Fig 2: Immunodepletion of CHIL1 in A-Neu supernatant attenuated the inhibitory effect on pro-inflammatory macrophages. (A) Schematic representation of the experimental design: bone marrow-derived macrophages were stimulated with LPS 10 ng/mL for 3 hours, washed with PBS, and then cultured in the presence of A-Neu supernatant (SUP) or A-Neu supernatant immunodepleted with anti-CHIL1 (SUP-anti-CHIL1). Expressions of CCL4, TNF and NOS2 in indicated groups were detected by RT-qPCR (B, n=6), CBA (C, D, n=4) and WB (E, n=3). The results were presented as mean ± SD. One-way ANOVA was used. *P < 0.05. A-Neu, activated neutrophil; N-Neu, non-activated neutrophil; SUP, activated neutrophil supernatant; SUP-anti-CHIL1, activated neutrophil supernatant immunodepletion CHIL1; Ccl4, C-C chemokine motif ligand 4; Tnf, tumor necrosis factor; Nos2, nitric oxide synthase 2; LPS, lipopolysaccharide.

Fig 3: The schematic diagram of the main content of this study. A-Neu secrete CHIL1 and reduce liver inflammation by inhibiting the pro-inflammatory macrophage response. A-Neu, activated neutrophil; N-Neu, non-activated neutrophil; Ccl4, C-C chemokine motif ligand 4; Tnf, tumor necrosis factor; Nos2, nitric oxide synthase 2; S1P, Sphingosine 1-phosphate; Chil1, Chitinase-like 1.

Fig 4: Microarray analysis was performed in N-Neu and A-Neu. (A) Microarray heatmap data showing the expression level of transcript between N-Neu and A-Neu. The mRNA expressions were expressed as follows: Bright green, under expression; black, no change; bright red, overexpression. n=3. Row-based Z-score normalized. (B) GO enrichment analysis of differentially expressed genes in S1P activated neutrophils. (C) Venn diagram of gene expression overlap between GO terms including Regulation of Cell Communication, Cytokine Secretion, Inflammatory Response and Extracellular Space clusters. (D) Volcano plot of A-Neu gene expression compared with N-Neu. The threshold values were fold change=|2| and P value = 0.05. Red dots and blue dots represent up-regulated and down-regulated genes, respectively. Gray dots indicate genes with no statistically significant differences. The arrow indicated the location of Chil1. A-Neu, activated neutrophil; N-Neu, non-activated neutrophil; Il1b, Interleukin 1 beta; Chil1, Chitinase-like 1; Ccl3, Chemokine (C-C motif) ligand 3; Il1a, Interleukin 1 alpha; Tnf, tumor necrosis factor.

Fig 5: Recombinant CHIL1 inhibited the pro-inflammatory phenotype of macrophages. (A) Schematic representation of the experimental design: bone marrow-derived macrophages were stimulated with LPS 10 ng/mL for 3 hours, washed with PBS, and then treated with Vehicle (PBS) or rCHIL1 (100 ng/mL) for 24 hours respectively. (B) Macrophages were harvested and the mRNA expression levels of genes Ccl4, Tnf and Nos2 were evaluated using RT-qPCR, presented relative to Gapdh, (control group, n=3; LPS group, n=6). The protein expressions of CCL4, TNF and NOS2 in liver were detected by CBA (C, D) and WB (E) n=4. The results were presented as mean ± SD. Two-way ANOVA was used. *P < 0.05. rCHIL1, Recombinant CHIL1; Ccl4, C-C chemokine motif ligand 4; Tnf, tumor necrosis factor; Nos2, nitric oxide synthase 2; LPS, lipopolysaccharide.