Malaria-associated L-arginine deficiency induces mast cell-associated disruption to intestinal barrier defenses against nontyphoidal Salmonella bacteremia

Abstract

Coinfection with malaria and nontyphoidal Salmonella serotypes (NTS) can cause life-threatening bacteremia in humans. Coinfection 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 reveal that malarial-parasite-infected mice, like humans, develop L-arginine deficiency, which is associated with intestinal mastocytosis, elevated levels of histamine, and enhanced intestinal permeability. Prevention or reversal of L-arginine deficiency blunts mastocytosis in ileal villi as well as bacterial translocation, measured as numbers of mesenteric lymph node CFU of noninvasive Escherichia coli Nissle and Salmonella enterica serotype Typhimurium, the latter of which is naturally invasive in mice. Dietary supplementation of malarial-parasite-infected mice with L-arginine or L-citrulline reduced levels of ileal transcripts encoding interleukin-4 (IL-4), a key mediator of intestinal mastocytosis and macromolecular permeability. Supplementation with L-citrulline 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 testing nutritional interventions to reduce malaria-associated mortality in humans.

Fig 1

Plasmodium yoelii infection is associated with increased intestinal permeability and hypoargininemia. (A) Lactulose/mannitol (L:M) ratios in urine from uninfected (UI) mice (white circles) and from P. yoelii nigeriensis-infected mice from 2 to 12 days postinfection (PI). Means are indicated as bars within each treatment group. Percentages are the proportions of infected mice with L:M ratios that exceeded the highest L:M ratio (dashed line) in uninfected mice. Each circle indicates the value for 1 mouse. (B) l-Arginine concentrations in sera 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 histidine and increased plasma histamine. (A) Histidine levels in sera of uninfected (UI) mice (white circles) and P. yoelii nigeriensis-infected mice (black circles) over time. Each circle indicates the value for 1 mouse; means of each treatment group are indicated with bars. (B) Histamine levels in plasma of uninfected mice and P. yoelii nigeriensis-infected mice from panel A at 10 days postinfection (10d PI).

Fig 3

Plasmodium yoelii infection is associated with ileal mastocytosis and increased tissue histamine. (A) Representative metachromatic MMCs in the ilea of P. yoelii nigeriensis-infected mice. Bars = 20 μm. (B) Mean numbers of MMCs (± standard errors of the means [SEM]) in ileal villi and crypts from toluidine blue-stained tissue sections from P. yoelii nigeriensis-infected mice (black bars) and uninfected (UI) controls (white bars) (3 mice per time point). *, P < 0.001 relative to UI controls. (C) Fold changes in threshold cycles (ΔΔC_T) of ileal Il-4 expression in P. yoelii nigeriensis-infected mice at 14 days postinfection relative to 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 in ileal villi and crypts from NASDCE-stained tissue sections from P. yoelii yoelii 17XNL-infected mice (black bars) and uninfected controls (white bars) (3 mice per time point). *, P < 0.001 relative to uninfected controls. (F) Mean concentrations (±SEM) of histamine in ilea of uninfected and P. yoelii yoelii 17XNL-infected mice from panel E. *, P < 0.05 relative to uninfected controls.

Fig 4

l-Arginine supplementation reduces noninvasive bacterial translocation, 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 at 14 days postinfection of P. yoelii nigeriensis-infected mice, which received different water supplements (3 to 4 mice per group), as indicated by + and −. nd, none detected. (B) Fold change in threshold cycles (ΔΔC_T) of ileal Il-4 expression in l-arginine-supplemented P. yoelii nigeriensis-infected mice at 14 days postinfection relative to expression in unsupplemented, infected controls by real-time PCR (3 mice per group). (C) Mean numbers (±SEM) of MMCs in the ileal villi and crypts of uninfected mice (white bars) and of unsupplemented and l-arginine-supplemented P. yoelii nigeriensis-infected mice (black bars) at 14 days postinfection determined from toluidine blue-stained tissue sections (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 numbers of altered villus and crypt MMCs in the ilea of P. yoelii-infected mice. (A) Mean numbers (±SEM) of E. coli Nissle CFU in the mesenteric lymph nodes at 14 days postinfection of P. yoelii yoelii 17XNL-infected mice, which received different water supplements (5 mice per group), as indicated by + and −. nd, none detected. (B) Fold change in threshold cycles (ΔΔC_T) of ileal Il-4 expression in l-citrulline-supplemented P. yoelii yoelii 17XNL-infected mice at 14 days postinfection relative to expression in unsupplemented, infected controls by real-time PCR (4 mice per group). (C) Mean numbers (±SEM) of MMCs in ileal villi and crypts of unsupplemented, P. yoelii yoelii 17XNL-infected mice and of l-citrulline- or l-alanine-supplemented, infected mice at 14 days postinfection determined from toluidine blue-stained tissue sections (5 mice per group). **, P < 0.001 relative to l-alanine day −3; *, P < 0.01 relative to l-alanine day 10; +, P < 0.001 relative to unsupplemented P. yoelii yoelii 17XNL-infected mice.

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) in mice infected with P. yoelii yoelii 17XNL (P) only, coinfected with P. yoelii yoelii 17XNL and S. Typhimurium (P+S), or coinfected and supplemented with 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 in the mesenteric lymph nodes at 14 days postinfection of P. yoelii yoelii 17XNL-infected mice that received different supplements (5 mice per group), as indicated by + and −. (C) Fold changes in threshold cycles (ΔΔC_T) of ileal Il-4 expression in l-arginine-supplemented P. yoelii yoelii 17XNL-coinfected mice (4 to 5 mice per group) at 14 days postinfection relative to expression in unsupplemented, coinfected controls (5 mice) by real-time PCR. (D) Mean numbers of (±SEM) of MMCs in ileal villi and crypts of unsupplemented, coinfected mice and of l-arginine- or l-alanine-supplemented, coinfected mice at 14 days postinfection (5 mice per group). **, P < 0.001 relative to unsupplemented mice; *, P < 0.05 relative to l-alanine-supplemented mice beginning 3 days prior to infection; +, P < 0.05 relative to l-alanine-supplemented mice beginning 10 days postinfection.

Fig 7

l-Citrulline supplementation reduces mast cell activation and S. Typhimurium translocation to the mesenteric lymph nodes in P. yoelii-infected mice. (A) Peripheral blood parasitemias (means ± SEM) in mice infected with P. yoelii yoelii 17XNL (P) only, coinfected with P. yoelii yoelii 17XNL and S. Typhimurium (P+S), or coinfected with l-citrulline supplementation beginning 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 postinfection in the mesenteric lymph nodes of P. yoelii yoelii 17XNL-infected mice which received different water supplements (5 mice per group), as indicated by + and −. (C) Fold changes in threshold cycles (ΔΔC_T) of ileal Il-4 expression in l-citrulline-supplemented P. yoelii yoelii 17XNL-coinfected mice (4 to 5 mice per group) at 14 days postinfection relative to expression in unsupplemented, coinfected controls (5 mice) by real-time PCR. (D) Mean numbers (±SEM) of MMCs in ileal villi and crypts of unsupplemented, coinfected mice and of l-citrulline- or l-alanine-supplemented, coinfected mice at 14 days postinfection (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 the l-alanine-supplemented group beginning 10 days postinfection; +, P < 0.05 relative to the l-alanine-supplemented group beginning 3 days prior to infection. (E) Intracellular junction staining of uninfected mouse ileum and of P. yoelii yoelii 17XNL-infected mouse ileum at 14 days postinfection with no supplement, 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-alanine beginning 3 days prior to infection (L-Ala d−3), or 3% l-alanine beginning 10 days postinfection (L-Ala d10). The top, middle, and bottom rows contain images of ZO-1 (green) staining, E-cadherin (E-cad) (red) staining, and merged images, respectively. DAPI (blue) was used for nuclear staining. Bar, 20 μm.

Fig 8

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