Targeting circulating labile heme as a defense strategy against malaria
Introduction
Malaria is an ancestral vector-borne disease, transmitted by the bite of female Anopheles mosquitoes. Upon injection into the dermis, Plasmodium sporozoites migrate to the liver, invading, developing and proliferating in hepatocytes (Prudencio et al, 2006). Clinical presentations occur later, during the blood stage of infection, as asexual stages of the parasite invade, develop, and proliferate in RBC.
The blood stage of Plasmodium infection is associated with a transient depletion of erythrophagocytic macrophages (Gazzinelli et al, 1988; Nahrendorf et al, 2021; Wu et al, 2023), which decouples RBC lysis from erythrophagocytosis and iron recycling (Wu et al, 2023). The ensuing intravascular hemolysis releases HB α2β2 tetramers, which disassemble into αβ dimers in plasma, releasing their non-covalently bound prosthetic heme groups (Pamplona et al, 2007; Gouveia et al, 2017). Labile heme refers to the pool of circulating heme, loosely bound to plasma proteins and macromolecules (Gouveia et al, 2017; Ramos et al, 2019), which fails to control the redox activity of the iron contained in the protoporphyrin ring.
Labile heme is an alarmin (Ferreira et al, 2008; Soares & Bozza, 2016) that catalyzes the pathogenesis of severe and often lethal outcomes of experimental malaria in mice (Pamplona et al, 2007; Ferreira et al, 2008; Gozzelino et al, 2010; Ramos et al, 2019; Wu et al, 2023). The pathogenic effects of labile heme are countered by the infected host, via the induction of heme oxygenase-1 (HO-1) (Pamplona et al, 2007; Ferreira et al, 2008; Gozzelino et al, 2010; Ramos et al, 2019), a stress responsive enzyme that cleaves the protoporphyrin ring of heme and generates equimolar amounts of biliverdin, iron, and carbon monoxide (Tenhunen et al, 1968; Gozzelino et al, 2010). This defense strategy was co-opted throughout evolution to prevent the onset of severe presentations of malaria in individuals carrying a single (i.e. hemizygous)-sickle HB mutation (i.e. sickle trait) (Ferreira et al, 2011).
Mammals express a number of plasma proteins that limit the release of heme from extracellular HB or restrain the redox activity of labile heme (Ferreira et al, 2008; De Simone et al, 2023). These include the HB scavenger haptoglobin (HP) and the heme scavengers, hemopexin (HPX), and ⍺1-microglobulin (De Simone et al, 2023), respectively. The observation that HP and HPX limit the extent of renal damage imposed by sterile intravascular hemolysis (Tolosano et al, 1999), suggested that HP and HPX might counter the pathogenesis of malaria-associated AKI, a major independent risk factor for P. falciparum malaria mortality, in children and adults (Sitprija, 1988; Trang et al, 1992; Mishra & Das, 2008; Plewes et al, 2017; Cruz et al, 2018; Katsoulis et al, 2021; Wu et al, 2023).
The HP protein complex is composed of two αβ dimers (∼100–160 kD), generated by proteolytic cleavage of a common precursor, linked by disulphide bridges (Polticelli et al, 2008; Andersen et al, 2017). Humans carry two allelic HP1 and HP2 variants, expressing three HP1-1, HP2-1, and HP2-2 genotypes, with affinities towards αβ HB dimers, in the range of KM∼10−12 M. Binding of HP to αβ HB (Andersen et al, 2012), restrains HB oxidation and the release of its prosthetic heme groups (Andersen et al, 2012, 2017; Schaer et al, 2013). HB/HP complexes are scavenged, via CD163, by erythrophagocytic macrophages, coupling HB disposal with heme catabolism by HO-1.
P. falciparum malaria has been associated with depletion of circulating HP in children (Trape et al, 1985), presumably because of the removal of HP/HB complexes by macrophages. Whether or not the allelic HP1 and HP2 variants are associated with P. falciparum malaria incidence and/or outcome is not clear. The HP1-1 genotype, with the highest HB affinity, was linked with P. falciparum malaria susceptibility and severe disease (Quaye et al, 2000), whereas other studies suggest the HP1-2 (Elagib et al, 1998) and HP2-2 (Atkinson et al, 2007) genotypes, with an intermediate and lowest HB affinity, respectively, are correlated with higher risk of severe P. falciparum malaria. Of note, genetic deletion of Hp in mice was associated with increased parasite burden (Hunt et al, 2001).
HPX is a 63-kD plasma protein that binds labile heme with the highest affinity (Kd < 10−12 M) of any protein described so far (Muller-Eberhard, 1970; Paoli et al, 1999) and neutralizes heme cytotoxicity (Larsen et al, 2010). Heme/HPX complexes are removed from the circulation via the low-density lipoprotein receptor-related protein 1 (LRP-1/CD91), expressed by circulating monocytes. At least one study suggests that the ratio of plasma heme to HPX is associated with P. falciparum malaria severity in children (Elphinstone et al, 2016). Other studies have also reported an association between plasma heme and extracellular HB with severe P. falciparum in children (Elphinstone et al, 2015) and P. vivax in adults (Mendonca et al, 2012).
To determine whether the HP/HB and/or HPX/heme scavenging systems are protective against malaria we combined the analyzes of a pediatric P. falciparum malaria case-control study (Sambo et al, 2010) with experimental models of malaria in mice carrying Hp and/or Hpx genetic deletions. We found that labile heme is an independent risk factor for cerebral and non-cerebral presentations of severe P. falciparum malaria and that HP and HPX act in an age-depended manner to prevent the pathogenesis of non-cerebral severe malaria in mice. Neither HP, HPX nor labile heme interfere with parasite burden, suggesting that the HP/HB and HPX/heme scavenging systems contribute to the establishment of disease tolerance to malaria (Medzhitov et al, 2012; Martins et al, 2019). These findings suggest that HP and/or HPX genetic variants may contribute to age-dependent increase in malaria susceptibility (Dondorp et al, 2008).
Results
Labile heme is an independent risk factor of severe P. falciparum malaria
We analyzed a case-control study of P. falciparum-infected children, ranging from 6 mo old to 13 yr old, hospitalized at Hospital Pediátrico David Bernardino, Luanda, Angola (Sambo et al, 2010). The original study included 130 cases of cerebral malaria (CM), 158 cases of severe non-cerebral, 142 cases of uncomplicated malaria and 319 children not infected by P. falciparum, selected randomly from the vaccination ward (Sambo et al, 2010). A subgroup of 58 cases of CM, 61 cases of severe non-cerebral malaria and 25 uncomplicated cases of malaria, for which serum was available, was evaluated for HP, HPX, total heme (i.e., HB-bound heme plus heme bound to other serum proteins and macromolecules), HB-bound heme and labile heme (i.e., fraction of total heme bound to serum proteins and macromolecules other than HB; Total heme - HB-bound heme) concentration in serum (Table 1; Fig 1A and B).
Baseline demographic and serological characteristics of P. falciparum-infected children according to disease severity.
(A, B) Representative UV–visible spectra of plasma from P. falciparum-infected individuals with uncomplicated malaria or developing CM, highlighting (A) Soret region (λ363–383 nm) corresponding to labile heme, with a peak at λ405 nm and (B) λ582 nm region corresponding to HB-bound heme. (C) Labile heme, haptoglobin (HP), hemopexin (HPX) concentrations in serum from P. falciparum-infected individuals stratified according to disease severity: Uncomplicated malaria (N = 25), cerebral malaria (CM; N = 58) or severe non-cerebral malaria (N = 61). Circles represent individuals and red lines indicate median values. P-values determined using a one-way ANOVA test and subsequent posthoc tests. NS, nonsignificant; *P < 0.05; ****P < 0.0001. (C, D) Contribution of each of the parameters to distinguish the indicated sub-groups (stratified as in (C)), controlling for age and sex. Values indicate regression coefficients of the standardized variable on a logit regression. Raw data in Table 1. (E) Correlation coefficients between log-transformed haptoglobin (HP; left panel) or hemopexin (HPX; right panel) versus labile heme concentration in serum of P. falciparum-infected individuals, stratified as in (C). Circles represent individual patients. P-values determined using a Spearman’s rank correlation coefficient test. Spearman’s correlation coefficients (r) are highlighted.
Source data are available for this figure.
CM and severe non-cerebral malaria were associated with a median concentration of labile heme in serum of 49.5 and 37.5 μM, respectively (Table 1; Fig 1C). This was significantly higher compared with the 21.4 μM median concentration of circulating labile heme in uncomplicated P. falciparum malaria (Table 1; Fig 1C). The median concentration of circulating labile heme was indistinguishable in CM versus severe non-cerebral malaria (Table 1; Fig 1C). These observations suggest that the accumulation of circulating labile heme is associated with the onset of CM and severe non-cerebral P. falciparum malaria, similar to experimental models of CM (Pamplona et al, 2007) and non-cerebral malaria (Seixas et al, 2009; Gouveia et al, 2017; Ramos et al, 2019) in mice.
The concentrations of labile and total heme in serum were major independent risk factors for P. falciparum CM versus uncomplicated malaria, when controlling for age and sex (Table 1, Fig 1D). This was also the case when comparing severe non-cerebral versus uncomplicated malaria (Table 1, Fig 1D). Moreover, labile heme remained significantly associated with P. falciparum CM versus uncomplicated malaria, even when controlling for parasitemia (P = 0.004).
Parasitemia was not a risk factor for CM versus uncomplicated P. falciparum malaria (Table 1, Fig 1D), similar to experimental rodent models of CM (Pamplona et al, 2007; Ferreira et al, 2008, 2011; Jeney et al, 2014). This suggests that the pathogenesis of P. falciparum CM is fueled, irrespectively of parasite burden, by the accumulation of circulating labile heme.
Parasitemia was an independent risk factor of severe P. falciparum non-cerebral versus uncomplicated malaria (Table 1, Fig 1D). This is consistent with the pathogenesis of life-threatening malaria anemia being fueled by the accumulation of high levels of labile heme in plasma owed to high parasite burdens and hemolysis (Seixas et al, 2009; Ramos et al, 2019; Ramos et al, 2022; Wu et al, 2023). Parasitemia was also an independent risk factor for severe non-cerebral malaria versus CM (Table 1, Fig 1D).
HP and HPX are not risk factors of severe P. falciparum malaria
The concentrations of HP and HPX in serum were indistinguishable in children that developed P. falciparum CM versus those with uncomplicated malaria (Table 1, Fig 1C). In contrast, severe non-cerebral malaria was associated with lower concentration of circulating HP, but not HPX, compared with uncomplicated malaria (Table 1, Fig 1C). This is consistent with severe non-cerebral malaria being related with extensive hemolysis and accumulation of extracellular HB, presumably leading to HP depletion. Circulating HP was associated (P = 0.005) with the distinction between severe non-cerebral, but not CM, and uncomplicated malaria, when controlling for age and sex (Table 1, Fig 1D). HPX was not a raw risk factor for CM or severe non-cerebral malaria (Table 1, Fig 1D).
HP and HPX are negatively correlated with labile heme in P. falciparum malaria
We asked whether HP and HPX are linked to the accumulation of labile heme in serum during P. falciparum malaria. In support of this hypothesis, children that developed CM showed a negative correlation between circulating HP and labile heme (Fig 1E). This was not observed in children that developed severe non-cerebral malaria or in uncomplicated malaria (Fig 1E). The negative correlation between HP and labile heme in children that developed CM remained significant after controlling for parasitemia (P < 0.013). These observations are consistent with HP regulating the levels of circulating labile heme in children that develop CM, irrespectively of parasite burden.
HPX was negatively correlated with circulating labile heme concentration in children that developed CM and severe non-cerebral malaria, but not in those with uncomplicated P. falciparum malaria (Fig 1E). The negative correlation between HPX and labile heme in CM (P < 0.0001) or in severe non-cerebral malaria (P < 0.0001) remained significant after controlling for parasitemia. This is consistent with HPX regulating the levels of labile heme in CM and severe non-cerebral patients, irrespectively of parasite burden.
Taken together, these observations are consistent with (1) hemolysis associated with P. falciparum malaria depleting circulating HP and HPX and (2) HP and HPX exerting some level of control over the accumulation of labile heme in serum, without preventing the onset of severe presentations of P. falciparum malaria.
HP and HPX control the accumulation of labile heme in experimental rodent malaria
The protective effect exerted by HP and HPX against sterile intravascular hemolysis in mice (Tolosano et al, 2002) led us to hypothesize that HP and HPX may be protective against malaria-associated intravascular hemolysis. We tested this hypothesis in 8–12 wk-old (i.e., adult) C57BL/6 mice infected with Plasmodium chabaudi chabaudi AS (Pcc), a non-lethal experimental model of severe non-cerebral malaria associated with high parasite burdens (Seixas et al, 2009; Jeney et al, 2014), intravascular hemolysis (Gouveia et al, 2017; Ramos et al, 2019) and severe anemia (Wu et al, 2023).
Pcc infection was associated with a relative increase in hepatic Hp and Hpx mRNA expression, compared with non-infected control mice, as assessed in a previously published RNA-seq data set (Fig 2A) (Ramos et al, 2022) and confirmed independently by qRT–PCR (Fig 2B). This was not linked however, with a corresponding increase in circulating HP and HPX levels (Fig 2C and D), presumably reflecting the “removal” of circulating HP/HB and HPX/heme complexes generated via intravascular hemolysis.
Mice were infected with Pcc (2 × 106 iRBC) and serum was collected at the peak of parasitemia (day 6–7 post infection). (A, B) Relative expression of hepatic haptoglobin (Hp) and hemopexin (Hpx) mRNA in C57BL/6 mice, not infected (NI) or infected with Pcc, determined by (A) bulk RNAseq (N = 4 per genotype) from a previously published dataset (Ramos et al, 2022) or by (B) qRT–PCR from whole liver, normalized to Arbp0 mRNA (N = 7 per genotype). (C, D, E, F) Serum concentrations of (C) haptoglobin (HP) and (D) hemopexin (HPX), (E) total heme (Left panel) and labile heme (Right panel), (F) Iron (Left panel) and transferrin saturation (Right panel), in Pcc-infected (1 × 105 iRBC, at the peak of parasitemia: days 8–9 post-infection) and non-infected control mice (N = 10–12 per indicated genotype). Data represented as mean ± SD from one or two independent experiments with similar trend. Dots correspond to individual mice. P-values determined by two-way ANOVA. NS, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Source data are available for this figure.
To address whether HP and/or HPX control the levels of circulating labile heme during Plasmodium infection we generated C57BL/6 mice carrying individual or combined germline Hp (Hp−/−), Hpx (Hpx−/−) or Hp and Hpx (Hp−/−Hpx−/−) gene deletions. These were confirmed by the quantification of circulating HP (Fig 2C) and/or HPX (Fig 2D) protein in serum.
Hpx deletion was associated with an increase in HP concentration in serum, as compared with age-matched control Hp+/+Hpx+/+ mice, both at steady state and after Pcc infection (Fig 2C). In contrast, Hp deletion had no impact on HPX concentration in serum, compared with age-matched control Hp+/+Hpx+/+ mice (Fig 2D). This suggests the existence of a crosstalk in the regulation of HP and HPX expression, whereby a reduction of HPX induces the expression of HP.
Labile heme concentration in serum was higher in Pcc-infected Hp−/−Hpx−/− versus Hp+/+Hpx+/+ mice (Fig 2E). The concentration of total and labile heme in serum was similar at steady state in adult Hp−/−Hpx−/− versus control Hp+/+Hpx+/+ mice (Fig 2E). This suggests that in adult mice the accumulation of labile heme in serum during malaria is controlled by HP and HPX.
We noticed a relatively lower accumulation of labile heme in serum during Pcc infection, compared with our previous studies (Gouveia et al, 2017; Ramos et al, 2019). This is likely attributed to the lower Pcc inoculum used in the present study.
Iron concentration in serum was higher in adult Hp−/−Hpx−/− versus Hp+/+Hpx+/+ mice, infected by Pcc, but not at steady state (Fig 2F). Transferrin saturation, a pathophysiologic parameter reporting on circulating iron transport, was also higher in adult Pcc-infected Hp−/−Hpx−/− versus Hp+/+Hpx+/+ mice (Fig 2F), but not at steady state. This suggests that HP and HPX control systemic iron metabolism during malaria in adult mice.
HP and HPX are not essential to survive malaria in adult mice
Next, we asked whether HP and/or HPX counter the development of malaria-associated AKI. Renal heme content was similar in adult Hp−/−Hpx−/− versus Hp+/+Hpx+/+ mice, both at steady state and after Pcc infection (Fig 3A). In contrast, renal iron overload was exacerbated in adult Hp−/−Hpx−/− versus age-matched control Hp+/+Hpx+/+ mice infected with Pcc but not at steady state (Fig 3A). Renal iron overload was not associated however, with the development of AKI, as assessed by blood urea nitrogen and creatinine concentration in serum at the peak of Pcc infection (Fig 3B). This was confirmed histologically by the extent and frequency of HB casts and proximal tubular necrosis (Fig 3C and D).
Adult 8–12 wk-old mice from the indicated genotypes were infected with Pcc and serum was collected, at the peak of parasitemia. (A) Renal heme (Left panel) and iron (Right panel) concentrations. Mice were infected with 2 × 106 iRBC and collected 7 d post infection. Data represented as mean ± SD from two independent experiments with a similar trend (N = 4–5 per genotype). Dots correspond to individual mice. (B) Blood urea nitrogen (left panel) and creatinine (right panel) concentrations in serum. Mice were infected with 1 × 105 iRBC and collected 8–9 d post infection. Data represented as mean ± SD from two independent experiments. with similar trend (N = 7–12 per genotype). Dots correspond to individual mice. (C) Kidney H&E staining, representative of N = 4 mice per genotype, corresponding to Pcc-infected (1 × 105 iRBC, at the peak of parasitemia: days 8–9 post-infection) and noninfected control mice. Top panels show whole kidney sections and bottom panels higher magnifications of the highlighted area. Arrowheads indicate renal proximal tubule epithelial single cell necrosis. GL, glomerulus; PT, proximal tubules. (C, D) Histopathological evaluation of the kidneys from (C) performed using digitalized whole-slide images, corresponding to whole kidney sections. Scores are represented as mean ± SD (n = 4–5 mice per genotype). Dots correspond to individual mice. Scores: 0 = No lesions; 1 = Single cell necrosis, discrete hemoglobin tubular casts; 2 = Mild; 3 = Moderate; 4 = Severe tubular cell necrosis, hemoglobin tubular casts. (E) Survival (left panel) and parasitemia (right panel) of Pcc-infected (1 × 105 iRBC) Hp+/+Hpx+/+, Hp−/−Hpx+/+, Hp+/+Hpx−/− and Hp−/−Hpx−/− mice (N = 7–11 mice per genotype), from two independent experiments with similar trend. Survival is represented in Kaplan–Meier plots and parasitemia as mean ± SD. P-values in (A, B, D) determined by two-way ANOVA. NS, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Source data are available for this figure.
Adult Pcc-infected Hp−/−Hpx−/− mice survived and cleared parasitemia, similar to age-matched control Pcc-infected Hp+/+Hpx+/+ mice (Fig 3E). This was also the case for Hp−/−Hpx−/−Hmox1+/− mice, lacking one Hmox1 allele (Fig 4A), suggesting that the extent of renal iron overload imposed by HP and HPX depletion is not sufficient to precipitate the pathogenesis of malaria AKI in adult mice.
Adult 8–12 wk-old mice from the genotypes indicated were infected with different Plasmodium strains (i.p., 1 × 105 iRBC). (A, B, C, D) Survival (left panels) and parasitemia (right panels) were monitored daily from day 3 post infection with: (A) P. chabaudi chabaudi (Pcc; N = 8–10 mice, two independent experiments), (B) P. berghei ANKA-GFP (PbANKA; N = 6–9 mice, two independent experiments), (C) P. berghei NK65 (PbNK65; N = 8–23 mice, five independent experiments), (D) P. yoelii yoelii 17XNL (Pyy17XNL; N = 8–11 mice, two independent experiments). Survival is represented in Kaplan–Meier plots. Percentage of infected RBCs (iRBC) was quantified by FACS when using PbANKA-GFP transgenic parasites, or by morphologic assessment of Giemsa-stained blood smears (four to five fields) when using other Plasmodium strains and is represented as mean ± SD.
Source data are available for this figure.
The virulence of other rodent-infective Plasmodium strains was also similar in adult Hp−/−Hpx−/− versus age-matched control Hp+/+Hpx+/+ mice, as assessed for P. berghei ANKA (Fig 4B) or P. berghei NK65 infection, with the latter failing to elicit experimental CM in Hp−/−Hpx−/− mice (Fig 4C). Similar findings were obtained for P. yoelii yoelii infection, which was not lethal to adult Hp−/−Hpx−/− mice nor to age-matched control Hp+/+Hpx+/+ mice (Fig 4D). This suggests that, in contrast to other components of the heme/iron detoxifying pathway, including HO-1 (Pamplona et al, 2007; Ferreira et al, 2008; Seixas et al, 2009; Jeney et al, 2014; Ramos et al, 2019, 2022; Wu et al, 2023), ferritin H chain (Gozzelino et al, 2012; Ramos et al, 2019) or ferroportin 1 (Zhang et al, 2018; Wu et al, 2023), HP and HPX are not essential to survive malaria in adult mice.
Compensatory heme scavenging mechanisms during malaria
We then asked whether other plasma proteins and/or macromolecules might scavenge labile heme in the absence of HP and/or HPX. To this aim, we used an ELISA-based assay that quantifies serum heme buffering capacity, that is, the relative capacity of serum proteins and macromolecules to scavenge labile heme (Gouveia et al, 2017). The assay is based on a heme-specific single-domain antibody (sdAb) that binds heme with an affinity of 10−7 M (Gouveia et al, 2017). Heme binding to this sdAb was inhibited when heme was preincubated with serum from control Hp+/+Hpx+/+ mice (Fig 5A). This effect was dose dependent, that is, the higher the serum dilution and/or the amount of heme, the higher was heme recognition by the sdAb (Fig 5A). Surprisingly, the heme buffering capacity of serum from adult Hp−/−Hpx−/− mice was indistinguishable from that of control Hp+/+Hpx+/+ mice, at steady state (Fig 5B). This suggests that circulating proteins and/or macromolecules other than HP and/or HPX can scavenge labile heme.
(A) Heme buffering capacity of serum from C57BL/6 mice at steady state, assayed by a heme-specific single domain Ab-based sandwich ELISA. Hemin, at the concentrations indicated, was preincubated with serially diluted serum from C57BL/6 mice. Each dot represents a single well in one experiment. (B) Comparison of the heme buffering capacity of serum from Hp+/+Hpx+/+ versus Hp−/−Hpx−/− mice, at steady state. (A) Increasing heme concentrations were pre-incubated with serum (1/250) in the same assay as in (A). Data shown as mean ± SD (N = 3 per genotype) from one experiment. (C) Serum concentrations of ⍺1-microglobulin (mg/dl) in Pcc-infected (1 × 105 iRBC, at the peak of parasitemia: days 8–9 post-infection) and non-infected control mice (N = 4 per indicated genotype). (D) α1-Microglobulin concentrations in serum from P. falciparum-infected individuals stratified according to disease severity: Uncomplicated malaria (N = 21), cerebral malaria (CM; N = 58) or severe non-cerebral malaria (N = 60). (C, E) Serum concentrations of albumin in the same mice as in (C). (D, F) α1-Microglobulin concentrations in serum from P. falciparum-infected individuals as in (D). (G, H, I) Serum concentrations of (G) low density lipoprotein (LDL); (H) Oxidized LDL. (G, H, I) Ratio of oxidized LDL/LDL calculated from (G) and (H). Data in (C, E, G, H, I) represented as mean ± SD (N = 4 mice per genotype) from one to two independent experiments with similar trend. Dots represent individual mice. Data in (D, F) are represented in violin plots where circles represent individuals and red line indicates median values. (D, F) In (C, E, G, H, I) P-values were determined by two-way ANOVA and in (D, F) by one-way ANOVA. NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Source data are available for this figure.
We next quantified other known serum heme-binding proteins and macromolecules, at the peak of Pcc infection. The concentration of the heme scavenger ⍺1-microglobulin (Allhorn et al, 2002) in serum from Pcc-infected mice was similar to that of control noninfected mice (Fig 5C). Pcc-infected Hp−/− mice had higher concentrations of ⍺1-microglobulin in serum, compared with noninfected genotype-matched controls (Fig 5C). This suggests that, in the absence of HP, ⍺1-microglobulin might take a predominant role in scavenging labile heme during malaria.
The median concentration of ⍺1-microglobulin in serum from children that developed severe non-cerebral P. falciparum malaria was significantly higher than that of children that developed uncomplicated P. falciparum malaria (Fig 5D). The median concentration of ⍺1-microglobulin in serum was indistinguishable in CM versus uncomplicated P. falciparum-infected children (Fig 5D). These observations suggest that ⍺1-microglobulin might “compensate” for the HP depletion and limit the accumulation of labile heme in serum in severe non-cerebral malaria.
Albumin concentration in serum from Pcc-infected mice was reduced by ∼50% (i.e., hypoalbuminemia), compared with noninfected controls (Fig 5E). This effect (i.e., hypoalbuminemia) was indistinguishable in Pcc-infected Hp−/−, Hpx−/− and Hp−/−Hpx−/− mice (Fig 5E). This suggests that the contribution of albumin to the heme buffering capacity of serum is reduced during Plasmodium infection.
The median concentration of albumin in serum was indistinguishable in uncomplicated P. falciparum-infected children versus CM versus children that developed severe non-cerebral P. falciparum malaria (Fig 5F).
The concentration of circulating low-density lipoprotein (LDL), a lipid/protein macromolecule that binds avidly to labile heme (Jeney et al, 2002), increased by ∼fivefold in adult Pcc-infected Hp+/+Hpx+/+, Hp−/−, Hpx−/− and Hp−/−Hpx−/− mice versus genotype and aged-matched noninfected controls (Fig 5G). The concentration of oxidized LDL was reduced by ∼30% (Fig 5H and I), accounting for a <10-fold lower ratio of oxidized versus total LDL in Pcc-infected versus noninfected genotype matched controls (Fig 5H and I). This suggests that Plasmodium infection is associated with major changes in the relative concentration and oxidation of plasma heme-binding proteins and macromolecules. To what extent this contributes to regulate the pathogenetic effects of labile heme during P. falciparum malaria remains, however, to be established.
HP and HPX are essential to prevent malaria mortality in ageing mice
In sharp contrast to adult mice (i.e., 8–12 wk), ageing (i.e., >30 wk) Hp−/−, Hpx−/− and Hp−/−Hpx−/− mice succumbed to Pcc infection, as compared with age-matched control Pcc-infected Hp+/+Hpx+/+ mice that survived (Fig 6A). This was not associated, however, with changes in parasite burden (Fig 6A), suggesting that HP and HPX are essential to establish disease tolerance to malaria (Medzhitov et al, 2012; Martins et al, 2019) in ageing, but not adult, mice.
(A) Ageing (>30 wk) mice from the indicated genotypes were infected with Pcc (i.p., 2 × 106 iRBCs). Survivals (left panel) are represented in the Kaplan–Meier plot and parasitemia (Right panel) by mean ± SD, monitored daily from day 3 post infection. Data pooled from three independent experiments (N = 7–12 mice per genotype), with a similar trend. (B) Quantification of labile heme in serum (Left panel) and renal heme (Right panel) at the peak of Pcc infection (2 × 106 iRBC; day 7 post-infection) in Hp+/+Hpx+/+ and Hp−/−Hpx−/− mice. Data shown as mean ± SD from one experiment (N = 4–5 mice per genotype). P-values determined using two-way ANOVA. (C) Kidney Perl’s Prussian blue staining (non-heme Fe3+) from N = 4–5 mice per genotype (non-infected or Pcc-infected: 2 × 106 iRBC, at the peak of infection: day 7 post-infection) in one experiment. Top panels show whole-kidney sections and bottom panels higher magnifications from the area highlighted. Arrowheads indicate Fe3+ (blue). GL, glomerulus; PT, proximal tubules. (C, D) Quantification of renal non-heme Fe3+ accumulation, detected in the same experiment as in (C). Data presented as mean ± SD (N = 4–5 mice per genotype). (E) Quantification of renal non-heme iron concentration, shown as mean ± SD (N = 4–5 mice per genotype). P-values in (D, E) determined using two-way ANOVA. NS, nonsignificant; *P < 0.05; **P < 0.01; ****P < 0.0001.
Source data are available for this figure.
HP and HPX control renal iron overload and AKI in ageing mice
The concentrations of labile heme in serum and total heme in the kidneys from ageing Pcc-infected Hp−/−Hpx−/− mice were in the range of control age-matched Pcc-infected Hp+/+Hpx+/+ mice (Fig 6B). However, ageing Pcc-infected Hp−/−Hpx−/− mice accumulated higher levels of iron (Fig 6C–E) in the kidneys, including ferric (Fe3+) iron in renal proximal tubules, when comparing with Pcc-infected Hp+/+Hpx+/+ controls (Fig 6C and D). This suggests that HP and HPX limit the accumulation of heme–iron in the kidneys of ageing mice, a major driving force in the pathogenesis of malaria AKI (Ramos et al, 2019; Wu et al, 2023).
Ageing alters renal response to Plasmodium infection
To further understand the age-dependent protective effect of HP and HPX against malaria AKI, we compared bulk RNAseq data from adult versus ageing naïve and Pcc-infected Hp+/+Hpx+/+ mice. Consistent with previously described (Ramos et al, 2019; Wu et al, 2023), Pcc infection in adult Hp+/+Hpx+/+ mice was associated with a robust transcriptional response in the kidneys, as compared with age-matched noninfected controls (Fig 7A). This integrated (i.e., parenchyma plus hematopoietic cells) response was characterized by the induction of 1,292 genes and repression of 609 genes (Fig 7A). Ageing Pcc-infected Hp+/+Hpx+/+ mice also showed a robust transcriptional response (Fig 7A). However, only 34% of the 1,536 genes induced and 1,313 genes repressed in ageing Pcc-infected Hp+/+Hpx+/+ mice were shared with those induced or repressed in adult Pcc-infected Hp+/+Hpx+/+ mice, respectively (Fig 7A). This suggests that ageing interferes per se with the integrated renal transcriptional response to malaria.
(A) Schematic representation of the experimental procedure used for renal bulk RNA-seq analyzes (Left panel). Euler plots of renal bulk RNA-seq data (Right panels) indicating the number and proportion of genes differentially expressed (All), induced (up-regulated) and repressed (down-regulated) in kidneys from Pcc-infected versus non infected (NI) male C57BL/6 mice. Red corresponds to genes differentially regulated, in a nonoverlapping manner (unique), in adult Pcc-infected male mice. Green corresponds to genes regulated, in an overlapping (shared) manner, in adult and ageing Pcc-infected mice. Blue corresponds to genes regulated, in a nonoverlapping manner (unique), in ageing Pcc-infected mice. (A, B) Manhattan plots of gProfiler renal bulk RNA-seq data from (A), depicting gene ontology (GO) analysis for biological processes (GO:BP; green), cellular components (GO:CC; orange), and molecular function (GO:MF; blue), Kyoto Encyclopedia of Genes and Genomes pathways (pink), Reactome (REAC, light green), transcription factors (TF; yellow), and WikiPathway (WP; gray). Left panel (red) corresponds to genes differentially regulated, in a nonoverlapping manner (unique), in adult Pcc-infected mice. Middle panel (green) corresponds to genes regulated, in an overlapping (shared) manner, in adult and ageing Pcc-infected mice. Right panel (blue) corresponds to genes regulated, in a nonoverlapping manner (unique), in ageing Pcc-infected mice. Data from N = 2 mice per experimental group from two independent experiments with similar trend.
Source data are available for this figure.
The unique gene expression “signature” of adult Pcc-infected mice was related to proinflammatory cytokines (e.g., interleukin 1 and 6) and regulation of lipid metabolism as well as carboxylic metabolic processes (Fig 7B). These were associated with transcriptional programs regulated by the transcription factors Paired Box 4 (Pax4), Kruppel-like factor 4 (KLF4; gut-enriched Krüppel-like factor or GKLF) or Forkhead Box N4 (FOXN4) (Fig 7B).
Consistent with previously described (Wu et al, 2023), the shared gene-expression “signature” between Pcc-infected adult and ageing mice (Fig 7B) was related to type I and II interferon responses, and to antigen processing and presentation (Fig 7B). This was associated with transcriptional programs regulated by the interferon regulatory factor (IRF) family of transcription factors including IRF-1, 2 and 9 (Fig 7B).
Ageing Pcc-infected mice also presented a unique gene-expression “signature,” related to metabolism of nitrogen compounds and primary metabolic processes and with nucleic acid metabolic processes and stress responses, including DNA damage responses but also with cell cycle regulation (Fig 7B). This was associated with transcriptional programs regulated by the transcription factors E2F transcription factor 1 (E2F1), ZF5, and FOXN4 (Fig 7B).
These observations reveal that ageing interferes with the integrated renal transcriptional responses to Plasmodium infection without however, precipitating the onset of malarial AKI. Whether these transcriptional responses emanate predominatly from parenchyma or hematopoietic-derived cells is not clear.
HP and HPX regulate the renal response to malaria in ageing mice
We asked whether HP and HPX shape the integrated renal transcriptional response to Plasmodium infection in ageing mice. Kidneys from ageing noninfected Hp−/−Hpx−/− mice showed a distinct gene expression “signature,” driven by the induction of 29 genes and repression of 79 genes, compared with age-matched control Hp+/+Hpx+/+ mice (Fig 8A and B). Gene ontology analysis suggests that kidneys from ageing mice up-regulated the expression of genes associated with epithelial barrier integrity (e.g., apical junction complex; tight junction; cell-cell junction; anchoring junction, etc.), cell proliferation (e.g., E2F-1, E2F-3), apoptosis, and stem cell self-renewal (e.g., Hippo signaling) as well as with host–microbe interactions (e.g., regulation of symbiont or viral entry into host, etc.) (Fig 8C). This suggests that HP and HPX regulate steady state renal physiology.
(A) Schematic representation of the experimental procedure used for renal bulk RNA-seq analyzes in ageing Hp−/−Hpx−/− versus control aged-matched Hp+/+Hpx+/+ mice at steady state. (B) Volcano plots displaying differential gene expression between whole kidneys from ageing Hp−/−Hpx−/− versus control aged-matched Hp+/+Hpx+/+ mice at steady state. (B, C) Gene ontology (GO) analysis of differentially expressed genes from (B). Top 20 significant up- or down-regulated GO terms depicted, for comparisons where more than 20 significant GO terms were found. (D) Schematic representation of the experimental procedure used for renal bulk RNA-seq analyzes in ageing Pcc-infected Hp−/−Hpx−/− versus control aged-matched Pcc-infected Hp+/+Hpx+/+ mice. (E) Volcano plots displaying differential gene expression between whole kidneys from ageing Pcc-infected Hp−/−Hpx−/− versus control aged-matched Pcc-infected Hp+/+Hpx+/+ mice. (F) Gene ontology (GO) analysis of differentially expressed genes from (E), displayed as in (C). Genes significantly up-regulated in kidneys from Hp−/−Hpx−/− mice are represented in red dots, whereas blue dots represent down-regulated genes. Grey dots represent not statistically significant genes. Analysis of same data as Fig 7, performed using gProfiler. Data from N = 2–3 mice per experimental group from 1 independent experiment.
Source data are available for this figure.
The kidneys from Pcc-infected Hp−/−Hpx−/− mice also showed a distinct gene-expression “signature” profile driven by the up-regulation of 98 genes and repression of 50 genes, compared with age-matched control Pcc-infected Hp+/+Hpx+/+ mice (Fig 8D and E). Gene ontology analysis suggests that among the up-regulated genes are mitochondrial genes, whereas the repressed genes were associated mainly with extracellular matrix (e.g., collagen trimer, extracellular region, extracellular space; extracellular matrix, structural constituent; external encapsulating structure, etc.) (Fig 8F). This suggests that ageing interferes with the renal transcriptional response to Plasmodium infection, associated with the onset of malarial AKI. To what extent the differential expression of these genes justifies how the combination of ageing and HP and HPX regulates the pathogenesis of malaria-associated AKI remains to be established functionally.
Circulating HPX and heme are associated with P. falciparum AKI
Ageing Pcc-infected Hp−/−Hpx−/− presented more extensive HB cast nephropathy, predominantly in the proximal tubules, as compared with Pcc-infected Hp+/+Hpx+/+ mice (Fig 9A and B). This is consistent with renal iron overload promoting the pathogenesis of malaria AKI (Ramos et al, 2019; Wu et al, 2023).
(A) Representative H&E staining of the kidney from ageing (>30 wk) Hp+/+Hpx+/+ and Hp−/−Hpx−/− mice infected with Pcc (i.p., 2 × 106 iRBCs, at the peak of infection: day 7 postinfection). Images are representative from N = 4–5 mice per condition. Top panels show whole-kidney section and bottom panels show higher magnifications from the rectangle highlighted in the top panel. (B) Asterisks indicate hemoglobin casts. GL, glomerulus; PT, proximal tubules. (A) Histopathological evaluation of kidneys from (A) was performed using digitalized whole-slide images, corresponding to whole-kidney sections. Scores are represented as mean ± SD (n = 4–5 mice per genotype). Dots correspond to individual mice. P-values determined by Two-Way ANOVA. *P < 0.05; ****P < 0.0001; NS, not significant. Scores: 0 = No lesions; 1 = Single cell necrosis, discrete hemoglobin tubular casts; 2 = Mild; 3 = Moderate; 4 = Severe tubular cell necrosis, hemoglobin tubular casts. (C, D) Spearman correlation coefficients between indicated variables and LNC2 and creatinine in serum of P. falciparum-infected individuals (same individuals as in Fig 1), corrected for multiple tests (Holm–Sidak).
Source data are available for this figure.
We asked whether the levels of circulating HP, HPX, and/or labile heme were associated with P. falciparum AKI in the case-control study described in Table 1 (Sambo et al, 2010). HPX was negatively correlated with lipocalin 2 (LCN2) (P = 0.0001) (Fig 9C) and with creatinine (P = 0.006) (Fig 9D) concentrations in serum, two serological markers of AKI. These negative correlations remained significant when controlling for parasitemia (P = 0.000061 for LNC2 and P = 0.008 for creatinine) (Fig 9C and D).
Total heme, HB–heme, and labile heme were positively correlated with the concentration of LCN2 in the serum of P. falciparum-infected children (Fig 9C). These positive correlations remained significant when controlling for parasitemia (P = 0.000001 for total heme, P = 0.004 for HB-Heme and P = 0.0006 or labile heme) (Fig 9C). Although total heme was also positively correlated with creatinine concentration in serum (Fig 9D), this was no longer significant for labile heme or HB–heme (Fig 9D).
These observations suggest that HPX acts irrespectively of parasite burden to prevent labile heme from partaking in the pathogenesis of malarial AKI, contributing to the establishment of disease tolerance (Medzhitov et al, 2012; Martins et al, 2019) to P. falciparum malaria. The association of labile heme with CM (Fig 1D) and malaria AKI (Fig 9C and D) is consistent with the proposed pathophysiological contribution of AKI to the development of brain dysfunction in P. falciparum malaria (Conroy et al, 2023).
Discussion
Having established that heme catabolism by HO-1 is protective against the pathogenesis of cerebral (Pamplona et al, 2007; Ferreira et al, 2008; Jeney et al, 2014) and non-cerebral (Seixas et al, 2009; Ramos et al, 2019) presentations of severe malaria in mice, we put forward that labile heme acts as a major driving force in the pathogenesis of severe presentations of malaria (Ferreira et al, 2008). However, two independent clinical studies have shown that microsatellite (GTn) polymorphisms in the human HMOX1 promoter (i.e., lower GTn repeats), enhancing HO-1 expression, are associated with increased P. falciparum malaria susceptibility in children and adults (Takeda et al, 2005; Walther et al, 2012). This questioned whether the protective effects of HO-1 in rodent malaria are extrapolatable to the human disease. In support of the latter, there are “additional” HMOX1 gene variants enhancing HO-1 expression that were associated with reduced susceptibility to P. falciparum CM in children (Sambo et al, 2010), consistent with experimental models of malaria in mice (Pamplona et al, 2007; Ferreira et al, 2008; Jeney et al, 2014).
The apparent discrepancy between the protective versus pathogenic effects of HO-1 in rodent models of malaria and human malaria, respectively, is likely explained by the opposing effects of HO-1, exerted at different stages of Plasmodium infection. Malaria transmission by Anopheles mosquitoes is associated with the induction of HO-1 by erythrophagocytic macrophages in the dermis, which limit the extent of damage imposed locally by microvascular bleeding (DeSouza-Vieira et al, 2020). Whether this impacts on the progression and outcome of malaria was, to the best of our knowledge, not established. As Plasmodium sporozoites migrate from the dermis to liver, HO-1 expression by Kupfer cells, and probably other cell compartments, becomes essential to establish the liver stage of malaria (Epiphanio et al, 2008). Thereafter, during the blood stage of infection, HO-1 acts in different cell compartments to prevent the developemnt of experimental CM (Pamplona et al, 2007; Ferreira et al, 2008; Jeney et al, 2014) and in renal proximal tubule epithelial cells (Ramos et al, 2022), to prevent the development of AKI, a major independent risk factor of malaria mortality in children and adults (Sitprija, 1988; Trang et al, 1992; Mishra & Das, 2008; Plewes et al, 2017; Cruz et al, 2018; Katsoulis et al, 2021; Wu et al, 2023). These findings suggest that heme catabolism by HO-1 exerts a dual role, promoting the initial stages of Plasmodium spp. infection whereas preventing, later on, the onset of severe presentations of malaria.
To probe the pathologic effect of labile heme in P. falciparum malaria we asked whether targeting extracellular HB and/or labile heme by HP and HPX, respectively, limit the accumulation of labile heme and/or prevent the pathogenesis of severe presentations of malaria. Our findings suggest that labile heme is a major risk factor for severe presentations of P. falciparum malaria in children (Table 1, Fig 1). This is not the case however, for HP nor HPX (Table 1, Fig 1C), suggesting that other heme-binding proteins and macromolecules might contribute to control the pathogenetic effects of circulating labile heme. In strong support of this notion, HP and HPX have little or no contribution to the heme-buffering capacity of mouse serum (Fig 5A and B). As the assay used to quantify heme-buffering capacity is based on a heme-binding sdAb with an affinity towards heme in the range of 10−7 M (Gouveia et al, 2017), our findings suggest that a number of plasma heme-binding proteins and/or macromolecules bind labile heme with an affinity higher than 10−7 M. These are likely to include circulating α1-microglobulin (De Simone et al, 2023), which accumulates to a higher extent in plasma from children developing non-cerebral severe presentations, when compared with uncomplicated P. falciparum malaria (Fig 5D). This is not observed for albumin (Fig 5F), which binds labile heme with an affinity in the range of 10−8 M (Adams & Berman, 1980; Severance & Hamza, 2009). However, this should not exclude albumin from acting as an intermediate low-affinity high-capacity heme scavenger (Bunn & Jandl, 1968; Ascenzi et al, 2005; De Simone et al, 2023) during P. falciparum malaria.
Other putative protective heme scavengers controlling the pathogenic effects of labile might include LDL, which binds labile heme (Jeney et al, 2002) with an affinity in the range of 10−11–10−12 M (Camejo et al, 1998). The marked increase in the concentration of LDL in serum of Pcc-infected mice (Fig 5G), suggests that LDL might provide an alternative heme-scavenging route during malaria. In support of this notion, labile heme binds and induces lipid peroxidation in LDL (Jeney et al, 2002), causing nonenzymatic cleavage of the protoporphyrin ring of heme and retaining iron (Balla et al, 1991). To what extent LDL contributes individually or collectively with albumin and/or α1-microglobuling to mitigate the pathogenic effects of labile heme during malaria remains however to be established.
We note that despite the accumulation of labile heme in plasma (Fig 2E and F), this was not associated with the accumulation of oxidized LDL (Fig 5H and I), at the peak of Pcc infection. This suggests that heme-driven LDL oxidation (Jeney et al, 2002) is actively prevented during Plasmodium infection, via a mechanism that remains to be established.
The age-dependent protective effect of HP and/or HPX against malaria AKI in mice (Figs 6–9) and the inverse correlation of HPX and heme with renal impairment in P. falciparum malaria (Fig 9C and D) are consistent with the age-dependent increase in susceptibility to renal impairment in P. falciparum malaria (Dondorp et al, 2008). Moreover, the age-dependent renal transcriptional response (Fig 7) and protective effect of HP and HPX (Fig 8) in Plasmodium-infected mice supports the idea of an age-dependent impairment of tissue damage control mechanisms (Soares et al, 2014) establishing disease tolerance to malaria (Martins et al, 2019; Ramos et al, 2019). This interpretation is contingent on the accuracy of the quantitative approach (i.e., microscopy) used to estimate peripheral parasitemia in clinical samples and on peripheral parasitemia reflecting parasite biomass. Of note, the notion of an age-dependent impairment of disease tolerance to malaria is in keeping with the age-dependent impairment of disease tolerance to bacterial sepsis (Sanchez et al, 2023 Preprint).
An open question raised by our study pertains to the possible pharmacologic use of HP and HPX in the treatment of severe P. falciparum malaria, presumably, as an adjunctive therapy with standard antimalarial drugs. Although this remains to be tested, such an approach warrants some considerations. Namely, sustained oxidation of extracellular HB can generate meth-(Fe3+)HB and ferryl-(Fe4+)HB. The latter forms covalently linked multimeric complexes (Vallelian et al, 2008) that can “escape” HP and activate endothelial cells (Silva et al, 2009; Nyakundi et al, 2019; Erdei et al, 2020), presumably therefore contributing to the pathogenesis of severe presentations of malaria.
Moreover, damaged RBC can release heme-containing microvesicles, described originally in sickle cell anemia (Camus et al, 2012) and thereafter in malaria (Pais et al, 2022). This suggests that a fraction of the heme released from damaged RBC might “escape” HPX, presumably therefore contributing to the pathogenesis of severe presentations of malaria, as demonstrated for experimental CM in mice (Pais et al, 2022).
More recently, heme-binding histidine-rich protein II (HRPII) nanomers, secreted by P. falciparum-infected RBC, were shown to induce vascular inflammation and edema (Nguyen et al, 2023). To what extent these partake in the pathogenesis of severe presentations of malaria is likely but remains to be tested experimentally.
In conclusion, circulating labile heme is a risk factor for severe presentations of P. falciparum malaria, consistent with the functional role of labile heme in the pathogenesis of severe malaria in mice (Pamplona et al, 2007; Ferreira et al, 2008; Gozzelino et al, 2010; Ramos et al, 2019; Wu et al, 2023). Whereas HP and HPX exert some level of control over the pathogenic effects of labile heme, other serum heme-binding proteins and/or macromolecules might partake in this defense mechanism that establishes disease tolerance to malaria. Identifying and characterizing such heme-binding proteins and/or macromolecules should contribute to the development of much needed therapeutic approaches against CM and non-cerebral malaria severe presentations of P. falciparum malaria.
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