Plasmodium falciparum malaria drives epigenetic reprogramming of human monocytes toward a regulatory phenotype

Abstract

In malaria-naïve children and adults, Plasmodium falciparum-infected red blood cells (Pf-iRBCs) trigger fever and other symptoms of systemic inflammation. However, in endemic areas where individuals experience repeated Pf infections over many years, the risk of Pf-iRBC-triggered inflammatory symptoms decreases with cumulative Pf exposure. The molecular mechanisms underlying these clinical observations remain unclear. Age-stratified analyses of uninfected, asymptomatic Malian individuals before the malaria season revealed that monocytes of adults produced lower levels of inflammatory cytokines (IL-1β, IL-6 and TNF) in response to Pf-iRBC stimulation compared to monocytes of Malian children and malaria-naïve U.S. adults. Moreover, monocytes of Malian children produced lower levels of IL-1β and IL-6 following Pf-iRBC stimulation compared to 4-6-month-old infants. Accordingly, monocytes of Malian adults produced more IL-10 and expressed higher levels of the regulatory molecules CD163, CD206, Arginase-1 and TGM2. These observations were recapitulated in an in vitro system of monocyte to macrophage differentiation wherein macrophages re-exposed to Pf-iRBCs exhibited attenuated inflammatory cytokine responses and a corresponding decrease in the epigenetic marker of active gene transcription, H3K4me3, at inflammatory cytokine gene loci. Together these data indicate that Pf induces epigenetic reprogramming of monocytes/macrophages toward a regulatory phenotype that attenuates inflammatory responses during subsequent Pf exposure. Trial Registration: ClinicalTrials.gov NCT01322581.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Monocytes of Malian adults exhibit reduced P. falciparum-inducible inflammatory cytokine production and increased IL-10 production.

PBMCs were collected cross-sectionally from an age-stratified cohort in Mali before the malaria season when all subjects were negative for Pf infection by PCR; and also, from malaria-naïve U.S. adults. Monocytes were isolated from thawed PBMCs and stimulated with Pf-iRBC lysate at a ratio of 1 monocyte to 30 iRBCs. After 24 hours, cell culture supernatants were analyzed to quantify secreted (A) TNF, (B) IL-1β, (C) IL-6 and (D) IL-10. Median values are indicated by a horizontal line. P-values are from pairwise Mann-Whitney tests with a Bonferroni adjustment for all 10 pairwise comparisons. Nonsignificant p-values are not shown.

Fig 2. Monocytes of Malian adults upregulate markers of M2 regulatory monocytes/macrophages.

(A-E) PBMCs from Malian children (aged 4–6 years; n = 9) and adults (n = 9) before the malaria season, as well as healthy malaria-naïve U.S. adults (n = 7) were analyzed ex vivo by flow cytometry with gating on live monocytes for surface expression of CD14, CD16, CD163 and CD206. (A, B) t-SNE analysis of monocytes for all subjects in each group. Expression levels of each marker are indicated by color scales. (C) t-SNE analyzed data (shown in A and B) subjected to gating for monocyte subsets based on CD14 and CD16 expression using concatenated data from all subjects. MFI of (D) CD163 and (E) CD206 surface expression on manually gated live monocytes. (F, G) PBMCs were stimulated with Pf-iRBCs and analyzed for arginase-1 expression in monocytes intracellularly. (F) Representative t-SNE plots showing expression of Arginase-1 in the clusters in three different groups of monocytes. Expression of Arginase-1 is indicated by a color scale. (G). Expression MFI level of intracellular Arginase-1 on manually gated live monocytes comparing three different groups. Data were analyzed by the Mann-Whitney test with Bonferroni adjustment, and the level of significance between groups is indicated by P values.

Fig 3. RNA-seq analysis of monocytes after Pf-iRBC stimulation reveals a regulatory signature in Malian adults that is distinct from children.

Monocytes isolated from PBMCs of Malian children (n = 8) and adults (n = 8) were stimulated in vitro with Pf-iRBC lysate at a monocyte:Pf-iRBC ratio of 1:5 for 24 hours and then total RNA was isolated for sequencing. From separate subjects (n = 4 subjects in each age group), total RNA from unstimulated monocytes was also sequenced. (A) Principal-component analysis of log2-normalized gene counts across all samples. (B-E) Heatmaps representing log2-normalized gene counts of Pf-iRBC stimulated monocytes from Malian children and adults. Each column represents one individual sample. Heatmaps represent gene sets with pre-specified functions: (B) M1/M2 monocyte/macrophage signature, (C) inflammation, (D) phagocytosis, and (E) antigen processing and presentation.

Fig 4. In vitro model of monocyte to macrophage differentiation during exposure to P. falciparum blood-stage parasites.

Elutriated monocytes from healthy U.S. adults were incubated for 24 hours with medium alone, uninfected red blood cells (RBC) or Pf-iRBC (monocyte:Pf-iRBC ratio 1:15). At 24 hours, supernatants and cells were collected from some replicate wells for cytokine analysis, and the ChIP assay, while monocytes in other replicate wells were washed and incubated for 3 additional days in human serum plus medium to allow monocytes to differentiate into macrophages (Mf). On day 5, the three populations of macrophages (Mf, RBC-Mf and Pf-iRBC-Mf) were either harvested for ChIP and cytokine gene expression analysis; or re-stimulated with Pf-iRBCs or LPS for 24 hours prior to supernatants and cells being collected for cytokine analysis.

Fig 5. Pre-exposure of monocytes to P. falciparum dampens subsequent inflammatory responses to P. falciparum and LPS.

Monocytes of U.S. adults were incubated with medium, RBC or Pf-iRBC. At 24 hours, cells were analyzed by (A) cytokine gene expression Taqman arrays (n = 3 subjects), and supernatants were analyzed by a bead-multiplexed assay (n = 9 subjects) to quantify (D) TNF, (E) IL-6 and (F) IL-1β. In the same experiment, replicate monocytes were incubated with medium, RBC or Pf-iRBC, washed at 24 hours and incubated for 3 additional days in human serum to permit macrophage (Mf) differentiation. On day 5, the three populations of macrophages were analyzed by (B) cytokine gene expression arrays (n = 3 subjects). Finally, in the same experiment, replicate monocytes were incubated with medium, RBC or Pf-iRBC, washed at 24 hours, incubated for 3 additional days in human serum to permit Mf differentiation, and then co-cultured with Pf-iRBC or LPS for 24 hours. Cells were harvested for (C) cytokine gene expression arrays (n = 3 subjects), and supernatants were analyzed (n = 9 subjects) to quantify TNF, IL-6 and IL-1β induced by (G-I) Pf-iRBC or (L-N) LPS (n = 14 subjects). ΔCt values (mean ±SE) for (J) TNF and (K) IL-6 at the indicated timepoints for the 3 subjects shown in A-C. (A-C) Heatmaps were generated from ΔCt values, with lower ΔCt values corresponding to higher gene expression. ΔCt values were normalized to 18S rRNA expression. (D-I and L-N) Lines represent median values. Data were analyzed by the Wilcoxon test with Bonferroni adjustment, and significance levels between the groups are indicated by P values. (J and K) Two-way ANOVA was performed followed by Tukey’s multiple comparisons test. Significance level between conditions (Pf-iRBC stim vs. Control Medium and Pf-iRBC stim vs. RBC stim, respectively) are indicated by P values at each timepoint.

Fig 6. P. falciparum exposure drives epigenetic reprogramming of monocyte/macrophages toward a regulatory phenotype.

Monocytes of U.S. adults (n = 4) were incubated with medium, RBC or Pf-iRBC for 24 hours and then analyzed by chromatin immunoprecipitation (ChIP) and RT-PCR to quantify H3K4me3 enrichment at (A) TNF and (D) IL-6 promoter sites. Replicate monocytes were incubated with medium, RBC or Pf-iRBC, washed at 24 hours, and incubated for 3 days in human serum to permit Mf differentiation. On day 5, cells were analyzed by ChIP and RT-PCR to quantify H3K4me3 enrichment at (B) TNF and (E) IL-6 promoter sites. Finally, replicate monocytes were incubated with medium, RBC or Pf-iRBC, washed at 24 hours, incubated for 3 days in human serum, co-cultured with Pf-iRBC for 24 hours, and analyzed by ChIP and RT-PCR to quantify H3K4me3 enrichment at (C) TNF and (F) IL-6 promoter sites. Kinetics of H3K4me3 enrichment at (G) TNF and (H) IL-6 promoter sites across indicated timepoints. Results are shown as means ±SEM fold enrichment of H3K4me3 antibody as percent of input. (A-F) One-way ANOVA with Tukey’s adjustment for multiple comparisons. P values indicate level of significance. (G, H) Two-way ANOVA with Tukey’s adjustment for multiple comparisons. P values indicate level of significance between Pf-iRBC vs. medium and Pf-iRBC vs. RBC, respectively.