Contribution of T Cells and in Protection of Young Susceptible Rats from Fatal Experimental Malaria

This information is current as Christine Pierrot, Estelle Adam, David Hot, Sophia Lafitte, of September 23, 2021. Monique Capron, James D. George and Jamal Khalife J Immunol 2007; 178:1713-1722; ; doi: 10.4049/jimmunol.178.3.1713 http://www.jimmunol.org/content/178/3/1713 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 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Contribution of T Cells and Neutrophils in Protection of Young Susceptible Rats from Fatal Experimental Malaria1

Christine Pierrot,* Estelle Adam,2* David Hot,† Sophia Lafitte,* Monique Capron,* James D. George,‡ and Jamal Khalife3*

In human malaria, children suffer very high rates of morbidity and mortality. To analyze the mechanisms involved in age- dependent protection against malaria, we developed an experimental model of infection in rats, where young rats are susceptible to Plasmodium berghei and adult rats control blood parasites and survive thereafter. In this study, we showed that protection of young rats could be achievable by adoptive transfer of spleen cells from adult protected rats, among which T cells could transfer partial protection. Transcriptome analysis of spleen cells transferring immunity revealed the overexpression of mainly expressed by and neutrophils. Evaluation of the role of neutrophils showed that these cells were able to transfer partial protection to young rats. This antiparasitic effect was shown to be mediated, at least in part, through the protein-1 Downloaded from . Further adoptive transfer experiments indicated an efficient cooperation between neutrophils and T cells in protecting all young recipients. These observations, together with those from in vitro studies in human malaria, suggest that the failure of children to control infection could be related not only to an immaturity of their adaptive immunity but also to a lack in an adequate innate immune response. The Journal of Immunology, 2007, 178: 1713–1722.

n human malaria caused by Plasmodium falciparum infec- were conducted under conditions of chronic exposure to malaria, http://www.jimmunol.org/ tion, there are at least 1–2 million deaths annually, mainly in and there have been no studies characterizing immune responses in I children under the age of 5 years. To understand this age- detail in children and adults after a primary exposure to malaria. related susceptibility and protection of humans to malaria infec- However, in newly infected individuals, Baird (18) observed that tion, several epidemiological studies have addressed the issue of protective immunity developed more rapidly in adults than in whether the immune response to malaria in children and adults is children. different. There may be several mechanisms potentially involved In experimental malaria, the most extensive work on immune in immune hyporesponsiveness in young mice and humans, such responses has been conducted using adult mouse models with no as developmental immaturity of APCs thus influencing establish-

clear studies on the effects of age. From these studies, it appears by guest on September 23, 2021 ment of effective T cell-APC interactions (1, 2), incomplete sig- that cellular and humoral responses are essential actors in the con- naling because of low expression of CD40L on T cells (3), and/or trol and clearance of malaria parasites (for review, see Refs. 19– impaired responsiveness to TLR ligands leading to a lack of ap- 21). Analysis of cellular immunity demonstrated that CD4ϩ T cells propriate cytokine production (4). Whether these deficiencies are still present in 1- to 5-year-old infants has not yet been established. (22–24) and B cells (22, 25) are associated with protection against Comparison of cell-mediated activity or circulating cytokine malaria through the control of growth of blood parasite stages. levels in children and adults in different areas of malaria transmis- Although there has been some controversy as to the function of sion found Th1-like responses in childhood tending to a Th2-like NK T cells (26–29), it has been reported that NK cells play a response in adulthood (5–10). Similarly, analysis of IgG sub- crucial role during the early phase of infection (30–32). In a recent classes indicated a predominance of IgG1, IgG3, and IgE during work, we showed that the mouse model was not suitable to study adulthood, features of a Th2-like response (11–17). These studies the effect of age on the course of malaria infection because dif- ferent parasitological and clinical parameters were found age-in- dependent (33). However the course and outcome of a primary infection in the rat was clearly dependent on age (33), where an *Institut National de la Sante´et de la Recherche Me´dicale Unite´547, Institut Pasteur ϩ de Lille, Lille, France; †Laboratoire des Biopuces, Institut Pasteur de Lille, Lille, increase in the number of CD8 T cells and NK T cells was as- France; and ‡GE Healthcare, Piscataway, NJ 08855 sociated with the control of infection in adult rats. In contrast, high Received for publication March 29, 2006. Accepted for publication November levels of circulating IL-10 and persistence of CD4ϩCD25ϩ T cells 8, 2006. were observed in young susceptible rats. In vivo neutralization of The costs of publication of this article were defrayed in part by the payment of page ␥ charges. This article must therefore be hereby marked advertisement in accordance IL-10 or administration IFN- was unable to protect young in- with 18 U.S.C. Section 1734 solely to indicate this fact. fected rats from death (34). Nevertheless, susceptibility of young 1 This work was supported by Institut National de la Sante´et de la Recherche Me´di- rats can be reversed by adoptive spleen cell transfer from immune cale Unite´547 and Institut Pasteur de Lille. C.P. is a member of Institut Pasteur de adult rats, indicating that the cellular response present in the spleen Lille and J.K. is a member of Centre National de la Recherche Scientifique. of adult host after parasite resolution is capable of controlling in- 2 Current address: Endotis Pharma, Bioincubateur, Parc Eurasante´, 70 rue du Dr Yersin, 59120 Loos, France. fection in young host. 3 Address correspondence and reprint requests Dr. Jamal Khalife, Institut National de la The studies reported here were designed to define in more detail Sante´et de la Recherche Me´dicale Unite´547, Institut Pasteur de Lille, 1 rue du Prof. the effector cells responsible for the transfer of protective immu- Calmette, 59019 Lille, France. E-mail address: [email protected] nity to young host, and to investigate the genes expressed in spleen Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 cells of adult protected rats capable of transferring protection. www.jimmunol.org 1714 PROTECTION OF YOUNG SUSCEPTIBLE RATS FROM MALARIA

Adoptive transfer revealed that T cells obtained from adult im- Results are expressed as absolute number of cells within 150 ϫ 106 trans- mune rats partially protect young recipient rats, whereas microar- ferred spleen cells. ray analysis indicated the presence of a more efficient pathway Microarray analysis in protection that involves neutrophils and defensin neutrophil protein-1 (NP-1).4 Total RNA was extracted using RNAplus (Qbiogene) from spleen cells of protected adult rats that have been shown to transfer protection to young rats and from spleens of uninfected age-matched rats. To ensure RNA Materials and Methods quality of each sample, integrity and purity were assessed by use of the Parasites and animals Agilent Bioanalyser (Agilent Technologies). Ten micrograms of purified total RNA was then used for linear amplification following the CodeLink Plasmodium berghei ANKA strain (uncloned line from I. Landau, Museum Expression System Protocol (GE Healthcare). Ten micrograms of the d’Histoire Naturelle, Paris, France) used in this study was described pre- resulting cRNA was hybridized to each CodeLink Rat UniSet I Bioarray viously (34). This parasite strain was adapted to Fischer rats by at least following the CodeLink Gene Expression System Protocol without any three successive passages (by i.p. injection). Four- and 8-wk-old F344 rats modification. Each bioarray contains 9911 rat sequences and 300 negative were purchased from Harlan and raised in the specific pathogen-free ani- bacterial control probes. The bioarrays were scanned using an Axon mal facility of the Institut Pasteur de Lille. Plasmodium infections were ␮ 7 GenePix 4000B scanner at 635 nm, 10 m resolution and a PMT value performed by i.p. injection of 10 parasitized RBC. Tail vein blood was of 600. The resulting image was analyzed using the CodeLink Expres- used to measure parasitemia on thin film blood smears stained with eosin- sion Analysis Software version 2.3, and data from nonexploitable and thiazine (Diff Quick II kit; Dade Behring). The absence of other infections control spots were filtered out. including rodent viruses, bacteria, and parasites was checked in infected The mean intensity of data from the three noninfected animals was used animals by the International Council for Laboratory Animal Science Virus as reference, and intensities of each animal (infected and noninfected) were Reference Centre (The Netherlands) and the Centre de De´veloppement des

compared with this reference to calculate M and A values. The SNOMAD Downloaded from Techniques Avance´es pour l’Expe´rimentation Animale (France). Animal (Standardization and NOrmalization of MicroArray Data) package (35) work was conducted in accordance with the guidelines of laboratory animal was then used in the statistical language R (36) to normalize data. MA-plot care published by the French Ethical Committee. was drawn and the “global loess” function was applied to the data to cor- Spleen cell and neutrophil preparations and transfer rect for bias (37). Genes with absolute mean M value for noninfected rat above 0.5 were removed from analysis. M values of the remaining genes of experiments the four infected rats were analyzed using the Linear Model for Microarray Spleens were removed from infected adult rats and from uninfected age- Data library in R (38). A classification of statistically significant modula- matched controls 1 wk after parasite clearance (day 25 postinfection (p.i.)). tions was obtained using moderated t statistic with empirical Bayes shrink- http://www.jimmunol.org/ Total spleen cells were prepared in RPMI 1640 medium (Invitrogen Life age of the SE (39). Because of multiple testing, obtained p values were Technologies) supplemented with 10% heat-inactivated FCS (AbCys). corrected using the Benjamini and Hochberg method (40) to control the RBC were lysed by hyperosmotic treatment (0.2 M ammonium chloride, false discovery rate. 10 mM sodium carbonate, 0.1 mM EDTA buffer). After three washes in RPMI 1640, spleen cell viability was evaluated by trypan blue dye exclu- Confirmation of increased gene expression by quantitative sion. Neutrophils were recovered in the peritoneal cavity 4 h after i.p. PCR (Q-PCR) injection of 5 ml of 5% thioglycollate (Difco Laboratories). Examination of cytospins and flow cytometry analysis showed that peritoneal cells con- For Q-PCR analyses, cDNA were prepared from total RNA using a Su- tained a mean of 92% neutrophils, 4% eosinophils, and 4% . perscript II kit (Invitrogen Life Technologies), according to the manufac- turer’s recommendations. Q-PCR was conducted on an Applied Biosys- All cells were resuspended in RPMI 1640 and used in transfer experiments by guest on September 23, 2021 by i.v. route at the day of infection. Spleen cells from age-matched unin- tems 7000 PCR apparatus using SYBR Green dye (Applied Biosystems) to fected rats were prepared and used as control. quantify the products over the course of the amplification reaction. Primers were designed using Primer Express software (ABI Prism) and were se- Spleen T cell enrichment and depletions lected on the basis of their melting point and whether they produce an amplicon in the range of 120–150 bp. Primers pairs used for Q-PCR are T cells were purified from whole spleen cells prepared from adult rats at shown in Table I. The cDNA samples were diluted with sterile Dnase-free day 25 p.i. To this end, we used Rat T cell Enrichment Columns (R&D water and used at the equivalent of 40 ng of total RNA used in transcription Systems) based on high-affinity negative selection, and followed the man- for quantification of target transcript levels. For each primer/cDNA com- ufacturer’s recommendations. Enriched population was checked by flow bination, reactions were conducted in triplicate with controls including ϩ cytometry analysis and TCR␣␤ cells ranged between 85 and 90%. primers alone or primers/RNA. Q-PCR mixtures (25 ␮l) contained 2.5 ␮l Depletions of T and B cells from spleen cells of Fischer F344 rats were of cDNA, 7.5 pmol of both primers, and 12.5 ␮lof2ϫ SYBR Green PCR performed by positive selection. The spleen cell suspension was incubated Master Mix containing the ROX-1 dye labeled. Reactions were done as with mouse IgG1 mAbs R73 (anti-␣␤ TCR) or OX33 (anti-CD45RA, ex- follows: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min pressed mainly on B cells) (I. Anegon, Institut National de la Sante´etde at 60°C. Dissociation curves were conducted at the end of each run to la Recherche Me´dicale Unite´437, Nantes, France). Unlabeled cells were verify the absence of DNA contamination. All data were analyzed with purified using magnetic beads coated with rat anti-mouse IgG1 (Dynal ABI Prism 7000 SDS software (Applied Biosystems). The amplification Biotech). Quality of the depletion was checked by flow cytometry analysis. threshold was set at 0.2 and the background fluorescence was determined during cycle 5–10. The mean of raw cycle threshold (Ct) values obtained Flow cytometry for the control ␤-actin transcript was used to adjust the Ct values of the ⌬ Spleen cells were analyzed by one- or two-color fluorescence-activated cell gene of interest for each sample/primer combination ( CT). We calculated ⌬CT ⌬ ϭ sorting immunophenotyping, performed using FITC-, PE-, or biotin-con- the ratio of infected rat:control as a 2 value (where CT CT of Ϫ jugated mAbs: mouse anti-TCR (R-73), anti-CD45RA (OX33), anti-CD25 infected rat CT of control rat) for each gene to evaluate the fold increase. (OX39), anti- (His48), anti-macrophages (ED2-like, His36) (BD Pharmingen), anti-CD4 (W3/25), anti-CD8 (OX8), anti-CD172a Synthesis of rat defensin NP-1 and administration to young rats (OX41), anti-CD134 (OX40), and anti-NKR-P1A (clone 3.2.3) (I. Anegon, The active rat defensin NP-1 is composed of a mature peptide of 32 aa Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 437, (VTCYCRRTRCGFRERLSGACGYRGRIYRLCCR) (41, 42). Given that ϫ 5 Nantes, France). Spleen cells (5 10 cells) were stained for 30 min on ice the Cys residues present in the mature peptide are fully dispensable for the with these different Abs. After two washes in staining buffer (PBS with 2% function of defensin (43) and that they are known to be involved in the FCS), streptavidin-allophycocyanin was added for 10 min on ice to detect oxidation, dimerization, and solubility of synthetic peptides, we replaced biotinylated mAb. Fluorescence analysis was performed with CellQuest them by Ser residues. The synthetic mature peptide with the following software (FACScan; BD Biosciences) on a total of 10,000 acquired events. sequence (VTSYSRRTRSGFRERLSGASGYRGRIYRLSSR) was then produced using Fmoc solid-phase synthesis (Genepep). The peptide was purified to homogeneity (purity Ͼ98%) and the molecular mass was con- 4 Abbreviations used in this paper: NP-1, neutrophil protein-1; p.i., postinfection; Q-PCR, quantitative PCR; Ct, cycle threshold; PGRP, peptidoglycan recognition firmed by mass spectrometry. The peptide dissolved in PBS LPS free (1 protein; MMP9, matrix metalloproteinase 9; PRG2, 2; MBP, major mg/ml/rat) was injected (i.p.) on the day of infection and at day 6 postin- basic protein; NRAMP1, natural resistance-associated protein 1; Hp, fection with the same dose. As control, young rats were injected either with haptoglobin. PBS alone or a control peptide (AKFEVNNPQVQRAFNELIRVVHQL The Journal of Immunology 1715

Table I. List of primers used for Q-PCR

GenBank Primer Accession No. Gene Description Primer Sequence Orientation

NM_130741.1 Lipocalin 2 5Ј-CAgCTACgATgTgCAAgTggC-3Ј Forward 5Ј-CgCTCCTTCAgTTCATCggA-3Ј Reverse NM_031619.1 PRG2 5Ј-CTggATTggAggCAggATTg-3Ј Forward 5Ј-CCTCgggTACACAgggTCAC-3Ј Reverse U16686 Defensin ratNP-1 5Ј-CTgAggAggCTCCAgACCAg-3Ј Forward 5Ј-CACAgCgTgTgCgTCTgC-3Ј Reverse NM_053373.1 PGRP 5Ј-gTgACCACACAgggCCCA-3Ј Forward 5Ј-TCAggAAgCCCTCAgACACC-3Ј Reverse NM_031055.1 MMP9 5Ј-ATCACggAggAAgCCAATTg-3Ј Forward 5Ј-TgTACACCCACATTTTgCgC-3Ј Reverse NM_031537.1 NRAMP1 5Ј-ACAgTgCAACAACAAgCCCA-3Ј Forward 5Ј-TTAACACACgTCgTCTgTgCC-3Ј Reverse NM_012582.1 Hp 5Ј-ATATCgggCTgATCAAgCTCA-3Ј Forward 5Ј-gTAAATCTAAAgTTGACATTCCGCC-3Ј Reverse NM_031144 ␤-Actin 5Ј-gACggTCAggTCATCACTATCg-3Ј Forward 5Ј-ACggATgTCAACgTCACACTTC-3Ј Reverse Downloaded from

LPESSLRKRKRSR) under the conditions described above. In parallel, a Contribution of B and T cells in protection of young set of experiments was performed to evaluate the direct effect of defensin susceptible rats on blood parasites. To this end, 107 infected RBC were preincubated with 300 ␮g of defensin or vehicle for1hat37°C before infection. The course Next, we evaluated the contribution of B and T cells because they of infection was compared with that observed in rats administered with 300 represent the dominant population in the spleen of adult immune http://www.jimmunol.org/ ␮g of defensin i.p. at the day of infection.

Statistical analysis The Mann-Whitney U test for nonparametric data was used for statistical comparisons of cellular numbers in spleen cells from protected rats and from age-matched uninfected controls.

Results Protection of young susceptible rats by transfer of spleen cells by guest on September 23, 2021 of adult protected rats The course of parasitemia and survival in young infected rats (4 wk old at the beginning of experiment) that received spleen cells from adult rats prepared 1 wk after resolution of infection (day 25 p.i.) were monitored. All infected young rats receiving 150 ϫ 106 immune spleen cells controlled blood parasite growth and sur- vived with no parasite recrudescence (Fig. 1A), and protection ap- peared to be dose dependent as 10 ϫ 106 transferred cells did not protect, 50 ϫ 106 protected 50% of animals, and Ͼ80 ϫ 106 con- ferred protection to all rats (Fig. 1B). In contrast, infected young rats that received 150 ϫ 106 spleen cells from uninfected healthy adult rats were still susceptible to infection.

Analysis of cell distribution in spleen cells transferring protection The distribution of spleen cells transferring protection was inves- tigated and compared with cells from uninfected age-matched rats. A comparison of either cell percentages or absolute numbers of CD45RAϩ, CD4ϩ, CD8ϩ, neutrophils, and NK T with those of age-matched controls did not show any significant differences (Fig. 2A). However, the number of macrophages His36ϩ present in the FIGURE 1. Protection of young rats infected with P. berghei by i.v. spleen decreased in adult rats after parasite clearance. By contrast, transfer of spleen cells from adult protected rats (obtained at day 25 p.i.). A, Course of parasitemia observed in young infected rats after transfer of a significantly lower number of cells expressing CD25 was observed 6 ϩ 150 ϫ 10 spleen cells purified either from adult protected rats or from among the CD4 T cells of protected adult rats, when compared with ϩ ϩ control uninfected rats. Data are represented as mean parasitemia Ϯ SEM CD4 CD25 T cells in uninfected age-matched controls (Fig. (n ϭ 12 rats). B, Course of parasitemia and cumulative survival at day 2B). With regard to the expression of OX40, a marker of activated 25 p.i. (insert) of young infected rats that received 10 ϫ 106,50ϫ 106, T cells, we observed a significant higher number within the spleen 80 ϫ 106,or150ϫ 106 spleen cells, respectively. For clarity of the figure, cells transferring protection when compared with uninfected age- the SEs are not shown. Data are representative of two experiments (n ϭ 6 matched rats (Fig. 2B). in each group). †, Represents death of all rats. 1716 PROTECTION OF YOUNG SUSCEPTIBLE RATS FROM MALARIA

FIGURE 2. A, Cellular distribution of spleen cells from adult protected rats upon resolution of blood parasites. B, Distribution of CD4ϩCD25ϩ and T OX40ϩ cells. Results are presented as absolute number of each cell population present within 150 ϫ 106 spleen cells used for adoptive transfer experiments. f, Represents spleen cells from adult protected rats; s, represents spleen cells from uninfected age-matched control rats. Data (mean ϩ SEM) are of three independent experiments (n ϭ 6 .p Ͻ 0.001 ,ء .(in each experiment Downloaded from rats transferring protection. When using whole spleen cells de- pleted in B cells before transfer to young rats, parasitemia levels were still low and comparable to those observed for the parental whole spleen cells (Fig. 3). In both cases, as expected, all rats recovered from infection. With respect to the role of T cells, young http://www.jimmunol.org/ infected rats receiving 80 ϫ 106 cells depleted in T cells developed high parasitemia and 37% of rats survived to infection, suggesting a partial contribution of T cells in protection. To confirm this role, adoptive transfer experiments with enriched T cells from adult immune rats were performed. Transfer of whole spleen cells, as expected, dramatically and rapidly decreased the level of infected FIGURE 4. Protection of P. berghei-infected young rats after transfer of RBC and all rats survived (Fig. 4A). However, young rats receiv- immune T-enriched cells. Course of parasitemia (A) and survival rate (B) ing enriched T cells (Ͼ85% purity) exhibited high parasitemia ϫ 6 observed in young infected rats after transfer of 40 10 immune spleen by guest on September 23, 2021 similar to that observed in the infected control group. After 15 days T cells (proportion of T cells in 80 ϫ 106 of whole spleen cells) or 80 ϫ p.i., the parasitemia decreased in the young rats receiving T cells, and 106 whole spleen cells obtained from adult protected rats upon resolution 58% eliminated their parasites and survived while all infected control of blood parasites. ϫ, Represents young infected control rats. Data are rats died (Fig. 4B). The fact that parasitemia was high and required mean ϩ SEM (n ϭ 12). †, Represents death of rats. more time to be cleared in the young rats receiving purified T cells likely suggest an activation by blood parasites and/or a crosstalk with cells of recipients. All together, although these data suggest a partial role of T cell in the transfer of protection, they support the role of other spleen cells in the early control of parasite growth.

Gene expression profiling of spleen cells transferring protection to young rats Having observed a dramatic difference in the control of para- sitemia levels between the transfer of purified T cells and that of whole spleen cells, it is conceivable that a rapid T-independent mechanism may be involved in the protection of young rats. To investigate the molecular underlying mechanisms and thereby the cells involved, we conducted a microarray analysis of the mRNA of spleen cells from immune adult rats obtained 1 wk after reso- lution of infection (day 25 p.i.) and compared them with unin- fected rat spleen cells. For this purpose, we used the GE Health- care Codelink Uniset Rat array. Gene expression data generated from spleen cells of adult protected rats (day 25 p.i.) allowed the identification of a number of genes that are up- and down-regu- lated upon resolution of infection when compared with the tran- scriptome obtained from age-matched uninfected rats. A complete list of genes has been deposited in National Center for Biotechnol- FIGURE 3. T depletion, but not B depletion of spleen cells before trans- fer, affects course of parasitemia and survival rate of young recipient rats ogy Information’s Gene Expression Omnibus (GEO; http://www. infected with P. berghei. Course of parasitemia and cumulative survival ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series ac- rate at day 25 p.i. (inset) observed in young rats receiving whole spleen cession no. GSE4552. Genes with significant modulation of ex- Ͻ cells, T-depleted or B-depleted spleen cells obtained from adult protected rats. pression ( p value 0.02) and with a log2-ratio (M value) above Data are mean ϩ SEM (n ϭ 8 in each group). †, Represents death of rats. 2.0 or below Ϫ2.0 (fold equal to or less than Ϫ4orՆ4) are listed h ora fImmunology of Journal The

Table II. Genes for which transcript levels change in spleen cells from adult immune rats following a primary infection with P. bergheia

Up-Regulated Genes Down-Regulated Genes

Ratio Fold Ratio Fold GenBank Accession No. Description (log2) Change p GenBank Accession No. Description (log2) Change p Genes with known Genes with known functions functions NM_130741.1 Lipocalin 2 4.2 17.8 0.01956 Y17319 CDK110 Ϫ3.2 Ϫ9.4 0.01952 Ϫ Ϫ NM_031619.1 PRG2, bone marrow 4.0 16.1 0.01952 NM_031644.1 PGD2 synthase 3.0 8.1 0.01995 U16686 Defensin ratNP-1 precursor 3.9 14.6 0.01952 X13016 MRC OX-45 surface IL-1␤ Ag Ϫ2.9 Ϫ7.5 0.01956 NM_053373.1 PGRP 2.9 7.5 0.01952 NM_012762.1 IL-1␤ converting enzyme (CASP1) Ϫ2.7 Ϫ6.4 0.01956 NM_031055.1 Matrix metalloproteinase 9 (MMP9) 2.8 7.1 0.01952 NM_019299.1 Clathrin heavy polypeptide Ϫ2.4 Ϫ5.2 0.01956 NM_031537.1 NRAMP1 2.3 5.1 0.01952 NM_031570.1 Ribosomal protein S7 (RPS7) Ϫ2.3 Ϫ5.0 0.01952 NM_012582.1 Hp 2.0 4.1 0.01952 NM_012862.1 Matrix GLA protein Ϫ2.3 Ϫ4.9 0.01956 NM_057153.1 Oxidation resistance 1 (OXR1) Ϫ2.3 Ϫ4.8 0.01952 NM_017150.1 Ribosomal protein L29 (RPL29) Ϫ2.2 Ϫ4.5 0.01980 NM_012771.1 Ϫ2.1 Ϫ4.4 0.01956 D25233 Retinoblastoma protein Ϫ2.0 Ϫ4.0 0.01956 NM_031122.1 HSP70-Interacting protein (ST13) Ϫ2.0 Ϫ4.0 0.01995 NM_017042.1 Calcineurin subunit A ␤ Ϫ2.0 Ϫ4.0 0.01980 NM_019165.1 IL-18 Ϫ2.0 Ϫ3.9 0.01995 Genes with unknown Genes with unknown functions functions AW919304 3.1 8.3 0.01952 AW916836 Ϫ2.7 Ϫ6.4 0.01995 AI170665 2.0 4.1 0.01980 AI411510 Ϫ2.4 Ϫ5.4 0.01952 AA964289 2.0 4.0 0.01956 AA819268 Ϫ2.4 Ϫ5.2 0.01956 AW913878 Ϫ2.4 Ϫ5.2 0.01956 AW916119 Ϫ2.3 Ϫ4.8 0.01956 BE115635 Ϫ2.3 Ϫ4.8 0.01952 AA891872 Ϫ2.2 Ϫ4.6 0.01995 AI232085 Ϫ2.2 Ϫ4.5 0.01995 BE113190 Ϫ2.2 Ϫ4.5 0.01956 AW916093 Ϫ2.2 Ϫ4.5 0.01956 AA945100 Ϫ2.1 Ϫ4.4 0.01995 BF564940 Ϫ2.1 Ϫ4.4 0.01956 AA943974 Ϫ2.0 Ϫ4.1 0.01956 AI230596 Ϫ2.0 Ϫ4.0 0.01952 AW918457 Ϫ2.0 Ϫ4.0 0.01956 BF543478 Ϫ2.0 Ϫ4.0 0.01956 AW919873 Ϫ2.0 Ϫ3.9 0.01956

a Average fold changes of genes in spleen of adult immune rats (n ϭ 4 from two separate infection experiments) were calculated in comparison with uninfected age-matched rats (n ϭ 3). Complete list of genes are accessible at http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?accϭGSE4552.

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Table III. Q-PCR analysis of increased gene expression in spleen cells of adult protected rats

Description Q-PCR Fold Increasea

Lipocalin 2 10.5 Ϯ 1.5 PRG2 18.4 Ϯ 1.3 Defensin ratNP-1 13.4 Ϯ 1.4 PGRP 3.8 Ϯ 0.4 MMP9 6.7 Ϯ 1.1 NRAMP1 3.7 Ϯ 0.5 Hp 11.7 Ϯ 4.1

a Fold increase was calculated as described in Material and Methods. Values represent average fold increase Ϯ SEM with n ϭ 6 rats.

in Table II. We have grouped the genes as up-regulated (10 genes in the left column, Table II) or down-regulated (31 genes in the right column, Table II) and have delineated known and unknown genes. Six of the known increased genes are mainly expressed by Downloaded from neutrophils (lipocalin 2, NM_130741; peptidoglycan recognition protein (PGRP), NM_053373; ␣ defensin ratNP-1, U16686, matrix metalloproteinase 9 (MMP9), NM_031055), eosinophils (proteo- glycan 2 (PRG2), NM_031619, referred to as major basic protein (MBP)), and macrophages (natural resistance-associated macro-

phage protein 1 (NRAMP1), NM_031537). We also observed an http://www.jimmunol.org/ increase of haptoglobin (Hp) (NM_012582.1), an acute phase pro- tein described as a marker of inflammation, infection, and trauma. It is important to point out that ␤ 1 and 2 that are mapped closely to the ␣ defensin gene (44) were not overexpressed. This suggests that the increase of NP-1 is specific and is not due to a general boost of defensins. Interestingly, we observed an increased expression of lipocalin (Ͼ17-fold), secreted mainly by neutrophils, described to bind MMP9 to protect its activity. Intriguingly, many genes that are associated with T lymphocyte activation or cyto- by guest on September 23, 2021 kines were not found to be enhanced in spleen cells from adult protected rats when compared with healthy uninfected rats, even if FIGURE 5. Role of neutrophils in the transfer of protection to P. Ն we consider the threshold 2-fold. This result could likely be ex- berghei-infected young rats. A, Course of parasitemia and cumulative sur- plained by the fact that the transcriptional profiling was performed vival rate at day 25 p.i. (inset) observed in young infected rats after transfer after parasite clearance, during the recovery period p.i. of 35 ϫ 106 neutrophils (polymorphonuclear cell; PMN) obtained from Expression of mRNA for many enzymes was decreased 4- to adult protected rats or from uninfected age-matched controls. B, Course of 9-fold when compared with controls. As shown in Table II, we parasitemia and cumulative survival rate at day 25 p.i. (inset) observed in young infected rats after transfer of 20 ϫ 106 neutrophils (PMN) and 40 ϫ observed an 8-fold decrease of PGD2 synthase (NM_031644) that 6 ϫ can be linked, at least in part, to the decrease of CASP1 10 T cells alone or combined, obtained from adult protected rats. , ϩ ϭ (NM_012762) because the product of the latter (IL-1␤) was shown Represents young infected control rats. Data are mean SEM (n 6in each group). †, Represents death of rats. to up-regulate the expression of PGD2 synthase (45). Other down- regulated genes include one transcript associated with protection against oxidative stress (oxidation resistance 1, NM_057153). This gene is targeted to the mitochondria, suggesting that the function cells did not show significant differences (see Fig. 2A), suggesting of these organelles may be impaired following Plasmodium an increase of cell activity rather than cell number. infection. Role of neutrophils in protection of young rats Confirmation of increased genes by Q-PCR Transcriptome analysis indicated that neutrophils of adult immune The transcriptional changes of several genes reflecting the activa- rats may be one of the cell populations that may participate in the tion of myeloid cells, and particularly neutrophils, were evaluated early defense of young susceptible rats. For this reason, we eval- using real-time PCR on independently generated RNA samples uated the role of thioglycolate-elicited peritoneal neutrophils ob- (␤-actin was used as an internal control for each sample). Results tained from adult protected rats in protection. As shown in Fig. 5A, presented in Table III show that lipocalin, PGRP, defensin rat neutrophils transferred to young rats exerted a significant dose- NP-1, MMP9, eosinophils PRG2, NRAMP1, and Hp transcript dependent antiparasite effect because the parasitemia level was sig- levels were significantly higher in the spleen cells of protected nificantly decreased when compared with either rats that received adult rats when compared with uninfected age-matched controls. peritoneal neutrophils from uninfected rats or vehicle. The fol- The increase in gene expression intensity did not correspond to a low-up of survival revealed that 33 and 75% of young rats receiv- significant increase in neutrophil or numbers. Indeed, ing 20 or 35 ϫ 106 neutrophils, respectively, resolved the infec- differential spleen cell count using stained-cytospin preparations tion. The mortality was 75 and 100% in young rats that received (data not shown) or flow cytometry analysis of neutrophil His48ϩ 35 ϫ 106 neutrophils from uninfected rats or vehicle, respectively The Journal of Immunology 1719

regulated, and because defensins are known to play a key role not only in the defense against microbial infections but also against malaria parasite stages in the mosquito (46, 47). Administration of 2 ϫ 1 mg/rat NP-1 (days 0 and 6 p.i.) allowed young rats to control parasitemia levels rapidly and protected all rats from death (Fig. 6). A single injection of 300 ␮g/rat of synthetic NP-1 did not affect growth of blood parasites nor confer protection to rats from death. However, a preincubation of infected RBC with 300 ␮g of NP-1 before infection led to a significant increase of protection levels (Fig. 6B).

Discussion There is a critical need to establish approaches for effective and rapid protection from infection of young hosts, because their im- mune responses are immature. In this study, the combination of cellular approaches and gene profiling of the host response to ex- perimental malaria showed that the protection of young rats from lethal infection can be accomplished in two ways: 1) the transfer of

T cells obtained from spleen cells of adult protected rats after Downloaded from parasite resolution, and 2) the transfer of peritoneal neutrophils elicited by thioglycollate from adult protected rats or the admin- istration of NP-1, known to be an antimicrobial peptide. The latter finding has implications for young hosts with deficient immune system who may generate poor T cell immunity against intracel-

lular infections. http://www.jimmunol.org/ Experimental malaria studies performed exclusively in adult hosts indicated that both CD4ϩ and CD8ϩ T cells play an impor- tant role in the defense against malaria. To address the role of these cells in an age-dependent model, where young susceptible rats can be protected by the transfer of whole spleen cells from adult pro- tected rats, TCRϩ cells were transferred to young infected rats. These experiments indicated that 58% of young rats recovered from infection after T cell transfer. In this context, our results demonstrated that expression of OX40, a marker restricted to rat- by guest on September 23, 2021 FIGURE 6. Synthetic rat defensin NP-1 protects young rats from a le- activated CD4ϩ T cells (48), was significantly increased in the thal infection with P. berghei. Course of parasitemia (A) and survival rate spleen of protected rats. The OX40 molecule is known to bind to (B) observed in young infected rats after administration of synthetic de- rat splenic B cells and dendritic cells via OX40L and to induce fensin. Each rat received at day 0 and day 6 p.i. 1 mg of defensin or control proliferation and IL-2, IFN-␥, IL-10, and IL-13 production (49), peptide (indicated by arrows). Survival rate of rats infected with P. berghei suggesting a possible involvement in cellular and humoral immune parasites preincubated with 300 ␮g of defensin1hat37°C was compared with that observed in rats administered with 300 ␮g of defensin i.p. at responses in the rat system. In parallel to this increase, analysis of B ϩ n ϭ CD25 cells, a marker for the regulating phenotype, showed that the the day of infection ( ). Data are mean SEM ( 6 in each group). ϩ ϩ †, Represents death of rats. CD4 CD25 cells were decreased in the spleen of adult protected rats when compared with uninfected age-matched rats. Together, these data suggest that a down-regulation of CD4ϩCD25ϩ along (Fig. 5A, inset). The role of other contaminating cells was excluded with an increase of OX40ϩ T cells in adult protected rats may be because the transfer of peritoneal cells from immune rats without involved, at least in part, in the transfer of protection to young rats. thioglycolate induction did not control the parasitemia levels (data Although the transfer of enriched T cells can protect young rats, not shown). surprisingly the parasitemia levels and the time required for par- Next, we conducted adoptive cotransfer of neutrophils and T asite clearance by these cells greatly differed from that obtained cells to see whether the protection levels observed with each cell after total spleen cell transfer. The slow and partial effect of the population may be enhanced. As can be seen from Fig. 5B, para- transfer of purified T cells may be attributed to a delay of activa- sitemia of cotransferred recipients was undetectable until day tion of the immune system, suggesting that they play the role of 13 p.i., followed by a low level of blood parasites between day 13 delivery vector to induce cell-mediated immunity of young recip- and 21 p.i. and a total clearance of parasites thereafter. Under the ient rats. Another explanation for this difference could be the pos- same conditions, young rats transferred with either neutrophils or sible implication of other cells that act more rapidly and provide a T cells were only partially protected (Fig. 5B, inset). These data quick control of blood parasite growth. The participation of B cells demonstrate that neutrophils together with T cells can actively or NK/NK T cells (data not shown) can be excluded because their control the early parasite growth and prevent rats from death. depletion before transfer did not affect the protection of young infected rats. These results contrast with cells required to confer Role of synthetic defensin NP-1 in protection of young rats resistance in infected adult mice (20, 21) and emphasize the need To further investigate the mechanism of protection at the molec- for caution when extrapolating immune mechanisms from adult to ular level, we decided to examine the effects of defensin NP-1 on young hosts. a lethal P. berghei challenge in young rats. This was motivated by The comparison of the transfer of T cells with that of whole the fact that the expression of rat defensin NP-1 mRNA was up- spleen cells from adult protected rats suggests that other cells than 1720 PROTECTION OF YOUNG SUSCEPTIBLE RATS FROM MALARIA

T cells are involved in the rapid protection of parasite growth and Interestingly, it has been shown that eosinophil proteins protection of young susceptible rats. At this stage, it is reasonable including eosinophil cationic protein and MBP are toxic for P. to propose that the nonlymphoid compartment of the spleen con- falciparum parasites (66). Subsequent studies revealed that MBP taining myeloid cells could be able to confer protection in adoptive together with hemozoin, produced in malarial infection, can di- transfer to young rats. Based on the genome-wide analysis of ex- rectly activate neutrophils (67, 68). With respect to Hp that is pression in spleens of adult protected rats, it appears that neutro- produced by different cell types including macrophages and eosin- phils may be involved in the transfer of an early control of blood ophils (69), there are several arguments that well support its role in parasites in young rats because several related genes were up- the transfer of protection. First, it has been shown in vitro to have regulated. The activation of neutrophils in infected rats is well a deleterious effect on the growth of P. falciparum (70). Second, its supported by a recent study in which transcriptome analysis of role in vivo for antiparasitic effect comes from experiments show- whole blood from humans infected with P. falciparum identified a ing that blood parasites burden were significantly greater in Hp gene expression profile related to neutrophil activity (50). Al- knock-out mice compared with wild-type mice (71). Finally, stud- though neutrophilia has been linked with acute malaria (51), their ies investigating its relationship with immune response revealed role in host defense to malaria has been relatively underinvesti- that Hp plays a modulating role on the Th1/Th2 balance by gated (52–54). To clarify the role of neutrophils in adoptive im- promoting a dominant Th1 cellular response (72), which is de- munity of infected rats, thioglycollate-elicited neutrophils from scribed to participate in defense against intracellular parasites. adult protected rats were used in transfer experiments. First, the Combining the in vivo data presented in this study, together transfer of these cells to adult-infected rats contributed to the con- with those obtained from human in vitro studies, it can be trol of parasitemia growth, suggesting an antiparasitic effect of strongly suggested that both neutrophils and eosinophils partic- Downloaded from these neutrophils (data not shown). With respect to young rats, ipate in natural immunity to human malaria until the adaptive adoptive transfer of these cells kept the parasitemia levels very low immune system becomes effective. and allowed to resolve the infection in 33–75% of rats according In summary, this study has demonstrated the efficient way to to the number of neutrophils administered. It is noteworthy that protect young rats infected with a lethal Plasmodium strain. It has the i.v. coadministration of neutrophils together with T cells provided us with specific insight on the role of neutrophils and

significantly increased the survival rate of young rats from fatal their effector proteins in the control of early phase blood par- http://www.jimmunol.org/ infection when compared with single administration of each cell asite growth, very likely associated with T cells to clear the population. This result suggests that donor T cells and donor infection. Further investigations during infection are now nec- neutrophils act, at least additively, to increase the protection. In essary to elucidate at the molecular levels why young rats ex- this context, neutrophils have been shown to prime in vivo ef- hibit heavy parasite burden leading to a fatal outcome, whereas fector T cells in response to bacterial Ags that are not directly adult rats control parasitemia levels and resolve completely the accessible to the classical presentation pathway (55). infection. The gene expression intensity showed a significant increase of NP-1, referred to as defensin. Many studies have shown that par- Acknowledgments asites are susceptible to mosquito defensins, dermaseptin S3, and We thank Dr. Jean Langhorne, Prof. David Dunne, and Dr. Laurent by guest on September 23, 2021 some synthetic peptides designed on the known structure of natural Renia for their comments and critical reading of the manuscript and (56, 57). In this study, we investigated the C. Godin for his technical assistance. capacity of synthetic defensin NP-1 to protect young rats from a lethal infection. Its injection revealed that all treated young rats Disclosures significantly reduced the peak of parasitemia, cleared total blood The authors have no financial conflict of interest. parasites within 3 wk, and survived the infection. Defensins are References cytotoxic peptides and are believed to permeabilize the membranes 1. Hunt, D. W., H. I. Huppertz, H. J. Jiang, and R. E. Petty. 1994. 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