Administration of L-citrulline prevents Plasmodium growth by inhibiting/ modulating T-regulatory cells during malaria pathogenesis

Background & objectives: Malaria affects around 228 million people all over the globe. Malaria causing parasite Plasmodium infection leads to activation of immune responses. The growth of parasite and immune activation requires semi essential amino acids like L-arginine. Malaria infection leads to condition of hyperargininemia and low availability of nitric oxide. However, the effect of L-arginine supplementation in malaria infected mice has not been explored in in-vivo studies. In this study we have compared the effect of oral supplementation of nitric oxide donor, L-arginine and L-citrulline, in malaria infected mice Methods: To examine the effect of oral supplementation of L-arginine and L-citrulline, Plasmodium berghei infected mice were divided in different groups and respective groups were fed with L- arginine and L-citrulline, parasitemia was measured on different days. Mice was sacrificed and immunophenotyping was done on 10 days post infection. Results: our results show that supplementation of L-arginine induces conducive environment for Plasmodium growth due to which the infected mice dies earlier than control wild type infected mice whereas L-citrulline supplementation inhibits parasite growth and mice survives for longer period of time. Flow cytometric analysis shows that supplementation of L-arginine increases cTLA-4 on T cell population, increases Treg cells leading to immunosuppression while supplementation of L-citrulline does not have effect on T cells population and number of Treg cell decrease compared to P. berghei infected mice. Interpretation & conclusion: our results show that L-citrulline can be a better alternative than L-arginine because of lower expression of inhibitory molecules and lower parasitemia as well as increased survival of infected mice

Effect of L-arginine on the growth of Plasmodium falciparum and immune modulation of host cells

Background & objectives: Malaria is a life-threatening disease caused by Plasmodium parasites. The life-cycle of Plasmodium species involves several stages both in mosquito and the vertebrate host. In the erythrocytic stage, Plasmodium resides inside the red blood cells (RBCs), where it meets most of its nutritional requirement by degrad- ing host′s haemoglobin. L-arginine is required for growth and division of cells. The present study was aimed to demonstrate the effect of supplementation of different concentrations of L-arginine and L-citrulline on the growth of parasite, and effect of the culture supernatant on the host′s peripheral blood mononuclear cells (PBMCs). Methods: To examine the effect of supplementation of L-arginine and L-citrulline, Plasmodium falciparum (3D7 strain) was cultured in RPMI 1640, L-arginine deficient RPMI 1640, and in different concentrations of L-arginine, and L-citrulline supplemented in arginine deficient RPMI 1640 medium. To have a holistic view of in vivo cell activation, the PBMCs isolated from healthy human host were cultured in the supernatant collected from P. falciparum culture. Results: Growth of the parasite was greatly enhanced in L-arginine supplemented media and was found to be concentration dependent. However, parasite growth was compromised in L-citrulline supplemented and L-arginine deficient media. The supernatant collected from L-arginine supplemented parasite media (sArg) showed increased FOXP3 and interleukin-10 (IL-10) expression as compared to the supernatant collected from L-citrulline supple- mented parasite media (sCit). Interpretation & conclusion: The in vitro culture results showed, decreased parasite growth, and decreased expression of programmed cell death-1 (PD-1) (a coinhibitory molecule) and IL-10 in the L-citrulline supplemented media as compared to L-arginine supplemented media. Hence, it was concluded that L-citrulline supplementation would be a better alternative than L-arginine to inhibit the parasite growth

Summary of 353 samples collected at three sites in India.

<p>Summary of 353 samples collected at three sites in India.</p

Age-dependent breadth and intensity of response to <i>P</i>. <i>vivax</i> and <i>P</i>. <i>falciparum</i>.

<p>Age-dependent A) breadth of response to 265 <i>P</i>. <i>vivax</i> and 373 <i>P</i>. <i>falciparum</i> antigens in children (n = 61) and adults (n = 129) from Raurkela and Nadiad. The box indicates the first and third quartiles, the line inside the box indicates the median, and whiskers represent the minimum and maximum values. B) Average of mean intensity of antibody binding to the same subset of <i>P</i>. <i>vivax</i> and <i>P</i>. <i>falciparum</i> antigens in children and adults, top of bars indicate the mean value and error bars represent 95% confidence interval of the mean. Kruskal–Wallis/Dunn adjusted p-values for pairwise comparison of groups are shown as asterisks: 0.03 (*), 0.002 (**), 0.0002 (***), <0.0001 (****).</p

Comparison of individuals with symptomatic and asymptomatic malaria.

<p>A) Breadth of response to 265 <i>P</i>. <i>vivax</i> antigens, and 373 <i>P</i>. <i>falciparum</i> antigens in symptomatic (<i>P</i>. <i>vivax</i> = 58; <i>P</i>. <i>falciparum</i> = 38) and asymptomatic (<i>P</i>. <i>vivax</i> = 16; <i>P</i>. <i>falciparum</i> = 9) malaria-positive adults at three sites in India. The box indicates the first and third quartiles, the line side of the box indicates the median, and whiskers represent the minimum and maximum values. B) Average of mean intensity of antibody binding to 265 <i>P</i>. <i>vivax</i> and 373 <i>P</i>. <i>falciparum</i> antigens in symptomatic and asymptomatic malaria-positive adults, top of bars indicate the mean value and error bars represent 95% confidence interval of the mean. C) Average of <i>P</i>. <i>vivax</i> and <i>P</i>. <i>falciparum</i> asexual parasitemia (number of asexual parasites per microliter) in symptomatic and asymptomatic malaria-positive adults. Kruskal–Wallis/Dunn adjusted p-values for pairwise comparison of groups are shown as asterisks: 0.03 (*), 0.002 (**), 0.0002 (***), <0.0001 (****).</p

Breadth of antibody response to <i>P</i>. <i>vivax</i> and <i>P</i>. <i>falciparum</i>.

<p>Breadth of response to A) 265 <i>P</i>. <i>vivax</i> and B) 373 <i>P</i>. <i>falciparum</i> antigens in samples collected from malaria-positive (Chennai = 45; Nadiad = 55; Raurkela = 74) and malaria-negative adults (Chennai = 25; Nadiad = 34; Raurkela = 32) at three sites in India. The box indicates the first and third quartiles, the line inside the box indicates the median, and whiskers represent the minimum and maximum values. Kruskal–Wallis/Dunn adjusted p-values for pairwise comparison of groups are shown as asterisks: 0.03 (*), 0.002 (**), 0.0002 (***), <0.0001 (****).</p

<i>Plasmodium falciparum</i> antigens recognized with greater intensity in asymptomatic individuals.

<p><i>Plasmodium falciparum</i> antigens recognized with greater intensity in asymptomatic individuals.</p

Serological profiles of malaria-positive and malaria-negative individuals from three sites in India.

<p>Heat map showing signal intensity of antibody binding to 265 <i>P</i>. <i>vivax</i> and 373 <i>P</i>. <i>falciparum</i> polypeptides in 236 malaria-positive and 117 malaria-negative samples collected from Raurkela, Nadiad and Chennai. Red indicates positive reactivity, white indicates no reactivity, and the gradient indicates intermediate reactivity. Samples for each site and <i>Plasmodium</i> antigens were ranked from left to right and top to bottom, respectively, by decreasing log₂(FOC).</p