Europe PMC

This website requires cookies, and the limited processing of your personal data in order to function. By using the site you are agreeing to this as outlined in our privacy notice and cookie policy.

Neutrophil paralysis in Plasmodium vivax malaria.

Author information

Affiliations

  • 1. Laboratório de Imunopatologia, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil.
      Authors
    • Leoratti FM 1
    (1 author)

ORCIDs linked to this article

Show all (6)

Plos Neglected Tropical Diseases, 26 Jun 2012, 6(6):e1710
https://doi.org/10.1371/journal.pntd.0001710 PMID: 22745844 PMCID: PMC3383745

This article is in the Europe PMC Open access subset. Refer to the copyright information in the article for licensing details.
Free full text in Europe PMC

Abstract 

Background

The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria.

Materials and methods

Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30-45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients.

Principal findings

Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8).

Conclusion

Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.

Free full text 

Logo of plosntdLink to Publisher's site
PLoS Negl Trop Dis. 2012 Jun; 6(6): e1710.
Published online 2012 Jun 26. https://doi.org/10.1371/journal.pntd.0001710
PMCID: PMC3383745
PMID: 22745844

Neutrophil Paralysis in Plasmodium vivax Malaria

Fabiana Maria de Souza Leoratti, 1 Silvia Cellone Trevelin, 2 Fernando Queiroz Cunha, 2 Bruno Coelho Rocha, 1 , 3 Pedro Augusto Carvalho Costa, 1 Humberto Doriguêtto Gravina, 1 , 3 Mauro Shugiro Tada, 4 Dhelio Batista Pereira, 4 Douglas Taylor Golenbock, 5 Lis Ribeiro do Valle Antonelli,# 1 and Ricardo T. Gazzinelli# 1 , 3 , 5 , *
Mauricio Martins Rodrigues, Editor
Author information Article notes Copyright and License information Disclaimer

Abstract

Background

The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria.

Materials and Methods

Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30–45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients.

Principal Findings

Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8).

Conclusion

Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.

Author Summary

Plasmodium vivax is responsible for approximately 60–80% of the malaria cases in the world, and contributes to significant social and economic instability in the developing countries of Latin America and Asia. The pathogenesis of P. vivax malaria is a consequence of host derived inflammatory mediators. Hence, a better understanding of the mechanisms involved in induction of systemic inflammation during P. vivax malaria is critical for the clinical management and prevention of severe disease. The innate immune receptors recognize Plasmodium sp. and initiate a broad spectrum of host defense mechanisms that mediate resistance to infection. However, the innate immune response is the classic “two-edged sword”, and clinical malaria is associated with high levels of circulating pro-inflammatory cytokines. Our findings show that both monocytes and neutrophils are highly activated during malaria. Monocytes produced high levels of IL-1β, IL-6 and TNF-α during acute malaria. On the other hand, neutrophils were a poor source of cytokines, but displayed an enhanced phagocytic activity and superoxide production. Unexpectedly, we noticed an impaired chemotaxis of neutrophils towards an IL-8 (CXCL8) gradient. We proposed that neutrophil paralysis is in part responsible for the enhanced susceptibility to bacterial infection observed in malaria patients.

Introduction

Malaria is a complex disease that affects approximately 300 million people every year. Among the different Plasmodium species that infect humans, P. falciparum is the main cause of deaths in sub- Saharan Africa. On the other hand, P. vivax is responsible for approximately 60–80% of the malaria cases in the world [1], [2], and contributes to significant political, social and economic instability in the developing countries of Latin America and Asia [1].

The innate immune system recognizes Plasmodium sp. by different pattern-recognition receptors and initiates a broad spectrum of defense mechanisms that mediate host resistance to infection [3], [4]. However, the innate immune response is the classic “two-edged sword”, and clinical malaria is associated with high levels of circulating pro-inflammatory cytokines. The outcome of infection depends on a balance between pro- and anti-inflammatory responses allowing the formation of an effective immune response, while limiting its pathogenic potential [5], [6], [7].

Toll like receptors (TLR) play an important role in recognition of pathogens through distinct pathogen-associated molecular patterns (PAMPs). Activation of TLR on monocytes, dendritic cells and neutrophils can induce changes in the expression of surface proteins and release inflammatory mediators such as cytokines and chemokines. The production of cytokines amplifies innate immune responses and shapes the development of acquired immunity. In addition, activated myeloid cells release high levels of reactive oxygen species (ROS) and antimicrobial peptides that efficiently kill invading pathogens [8], [9].

It is noteworthy that glycosylphosphatidylinositol anchors and DNA from Plasmodium parasites are important PAMPs that activate TLR during malaria [10], [11], [12], [13]. Some in vitro studies show that phagocytosis of opsonized hemozoin (HZ) decreases expression of HLA-DR in monocytes [14], [15]. On the other hand, study has demonstrated that DNA bound to HZ induces monocytes to produce high levels of cytokines and contribute to dendritic cell maturation [16]. While other report evaluated activation of polymorphonuclear cells and observed elevated levels of myeloperoxidase, lysozyme and lipocalin in patients with severe malaria [17], the involvement of neutrophils in malaria pathogenesis has been poorly investigated.

Our findings show that both monocytes and neutrophils are highly activated during malaria. Monocytes produced high levels of IL-1β, IL-6 and TNF-α in response to TLR agonists during acute malaria and seem to be the main source of pro-inflammatory cytokines in the blood. On the other hand, neutrophils were a poor source of cytokines, but displayed an enhanced phagocytic activity and superoxide production. Interestingly, we noticed an enhanced expression of G-protein receptor protein kinase (GRK2) associated with decreased levels of CXCR2 and impaired chemotaxis of neutrophils towards an IL-8 (CXCL8) gradient. Our findings indicate a mechanism by which malaria patients may become more susceptible to bacterial infection.

Methods

Ethics statement

All protocols and consent forms were approved by the Institutional Research Board from University of Massachusetts Medical School (IRB-UMMS 10268), the Ethical Committees on Human Experimentation from Centro de Pesquisa em Medicina Tropical (CEP-CEPEM 095/2009) and Centro de Pesquisas René Rachou – Fundação Oswaldo Cruz (CEP-CPqRR 2004), as well as by the National Ethical Committee (CONEP 15652) from Ministry of Health, Brazil. A signed informed consent was obtained from each subject prior to enrollment in the study.

Patients

Patients were recruited and examined at CEPEM in Porto Velho, a malaria endemic area in the Amazon region of Brazil. Up to 100 ml of peripheral blood was collected immediately after confirmation of P. vivax infection by thick blood smear film and 30–45 days after chemotherapy (n = 26, ranging from 18 to 66 years old [35±9.5]). Patients were treated for 10 days with chloroquine and primaquine. P. vivax infection was confirmed by polymerase chain reaction (PCR) analysis [18]. The clinical manifestations of malaria were fever, myalgia, chills, arthralgia, nausea, vomiting or diarrhea, but no patient had complicated malaria. Peripheral blood was also collected from 15 healthy donors (HD) ranging from 21 to 56 years old [32±8] living Porto Velho and negative for P. vivax infection by thick blood film and PCR. All the experiments were done using fresh cells.

Cellular immunophenotyping

Whole blood was stained with antibodies from ebioscience: anti-CD14-FITC (clone 61D3), anti-TLR2-PE (clone T2.5), anti-TLR4-PE (clone HTA125), anti-HLA-DR-PE (clone L243), anti-CD14-APC (clone 61D3), anti-CD62L-APC (clone DREG56); BD Bioscience-Pharmingen: anti-CD66b-FITC (clone G10F5), anti-CD88-PE (clone C85-4124), CXCR2-PE (clone 6C6), anti-CD16-PercpCy5.5 (clone3G8), anti-CD15-FITC (clone HI98); R&D: CCR2-PE (clone 48607) Subsequently, red blood cells (RBC) were lysed with FACS lysing solution (BD Biosciences) following manufacturer's instructions, washed with phosphate buffered saline (PBS) and maintained in paraformaldehyde 2% (PFA) until the acquisition on an FACScan upgraded with a second laser (5 colors). The software used for acquisition was CellQuest Pro from BD and Rainbow from Cytek. Data analyzed using FlowJo version 9.3.2 (TreeStar)

Purification of monocytes and neutrophils

Peripheral blood mononuclear cells (PBMC) and neutrophils were enriched on-site by gradient centrifugation over Ficoll-Paque™ plus (GE-Healthcare). Isolated PBMC were washed twice in RPMI, ressuspended at 5×107/ml in PBS supplemented with 2% heat- inactivated fetal bovine serum (FBS) and 1 mM EDTA. Monocytes were purified from PBMC with the Human Monocyte Enrichment Kit (STEMCELL-Technologies) following manufacturer's instructions. The cell layer containing the granulocytes were collected just above the red blood cells phase and the cell suspension was lysed with NH4Cl 0.15 M with KHCO3 0.1 M and Na2EDTA 0.1 mM. Cells were ressuspended in PBS (2% FBS, 1 mM EDTA) at final concentration 5×107/ml. Purification of neutrophils were performed with the Human Neutrophil Enrichment Kit (STEMCELL-Technologies) following manufacturer's instructions. Purity of monocytes and neutrophils was measured by flow cytometry after staining with mAbs specific for CD14, CD66b and CD16. Purified monocytes were CD14+CD16−CD66b− and neutrophils were CD14−CD16+CD66b+. Cell preparations reached over 98% of purity and viability of 90–100%.

Tissue culture assays

Purified monocytes and neutrophils were plated in 96-well cell culture plates at a final concentration 2×105/well in RPMI1640 (Sigma Aldrich R6504) (10% FBS and 100 µg/ml streptomycin/100 U/ml penicillin) in the presence or absence of Toll like receptors agonists: 100 ng/ml lipopolysaccharide (LPS - TLR4 agonist, Sigma-Aldrich) or 100 ng/ml Pam2CSK4 (Pam - TLR2/TLR1 agonist, InvivoGen). Supernatants were harvested after 48 hours of culture and kept at −20°C until cytokine measurement.

Cytokine measurements

IL-8 (CXCL8), IL-1β, IL-6, IL-10 and TNF-α were quantified in cryopreserved plasma and supernatants from monocyte and neutrophil cultures using the Cytometric Bead Array kit (CBA, BD Biosciences Pharmingen) following manufacturer's instructions. The limits for cytokine detection were 3.6 pg/ml (IL-8), 7.2 pg/ml (IL-1β, 2.5 pg/ml (IL-6), 3.3 pg/ml (IL-10) and 3.7 pg/ml (TNF-α).

Phagocytosis and superoxide assays

Phagocytosis was assessed by incubating neutrophils for 20 min with opsonized zymosan (5×106 particles/cell) in a glass slide. Cells were stained by the May-Grünwald-Giemsa (Rosenfeld) method (Laborclin Pinhais, PR, BR). The number of cells containing phagocytic vacuole was determined by counting 100 neutrophils. Nitro Blue Tetrazolium (NBT) assay was performed using peripheral blood. Briefly, 25 µl of blood sample were added to the same volume of NBT (1 mg/ml) at 37°C for 15 min and an additional 15 min at room temperature. Percentage of cells reducing NBT was established by counting 100 neutrophils.

Chemotaxis assay

Chemotaxis was performed with purified neutrophils using a 48-well microchamber (NeuroProbe, Gaithersburg, MD) with a 5 µm pore polycarbonate membrane. Fifty thousand neutrophils were allowed to migrate toward IL-8 (CXCL8) (10 ng/ml), CCL2 (1 ng/ml), or medium alone. After 1 h incubation, at 37°C with 5% CO2 the membrane was removed, fixed and stained. The number of neutrophils that migrated through the membrane was counted under a light microscope on at least 5 randomly selected fields.

Real-Time PCR

Total RNA from neutrophils was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA to cDNA was carried out by reverse transcription (Superscript II, Gibco Life Technologies Grand Island, NY). Real Time PCR (qPCR) analysis of CXCR2 receptor mRNA profile was performed with the Platinum SYBR® Green qPCR SuperMix-UDG kit (Invitrogen-Life Technologies, USA) and the reactions were processed in ABI Prism 7500 equipment (Applied Biosystems, Warrington, UK) was performed in an ABI Prism 7500. The reactions were carried out in a final volume of 10 µl containing 6.25 µl of SYBR Green, 0.5 µM of each primer (designed on the basis of the mRNA sequence of the receptor gene available at the GenBank) and 1 µl of cDNA. RT-PCR cycles were 95°C for 15 s and 60°C for 1 minute and the dissociation curve was constructed by increasing temperatures from 60°C to 90°C. The primers used were: CXCR2 sense: 5′- AGATGCTGAGACATATGAATTT -3′ and CXCR2 antisense: 5′- CTTTTCTACTAGATGCCGC-3′, β-Actin sense: 5′-GCTCGTCGTCGACAACGGCTC -3′ and β-Actin antisense: 5′- CAAACATGATCTGGGTCATC -3′. The expression of β-actin mRNA was used as a control for all samples. The relative level of gene expression was determined by the comparative threshold cycle (Ct) method using the formula −2ΔΔCt whereby data for each sample were normalized to β-actin mRNA levels.

Immunofluorescence assay for GRK2

Neutrophils purified on glass slides were fixed with 4% paraformaldehyde in a wet chamber at room temperature. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 (Amersham Pharmacia Biotech, CA) and blocked with 1% bovine serum albumin (Sigma-Aldrich) in PBS containing normal goat serum (1[ratio]50). Cells were then incubated overnight with polyclonal rabbit anti-GRK-2 antibody (1[ratio]200, clone C-15, Santa Cruz Biotechnology) followed by incubation with red fluorescent Alexa Fluor 594 (goat anti-rabbit IgG (H+L); 1[ratio]400, Life Technologies). Nuclear material was stained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Images of stained cells were captured using an epi-fluorescence microscope (BX-40; Olympus, Japan). The mean fluorescence intensity was determined from a linear measurement of fluorescence of individual cells using Image J software (Imaging process and analysis in Java, National Institute of Mental Health). Ten cells of five randomly chosen fields of each slide were analyzed.

Statistical analysis

Statistical analysis was performed using Prism software, version 5.0 (GraphPad). The results were analyzed using two-tailed paired test. Wilcoxon testing was used when data did not fit a Gaussian distribution. The results were analyzed using unpaired t test when two groups were compared. Mann-Whitney (MW) test was used when a normality test failed. Differences were considered to statistically significant, when p≤0.05.

Results

High levels of circulatory cytokines in P. vivax malaria patients

High levels of the pro-inflammatory cytokines, IL-1β, IL-6, IL-8, were found when comparing the sera from the same patients presenting the active disease and after anti-malarial therapy and parasitological cure or to HD. ( Figure 1 ). The levels of TNF-α in the sera did not differ significantly. The levels of IL-10 were also higher before treatment than after parasitological cure. No significant differences in the level of circulating cytokines were found between HD (IL-8 median: 9.8; interquartile (IQR): 1.8- 21; IL-1β median: 0.2 IQR: 0.13–2.8; IL-6 median: 0.14; IQR:0–5; IL-10 median: 0–7.1; IQR: 0–7 and TNF-α median: 5.4; IQR 0–21) and malaria patients who had completed the chemotherapy.

An external file that holds a picture, illustration, etc. Object name is pntd.0001710.g001.jpg
High levels IL-1β IL-6, IL-8 and IL-10 in plasma from patients infected with P. vivax.

The cytokines IL-8 (CXCL8), IL-1β, IL-6, IL-10, TNF-α were measured in the plasma of P. vivax-infected subjects (n = 26), before (closed circles) and 30–45 days after treatment (open circles). Dotted lines represent medians of given measurements from healthy donors (HD; n = 13). Levels of cytokines were measured employing the Cytometric Bead Array (CBA). Significant differences are indicated with p-values using Wilcoxon signed rank test when the data failed the normality test.

Monocytes and neutrophils activation during P. vivax infection

The expression of various activation markers was analyzed on monocytes ( Figure 2A ) and neutrophils ( Figure 2B ). Monocytes were gated taking in consideration the expression of CD14, as shown in the representative density plot ( Figure 2A ). Lower expression of HLA-DR (median fluorescence intensity-MFI) was observed on monocytes from 9 out of 12 P. vivax-infected patients, when compared to the same patients after anti-malarial treatment. To assess the neutrophil population, CD66b+CD16+ cells were gated. As CD66b is GPI-anchores highly glycoslated protein, widely expressed on human neutrophils and to regulate neutrophil acitvation. Lower levels of CD62L (8 out of 12 subjects) and CD88 (all patients) were found on neutrophils from malaria patients before treatment ( Figure 2B ). The lower expression of HLA-DR as well as CD62L is a cell adhesion molecule, and CD88 is a G protein-coupled receptor for C5a, have been previously described in activated monocytes and neutrophils, respectively [14], [19]. The levels of these activation markers return to normal levels (comparable to the median of HD (monocytes: HLA-DR (median: 278; IQR: 143–323); neutrophils: CD88 (median: 27; IQR: 17–32) and CD62L (median: 601; IQR: 439–666); in cells from P. vivax-patients submitted to anti-malarial therapy.

An external file that holds a picture, illustration, etc. Object name is pntd.0001710.g002.jpg
Systemic activation of monocytes and neutrophils during P. vivax malaria.

Mean fluorescence intensity (MFI) of HLA-DR, TLR2 and TLR4 was evaluated on monocytes (A) and CD62L, CD88, TLR2 and TLR4 on neutrophils (B) in whole blood from P. vivax-infected subjects (n = 12), before (closed circles) and 30–45 days after treatment (open circles). Representative density plots showing the gate strategy for monocytes CD14+ (A) and neutrophils CD66b+CD16+ (B) are shown. Significant differences are indicated with p-values using Wilcoxon signed rank test when the data failed the normality test.

Monocytes are a major source of cytokines during P. vivax malaria

The expression of TLR2 was lower on both monocytes (8 out of 11 subjects) and neutrophils (10 out of 11 subjects) from malaria patients, whereas expression of TLR4 was only affected on neutrophils (8 out of 12) from infected individuals ( Figures 2A and 2B ). After parasitological cure, the surface expression of TLR returned to levels comparable to cells from HD (monocytes: TLR2 (median: 256; IQR: 241–276) and TLR4 (median: 11; IQR: 9–17; neutrophils: TLR2 (median 46; IQR: 36–62) and TLR4 (median: 3.5; IQR: 2.9–6.4)). Two millions per ml (2×105/well) of highly purified monocytes or neutrophils were cultured with or without TLR agonists and cytokines measured in the tissue culture supernatants ( Figure 3A and B ). Cell preparations reached over 98% of purity, as shown in the representative dot plots presented in Figure 3 . Except for IL-8, neutrophils were over all a poor source of cytokines. No detectable levels of IL-1β, IL-6, IL-10 and TNF-α were found in culture supernatants of neutrophils stimulated or not with LPS or Pam ( Figure 3B ), even when neutrophils were used at a concentration of 1×107/ml (1×106/well). On the other hand, upon stimulation with LPS or Pam, high levels of IL-1β, IL-6, and TNF-α were produced by monocytes from malaria patients. ( Figure 3A ). Interestingly, the levels of LPS-induced IL-10 were lower in culture supernatants of monocytes derived from patients with ongoing P. vivax infection. After parasitological cure the cytokine production by monocytes returned to levels comparable to those produced by monocytes from HD (median: 1692; IQR: 1200–5265). IL-8 (CXCL8) was the only cytokine produced in high levels by neutrophils. Despite the down-modulation of TLR2 and TLR4, stimulation with TLR agonists increased the levels of IL-8 produced by neutrophils ( Figure 3B ) from malaria patients before (medium versus LPS, p = 0.0010 or Pam, p = 0.0024) and after treatment (medium vs LPS, p = 0.0002; or Pam, p = 0.0017). IL-8 was also produced by monocytes and the levels of cytokine did not differ between patients, before and after treatment ( Figure 3A ).

An external file that holds a picture, illustration, etc. Object name is pntd.0001710.g003.jpg
TLR agonists induce production of high IL-1β, IL-6 and TNF-α levels by monocytes from P. vivax-infected subjects.

Purified monocytes (A) or neutrophils (B) from P. vivax-infected subjects before (closed circles; n = 13) and 30–45 days after treatment (open circles; n = 13) were cultured for 48 hours in the absence or presence of LPS or Pam. Levels of IL-1β, IL-6, IL-10, TNF-α, and IL-8 (CXCL8) were measured in supernatant of monocyte (A) and neutrophil (B) cultures. Levels of cytokines were measured employing the Cytometric Bead Array (CBA). Significant differences are indicated p-values using paired t test or Wilcoxon signed rank test when a normality test failed.

Enhanced oxidative and phagocytic activity by neutrophils from malaria patients

The O2 − production by neutrophils was increased during acute malaria ( Figure 4 ). Higher percentage of NBT positive cells was observed in the blood of P. vivax-infected patients ( Figure 4 , left panel). The function of neutrophils was further investigated by assessing their phagocytic activity, which was also higher in cells from P. vivax-infected individuals ( Figure 4 , right panel).

An external file that holds a picture, illustration, etc. Object name is pntd.0001710.g004.jpg
Neutrophils from P. vivax-infected patients produce high levels of superoxide and display enhanced phagocytic function.

Neutrophils were isolated from P. vivax-infected patients (closed circles; n = 15) or healthy donors (open circles; n = 15), and the frequencies of neutrophils reducing NBT (left panel) as well as cell containing zymosan (right panel) were quantified. Significant differences are indicated with p-values using unpaired t test or Mann-Whitney test when a normality test failed.

Impaired chemotaxis by neutrophils from malaria patients

The ability of purified neutrophils to migrate towards CXCR1, CXCR2 and CCR2 ligands (IL-8 and CCL2) was evaluated by using a modified Boyden chamber assay [20]. Neutrophils from malaria patients showed impaired chemotaxis to IL-8, when compared to HD (median: 31; IQR: 28–39) ( Figure 5A ). The poor migration of neutrophils from malaria patients was associated with decreased expression of the chemokine receptor CXCR2, as indicated by qPCR ( Figure 5C ) and FACS ( Figure 5B ). Chemotaxis to CCL2 and expression of CCR2 by neutrophils were minimal and not altered in malaria patients ( Figures 5A and 5B ).

An external file that holds a picture, illustration, etc. Object name is pntd.0001710.g005.jpg
Malaria impairs neutrophils response to CXCR1 and CXCR2 ligand.

Neutrophils were isolated from P. vivax-infected patients (closed circles; n = 15) or healthy donors (open circles; n = 15), and chemotaxis towards IL-8 (CXCL8) and CCL2 was assessed (A). MFI of CXCR1, CXCR2 and CCR2 on neutrophils were evaluated by flow cytometry and representative histograms of CXCR2 expression are shown (B). CXCR2 message was measured by qPCR (C). Significant differences are indicated with p-values using unpaired t test or Mann-Whitney test when a normality test failed.

Enhanced expression of GRK2 in neutrophils from malaria patients

GRK2 has been described to down-modulate expression of chemokine receptors. Importantly, continuous and excessive activation of neutrophils results in an increased expression of GRKs, which phosphorylate G protein–coupled receptors (GPCR) leading to receptor desensitization [21]. GRK2 expression was measured by immunofluorescence in neutrophils purified from blood of P. vivax-infected patients and HD (median: 12.6; IQR: 4.7–23). Compared with HD, a significant increase in GRK2 expression was observed in neutrophils from P. vivax-infected patients ( Figure 6A and 6B ).

An external file that holds a picture, illustration, etc. Object name is pntd.0001710.g006.jpg
GRK2 expression is enhanced in neutrophils during acute malaria.

Neutrophils isolated from P. vivax-infected patients (closed circles; n = 11) or healthy donors (closed circles; n = 12) were stained for GRK2 and mean fluorescence intensity (MFI) of GRK2 was quantified (A). Representative fluorescence microscopy illustrating GRK2 expression in neutrophils from a healthy donor and a P. vivax-infected patient (B). Significant difference is indicated with p-values using unpaired t test.

Discussion

The pathogenesis of P. vivax malaria is a consequence of host derived inflammatory mediators [1], [22]. Hence, a better understanding of the mechanisms involved in induction of systemic inflammation during P. vivax malaria is critical for the clinical management and prevention of severe disease. Neutrophils are the most abundant leukocyte population in the peripheral blood and thus being in direct contact with red blood cells, the main cells population that is parasitized by Plasmodium species. Surprisingly, very few studies have addressed the role of the polymorphonuclear cells (PMNs) during malaria. In this study, we investigated the activation and function of neutrophils during acute episodes of P. vivax malaria. Neutrophils were shown to be highly activated presenting enhanced phagocytic activity as well as superoxide production. In contrast, neutrophils were found to be poor sources of cytokines during malaria. It is noteworthy that we described for the first time an impaired chemotaxis by circulatory neutrophils from malaria patients. Importantly, different studies have described an association of severe malaria and bacterial infections [23], [24]. Thus, our results suggest that the failure of neutrophils to migrate to the sites of infection may represent an important mechanism that leads to enhanced susceptibility of malaria patients to secondary bacterial infections.

We first noticed that both circulatory neutrophils (decreased expression of CD62L and CD88) and monocytes (decreased expression of HLA-DR) from malaria patients were highly activated. We then asked, what would be the primary source of pro-inflammatory cytokines during P. vivax-malaria? We found that in the blood, monocytes are an important source of cytokine compared with neutrophils. Despite the fact that neutrophils are an important source of cytokines [25], we found that except for IL-8 (CXCL8), the neutrophils from malaria patients produced none or very small amounts of pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNF-αin response to TLR agonists. Importantly, PBMCs from individuals experimentally or naturally infected with P. falciparum are hyperresponsive and produce high amounts of pro-inflammatory cytokines once activated with TLR agonists [6], [7]. Here, we observed that highly purified monocytes (but not neutrophils) derived from P. vivax malaria patients were primed and produce high levels of IL-1β, IL-6 and TNF-α upon TLR stimulation. In contrast, the monocytes from malaria patients produced low levels of IL-10, even when activated with TLR agonists. Thus, we favor the hypothesis that during malaria monocytes differentiate into an inflammatory stage producing high levels of pro-inflammatory cytokines and low levels of IL-10.

On the other hand, circulatory neutrophils from malaria-patients displayed enhanced phagocytic activity and constitutively released high levels of superoxide. The process of neutrophil activation could involve phagocytosis of opsonized parasites, which would in turn trigger the antibody dependent respiratory burst [26]. Alternatively, phagocytosis of the malaria pigment (hemozoin) may also activate neutrophils [27]. The enhanced effector function of neutrophils may account for a more efficient up-take and destruction of free parasites and infected erythrocytes [26]. On the other hand, activated neutrophils have been shown to cause damage of endothelial cells, in a process that is mediated by sera of malaria patients [28]. Therefore, enhanced effector functions in neutrophils could be involved in both host resistance and pathogenesis of P. vivax malaria.

Unexpectedly, we found an altered migration towards IL-8 gradient, which was associated with a decreased expression of CXCR2 on neutrophils from P. vivax-infected patients. Functional studies showed that upon phagocytosis of bacteria by neutrophils there is reduction in expression of CXCR1 and CXCR2 [29]. Down-regulation of CXCR2 in severe sepsis also results in failure of neutrophil migration that is associated with enhanced susceptibility to bacterial infection [30]. Furthermore, expression of CD88 (C5a receptor) and CD62L (L-selectin) is decreased on the surface of neutrophils from P. vivax-malaria patients. CD88 is a G protein-coupled receptor involved in recruitment [19], [31], whereas CD62L is a key molecule that mediates cytoadherence of leukocytes [32]. Thus, altogether, our data strongly suggest that systemic activation neutrophils, leads to failure of extravasation and chemotaxis from blood to the tissues. Since IL-8 mediates chemotaxis and stimulate neutrophils to release specific granules and proteases to fight microbial infections [33], [34], the impairment of neutrophil migration to the site of infection would prevent front line cells to promote an inflammation to effectively kill infectious pathogens allowing secondary infections.

Production of IL-8 has been assessed in several cell type upon stimulation, several medical conditions and even constitutively [35], [36], [37]. The observed high levels of circulatory IL-8 may mediate desensitization and/or down-regulation of CXCR2 in acutely infected malaria patients. In addition, TNF-α [38], nitric oxide [30], heme-oxygenase products [39] and TLR ligands [20], cause the heterologous desensitization of CXCR2 via GRK2 induction. As previous described in vivax malaria [40], [41], we did not find a high level of TNFα in serum, the monocyte stimulated with TLR agonists produced high amounts of TNF-α that may contribute to CXCR2 desensitization. In addition, Plasmodium can be recognized by TLR2, TLR4 and TLR9 [10], [42] and induce down-regulation of CXCR2 via GRK2. Importantly, CD88 is also desensitized via GRK2 [43]. Thus, decreased expression of CXCR2 and CD88 on neutrophils from malaria patients may be a consequence of an enhanced expression of GRK2.

GRKs constitute a group of serine/threonine protein kinases that are key modulators of protein-coupled receptor signaling (GPCR) [44]. A major mechanism for desensitization of activated GPCR is their phosphorylation by GRKs [45]. Of note both CD88 and CXCR2 are GPCRs. Deficient expression of GRK and regulation of chemokine receptors in GRK2+/− mice results in enhanced migration of lymphocytes and chemotaxis toward CCL4, the CCR5 ligand [46]. It was also described that transcription of GRK2 and GRK5 is upregulated upon LPS-mediated activation, leading to reduced expression of chemokine receptor and neutrophils chemotaxis [47]. GRK2 and GRK5 expression are enhanced in sepsis patients [48] and in rodent models of severe sepsis [20], which are associated with impaired migration of neutrophils and enhanced susceptibility to secondary microbial infection.

For many years, P. vivax malaria was considered a benign and self-limited disease, especially when compared to P. falciparum infection [49]. However, recent studies highlighted the association of P. vivax malaria with life-threatening manifestations, such as respiratory distress, severe thrombocytopenia and anemia, as well as neurological manifestations [1], [22], [50], [51], [52], [53], [54]. A main hypothesis of our research group is that secondary infections, in malaria primed individuals, is a main cause of severe disease. In this regard, pro-inflammatory priming during malaria would result in dramatic decrease in the threshold to initiate a septic shock [7], due to an over-reaction to secondary infections, particularly in the case of bacteria that have extremely potent TLR agonists.

Importantly, areas of the world with the highest incidence and prevalence of malaria also have a high incidence of bacterial infections, including Salmonella, Pneumococcus and Meningococcus [23], [55]. Furthermore, a recent study highlights that severe malaria as indicated by respiratory distress, anemia and mortality, is 8.5 times more elevated in children with both malaria and bacteremia as compared to infection with P. falciparum alone [24]. Thus, co-infection with bacteria is not only common, but as we might predict, it is an important factor influencing outcome of disease and development of severe disease [24], [56]. Here, we demonstrate for the first time that circulatory neutrophils from malaria patients display a decreased expression of chemokine receptors and adhesion molecules, which culminates in impaired chemotaxis. Hence, our results suggest that a failure of these PMNs to migrate to peripheral tissues is an important mechanism leading to enhance susceptibility to bacterial infection during malaria.

Acknowledgments

We are grateful to Dr. Marco Cassatella for all advice and for critically reading this manuscript, to Dr. Luiz Hildebrando Pereira da Silva for supporting the development of this work in the endemic area of Porto Velho and also to the nurses for excellent technical assistance, and to the Program for Technological Development in Tools for Health–PDTIS-FIOCRUZ for use of its facilities.

Footnotes

The authors have declared that no competing interests exist.

This work was supported by National Institutes of Health (R21 AI080907 and RO1 AI079293) to RTG and DTG, Malaria Network (CNPq-555646/2009-2/FAPEMIG/MS-CBB-APQ-01153-10), National Institute of Science and Technology for Vaccines (CNPq-573547/2008-4/FAPEMIG/MS-CBB-APQ 00077-09), and the Program for Technological Development in Tools for Health from Fundação Oswaldo Cruz (PDTIS-FIOCRUZ- RVR22). RTG, FQC and LRVA are research fellows from CNPq; DTG and FMSL are research fellows from FAPEMIG and FIOCRUZ, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1. Mueller I, Galinski MR, Baird JK, Carlton JM, Kochar DK, et al. Key gaps in the knowledge of Plasmodium vivax, a neglected human malaria parasite. Lancet Infect Dis. 2009;9:555–566. [Abstract] [Google Scholar]
2. WHO. 2010. World Malaria Report.
3. Gazzinelli RT, Denkers EY. Protozoan encounters with Toll-like receptor signalling pathways: implications for host parasitism. Nat Rev Immunol. 2006;6:895–906. [Abstract] [Google Scholar]
4. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell. 2010;140:805–820. [Abstract] [Google Scholar]
5. Kwiatkowski D, Hill AV, Sambou I, Twumasi P, Castracane J, et al. TNF concentration in fatal cerebral, non-fatal cerebral, and uncomplicated Plasmodium falciparum malaria. Lancet. 1990;336:1201–1204. [Abstract] [Google Scholar]
6. McCall MB, Netea MG, Hermsen CC, Jansen T, Jacobs L, et al. Plasmodium falciparum infection causes proinflammatory priming of human TLR responses. J Immunol. 2007;179:162–171. [Abstract] [Google Scholar]
7. Franklin BS, Parroche P, Ataide MA, Lauw F, Ropert C, et al. Malaria primes the innate immune response due to interferon-gamma induced enhancement of toll-like receptor expression and function. Proc Natl Acad Sci U S A. 2009;106:5789–5794. [Abstract] [Google Scholar]
8. Babior BM. Oxidants from phagocytes: agents of defense and destruction. Blood. 1984;64:959–966. [Abstract] [Google Scholar]
9. Greenberg S, Grinstein S. Phagocytosis and innate immunity. Curr Opin Immunol. 2002;14:136–145. [Abstract] [Google Scholar]
10. Krishnegowda G, Hajjar AM, Zhu J, Douglass EJ, Uematsu S, et al. Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem. 2005;280:8606–8616. [Europe PMC free article] [Abstract] [Google Scholar]
11. Parroche P, Lauw FN, Goutagny N, Latz E, Monks BG, et al. Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9. Proc Natl Acad Sci U S A. 2007;104:1919–1924. [Europe PMC free article] [Abstract] [Google Scholar]
12. Franklin BS, Ishizaka ST, Lamphier M, Gusovsky F, Hansen H, et al. Therapeutical targeting of nucleic acid-sensing Toll-like receptors prevents experimental cerebral malaria. Proc Natl Acad Sci U S A. 2011;108:3689–3694. [Europe PMC free article] [Abstract] [Google Scholar]
13. Sharma S, DeOliveira RB, Kalantari P, Parroche P, Goutagny N, et al. Innate immune recognition of an AT-rich stem-loop DNA motif in the Plasmodium falciparum genome. Immunity. 2011;35:194–207. [Europe PMC free article] [Abstract] [Google Scholar]
14. Schwarzer E, Alessio M, Ulliers D, Arese P. Phagocytosis of the malarial pigment, hemozoin, impairs expression of major histocompatibility complex class II antigen, CD54, and CD11c in human monocytes. Infect Immun. 1998;66:1601–1606. [Europe PMC free article] [Abstract] [Google Scholar]
15. Skorokhod OA, Alessio M, Mordmuller B, Arese P, Schwarzer E. Hemozoin (malarial pigment) inhibits differentiation and maturation of human monocyte-derived dendritic cells: a peroxisome proliferator-activated receptor-gamma-mediated effect. J Immunol. 2004;173:4066–4074. [Abstract] [Google Scholar]
16. Jaramillo M, Plante I, Ouellet N, Vandal K, Tessier PA, et al. Hemozoin-inducible proinflammatory events in vivo: potential role in malaria infection. J Immunol. 2004;172:3101–3110. [Abstract] [Google Scholar]
17. Mohammed AO, Elghazali G, Mohammed HB, Elbashir MI, Xu S, et al. Human neutrophil lipocalin: a specific marker for neutrophil activation in severe Plasmodium falciparum malaria. Acta Trop. 2003;87:279–285. [Abstract] [Google Scholar]
18. Kimura M, Kaneko O, Liu Q, Zhou M, Kawamoto F, et al. Identification of the four species of human malaria parasites by nested PCR that targets variant sequences in the small subunit rRNA gene. Parasitol Int. 1997;46:91–95. [Google Scholar]
19. Fortunati E, Kazemier KM, Grutters JC, Koenderman L, Van den Bosch JM. Human neutrophils switch to an activated phenotype after homing to the lung irrespective of inflammatory disease. Clin Exp Immunol. 2009;155:559–566. [Abstract] [Google Scholar]
20. Alves-Filho JC, Freitas A, Souto FO, Spiller F, Paula-Neto H, et al. Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis. Proc Natl Acad Sci U S A. 2009;106:4018–4023. [Europe PMC free article] [Abstract] [Google Scholar]
21. Gainetdinov RR, Bohn LM, Walker JK, Laporte SA, Macrae AD, et al. Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice. Neuron. 1999;24:1029–1036. [Abstract] [Google Scholar]
22. Karunaweera ND, Wijesekera SK, Wanasekera D, Mendis KN, Carter R. The paroxysm of Plasmodium vivax malaria. Trends Parasitol. 2003;19:188–193. [Abstract] [Google Scholar]
23. Bronzan RN, Taylor TE, Mwenechanya J, Tembo M, Kayira K, et al. Bacteremia in Malawian children with severe malaria: prevalence, etiology, HIV coinfection, and outcome. J Infect Dis. 2007;195:895–904. [Abstract] [Google Scholar]
24. Were T, Davenport GC, Hittner JB, Ouma C, Vulule JM, et al. Bacteremia in Kenyan children presenting with malaria. J Clin Microbiol. 2011;49:671–676. [Europe PMC free article] [Abstract] [Google Scholar]
25. Cassatella MA. Neutrophil-derived proteins: selling cytokines by the pound. Adv Immunol. 1999;73:369–509. [Abstract] [Google Scholar]
26. Joos C, Marrama L, Polson HE, Corre S, Diatta AM, et al. Clinical protection from falciparum malaria correlates with neutrophil respiratory bursts induced by merozoites opsonized with human serum antibodies. PLoS One. 2010;5:e9871. [Europe PMC free article] [Abstract] [Google Scholar]
27. Huy NT, Trang DT, Kariu T, Sasai M, Saida K, et al. Leukocyte activation by malarial pigment. Parasitol Int. 2006;55:75–81. [Abstract] [Google Scholar]
28. Hemmer CJ, Vogt A, Unverricht M, Krause R, Lademann M, et al. Malaria and bacterial sepsis: similar mechanisms of endothelial apoptosis and its prevention in vitro. Crit Care Med. 2008;36:2562–2568. [Abstract] [Google Scholar]
29. Doroshenko T, Chaly Y, Savitskiy V, Maslakova O, Portyanko A, et al. Phagocytosing neutrophils down-regulate the expression of chemokine receptors CXCR1 and CXCR2. Blood. 2002;100:2668–2671. [Abstract] [Google Scholar]
30. Rios-Santos F, Alves-Filho JC, Souto FO, Spiller F, Freitas A, et al. Down-regulation of CXCR2 on neutrophils in severe sepsis is mediated by inducible nitric oxide synthase-derived nitric oxide. Am J Respir Crit Care Med. 2007;175:490–497. [Abstract] [Google Scholar]
31. Azeredo EL, Neves-Souza PC, Alvarenga AR, Reis SR, Torrentes-Carvalho A, et al. Differential regulation of toll-like receptor-2, toll-like receptor-4, CD16 and human leucocyte antigen-DR on peripheral blood monocytes during mild and severe dengue fever. Immunology. 2010;130:202–216. [Abstract] [Google Scholar]
32. Ley K. Integration of inflammatory signals by rolling neutrophils. Immunol Rev. 2002;186:8–18. [Abstract] [Google Scholar]
33. Larsen CG, Anderson AO, Appella E, Oppenheim JJ, Matsushima K. The neutrophil-activating protein (NAP-1) is also chemotactic for T lymphocytes. Science. 1989;243:1464–1466. [Abstract] [Google Scholar]
34. Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokines–CXC and CC chemokines. Adv Immunol. 1994;55:97–179. [Abstract] [Google Scholar]
35. Srivastava MD, Srivastava R, Srivastava BI. Constitutive production of interleukin-8 (IL-8) by normal and malignant human B-cells and other cell types. Leuk Res. 1993;17:1063–1069. [Abstract] [Google Scholar]
36. Wurm S, Neumeier M, Weigert J, Wanninger J, Gerl M, et al. Insulin induces monocytic CXCL8 secretion by the mitogenic signalling pathway. Cytokine. 2008;44:185–190. [Abstract] [Google Scholar]
37. Wong CK, Wong PT, Tam LS, Li EK, Chen DP, et al. Activation profile of Toll-like receptors of peripheral blood lymphocytes in patients with systemic lupus erythematosus. Clin Exp Immunol. 2010;159:11–22. [Abstract] [Google Scholar]
38. Secher T, Vasseur V, Poisson DM, Mitchell JA, Cunha FQ, et al. Crucial role of TNF receptors 1 and 2 in the control of polymicrobial sepsis. J Immunol. 2009;182:7855–7864. [Abstract] [Google Scholar]
39. Freitas A, Alves-Filho JC, Trevelin SC, Spiller F, Suavinha MM, et al. Divergent role of heme oxygenase inhibition in the pathogenesis of sepsis. Shock. 2011;35:550–559. [Abstract] [Google Scholar]
40. Andrade BB, Reis-Filho A, Souza-Neto SM, Clarencio J, Camargo LM, et al. Severe Plasmodium vivax malaria exhibits marked inflammatory imbalance. Malar J. 2010;9:13. [Europe PMC free article] [Abstract] [Google Scholar]
41. Goncalves RM, Salmazi KC, Santos BA, Bastos MS, Rocha SC, et al. CD4+ CD25+ Foxp3+ regulatory T cells, dendritic cells, and circulating cytokines in uncomplicated malaria: do different parasite species elicit similar host responses? Infect Immun. 2010;78:4763–4772. [Europe PMC free article] [Abstract] [Google Scholar]
42. Coban C, Ishii KJ, Kawai T, Hemmi H, Sato S, et al. Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin. J Exp Med. 2005;201:19–25. [Europe PMC free article] [Abstract] [Google Scholar]
43. Langkabel P, Zwirner J, Oppermann M. Ligand-induced phosphorylation of anaphylatoxin receptors C3aR and C5aR is mediated by “G protein-coupled receptor kinases. Eur J Immunol. 1999;29:3035–3046. [Abstract] [Google Scholar]
44. Penela P, Ribas C, Mayor F., Jr Mechanisms of regulation of the expression and function of G protein-coupled receptor kinases. Cell Signal. 2003;15:973–981. [Abstract] [Google Scholar]
45. Krupnick JG, Benovic JL. The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annu Rev Pharmacol Toxicol. 1998;38:289–319. [Abstract] [Google Scholar]
46. Vroon A, Heijnen CJ, Lombardi MS, Cobelens PM, Mayor F, Jr, et al. Reduced GRK2 level in T cells potentiates chemotaxis and signaling in response to CCL4. J Leukoc Biol. 2004;75:901–909. [Abstract] [Google Scholar]
47. Fan J, Malik AB. Toll-like receptor-4 (TLR4) signaling augments chemokine-induced neutrophil migration by modulating cell surface expression of chemokine receptors. Nat Med. 2003;9:315–321. [Abstract] [Google Scholar]
48. Arraes SM, Freitas MS, da Silva SV, de Paula Neto HA, Alves-Filho JC, et al. Impaired neutrophil chemotaxis in sepsis associates with GRK expression and inhibition of actin assembly and tyrosine phosphorylation. Blood. 2006;108:2906–2913. [Abstract] [Google Scholar]
49. Singh H, Parakh A, Basu S, Rath B. Plasmodium vivax malaria: is it actually benign? J Infect Public Health. 2011;4:91–95. [Abstract] [Google Scholar]
50. Anstey NM, Handojo T, Pain MC, Kenangalem E, Tjitra E, et al. Lung injury in vivax malaria: pathophysiological evidence for pulmonary vascular sequestration and posttreatment alveolar-capillary inflammation. J Infect Dis. 2007;195:589–596. [Europe PMC free article] [Abstract] [Google Scholar]
51. Obaldia N., 3rd Clinico-pathological observations on the pathogenesis of severe thrombocytopenia and anemia induced by Plasmodium vivax infections during antimalarial drug efficacy trials in Aotus monkeys. Am J Trop Med Hyg. 2007;77:3–13. [Abstract] [Google Scholar]
52. Anstey NM, Russell B, Yeo TW, Price RN. The pathophysiology of vivax malaria. Trends Parasitol. 2009;25:220–227. [Abstract] [Google Scholar]
53. Alexandre MA, Ferreira CO, Siqueira AM, Magalhaes BL, Mourao MP, et al. Severe Plasmodium vivax malaria, Brazilian Amazon. Emerg Infect Dis. 2010;16:1611–1614. [Europe PMC free article] [Abstract] [Google Scholar]
54. Bassat Q, Alonso PL. Defying malaria: Fathoming severe Plasmodium vivax disease. Nat Med. 2011;17:48–49. [Abstract] [Google Scholar]
55. Berkley JA, Maitland K, Mwangi I, Ngetsa C, Mwarumba S, et al. Use of clinical syndromes to target antibiotic prescribing in seriously ill children in malaria endemic area: observational study. Bmj. 2005;330:995. [Europe PMC free article] [Abstract] [Google Scholar]
56. Berkley JA, Bejon P, Mwangi T, Gwer S, Maitland K, et al. HIV infection, malnutrition, and invasive bacterial infection among children with severe malaria. Clin Infect Dis. 2009;49:336–343. [Europe PMC free article] [Abstract] [Google Scholar]
Articles from PLOS Neglected Tropical Diseases are provided here courtesy of PLOS

Full text links 

Read article at publisher's site: https://doi.org/10.1371/journal.pntd.0001710

Read article for free, from open access legal sources, via Unpaywall: https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0001710&type=printableUnpaywall

Citations & impact 

Impact metrics

44
Citations
Jump to Citations

Citations of article over time

Alternative metrics

Altmetric item for https://www.altmetric.com/details/898964
Altmetric
Discover the attention surrounding your research
https://www.altmetric.com/details/898964

Article citations

Go to all (44) article citations

Similar Articles 

To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.

Funding 

Funders who supported this work.

NIAID NIH HHS (2)