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Malaria-Associated L-Arginine Deficiency Induces Mast Cell-AssociatedDisruption to Intestinal Barrier Defenses against NontyphoidalSalmonella Bacteremia

Jennifer Y. Chau,a Caitlin M. Tiffany,a Shilpa Nimishakavi,b Jessica A. Lawrence,a Nazzy Pakpour,a Jason P. Mooney,a

Kristen L. Lokken,a George H. Caughey,b Renee M. Tsolis,a Shirley Luckharta

Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, California, USAa; Cardiovascular Research Institute andDepartment of Medicine, University of California at San Francisco and Veterans Affairs Medical Center, San Francisco, California, USAb

Coinfection with malaria and nontyphoidal Salmonella serotypes (NTS) can cause life-threatening bacteremia in humans. Coin-fection with malaria is a recognized risk factor for invasive NTS, suggesting that malaria impairs intestinal barrier function.Here, we investigated mechanisms and strategies for prevention of coinfection pathology in a mouse model. Our findings revealthat malarial-parasite-infected mice, like humans, develop L-arginine deficiency, which is associated with intestinal mastocyto-sis, elevated levels of histamine, and enhanced intestinal permeability. Prevention or reversal of L-arginine deficiency bluntsmastocytosis in ileal villi as well as bacterial translocation, measured as numbers of mesenteric lymph node CFU of noninvasiveEscherichia coli Nissle and Salmonella enterica serotype Typhimurium, the latter of which is naturally invasive in mice. Dietarysupplementation of malarial-parasite-infected mice with L-arginine or L-citrulline reduced levels of ileal transcripts encodinginterleukin-4 (IL-4), a key mediator of intestinal mastocytosis and macromolecular permeability. Supplementation with L-citrul-line also enhanced epithelial adherens and tight junctions in the ilea of coinfected mice. These data suggest that increasing L-arginine bioavailability via oral supplementation can ameliorate malaria-induced intestinal pathology, providing a basis for test-ing nutritional interventions to reduce malaria-associated mortality in humans.

Half of the global population is at risk for malaria, which resultsin nearly 1 million deaths annually, 86% of which are of chil-

dren (1). The majority of cases are in sub-Saharan Africa, wherethere is a high prevalence of coinfection with nontyphoidal Sal-monella serotypes (NTS) during the rainy season (2–5). Whileinfections with NTS are normally self-limiting in immunocompe-tent children, coinfection with malaria can predispose to the de-velopment of deadly NTS bacteremia (6–9). During malaria infec-tion, sequestration of parasitized red blood cells (RBCs) andcapillary blockage are prominent in intestinal villi (10) and areassociated with ischemia, malabsorption, and increased gastroin-testinal (GI) permeability (11, 12). These phenomena are not re-stricted to severe malaria; up to 50% of Nigerian children withuncomplicated malaria have GI disturbances (13). The mecha-nisms that underlie malaria-associated GI pathology and enhancethe risk of bacteremia are incompletely understood (14), althoughrecent data indicate that malaria-induced heme oxygenase-1(HO-1) contributes to impaired resistance to NTS by reducing theproduction of reactive oxygen species (15). Beyond these findings,knowledge is limited and therapeutic options for coinfection arefew in the face of high antibiotic resistance in areas of malariaendemicity (16). To support the development of novel therapeuticinterventions for invasive bacterial disease, we developed a mu-rine model of coinfection with Plasmodium yoelii and the NTSstrain Salmonella enterica serotype Typhimurium ATCC 14028. Inthis model, coinfected mice have higher levels of S. Typhimuriumin their mesenteric lymph nodes, spleens, and livers than do miceinfected with S. Typhimurium alone (17). Based on the sum of ourobservations, this model recapitulates enhanced bacterial translo-cation from the gut, an important feature of pediatric NTS-ma-laria parasite coinfection.

Several features of malaria pathology suggest that the increased

risk of developing bacteremia during malaria parasite and NTScoinfection results from malaria-induced damage to the intestinalepithelium. Patients infected with Plasmodium falciparum sufferfrom increased GI permeability during the acute and late stages ofinfection (12). A hallmark of malaria parasite infection, hy-poargininemia, may contribute to this pathology. During infec-tion, L-arginine bioavailability drops due to enhanced destructionby host- and parasite-encoded arginases, utilization by host nitricoxide synthase (NOS), and increased scavenging of NO by cell-free hemoglobin from lysed RBCs (18–20). Reduced bioavailabil-ity of L-arginine is predicted to potentiate allergic inflammation(21), which can be defined by altered epithelial permeability andrepair, mast cell activation, and prostaglandin E2 deficiency.These physiological changes have been associated with P. falcipa-rum infection (11, 12, 22, 23), suggesting that malaria-associatedL-arginine deficiency potentiates allergic inflammation intesti-nally and perhaps systemically.

Mast cells are the major source of histamine (24, 25), whichrises in plasma during parasite infection (26–28), consistent withmast cell activation. Mast cells are also primary regulators of theintegrity and function of the intestinal epithelial barrier (29), andthis biology is dependent on inducible changes to intracellular

Received 25 March 2013 Returned for modification 30 April 2013Accepted 3 May 2013

Published ahead of print 20 May 2013

Editor: J. H. Adams

Address correspondence to Shirley Luckhart, [email protected].

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00380-13

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tight junctions (TJs) that are comprised of occludin and claudinsthat form scaffolds with cytoplasmic zonula occludens 1 and 2(ZO-1 and ZO-2, respectively) and to adherens junctions that aredefined principally by E-cadherin. In the most prominent modelsfor infection-associated mast cell regulation of the intestinal bar-rier, specifically, GI helminth infection in mice, mucosal mast cells(MMCs) are recruited in response to interleukin-4 (IL-4) (30, 31)and then traffic from the submucosa to the villus tips and return tothe crypts once infection is resolved (32). This movement is ac-companied by stereotypical changes in expression of mast cellproteases (MCPs) (32). MCPs can function directly to regulatecytokine levels (e.g., IL-6 and tumor necrosis factor alpha [TNF-�]) by enzymatic degradation (33–36) and can also induce theredistribution of occludin and ZO-1 to alter the intestinal barrier(37, 38).

Hypoargininemia in malaria has been targeted previously fortherapeutic intervention and has been considered for clinical de-velopment for treatment of severe malaria. In particular, intrave-nous (i.v.) administration of L-arginine can improve endothelialfunction in the context of malaria, perhaps due to reduced endo-thelial activation and parasite sequestration (39). However, i.v.administration of L-arginine requires medical support facilitiesand is difficult under field conditions. In contrast, oral supple-mentation is noninvasive and has been used to increase nutrientuptake, improve recovery from intestinal ischemia, enhancewound healing, and improve intestinal barrier function (40–43).A recent meta-analysis of 54 clinical trials involving perioperativeoral L-arginine therapy reported substantial decreases in hospitalstays and infectious complications, including sepsis (44).

Based on these observations, we proposed that low L-argininebioavailability during malaria infection potentiates mucosal mas-tocytosis and consequent degradation of intestinal epithelial junc-tions by mast cell proteases, yielding a leaky gut that facilitatestransepithelial transit of enteric bacteria. In support of our hy-potheses, we show here that oral L-arginine and L-citrulline sup-plementation of P. yoelii-infected mice reduces translocation ofnonpathogenic Escherichia coli Nissle and S. Typhimurium, re-duces ileal expression of IL-4 and MMC recruitment to ileal villi,and enhances epithelial junction morphology. These data suggestthat oral supplementation to increase L-arginine bioavailabilitymay be a useful strategy to reduce intestinal allergic inflammationand the risk of NTS bacteremia in children with malaria.

MATERIALS AND METHODSAnimals. Six- to 8-week-old female CBA/J mice were purchased fromJackson West. Female CD-1 mice were purchased from Harlan Laborato-ries. All mice were maintained in standard cages and received standardrodent chow (PMI lab chow number 5001) and sterile drinking water adlibitum, which were supplemented according to experiment. All micewere monitored daily, and signs of morbidity or unusual weight loss led toeuthanasia and exclusion from the study. All animal experiments werereviewed and deemed to be in accord with all relevant institutional poli-cies and federal guidelines by the UC—Davis Institutional Animal Careand Use Committee.

Parasites and bacteria. Stocks of P. yoelii nigeriensis (ATCC Malariaand Research Reference Reagent Resource) and P. yoelii yoelii 17XNL(kindly provided by A. Rodriguez) were expanded in CD-1 mice. Exper-imental mice were inoculated on day 0 with 0.1 ml of CD-1 RBCs (unin-fected controls) or with RBCs intraperitoneally infected with 107 para-sites. Studies included oral inoculation with E. coli Nissle, which isresistant to streptomycin and ampicillin, and Salmonella enterica serotype

Typhimurium strain IR715(pHP45�), which is resistant to streptomycin,ampicillin, and nalidixic acid. For coinfection, mice received 20 mg ofstreptomycin intragastrically at 9 days following malaria infection, whichwas 24 h prior to bacterial inoculation. Mice were inoculated by gastricgavage 10 days after parasite infection with 0.1 ml of sterile Luria-Bertani(LB) broth (as a control) or with 0.1 ml of an overnight culture (37°C) of108 CFU of E. coli or S. Typhimurium grown in LB broth.

Water supplementation with amino acids. Mice supplemented withL-arginine (Sigma-Aldrich) received 2.5% L-arginine and 1% sucrose insterile drinking water daily starting 3 days prior to malaria parasite infec-tion or 10 days after parasite infection. Control mice for L-arginine sup-plementation received 1% sucrose in drinking water beginning 3 daysprior to parasite infection or 5% L-alanine and 1% sugar in drinking water3 days prior to parasite infection or 10 days after parasite infection. Micesupplemented with L-citrulline (Kyowa Hakko USA, Inc., Sigma-Aldrich)received 2% L-citrulline in sterile drinking water daily starting 3 days priorto malaria parasite infection or 10 days after parasite infection. Controlmice received unsupplemented, sterile drinking water or 3% L-alanine insterile drinking water starting 3 days prior to malaria parasite infection or10 days after parasite infection. All solutions were sterilized by passagethrough a 0.2-�m filter (Nalgene; Thermo Fisher Scientific).

Histopathology of ileum. Samples were collected for histopathologyat the time of necropsy. Tissues were formalin fixed and embedded inparaffin. From these tissue blocks, 5-�m sections were cut, deparaffinizedin xylene, rehydrated in graded solutions of alcohol, and stained withtoluidine blue. Additional sections were subjected to enzyme histochem-ical staining to identify cells with naphthol AS-D chloroacetate esterase(NASDCE) activity, which detects chymases in mast cell secretory gran-ules (45). For each mouse examined, mast cells were enumerated for anaverage of 6 ileal villi in different fields using a Nikon Eclipse E800 micro-scope at a �20 magnification.

Immunofluorescence staining of ZO-1 and E-cadherin in the ileum.Unstained 5-�m sections of paraffin-embedded mouse ileum were placedon microscope slides, treated with xylene to remove paraffin, and thentreated with graded alcohol solutions to rehydrate the tissues. To enhanceantigen retrieval, the slides were boiled in 1 mM citrate buffer (0.1 M citricacid and 0.1 M trisodium citrate-dihydrate). Slides were incubated in 5%skimmed milk to block nonspecific binding sites. After a series of phos-phate-buffered saline (PBS) washes, primary antibodies or isotype con-trols (1:100 rabbit anti-ZO-1, 1:50 monoclonal mouse anti-E-cadherin,1:100 rabbit primary isotype control, 1:50 mouse primary isotype control;Invitrogen, Abcam) were added, and the mixtures were incubated over-night in a humidified chamber. Following this incubation, antibody solu-tion was removed with PBS washes and fluorochrome-labeled secondaryantibodies (1:150 donkey anti-rabbit Alexa Fluor 488 or 1:150 chickenanti-mouse Alexa Fluor 594; Invitrogen) were applied, and these mixtureswere incubated for 1 h in a humidified chamber in the dark. DAPI (4=,6-diamidino-2-phenylindole) was used as a nuclear counterstain. After afinal PBS wash, the coverslips were mounted with ProLong Gold antifadereagent (Invitrogen). Slides were viewed using an Olympus FluoViewFV1000 confocal microscope (Olympus) with a 40� objective. OlympusFluoView FV1000 software was used to analyze stained tissue. Five fieldswere viewed per slide on 3 slides per group of mice.

Microbial readouts for infection. Daily percentages of parasitemiawere calculated beginning 2 days following P. yoelii infection by countingthe number of infected RBCs and dividing this number by the total num-ber of RBCs in thin blood films stained with Giemsa (Sigma-Aldrich). Inorder to determine the numbers of viable E. coli Nissle or S. Typhimuriumcells, cecal or colon contents (to confirm equivalent colonizations or in-fections) and mesenteric lymph nodes (to assess bacterial translocation)were aseptically removed and homogenized in cold PBS using an UltraTurrax T25 basic mixer (IKA Works, Inc.). Serial dilutions of the homog-enates were plated on selective media (LB agar plus ampicillin or LB agarplus nalidixic acid) and grown overnight at 37°C. Bacterial colonies werecounted to determine numbers of CFU per gram of tissue.

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Quantification of tissue and plasma histamine levels. Blood col-lected by cardiac puncture was placed into Microtainer tubes with EDTA(BD Biosciences) and spun at 1,600 � g for 20 min, and plasma wascollected and stored at �20°C. Plasma histamine concentrations weredetermined by histamine enzyme immunoassay (Cayman Chemicals) perthe manufacturer’s instructions. Ileum tissue samples were snap-frozen inliquid nitrogen and stored at �20°C. Samples were then sonicated in 0.1M perchloric acid and spun at 9,500 � g for 10 min, and supernatants werebrought to pH 7 with 1.5 M NaOH. Tissue histamine concentrations weredetermined by histamine enzyme immunoassay (Oxford Biomedical Re-search) per the manufacturer’s instructions.

RNA extraction and quantitative real-time PCR for Il-4 and Gapdh.At the time of necropsy, tissues were flash frozen in liquid nitrogen andstored at �80°C. RNA was extracted from tissue using TRIzol reagent(Invitrogen) according to the manufacturer’s protocol. To remove copu-rifying genomic DNA, all RNA samples were treated with Turbo DNase(Invitrogen). cDNA was synthesized from RNA using a QuantiTect re-verse transcription kit according to the manufacturer’s instructions (Qia-gen). cDNA samples were preamplified using Advantage 2 DNA polymer-ase mix (Clontech) with 5 �l 10� Advantage 2 buffer, 2 �l 10 mMdeoxynucleoside triphosphate (dNTP) mix, 1 �l 50� Advantage 2 poly-merase mix, 0.41 �l of each TaqMan probe-based gene expression assaymixtures for mouse Il-4 or Gapdh (Applied Biosystems, Carlsbad CA),and 20 �l of a cDNA sample normalized to 100 ng/�l (NanoDrop Tech-nologies, Wilmington, DE) and brought up to a final volume of 50 �l withmolecular-grade water. PCR preamplification was performed on anGeneAmp 9700 PCR system (Applied Biosystems) with temperature cy-cling conditions as follows: 94°C for 1 min, 25 cycles of 94°C for 15 s, 55°Cfor 15 s, and 70°C for 45 s, and then 70°C for 5 min. Real-time PCR wasperformed with TaqMan probe-based gene expression assays for mouseIl-4 (Applied Biosystems; efficiency, 91%) and mouse Gapdh (AppliedBiosystems; efficiency, 93.6%) using a 7900HT fast real-time PCR instru-ment (Applied Biosystems). Reaction mixtures of 12 �l containing 6 �luniversal master mix (Applied Biosystems), 0.2 �l of a TaqMan probe-based gene expression assay mixture, 0.8 �l of molecular-grade water, and5 �l of preamplified cDNA (normalized to 100 ng/�l) were analyzed intriplicate to confirm uniform amplification using the following cyclingconditions: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15s and 60°C for 1 min. The comparative threshold cycle (CT) method wasused to analyze data and target gene transcription levels of each samplenormalized to levels of Gapdh mRNA and are represented as fold changesover the gene expression in control animals.

Lactulose-mannitol assay for intestinal permeability. For these stud-ies, mice were either infected with P. yoelii-infected RBCs or mock in-fected with uninfected RBCs. At 2, 4, 6, 8, 10, and 12 days after parasiteinfection, mice were administered 0.25 ml of 20-mg/ml lactulose and50-mg/ml mannitol in sterile water via gavage needle. Mice were fasted for4 h prior to sugar solution oral gavage. Urine was collected from individ-ual mice beginning 1 h postgavage and continued hourly for the next 24 hin Eppendorf tubes containing 100 �l paraffin oil (to prevent desiccation)and 100 �l 10% thymol (antimicrobial and preservative) and then storedat �80°C. Mice were restrained by the dorsum scruff method, and gentlepressure was applied to their abdomen until urine was expelled. Urinesamples were obtained by placing the Eppendorf collection tube directlyover the urogenital area, avoiding the anal region. Any samples that cameinto contact with feces was discarded and not used. Urine samples werediluted in acetonitrile and centrifuged, and the supernatant was furtherdiluted with acetonitrile. Samples were analyzed for lactulose and manni-tol as described previously (46) using a liquid chromatography/mass spec-trometry system consisting of a Q-Trap 2000 triple-quadrupole/ion trapmass spectrometer (Applied Biosystems).

Serum amino acid analysis. Whole blood was collected from miceupon necropsy, and collected serum was precipitated with sulfosalicylicacid. The resulting supernatant was filter sterilized and analyzed using the

L-8900 Hitachi analyzer (Hitachi High-Tech) in a lithium citrate buffersystem as described previously (47, 48).

Statistical analyses. All data were analyzed using GraphPad InStat(GraphPad Software, Inc.). Normality was determined using the Kolm-ogorov-Smirnov test. Differences among groups with regard to CFU(bacterial translocation) and mast cell quantitation were determined byone-way analysis of variation (ANOVA) followed by Tukey’s multiple-comparison test or the Newman-Keuls test. Gene expression in tissuefrom supplemented mice and unsupplemented controls was analyzed bythe Wilcoxon signed-rank test or one-sample Student t test. P values of0.05 or less were considered significant.

RESULTSPlasmodium yoelii-infected mice exhibit early and sustained in-testinal permeability and hypoargininemia. For these and theremaining studies, mice were infected with P. yoelii nigeriensis(N67) (17) or with P. yoelii yoelii 17XNL (kindly provided by A.Rodriguez). Although these closely related subspecies differ ingrowth rate (N67’s growth is fast; 17XNL’s is slow) and lethality(N67 is lethal; 17XNL is nonlethal), infections with these subspe-cies yield peak parasitemias in inbred (C57BL/6) and outbred(CD-1, Kunming) mice by 11 days postinfection (49) and inCBA/J mice by 10 days postinfection. Neither parasite was lethal inCBA/J mice. Hence, we refer to these parasites as P. yoelii in thetext and identify subspecies in the figure legends.

To monitor intestinal permeability in mice infected with P.yoelii over a time course that captured rising and peak parasitemia(2 to 10 days) and at a point during declining parasitemia (12days), we quantified the ratio of excreted lactulose to excretedmannitol (L:M) in urine following oral gavage (50), a protocolanalogous to that used to establish increased intestinal permeabil-ity in P. falciparum-infected humans (12). In the intestine, lactu-lose passes through TJs and extrusion zones of the intervillousspaces, whereas mannitol crosses the cell membrane. Therefore, aloss of mucosal integrity should increase lactulose absorption,while loss of absorptive area should decrease the passage of man-nitol (51). At all times postinfection, P. yoelii-infected mice hadgreater L:M ratios in urine than did uninfected controls, withacute increases at 2 days and 4 days postinfection that persisted in30 to 40% of the mice for 10 days (Fig. 1A).

To assess hypoargininemia, we quantified L-arginine in the seraof P. yoelii-infected mice over time. Mean serum L-arginine waslowest at 2 days postinfection, temporally consistent with the ob-served acute increase in permeability (Fig. 1A), and never recov-ered to levels observed in uninfected mice (Fig. 1B).

Mast cells are recruited to the ileum during P. yoelii infec-tion. To determine whether our infected-mouse phenotype wasconsistent with observations of allergic inflammation in othermodels (52, 53) and with patterns of increased histidine/hista-mine levels during P. falciparum infection (26–28), we examinedmarkers associated with basophil and mast cell activation in P.yoelii-infected mice. Serum histidine levels were elevated relativeto those in uninfected controls at the earliest detection of periph-eral parasitemia (2 days postinfection) (Fig. 2A), increasing morethan 2-fold by 10 days postinfection (Fig. 2A). Plasma histaminewas similarly elevated postinfection (Fig. 2B). Consistent with theobserved elevation of histamine levels, increased densities ofmetachromatic (toluidine blue) MMCs (Fig. 3A) were evident inthe ileal villi and crypts of parasite-infected mice from the earliestdetection of parasitemia at 2 days, with the highest densities at 10days (Fig. 3B), when mean parasitemia peaked at �25% of RBCs

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infected. Given that the rise in ileal MMCs was positively corre-lated with rising parasitemia and elevated numbers of transcriptsencoding IL-4 in the same tissue (Fig. 3C), we examined whetherMMC activation was similarly evident in mice with a lower meanpeak parasitemia (�9%). At lower peak levels of parasitemia, thelevels of early signals from circulating parasites were reduced rel-ative to those associated with a higher peak parasitemia (Fig. 3D).As with increased ileal MMCs in mice with 25% peak parasitemia(Fig. 3B), mice with 9% peak parasitemia exhibited increasednumbers of NASDCE-positive MMCs at 2 days and 4 days in boththe villi and the crypts relative to those in uninfected mice, al-though patterns through 14 days were more variable (Fig. 3E) thanthose observed in mice with higher peak parasitemia (Fig. 3B). Inaddition, mice with 9% peak parasitemia tended to have highernumbers of crypt MMCs than villus MMCs (Fig. 3E), unlike micewith 25% peak parasitemia, which had comparable levels of villusand crypt MMCs (Fig. 3B). Given that MMCs traffic from thesubmucosa to the villus tips and return to the crypts when hel-minth infection is resolved (32), we reasoned that the rise in villusMMCs at 2 and 4 days postinfection was associated with increasedhistamine levels in the ileum. Indeed, histamine levels in the ilea ofmice from Fig. 3E were significantly elevated relative to those ofcontrols from 2 to 10 days postinfection (Fig. 3F).

Oral supplementation with L-arginine reduces E. coli Nissletranslocation and mucosal mast cell activation during P. yoeliiinfection. Given that hypoargininemia, increased intestinal per-meability, and mucosal mastocytosis were coincident in P. yoelii-infected mice, we reasoned that oral supplementation with L-argi-nine could reverse these phenomena if they were causally linked.To examine the effect of L-arginine supplementation on entericbacterial translocation in P. yoelii-infected mice, we first used thehuman commensal E. coli strain Nissle (54). Unlike S. Typhimu-rium, this commensal probiotic bacterial strain is nonpathogenicand noninvasive in the mouse and, therefore, provided an inde-pendent assessment of intestinal permeability. Two different sup-plementation regimens were used for the 14-day parasite infectionperiod to determine how effects varied with treatment timing andduration. One group received water ad libitum with 2.5% L-argi-nine (and 1% sucrose to mask the bitter taste) from 3 days prior toP. yoelii infection through 14 days, while the second group re-ceived the same supplemented water starting 10 days after parasiteinfection. Both groups were orally inoculated with E. coli Nissle at10 days postinfection, when P. yoelii-infected mice had a meanparasitemia of �17%.

Relative to controls (water only, 1% sucrose in water), para-site-infected mice provided with L-arginine-supplemented waterfrom 3 days prior to malaria infection had reduced numbers of E.coli Nissle CFU in their mesenteric lymph nodes, while micestarted on supplementation at 10 days after malaria infection hadno detectable CFU in their mesenteric lymph nodes (Fig. 4A). Thiswas particularly notable in light of the fact that probiotic E. coliNissle has been observed to upregulate ZO-1 expression and en-hance intestinal barrier function (55). In addition, ileal Il-4 ex-pression relative to that in controls was reduced by 75% in mice

FIG 1 Plasmodium yoelii infection is associated with increased intestinal perme-ability and hypoargininemia. (A) Lactulose/mannitol (L:M) ratios in urine fromuninfected (UI) mice (white circles) and from P. yoelii nigeriensis-infected micefrom 2 to 12 days postinfection (PI). Means are indicated as bars within eachtreatment group. Percentages are the proportions of infected mice with L:Mratios that exceeded the highest L:M ratio (dashed line) in uninfected mice.Each circle indicates the value for 1 mouse. (B) L-Arginine concentrations insera of uninfected mice (white circles) and P. yoelii nigeriensis-infected mice(black circles) from 2 to 13 days postinfection.

FIG 2 Plasmodium yoelii infection is associated with increased serum histidineand increased plasma histamine. (A) Histidine levels in sera of uninfected (UI)mice (white circles) and P. yoelii nigeriensis-infected mice (black circles) overtime. Each circle indicates the value for 1 mouse; means of each treatmentgroup are indicated with bars. (B) Histamine levels in plasma of uninfectedmice and P. yoelii nigeriensis-infected mice from panel A at 10 days postinfec-tion (10d PI).

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supplemented with L-arginine from 3 days prior to infection andby 50% in mice supplemented starting 10 days after P. yoelii infec-tion (Fig. 4B). In support of a causal link between Il-4 expressionand mucosal mastocytosis, mast cell numbers in ileal villi andcrypts were reduced in both L-arginine-supplemented groups rel-ative to numbers in controls (1% sucrose in water) (Fig. 4C). Inthe absence of P. yoelii infection, L-arginine-supplemented andunsupplemented mice had equivalent (and nearly undetectable)numbers of E. coli Nissle CFU in mesenteric lymph nodes andmast cells in ileal villi and crypts (data not shown), indicating thatthe effects of L-arginine supplementation were dependent on P.yoelii infection.

Oral supplementation with L-citrulline blocks E. coli Nissletranslocation and reduces mucosal mast cell activation duringP. yoelii infection. L-Arginine is metabolized by the liver to orni-thine and polyamines, resulting in increased blood urea nitrogen,which is excreted by the kidneys. Under conditions of high L-argi-nine supplementation, adequate hydration is necessary to avoidadverse effects on the kidneys (56). L-Arginine also has a bittertaste and requires the addition of sugar for oral supplementation.Conversely, L-citrulline is tasteless, readily taken up by the intes-tine, metabolized to L-arginine by the kidneys, and then returnedto circulation (57, 58). Accordingly, we attempted to determinewhether L-citrulline supplementation could provide the benefitsof L-arginine in the context of P. yoelii infection. As with L-argin-

ine, two different supplementation regimens were used for the14-day infection period. For these studies, however, control micereceived water ad libitum with 3% L-alanine (isonitrogenous to2% L-citrulline) 3 days prior to or 10 days following infection withP. yoelii, when both groups were inoculated with E. coli Nissle.L-Alanine is not converted to L-arginine and is used as an isoni-trogenous control to ensure that the resulting phenotype is not aproduct of extra nitrogen content (59–61). In these studies, P.yoelii-infected mice had a mean parasitemia of �9% at 10 dayspostinfection.

Relative to matched L-alanine-supplemented controls, miceprovided with L-citrulline from 3 days before infection and start-ing 10 days postinfection had no detectable E. coli Nissle CFU intheir mesenteric lymph nodes (Fig. 5A). These data suggested thatL-citrulline was more broadly active than L-arginine (Fig. 4A) inreversing intestinal permeability in P. yoelii-infected mice. Unex-pectedly, L-citrulline did not alter ileal Il-4 expression (Fig. 5B). Incontrast, mast cell numbers in ileal villi and crypts were reduced inboth L-citrulline groups relative to numbers in L-alanine controlgroups (Fig. 5C).

L-Arginine supplementation decreases bacterial transloca-tion and mucosal mast cell activation in mice coinfected with P.yoelii and S. Typhimurium. L-Arginine and L-citrulline altered P.yoelii-dependent intestinal permeability and mucosal mastocyto-sis in mice colonized with E. coli Nissle. However, S. Typhimu-

FIG 3 Plasmodium yoelii infection is associated with ileal mastocytosis and increased tissue histamine. (A) Representative metachromatic MMCs in the ilea ofP. yoelii nigeriensis-infected mice. Bars � 20 �m. (B) Mean numbers of MMCs (� standard errors of the means [SEM]) in ileal villi and crypts from toluidineblue-stained tissue sections from P. yoelii nigeriensis-infected mice (black bars) and uninfected (UI) controls (white bars) (3 mice per time point). *, P 0.001relative to UI controls. (C) Fold changes in threshold cycles (CT) of ileal Il-4 expression in P. yoelii nigeriensis-infected mice at 14 days postinfection relativeto expression in uninfected controls by real-time PCR (3 mice per group). (D) Mean percentages of parasitemia (percentages of total RBCs parasitized � SEM)over time in mice infected with P. yoelii nigeriensis (solid line) or with P. yoelii yoelii 17XNL (dashed line) (5 mice per time point). (E) Mean (�SEM) MMCs inileal villi and crypts from NASDCE-stained tissue sections from P. yoelii yoelii 17XNL-infected mice (black bars) and uninfected controls (white bars) (3 mice pertime point). *, P 0.001 relative to uninfected controls. (F) Mean concentrations (�SEM) of histamine in ilea of uninfected and P. yoelii yoelii 17XNL-infectedmice from panel E. *, P 0.05 relative to uninfected controls.




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rium coinfection and the associated impact on the host immunesystem (17, 62) may alter the relative efficacies of these amino acidsupplements. To test this possibility, we examined both supple-ments in mice coinfected with P. yoelii and S. Typhimurium, ourmodel for human malaria parasite-NTS coinfection. Mice werecoinfected with S. Typhimurium at the peak of parasitemia (10days postinfection) to mimic probable infection patterns in areasof endemicity with seasonal malaria that tracks with rainfall pat-terns. In these areas, NTS infections are most frequent at the endof the rainy season, after malaria parasite infection has peaked andthe prevalence of anemia is high (63, 64).

As observed in mice coinfected simultaneously (17), peripheralblood parasitemias in mice inoculated with S. Typhimurium fol-lowing P. yoelii infection were not different from those in miceinfected only with P. yoelii or from L-arginine-supplemented,coinfected mice (Fig. 6A). Supplementation with 2.5% L-argininefollowed the regimen used for E. coli Nissle coinfection, exceptthat matched 5% L-alanine-supplemented groups (isonitrog-enous to 2.5% L-arginine) were added as controls. Both groups of

L-arginine-supplemented coinfected mice had significantly fewerS. Typhimurium CFU in their mesenteric lymph nodes than un-supplemented and L-alanine-supplemented coinfected controls(Fig. 6B), indicating that the beneficial effect of L-arginine on ma-laria-dependent pathology could be extended to coinfection withS. Typhimurium. This reduction was not expected to approachreductions observed for E. coli Nissle because S. Typhimurium isnaturally invasive in mice. A reduction nonetheless was consistentwith observed effects on noninvasive E. coli Nissle and suggestedthat a similar clinical intervention in children, where S. Typhimu-rium is not naturally invasive, could reduce the clinical impact ofcoinfection.

As observed for L-arginine supplementation in mice coinfectedwith E. coli Nissle, ileal Il-4 expression was significantly decreasedin both groups of L-arginine-supplemented coinfected mice rela-tive to that in controls (Fig. 6C). As expected, L-arginine supple-mentation also reduced mast cell numbers in the intestinal villiof coinfected mice relative to numbers in unsupplemented and

FIG 4 L-Arginine supplementation reduces noninvasive bacterial transloca-tion, Il-4 expression, and villus MMCs in the ilea of P. yoelii-infected mice. (A)Mean numbers (�SEM) of E. coli Nissle CFU in the mesenteric lymph nodes at14 days postinfection of P. yoelii nigeriensis-infected mice, which received dif-ferent water supplements (3 to 4 mice per group), as indicated by � and �. nd,none detected. (B) Fold change in threshold cycles (CT) of ileal Il-4 expres-sion in L-arginine-supplemented P. yoelii nigeriensis-infected mice at 14 dayspostinfection relative to expression in unsupplemented, infected controls byreal-time PCR (3 mice per group). (C) Mean numbers (�SEM) of MMCs inthe ileal villi and crypts of uninfected mice (white bars) and of unsupple-mented and L-arginine-supplemented P. yoelii nigeriensis-infected mice (blackbars) at 14 days postinfection determined from toluidine blue-stained tissuesections (5 mice per group). *, P 0.05 relative to uninfected mice; **, P 0.01 relative to uninfected mice; �, P 0.05 relative to P. yoelii nigeriensis-infected mice given 1% sucrose.

FIG 5 L-Citrulline reduces noninvasive bacterial translocation and numbersof altered villus and crypt MMCs in the ilea of P. yoelii-infected mice. (A) Meannumbers (�SEM) of E. coli Nissle CFU in the mesenteric lymph nodes at 14days postinfection of P. yoelii yoelii 17XNL-infected mice, which received dif-ferent water supplements (5 mice per group), as indicated by � and �. nd,none detected. (B) Fold change in threshold cycles (CT) of ileal Il-4 expres-sion in L-citrulline-supplemented P. yoelii yoelii 17XNL-infected mice at 14days postinfection relative to expression in unsupplemented, infected controlsby real-time PCR (4 mice per group). (C) Mean numbers (�SEM) of MMCs inileal villi and crypts of unsupplemented, P. yoelii yoelii 17XNL-infected miceand of L-citrulline- or L-alanine-supplemented, infected mice at 14 dayspostinfection determined from toluidine blue-stained tissue sections (5 miceper group). **, P 0.001 relative to L-alanine day �3; *, P 0.01 relative toL-alanine day 10; �, P 0.001 relative to unsupplemented P. yoelii yoelii17XNL-infected mice.

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L-alanine-supplemented mice (Fig. 6D). Unlike parasite-infectedmice colonized with E. coli Nissle (Fig. 4C), coinfected mice sup-plemented with L-arginine exhibited increased levels of crypt-as-sociated mast cells from 3 days prior to P. yoelii infection relativeto levels in all other groups (Fig. 6D). Given that activated mastcells move from the villi back to the crypts following resolution ofhelminth infection (32), increased numbers of crypt-associatedmast cells in this group suggested perhaps an effect on reducingmast cell-dependent barrier damage that was greater than thatshown by controls and mice treated with L-arginine starting onday 10.

L-Citrulline supplementation decreases bacterial transloca-tion from the intestine and mucosal mast cell activation in micecoinfected with malaria parasites and S. Typhimurium. Basedon the success of L-arginine supplementation in mice coinfectedwith P. yoelii and S. Typhimurium, we repeated identical assays

with L-citrulline supplementation and with 3% L-alanine as anisonitrogenous control. Peripheral blood parasitemias in mice in-oculated with S. Typhimurium following P. yoelii infection werenot significantly different from those of coinfected mice supple-mented with L-citrulline (Fig. 7A). Both groups of L-citrulline-supplemented coinfected mice had significantly fewer S. Typhi-murium CFU in their mesenteric lymph nodes than controls (Fig.7B). The maximal reduction of 0.7 log CFU by L-citrulline supple-mentation relative to L-alanine supplementation was similar inscale to the maximal reduction by L-arginine supplementation(Fig. 6B), indicating that the increased efficacy observed for L-cit-rulline relative to L-arginine in the context of E. coli Nissle coloni-zation (Fig. 5A versus 4A) was not achieved in the context of S.Typhimurium coinfection. As observed for L-arginine-supple-mented mice coinfected with S. Typhimurium (Fig. 6C), ileal Il-4expression was significantly decreased in both groups of L-citrul-line-supplemented, coinfected mice relative to that in controls(Fig. 7C). In contrast to effects of L-arginine supplementation onmastocytosis (Fig. 6D), however, both L-citrulline treatment reg-imens reduced mast cell numbers in the intestinal villi and in-creased mast cell numbers in the intestinal crypts of coinfectedmice relative to numbers in coinfected mice supplemented withL-alanine (Fig. 7D).

Infection with S. Typhimurium induces inflammation in themouse intestine (65, 66), suggesting that observed reductions inbacterial translocation may be less clearly associated with epithe-lial barrier integrity and perhaps more dependent on other phe-notypic effects of supplementation in the context of P. yoelii in-fection. To address this possibility, we examined ZO-1 andE-cadherin staining in the ilea of unsupplemented, coinfectedcontrol mice and in coinfected mice supplemented with L-citrul-line or L-alanine. In coinfected control mice with no supplement(Fig. 7E), apical ZO-1 and E-cadherin staining were discontin-uous and similar to those in ileal tissues from both groups ofL-alanine-supplemented, coinfected mice (Fig. 7E) (L-Ala day �3and day 10). However, in both groups of L-citrulline-supplementedmice (Fig. 7E) (L-Cit day �3, day 10), apical ZO-1 and E-cadherinstaining were markedly enhanced and accompanied by notable ZO-1staining around subepithelial capillaries (Fig. 7E) (L-Cit day �3, day10). This phenomenon may be linked to a reversal in mastocytosis.Mast cell-derived histamine can induce gaps in the venular endo-thelium such that a reversal of mastocytosis by L-citrulline supple-mentation after infection may be accompanied by active repair ofthe endothelial junctional barrier (67).

DISCUSSION

The data presented here demonstrate that oral supplementationto enhance L-arginine bioavailability in the context of malaria par-asite infection can reduce mast cell-associated damage to the in-testinal barrier and transepithelial translocation of enteric bacte-ria, including a causative agent of NTS bacteremia in coinfectedchildren. Intravenous infusion of L-arginine has been used to re-duce pathology associated with severe malaria, but this applica-tion is invasive and expensive and requires medical monitoring(39, 68). However, oral supplementation has been used to en-hance livestock production (40–43) and to treat clinical cases oftrauma and sepsis (44). Here, we demonstrate that a noninvasive,immune-enhancing oral supplement can reduce the impact of abacterial coinfection that is of major public health importance incountries where malaria is endemic.

FIG 6 L-Arginine supplementation reduces S. Typhimurium translocation,Il-4 expression, and numbers of altered villus and crypt MMCs in the ilea of P.yoelii-infected mice. (A) Peripheral blood parasitemias (means � SEM) inmice infected with P. yoelii yoelii 17XNL (P) only, coinfected with P. yoeliiyoelii 17XNL and S. Typhimurium (P�S), or coinfected and supplementedwith L-arginine beginning 3 days prior to parasite infection (P�S L-Arg d�3)(5 mice per group). (B) Mean numbers (�SEM) of S. Typhimurium CFU inthe mesenteric lymph nodes at 14 days postinfection of P. yoelii yoelii 17XNL-infected mice that received different supplements (5 mice per group), as indi-cated by � and �. (C) Fold changes in threshold cycles (CT) of ileal Il-4expression in L-arginine-supplemented P. yoelii yoelii 17XNL-coinfected mice(4 to 5 mice per group) at 14 days postinfection relative to expression inunsupplemented, coinfected controls (5 mice) by real-time PCR. (D) Meannumbers of (�SEM) of MMCs in ileal villi and crypts of unsupplemented,coinfected mice and of L-arginine- or L-alanine-supplemented, coinfectedmice at 14 days postinfection (5 mice per group). **, P 0.001 relative tounsupplemented mice; *, P 0.05 relative to L-alanine-supplemented micebeginning 3 days prior to infection; �, P 0.05 relative to L-alanine-supple-mented mice beginning 10 days postinfection.




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In our model, P. yoelii infection is associated with increasedacute and sustained intestinal permeability and hypoargininemia(Fig. 1A and B) as well as with circulating and tissue histaminelevels and ileal mastocytosis, which rise with parasitemia (Fig. 2Band 3B, E, and F). Supplementation with L-arginine and L-citrul-line decreased intestinal permeability to E. coli Nissle and S. Ty-phimurium, with indication of enhanced efficacy of L-citrullinerelative to L-arginine supplementation (e.g., compare Fig. 4A and5A and Fig. 6D and 7D). Supplementation also decreased mast celllocalization to the ileal villi (Fig. 4C, 5C, 6D, and 7D). Given thatintestinal helminth infections in mice are associated with mast celltrafficking into the villus tips and then back to the submucosa atthe resolution of infection and inflammation (32), the altered lo-calization of mast cells with L-arginine and L-citrulline treatmentsuggests that hypoargininemia is functionally associated withmast cell activation and ileal mastocytosis. Further, these data,along with concordant effects of treatment regimen on the mor-phology of TJs and adherens junctions (Fig. 7E), are additionalindications that supplementation with L-citrulline strengthenedthe intestinal barrier in the context of coinfection. While we didnot confirm that L-citrulline supplementation resulted in in-creased levels of plasma L-arginine, clinical studies have confirmedthis phenomenon (57).

The implicit assumptions connecting observations in ourmodel are well supported by the literature and are highlightedbelow. In particular, published data indicate that (i) activation ofearly innate responses precedes mast cell activation, (ii) mast cellactivation is potentiated by low L-arginine bioavailability, and (iii)intestinal mastocytosis does not afford protection against S. Ty-phimurium but rather enhances epithelial permeability to trans-locating bacteria. Further, our previous data affirm that the im-pact of P. yoelii infection on bacterial translocation is rapid (17),confirming that effects of P. yoelii within the first 2 to 4 dayspostinfection are functionally significant.

One of the earliest effects of malaria infection is the elaborationof translationally controlled tumor protein (TCTP) by growingparasites (Fig. 8). Secreted P. falciparum TCTP (PfTCTP) can in-duce histamine release by basophils in vitro (69), and basophilsfrom PfTCTP-positive patients with both mild and severe malariaexhibited higher unstimulated activation (CD203c mean fluores-cence intensity) than did basophils from PfTCTP-negative pa-tients (70). These observations suggest that rapid activation ofbasophils, an important innate cellular source for IL-4 (71), canoccur early in malaria parasite infection. Murine malaria parasitesencode TCTP that is highly homologous to PfTCTP, suggestingsimilar biological means of infection across mammalian hosts(72). Basophils accumulate at sites of inflammation, which in thecontext of P. falciparum infection are defined by sites of endothe-lial activation associated with increased sequestration of infectedRBCs in the microvasculature (73); in P. yoelii infection, seques-tration occurs in postcapillary venules throughout the body (74).Both parasite sequestration and endothelial transmigration of ba-sophils are mediated by the adhesion molecules ICAM-1 and/orVCAM-1 (74, 75), which are upregulated under conditions of lowNO availability (76). Given that hypoargininemia occurs acutelyfollowing P. yoelii infection (Fig. 1B), TCTP-activated basophilsmay be recruited quickly to microcapillaries in the villus tips of theintestine, which along with skin and brain have been implicated asmajor parasite sequestration sites for P. falciparum (10). Synthesisof ileal IL-4 and histamine, which rises with increasing parasitemia

FIG 7 L-Citrulline supplementation reduces mast cell activation and S. Ty-phimurium translocation to the mesenteric lymph nodes in P. yoelii-infectedmice. (A) Peripheral blood parasitemias (means � SEM) in mice infected withP. yoelii yoelii 17XNL (P) only, coinfected with P. yoelii yoelii 17XNL and S.Typhimurium (P�S), or coinfected with L-citrulline supplementation begin-ning 3 days prior to parasite infection (P�S L-Cit d�3) (5 mice per group).(B) Mean numbers (�SEM) of S. Typhimurium CFU at 14 days postinfectionin the mesenteric lymph nodes of P. yoelii yoelii 17XNL-infected mice whichreceived different water supplements (5 mice per group), as indicated by �and �. (C) Fold changes in threshold cycles (CT) of ileal Il-4 expression inL-citrulline-supplemented P. yoelii yoelii 17XNL-coinfected mice (4 to 5 miceper group) at 14 days postinfection relative to expression in unsupplemented,coinfected controls (5 mice) by real-time PCR. (D) Mean numbers (�SEM) ofMMCs in ileal villi and crypts of unsupplemented, coinfected mice and ofL-citrulline- or L-alanine-supplemented, coinfected mice at 14 days postinfec-tion (5 mice per group). **, P 0.001 relative to the unsupplemented group;*, P 0.05 relative to the unsupplemented group; ��, P 0.01 relative to theL-alanine-supplemented group beginning 10 days postinfection; �, P 0.05relative to the L-alanine-supplemented group beginning 3 days prior to infec-tion. (E) Intracellular junction staining of uninfected mouse ileum and of P.yoelii yoelii 17XNL-infected mouse ileum at 14 days postinfection with nosupplement, 2% L-citrulline beginning 3 days prior to infection (L-Cit d�3),2% L-citrulline beginning 10 days postinfection (L-Cit d10), 3% L-alaninebeginning 3 days prior to infection (L-Ala d�3), or 3% L-alanine beginning 10days postinfection (L-Ala d10). The top, middle, and bottom rows containimages of ZO-1 (green) staining, E-cadherin (E-cad) (red) staining, andmerged images, respectively. DAPI (blue) was used for nuclear staining. Bar,20 �m.

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and increased basophil activation, results in rapid mast cell recruit-ment, as observed in P. yoelii-infected mice (Fig. 3B and E).

Mast cell activation in situ following recruitment to the intes-tine is predicted to be potentiated by hypoargininemia and lowNO availability (21, 77, 78). Hence, low NO availability can en-hance parasite sequestration and basophil transmigration, as wellas mast cell activation, a panoply of effects that can result in in-creased intestinal permeability (79) akin to that observed in bothacute and later stages of P. falciparum infection (12). Althoughmast cell activation in the intestine can be protective during hel-minth infection (37), mast cell-deficient mice die more rapidlyafter infection with S. Typhimurium. In addition, adoptive trans-fer of mast cells does not enhance survival after infection with S.Typhimurium (80), indicating that the protective role of mastcells during bacterial infection is pathogen specific. Here, our datasuggest that decreased paracellular permeability can reduce S. Ty-phimurium translocation by mechanisms to be established. Ofnote, the S. Typhimurium SPI1 effectors SopB, SopE, SopE2, andSipA decrease ZO-1 expression levels and alter the localization ofoccludins to alter host epithelial TJs (81). The S. TyphimuriumT3SS effector AvrA, in contrast, stabilizes TJs based on infectionwith an avrA mutant that reduces expression of ZO-1, claudin-1,and occludins in intestinal epithelial cells (82). Perhaps alterationof TJ and adherens junction morphology by L-citrulline and L-arginine in the context of P. yoelii infection confounds S. Typhi-murium manipulation of this host biology, reducing successfultranslocation.

Porcherie et al. (83) recently reported that a unique neutrophilpopulation that homed to the brain was responsible for the devel-opment of IgE-dependent experimental cerebral malaria (ECM)in Plasmodium berghei-infected mice. Further, those authors dem-

onstrated that C57BL/6 mice genetically depleted of mast cells, orimmunologically depleted of basophils, exhibited no significantdifferences in parasitemia or survivorship. This led the authors toconclude that “mast cells and basophils are not involved in ma-laria pathogenesis.” While the data and conclusions presented inthis model of ECM are both novel and well supported, other mastcell-associated aspects of malaria pathology, including changes inintestinal permeability, remain to be investigated fully. However,we recognize that mast cell-independent explanations for thebeneficial effects of increased L-arginine bioavailability are alsoconsistent with our data. In particular, polyamines, which areproducts of L-arginine metabolism, can upregulate expression ofE-cadherin (84), suggesting that L-arginine supplementation mayimprove epithelial barrier integrity directly. Polyamines also in-crease epithelial cell migration (85), which is critical for resealingdefects that occur under some pathological conditions. Alterna-tively, metabolism of L-arginine by arginase and ornithine amino-transferase can produce L-ornithine and L-proline, which also in-crease epithelial migration and restitution in the Citrobacterrodentium model of colitis (86). The effects of altered Il-4 expres-sion on intestinal permeability may be similarly mast cell indepen-dent. Berin et al. (87) reported that IL-4 treatment of a human T84intestinal epithelial cell monolayer increased both transcellularand paracellular transport, suggesting that enhanced productionof IL-4 under allergic conditions would increase access of luminalantigens to the mucosal immune system. In a subsequent study,Ceponis et al. (88) reported that IL-4-dependent transepithelialpermeability in T84 monolayers was dependent on phosphatidyl-inositol 3-kinase signaling.

In sum, our data indicate that oral supplementation to im-prove L-arginine bioavailability may provide a strategy to reducethe public health impact of bacteremia in children coinfected withmalaria. Critical follow-up studies will focus on whether geneticand immunological ablation of mast cells in the context of P. yoeliiinfection can prevent mucosal mastocytosis and improve themorphology and function of the intestinal epithelial barrier. Thesequestions are particularly interesting because parasitic infectionsare often associated with increased allergic inflammation, al-though whether these responses are protective is not clear. What isclear, however, is that intestinal helminths and some bacterial in-fections are directly controlled by mucosal mastocytosis. Despitethe obvious connections of malaria to allergic inflammation, therehas been no recognition that these processes may explain the long-standing observations of GI disturbances in clinical malaria. Ad-ditionally, while there is significant recognition of the role of L-arginine in regulating key allergic effectors, L-arginine deficiencyhas not been connected to chronic allergic inflammation in ma-laria or specifically to chronic allergic inflammation in the intes-tine. In this regard, our work will add to potential mechanisms bywhich L-arginine supplementation can benefit patients with severeforms of malaria. In particular, the identification of significantand innovative links among nutrition, immunity, malaria, andenteric diseases can be targeted with a simple intervention to mit-igate an important malaria-associated coinfection.

ACKNOWLEDGMENTS

These studies were supported by a grant from the Bill & Melinda GatesFoundation through the Grand Challenges Explorations initiative to S.L.,by NIH grant NIAID R21 A1082320 to R.M.T., and by NIH grant NLHBIP01 HL024136 to G.H.C.

FIG 8 Model for malaria-induced intestinal permeability to S. Typhimurium.Circulating malaria parasites release TCTP, which activates basophils to re-lease histamine and IL-4. Synthesis of ileal IL-4 and histamine rises with in-creasing parasitemia and basophil activation, resulting in rapid mast cell re-cruitment. Mast cell activation in situ following recruitment to the intestine ispotentiated by hypoargininemia and predicted low NO availability. Hence,low L-arginine and low NO availability enhance parasite sequestration to acti-vated endothelium and basophil transmigration, as well as mast cell activation,a panoply of effects that can degrade the intestinal epithelial barrier to S.Typhimurium.




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We thank Kong Wai Cheung, Maria Winter, Nicola Schlatenberg,Hannah Smithers, Kim Nguyen, and Vladimir Tolstikov for their gener-ous assistance in these studies. We also acknowledge Edwin E. Lewis,Gordon Douglas, and Paul Fitzgerald for their advice and expertise.

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