Fig 2: Chitinase 3-like-1 (Chi3l1) contributes to acetaminophen-induced liver injury (AILI) by promoting hepatic platelet recruitment.(A) Immunohistochemical (IHC) staining to detect platelets (CD41+) in healthy liver biopsies (Normal) and those from patients with AILI (Patient). Scale bar, 250 µm (n = 10/group). (B) Male C57B/6 mice treated with PBS or acetaminophen (APAP). Intravital microscopy analyses were performed around 3 hr post-APAP. M?s (cyan) and platelets (white) in liver sinusoids (red) are indicated. Representative images were chosen from intravital microscopy videos: https://bcm.box.com/s/15hmtryyrdl302mihrsm034ure87x4ea (Supplementary video 1, PBS treatment) and https://bcm.box.com/s/tuljfmstvv4lvoksx16fkxkpirkekynz (Supplementary video 2; n = 6–7 mice/group, 4–15 videos/mouse). (C–E) Male C57B/6 (wild-type [WT]) mice were treated with control IgG (Ctrl IgG) or an anti-CD41 antibody (a-CD41 Ab) either 3 hr before or 3 hr after APAP administration. (C) Serum levels of ALT and (D) liver histology with necrotic areas outlined were evaluated 24 hr after APAP treatment (n = 5 mice/group in C, D). Scale bar, 250 µm. (E) Male C57B/6 (WT) and Chil1-/- mice were treated with APAP. Additionally, Chil1-/- mice were divided into two groups treated with either PBS or recombinant mouse Chi3l1 (rmChi3l1) simultaneously with APAP. Immunofluorescence (IF) staining was performed to detect intrahepatic platelets (CD41+) 3 hr after APAP treatment (n = 3 mice/group). Scale bar, 25 µm. Two-tailed, unpaired Student’s t-test was performed in A–C. One-way ANOVA were performed in E.

Fig 3: Evaluation of the therapeutic potential of targeting chitinase 3-like-1 (Chi3l1) in the treatment of acetaminophen-induced liver injury (AILI).(A–C) Male C57B/6 mice were treated with acetaminophen (APAP) for 3 hr, followed by intraperitoneally (i.p.) injection of either a control IgG (Ctrl IgG) or an anti-mouse Chi3l1 Ab (a-mChi3l1 Ab, C59). (A) Immunofluorescence (IF) staining for intrahepatic platelets (CD41+) was performed 6 hr after APAP treatment (n = 3 mice/group). Scale bar, 25 µm. (B) Serum levels of ALT and (C) liver histology were evaluated 24 hr after APAP treatment (n = 4–6 mice/group). Scale bar, 250 µm. (D–F) Chil1-/- mice were treated with APAP plus PBS or recombinant human Chi3l1 (rhChi3l1) for 3 hr as indicated and APAP plus rhChi3l1 treatment group were either without treatment or treated with a control IgG (Ctrl IgG) or an anti-human Chi3l1 Ab (a-hChi3l1 Ab, C7). (D) IF staining was performed to identify intrahepatic platelets (CD41+) 6 hr after APAP treatment. Scale bar, 25 µm. (E) Serum levels of ALT and (F) liver histology were evaluated 24 hr after APAP treatment. Scale bar, 250 µm (n = 5–10 mice/group in D–F). Two-tailed, unpaired Student’s t-test was performed in B. One-way ANOVA were performed in E.

Fig 4: Two-dimensional electrophoresis of neutrophil lysates. In the first dimension, proteins were separated by isoelectric focusing followed by SDS-PAGE in the second dimension and subsequently stained with Coomassie blue [A]. The rectangle marks the spot with an apparent isoelectric point of pH 8.5 and molecular weight of 40 kDa identified as chitinase-3-like protein 1 protein by MALDI-TOF mass spectrometry. Immunoblotting of neutrophil proteins separated by two-dimensional electrophoresis and incubated with patient sera [B] or sera from healthy controls [C] followed by enhanced chemiluminescence detection after incubation with peroxidase-conjugated IgG. Arrow marks the reactive spot that was identified as CHI3L1. Circles mark the known neutrophil antigenic targets proteinase 3 [PR3] and lactoferrin [LTF].

Fig 5: The effect of lenti-synd4 shRNAs on the migration of HUVECs treated with YKL-40 at 24 hours and 32 hours. The representative photomicrographs of wound healing assay for control, YKL-40 group, YKL-40+lenti-synd4 shRNAs group and YKL-40+ lenti-null group (A). Photos were taken at 24 hours and 32 hours. The relative migration rate was calculated according to the ratio of (initial wound area-terminal wound area)/initial wound area (B-C). Data were presented as mean ± S.D. (n=3 per group).*** p<0.001, ** p<0.01 and *p<0.05 (the scale bar=200um, 40×magnification).