Fig 2: Ninj1 deficiency in KCs restrained neutrophils but not macrophage infiltration, and inhibited intrahepatic inflammation post-IR. Chimeric mice were established as described in the Materials and Methods section. WT→WT and KO→WT mice were subjected to 90 minutes of warm ischemia or a sham procedure, and liver tissues and blood specimens were collected at 24 hours after reperfusion. (A and B) Representative immunofluorescence staining of CD11b (red) and 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue) in livers from each group, quantification of CD11b+ cells per high-power field. Scale bar: 100 μm. (C and D) Representative immunofluorescence staining of LY6G (red) and DAPI (blue) in livers from each group, quantification of LY6G+ cells per high-power field. Scale bar: 100 μm. (E) Quantitative RT-PCR analysis of liver inflammatory cytokines and chemokine gene induction (IL6, IL1β, TNF-α, CXCL-1, CXCL-2, CCL1, CCL2, CCL3, and CCL5). (F) Enzyme-linked immunosorbent assay of inflammatory cytokine and chemokine levels in serum. All results are representative of at least 2 independent experiments; n = 6 mice/group. ∗P < .05. All data are presented as the means ± SD. LY6G, lymphocyte antigen 6 complex locus g.

Fig 3: Ninj1 deficiency attenuated IR-induced intrahepatic inflammation and neutrophil infiltration by DUSP1-dependent inhibition of p38 and JNK pathways in vivo. WT→WT and KO→WT mice were treated with either DUSP1-siRNA or NS-siRNA before the onset of liver ischemia, as described in the Materials and Methods section. The liver tissues and blood specimens were collected at 24 hours after reperfusion. (A) Western blot analysis of DUSP1, p-P38, T-p38, p-JNK, T-JNK, and β-actin expression. Relative intensity was analyzed by ImageJ software (National Institutes of Health). (B) Quantitative RT-PCR analysis of liver inflammatory cytokine and chemokine gene induction (IL6, IL1β, TNF-α, and CXCL-1). (C) Enzyme-linked immunosorbent assay (ELISA) of inflammatory cytokine and chemokine levels in serum. All results are representative of at least 2 independent experiments. (D and E) Representative immunofluorescence staining of LY6G (red) and 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue) in livers from each group, quantification of LY6G+ cells per high-power field (HPF), Scale bar: 100 μm; n = 6 mice/group. ∗P < .05. All data are presented as the means ± SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. T-p38, Total-p38; T-JNK, Total-JNK; LY6G, lymphocyte antigen 6 complex locus g.