Immune dysfunction is a prominent feature of decompensated cirrhosis and contributes to 50% of cirrhotic deaths (35)

Immune dysfunction is a prominent feature of decompensated cirrhosis and contributes to 50% of cirrhotic deaths (35). IL4-induced differentiation Next we studied the phenotype of wild-type and knock-out macrophages using peritoneal macrophages isolated from untreated mice. We confirmed that 95 3% of those cells express the F4/80 macrophage marker (Fig. 2and = 4C8 per group, *, 0.05, **, 0.01 compared with WT. 0.01 compared with WT, = 4C6 per group. after low dose (2 mg/kg) intraperitoneal LPS injection. Fig. 2shows that a number of macrophage-specific markers were unchanged in the livers; the M2 differentiation Mevalonic acid marker Mrc1 (CD206) expression was significantly lower in PRMT1 myeloid KO mice at 24 h post LPS. This suggests lack of M2 differentiation. Immunohistochemical staining of liver sections 24 h post LPS confirmed that although the number of macrophages is the same in wild-type and knock-out mice (F4/80 staining), Mevalonic acid Mrc1 expression is not detectable in sections from PRMT1 KO mice (Fig. 2and = 4, **, 0.01. = 4C6, *, 0.05, **, 0.01. 0.01 compared with WT, = 4C6 per group. and 0.05, **, 0.01 compared with WT, = 4C6. = 4, *, 0.05, **, 0.01. Next we examined LPS responses under M1 or M2 differentiation conditions. IL4-mediated differentiation of macrophages is known to suppress cytokine production in response to LPS challenge. It is an important step in infection clearance to avoid excessive cytokine production. We found that KO macrophages produced higher levels of IL6 and TNF when differentiated with IL4 prior to LPS stimulation (Fig. 3data, as in Fig. 1, and neutralization assay (Fig. 3shows relative PRMT1 levels in human blood monocytes isolated from control subjects and subsequently differentiated with Mevalonic acid CSF-1 (colony-stimulating factor 1) for 5 days. To determine whether PRMT1 also increases after monocyte differentiation we directly compared blood monocytes and peritoneal macrophages from patients with liver cirrhosis and ascites; a similar increase was observed when comparing PRMT1 expression in monocytes and macrophages from the same patients (Fig. 4differentiated monocyte-derived macrophages from healthy individuals (Fig. 4= 4C6. Data are presented as mean S.D. = 4. Data are presented as mean S.D. **, 0.01 compared with day 0. = 13). Data are presented as mean S.D. **, 0.01. = 12). **, 0.01 by paired test. 0.01, ***, 0.001 between paired monocytes and macrophages, ###, 0.001 for H4R3me2a pulldown signal in macrophages compared with IgG. 0.01. = 4. Data are presented as mean S.D. The PRMT1 effect on M2 differentiation is PPAR dependent To examine whether a defect in alternative macrophage differentiation can be explained by this loss of PPAR induction, we isolated peritoneal macrophages from wild-type and myeloid PRMT1 knock-out mice and treated them with IL4 in the presence or absence of the PPAR agonist GW1929 or the PPAR antagonist GW9662 (Fig. 5, and and and 3. Data are presented as mean S.D. ***, 0.001 compared with wild type, *, 0.05 compared with DMSO control. shows average percentage of Mrc1-positive cells calculated from 10 random fields from each of = 3 mice per condition **, 0.01. and compare relative mRNA levels in macrophages differentiated with IL4 for 24 h in the presence or absence of AMI-1 and PPAR antagonist GW9662 (1 m). Data are presented as mean S.D. = 3. *, 0.05, **, 0.01 compared with untreated. Next we examined whether only histone arginine methylation induced during monocyte to macrophage differentiation might be important for PRMT1-dependent Mrc1 and other gene expression. To investigate if transient MAPKKK5 inhibition of PRMT1 activity during monocyte to macrophage differentiation can prevent alternative activation as seen in the PRMT1 knock-out phenotype, we isolated blood monocytes from healthy individuals and differentiated them with CSF-1 in the presence or absence of the PRMT1 inhibitor AMI-1. AMI-1 was present only during the differentiation step and was removed prior to further manipulations. Next we assessed the expression of macrophage genes after IL4-induced differentiation. We found that macrophages that were treated with AMI-1 during differentiation are deficient in PPAR-dependent.