Hemolysis and Heme Oxygenase-1 Induction Malaria Is Related to Plasmodium Falciparum Prolonged Neutrophil Dysfunction After

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Prolonged Neutrophil Dysfunction after Plasmodium falciparum Malaria Is Related to Hemolysis and Heme Oxygenase-1 Induction

This information is current as Aubrey J. Cunnington, Madi Njie, Simon Correa, Ebako N. of September 26, 2021. Takem, Eleanor M. Riley and Michael Walther J Immunol published online 24 October 2012 http://www.jimmunol.org/content/early/2012/10/24/jimmun ol.1201028 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 24, 2012, doi:10.4049/jimmunol.1201028 The Journal of Immunology

Prolonged Neutrophil Dysfunction after Plasmodium falciparum Malaria Is Related to Hemolysis and Heme Oxygenase-1 Induction

Aubrey J. Cunnington,*,† Madi Njie,† Simon Correa,† Ebako N. Takem,† Eleanor M. Riley,* and Michael Walther†,‡

It is not known why people are more susceptible to bacterial infections such as nontyphoid Salmonella during and after a malaria infection, but in mice, malarial hemolysis impairs resistance to nontyphoid Salmonella by impairing the neutrophil oxidative burst. This acquired neutrophil dysfunction is a consequence of induction of the cytoprotective, heme-degrading enzyme heme oxygenase-1 (HO-1) in neutrophil progenitors in bone marrow. In this study, we assessed whether neutrophil dysfunction occurs in

humans with malaria and how this relates to hemolysis. We evaluated neutrophil function in 58 Gambian children with Plasmo- Downloaded from dium falciparum malaria [55 (95%) with uncomplicated disease] and examined associations with erythrocyte count, haptoglobin, hemopexin, plasma heme, expression of receptors for heme uptake, and HO-1 induction. Malaria caused the appearance of a dominant population of neutrophils with reduced oxidative burst activity, which gradually normalized over 8 wk of follow- up. The degree of neutrophil impairment correlated signiﬁcantly with markers of hemolysis and HO-1 induction. HO-1 expression was increased in blood during acute malaria, but at a cellular level HO-1 expression was modulated by changes in surface

expression of the haptoglobin receptor (CD163). These ﬁndings demonstrate that neutrophil dysfunction occurs in P. falciparum http://www.jimmunol.org/ malaria and support the relevance of the mechanistic studies in mice. Furthermore, they suggest the presence of a regulatory pathway to limit HO-1 induction by hemolysis in the context of infection and indicate new targets for therapeutic intervention to abrogate the susceptibility to bacterial infection in the context of hemolysis in humans. The Journal of Immunology, 2012, 189: 000–000.

lasmodium falciparum caused an estimated 655,000 for treatment of neurosyphilis was frequently complicated by NTS deaths and 216 million cases of malaria globally in 2010 bacteremia even when NTS infection was otherwise very rare (8), (1), but this almost certainly underestimates the indirect and quinine alone often cured endemic malaria–NTS coinfection

P by guest on September 26, 2021 health burden (2), which includes increased susceptibility to (9). NTS bacteremia incidence was found to be more closely re- Gram-negative bacterial infections (3, 4), particularly nontyphoid lated to malaria incidence than to stool carriage of NTS (6), and in Salmonella (NTS) (3, 5, 6). In areas with high malaria transmis- Kenyan children, sickle cell trait was found to have a protective sion, these indirect effects of malaria infection may explain more effect against bacteremia, which was dependent on the protection than half of the child mortality (2) and community-acquired it affords against malaria (4). Several studies have shown that bacteremia (4). The incidence of NTS closely reflects that of susceptibility to NTS is greatest in the context of severe malarial malaria (4, 6, 7), and there is compelling evidence that P. falcip- anemia (5, 6), whereas others have found that the greatest risk arum malaria increases susceptibility to NTS bacteremia in occurred in children with recent rather than current malaria in- humans. In The Gambia, the incidence of NTS bacteremia has fection (10, 11). declined dramatically over the past 30 y, mirroring the decline Most mechanisms that have been proposed to account for the in the incidence of malaria (7); this observation has since been susceptibility to NTS that occurs in malaria involve monocyte confirmed in Kenya (4). In the pre-antibiotic era, malaria therapy and macrophage dysfunction. Malaria may impair monocyte and macrophage function through direct adhesion of infected RBCs (12), through the accumulation of hemozoin within these cells *Department of Immunology and Infection, Faculty of Infectious and Tropical Dis- (13), or by impairment of systemic IL-12 production (14). How- eases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom; †Medical Research Council Laboratories, Banjul, Gambia; and ever, other studies in mice have demonstrated that hemolysis— ‡Immune Regulation Section, Laboratory of Malaria Immunology and Vaccinology, caused by malaria or in any other way—increases susceptibility to Division of Intramural Research, National Institute of Allergy and Infectious Dis- NTS and some other bacterial infections, whereas blood loss eases, National Institutes of Health, Rockville, MD 20852 alone does not (15–17). We have recently shown in a mouse model Received for publication April 9, 2012. Accepted for publication October 4, 2012. of malarial anemia that resistance to Salmonella typhimurium is This work was supported by a clinical research training fellowship from the Medical impaired as a result of neutrophil dysfunction (rather than Research Council (U.K.) (G0701427 to A.J.C.) and by Medical Research Council Institute funding to the Medical Research Council Laboratories, Gambia. monocyte/macrophage dysfunction) caused by liberation of heme Address correspondence and reprint requests to Dr. Aubrey J. Cunnington, London during hemolysis and by induction of the cytoprotective heme- School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K. catabolizing enzyme heme oxygenase-1 (HO-1) (18). In this E-mail address: [email protected] model system, HO-1 induction in myeloid progenitor cells in the The online version of this article contains supplemental material. bone marrow leads to production of granulocytes with reduced Abbreviations used in this article: CRP, C-reactive protein; HO, heme oxygenase; oxidative burst activity, and their mobilization into the blood is HO-1, heme oxygenase-1; LB, Luria–Bertani; MFI, median fluorescence intensity; NTS, nontyphoid Salmonella; PfHRP-2, P. falciparum histidine rich protein-2; qRT- enhanced by both hemolysis-derived heme and the response to PCR, quantitative RT-PCR. bacterial coinfection. This results in the accumulation of func-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201028 2 IMPAIRED NEUTROPHIL FUNCTION IN MALARIA tionally impaired granulocytes in the circulation that are able to was used for assessment of neutrophil oxidative burst and degranulation phagocytose S. typhimurium but not able to kill the bacteria ef- (350ml), intracellular and cell surface flow cytometry (400 ml), and for fectively, providing a new niche for bacterial replication. We neutrophil isolation (1.25 ml). On some occasions, there was insufficient blood available to perform all assays. Single blood samples (handled as found that normal resistance to S. typhimurium was restored by described earlier) were obtained from six healthy Gambian children and 10 inhibition of heme oxygenase (HO) with the competitive inhib- healthy Gambian adults, all without current or recent malaria, recruited itor tin protoporphyrin, a drug that can be used to treat hyper- from Brefet village where malaria transmission is now extremely low (25). bilirubinemia in newborns (19), suggesting that HO inhibitors might Laboratory reagents represent a novel therapeutic intervention to abrogate the susceptibility to NTS induced by malaria. All reagents were obtained from Sigma unless specified otherwise. Humans and mice with genetic deficiency of subunits of the GFP-expressing Salmonella enterica serovar Typhimurium pfpv 12023 (S. typhimurium) was a gift from Prof. David Holden (Imperial College, phagocytic NADPH oxidase, a complex enzyme that catalyzes the London, U.K.), grown to late log-phase in Luria–Bertani (LB) broth generation of superoxide radicals in phagocytic cells, are known supplemented with ampicillin, and kept as frozen stock at 270˚C in 10% to be susceptible to NTS infection (20, 21), and the importance glycerol. of the neutrophil oxidative burst for killing of serum-opsonized Neutrophil oxidative burst and degranulation assays Salmonella by blood leukocytes from African children has been demonstrated in vitro (22). Impairment of the neutrophil oxida- The neutrophil oxidative burst was assessed in minimally manipulated tive burst in humans with malaria would thus be a compelling whole blood using a modification of the assay described by Richardson et al. m m explanation for susceptibility to NTS bacteremia. In the current (26). Briefly, 50- l aliquots of blood were mixed with 50 l PMA (final concentration 1 mM) or PBS (as control) and incubated for 15 min at 37˚C study, we investigated whether the same mechanism may apply in in a water bath. Next, 25 ml PBS (unstained sample) or staining mixture Downloaded from humans by examining neutrophil function in a cohort of children [dihydrorhodamine 123, PECy7 anti-CD11b (ICRF44; eBioscience), and with predominantly uncomplicated malaria. Despite the fact that allophycocyanin anti-CD15 (VIMC6; Miltenyi Biotec) (unstimulated and this population would not be considered at high risk of NTS stimulated samples)] was added and incubated for 5 min at 37˚C in the dark. Ammonium chloride RBC lysis buffer was added for 5 min at room coinfection, we found that malaria caused a marked abnormality temperature, shielded from light, before washing in PBS and resuspending of function in a large proportion of neutrophils, with impairment cells in 1% paraformaldehyde in PBS. Samples were stored at 4˚C pro- of oxidative burst capacity but not degranulation. The severity of tected from light and analyzed on the day of collection using a three-laser/ http://www.jimmunol.org/ the impairment of the neutrophil oxidative burst was strongly nine-channel CyAn ADP flow cytometer with Summit 4.3 software (Dako) associated with hemolysis and prior induction of HO-1, but the after calibration of the FL-1 voltage with fluorescent beads (Spherotech). Data were analyzed in FlowJo 7.6 (Tree Star). The magnitude of the ox- duration of impairment was much longer than expected, lasting idative burst was quantified by the rhodamine median fluorescence in- up to 8 wk postinfection. tensity (MFI), and degranulation was quantified by the fold increase in surface CD11b MFI from the unstimulated to the stimulated sample. Neutrophils were divided into rhodamineHi and rhodamineLo populations Materials and Methods at the midpoint of the nadir between peaks. Study subjects and procedures Flow cytometry for cell surface receptors and intracellular

The study and all procedures were approved by the Gambian government/ by guest on September 26, 2021 Medical Research Council Laboratories Joint Ethics Committee and the HO-1 expression London School of Hygiene and Tropical Medicine Ethics Committee. All Whole blood was subjected to ammonium chloride RBC lysis and, after human samples were collected with written informed consent from the washing, cell pellets were resuspended in surface marker Ab mixture [FITC participant or from the parent or legal guardian of child participants. Be- anti-CD91 (A2Mr a-2; AbD Serotec), PE anti-CD16b (CLB-gran11.5; BD tween September and December 2010, 58 Gambian children with P. fal- Pharmingen), PerCP anti-CD14 (MwP9; BD Pharmingen), allophycocyanin ciparum malaria (defined by compatible clinical symptoms and .5000 anti-CD163 (215927; R&D Systems)] or a similar mixture instead con- asexual parasites/ml blood) were recruited within a longitudinal study in- taining the corresponding manufacturer-matched isotype control Abs for vestigating clinical, immunological, and parasitological factors in mild and CD91 (mouse IgG1) and CD163 (11711; mouse IgG1). Cells were per- severe malaria, details of which have been published (23). Briefly, subjects meabilized with Cytofix/Cytoperm (BD Biosciences) before intracellular were recruited, without selection for disease severity, from three peri-urban staining with polyclonal anti–HO-1 (SPA-895; Assay Designs) or an health centers: the Medical Research Council Gate Clinic, Brikama equivalent concentration of polyclonal control rabbit serum (Covance), Health Center, and the Jammeh Foundation for Peace Hospital, Ser- followed by PE–Cy7–conjugated secondary Ab [F(ab9)2 anti-rabbit IgG; ekunda. Initial parasitemia (to determine eligibility for inclusion in the Santa Cruz Biotechnology]. The expression of HO-1, CD163, and CD91 study) was estimated from Field’s stained thick blood films and subse- were quantified as the ratio of MFI to the respective isotype control Ab for quently accurately counted from 50 fields on Giemsa-stained thin blood the same sample. smears. All children underwent full clinical examination and were man- aged in accordance with Gambian government guidelines, treated with Neutrophil isolation and Salmonella phagocytosis and killing artemether–lumefantrine for 3 d. Severe malaria was defined using mod- assays ified World Health Organization criteria (24): severe anemia, defined as hemoglobin ,6 g/dl; lactic acidosis defined as blood lactate .7 mmol/l; CD15+ cells were isolated from whole blood, after RBC lysis and labeling cerebral malaria defined as a Blantyre coma score #2 in the absence of with allophycocyanin anti-CD15, using anti-CD15 magnetic beads and MS hypoglycemia, with the coma lasting at least for 2 h; severe prostration columns (all from Miltenyi Biotec) according to the manufacturer’s defined as inability to sit unsupported (children .6 mo) or inability to suck instructions. CD15+ cells were resuspended in RPMI 1640 plus 2 mM 7 (children #6 mo). Children suspected to have concomitant bacterial L-glutamine at a concentration of 10 /ml. GFP-expressing S. typhimurium infections were excluded. For this study, children underwent standardized (concentration confirmed by serial dilution) were opsonized in 10% pooled assessment on the day of presentation (day 0) and on days 7, 28, and 56, healthy Gambian adult serum [derived from 10 donors, as has been de- and blood was collected for: thick blood film, full blood count (EDTA), scribed by others (22)] for 20 min in the dark at room temperature. immunological assays (sodium heparin), and RNA (PaxGene tube) (days Neutrophils and S. typhimurium were mixed continuously at a ratio of 50:1 0 and 28). On day 0, a thin blood film was prepared, and sickle cell status, at 60 rpm at 37˚C. Bacterial counts were assessed at time 0 and 120 min by blood lactate, and glucose were determined. Blood samples were stored on 10-fold dilutions of aliquots of the neutrophil–S. typhimurium suspension ice, transported to the laboratory within 2 h of sample collection, and in 1% Triton, and plating onto LB agar, with CFUs counted 16–18 h later. processed within 4 h of collection. Full blood count was performed using Bacterial killing was quantified as the percentage reduction in bacterial a Medonic instrument (Clinical Diagnostics Solutions). Sickle cell status count between time 0 and 120 min. Phagocytosis was assessed after 15 min was determined by metabisulfite test and confirmed on cellulose acetate of incubation by removing the neutrophil–S. typhimurium suspension di- electrophoresis. The proportion of immature neutrophils and hemozoin rectly into PBS 4% paraformaldehyde and analyzing by flow cytometry. (malaria pigment) containing neutrophils and monocytes was determined To control for autofluorescence and surface binding of bacteria without from the day 0 Giemsa-stained thin blood film. Heparinized whole blood phagocytosis, control samples were prepared in an identical manner except The Journal of Immunology 3 that neutrophils and S. typhimurium were both fixed with 4% formaldehyde Prolonged impairment of the neutrophil oxidative burst before mixing together. The proportion of cells phagocytosing bacteria was determined by subtraction of the proportion of GFP+ cells in the fixed- We assessed the PMA-stimulated oxidative burst of neutrophils control samples from that in the respective unfixed sample. using a whole-blood flow cytometric assay based on the oxidation of dihydrorhodamine 123 to its fluorescent derivative rhodamine, ELISAs where the magnitude of the oxidative burst is quantified by the Plasma levels of P. falciparum histidine rich protein-2 (PfHRP-2; (Cellabs), rhodamine fluorescence intensity (26). The assay was modified to hemopexin, haptoglobin (both Genway), C-reactive protein (CRP; R&D allow simultaneous assessment of degranulation based on upreg- Systems), and HO-1 (Enzo Life Sciences) were measured by ELISA. All ELISA assays were performed according to the manufacturer’s instruc- ulation of CD11b (32) and surface staining of CD15 to identity tions, and samples for each assay were performed in a single batch. neutrophils. We found that neutrophils (Fig. 1A) from subjects with acute malaria (day 0) showed an abnormal, bimodal distri- Heme assay bution of oxidative burst activity (Fig. 1B), with distinct pop- Total plasma heme (i.e., plasma hemoglobin plus free- and protein bound- ulations of rhodamineHi and rhodamineLo cells, whereas CD11b heme) was measured using a colorimetric heme assay kit (QuantiChrom expression showed a unimodal distribution (Fig. 1C). Overall, heme; BioAssay Systems). neutrophil rhodamine MFI increased significantly over time (Fig. Quantitative RT-PCR 1D), but in view of the bimodal distribution of neutrophil rhodamine fluorescence, we also compared the proportion of cells that Total RNA was extracted from PAX tubes using PAXgene blood RNA kits Lo Lo (Qiagen) according to the manufacturer’s instructions and converted into were rhodamine and the rhodamine MFI of the rhodamine cDNA using a reverse transcription reagent kit (Invitrogen). HMOX1 cells over time. The proportion of rhodamineLo cells decreased (141250) gene expression was determined by quantitative RT-PCR (qRT- significantly during the convalescent period (Fig. 1E) but Downloaded from PCR) on a DNA Engine Opticon (MJ Research) using a TaqMan Probe kit remained above that of healthy controls for at least 56 d; the with primers (all Metabion) as described by Hirai et al. (27). 18S rRNA Lo was used as an endogenous reference gene, as its expression has been rhodamine MFI of the rhodamine cells also significantly in- shown to be stable in acute and convalescent samples from malaria cases creased over time (Fig. 1F). In contrast, there was no evidence of regardless of disease severity (23), and was amplified with a commercial abnormalities in neutrophil degranulation as assessed by CD11b kit (rRNA primers and VIC labeled probe; Applied Biosystems). Data expression (Fig. 1G). Of interest, the rhodamine MFI of the rho- were analyzed using Opticon Monitor 3 analysis software (Bio-Rad). damineHi cells was higher on days 0 and 7 after presentation http://www.jimmunol.org/ HMOX1 expression was quantified as the ratio of the transcript number of , the HMOX1 to 18S rRNA. (p = 0.04 and p 0.001 respectively, Wilcoxon matched pairs test) than on day 56 (Fig. 1H), suggesting that the oxidative burst Estimation of total parasite biomass is primed in these neutrophils (33). These findings are consistent Total parasite biomass was calculated from plasma PfHRP-2 concentration with our observations in mice that hemolysis and infection can using the method of Dondorp et al. (28). This assumes that PfHRP-2 prime the oxidative burst of mature, circulating neutrophils while concentration is an integral of all PfHRP-2 released in preceding rounds simultaneously mobilizing immature neutrophils with impaired of schizogony (when infected erythrocytes rupture to release merozoites) and is therefore a reliable indicator of cumulative hemolysis since the start oxidative burst activity from the bone marrow (18). However the duration of these neutrophil abnormalities after P. fa lc ipa rum in-

of the infection (28). We modified the calculation to account for the rel- by guest on September 26, 2021 atively higher blood volume at lower body weight in small children (29). fection was longer than we expected. We assessed whether a To account for variation in size of children, parasite biomass was expressed similar abnormality was present in monocytes in peripheral blood as parasites per kilogram body weight. (Supplemental Fig. 1A) but found that the oxidative burst appeared Statistics to be slightly enhanced on day 0 compared with other time points (Supplemental Fig. 1B, 1C). The study was designed to detect a 30% difference in neutrophil oxidative burst activity between samples at day 0 and day 28 with 80% power at the 0.05 significance level, allowing for 15% loss to follow-up. Statistical analysis was Hemolysis and neutrophil dysfunction performed using PASW Statistics 18 (SPSS). Variables were examined for We have previously shown that hemolysis-derived heme impairs normality of distribution, and most were found to be nonnormal. Two-tailed neutrophil function during malaria infection in mice through two nonparametric tests for paired (Wilcoxon matched pairs test) or related re- peated measures (Friedman’s two-way ANOVA) were used to compare related mechanisms: mobilization of functionally immature neu- longitudinal data at different time points, and correlation was tested with trophils from bone marrow, and impairment of the oxidative burst Spearman’s rho correlation. To normalize distribution for general linear capacity of developing neutrophils due to HO-1 induction in bone model analysis, some variables were log10 transformed or converted to marrow progenitors (18). RBC destruction in malaria is multifac- binary variables, as described in the text. Haptoglobin concentrations showed a bimodal distribution and were thus converted to a binary vari- torial, but the severity of intravascular hemolysis can be inferred able (,0.349 mg/ml, the lowest value observed in healthy control sam- from levels of the plasma proteins haptoglobin and hemopexin (34), ples, or $0.349 mg/ml). Sample volumes did not allow for some assays to which provide sequential lines of defense against heme-mediated be performed at time points after day 0, in which case values from six toxicity by binding cell-free hemoglobin and cell-free heme, healthy control children were presented for comparison, but not for formal respectively (35). Only once haptoglobin is depleted do levels of statistical analysis. hemopexin begin to fall, indicating that heme is being released from cell-free hemoglobin (36). Results To assess the extent of hemolysis in study participants, we Subjects measured erythrocyte count and total parasite biomass (Table I), Fifty-eight children with P. falciparum malaria were recruited to total plasma heme (Fig. 2A), haptoglobin (Fig. 2B), and hemo- the study, 55 (94.8%) of whom had uncomplicated malaria (Table pexin (Fig. 2C) and examined their correlation with the proportion I). Four children had recurrent episodes of parasitemia during the and function of rhodamineLo neutrophils (Table II). Although only course of follow-up and were excluded from longitudinal analy- 13 (22.4%) children were actually anemic by Gambian reference ses; another 13 children were lost to follow-up or withdrew con- standards (Hb ,9.5 g/dl) (30), total plasma heme levels were sent. Thus, at days 7, 28, and 56, the number (%) of children in significantly greater on day 0 than on day 28 (Fig. 2A). Levels of follow-up were 52 (89.6%), 46 (79.3%), and 41 (70.1%), re- haptoglobin showed a bimodal distribution (Fig. 2B), consistent spectively. with the expected depletion of haptoglobin by hemolysis in some 4 IMPAIRED NEUTROPHIL FUNCTION IN MALARIA

Table I. Demographic, clinical, and laboratory characteristics at recruitment

Variable Category na (%) Median (IQR) Normal Range Sex Male 36 (62) Female 22 (38) Ethnicity Mandingo 25 (43) Fula 10 (17) Wolof 7 (12) Manjago 5 (9) Jola 5 (9) Serere 4 (7) Aku 1 (2) Fanti 1 (2) Age (y) 58 8 (4–12) Severity Uncomplicated 55 (95) Severe 3 (5) Prostration 2 Lactic acidosis 1 Plasmodium species P. falciparum 55 (95) P. falciparum and P. malariae 3 (5) Sickle cell screen Negative 53 (91) AS 1 (2) Downloaded from Not done 4 (7) Hemoglobin, g/dl 58 11.5 (9.98–12.5) 9.5–14.4b Erythrocyte count, 31012/l 58 4.26 (3.83–4.66) Mean corpuscular volume, fl 56 76.9 (73.5–80.7) 67.8–90.0b Leukocyte count, 3109/1 57 8.50 (6.74–10.3) 4.1–11.1b Percent neutrophils 58 74.8 (66.3–83.0) 35–75c Percent bandsd 58 1.39 (0.57–2.75) 0–10c http://www.jimmunol.org/ Percent immatured,e 58 5.32 (2.71–10.8) 0c Percent pigmentedd 58 1.22 (0.42–4.08) Percent of monocytes with pigment 58 20 (0–50) Parasite density, parasites/ml 57 92,800 (28,200–219,000) Parasite biomass, parasites/kg 55 1.23 3 1010 (5.21 3 109 to 2.14 3 1010) CRP, mg/l 46 106.4 (64.5–234.3) Lactate, mmol/l 42 2.0 (1.6–2.45) aData were not available for every variable for every subject. b90% reference interval for Gambian children aged 6–12 y (30), where available, for laboratory parameters. cGeneral pediatric reference interval (31), where available, for laboratory parameters. dPercentage of all neutrophils. by guest on September 26, 2021 eNeutrophil precursors less mature than band forms. AS, Hemoglobin AS heterozygote; IQR, interquartile range. subjects (36), but also increased production of haptoglobin as part other, we analyzed their effects on rhodamineLo neutrophil MFI of the acute-phase response in other subjects (37, 38). Hemopexin using a general linear model. After stepwise elimination of the least levels, however, were relatively normally distributed and very significant variables in the model, only parasite biomass remained similar to healthy controls (Fig. 2C), suggesting that in these significantly associated with rhodamineLo neutrophil MFI (F = subjects with predominantly uncomplicated malaria, hemolysis 16.036, p , 0.001). Overall, these results are consistent with the does not liberate sufficient cell-free heme to deplete plasma acute-phase inflammatory response being the primary determinant hemopexin (35, 36). of release of rhodamineLo neutrophils into the circulation in chil- Because rhodamineLo cells may be similar to the functionally dren with uncomplicated malaria, but parasite burden and conse- immature granulocytes released into the circulation during malaria quent hemolysis being the major determinant of the impairment of and NTS infection in mice (18), we assessed whether their frequency the oxidative burst in rhodamineLo neutrophils. Neither variable was was associated with markers of inflammation, hemolysis, and neu- associated with the proportion of pigment containing neutrophils. trophil characteristics (Table II). The proportion of rhodamineLo cells was significantly correlated with CRP concentration, which Factors associated with HO-1 induction would be consistent with their mobilization as part of an inflam- In malaria-infected mice, we found that heme-mediated HO-1 in- matory response (39), but did not correlate directly with measures of duction in neutrophil progenitors in bone marrow was necessary to hemolysis or hemozoin uptake. There was a nonsignificant positive impair the oxidative burst of developing neutrophils (18). Because correlation between the proportion of immature neutrophils and there were no clinical indications for bone marrow aspiration in the proportion of rhodamineLo cells (Table II), suggesting that any of the study subjects, our analysis of the HO-1 pathway was the rhoadmineLo population is not only due to morphologically im- restricted to parameters measurable in peripheral blood, namely mature neutrophils, but must also include morphologically mature plasma HO-1, whole-blood HMOX1 gene expression, and HO-1 but functionally abnormal neutrophils. By contrast, the magnitude protein expression in peripheral blood cells. Also, because the of the oxidative burst among rhodamineLo neutrophils (rhodamineLo induction of cellular HO-1 by haptoglobin–hemoglobin or heme– MFI) showed significant univariate correlations with erythrocyte hemopexin complexes depends on the presence of surface recep- count and haptoglobin concentration and significant negative cor- tors for their uptake (CD163 and CD91, respectively) (37, 40, 41), relations with total parasite biomass, CRP, and total plasma heme. we examined CD163 and CD91 expression on monocytes and Because these variables are likely to be highly correlated with each neutrophils. The Journal of Immunology 5

FIGURE 1. P. falciparum malaria causes prolonged impairment of the neutrophil oxidative burst. (A) Representative FACS plots showing the gating of the neutrophil population based on forward scatter and side scatter characteristics followed by selection of single cells based on pulse width and forward scatter and selection of the CD15+ population. (B and C)Rhodamine(B) and CD11b (C)fluorescence of unstimulated (filled histogram) and PMA- stimulated (unfilled histogram) neutrophils on days 0, 7, 28, and 56 after presentation with

P. falciparum malaria. Representative plots Downloaded from from a healthy control child are also shown for comparison. RhodamineHi and rhodamineLo populations of neutrophils were deﬁned for each sample by partition at the nadir of the bimodal distribution, and percentages of the total cells in each population are shown (B). (D–H) Longitudinal analyses of neutrophil http://www.jimmunol.org/ function compared using Friedman’s two- way ANOVA for all subjects with valid data at all time points. Healthy controls are also shown for comparison but not included in the statistical analysis (D, E, G). Horizontal lines represent medians. n stated for valid data at every time point. (D) Rhodamine MFI for all neutrophils (rhodamineHi and rhodamineLo

considered together as a single population), by guest on September 26, 2021 n = 32. (E) Proportion of neutrophils that are rhodamineLo, n = 28. (F) Rhodamine MFI of rhodamineLo neutrophils, n = 28. (G) Degran- ulation of all neutrophils, assessed by fold change in surface CD11b (PMA-stimulated CD11b MFI/unstimulated CD11b MFI), n = 29. (H) Rhodamine MFI of rhodamineHi neutrophils, n = 32.

As previously reported in mice (18) and humans (42), HO-1 subjects (Fig. 3F). CD91 expression did not change significantly expression (assessed by fluorescence intensity) in circulating over time on monocytes (Fig. 3G) or neutrophils (Fig. 3H), al- neutrophils was not increased in acute malaria infection compared though when all subjects were considered together, there did ap- with convalescence (data not shown). In contrast, monocyte HO-1 pear to be very low level CD91 expression on neutrophils at all expression was higher on day 0 than on days 7 or 28 (Fig. 3A). time points. Plasma HO-1 was higher in subjects on day 0 than in healthy To explore further the likely pathways of HO-1 induction during control children (Fig. 3B), and as we have previously reported malaria infection, we constructed a simple conceptual model (Fig. (42), whole-blood HMOX1 expression was significantly higher on 4), beginning with the malaria parasite as the cause of hemolysis, day 0 than after recovery on day 28 (Fig. 3C). inflammation, and tissue hypoxia/ischemia (43)—all of which In control subjects, CD163 and CD91 were expressed on the may induce HO-1 expression (44)—and analyzed univariate cor- surface of monocytes but were not detectable on neutrophils (Fig. relations between the various measures of HO-1 induction. As 3D). In children with malaria, monocyte CD163 expression was expected, parasite biomass was strongly correlated with total plasma significantly lower at day 0 than on days 7 and 28 (Fig. 3E), heme and CRP. Plasma HO-1 correlated much more strongly with whereas CD163 remained undetectable on neutrophils from most CRP and lactate than with plasma heme, supporting the idea that it 6 IMPAIRED NEUTROPHIL FUNCTION IN MALARIA

9.378, p = 0.003). In other words, monocyte HO-1 induction would closely correlate with total plasma heme, but downregulation of surface CD163 prevents the uptake of hemoglobin– haptoglobin complexes and hence limits HO-1 induction. This explains in part the discordance between different measures of the HO-1 induction pathway in blood. In summary, this analysis demonstrates that HO-1 protein expression in myeloid cells can be increased by hemoglobin and heme liberated during malarial hemolysis, although this effect is limited by reductions in surface CD163 expression. Neutrophil oxidative burst and prior HO-1 induction In mice, we had observed that suppression of the oxidative burst of circulating neutrophils by hemolysis required the release of immature neutrophils from bone marrow, requiring either a lag time or an additional stimulus (such as NTS infection) to cause these cells rapidly to enter the circulation (18). Having observed that an abnormal population of neutrophils was present for a prolonged

period of time after P. falciparum, we looked for evidence of an Downloaded from association between HO-1 induction on day 0 and neutrophil oxidative burst on day 7. We used the ratio of day 0 to day 28 whole-blood HMOX1(HMOX1day0/day28) expression as an indicator of induction in acute malaria. We found a signiﬁcant negative correlation between HMOX1day0/day28 and the rhodamine MFI of Lo

rhodamine neutrophils on day 7 (Spearman’s correlation coef- http://www.jimmunol.org/ ficient = 20.352, p = 0.028, n = 39). There was no significant correlation with the proportion of rhodamineLo cells on day 7 2 FIGURE 2. Indicators of hemolysis in subjects with P. falciparum (Spearman’s correlation coefficient = 0.101, p = 0.542, n = 39). malaria. (A) Total plasma heme on day 0 and day 28 compared using Although the kinetics of the process of HO-1 induction, sup- Wilcoxon matched pairs test for those with data at both time points (n = pression of oxidative burst capacity in developing neutrophils, and 32). (B) Distribution of plasma haptoglobin levels at day 0, n = 57. For subsequent release of the functionally immature neutrophils into comparison, levels in six healthy control children are also shown. (C) the circulation are unknown, the observed association between Distribution of plasma hemopexin levels at day 0, n = 49. For comparison, HO-1 induction during acute disease and neutrophil dysfunction levels in six healthy control children are also shown. during early convalescence is consistent with this sequence of by guest on September 26, 2021 events. may be released in response to harmful and inflammatory stimuli. Surprisingly, however, neither whole-blood HMOX1 expression Salmonella phagocytosis and killing nor monocyte-specific HO-1 correlated significantly with total As we have observed that neutrophil killing (but not phagocytosis) plasma heme, CRP, or lactate. As the haptoglobin receptor CD163 of S. typhimurium is defective in malaria-infected mice (18), we has been reported to be downregulated during inflammation (45) assessed the ex vivo killing and phagocytosis of serum-opsonized and to be shed from the cell surface during acute, uncomplicated S. typhimurium by neutrophils isolated from whole blood of malaria infections (46), we explored whether monocyte-specific subjects and controls from whom sufficient blood remained after HO-1 expression might be confounded by changes in expression the preceding assays (Fig. 5A–C). Bacterial killing, calculated as of CD163 using a general linear model controlling for the effect of the reduction in the viable bacterial count 2 h after coculture, was CD163 expression. This revealed a significant interaction between slightly higher on day 0 than at subsequent time points (Fig. 5A). total plasma heme and CD163 expression, but strong independent There was no significant correlation between bacterial killing associations between monocyte HO-1 and total plasma heme (F = on day 0 and either the proportion of rhodamineLo neutrophils, 15.1, p , 0.001) and monocyte HO-1 and CD163 expression (F = the rhodamine MFI of the rhodamineLo neutrophils, total plasma

Table II. Association of neutrophil dysfunction with hemolysis on day 0

Rhodamine MFI of RhodamineLo Percent RhodamineLo Neutrophils Neutrophils

Variable na Correlation Coefficient pna Correlation Coefficient p Parasite biomass/kg 53 0.208 0.134 53 20.539 ,0.001 Erythrocyte count 56 20.001 0.992 56 0.280 0.037 Haptoglobin 55 0.004 0.976 55 0.292 0.031 Total plasma heme 54 0.222 0.106 54 20.268 0.050 CRP 44 0.350 0.020 44 20.452 0.002 Immature neutrophil percent 56 0.232 0.085 56 0.031 0.822 Pigmented neutrophil percent 56 20.017 0.898 56 0.180 0.185 Indicators of hemolysis, inflammation, and neutrophil characteristics were assessed for correlation with the proportion of rhodamineLo neutrophils and with the rhodamine MFI of rhodamineLo neutrophils on day 0 using Spearman’s correlation. aData were not available for every variable for every subject. The Journal of Immunology 7

FIGURE 3. Factors associated with HO-1 expression in P. falciparum malaria. (A–C) HO-1 induction. (A) Representative FACS analysis of HO-1 induction in monocytes showing exclusion of RBCs, gating on single cells, and subsequent def- inition of monocytes as CD14+. Histograms show fluorescence of monocytes stained intracellularly with control Ab (filled) or anti–HO-1 Ab (unfilled) followed by a secondary detection Ab. Quantitative data for HO-1 expression in monocytes (ratio of anti–HO-1 to control Ab fluorescence) for all subjects on days 0, 7, and 28 are shown in the right-hand panel, compared using Friedman’s two-way ANOVA (n =20).(B) The distribution of plasma HO-1 on day 0, n = 57. For comparison, levels in six healthy control children are also shown. (C) Whole-blood Downloaded from HMOX1 RNA expression, measured by qRT-PCR, compared between samples on day 0 and day 28 using Wilcoxon matched pairs test for those with data at both time points (n = 42). (D–H) CD163 and CD91 expression on monocytes and neutrophils. (D) Representative flow cytometry analysis of 2 healthy control subject neutrophils (CD14 CD16b+) http://www.jimmunol.org/ and monocytes (CD14+CD16b2) showing staining with respective control (filled histogram) and anti- CD91 or anti-CD163 Abs (unfilled histograms). Quantitative data for expression (ratio to control Ab fluorescence) of CD163 in monocytes (E)and neutrophils (F) and CD91 in monocytes (G)and neutrophils (H) compared using Friedman’s two- way ANOVA for all subjects with valid data at all time points (CD163, n = 19; CD91, n = 20). by guest on September 26, 2021 Horizontal bars represent medians. n stated for those with valid data at all time points.

heme, or parasite biomass (data not shown). Bacterial phagocy- ditions associated with hemolytic anemia—sickle cell disease (48) tosis, determined by flow cytometric analysis of the proportion of and acute bartonellosis (49)—also predispose to NTS bacteremia. GFP+ neutrophils after 15-min coculture (Fig. 5B), did not vary This is likely a result of both the nature of the defect in host defense significantly over time after infection (Fig. 5C). However, induced by malaria and the prevailing epidemiology of invasive phagocytosis at day 0 was inversely correlated with parasite bio- bacterial infection: NTS is one of the most common causes of mass (n = 26, Spearman’s correlation coefficient = 20.512, p = bacteremia in Sub-Saharan Africa (50, 51). In a mouse model, we 0.008), and with total plasma heme (n = 28, Spearman’s corre- recently showed that hemolysis due to malaria or phenylhydrazine lation coefficient = 20.441, p = 0.019) (Fig. 5D). Taken together treatment impaired resistance to S. typhimurium, which could be with the neutrophil oxidative burst assay data (Fig. 1F, 1H), these recapitulated by treatment with hemin and abrogated by treatment data indicate that although there may be some degree of (heme- with the HO inhibitor tin protoporphyrin (18). We found that bac- mediated) priming of neutrophil function in children with acute teria accumulated in circulating neutrophils and that these neu- malaria (day 0), which enhances bacterial killing during acute trophils were defective in killing S. typhimurium,associatedwith illness (47), phagocytosis of S. typhimurium by circulating neu- impairment of their oxidative burst response, which is an essential trophils becomes increasingly impaired with increasing parasite mechanism for killing S. typhimurium (21). This was due to heme- burden and increasingly severe hemolysis, and the ability of mediated induction of HO-1 in granulocyte precursors in bone neutrophils to kill S. typhimurium once they are phagocytosed marrow, causing neutrophils leaving the bone marrow to have might also become impaired. a reduced capacity to mount an effective oxidative burst. It is not known whether bacteria accumulate preferentially in neutrophils Discussion in humans with malaria and NTS coinfections, but in the pre- Although the association between malaria infection and suscep- antibiotic era, neutrophils and NTS were often found colocalized tibility to NTS bacteremia has been recognized for almost a century in abscesses that formed at the site of i.m. quinine injection in (9), the mechanism has been elusive. The strongest association coinfected individuals (9). Although neutrophil function has not is with severe malarial anemia (5, 6), and at least two other con- been extensively studied in malaria, there are several case reports 8 IMPAIRED NEUTROPHIL FUNCTION IN MALARIA

FIGURE 4. A conceptual model of the pathways leading to HO-1 induction in acute P. falciparum malaria. The biomass of P. falciparum parasites within the subject was considered to be the quantifiable cause of hemolysis, inflammation, and tissue hypoxia/ischemia, all of which are stimuli for inductionof HO-1. The variable measured to quantify each step in the pathway is indicated in italics. Associations between variables were tested using Spearman’s correlation as indicated by lines with arrows showing the hypothesized relationship from cause to effect. The strength of correlation is indicated by Spearman’s rho adjacent to significant correlation lines. Significant correlations are denoted by solid lines, and line thickness indicates the significance of the correlation (thin line 0.01 # p , 0.05, medium line 0.001 # p , 0.01, heavy line p , 0.001), whereas nonsignificant correlations are denoted by dashed lines. Downloaded from of patients with severe malarial hemolysis spontaneously devel- with plasma lactate and CRP concentrations than with plasma oping fungal sepsis (52–54), which is typically associated with heme concentrations, suggesting that plasma HO-1 may be pre- neutropenia and neutrophil dysfunction. dominantly a response to inflammation and hypoxia and a marker The current study was designed to determine whether Gambian of cell damage. As noted previously, intracellular HO-1 protein children with P. falciparum malaria have evidence of neutrophil expression was not significantly upregulated in acute malaria in http://www.jimmunol.org/ dysfunction similar to that observed in mice infected with Plas- circulating neutrophils (42), presumably because they lack CD163 modium yoelii 17XNL. We hypothesized that the neutrophil oxi- expression, whereas monocytes did show evidence of increased dative burst would be impaired in children with malaria, and the HO-1 protein expression in acute malaria. However, there was not severity of this impairment would be related to hemolysis and a significant univariate association between total plasma heme HO-1 induction. However, we also predicted that the impairment concentration and HO-1 expression in monocytes, which could be of neutrophil function would be relatively mild because declining explained statistically by the reduced levels of monocyte surface malaria transmission in The Gambia has led to a decrease in the CD163 expression in acute malaria. This explanation is fully incidence of severe malarial anemia and malaria–NTS coinfection consistent with the subjects in this study having only mild he-

(7). Overall, our results are consistent with our hypothesis: the molysis [most had hemoglobin values within the normal range for by guest on September 26, 2021 oxidative burst activity of circulating neutrophils was profoundly their age (30), none had severe malarial anemia, and only half had abnormal in subjects with acute P. falciparum malaria and most low haptoglobin levels] and with the assumption (as hemopexin severely impaired in children with the highest parasite burdens levels were not depleted) that very little of the total circulating and greatest hemolysis, albeit the magnitude of this impairment heme represents cell-free heme. In this case, HO-1 induction due did not translate into a clinically significant defect in neutrophil to hemolysis would be expected to proceed predominantly through killing of S. typhimurium in vitro. We also found that these ab- CD163-mediated uptake of haptoglobin–hemoglobin complexes, normalities persisted for at least 56 d and that bacterial phago- and reduction in surface CD163 would be expected to limit HO-1 cytosis and killing appeared to deteriorate during the early induction (45). In contrast, severe hemolysis would be expected to convalescent period (up to 28 d), findings that may be consistent generate cell-free heme and lead to HO-1 induction and heme with descriptions of increased susceptibility to NTS bacteremia in catabolism in cells expressing the surface receptor (CD91) for children who have recently had malaria (6, 10) and the gradual heme–hemopexin complexes, which appears to be invariantly emergence of dysfunctional neutrophils from bone marrow after expressed during infection. Indeed, we previously found elevated HO-1 induction during the acute infection (18). To facilitate carboxyhemoglobin levels, an indirect measure of HO activity, comparison with our studies in mice (18), we assessed neutrophil only in children with severe malarial anemia suggesting that heme function using PMA as the stimulus for the oxidative burst. catabolism is constrained in acute malaria and only detectably Although this is not a physiological stimulus, the advantages of increased in cases with the most severe hemolysis (59). It is this method are i) that it produces a strong oxidative burst (55, conceivable that by reducing CD163 expression in the context 56), which is clearly distinguished from any low-level activation of infection, monocytes are rendered relatively resistant to HO-1 caused by malaria infection per se; ii) it is not dependent on induction by hemolysis, perhaps preventing HO-1–mediated im- phagocytosis (which might also be impaired by malaria) (55, 56); pairment of their normal inflammatory responses (45, 60). How- and iii) variations in the magnitude of the PMA-induced oxidative ever, it is currently unknown whether either CD163 or CD91 burst are directly related to the ability of humans to survive in- expression is required for HO-1 induction in immature myeloid fections (57). cells and their progenitors in human bone marrow. If HO-1 in- Consistent with data from mice and humans (18, 42, 58), we duction in bone marrow is responsible for the observed neutrophil observed induction of HO-1 during acute malaria. Although dis- dysfunction [as it is in mice (18)], either it may be independent of secting the causal and consequential pathways of HO-1 induction CD163 or CD163 may not be downregulated in the bone marrow is difficult in an observational study, we constructed a conceptual to the same extent as in blood monocytes. We did not examine the model of likely pathways leading to HO-1 induction based on effect of HMOX1 promoter (GT)n length polymorphisms in this existing literature (Fig. 4) (44) and used this model to guide our study because the majority of subjects had uncomplicated malaria statistical analysis. Plasma HO-1 levels correlated more strongly with mild hemolysis, and we expected that under these circum- The Journal of Immunology 9

stances, genetic polymorphisms would have a relatively small effect and a much larger sample size would have been required (42). In apparent contradiction of our ﬁndings in mice, killing of S. typhimurium by neutrophils was not noticeably impaired on day 0, but deteriorated over the next 4 wk. This ﬁnding is perhaps not surprising because none of the children had severe hemolysis or severe anemia, which are the major risk factors for malaria–NTS coinfection (5, 6), suggesting that their bactericidal capacity should not be seriously impaired. A possible explanation is that despite the accumulation of a rhodamineLo neutrophil population, the oxidative burst of the rhodamineHi neutrophil population was higher on days 0 and 7 than at later time points, and enhanced bactericidal activity among rhodamineHi cells may initially com- pensate for the lack of killing among the rhodamineLo population, particularly if the rhodamineHi cells showed preferential phagocytosis of the opsonized S. typhimurium. Unfortunately, our assays could not simultaneously assess phagocytosis and oxidative burst

in the same cells. We predict, however, that bacterial killing would Downloaded from be seriously impaired in children with severe hemolytic anemia. The acquired defect of neutrophil function that we observed in children with malaria in this study might be considered analogous to the neutrophil defect observed in female carriers of X-linked chronic granulomatous disease, where around 50% of neutrophils

have defective oxidative burst activity due to random inactiva- http://www.jimmunol.org/ tion of the X-chromosome but there is not increased susceptibility to infection (61), and in vitro bactericidal activity may be normal (62). However, in some carriers, inactivation of the X- chromosome becomes skewed, and when ,15% of neutrophils are able to make a normal oxidative burst, susceptibility to infections is markedly increased (20). Consistent with this, in a large Eu- ropean registry of chronic granulomatous disease patients, Sal- monella has been reported as by far the most common cause of septicemia (whereas fungi and Staphylococcus aureus are the by guest on September 26, 2021 most common causes of chronic lung and deep tissue infections, due to the persistent defect in oxidative burst activity) (20). We propose that in patients with malaria, a threshold proportion of abnormal neutrophils in blood may be required to produce susceptibility to NTS, and when this threshold is exceeded, the degree of susceptibility may then also be determined by the magnitude of the impairment of oxidative burst capacity. Factors that may determine whether the proportion of dysfunctional neutrophils exceeds this putative threshold may include the duration of infection and the severity of the inflammatory response, which may both influence the mobilization of dysfunctional neutrophils from FIGURE 5. Ex vivo killing and phagocytosis of S. typhimurium by bone marrow (18); the severity of hemolysis, as cell-free heme neutrophils from children with P. falciparum malaria. (A) Killing of itself promotes neutrophil mobilization (18); and possibly host S. typhimurium. Neutrophils isolated on days 0, 7, 28, and 56 after presen- factors such as age and genetic background. The strong correlation tation with P. falciparum malaria were mixed with S. typhimurium at a ratio we observed between impaired neutrophil oxidative burst and total of 50:1 and killing was expressed as the percentage reduction in bacterial parasite biomass allows us to predict that children with high numbers after 2-h incubation. Statistical comparison using Friedman’s two- parasite burden (who are also most likely to have severe hemo- way ANOVA for all subjects with valid data at all time points, n =18. lysis) would have the most impaired oxidative burst. These chil- B Data from control subjects shown for comparison. ( ) Representative flow dren may also have depleted hemopexin levels and accumulate cytometry plots showing phagocytosis of GFP+ S. typhimurium by neu- cell-free heme, which can itself mobilize neutrophils from bone trophils isolated on day 0. RBCs and debris were excluded based on forward scatter and side scatter characteristics, then single cells were selected based marrow (18), perhaps increasing the proportion of abnormal on pulse width and forward scatter characteristics (upper row). The proportion of GFP+CD15+ cells was determined in samples where both neutrophils and S. typhimurium were fixed in 4% formaldehyde prior to incubation (to control for surface binding without phagocytosis) and in proportion of GFP+ cells in formaldehyde fixed samples from that in unfixed samples (lower row). (C) Phagocytosis of S. typhimurium. Neu- unfixed samples. Statistical comparison using Friedman’s two-way trophils isolated on days 0, 7, 28, and 56 after presentation with P. falcip- ANOVA for all subjects with valid data at all time points, n = 7. Data from arum malaria were mixed with S. typhimurium at a ratio of 50:1 and control subjects shown for comparison. (D) Correlation of phagocytosis phagocytosis expressed as the percentage of GFP+ neutrophils after 15-min (on day 0) with parasite biomass on day 0 (left-hand panel, n = 26) and total incubation. The percentage of GFP+ cells was calculated by subtracting the plasma heme on day 0 (right-hand panel, n = 28). 10 IMPAIRED NEUTROPHIL FUNCTION IN MALARIA neutrophils above a threshold required to induce susceptibility to Disclosures NTS. The authors have no financial conflicts of interest. The prolonged duration of abnormal neutrophil oxidative burst activity, extending up to 8 wk after acute infection in some subjects, was unexpected. This is unlikely to be due to antimalarial treat- References 1. World Health Organization. 2011. World Malaria Report 2011. World Health ment, because artemisinin-based treatments cause mild enhance- Organization, Geneva. ment of the oxidative burst and suppression of phagocytosis, the 2. Snow, R. W., E. L. Korenromp, and E. Gouws. 2004. Pediatric mortality in opposite of what we observed (63). Two possible explanations are Africa: plasmodium falciparum malaria as a cause or risk? Am. J. Trop. Med. Hyg. 71(2, Suppl):16–24. the effect of persisting hemozoin and fundamental alteration in 3. Berkley, J. A., P. Bejon, T. Mwangi, S. Gwer, K. Maitland, T. N. Williams, myelopoiesis. Hemozoin is an insoluble hemin polymer, the end S. Mohammed, F. Osier, S. Kinyanjui, G. Fegan, et al. 2009. HIV infection, product of hemoglobin digestion inside the parasitized red cell, malnutrition, and invasive bacterial infection among children with severe malaria. Clin. Infect. Dis. 49: 336–343. and is able to induce HO-1 (and impair the oxidative burst in 4. Scott, J. A., J. A. Berkley, I. Mwangi, L. Ochola, S. Uyoga, A. Macharia, phagocytes) but is not catabolized (13, 64). Prolonged persistence C. Ndila, B. S. Lowe, S. Mwarumba, E. Bauni, et al. 2011. Relation between falciparum malaria and bacteraemia in Kenyan children: a population-based, of hemozoin in phagocytic cells in the bone marrow (65) could case-control study and a longitudinal study. Lancet 378: 1316–1323. cause prolonged HO-1 induction and abnormal development of 5. Bronzan, R. N., T. E. Taylor, J. Mwenechanya, M. Tembo, K. Kayira, neutrophils (18). Associated with this or through separate mech- L. Bwanaisa, A. Njobvu, W. Kondowe, C. Chalira, A. L. Walsh, et al. 2007. Bacteremia in Malawian children with severe malaria: prevalence, etiology, HIV anisms, a novel myeloid progenitor phenotype may be generated coinfection, and outcome. J. Infect. Dis. 195: 895–904. in P. falciparum malaria, as has been observed in rodent malaria 6. Mabey, D. C., A. Brown, and B. M. Greenwood. 1987. Plasmodium falciparum infection (66), and may lead to sustained abnormal granulopoiesis. malaria and Salmonella infections in Gambian children. J. Infect. Dis. 155: Downloaded from 1319–1321. The major limitations of our study are that most subjects had 7. Mackenzie, G., S. J. Ceesay, P. C. Hill, M. Walther, K. A. Bojang, J. Satoguina, relatively mild hemolysis and that we did not have bone marrow G. Enwere, U. D’Alessandro, D. Saha, U. N. Ikumapayi, et al. 2010. A decline in samples to confirm HO-1 induction in neutrophil progenitors. To the incidence of invasive non-typhoidal Salmonella infection in The Gambia temporally associated with a decline in malaria infection. PLoS ONE 5: e10568. recruit a significant number of subjects with severe malarial he- 8. Hayasaka, C. 1933. Im Verlauf einer Malariakur durch Bacillus enteritidis molysis would require a much larger study, probably conducted in Ga¨rtner entstandene Meningitis und Sepsis. Tohoku J. Exp. Med. 21: 466–504. 9. Giglioli, G. 1929. Paratyphoid C an endemic disease of British Guiana: a clinical a higher-transmission setting. To study prospectively whether the and pathological outline. B. paratyphosum C as a pyogenic organism: case http://www.jimmunol.org/ severity of neutrophil dysfunction at recruitment correlated with reports. Proc. R. Soc. Med. 23: 165–177. susceptibility to NTS bacteremia during convalescence would 10. Brent, A. J., J. O. Oundo, I. Mwangi, L. Ochola, B. Lowe, and J. A. Berkley. 2006. Salmonella bacteremia in Kenyan children. Pediatr. Infect. Dis. J. 25: 230– require an even larger and more logistically complex study. To 236. obtain bone marrow aspirates from children with malaria would be 11. Nadjm, B., B. Amos, G. Mtove, J. Ostermann, S. Chonya, H. Wangai, J. Kimera, difficult to justify ethically unless appropriate sedation and anal- W. Msuya, F. Mtei, D. Dekker, et al. 2010. WHO guidelines for antimicrobial treatment in children admitted to hospital in an area of intense Plasmodium gesia could be provided without additional risk of complications. falciparum transmission: prospective study. BMJ 340: c1350. Nevertheless, our findings have a number of important impli- 12. Urban, B. C., and D. J. Roberts. 2002. Malaria, monocytes, macrophages and myeloid dendritic cells: sticking of infected erythrocytes switches off host cells. cations. First, we show that the oxidative burst capacity of a large Curr. Opin. Immunol. 14: 458–465. proportion of neutrophils is markedly abnormal in children with 13. Schwarzer, E., F. Turrini, D. Ulliers, G. Giribaldi, H. Ginsburg, and P. Arese. by guest on September 26, 2021 P. falciparum malaria, supporting the translation of findings in a 1992. Impairment of macrophage functions after ingestion of Plasmodium falciparum-infected erythrocytes or isolated malarial pigment. J. Exp. Med. 176: mouse model (18). Second, neutrophil function recovers only very 1033–1041. slowly over the 2 mo after treatment, providing an explanation for 14. Roux, C. M., B. P. Butler, J. Y. Chau, T. A. Paixao, K. W. Cheung, R. L. Santos, the association of susceptibility to NTS with recent malaria (10, S. Luckhart, and R. M. Tsolis. 2010. Both hemolytic anemia and malaria parasite-specific factors increase susceptibility to Nontyphoidal Salmonella 11). In the mouse model, hemolysis-induced neutrophil dysfunc- enterica serovar typhimurium infection in mice. Infect. Immun. 78: 1520–1527. tion could be abrogated by competitive inhibition of HO with tin 15. Kaye, D., and E. W. Hook. 1963. The influence of hemolysis or blood loss on protoporphyrin (18), but using this treatment in acute malaria susceptibility to infection. J. Immunol. 91: 65–75. 16. Kaye, D., and E. W. Hook. 1963. The influence of hemolysis on susceptibility to would be challenging because HO-1 is also important for toler- Salmonella infection: additional observations. J. Immunol. 91: 518–527. ance to cytotoxic effects of cell-free heme in mouse models (58, 17. Kaye, D., J. G. Merselis, Jr., and E. W. Hook. 1965. Influence of Plasmodium berghei infection on susceptibility to Salmonella infection. Proc. Soc. Exp. Biol. 67). Alternative therapeutic strategies would be administration Med. 120: 810–813. of tin protoporphyrin upon completion of antimalarial treatment, 18. Cunnington, A. J., J. B. de Souza, M. Walther, and E. M. Riley. 2012. Malaria with the aim of restoring neutrophil function during convalescence impairs resistance to Salmonella through heme- and heme oxygenase-dependent dysfunctional granulocyte mobilization. Nat. Med. 18: 120–127. and preventing the susceptibility to NTS caused by recent malaria, 19. Kappas, A., G. S. Drummond, T. Manola, S. Petmezaki, and T. Valaes. 1988. Sn- or prioritization of children at greatest risk of persistent neutrophil protoporphyrin use in the management of hyperbilirubinemia in term newborns dysfunction for prophylactic antibiotic treatment. Third, we pro- with direct Coombs-positive ABO incompatibility. Pediatrics 81: 485–497. 20. van den Berg, J. M., E. van Koppen, A. Ahlin, B. H. Belohradsky, pose the downregulation of the haptoglobin receptor CD163 on E. Bernatowska, L. Corbeel, T. Espanõl, A. Fischer, M. Kurenko-Deptuch, the surface of blood monocytes during acute P. falciparum malaria R. Mouy, et al. 2009. Chronic granulomatous disease: the European experience. PLoS ONE 4: e5234. as a novel host-protective homeostatic response to hemolysis and 21. Mastroeni, P., A. Vazquez-Torres, F. C. Fang, Y. Xu, S. Khan, C. E. Hormaeche, inflammation, which may prevent HO-1 induction from impairing and G. Dougan. 2000. Antimicrobial actions of the NADPH phagocyte oxidase monocyte function. Further experimental studies are needed to and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo. J. Exp. Med. 192: 237–248. confirm the effects of manipulating CD163 expression during 22. Gondwe, E. N., M. E. Molyneux, M. Goodall, S. M. Graham, P. Mastroeni, infections, but manipulation of this axis would hold promise for M. T. Drayson, and C. A. MacLennan. 2010. Importance of antibody and both the modulation of inflammation and optimization of iron complement for oxidative burst and killing of invasive nontyphoidal Salmonella by blood cells in Africans. Proc. Natl. Acad. Sci. USA 107: 3070–3075. reutilization during chronic infections. 23. Walther, M., D. Jeffries, O. C. Finney, M. Njie, A. Ebonyi, S. Deininger, E. Lawrence, A. Ngwa-Amambua, S. Jayasooriya, I. H. Cheeseman, et al. 2009. Distinct roles for FOXP3 and FOXP3 CD4 T cells in regulating cellular im- Acknowledgments munity to uncomplicated and severe Plasmodium falciparum malaria. PLoS We are grateful to David Conway who helped to set up and manage Pathog. 5: e1000364. the recruitment process used in the study, to David Jeffries for statistical 24. WHO. 2000. Severe falciparum malaria. World Health Organization, Communi- cable Diseases Cluster. Trans. R. Soc. Trop. Med. Hyg. 94(Suppl 1): S1/1–S1/18. advice, and to the clinic, laboratory, and administrative staff, field workers, 25. Ceesay, S. J., C. Casals-Pascual, D. C. Nwakanma, M. Walther, N. Gomez- and subjects who participated in the study. Escobar, A. J. Fulford, E. N. Takem, S. Nogaro, K. A. Bojang, T. Corrah, et al. The Journal of Immunology 11

2010. Continued decline of malaria in The Gambia with implications for elim- and severity of malaria in children in Ghana. Clin. Vaccine Immunol. 15: 1456– ination. PLoS ONE 5: e12242. 1460. 26. Richardson, M. P., M. J. Ayliffe, M. Helbert, and E. G. Davies. 1998. A simple 47. Kowanko, I. C., A. Ferrante, G. Clemente, and L. M. Kumaratilake. 1996. Tumor flow cytometry assay using dihydrorhodamine for the measurement of the necrosis factor primes neutrophils to kill Staphylococcus aureus by an oxygen- neutrophil respiratory burst in whole blood: comparison with the quantitative dependent mechanism and Plasmodium falciparum by an oxygen-independent nitrobluetetrazolium test. J. Immunol. Methods 219: 187–193. mechanism. Infect. Immun. 64: 3435–3437. 27. Hirai, H., H. Kubo, M. Yamaya, K. Nakayama, M. Numasaki, S. Kobayashi, 48. Wright, J., P. Thomas, and G. R. Serjeant. 1997. Septicemia caused by Salmo- S. Suzuki, S. Shibahara, and H. Sasaki. 2003. Microsatellite polymorphism in nella infection: an overlooked complication of sickle cell disease. J. Pediatr. 130: heme oxygenase-1 gene promoter is associated with susceptibility to oxidant- 394–399. induced apoptosis in lymphoblastoid cell lines. Blood 102: 1619–1621. 49. Bennett, I. L., and E. W. Hook. 1959. Infectious diseases (some aspects of 28. Dondorp, A. M., V. Desakorn, W. Pongtavornpinyo, D. Sahassananda, salmonellosis). Annu. Rev. Med. 10: 1–20. K. Silamut, K. Chotivanich, P. N. Newton, P. Pitisuttithum, A. M. Smithyman, 50. Berkley, J. A., B. S. Lowe, I. Mwangi, T. Williams, E. Bauni, S. Mwarumba, N. J. White, and N. P. Day. 2005. Estimation of the total parasite biomass in C. Ngetsa, M. P. Slack, S. Njenga, C. A. Hart, et al. 2005. Bacteremia among acute falciparum malaria from plasma PfHRP2. PLoS Med. 2: e204. children admitted to a rural hospital in Kenya. N. Engl. J. Med. 352: 39–47. 29. Raes, A., S. Van Aken, M. Craen, R. Donckerwolcke, and J. Vande Walle. 2006. 51. Morpeth, S. C., H. O. Ramadhani, and J. A. Crump. 2009. Invasive non-Typhi A reference frame for blood volume in children and adolescents. BMC Pediatr. Salmonella disease in Africa. Clin. Infect. Dis. 49: 606–611. 6: 3. 52. Soesan, M., P. A. Kager, M. A. Leverstein-van Hall, and J. J. van Lieshout. 1993. 30. Adetifa, I. M., P. C. Hill, D. J. Jeffries, D. Jackson-Sillah, H. B. Ibanga, G. Bah, Coincidental severe Plasmodium falciparum infection and disseminated candi- S. Donkor, T. Corrah, and R. A. Adegbola. 2009. Haematological values from diasis. Trans. R. Soc. Trop. Med. Hyg. 87: 288–289. a Gambian cohort—possible reference range for a West African population. Int. 53. Eckerle, I., D. Ebinger, D. Gotthardt, R. Eberhardt, P. A. Schnabel, W. Stremmel, J. Lab. Hematol. 31: 615–622. T. Junghanss, and C. Eisenbach. 2009. Invasive Aspergillus fumigatus infection 31. Soldin, S. J., C. Brugnara, and E. C. Wong. 2007. Pediatric Reference Intervals. after Plasmodium falciparum malaria in an immuno-competent host: case report AACC Press, Washington, DC. and review of literature. Malar. J. 8: 167. 32. de Haas, M., J. M. Kerst, C. E. van der Schoot, J. Calafat, C. E. Hack, 54. Hocqueloux, L., F. Bruneel, C. L. Pages, and F. Vachon. 2000. Fatal invasive J. H. Nuijens, D. Roos, R. H. van Oers, and A. E. von dem Borne. 1994. aspergillosis complicating severe Plasmodium falciparum malaria. Clin. Infect. Downloaded from Granulocyte colony-stimulating factor administration to healthy volunteers: Dis. 30: 940–942. analysis of the immediate activating effects on circulating neutrophils. Blood 84: 55. Avendanõ, A., I. Sales-Pardo, L. Marin, P. Marin, and J. Petriz. 2008. Oxidative 3885–3894. burst assessment and neutrophil-platelet complexes in unlysed whole blood. J. 33. Sheppard, F. R., M. R. Kelher, E. E. Moore, N. J. McLaughlin, A. Banerjee, and Immunol. Methods 339: 124–131. 56. Lun, A., M. Schmitt, and H. Renz. 2000. Phagocytosis and oxidative burst: C. C. Silliman. 2005. Structural organization of the neutrophil NADPH oxidase: reference values for flow cytometric assays independent of age. Clin. Chem. 46: phosphorylation and translocation during priming and activation. J. Leukoc. Biol. 1836–1839. 78: 1025–1042. 57. Kuhns, D. B., W. G. Alvord, T. Heller, J. J. Feld, K. M. Pike, B. E. Marciano, 34. Fendel, R., C. Brandts, A. Rudat, A. Kreidenweiss, C. Steur, I. Appelmann,

G. Uzel, S. S. DeRavin, D. A. Priel, B. P. Soule, et al. 2010. Residual NADPH http://www.jimmunol.org/ B. Ruehe, P. Schro¨der, W. E. Berdel, P. G. Kremsner, and B. Mordmu¨ller. 2010. oxidase and survival in chronic granulomatous disease. N. Engl. J. Med. 363: Hemolysis is associated with low reticulocyte production index and predicts 2600–2610. blood transfusion in severe malarial anemia. PLoS ONE 5: e10038. 58. Seixas, E., R. Gozzelino, A. Chora, A. Ferreira, G. Silva, R. Larsen, S. Rebelo, 35. Ferreira, A., J. Balla, V. Jeney, G. Balla, and M. P. Soares. 2008. A central role C. Penido, N. R. Smith, A. Coutinho, and M. P. Soares. 2009. Heme oxygenase-1 J. Mol. for free heme in the pathogenesis of severe malaria: the missing link? affords protection against noncerebral forms of severe malaria. Proc. Natl. Acad. Med. 86: 1097–1111. Sci. USA 106: 15837–15842. 36. Muller-Eberhard, U., J. Javid, H. H. Liem, A. Hanstein, and M. Hanna. 1968. 59. Cunnington, A. J., S. F. Kendrick, B. Wamola, B. Lowe, and C. R. Newton. 2004. Plasma concentrations of hemopexin, haptoglobin and heme in patients with Carboxyhemoglobin levels in Kenyan children with Plasmodium falciparum various hemolytic diseases. Blood 32: 811–815. malaria. Am. J. Trop. Med. Hyg. 71: 43–47. 37. Nielsen, M. J., and S. K. Moestrup. 2009. Receptor targeting of hemoglobin 60. Rushworth, S. A., D. J. MacEwan, and M. A. O’Connell. 2008. mediated by the haptoglobins: roles beyond heme scavenging. Blood 114: 764– Lipopolysaccharide-induced expression of NAD(P)H:quinone oxidoreductase 1 771. and heme oxygenase-1 protects against excessive inflammatory responses in by guest on September 26, 2021 38. O’Donnell, A., F. J. Fowkes, S. J. Allen, H. Imrie, M. P. Alpers, D. J. Weatherall, human monocytes. J. Immunol. 181: 6730–6737. and K. P. Day. 2009. The acute phase response in children with mild and severe 61. Wolach, B., Y. Scharf, R. Gavrieli, M. de Boer, and D. Roos. 2005. Unusual late malaria in Papua New Guinea. Trans. R. Soc. Trop. Med. Hyg. 103: 679–686. presentation of X-linked chronic granulomatous disease in an adult female with 39. Summers, C., S. M. Rankin, A. M. Condliffe, N. Singh, A. M. Peters, and a somatic mosaic for a novel mutation in CYBB. Blood 105: 61–66. E. R. Chilvers. 2010. Neutrophil kinetics in health and disease. Trends Immunol. 62. Repine, J. E., C. C. Clawson, J. G. White, and B. Holmes. 1975. Spectrum of 31: 318–324. function of neutrophils from carriers of sex-linked chronic granulomatous dis- 40. Delanghe, J. R., and M. R. Langlois. 2001. Hemopexin: a review of biological ease. J. Pediatr. 87: 901–907. aspects and the role in laboratory medicine. Clin. Chim. Acta 312: 13–23. 63. Wenisch, C., B. Parschalk, K. Zedwitz-Liebenstein, W. Wernsdorfer, and 41. Hvidberg, V., M. B. Maniecki, C. Jacobsen, P. Højrup, H. J. Møller, and W. Graninger. 1997. The effect of artemisinin on granulocyte function assessed S. K. Moestrup. 2005. Identification of the receptor scavenging hemopexin-heme by flow cytometry. J. Antimicrob. Chemother. 39: 99–101. complexes. Blood 106: 2572–2579. 64. Schwarzer, E., F. De Matteis, G. Giribaldi, D. Ulliers, E. Valente, and P. Arese. 42. Walther, M., A. De Caul, P. Aka, M. Njie, A. Amambua-Ngwa, B. Walther, 1999. Hemozoin stability and dormant induction of heme oxygenase in I. M. Predazzi, A. Cunnington, S. Deininger, E. N. Takem, et al. 2012. HMOX1 hemozoin-fed human monocytes. Mol. Biochem. Parasitol. 100: 61–72. gene promoter alleles and high HO-1 levels are associated with severe malaria in 65. Ha¨nscheid, T., T. J. Egan, and M. P. Grobusch. 2007. Haemozoin: from mela- Gambian children. PLoS Pathog. 8: e1002579. tonin pigment to drug target, diagnostic tool, and immune modulator. Lancet 43. Mackintosh, C. L., J. G. Beeson, and K. Marsh. 2004. Clinical features and Infect. Dis. 7: 675–685. pathogenesis of severe malaria. Trends Parasitol. 20: 597–603. 66. Belyaev, N. N., D. E. Brown, A. I. Diaz, A. Rae, W. Jarra, J. Thompson, 44. Ryter, S. W., J. Alam, and A. M. Choi. 2006. Heme oxygenase-1/carbon mon- J. Langhorne, and A. J. Potocnik. 2010. Induction of an IL7-R(+)c-Kit(hi) oxide: from basic science to therapeutic applications. Physiol. Rev. 86: 583–650. myelolymphoid progenitor critically dependent on IFN-gamma signaling dur- 45. Van Gorp, H., P. L. Delputte, and H. J. Nauwynck. 2010. Scavenger receptor ing acute malaria. Nat. Immunol. 11: 477–485. CD163, a Jack-of-all-trades and potential target for cell-directed therapy. Mol. 67. Pamplona, A., A. Ferreira, J. Balla, V. Jeney, G. Balla, S. Epiphanio, A. Chora, Immunol. 47: 1650–1660. C. D. Rodrigues, I. P. Gregoire, M. Cunha-Rodrigues, et al. 2007. Heme 46. Kusi, K. A., B. A. Gyan, B. Q. Goka, D. Dodoo, G. Obeng-Adjei, M. Troye- oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental Blomberg, B. D. Akanmori, and J. P. Adjimani. 2008. Levels of soluble CD163 cerebral malaria. Nat. Med. 13: 703–710.