Malaria pigment hemozoin drives M1 pro-inflammatory macrophage polarization in vitro

Abstract

Severe malaria, a high burden parasitic disease, is characterized by hyperproduction of proinflammatory cytokines, most likely generated by M1-polarized macrophages. Malaria pigment or hemozoin (HZ), a byproduct of heme detoxification in intra-erythrocytic parasites, is internalized by circulating monocytes and tissue macrophages, modulating their functions. Although the immunomodulatory properties of HZ have been described, its specific role in M1/M2 macrophage polarization remains unclear. This study aims to fill this gap by elucidating whether HZ modulates M1/M2 polarization, contributing to the strong inflammatory response in severe malaria. Primary human monocyte derived macrophages (MDM) and THP-1 cells differentiated into macrophages (dTHP-1) were stimulated with M1 or M2 signals in the presence of native HZ. Gene expression and protein secretion of TNF-α, IL-1β, CXCL8, IL-6, IL-10 and PPARG were evaluated by Real-Time PCR and ELISA, respectively. STAT6 phosphorylation was evaluated by western blot analysis. MDM and dTHP-1 showed a different polarisation response to classical M1/M2 stimuli and to HZ treatment. In both non-polarized (M0) MDM and dTHP-1, HZ induced an M1/pro-inflammatory phenotype, increasing gene expression and protein secretion of CXCL8, TNF-α, and IL-1β. In the presence of M1- or M2-polarizing stimuli, HZ further increased CXCL8 and IL-1β in MDM but not in dTHP-1, where TNF-α secretion was even reduced. HZ did not affect M2 markers (PPARG and IL10 expression, STAT6 phosphorylation) in any condition. This is the first in vitro study investigating the effect of HZ on macrophage polarization, showing its ability to promote M1 pro-inflammatory differentiation. Results vary across experimental models, emphasizing the importance of considering model-specific effects. Clarifying HZ's role remains crucial for understanding malaria pathogenesis and developing new immunomodulatory therapies.

Keywords: Hemozoin (HZ); Innate immunity; Macrophage polarization; Malaria; Malaria pigment.

Fig. 1

Macrophage polarization. Macrophage polarization markers evaluated as mRNA expression levels (panels A,C) and protein secretion in the supernatants (panels B,D) in MDM (filled columns—panels A,B) and dTHP-1 cells (striped columns—panels C,D) after 24 h of incubation with polarizing stimuli. Gene expression of CXCL8, TNFA, IL1B, IL6, IL10 and PPARG was evaluated by Real-Time PCR analysis. Results are expressed as fold change calculated by the ΔΔCt method. Data are presented as mean ± standard deviation from four different donors for MDM and from at least three independent experiments for dTHP-1. CXCL8, TNF-α, IL-1β, IL-6 and IL-10 protein levels were measured by ELISA and expressed as % of the control, with data represented as the mean ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post-hoc test, comparing each gene or protein against M0 (untreated macrophages). Statistical significance is denoted as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 2

STAT6 phosphorylation. Western Blot analysis of STAT6 phosphorylation in primary MDM (A) and dTHP-1 cells (C) in control cells or cells treated with hemozoin (HZ). Relative expressions of total STAT6 and pSTAT6 normalized on β-actin in MDM (B) and dTHP-1 cells (D) were assessed in M2-like macrophages. Data are the mean ± SD from three different donors for MDM and from three independent experiments for dTHP-1. Statistical analysis was performed using an unpaired two-tailed t-test for each transcription factor and comparing the data versus the control M2-macrophages not incubated with HZ.

Fig. 3

HZ phagocytosis by M0, M1, M2 macrophages. Representative images of MDM (A) or dTHP-1 (B) after 24 h of incubation with polarizing stimuli in the presence or absence of HZ. Images were captured by using a Nikon Eclipse Ti-Series with 20× objective and the digital camera Nikon Digital Sight. Scale bar: 100 μm.

Fig. 4

Effect of HZ on M0 macrophages. Macrophage polarization markers evaluated as mRNA expression levels (panels A,C) and protein secretion in the supernatants (panels B, D) in MDM (full columns—panels A,B) and dTHP-1 cells (striped columns—panels C,D) after 24 h incubation with HZ alone in the absence of other polarizing stimuli. Gene expression of CXCL8, TNFA, IL1B, IL6, IL10 and PPARG was evaluated by Real-Time PCR analysis. Results are expressed as fold change calculated by the ΔΔCt method. Data are presented as mean ± standard deviation from four different donors for MDM and from at least three independent experiments for dTHP-1. CXCL8, TNF-α, IL-1β, IL-6 and IL-10 protein levels were measured by ELISA and expressed as % of the control, with data represented as the mean ± standard deviation. Statistical analysis was performed using an unpaired two-tailed t-test for each gene or protein in comparison to control (M0 - unpolarized macrophages). Statistical significance is denoted as follows: *p < 0.05; ** p < 0.01; ***p < 0.001.

Fig. 5

Effect of HZ on M1 macrophages. Macrophage polarization markers evaluated as mRNA expression levels (panels A,C) and protein secretion in the supernatants (panels B,D) in MDM (filled columns—panels A,B) and dTHP-1 cells (striped columns—panels C,D) after 24 h incubation with HZ and M1-polarizing stimuli. Gene expression of CXCL8, TNFA, IL1B, IL6, IL10 and PPARG was evaluated by Real-Time PCR analysis. Results are expressed as fold change calculated by the ΔΔCt method. Data are presented as mean ± standard deviation from four different donors for MDM and from at least three independent experiments for dTHP-1. CXCL8, TNF-α, IL-1β, IL-6 and IL-10 protein levels were measured by ELISA and expressed as % of the control, with data represented as the mean ± standard deviation. Statistical analysis was performed using an unpaired two-tailed t-test for each gene or protein, comparing the data versus the control (M1-stimulated macrophages). Statistical significance is denoted as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 6

HZ effect on dTHP-1 polarized with M1 stimuli used for MDM. M1 macrophage polarization markers assessed as mRNA expression levels (A) and protein secretion levels in cell supernatants (B) in dTHP-1 macrophages after 24 h incubation with HZ and M1-polarizing stimuli used for MDM (IFN-γ + TNF-α). Gene expression of CXCL8, IL1B and IL6 was evaluated by Real-Time PCR analysis. Results are expressed as fold change calculated by the ΔΔCt method. Data are presented as mean ± standard deviation from three independent experiments for dTHP-1. CXCL8, IL-1β and IL-6 protein levels were measured by ELISA and expressed as % of the control, with data represented as the mean ± standard deviation. Statistical analysis was performed using an unpaired two-tailed t-test for each gene or protein, comparing the data to the control (M1-stimulated macrophages). Statistical significance is denoted as follows: *p < 0.05.

Fig. 7

Effect of HZ on M2 macrophages. Macrophage polarization markers evaluated as mRNA expression levels (panels A,C) and protein secretion in the supernatants (panels B,D) in MDM (filled columns—panels A,B) and dTHP-1 cells (striped columns—panels C,D) after 24 h of incubation with HZ and M2-polarizing stimuli. Gene expression of CXCL8, TNFA, IL1B, IL6, IL10 and PPARG was evaluated by Real-Time PCR analysis. Results are expressed as fold change calculated by the ΔΔCt method. Data are presented as mean ± standard deviation from four different donors for MDM and from at least three independent experiments for dTHP-1. CXCL8, TNF-α, IL-1β, IL-6 and IL-10 protein levels were measured by ELISA and expressed as % of the control, with data represented as the mean ± standard deviation. Statistical analysis was performed using an unpaired two-tailed t-test for each gene or protein and comparing the data versus the control (M2-stimulated macrophages). Statistical significance is denoted as follows: *p < 0.05; **p < 0.01.

Fig. 8

Schematic representation of the results. Macrophage polarization markers evaluated as mRNA expression levels (gene) and protein secretion in the supernatants (protein) in M0/M1/M2 macrophages (MDM or dTHP-1 cells) in the presence of hemozoin.