|Year : 2022 | Volume
| Issue : 1 | Page : 45-51
Administration of L-citrulline prevents Plasmodium growth by inhibiting/ modulating T-regulatory cells during malaria pathogenesis
Vikky Awasthi, Rubika Chauhan, Jyoti Das
Parasite-Host biology, ICMR-National Institute of Malaria Research, New Delhi, India
|Date of Submission||09-Sep-2020|
|Date of Acceptance||18-Dec-2020|
|Date of Web Publication||07-Jun-2022|
Parasite-Host Biology, National Institute of Malaria Research, Sector-8, Dwarka, New Delhi110077
Source of Support: None, Conflict of Interest: None
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.
Keywords: Malaria, Plasmodium, L-arginine, L-citrulline, T regulatory cells.
|How to cite this article:|
Awasthi V, Chauhan R, Das J. Administration of L-citrulline prevents Plasmodium growth by inhibiting/ modulating T-regulatory cells during malaria pathogenesis. J Vector Borne Dis 2022;59:45-51
|How to cite this URL:|
Awasthi V, Chauhan R, Das J. Administration of L-citrulline prevents Plasmodium growth by inhibiting/ modulating T-regulatory cells during malaria pathogenesis. J Vector Borne Dis [serial online] 2022 [cited 2022 Jun 25];59:45-51. Available from: https://www.jvbd.org/text.asp?2022/59/1/45/325640
| Introduction|| |
Malaria is socioeconomic burden as causes millions of death across the globe. Plasmodium, causative agent of malaria, has two hosts. Mosquito is the primary host and serve as a vector for malaria transmission. When infected mosquito bites a secondary host sporozoite enters and reaches liver, undergoes rapid multiplication, producing merozoites capable of infecting RBCs,. Innate cells like macrophages, monocytes and dendritic cell act against them but as the disease progress these cells are modulated by parasite which affects the adaptive immunity. There is marked inflammation as the disease progresses. Several groups have attributed inefficiency of immune cells on hemozoins, T cell exhaustion due to expression of PD-1, and antigenic variants of the parasite.
Nitric oxide has been shown to be play protective role in malaria. Nitric oxide mediates host resistance and kills P. falciparum in vitro. Nitric oxide synthase (NOS) when uses L-arginine, L-citrulline and nitric oxide are formed. When L-arginine is acted upon by an enzyme arginase ornithine and urea are formed. Ornithine is utilized for polyamine synthesis, required for cell proliferation. One of the manifestations of the malaria disease is generation of hypo-argininemia condition. This condition generated during the malaria results in immune dysfunction. Low level of arginine results in down regulation of CD3z chain which further dampens the downstream signaling and thus the activation of T cell get compromised.
A number of interventions can be devised to correct arginine deficiency and improve nitric oxide level in blood. It has been shown that intravenous supplement of arginine improves nitric oxide level in blood hence, it plays protective role in the disease,,. The route of administration of arginine plays an important role in limiting the parasite growth. Intravenous injection of L-arginine was found to be effective. Though for any drug to be effective, oral route of administration is preferred. Pretreatment with arginine by oral route have been shown to increase the survival of mice infected with lethal Plasmodium yoelii by increasing T-regulatory cells.
On the other hand, L-citrulline, a non-protein amino acid can be converted to L-arginine and has been documented to modulate the immune cells. Therefore, L-citrulline can act as an arginine precursor for NO synthesis. Though L-citrulline is not taken up by the either liver and or used by the intestine. Administration of L-citrulline may be utilized to deliver nitrogen making it available for protein homeostasis in peripheral tissue and as an arginine precursor synthesized de novo in the kidneys, in endothelial and immune cells. Citrulline is efficiently absorbed when orally administered and could be used either as a method to deliver arginine to the systemic circulation or as a protein anabolic agent in specific clinical situations arginine can be recycled from citrulline.
The effect of arginine and citrulline in malaria parasite infected mice on immune system in context with costimulation by oral administration has not been explored yet. In this study, we show that L-arginine and L-citrulline when fed orally has opposite effect on malaria pathogenesis. Administration of L-arginine to malaria infected mice results in increased T regulatory cells and early death due to increased parasitemia. Whereas, administration of L-citrulline to malaria infected mice was found to be more effective in reducing growth of parasite, with increases survival rate and reduced T-regulatory cells.
| Material & Methods|| |
Animals and infection
Female Balb/c mice at 7–9 weeks of age. Cryopreserved Plasmodium berghei NK65 parasites (obtained from the parasite bank of the National Institute of Malaria Research, New Delhi, India) were passaged once through mice before use in experimental animals. Mice were infected with 5x105 syngenic parasitized erythrocytes (pRBCs) by intra-peritoneal injection. Infected mice were orally fed with the L-arginine and L-citrulline dissolved in water daily. The concentration of L-arginine and L-citrulline was 1.2 mg/g.
Determination of parasitemia
To determine the course of infection, blood smears were prepared from tail vein at various time points as indicated in figure legends and stained with Jaswant Singh Battacharya (JSB) 1 and 2 stains. Parasitemia was determined by counting the percentage of infected cells per 5000 RBCs per slide.
The animals in this study were used in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
Measurement of nitric oxide
To determine the level of nitric oxide in serum, Mice blood was collected without heparin. Serum was collected and subjected to nitric oxide level test by Griess reagent. Absorbance was measured within 30 min in a plate reader with a filter 550nm.
Single cell suspensions of splenocytes were prepared by smashing spleens of infected and control mice. RBCs were lysed with RBC lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) and the cells were washed and then resuspended in RPMI 1640 medium supplemented with 10% FBS (Hyclone, USA), 2 mM L-glutamine (Gibco-BRL, Life technologies, USA) and 100 IU/ ml penicillin-streptomycin (Gibco-BRL).
For flow cytometric analyses, on the 10th day after infection, spleen cells from mice were resuspended in 50 μl of FACS buffer (PBS, 2% FCS) and surface-stained at 4°C with anti-mouse CD4 (GK1.5), -CD8 (53-6.7), -CD19 (1D3), -CD11b (M1/70), -CD11c (N418),CD25 (PC61.5), (all from e-Biosciences). Stained cells were acquired with a FACSFortessa (BD Biosciences) and analyzed by FlowJo (Tree star) software.
Splenocytes (1x106 per ml) from mice treated or untreated with MSCs and infected with Plasmodium were suspended in FACS buffer (PBS with 2% FBS). Surface staining was carried out by incubating with the fluorochrome-labelled antibodies anti-CD4-FITC (fluorescein isothiocyanate) and anti-CD25-APC (allophycocyanin) at 4°C for 30 min in the dark. Cells were fixed for 30 min at RT, in the dark with 2% paraformaldehyde and then treated with Perm/Wash solution (e-bioscience) before incubation with PE (phycoerythrin)-conjugated anti-FOXP-3 antibody (e-bioscience) in Perm/Wash solution at 4°C for 1 hr. Finally, cells were washed in Perm/Wash solution and then washed in FACS buffer and resuspended in FACS buffer. Cells were acquired using LSR Fortessa (BD, Biosciences, USA).
Spleens from the infected and control Balb/c mice were analyzed by histopathology. Spleen tissue sections were cut into 5 microns by a microtome and stained by H&E stain. Hemozoin content was analyzed by microscopy as brown pigments at 100X as described elsewhere.
| Results|| |
Nitric oxide level during malaria pathogenesis
Nitric oxide plays an important role to control malaria pathogenesis. In our previous studies we observed that nitric oxide level become diminished in cells treated with Plasmodium antigens. We were further surprised to note that the production of nitric oxide decreases as the concentration of antigen was increased. To mimic the in-vitro results, we determined the level of nitric oxide in the serum of Plasmodium berghei infected mice, which causes lethal infection in mice, at different time points. Nitric oxide level increases initially and become more than 2-fold at 7th day of post infection then nitric oxide level decreases to basal level [Figure 1]A but percentage of parasitemia keeps on increasing and ultimately leads to death suggesting the crucial role of nitric oxide in protection as decreased level of nitric oxide is unable to control malaria pathogenesis.
|Figure 1: Nitric oxide level and malaria pathogenesis. Balb/c mice were infected with 5×105 Plasmodium berghei (Pb) parasitized erythrocytes via intraperitoneal injection (A) Serum was collected from infected mice and level of nitric oxide was determined. Infected mice were divided into 3 groups: Pb-infected, Pb-infected mice fed with L-arginine and another group consisted of infected mice fed with L-citrulline. Uninfected mice were used as control. Blood collected from tails was used to prepare smears to determine parasitic load (B) Survival of mice (C) Immunohistochemistry of spleen of Balb/c mice (Uninfected, infected and arginine fed and citruine fed mice (D). Black arrows indicate the parasitized RBC in spleen at 1000× original magnification. Data is shown as representative of four independent experiments consisting of three mice in each group|
Click here to view
Effect of L-arginine and L-citrulline on growth of malarial parasite and survival
Disease progression in case of malaria can be monitored by determining percentage of parasitemia in the thin blood smear of the infected mice. Blood collected from tail vein of the Plasmodium berghei (Pb) infected mice was used to determined parasitemia in control mice (without L-arginine or L-citrulline), L-arginine and L-citrulline administered in Plasmodium infected mice. We observed that the percentage of parasitemia in the L-citrulline fed mice was lower than control and L-arginine fed mice [Figure 1]B. The Survival of the L-citrulline administered mice was enhanced by about 10 days [Figure 1]C. Parasitic load was further confirmed by accumulation of infected RBCs by histology of spleen. Spleen from all different groups of mice was isolated and 0.5um sections were cut and stained with H&E stain. [Figure 1]D shows that the parasitic burden is highest in L-arginine fed mice while the parasitic burden in L-citrulline fed mice was lower as compared to control increase in parasitemia in L-arginine fed mice is in coordination with our previous in vitro findings.
Effect of L-arginine and L-citrulline administration on T-Lymphocytes in Plasmodium infected mice
Plasmodium infection in mice leads to splenomegaly due to infiltration of immune cells. Activation of these different immune cells determines the outcome of disease where cell mediated immunity plays a major role. To understand the effect of malaria infection on T cells, spleen was isolated from all the different groups on day 10th post infection. Splenocytes were isolated and stained with different T cell markers. As shown in [Figure 2], no effect on percentage of CD4+ T cells was observed in L-arginine fed mice compared to Pb infected mice. Whereas administration of L-citrulline was able to restore both CD4+ and CD8+ T cells.
|Figure 2: Effect of supplementation of arginine and citrulline in Pb-infected mice on T lymphocytes Splenocytes were harvested at day 10 post-infection from Pb-infected mice either fed with arginine and citrulline. The number of CD4+ and CD8+ T cells from Pb-infected mice fed with arginine and citrulline was determined by staining with anti-CD4 and anti CD8 antibody for cell surface markers and analysed by flow cytometry. Data shown are representative of four independent experiments.|
Click here to view
Effect of L-arginine and L-citrulline administration on CTLA-4 in Plasmodium infected mice
CTLA-4 is considered as a checkpoint during T cell activation as CTLA-4 inhibits T cell functions directly by binding to CD28 that provides positive signals for activation. Blockade of CTLA-4 is shown to enhance the pathogen-specific T cells function. In other words, we can say enhancedCTLA-4 expression is associated with T cell exhaustion. Therefore, mice of different groups were analyzed for CTLA-4 expression on both CD4 and CD8 T cells. Expression of CTLA-4 in mice fed with L-arginine showed significantly increased expression as compared to Pb infected mice on both CD4+ and CD8+ T cells [Figure 3]A, [Figure 3]B. Whereas L-citrulline fed mice has no effect on CTLA-4 expressed on and remains as low as basal level on both CD4+ and CD8+ T cells. These results suggest that L-arginine drives T cells towards cell exhaustion while L-citrulline doesn’t support suppression of effector T cells.
|Figure 3: Expression of CTLA-4 on CD4+ and CD8+ T cells: Pb-infected mice were fed with infected mice were sacrificed; spleen cells were harvested for flow cytometry analysis. Flow Cytometric analysis of CTLA4 molecule on CD 4+ cells (A)and CD 8+Tcell (B) was determined by staining with specific antibodies for cell surface markers. Data is shown as representative of our independent experiments.|
Click here to view
Effect of L-arginine and L-citrulline administration on PD-1 expression in Plasmodium infected mice
Programmed death-1, is one of well studied inhibitory receptors in infections because of its role in T-cell exhaustion. Expression of PD-1 get upregulated on both CD4+ and CD8+ T cells, as well as B cells and other immune cells during acute as well as chronic phase of infection. PD-1 interact with either PD-L1/PD-L2 to modulate the T cell responses during various infectious models. Therefore, the expression of PD-1 molecule was analyzed using splenocytes of mice from different groups. Expression of PD-1 in Pb infected mice showed significant increase on both CD4 and CD8 T cells. Expression of PD-1 in mice fed with L-arginine was further increased marginally as compared to Pb infected mice on both CD4+ and CD8+ T cells [Figure 4]A, [Figure 4]B. Surprisingly, L-citrulline fed mice do no show effect of PD-1 expression on CD4+T cells but expression is down regulated to basal level in case of CD8+ T cells compared to expression in Pb infected mice.
|Figure 4: Expression of PD-1on CD4+ and CD8+ T cells: Splenocytes harvested from different groups were stained with anti-CD4 and anti CD8 antibody and anti PD-1 antibody. Expression of the PD-1 was analyzed on the surface of CD 4+ cells (A)and CD 8+Tcell (B)by flow cytometry. Data is shown are representative of four independent experiments.|
Click here to view
Effect on regulatory T cells in different groups of mice
The role of CTLA-4 and PD-1 has been shown to be involved in regulating the functions of Foxp3+ CD4+ regulatory T (Treg) cells. We further evaluated the prevalence of Treg cells in spleen by flow cytometry by intracellular staining of Foxp3 [Figure 5]. Mice which were given L-citrulline have low proportion of Treg cells while the percentage of these cells increases by3 folds in Pb infected and in the group fed with L-arginine percentage of Treg further increase 2 folds which clearly indicates that L-arginine leads to immunosuppression aiding parasite growth.
|Figure 5: Enumeration of Treg cells: Presence of Treg cells was determined by intracellular staining of FoxP3. The percentage of Treg cells in different groups of mice L-arginine, L-citrulline and control was determined by multicolor staining for CD4 and CD25. Data is shown as representative of four independent experiments.|
Click here to view
| Discussion|| |
Malaria parasite manipulates our immune system to survive and multiply in host. Several research groups have demonstrated that nitric oxide impedes parasitic growth. However, role of nitric oxide donor and its impact on immune system has not been very well characterized. A research group has shown that intravenous injection of L-arginine before plasmodium infection leads to increase in Treg cell. On the other had increased Treg cells has been shown to have detrimental effect on the outcome of disease. In this study we have compared L-arginine and L-citrulline to determine the effect on immune system in malaria pathogenesis using mouse model of malaria. We observed that oral feeding of L-arginine in Plasmodium berghei infected mice parasitemia increased and mice died earlier than the control mice. We also fed L-arginine to Plasmodium chabaudi, a self-resolving strain of malaria parasite infected mice. We found that mice died in the presence of L-arginine (unpublished result). The survival of mice is compromised may be attributed to increased parasitemia. The histological studies also confirm increased parasite burden in L-arginine fed mice. Spleen is site of major immune activation and results in splenomegaly. The flow cytometric analysis on 10th day was done so that we could analyze the immunological response when the parasitemia among all the groups were high, besides it also ensured the inclusion of L arginine fed mice which died on 11th day post infection. The cytometric analysis shows that T cells decreased in L-arginine fed mice but drastically increased in L-citrulline fed mice. Activation of Lymphocytes are influenced by expression of co-stimulatory/inhibitory molecules. The role of these molecules has been well established in malaria. The expression of PD-1 and CTLA-4 co-inhibitory molecules is low on CD8 cells in L-citrulline fed mice while in L-arginine fed mice the expression of these molecules is high. Several studies have shown that high expression of these molecules is responsible for immune downregulation leading to immunosuppression. Blockade of PD-1 or CTLA-4 have been shown to be beneficial. Severe malaria in children have been shown to ameliorate with increased CTLA-4 expression on T cells. Butler and et al have shown that blockade of these molecule plays a protective role.
CTLA-4 and PD-1 has been shown to be involved in regulating the functions of Foxp3+ CD4+ regulatory T (Treg) cells. Regulatory T cells characterized by co-expression of CD4, CD25 and FOXP3 markers, were induce by malaria infection that was further increased by the administration of L-arginine leading to immunosuppression. As the infection progresses the percentage of these cells increases,,. These cells suppress the immune system. Study suggests that suppression of protective immunity may be responsible for parasite persistence. Increased regulatory T cells have been clearly been shown to be associated with disease severity. Mice which were given L-citrulline have low proportion of Treg cells and were able to survive. Our study highlights the benefits of using L citrulline as nitric oxide donor, over L arginine during malaria.
| Conclusion|| |
Group of Plasmodium infected mice that was fed with L-citrulline has enhanced survival and the parasitemia remained low as compared to mice fed with L-arginine. Increased parasitemia in mice fed with L-arginine seems to be a result of increased expression of co-inhibitory molecules like PD-1 and CTLA-4 and increased regulatory T cells. In the L-citrulline fed group Treg cells were found to be less. Decreased Treg in L-citrulline may have led to increased immune response to curb the growth of the parasite. Our result emphasizes on use of L-citrulline, a nitric oxide donor, as a supplement in chemotherapy, since it slows down or retard the parasite growth.
| Acknowledgements|| |
This work was supported by an intramural grant of the ICMR- National Institute of Malaria Research (NIMR), and Department of Biotechnology (DBT), Govt. of India.
Conflict of interest: None
| References|| |
World Health Organization (WHO), World Malaria Report 2019.
Kappe SH, Kaiser K, Matuschewski K. The Plasmodium sporozoite journey: a rite of passage. Trends Parasitol
Graewe S, Stanway RR, Rennenberg A, Heussler VT. Chronicle of a death foretold: Plasmodium liver stage parasites decide on the fate of the host cell. FEMS Microbiol Rev. 36:
Sponaas AM, Cadman E T, Voisine C, Harrison V, Boonstra A, O’Garra A, Langhorne J. Malaria infection changes the ability of splenic dendritic cell populations to stimulate antigen-specific T cells. J Exp Med
2006; 203(6): 1427–1433.
Millington OR, Gibson VB, Rush CM, Zinselmeyer B H, Phillips RS, Garside P, Brewer JM. Malaria impairs T cell clustering and immune priming despite normal signal 1 from dendritic cells. PLoS Pathog
2007; 3(10): 1380-1387.
Schofield L. Intravascular infiltrates and organ-specific inflammation in malaria pathogenesis. Immunol Cell Biol
Butler N S, Moebius J, Pewe L L, Traore B, Doumbo OK, Tygrett LT, et al
. Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection. Nat Immunol
2011; 13(2): 188–195.
Freeman, GJ Sharpe A H. A new therapeutic strategy for malaria: targeting T cell exhaustion. Nat Immunol 2012;
Jemmely NY, Niang M, Preiser PR,. Small variant surface antigens and Plasmodium evasion of immunity. Future Microbiol 2010; 5
Prystowsky J H, Clevenger C V, Zheng ZS. Inhibition of ornithine decarboxylase activity and cell proliferation by ultraviolet B radiation in EGF-stimulated cultured human epidermal keratinocytes. J Invest Dermatol
1993; 101: 54–58.
Pan YY, Liu J, Li Y, YanJ, Cao YM. [L-arginine enhances Th1 immune response against Plasmodium yoelii 17XL infection in DBA/2 mice via activation of dendritic cells]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi
Morris C R, Gladwin MT, Kato G J. Nitric oxide and arginine dysregulation: a novel pathway to pulmonary hypertension in hemolytic disorders. Curr Mol Med
2008; 8(7): 620–632.
Vasquez-Dunddel D, Pan F, Zeng Q, Gorbounov M, Albesiano E, Fu J, Blosser R L et al
. STAT3 regulates arginase-I in myeloid-derived suppressor cells from cancer patients. J Clin Invest
2013; 123(4): 1580–1589.
Munder M. Arginase: an emerging key player in the mammalian immune system. Br J Pharmacol
2009; 158(3): 638–651.
Martins YC, Zanini G M, Frangos JA, Carvalho L J. Efficacy of different nitric oxide-based strategies in preventing experimental cerebral malaria by Plasmodium berghei ANKA. PLoS One
2012; 7(2): e32048.
Bitonti A J, Dumont J A, Bush TL., Edwards ML, Stemerick DM, McCann P P, Sjoerdsma A. Bis(benzyl)polyamine analogs inhibit the growth of chloroquine-resistant human malaria parasites (Plasmodium falciparum) in vitro and in combination with alpha-difluoromethylornithine cure murine malaria. Proc Natl Acad Sci U S A
1989; 86(2): 651–655.
Zhu X, PanY, Li Y, Cui L, Cao Y. Supplement of L-Arg improves protective immunity during early-stage Plasmodium yoelii 17XL infection. Parasite Immunol
Curis E, Nicolis I, Moinard C, Osowska S, Zerrouk N, Benazeth S, Cynober L. Almost all about citrulline in mammals. Amino Acids
2005; 29(3): 177–205.
Amante F H, Stanley A C, Randall L M, Zhou Y, Haque A, Mc-Sweeney K, et al
. A role for natural regulatory T cells in the pathogenesis of experimental cerebral malaria. Am J Pathol
Hansen DS, Schofield L. Natural regulatory T cells in malaria: host or parasite allies? PLoS Pathog
2010; 6(4): e1000771.
Kong Y Y, Fuchsberger M, Xiang S D, Apostolopoulos V, Plebanski M. Myeloid derived suppressor cells and their role in diseases. Curr Med Chem
2013; 20(11): 1437–1444.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]