Journal: bioRxiv

Article Title: Complement activation at injury sites drives the phagocytosis of necrotic cell debris and resolution of liver injury

doi: 10.1101/2024.08.23.609344

Figure Lengend Snippet: (A-B) Representative immunofluorescence images of liver cryosections from control mice or mice challenged with acetaminophen (APAP; 600 mg/kg) for 24h. Green: (i)C3b; red: fibrin(ogen); Orange: IgM; Cyan: C1q. Scale bar represents 50 µm. (C-F) Quantification area percentage stained with fibrin(ogen) (C), IgM (D), C1q (E) and (i)C3b (F) in liver cryosections of control mice and mice receiving an APAP overdose for 24, 48 or 72h. (G) Pearson’s correlation coefficient of (i)C3b and nuclei (Hoechst) or fibrin(ogen) in the injured livers 24, 48 and 72h after APAP overdose. (H) Pearson’s correlation coefficient between (i)C3b, IgM and C1q in the injured livers 24h after APAP overdose. (I) C3 levels in the serum of control mice or challenged with APAP for 24h. (J) C3a levels in the serum of control mice or challenged with APAP for 24h. Image quantifications were pooled from 8 fields of view. Each dot represents a single mouse. Data are represented as mean ± SEM. *p≤0.05 compared to control; #p≤0.05 between indicated groups. APAP = acetaminophen.

Article Snippet: Sections were incubated overnight at 4°C with 10 µg/ml polyclonal rabbit anti-human/mouse fibrin(ogen) (Dako), 5 µg/ml rat anti-mouse C3b/iC3b (clone 3/26, Hycult Biotec) and 5 µg/ml rabbit anti-mouse C1q (clone 4.8, Abcam).

Techniques: Immunofluorescence, Control, Staining

Journal: bioRxiv

Article Title: Complement activation at injury sites drives the phagocytosis of necrotic cell debris and resolution of liver injury

doi: 10.1101/2024.08.23.609344

Figure Lengend Snippet: (A) Representative immunofluorescence images of liver cryosections from WT and Rag2 -/- mice 24h after an overdose of acetaminophen (APAP; 600 mg/kg). Red: IgM; Cyan: C1q; Green: (i)C3b. Scale bar represents 100 µm. (B) Quantification of area percentage stained with IgM in liver cryosections of WT and Rag2 -/- mice 24h after APAP overdose. (C) Mean fluorescence intensity (MFI) of IgM staining at the necrotic injury sites in liver cryosections. (D) Quantification of area percentage stained with fibrin(ogen) in liver cryosections of WT and Rag2 -/- mice 24h APAP overdose. (E) Quantification of C1q deposition in the liver of WT and Rag2 -/- mice 24h after APAP overdose, normalized to the degree of fibrin(ogen) staining. (F) Mean fluorescence intensity of C1q staining at the necrotic injury sites in liver cryosections. (G) Quantification of (i)C3b deposition in the liver of WT and Rag2 -/- mice 24h after APAP overdose, normalized to the degree of fibrin(ogen) staining. (H) Mean fluorescence intensity of (i)C3b staining at the necrotic injury site of liver cryosections. Image quantifications were pooled from 8 fields of view. Each dot represents a single mouse. Data are represented as mean ± SEM. *p≤0.05 compared to WT mice. APAP= acetaminophen, MFI = Mean fluorescence intensity.

Article Snippet: Sections were incubated overnight at 4°C with 10 µg/ml polyclonal rabbit anti-human/mouse fibrin(ogen) (Dako), 5 µg/ml rat anti-mouse C3b/iC3b (clone 3/26, Hycult Biotec) and 5 µg/ml rabbit anti-mouse C1q (clone 4.8, Abcam).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Complement activation at injury sites drives the phagocytosis of necrotic cell debris and resolution of liver injury

doi: 10.1101/2024.08.23.609344

Figure Lengend Snippet: (A) Representative image of neutrophils (green) and necrotic debris (pHRodo; red) in the liver 6h after FTI. pHRodo-SE was applied on top of the lesion 6h before imaging. Scale bar represents 50 µm, scale bar zoomed image represents 20 µm. (B) Quantification of the percentage neutrophils carrying pHRodo labeled debris at different areas of the injury. (C) Representative immunofluorescence stainings of liver cryosections 6h post burn injury. Green: (i)-C3b; Cyan: C1q. Scale bar represents 200 µm. (D-G) Flow cytometry of non-parenchymal cells isolated from liver FTI sites showing the percentage (D) pHrodo neutrophils (Ly6G), (E) pHrodo non-classical monocytes (Ly6C / CX 3 CR1), (F) pHrodo classical monocytes (Ly6C / CX 3 CR1 / CCR2) and macrophages (F4/80). Data are represented as mean ± SEM. Each dot represents a single mouse. *p≤0.05 compared to WT.

Article Snippet: Sections were incubated overnight at 4°C with 10 µg/ml polyclonal rabbit anti-human/mouse fibrin(ogen) (Dako), 5 µg/ml rat anti-mouse C3b/iC3b (clone 3/26, Hycult Biotec) and 5 µg/ml rabbit anti-mouse C1q (clone 4.8, Abcam).

Techniques: Imaging, Labeling, Immunofluorescence, Flow Cytometry, Isolation

Journal: bioRxiv

Article Title: Complement activation at injury sites drives the phagocytosis of necrotic cell debris and resolution of liver injury

doi: 10.1101/2024.08.23.609344

Figure Lengend Snippet: (A) 3D reconstruction image of human neutrophils stained with Calcein (green) phagocytosing pHrodo-labeled necrotic debris (red). Scale bar represents 10 µm. (B) Percentage of bone-marrow derived mouse neutrophils phagocytosing necrotic debris opsonized with serum, C3-depleted serum (from C3 -/- mice) or antibody-depleted serum (from Rag2 -/- mice). (C) Volume of necrotic debris phagocytosed by bone-marrow derived mouse neutrophils which was opsonized with serum, C3 serum or C1q serum. (D) Percentage of human neutrophils phagocytosing necrotic debris opsonized with serum, C1q-depleted serum or C3-depleted serum. (E) Volume of necrotic debris phagocytosed by human neutrophils which was opsonized with serum, C1q-depleted serum or C3-depleted serum. (F-J) Gene expression of human neutrophils incubated with unopsonized, serum-opsonized or C3-depleted serum-opsonized human necrotic debris. Data are normalized to the average expression of 3 housekeeping genes (GAPDH, 18s and CDKN1A) and represented as 2 −ΔΔCt relative to the unopsonized group. 10 µM latrunculin is used as a negative control. Data are represented as mean ± SEM. Each dot represents an independent experiment. *p≤0.05 compared to serum group or between indicated groups.

Article Snippet: Sections were incubated overnight at 4°C with 10 µg/ml polyclonal rabbit anti-human/mouse fibrin(ogen) (Dako), 5 µg/ml rat anti-mouse C3b/iC3b (clone 3/26, Hycult Biotec) and 5 µg/ml rabbit anti-mouse C1q (clone 4.8, Abcam).

Techniques: Staining, Labeling, Derivative Assay, Expressing, Incubation, Negative Control