Fig 1.
Expression of IL4I1 throughout macrophage differentiation.
BM cells at day 0 or during macrophage differentiation in the presence of M-CSF (20 ng/mL) for the indicated periods of time were collected. Levels of IL4I1 gene and IL4I1 protein expression were assessed by q-PCR (A) and western blotting (B), respectively. IL4I1 expression in medium (m) and total cellular proteins (c) from BMDMs were assessed by western blotting (C). Levels of IL4I1 gene and IL4I1 protein expression in primary monocytes and macrophages were assessed by q-PCR (D) and western blotting (E), respectively. All representative data are presented as means ± S.D. for three independent experiments. Significance was calculated by two tailed unpaired Student's t-test. Asterisks indicate significant differences compared to BM cells at day 0; *p<0.05, **p<0.01, ***p<0.001.
Fig 2.
Induction of IL4I1 expression in macrophages.
BMDMs were treated with LPS (100 ng/mL), IFN-γ (15 ng/mL), poly(I:C) (1 μg/mL), CpG (0.3 μM), or IL-4 (10 ng/mL) for the indicated amounts of time or were left untreated. Levels of IL4I1 expression were assayed by q-PCR (A–C, G–H) and western blotting (D–F, I–J), respectively. β-actin was used as a loading control throughout. Representative data are presented as means ± S.D. of three or four independent experiments. Significance was calculated by two tailed unpaired Student's t-test. Asterisks indicate significant differences compared to untreated cells; *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
IL4I1 promotes M2 macrophage phenotypes.
BMDMs were transfected with siRNA to target IL4I1 or with scrambled siRNA for 24 h. The silencing efficiency was evaluated by q-PCR (A) and western blotting (B), respectively. Representative data are presented as means ± S.D. of three independent experiments. Significance was calculated by one-way ANOVA with multiple comparison post test (Bonferroni). Asterisks indicate significant differences compared to scrambled siRNA transfections; ***p<0.001. BMDMs were transfected with siRNA against IL4I1 or scrambled siRNA for 24 h, and then were treated with IL-4 (20 ng/mL) or were untreated for 24 h. Expression of Arg-1 and IL4I1 was assayed by western blotting (C) and IL-10 expression was assayed by ELISA (F). The gene expression levels of the M2 markers Fizz-1, Arg-1, YM-1, and MR were assayed by q-PCR (E). BMDMs were infected with IL4I1-encoding recombinant retrovirus or control, then were treated with IL-4 (20 ng/mL) or were untreated for 24 h. Levels of gene expression for the M2 markers Fizz-1, Arg-1, YM-1, and MR were assayed by q-PCR (D) and IL-10 expression was assayed by ELISA (F). Representative data are presented as means ± S.D. of four independent experiments. Significance was calculated by one-way ANOVA with multiple comparison post test (Bonferroni). Asterisks indicate statistically significant differences, either compared to controls, or between two conditions that are linked by a bar (*p<0.05, **p<0.01, ***p<0.001). BMDMs were stimulated with IL-4 (20 ng/mL) or left untreated for 5, 30, or 120 min. The phosphorylation state of STAT-3 and STAT-6 was assessed by western blotting (G); blots are representative of four experiments. Total STAT-6, total STAT-3, and GADPH were used as loading controls.
Fig 4.
IL4I1 limits acquisition of M1 phenotypes.
BMDMs were infected with IL4I1-recombinant or control retrovirus, then were either untreated or treated with LPS (100 ng/mL) for 24 h. Levels of TNF-α, IL-1β, IL-12p40, and iNOS mRNA transcript expression were assayed by q-PCR (A) and those of TNF-α, IL-1β, and IL-12p40 protein expression were assayed by ELISA (C). BMDMs were transfected with siRNA against IL4I1 or scrambled siRNA for 24 h, then were untreated or treated with LPS (100 ng/mL) for 24 h. Levels of TNF-α, IL-1β, IL-12p40, and iNOS gene expression were assayed by q-PCR (B) and those of TNF-α, IL-1β, and IL-12p40 protein expression were assayed by ELISA (D). Representative data are presented as means ± S.D. of five independent experiments. Significance was calculated by one-way ANOVA with multiple comparison post test (Bonferroni). Asterisks indicate statistically significant differences, either compared to control, or between two conditions that are linked by a bar; *p<0.05, **p<0.01, ***p<0.001.
Fig 5.
IL4I1 promotes immunoregulatory functions of macrophages that were alternatively activated by IL-4.
BMDMs were transfected with siRNA against IL4I1 or scrambled siRNA for 24 h, then were treated with LPS (50 ng/mL), IL-4 (20 ng/mL), or were untreated for 24 h. After 24 h, supernatants were collected and 1 μg/mL OVA323–339 was added. CFSE-labeled splenocytes from DO11.10 mice were cultured with 1 μg/mL soluble OVA323–339 for 72 h. Cell-division was monitored based on levels of CFSE dilution that were measured using flow cytometry (A–B). Data are representative of three independent experiments. Protein expression levels of IFN-γ in supernatants from DO11.10 splenocyte cultures were assayed using ELISA (C). Insoluble extracts of RAW264.7 cells that were transiently transfected with indicated doses of pcDNA-IL4I1 or empty vector were assayed for specific L-tryptophan substrates at 10 mM (final concentration) under atmospheric oxygen (D). ROS assays from RAW264.7 cells transiently transfected with pcDNA4-IL4I1 or pcDNA4 vector in the presence of 50 ng/mL LPS for the indicated times (E). Representative data are presented as means ± S.D. of four independent experiments. Significance was calculated by two-way ANOVA with multiple comparison post test (Bonferroni). Asterisks indicate significant differences compared to controls, or between two conditions that are linked by a bar; *p<0.05, **p<0.01, ***p<0.001.
Fig 6.
Effective inhibitors of IL4I1 include L-1-MT, DPI, anti-IL-10Rα blocking antibody, and L-NMMA.
BMDMs infected with IL4I1-recombinant or control retrovirus were pretreated with the inhibitors mentioned above or were untreated for 4 h. CFSE-labeled splenocytes from DO11.10 mice (n = 6) were cultured in a mixture of IL4I1-overexpressing macrophage-derived supernatant and conventional T cell medium (1:1) with 1 μg/mL soluble OVA323–339 for 72 h. Cell division monitored based on levels of CFSE dilution that were measured using flow cytometry (A–B). Data are representative of three independent experiments. Levels of IFN-γ protein expression from the culture supernatants described above from DO11.10 splenocyte cultures were assayed by ELISA (C). Representative data are presented as means ± S.D. of three independent experiments. Significance was calculated by two-way ANOVA with multiple comparison post test (Bonferroni). Asterisks indicate significant differences compared to controls, or between two conditions that are linked by a bar; *p<0.05, **p<0.01, ***p<0.001.