Fig 2: 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 3: CHI3L1 binds to ACTN4 and NFKB1, and promote the activation of NF-?B pathway. (A) Schematic of the procedure used to detect biotin-hCHI3L1-binding proteins using HuProt 20K human proteome microarrays. (B) ACTN1, 4, NFKB1, 2, and NFKBIB were identified as CHI3L1-binding proteins in the proteome microarrays. (C) The IF analysis of the expression levels of CHI3L1 and NF-?B p65 subunit in peritumor and intratumor regions; Scale bars represent 100 µm. (D) Western blot analysis of the expression levels of CHI3L1 in U118MG and U251MG cells after treated with TNF? (200 ng/mL) for 0-96 h. (E) Chi3l1+/+ and Chi3l1-/- BMDMs were treated with TNF? (50 and 200 ng/mL), and phosphorylation of p65 were assessed via western blot. (F-G) U87MG and A172 cells transfected with shCtrl and shCHI3L1 were treated with TNF?, and the phosphorylation of p65 were detected using western blot. (H-I) Cytoplasmic and nuclear proteins were isolated from BMDMs and U87MG after treated with TNF? (50 ng/mL) and applied to western blot to detect the expression of p65 and ACTN4. (J) Lysates from U87MG and A172 cells were immunoprecipitated with IgG or anti-CHI3L1 antibody, and then immunoblotted as indicated. (K) Cytoplasmic and nuclear proteins were isolated from U87MG cells. The lysates were immunoprecipitated with IgG or anti-CHI3L1 antibody, and then immunoblotted as indicated. (L-N) Co-localization of CHI3L1 and ACTN4, NFKB1, or p65 in U87MG or A172 cells observed by confocal microscope. (O) CHI3L1 and ACTN4 enhance NF-kB activation by using a dual-luciferase reporter assay. (P) Dox-inducible CHI3L1 expression enhanced enhanced NF-kB activation by TNFa in U251 cells. (Q) Pearson correlations between CHI3L1 expression and ACTN4, NFKB1, NFKB2, RelA (p65), and NFKBIB in TCGA and CGGA glioma cohorts.

Fig 4: Chitinase 3-like-1 (Chi3l1) is upregulated and plays a critical role in acetaminophen-induced liver injury (AILI).(A) Immunohistochemical (IHC) staining for Chi3l1 in normal liver biopsies (Normal) and those from patients with AILI (Patient). Images shown are representative of 10 samples/group. Scale bar, 250 µm. (B) Enzyme-linked immunosorbent assay (ELISA) analysis of Chi3l1 in serum of healthy individuals (Normal, n = 6) and those from patients with AILI (Patient, n = 29). Data were presented as median+interquartile range. (C, D) Male C57B/6 mice treated with PBS or acetaminophen (APAP). (C) Chil1 mRNA in liver homogenates and (D) Chi3l1 protein levels in serum were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and ELISA at 3 and 24 hr, respectively (n = 4 mice/group). (E, F) Male C57B/6 (wild-typr [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 (n = 4–10 mice/group). (E) Serum levels of ALT and (F) liver histology with necrotic areas outlined were evaluated 24 hr after APAP treatment. Scale bar, 250 µm. Mann-Whitney test was performed in B. Two-tailed, unpaired Student’s t-test was performed in C, D. One-way ANOVA were performed in E.

Fig 5: CHI3L1 interacts with CD44 to drive M2 TAMs polarization. (A-B) Western blot of CHI3L1, p65, and ACTN4 expression after TNF? treatment in U87MG and A172 cells, which were pretreated with dimethyl sulfoxide (DMSO), CB, (-)-B, PF, and Y. (C-D) Cytoplasmic and nuclear CHI3L1, p65, and ACTN4 were analyzed using western blot. (E) mRNA expression levels of CHI3L1 between the control and CB pretreated group in U87MG and A172 cell lines. (F) Co-immunofluorescent staining of CHI3L1 and CD206 in frozen sections of human gliomas. (G-H) Macrophages derived from THP1 induced by PMA (10 ng/mL) for 24 h, and identified by morphologic evaluation and mRNA expression of CD68. (I) M2 markers (CD206 and CD163) expression in macrophages treated with IL-4 (100 ng/mL), rhCHI3L1 (500 ng/mL), and the culture supernatant (cs) of U87MG and A172 cells measured by IF. (J-K) mRNA expression of M1 and M2 markers quantitated by qRT-PCR in macrophages treated with IL4, and the culture supernatant of U87MG and A172 cells. (L-N) Migration of U118MG cells induced by M2 macrophages. (O-V) mRNA expression of M1 and M2 markers in Cd44+/+ and Cd44-/- BMDMs treated with IL-4 and rmCHI3L1 (500 ng/mL). (W) Western blot of phosphorylation of AKT and p38 in Cd44+/+ and Cd44-/- BMDMs pretreated with rmCHI3L1.