Plasma biomarkers of hemoglobin loss in Plasmodium falciparum-infected children identified by quantitative proteomics

Abstract

Anemia is common among young children infected with Plasmodium falciparum and severe malarial anemia (SMA) is a major cause of their mortality. Two major mechanisms cause malarial anemia: hemolysis of uninfected as well as infected erythrocytes and insufficient erythropoiesis. In a longitudinal birth cohort in Mali, we commonly observed marked hemoglobin reductions during P falciparum infections with a small proportion that progressed to SMA. We sought biomarkers of these processes using quantitative proteomic analysis on plasma samples from 9 P falciparum-infected children, comparing those with reduced hemoglobin (with or without SMA) vs those with stable hemoglobin. We identified higher plasma levels of circulating 20S proteasome and lower insulin-like growth factor-1 (IGF-1) levels in children with reduced hemoglobin. We confirmed these findings in independent enzyme-linked immunosorbent assay-based validation studies of subsets of children from the same cohort (20S proteasome, N = 71; IGF-1, N = 78). We speculate that circulating 20S proteasome plays a role in digesting erythrocyte membrane proteins modified by oxidative stress, resulting in hemolysis, whereas decreased IGF-1, a critical factor for erythroid maturation, might contribute to insufficient erythropoiesis. Quantitative plasma proteomics identified soluble mediators that may contribute to the major mechanisms underlying malarial anemia. This study was registered at www.clinicaltrials.gov as #NCT01168271.

Graphical abstract

Figure 1.

Quantitative proteomic of children’s plasma with hemoglobin drop. Venn diagram indicating the number of differentially expressed proteins in children with hemoglobin drop with and without SMA.

Figure 2.

Networks of differentially expressed plasma proteins in children with reduced hemoglobin. Comparison of differentially expressed plasma proteins in children with hemoglobin drop (A,B) with SMA and (C,D) without SMA to control malaria-infected children by Ingenuity Pathway Analysis. Analyses in panels A and C included differentially expressed proteins defined as log₂ fold change <−0.3 or >0.3, P < .05, and analyses displayed in panels B and D included differentially expressed proteins defined as log₂ fold change ≤-1.0 or ≥1.0. Green and red symbols represent proteins that were down- and upregulated, and gray symbols represent similarly expressed proteins identified in the study. Solid and broken lines represent direct and indirect interactions respectively. Proteins selected for validation study are marked with blue circles.

Figure 2.

Networks of differentially expressed plasma proteins in children with reduced hemoglobin. Comparison of differentially expressed plasma proteins in children with hemoglobin drop (A,B) with SMA and (C,D) without SMA to control malaria-infected children by Ingenuity Pathway Analysis. Analyses in panels A and C included differentially expressed proteins defined as log₂ fold change <−0.3 or >0.3, P < .05, and analyses displayed in panels B and D included differentially expressed proteins defined as log₂ fold change ≤-1.0 or ≥1.0. Green and red symbols represent proteins that were down- and upregulated, and gray symbols represent similarly expressed proteins identified in the study. Solid and broken lines represent direct and indirect interactions respectively. Proteins selected for validation study are marked with blue circles.

Figure 3.

Comparison of c-20S proteasome concentrations and activity between plasma samples of malaria-infected and uninfected children. (A) C-20S proteasome concentration, (B) caspase-like, (C) chymotrypsin-like, and (D) trypsin-like activities were measured in plasma samples from children with hemoglobin drop with and without SMA, stable hemoglobin, SM, and uninfected children (n = 17, 20, 19, 6, and 9 respectively). Significant differences (Holm adjusted P value) are indicated in the figure.

Figure 4.

ELISA analysis of (A) eNAMPT, (B) IL-18, (C) α-1-antitrypsin, and (D) IGF-1 levels in plasma samples of malaria-infected and uninfected children. Plasma levels were compared between plasma samples from children with hemoglobin drop with and without SMA, stable hemoglobin, SM, and uninfected children (eNAMPT and IL-18: n = 16, 20, 19, 6, and 18; AIAT and IGF-1: n = 14, 21, 18, 7, and 18, respectively). Significant differences (Holm adjust P value) are indicated in the figure.

Figure 5.

Model of acute hemoglobin drop. Based on quantitative plasma proteomic and validation studies, an acute drop in hemoglobin during malaria is associated with changes in levels of several soluble mediators. These observations were integrated with processes described in the literature and presented in the model. C-20S proteasome levels are significantly higher in malaria-infected children with hemoglobin drop vs children with stable hemoglobin, and 20S proteasome chymotrypsin-like activity is higher compared with uninfected children. Previously, malaria infection has been associated with oxidative stress, in particularly among children with SMA, which results in red blood cell membrane modification. In this model, we propose that modified membrane proteins are then subjected to degradation by c-20S proteasome, leading to hemolysis and decreased hemoglobin. In parallel, levels of IGF-1, one of the growth factors required for erythroid proliferation, are significantly reduced, which may be due to increased IL-18 levels, to increased chymotrypsin-like activity, or to both. The reduction in erythroid proliferation results in reduced hemoglobin levels, in addition to that caused by hemolysis. Factors associated with hemoglobin drops in the study are in green (increased) or red (decreased); dashed lines indicate associations described here and solid lines indicate previously described relationships.