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: International journal of biological sciences

Article Title: Suppressing MDSC Infiltration in Tumor Microenvironment Serves as an Option for Treating Ovarian Cancer Metastasis.

doi: 10.7150/ijbs.70013

Figure Lengend Snippet: Figure 7. The C5a receptor inhibitor suppresses MDSC infiltration in TME against cancer metastasis and tumorigenesis. A. Schematic diagram of subcutaneous ID8-luciferase tumors in C57BL/6 mice with CCX168 treatment. The ID8-luciferase cells (5x105) were inoculated into 6-week C57BL/6 mice subcutaneously. After inoculation, the CCX168 (10 mg/kg) or normal saline as control was given by oral gavage every day. B-D. Tumor growth curves, post-dissection measured tumor weights, and volumes of C57BL/6 mice treated with CCX168 or normal saline as control. ID8-luciferase cells (5x106) were inoculated s.c. into 6-week C57BL/6 mice (n=6 per group). The C57BL/6 mice were treated with CCX168 (C5aR inhibitor, 10 mg/kg) or by oral gavage every day. E. The flow cytometry analysis showed fraction change of MDSCs (CD11b+, Gr-1+) in ID8 mice tumor microenvironment. F. Bioluminescence imaging of intraperitoneal ID8-luciferase tumors in C57BL/6 mice with CCX168 treatment. The ID8-luciferase cells (5x105) were inoculated i.p. into 6-week C57BL/6 mice. After 9-day inoculation, the CCX168 (10 mg/kg) or normal saline as control was given by oral gavage every day. Statistic plot of total flux in bioluminescence imaging. G. The graphic illustration of LncOVM-mediated PPIP5K2 regulating complement C5 secretion through the Golgi complex and MDSC recruitment in tumor microenvironment.

Article Snippet: Primary antibodies including: P230 (Biolegend, 611280, 1:100), Golph3 (Abcam, ab91492, 1:100), PPIP5K2 (Abcam, ab204374, 1:100), C5aR (Proteintech, 21316-1-AP), Ly-6G/Ly-6C (Gr-1) (Biolegend, 108401,1:100), CD45 (Biolegend, 103132, 1:100), CD11c (BD, 553801, 1:100), F4/80 (Biolegend, 123116, 1:100), SMA (ZSGB-BIO, zm-0003, 1:100) incubated overnight at 4°C.

Techniques: Luciferase, Saline, Control, Dissection, Flow Cytometry, Imaging

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