Inhibitor of apoptosis proteins, NAIP, cIAP1 and cIAP2 expression during macrophage differentiation and M1/M2 polarization

Abstract

Monocytes and macrophages constitute the first line of defense of the immune system against external pathogens. Macrophages have a highly plastic phenotype depending on environmental conditions; the extremes of this phenotypic spectrum are a pro-inflammatory defensive role (M1 phenotype) and an anti-inflammatory tissue-repair one (M2 phenotype). The Inhibitor of Apoptosis (IAP) proteins have important roles in the regulation of several cellular processes, including innate and adaptive immunity. In this study we have analyzed the differential expression of the IAPs, NAIP, cIAP1 and cIAP2, during macrophage differentiation and polarization into M1 or M2. In polarized THP-1 cells and primary human macrophages, NAIP is abundantly expressed in M2 macrophages, while cIAP1 and cIAP2 show an inverse pattern of expression in polarized macrophages, with elevated expression levels of cIAP1 in M2 and cIAP2 preferentially expressed in M1. Interestingly, treatment with the IAP antagonist SMC-LCL161, induced the upregulation of NAIP in M2, the downregulation of cIAP1 in M1 and M2 and an induction of cIAP2 in M1 macrophages.

Fig 1. Validation of the macrophage differentiation and polarization THP-1 model.

THP-1 monocytes were incubated with 10ng/mL PMA for 24 h to obtain M0 macrophages and then cultured in regular media for 24 h. Cells were then treated with 20 ng/mL IFNγ and 100 ng/mL LPS or 20 ng/mL IL-4 for 48 h for the induction of the M1 or M2 states, respectively. A: Phase contrast microscopy images of THP-1 monocytes, and M0-, M1- and M2-macrophages. B: The expression of the indicated genes was examined by RT-qPCR. Values are normalized to internal controls (HPRT1 and GAPDH) in each group and then the fold change was calculated taking the expression in M1 as the base line in the study of CXCL10 and CD163, M2 expression as the base line in the CD206 analysis and monocyte expression as the baseline in CD14 analysis. Data represent the mean and standard deviation of three independent experiments. *** P<0.001 (ANOVA with Bonferroni post hoc).

Fig 2. NAIP expression in monocytes, M0-, M1- and M2-macrophages.

A, B: The protein level of NAIP was assessed by western blotting of cell extracts from undifferentiated monocytes and M0 or polarized M1/M2 THP-1 macrophages. HSC-70 was used as loading control. A: Representative western blot of one of three experiments with similar results. B: Quantification of NAIP protein abundance in western blots normalized to HSC-70 of three independent experiments. C: NAIP mRNA expression of NAIP was was analysed by RT-qPCR using primers that span exon 4 or between exons 16 and 17 of NAIP. Values are normalized to internal controls (HPRT1 and GAPDH) and presented as mean values ± s.d. of fold-change of three independent experiments. *P<0.05 ***P< 0.001 (ANOVA with Bonferroni post hoc).

Fig 3. NAIP expression in polarized primary human macrophages.

A: NAIP mRNA levels from PBMC-derived monocytes or M1 and M2 macrophages were analyzed by RT-qPCR. Values are normalized to internal controls (HPRT1 and GAPDH) and presented as mean values ± s.d. of fold-change of three independent experiments. B: Protein levels of NAIP in the indicated cell populations were assessed by western blotting. Representative western blot from samples of one of the donors. Graph represents the quantification of the NAIP protein, normalized to HSC-70 of experiments from four different healthy donors. C: Representative confocal microscopy images of monocytes, M1 and M2 macrophages from volunteer 1. Note the amoeboid-like shape in M1 and M2 macrophages. Bar, 10μm. D: Mean fluorescence intensity in monocytes and M1 and M2 macrophages from two healthy donors. 10 randomly selected cells in each case were analyzed for their mean fluorescence intensity per cell area using the ImageJ program. *Significant difference (P = 0.0163), **significant difference (P = 0.0037), ***significant difference (P = 0.0005).

Fig 4. cIAP1 and cIAP2 expression in unpolarized (THP-1/monocytes and M0-) and polarized M1- and M2-macrophages.

A, B: Polarized and unpolarized THP-1 cell proteins were extracted and cIAP1/2 protein abundance was assessed by western blotting using the RIAP1 antibody. HSC-70 was used as loading control. A: Representative western blot of one of three experiments with similar results. B: Quantification of cIAP1 and cIAP2 protein abundance in western blots normalized to HSC-70 of three independent experiments. C: Total RNA was extracted, reverse transcribed and cIAP1 and cIAP2 mRNA were analysed by RTq-PCR. Values are normalized to internal controls (HPRT1 and GAPDH) and presented as mean values ± s.d. of fold-change of three independent experiments. *P<0.05 ***P< 0.001 (ANOVA with Bonferroni post hoc).

Fig 5. Smac mimetics modulate IAP expression during macrophage polarization.

THP-1 M0 macrophages were treated with 1μM LCL161 for 24 h prior to polarization into M1 or M2. A, B: The levels of NAIP, cIAP1 and cIAP2 were determined by western blotting. HSC-70 was used as loading control. A: Representative western blot from one of three experiments with similar results. B: Quantification of NAIP, cIAP1 and cIAP2 protein abundance normalized to the corresponding HSC-70 in three independent experiments. C: Determination of NAIP, cIAP1 and cIAP2 mRNA levels by RT-qPCR. NAIP mRNA expression was evaluated using primers spanning exon 4 or between exons 16 and 17. Values are normalized to internal controls (HPRT1 and GAPDH) and presented as mean values ± s.d. of fold-change of three independent experiments. *P<0.05 **P<0.01 ***P< 0.001 (ANOVA with Bonferroni post hoc).

Fig 6. SMCs alters the markers expression profile of polarized macrophages.

THP-1 M0 macrophages were treated with 1μM LCL161 for 24 hours and then induced to polarized into M1 or M2 states. A: CD206, CXCL10 and CD163 polarization marker genes were examined by semi-quantitative RT-PCR. mRNA expression was normalized to GAPDH in each group and then calculated as fold change against the expression of the control group. Data represent the mean and standard deviation of three independent experiments. B: Cells were subsequently analyzed by flow cytometry for the expression of the macrophage markers CD14 and CD11b and of the M1 (CD86) and M2 (CD206) polarization markers. Data represents mean MFI of three independent experiments. *P<0.05 *** P<0.0001 (ANOVA with Bonferroni post Hoc).