SNE plot showing MP clusters generated by scRNA-seq data, and a violin plot showing the C5aR1 expression in all Mo/Mφ populations. (E) GO and KEGG analyses of genes up-regulated in stage II hepatocytes as revealed by scRNA-seq. FLR, future liver remnant; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; Mo, monocytes; MP, Mo/Mφ population; Mφ, macrophages; scRNA-seq, single-cell RNA sequencing; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: For C5a blockade, PMX-53, a specific C5aR1 antagonist, was used (HY-106178, MedChemExpress, Shanghai, China).

Techniques: Generated, Expressing, Single Cell, RNA Sequencing

Journal: Hepatobiliary Surgery and Nutrition

Article Title: EREG-secreting THBS1 + tissue monocytes are recruited by C5a to promote rapid liver regeneration in patients and mice during the ALPPS procedure

doi: 10.21037/hbsn-24-391

Figure Lengend Snippet: THBS1 + tissue monocytes were recruited through C5a. (A) Multiplexed immunofluorescence staining, showing the distribution of C5aR1 + monocytes (CD14 + C5aR1 + ). (B) Immunohistochemical staining of C5a at ALPPS stage I (upper left) and stage II (lower left). The H-score shows the up-regulation of C5a expression in hepatocytes from ALPPS stage I to stage II. *, P<0.05. (C) Liver samples from the murine ALPSS model on POD1 and POD5 (left). The boxplot represents comparisons of the FLR to body weight ratio between the control group and PMX-53 group on POD1 and POD5. (D) Immunohistochemical staining of Ki-67 for the control group and PMX-5 group on POD1 and POD5 (left). The boxplot shows comparisons of the ratios of Ki-67 + cells between the control and PMX-53 group on POD1 and POD5 (right). (E) Boxplot showing the EREG expression in the control and PMX-53 group on POD1 and POD5. (F) Schematic diagram of the ALPPS-induced liver regeneration. *, P<0.05 (actual P values were reported in results). ALPPS, associating liver partition and portal vein ligation for staged hepatectomy; EREG, epiregulin; IF, immunofluorescence; IHC, immunohistochemistry; FLR, future liver remnant; POD, post-operation day.

Article Snippet: For C5a blockade, PMX-53, a specific C5aR1 antagonist, was used (HY-106178, MedChemExpress, Shanghai, China).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Expressing, Control, Ligation, Immunohistochemistry

Journal: bioRxiv

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs and CD8 + T cells

doi: 10.1101/2024.10.15.618536

Figure Lengend Snippet: The immune-dependent and SERT-independent anti-tumor effects of citalopram in HCC . ( A , B ) Mouse HCC cells, Hepa1-6 or Hep53.4, were subcutaneously injected into Rag1 -/- C57BL/6 or immunocompetent C57BL/6 mice (n = 5-7 per group). When bore visible tumors, 5 mg/kg citalopram was treated daily for 15 or 25 days. Tumors were excised after mice were sacrificed, and the tumor weight was measured. ( C , D ) Western blotting showed the knockdown efficiency of SERT in Hepa1-6 and Hep53.4 cells. ( E , F ) shControl and sh Slc6a4 Hepa1-6 and Hep53.4 cells were subcutaneously injected into Rag1 -/- C57BL/6 or immunocompetent C57BL/6 mice (n = 5-6 per group). When bore visible tumors, 5 mg/kg citalopram was treated daily for 15 or 25 days. Tumors were excised after mice were sacrificed, and the tumor weight was measured. In all panels, * p < 0.05, ** p < 0.01, *** p < 0.001. Values as mean ± SD and compared by one-way ANOVA multiple comparisons with Tukey’s method (for bar chart comparison) and two-way ANOVA with Dunnett’s multiple comparisons (for survival curve comparison).

Article Snippet: For the C5aR1 knockdown study, C57BL/6J- C5ar1 em1Cya mice were purchased from Cyagen Biosciences (S-KO-01274, Suzhou, China).

Techniques: Injection, Western Blot, Knockdown, Comparison

Journal: bioRxiv

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs and CD8 + T cells

doi: 10.1101/2024.10.15.618536

Figure Lengend Snippet: C5aR1 is a direct target of citalopram. ( A ) Gene Set Enrichment Analysis (GSEA) identified GLUT1 and C5aR1 as two top hits related to SSRI-induced gene changes. ( B ) GSEA of HCC RNAseq 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 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 concentration 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 h, 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 ANOVA multiple comparisons with Tukey’s method among groups ( G , J ).

Article Snippet: For the C5aR1 knockdown study, C57BL/6J- C5ar1 em1Cya mice were purchased from Cyagen Biosciences (S-KO-01274, Suzhou, China).

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

Journal: bioRxiv

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs and CD8 + T cells

doi: 10.1101/2024.10.15.618536

Figure Lengend Snippet: Citalopram targets C5aR1 + TAMs ( 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; three 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, F, H ) GLUT1 KD Hepa1-6 cells admixed with C5ar1 +/- and C5ar1 -/- BMDMs from donor (d) mice were subcutaneously implanted into syngeneic recipient (r) mice. The therapeutic effect of citalopram ( E ), C5a deposition ( F ), and macrophage phagocytosis ( H ) in this model were analyzed. Scale bar, 50 μm. ( G ) The phagocytic capacity of macrophages isolated from GLUT1 KD Hepa1-6 tumors in C5ar1 +/- and C5ar1 -/- C57BL/6 host. ( I-K ) Flow cytometry showed the infiltration of CD45 + CD11b + F4/80 + macrophages ( I ), CD206 + TAMs and CD11b + TAMs ( J ), tumor-infiltrating lymphocytes ( K ) in tumor tissues from orthotopic xenograft model, which generated in immunocompetent C57BL/6 mice with Hepa1-6 cells (n = 5 per group). ( L, M ) Measurement of CD8 + T cell function in tumor tissues from the groups mentioned in C and E . ( N ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/- and C5ar1 -/- C57BL/6 host upon CD8 + T cell deletion (n = 7). ( O ) 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 as mean ± SD and compared by two-way ANOVA with Dunnett’s multiple comparisons ( B , C , E , N ), Student’s t test ( G , H , I - L ), one-way ANOVA multiple comparisons with Tukey’s method ( B , M ), and the Spearman’s rank correlation methods ( O ).

Article Snippet: For the C5aR1 knockdown study, C57BL/6J- C5ar1 em1Cya mice were purchased from Cyagen Biosciences (S-KO-01274, Suzhou, China).

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

Journal: bioRxiv

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs and CD8 + T cells

doi: 10.1101/2024.10.15.618536

Figure Lengend Snippet: Citalopram activates CD8 + T cells. ( A , B ) Measurement of CD8 + T cell function and glycolysis in orthotopic tumor tissues from WT C57BL/6 mice (n = 5 per group). ( C , D ) Measurement of CD8 + T cell function and glycolysis in orthotopic tumor tissues from MASH mice (n = 5 per group); Basal ECAR indicates glycolysis after the addition of glucose, and ΔECAR represents the difference between oligomycin-induced ECAR and 2-DG-induced ECAR. ( E ) Serum 5-HT levels in GLUT1 KD Hepa1-6 tumor-bearing mice fed with chow diet or CDAHFD, with the presence or absence of citalopram treatment (n = 5 per group). ( F ) Serum TNF-α, IL-1β, and IL-6 levels in GLUT1 KD Hepa1-6 tumor-bearing mice fed with chow diet or CDAHFD, with the presence or absence of citalopram treatment (n = 5 per group). ( G ) Serum 5-HT levels in WT C57BL/6 and Tph1 -/- mice, with the presence or absence of citalopram treatment (n = 5 per group). ( H ) Tumor growth of WT and Tph1 -/- mice after subcutaneous injection of Hepa1-6 cells and treatment with citalopram. ( I ) Measurement of CD8 + T cell function in tumor tissues from the groups mentioned in H . ( J ) The therapeutic effect of citalopram on GLUT1 KD Hepa1-6 tumor was tested in the presence or absence of CD4 + T or CD8 + T cell deletion. In all panels, * p < 0.05, ** p < 0.01, *** p < 0.001; ns, non-significant. Values as mean ± SD and compared by the Student’s t test ( A - F ), one-way ANOVA multiple comparisons with Tukey’s method ( I ), and two-way ANOVA with Dunnett’s multiple comparisons ( H , J ).

Article Snippet: For the C5aR1 knockdown study, C57BL/6J- C5ar1 em1Cya mice were purchased from Cyagen Biosciences (S-KO-01274, Suzhou, China).

Techniques: Cell Function Assay, Injection

Journal: Oncology reports

Article Title: C5aR is frequently expressed in metastatic renal cell carcinoma and plays a crucial role in cell invasion via the ERK and PI3 kinase pathways.

doi: 10.3892/or.2015.3800

Figure Lengend Snippet: Figure 1. Immunohistochemistry of C5aR in the RCC specimens. FFPE samples of RCC were stained with the anti-C5aR antibody. Representative examples of C5aR-negative RCC without metastasis (A) and C5aR-expressing mRCC (B) are shown. Original, x200 magnification. C5aR, C5a receptor; RCC, renal cell carcinoma; mRCC, metastatic RCC; FFPE, formalin-fixed paraffin-embedded.

Article Snippet: Establishment of C5aR stably expressing Renca cells and in vivo study. pCMV-C5aR that encodes full-length murine C5aR was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Immunohistochemistry, Staining, Expressing, Formalin-fixed Paraffin-Embedded

Journal: Oncology reports

Article Title: C5aR is frequently expressed in metastatic renal cell carcinoma and plays a crucial role in cell invasion via the ERK and PI3 kinase pathways.

doi: 10.3892/or.2015.3800

Figure Lengend Snippet: Figure 3. C5a elicits cytoskeletal rearrangement and changes in cellular mor- phology in the C5aR-expressing Renca cells. Renca/empty cells (A-D) and Renca/C5aR cells (E-G) were incubated with C5a (10 nM) and fixed at the indicated time-points. F-actin was visualized by immunofluorescent staining with Alexa 488-conjugated phalloidin (green), and nuclei with TO-PRO-3 (red). Scale bars, 20 µm. Arrowheads and the arrow indicate membrane ruffing and lamellipodia, respectively. C5aR, C5a receptor.

Article Snippet: Establishment of C5aR stably expressing Renca cells and in vivo study. pCMV-C5aR that encodes full-length murine C5aR was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Incubation, Staining, Membrane

Journal: Oncology reports

Article Title: C5aR is frequently expressed in metastatic renal cell carcinoma and plays a crucial role in cell invasion via the ERK and PI3 kinase pathways.

doi: 10.3892/or.2015.3800

Figure Lengend Snippet: Figure 4. C5a-C5aR axis triggers ERK and PI3K-dependent cellular inva- sion. (A) Renca-derived cells were stimulated with or without 10 nM C5a for 15 min after serum starvation for 24 h. Samples were harvested and subjected to immunoblotting using antibodies indicated. (B and C) Invasion assays were carried out using (B) Renca/empty and Renca/C5aR cells or (C) Renca/C5aR cells only. (C) Medium in both the upper and lower wells were mixed with the indicated kinase inhibitors or vehicle (DMSO). U0126 was used at 20 µM and PI-103 was used at 0.5 µM. Data are presented as the mean ± SD. *P<0.02 and **P<0.01. C5aR, C5a receptor.

Article Snippet: Establishment of C5aR stably expressing Renca cells and in vivo study. pCMV-C5aR that encodes full-length murine C5aR was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Derivative Assay, Western Blot

Journal: Nature Communications

Article Title: Schwann cell C5aR1 co-opts inflammasome NLRP1 to sustain pain in a mouse model of endometriosis

doi: 10.1038/s41467-024-54486-6

Figure Lengend Snippet: Representative images of SOX10 and C5aR1 (#orb393229, rabbit polyclonal, Biorbyt Ltd, 1:100) co-expression in sciatic ( a ) and trigeminal ( b ) nerve tissue from C57BL/6 J female (B6) mice, ( n = 4 independent experiments) (Scale bar, 50 μm). (c) C5aR1 mRNA relative expression in primary mouse and human Schwann cells ( n = 3 independent experiments). d Schematic representation of AAV-(loxP-shRNA)- C5aR1 vector pre- and post-Cre switch. e Representative images and cumulative data (Rcoloc) of SOX10 and C5aR1 (#orb393229, rabbit polyclonal, Biorbyt Ltd, 1:100) co-expression in sciatic and trigeminal nerve tissue in Plp -AAV- C5aR1 and Control mice ( n = 4 independent experiments) (Scale bar, 50 μm). f Time-dependent periorbital (PMA), hind paw (HMA), and abdominal (AMA) mechanical allodynia in endometriotic (endo) or Sham Plp -AAV- C5aR1 and Control mice. ( n = 8 mice per group). g Representative images and cumulative data of F4/80 + cells in sciatic and trigeminal nerve tissue in endo or Sham Plp -AAV- C5aR1 and Control mice. ( n = 4 independent experiments) (Scale bar, 50 μm, dashed lines, perineurium ). h Representative images and cumulative data of transmigrated macrophages after stimulation of human and mouse Schwann cells with C5a or vehicle (Veh) and in the presence of DF2593A (DF25) or Veh ( n = 6 independent experiments). Data are mean ± s.e.m. c , Student’s t-test, f , 2-way, g, h 1-way ANOVA, Bonferroni correction; **** P < 0.0001 vs. Sham/Control, #### P < 0.0001 vs. Endo/Control. Source data are provided as a Source Data file.

Article Snippet: Representative images of SOX10 and C5aR1 (#orb393229, rabbit polyclonal, Biorbyt Ltd, 1:100) co-expression in sciatic ( a ) and trigeminal ( b ) nerve tissue from C57BL/6 J female (B6) mice, ( n = 4 independent experiments) (Scale bar, 50 μm). (c) C5aR1 mRNA relative expression in primary mouse and human Schwann cells ( n = 3 independent experiments). d Schematic representation of AAV-(loxP-shRNA)- C5aR1 vector pre- and post-Cre switch. e Representative images and cumulative data (Rcoloc) of SOX10 and C5aR1 (#orb393229, rabbit polyclonal, Biorbyt Ltd, 1:100) co-expression in sciatic and trigeminal nerve tissue in Plp -AAV- C5aR1 and Control mice ( n = 4 independent experiments) (Scale bar, 50 μm). f Time-dependent periorbital (PMA), hind paw (HMA), and abdominal (AMA) mechanical allodynia in endometriotic (endo) or Sham Plp -AAV- C5aR1 and Control mice. ( n = 8 mice per group). g Representative images and cumulative data of F4/80 + cells in sciatic and trigeminal nerve tissue in endo or Sham Plp -AAV- C5aR1 and Control mice. ( n = 4 independent experiments) (Scale bar, 50 μm, dashed lines, perineurium ). h Representative images and cumulative data of transmigrated macrophages after stimulation of human and mouse Schwann cells with C5a or vehicle (Veh) and in the presence of DF2593A (DF25) or Veh ( n = 6 independent experiments).

Techniques: Expressing, shRNA, Plasmid Preparation, Control

Journal: The Journal of Immunology Author Choice

Article Title: Endoplasmic Reticulum Stress of Neutrophils Is Required for Ischemia/Reperfusion–Induced Acute Lung Injury

doi: 10.4049/jimmunol.1500073

Figure Lengend Snippet: C5a induces ER stress of neutrophil. (A) Eight-week-old C57BL/6 male mice (six mice per group) were subjected to intestinal IR. BALF was collected 60 min after the procedures. C5a was measured using ELISA. Data are from three independent experiments. (B) Neutrophils prepared from mice peripheral blood or murine myeloid cell line 32Dcl3 were treated with C5a for 4 h and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. (C) Neutrophils prepared from mice peripheral blood were treated with C5a alone or combined with pertussis toxin (PTX) for 4 h and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. (D) Neutrophils prepared from mouse peripheral blood were treated with fMLF (200 nM) or PMA (100 nM) for 60 min and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. (E) Supernatants from cultured neutrophils described as in (B) were collected, and MPO was measured using ELISA. (F) Murine myeloid cells, 32Dcl3, were transiently transfected with siXBP1 overnight, followed by 4 h of C5a treatment or 4 h of starvation and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. Quantification is expressed as mean ± SD of three independent experiments. *p < 0.05.

Article Snippet: The anti-mouse complement 5a (C5a) receptor (C5aR) Ab was obtained from OriGene (Rockville, MD).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Transfection

Journal: The Journal of Immunology Author Choice

Article Title: Endoplasmic Reticulum Stress of Neutrophils Is Required for Ischemia/Reperfusion–Induced Acute Lung Injury

doi: 10.4049/jimmunol.1500073

Figure Lengend Snippet: C5a interacting with C5aR induces ER stress in neutrophils. (A) Neutrophils prepared from mouse peripheral blood or murine myeloid cell line 32Dcl3 were lysed for immunoblotting with C5aR Ab. Images shown are representative of three experiments. Band intensities were determined with ImageJ. (B) Murine myeloid cells (32Dcl3) were transiently transfected with siC5aR overnight and then lysed. Lysates were analyzed by immunoblotting with C5aR Ab. Images shown are representatives of three experiments. (C) Murine myeloid cells (32Dcl3) were transiently transfected with siC5aR overnight, followed by treatment with C5a and/or C5a neutralizing Ab for 4 h and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. (D) Supernatants from cultured neutrophils described as in (C) were collected, and MPO was measured using ELISA. Data are expressed as mean ± SD of three independent experiments. *p < 0.05.

Article Snippet: The anti-mouse complement 5a (C5a) receptor (C5aR) Ab was obtained from OriGene (Rockville, MD).

Techniques: Western Blot, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Immunology Author Choice

Article Title: Endoplasmic Reticulum Stress of Neutrophils Is Required for Ischemia/Reperfusion–Induced Acute Lung Injury

doi: 10.4049/jimmunol.1500073

Figure Lengend Snippet: Inhibition of ER stress in neutrophils decreases ALI in vivo. (A) xbpf/f MRP8-cre mice (KO) and XBP1f/f mice were used to establish IR-induced ALI model. Lungs samples were fixed and embedded in paraffin. Tissue blocks were sectioned at 5 μm and stained with H&E. Morphology was examined using light microscopy. Pathological scores were given by an experienced pathologist. (B) Sections were stained with Ly6G (1A8) Ab. (C) Neutrophils were prepared from the BALF of normal or ALI xbpf/f MRP8-cre mice and XBP1fl/fl mice, and they were subjected to immunofluorescence staining with indicated Abs. Images shown are representatives of three experiments. (D) Neutrophils were prepared from the BALF of normal or ALI xbpf/f MRP8-cre mice and XBP1fl/fl mice and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. (E) BALF was collected from xbpf/f MRP8-cre mice (KO/N) and XBP1fl/fl mice (fl/fl). TNF-α, IL-6, C5a, and MPO were measured using ELISA. Quantification is expressed as mean ± SD of three independent experiments. Scale bars, 50 μm. *p < 0.05.

Article Snippet: The anti-mouse complement 5a (C5a) receptor (C5aR) Ab was obtained from OriGene (Rockville, MD).

Techniques: Inhibition, In Vivo, Staining, Light Microscopy, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay