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: DMAG induces the differentiation of HEL cells. a The expression of CD41 and CD61 was detected by flow cytometry after the HEL cells were treated with DMAG (10, 20 and 40 µM) for 6 days. The histogram shows the percentage of CD41+CD61+ cells in each group. The data represent the mean ± SD of three independent experiments; ***p < 0.001, compared to the control group. b The expression of CD41 and CD42b was measured by flow cytometry after the HEL cells were treated with DMAG (10, 20 and 40 µM) for 6 days. The histogram shows the percentage of CD41+CD42b+ cells in each group. The data represent the mean ± SD of three independent experiments; **p < 0.01, ***p < 0.001, compared to the control group. c Flow cytometry analysis of DNA ploidy after the cells treated with DMAG (10, 20 and 40 µM) for 6 days. The histogram shows the percentage of DNA ploidy in each group. The data represent the mean ± SD of three independent experiments; **p < 0.01, ***p < 0.001, compared to the control group. d HCS analysis of DNA ploidy after the cells treated with or without DMAG (10, 20 and 40 µM) for 6 days. Cells were marked with blue, turquoise, green, orange and red. The different colors represent different phases of the cell cycle. Blues indicate G0/G1 (2 N) cells. Turquoises indicate S (2 N) cells. Greens represent G2 (= 4 N) cell. Oranges represent early M (= 4 N) cells. Reds represent late M (2 N) cells. The histogram shows the percentage of DNA ploidy in each group. The data represent the mean ± SD of three independent experiments; ***p < 0.001, compared to the control group

Fig 4: Chitinase 3-like-1 (Chi3l1)/CD44 signaling in M?s upregulates podoplanin expression and platelet adhesion.(A) Male WT, Chil1-/-, Cd44-/- mice were treated with acetaminophen (APAP) (n = 4 mice/group). After 3 hr, mice were sacrificed and M?s were isolated to measure mRNA levels of various adhesion molecules, including selectin P ligand (Selplg), Cd40, melanoma cell adhesion molecule (Mcam), Fc receptor (Fcr), intercellular adhesion molecule 1 (Icam1), lymphocyte function-associated antigen 1 (Lfa1), von Willebrand factor (Vwf), and podoplanin (Pdpn). (B, C) Wild-type (WT) mice were treated with APAP. Chil1-/- and Cd44-/- mice were treated with PBS or rmChi3l1 followed by APAP challenge simultaneously and mice were sacrificed 3 hr after APAP (n = 3 mice/group). (B) M?s were isolated and mRNA levels of Pdpn in M?s were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (C) Immunofluorescence (IF) staining of liver sections for podoplanin and F4/80 is shown and the proportions of M?s that express Pdpn were quantified, Scale bar, 25 µm. (D–F) Chil1-/- mice reconstituted with rmChi3l1 were treated with either Ctrl IgG or a-podoplanin Ab for 16 hr and subsequently challenged with APAP. (D) Serum levels of ALT and (E) liver histology were evaluated 24 hr after APAP treatment (n = 6 mice/group). Scale bar, 250 µm. (F) IF staining for intrahepatic platelets (CD41+) and M?s (F4/80+) was performed 3 hr after APAP (n = 3 mice/group). Scale bar, 25 µm. One-way ANOVA were performed in A–C. Two-tailed, unpaired Student’s t-test was performed in D.

Fig 5: 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.