Plasmodium Berghei ANKA Infection Research Articles

Neurons upregulate PD-L1 via IFN/STAT1/IRF1 to alleviate damage by CD8+ T cells in cerebral malaria

BackgroundCerebral malaria (CM) is the most lethal complication of malaria, and survivors usually endure neurological sequelae. Notably, the cytotoxic effect of infiltrating Plasmodium-activated CD8+ T cells on cerebral microvasculature endothelial cells is a prominent feature of the experimental CM (ECM) model with blood–brain barrier disruption. However, the damage effect of CD8+ T cells infiltrating the brain parenchyma on neurons remains unclear. Based on the immunosuppressive effect of the PD-1/PD-L1 pathway on T cells, our previous study demonstrated that the systemic upregulation of PD-L1 to inhibit CD8+ T cell function could effectively alleviate the symptoms of ECM mice. However, it has not been reported whether neurons can suppress the pathogenic effect of CD8+ T cells through the PD-1/PD-L1 negative immunomodulatory pathway. As the important inflammatory factor of CM, interferons can induce the expression of PD-L1 via different molecular mechanisms according to the neuro-immune microenvironment. Therefore, this study aimed to investigate the direct interaction between CD8+ T cells and neurons, as well as the mechanism of neurons to alleviate the pathogenic effect of CD8+ T cells through up-regulating PD-L1 induced by IFNs.MethodsUsing the ECM model of C57BL/6J mice infected with Plasmodium berghei ANKA (PbA), morphological observations were conducted in vivo by electron microscope and IF staining. The interaction between the ECM CD8+ T cells (immune magnetic bead sorting from spleen of ECM mice) and primary cultured cortical neurons in vitro was observed by IF staining and time-lapse photography. RNA-seq was performed to analyze the signaling pathway of PD-L1 upregulation in neurons induced by IFNβ or IFNγ, and verified through q-PCR, WB, IF staining, and flow cytometry both in vitro and in vivo using IFNAR or IFNGR gene knockout mice. The protective effect of adenovirus-mediated PD-L1 IgGFc fusion protein expression was verified in ECM mice with brain stereotaxic injection in vivo and in primary cultured neurons via viral infection in vitro.ResultsIn vivo, ECM mice showed infiltration of activated CD8+ T cells and neuronal injury in the brain parenchyma. In vitro, ECM CD8+ T cells were in direct contact with neurons and induced axonal damage, as an active behavior. The PD-L1 protein level was elevated in neurons of ECM mice and in primary cultured neurons induced by IFNβ, IFNγ, or ECM CD8+ T cells in vitro. Furthermore, the IFNβ or IFNγ induced neuronal expression of PD-L1 was mediated by increasing STAT1/IRF1 pathway via IFN receptors. The increase of PD-L1 expression in neurons during PbA infection was weakened after deleting the IFNAR or IFNGR. Increased PD-L1 expression by adenovirus partially protected neurons from CD8+ T cell-mediated damage both in vitro and in vivo.ConclusionOur study demonstrates that both type I and type II IFNs can induce neurons to upregulate PD-L1 via the STAT1/IRF1 pathway mediated by IFN receptors to protect against activated CD8+ T cell-mediated damage, providing a targeted pathway to alleviate neuroinflammation during ECM.Graphical

IL-15 Complex-Induced IL-10 Enhances Plasmodium-specific CD4+ T Follicular Helper Differentiation and Antibody Production.

Malaria, which results from infection with Plasmodium parasites, remains a major public health problem. Although humans do not develop long-lived, sterilizing immunity, protection against symptomatic disease develops after repeated exposure to Plasmodium parasites and correlates with the acquisition of humoral immunity. Despite the established role Abs play in protection from malaria disease, dysregulated inflammation is thought to contribute to the suboptimal immune response to Plasmodium infection. Plasmodium berghei ANKA (PbA) infection results in a fatal severe malaria disease in mice. We previously demonstrated that treatment of mice with IL-15 complex (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) induces IL-10 expression in NK cells, which protects mice from PbA-induced death. Using a novel MHC class II tetramer to identify PbA-specific CD4+ T cells, in this study we demonstrate that IL-15C treatment enhances T follicular helper (Tfh) differentiation and modulates cytokine production by CD4+ T cells. Moreover, genetic deletion of NK cell-derived IL-10 or IL-10R expression on T cells prevents IL-15C-induced Tfh differentiation. Additionally, IL-15C treatment results in increased anti-PbA IgG Ab levels and improves survival following reinfection. Overall, these data demonstrate that IL-15C treatment, via its induction of IL-10 from NK cells, modulates the dysregulated inflammation during Plasmodium infection to promote Tfh differentiation and Ab generation, correlating with improved survival from reinfection. These findings will facilitate improved control of malaria infection and protection from disease by informing therapeutic strategies and vaccine design.

Blockage of mechanosensitive Piezo1 channel alleviates the severity of experimental malaria-associated acute lung injury

BackgroundMalaria-associated acute lung injury (MA-ALI) is a well-recognized clinical complication of severe, complicated malaria that is partly driven by sequestrations of infected red blood cells (iRBCs) on lung postcapillary induced impaired blood flow. In earlier studies the mechanosensitive Piezo1 channel emerged as a regulator of mechanical stimuli, but the function and underlying mechanism of Piezo1 impacting MA-ALI severity via sensing the impaired pulmonary blood flow are still not fully elucidated. Thus, the present study aimed to explore the role of Piezo1 in the severity of murine MA-ALI.MethodsHere, we utilized a widely accepted murine model of MA-ALI using C57BL/6 mice with Plasmodium berghei ANKA infection and then added a Piezo1 inhibitor (GsMTx4) to the model. The iRBC-stimulated Raw264.7 macrophages in vitro were also targeted with GsMTx4 to further explore the potential mechanism.ResultsOur data showed an elevation in the expression of Piezo1 and number of Piezo1+-CD68+ macrophages in lung tissues of the experimental MA-ALI mice. Compared to the infected control mice, the blockage of Piezo1 with GsMTx4 dramatically improved the survival rate but decreased body weight loss, peripheral blood parasitemia/lung parasite burden, experimental cerebral malaria incidence, total protein concentrations in bronchoalveolar lavage fluid, lung wet/dry weight ratio, vascular leakage, pathological damage, apoptosis and number of CD68+ and CD86+ macrophages in lung tissues. This was accompanied by a dramatic increase in the number of CD206+ macrophages (M2-like subtype), upregulation of anti-inflammatory cytokines (e.g. IL-4 and IL-10) and downregulation of pro-inflammatory cytokines (e.g. TNF-α and IL-1β). In addition, GsMTx4 treatment remarkably decreased pulmonary intracellular iron accumulation, protein level of 4-HNE (an activator of ferroptosis) and the number of CD68+-Piezo1+ and CD68+-4-HNE+ macrophages but significantly increased protein levels of GPX4 (an inhibitor of ferroptosis) in experimental MA-ALI mice. Similarly, in vitro study showed that the administration of GsMTx4 led to a remarkable elevation in the mRNA levels of CD206, IL-4, IL-10 and GPX-4 but to a substantial decline in CD86, TNF-α, IL-1β and 4-HNE in the iRBC-stimulated Raw264.7 cells.ConclusionsOur findings indicated that blockage of Piezo1 with GsMTx4 alleviated the severity of experimental MA-ALI in mice partly by triggering pulmonary macrophage M2 polarization and subsequent anti-inflammatory responses but inhibited apoptosis and ferroptosis in lung tissue. Our data suggested that targeting Piezo1 in macrophages could be a promising therapeutic strategy for treating MA-ALI.Graphical

N-(coumarin-3-yl)cinnamamide Promotes Immunomodulatory, Neuroprotective, and Lung Function-Preserving Effects during Severe Malaria.

Plasmodium berghei ANKA (PbA) infection in mice resembles several aspects of severe malaria in humans, such as cerebral malaria and acute respiratory distress syndrome. Herein, the effects of N-(coumarin-3-yl)cinnamamide (M220) against severe experimental malaria have been investigated. Treatment with M220 proved to protect cognitive abilities and lung function in PbA-infected mice, observed by an object recognition test and spirometry, respectively. In addition, treated mice demonstrated decreased levels of brain and lung inflammation. The production and accumulation of microglia, and immune cells that produce the inflammatory cytokines TNF and IFN-γ, decreased, while the production of the anti-inflammatory cytokine IL-10 by innate and adaptive immune cells was enhanced. Treatment with M220 promotes immunomodulatory, neuroprotective, and lung function-preserving effects during experimental severe malaria. Therefore, it may be an interesting therapeutic candidate to treat severe malaria effects.

Interleukin-33 exerts pleiotropic immunoregulatory effects in response to Plasmodium berghei ANKA (PbA) infection in mice

Objective: To determine the involvement and the modulatory effects of IL-33 during Plasmodium berghei ANKA (PbA) infection. Methods: PbA infection in male ICR mice was utilized as a model of malaria. Systemically circulating IL-33 levels were determined in blood plasma by enzyme-linked immunosorbent assay (ELISA). After 24 hours post-inoculation of PbA, recombinant IL-33 and ST2, and antibodies against IL-33 and IgG treatments were administered daily for 3 days. Tissue expression and localization of IL-33 were assessed in organs generally affected by malaria via immunohistochemistry. Moreover, histopathological examination was performed to assess the effects of the treatments. Results: The levels of systemic IL-33 were elevated at the critical phase of PbA infection. Likewise, immunohistochemical analysis revealed a significant upregulation of IL-33 expression at the critical phase in the brain, lungs, and spleen of PbA-infected mice as compared to healthy controls. Treatment with IL-33 protected against experimental cerebral malaria development and reduced pathological features in the brain and lungs of the PbA-infected mice. Conclusions: A potential critical role and involvement of IL-33 in PbA infection may hint at the resolution of immunopathological sequelae associated with malaria.

A high-fat diet protects C57BL/6 mice from Plasmodium berghei ANKA infection in an experimental malaria study

IntroductionIt is well known that dietary changes have a significant impact on the immune system, and modifications in lipid balance may contribute to disease progression in several cases. Malaria is still a major global health concern, and the development of the disease has already been linked to the host’s nutritional status, so it’s critical to understand how environmental factors, such as dietary variations, can influence the outcome of infection. We therefore investigated the effect of a short-term diet in a murine model of experimental cerebral malaria.MethodsFor this, male C57BL/6 mice were fed a high fat diet containing 60% of the calories from lipids for 5 days. Following this period, the animals were infected with Plasmodium berghei ANKA, and parasitemia, survival, and neurological scores were compared. Considering that one of the first elimination routes of the intracellular parasite is oxidative stress, the antioxidant N-acetylcysteine was administered to assess whether the protection would be reversed.Results and discussionAnimals fed a hyperlipidic diet reacted the same way to infection even after NAC administration. Unlike the control group, which died after eight days of infection with roughly 7% parasitized red blood cells, the hyperlipidic diet group was resistant to infection, with no clinical signs and no increase in blood parasitemia. Several proinflammatory cytokines such as TNF-α IFN-γ and IL-6 were increased in the spleen of both infected groups, regardless of their diet. The provision of a high-fat diet to mice for as little as 5 days completely prevents Plasmodium berghei ANKA infection in C57BL/6 mice, while the treatment of an antioxidant failed to reverse the parasite protection.

Human ApoE2 protects mice against Plasmodium berghei ANKA experimental cerebral malaria.

Cerebral malaria (CM) is a severe neurological complication of Plasmodium falciparum infection with acute brain lesions. Genetic variations in both host and parasite have been associated with susceptibility to CM, but the underlying molecular mechanism remains unclear. Here, we demonstrate that variants of human apolipoprotein E (hApoE) impact the outcome of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). Mice carrying the hApoE2 isoform have fewer intracerebral hemorrhages and are more resistant to ECM than mice bearing the hApoE3, hApoE4, or endogenous murine ApoE (mApoE). hApoE2 mice infected with PbA showed increased splenomegaly and IFN-γ levels in serum but reduced cerebral cell apoptosis that correlated with the survival advantage against ECM. In addition, upregulated expression of genes associated with lipid metabolism and downregulated expression of genes linked to immune responses were observed in the brain tissue of hApoE2 mice relative to ECM-susceptible mice after PbA infection. Notably, serum cholesterol and the cholesterol content of brain-infiltrating CD8+ T cells are significantly higher in infected hApoE2 mice, which might contribute to a significant reduction in the sequestration of brain CD8+ T cells. Consistent with the finding that fewer brain lesions occurred in infected hApoE2 mice, fewer behavioral deficits were observed in the hApoE2 mice. Finally, a meta-analysis of publicly available data also showed an increased hApoE2 allele in the malaria-endemic African population, suggesting malaria selection. This study shows that hApoE2 protects mice from ECM through suppression of CD8+ T cell activation and migration to the brain and enhanced cholesterol metabolism.IMPORTANCECerebral malaria (CM) is the deadliest complication of malaria infection with an estimated 15%-25% mortality. Even with timely and effective treatment with antimalarial drugs such as quinine and artemisinin derivatives, survivors of CM may suffer long-term cognitive and neurological impairment. Here, we show that human apolipoprotein E variant 2 (hApoE2) protects mice from experimental CM (ECM) via suppression of CD8+ T cell activation and infiltration to the brain, enhanced cholesterol metabolism, and increased IFN-γ production, leading to reduced endothelial cell apoptosis, BBB disruption, and ECM symptoms. Our results suggest that hApoE can be an important factor for risk assessment and treatment of CM in humans.

Thymic atrophy induced by Plasmodium berghei ANKA and Plasmodium yoelii 17XL infection

The thymus is the anatomical site where T cells undergo a complex process of differentiation, proliferation, selection, and elimination of autorreactive cells which involves molecular signals in different intrathymic environment. However, the immunological functions of the thymus can be compromised upon exposure to different infections, affecting thymocyte populations. In this work, we investigated the impact of malaria parasites on the thymus by using C57BL/6 mice infected with Plasmodium berghei ANKA and Plasmodium yoelii 17XL; these lethal infection models represent the most severe complications, cerebral malaria, and anemia respectively. Data showed a reduction in the thymic weight and cellularity involving different T cell maturation stages, mainly CD4−CD8− and CD4+CD8+ thymocytes, as well as an increased presence of apoptotic cells, leading to significant thymic cortex reduction. Thymus atrophy showed no association with elevated serum cytokines levels, although increased glucocorticoid levels did. The severity of thymic damage in both models reached the same extend although it occurs at different stages of infection, showing that thymic atrophy does not depend on parasitemia level but on the specific host-parasite interaction.

Effects of anti-tau immunotherapy on reactive microgliosis, cerebral endotheliopathy, and cognitive function in an experimental model of cerebral malaria.

Cerebral malaria (CM), a potentially fatal encephalopathy caused primarily by infection with Plasmodium falciparum, results in long-term adverse neuro-psychiatric sequelae. Neural cell injury contributes to the neurological deficits observed in CM. Abnormal regulation of tau, an axonal protein pathologically associated with the formation of neurofibrillary lesions in neurodegenerative diseases, has been linked to inflammation and cerebral microvascular compromise and has been reported in human and experimental CM (ECM). Immunotherapy with a monoclonal antibody to pathological tau (PHF-1 mAB) in experimental models of neurodegenerative diseases has been reported to mitigate cognitive decline. We investigated whether immunotherapy with PHF-1 mAB prevented cerebral endotheliopathy, neural cell injury, and neuroinflammation during ECM. Using C57BL/6 mice infected with either Plasmodium berghei ANKA (PbA), which causes ECM, Plasmodium berghei NK65 (PbN), which causes severe malaria, but not ECM, or uninfected mice (Un), we demonstrated that when compared to PbN infection or uninfected mice, PbA infection resulted in significant memory impairment at 6 days post-infection, in association with abnormal tau phosphorylation at Ser202 /Thr205 (pSer202 /Thr205 ) and Ser396-404 (pSer396-404 ) in mouse brains. ECM also resulted in significantly higher expression of inflammatory markers, in microvascular congestion, and glial cell activation. Treatment with PHF-1 mAB prevented PbA-induced cognitive impairment and was associated with significantly less vascular congestion, neuroinflammation, and neural cell activation in mice with ECM. These findings suggest that abnormal regulation of tau protein contributes to cerebral vasculopathy and is critical in the pathogenesis of neural cell injury during CM. Tau-targeted therapies may ameliorate the neural cell damage and subsequent neurocognitive impairment that occur during disease.

Administration of soluble gp130Fc disrupts M-1 macrophage polarization, dendritic cell activation, MDSC expansion and Th-17 induction during experimental cerebral malaria

Regulatory effect of IL-6 on various immune cells plays a crucial role during experimental cerebral malaria pathogenesis. IL-6 neutralization can restore distorted ratios of myeloid dendritic cells and plasmacytoid dendritic cells as well as the balance between Th-17 and T-regulatory cells. IL-6 can also influence immune cells through classical and trans IL-6 signalling pathways. As trans IL-6 signalling is reportedly involved during malaria pathogenesis, we focused on studying the effects of trans IL-6 signalling blockade on various immune cell populations and how they regulate ECM progression. Results show that administration of sgp130Fc recombinant chimera protein lowers the parasitemia, increases the survivability of Plasmodium berghei ANKA infected mice, and restores the distorted ratios of M1/M2 macrophage, mDC/pDC, and Th-17/Treg. IL-6 trans signalling blockade has been found to affect both expansion of myeloid derived suppressor cells (MDSCs) and expression of inflammatory markers on them during Plasmodium berghei ANKA infection indicating that trans IL-6 signalling might regulate various immune cells and their function during ECM. In this work for the first time, we delineate the effect of sgp130Fc administration on influencing the immunological changes within the host secondary lymphoid organ during ECM induced by Plasmodium berghei ANKA infection.

Immunomodulatory effects of testosterone and letrozole during Plasmodium berghei ANKA infection.

Malaria is one of the leading health problems globally. Plasmodium infection causes pronounced sexual dimorphism, and the lethality and severity are more remarkable in males than in females. To study the role of testosterone in the susceptibility and mortality of males in malaria, it is common to increase its concentration. However, this strategy does not consider the enzyme CYP19A1 aromatase, which can transform it into oestrogens. To avoid the interference of oestrogens, we inhibited in vivo CYP19A1 aromatase with letrozole and increased the testosterone level by exogen administration before infection with Plasmodium berghei ANKA. We measured the impact on free testosterone, 17β-oestradiol and dehydroepiandrosterone levels in plasma; additionally, we evaluated parasitaemia, body temperature, body mass, glucose levels and haemoglobin concentration. Furthermore, we evaluated the effects of testosterone on the immune response; we quantified the CD3+/CD4+, CD3+/CD8+, CD19+, Mac-3+ and NK cells in the spleen and the plasma concentrations of the cytokines IL-2, IL-4, IL-6, IFN-, IL-10, TNF-α and IL-17A. Finally, we quantified the levels of antibodies. We found that mice treated with the combination of letrozole and testosterone and infected with Plasmodium berghei ANKA had increased concentrations of free testosterone and DHEA but decreased levels of 17β-oestradiol. As a result, parasitaemia increased, leading to severe anaemia. Interestingly, testosterone increased temperature and decreased glucose concentration as a possible testosterone-mediated regulatory mechanism. The severity of symptomatology was related to critical immunomodulatory effects generated by free testosterone; it selectively increased CD3+CD8+ T and CD19+ cells but decreased Mac-3+. Remarkably, it reduced IL-17A concentration and increased IL-4 and TNF-α. Finally, it increased IgG1 levels and the IgG1/IgG2a ratio. In conclusion, free testosterone plays an essential role in pathogenesis in male mice by increasing CD8+ and decreasing Mac3+ cells and mainly reducing IL-17A levels, which is critical in the development of anaemia. Our results are important for understanding the mechanisms that regulate the exacerbated inflammatory response in infectious diseases and would be useful for the future development of alternative therapies to reduce the mortality generated by inflammatory processes.

Cell-specific MHC class I deletion on CD11c+ APCs abrogates CD8 T Cell infiltration into the brain

Abstract Cerebral malaria (CM) is a severe complication of P. falciparum infection and a leading cause of death, globally. Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice serves as a murine model of CM called experimental cerebral malaria (ECM) due to the neurological and neuropathological features that simulate the infection of human CM. A putative mechanism of disease in human CM requires CD8 T cell to infiltration into the brain and blood-brain barrier disruption. The specific antigen-presenting cell type(s) that prime this CNS infiltrating CD8 T cell response capable of inducing lethal blood-brain barrier disruption has not been defined. To answer this, single class I expressing K bloxP transgenic mice were generated. Crossing these mice to class I deficient CD11c-cre or LysM-cre mice created the LysM-cre K bcKO and CD11c-cre K bcKO mice, respectively. PbA infected K bloxP and LysM-cre K bcKO were susceptible to ECM. In contrast, CD11c-cre K bcKO mice survived acute PbA infection and avoided neurological manifestations. We therefore employed spectral flow cytometry to analyze splenocytes and brain infiltrating immune cells during acute PbA infection in these genotypes. We observed a reduction in activated CD8 T cells in the spleens of PbA infected CD11c-cre K bcKO mice versus the K bloxP mice and the LysM-cre K bcKO. This reduced peripheral CD8 T cell activation in PbA infected CD11c-cre Kb cKO mice also corresponded with reduced brain infiltration of CD8 T cells. We conclude a CD11c+ APC is therefore required for priming and potentially recruitment of CD8 T cells to the brain during lethal ECM pathology. 5R01NS103212-06

Evaluation of Experimental Cerebral Malaria of Curcumin and Kaempferol in Plasmodium berghei ANKA-Infected Mice

Background: Cerebral malaria (CM) is one of the most severe complications of Plasmodium falciparum infection and the leading cause of death from malaria in endemic areas. Natural products with antioxidant and anti-inflammatory activities have become valuable alternative therapeutic options in CM treatment. Therefore, this study aimed to investigate the neuroprotective effects of curcumin and kaempferol in experimental cerebral malaria (ECM) in mice infected with Plasmodium berghei ANKA (PbA). Methods: After PbA infection, mice were divided into 9 groups, namely Group I (negative control (NC)) with 0.5% HPMC, Group II received chloroquine 20 mg/kg, Group III (normal) with aquadest, Groups IV, V, and VI received curcumin at doses of 20, 40, and 80 mg/kg, respectively, Groups VII, VIII, and IX received kaempferol at doses of 20, 40, and 80 mg/kg, respectively. The antimalarial activity was evaluated using Peter's four-day suppressive test. This was conducted to determine the % parasitemia, survival rate, AST and ALT, blood-brain barrier (BBB) leakage, and neurobehavioral disorders in mice with CM. Results: The results showed that all treatments had significant antimalarial activity, with the % suppression depending on the dose. It also indicates that PbA-infected mice had a survival rate of 11-19 days after infection, which was higher than those in the NC group. This suggested that curcumin and kaempferol have a protective effect on the survival of PbA-infected mice. Furthermore, they significantly reduced the AST and ALT concentrations in the sample compared to the NC group. The same was observed in cerebral vessel extravasation, where the Evans Blue stain assay showed significantly less dye extravasation in the brains of PbA-infected mice treated with curcumin and kaempferol. This indicated better-protected integrity of the BBB. Additionally, the results also demonstrated a decrease in neurological disorders arising during ECM in the group treated with curcumin and kaempferol. Conclusion: Considering these results, it is concluded that treatments with curcumin and kaempferol could improve animal survival, prevent AST and ALT elevations, as well as protect the BBB and neurobehavioral disorders associated with CM in PbA-infected mice.

Phytochemical Analysis, Antimalarial Properties, and Acute Toxicity of Aqueous Extracts of Trisamo and Jatu-Phala-Tiga Recipes.

Drug resistance remains a significant problem that threatens antimalarial drug treatment. Hence, the challenge is to find new effective antimalarial drugs. Based on our previous study, aqueous extracts of trisamo (TSM) and jatu-phala-tiga (JPT) had good in vitro antimalarial activities, and these recipes contain multiple beneficial pharmacological effects that could be useful for malaria therapy. Therefore, this study aimed to investigate the antimalarial activity and toxicity of the aqueous extracts of TSM and JPT in mouse models. The aqueous extractions were carried out using the decoction method. Compound identification was conducted using LC-QTOF-MS analysis. The antimalarial activities of TSM and JPT at doses 200, 400, and 600 mg/kg were evaluated against Plasmodium berghei ANKA infection using a four-day suppressive test. The toxic effects of oral administration of the extracts at 2 g/kg dose were determined using an acute toxicity test. The chemical constituents of TSM contained 83 compounds, whereas JPT contained 84 compounds. All doses of the extracts exhibited a significant suppression (p < 0.05) of the parasite compared to the negative control in a four-day test. The maximum activities were observed at 600 mg/kg dose with 67.02% suppression for TSM and 79.34% for JPT, followed by 400 mg/kg dose (57.63% for TSM and 64.79% for JPT) and then 200 mg/kg dose (52.35% for TSM and 54.46% for JPT). In addition, there were no significant differences (p < 0.05) in the RBC, MCV, and MCH levels of mice receiving JPT extract compared to the uninfected control. The WBC level of mice receiving 400 and 600 mg/kg of TSM, and 200 and 400 mg/kg of JPT, was significantly (p < 0.05) lower than the infected control, and the extracts did not significantly prevent the loss of platelets. For the acute toxicity test, there were no signs of toxicity or deaths in mice, and there were no differences in the histology, weight, or enzyme biochemistry of the liver and kidney between the extract and vehicle groups. However, the platelet count in the extract-treated mice was significantly higher than that in the control group. In conclusion, this study suggests that aqueous extracts of TSM and JPT have potent antimalarial activities and could be promising as new candidates for antimalarial drug development.

Investigation of Plasma-Derived Lipidome Profiles in Experimental Cerebral Malaria in a Mouse Model Study.

Cerebral malaria (CM), a fatal complication of Plasmodium infection that affects children, especially under the age of five, in sub-Saharan Africa and adults in South-East Asia, results from incompletely understood pathogenetic mechanisms. Increased release of circulating miRNA, proteins, lipids and extracellular vesicles has been found in CM patients and experimental mouse models. We compared lipid profiles derived from the plasma of CBA mice infected with Plasmodium berghei ANKA (PbA), which causes CM, to those from Plasmodium yoelii (Py), which does not. We previously showed that platelet-free plasma (18k fractions enriched from plasma) contains a high number of extracellular vesicles (EVs). Here, we found that this fraction produced at the time of CM differed dramatically from those of non-CM mice, despite identical levels of parasitaemia. Using high-resolution liquid chromatography-mass spectrometry (LCMS), we identified over 300 lipid species within 12 lipid classes. We identified 45 and 75 lipid species, mostly including glycerolipids and phospholipids, with significantly altered concentrations in PbA-infected mice compared to Py-infected and uninfected mice, respectively. Total lysophosphatidylethanolamine (LPE) levels were significantly lower in PbA infection compared to Py infection and controls. These results suggest that experimental CM could be characterised by specific changes in the lipid composition of the 18k fraction containing circulating EVs and can be considered an appropriate model to study the role of lipids in the pathophysiology of CM.

Lymphotoxin-α Orchestrate Hypoxia and Immune factors to Induce Experimental Cerebral Malaria: Inhibition Mitigates Pathogenesis, Neurodegeneration, and Increase Survival.

Knockdown studies have shown lymphotoxin-α (Lt-α) as a critical molecule for Experimental cerebral malaria (ECM) pathogenesis. We investigated the role of lymphotoxin-α in regulating active caspase-3 and calpain1. T cell infiltration into the brains, and subsequent neuronal cell death are the essential features of Plasmodium berghei ANKA(PbA)-induced ECM. Our results showed increased Lt-α levels during ECM. Treatment of naïve mice with serum from ECM mice and exogenous Lt-α was lethal. We inhibited Lt-α in vivo during PbA infection by injecting the mice with anti-Lt-α antibody. Inhibition of Lt-α mitigated neuronal cell death and increased mice's survival until 30-day post-infection (p.i.) compared to only 15days survival of PbA control mice.

Interleukin-1b and Interferon-g are Associated with Malaria-Induced Hypoinsulinemic Hypoglycemia in Plasmodium berghei Anka-Infected Mice

Malaria-induced hypoglycemia is recognized as a serious complication of malaria and has one of the strongest associations with mortality in children. It has been speculated that oxidative stress and pro-inflammatory response during parasite infection were involved in its pathophysiology. Hence, this study aimed to investigate the development of malaria-induced hypoglycemia during Plasmodium berghei ANKA (PbANKA) infection with particular attention to the involvement of c-peptide, interleukin-1b (IL-1b), and interferon-g (IFN-g). ICR mice were infected with 1×107 parasitized erythrocytes of PbANKA, and parasitemia was monitored, and the development of hypoglycemia was assessed by measuring plasma glucose levels. The change of c-peptide level was evaluated. The pro-inflammatory response of IL-1b and IFN-g were also quantified in plasma. It was found that PbANKA infection resulted in hypoglycemia as indicated by a significantly (P &lt; 0.05) decrease in plasma glucose levels on day 4 post-infection and associated with parasitemia. The c-peptide was slightly increased at day 2 post-infection, and then significantly (P &lt; 0.05) decreased since day 4. Furthermore, we observed a significantly (P &lt; 0.05) increased IL-1b, firstly responded, at day 2 post-infection followed by increasing the IFN-g level at day 4 in PbANKA-induced hypoglycemia. Our findings support the idea that hypoinsulinemic hypoglycemia in the PbANKA infected mice may be involved in the high IL-1b and IFN-g against the parasite infection.

Designing and development of phthalimides as potent anti-tubulin hybrid molecules against malaria

Constant emergence of drug-resistant Plasmodium falciparum warrants urgent need for effective and inexpensive drugs. Herein, phthalimide (Pht) analogs possessing the bioactive scaffolds, benzimidazole and 1,2,3-triazole, were evaluated for in vitro and in vivo anti-plasmodial activity without any apparent hemolysis, or cytotoxicity. Analogs 4(a-e) inhibited the growth of 3D7 and RKL-9 strains at submicromolar concentrations. Defects were observed during parasite egress from or invasion of the red blood cells. Mitochondrial membrane depolarization was measured as one of the causes of cell death. Phts 4(a-e) in combination with artemisinin exhibited two-to three-fold increased efficacy. Biophysical and biochemical analysis suggest that Pht analogs mediate plasmodial growth inhibition by interacting with tubulin protein of the parasite. Lastly, Phts 4(a-e) significantly decreased parasitemia and extended host survival in murine model Plasmodium berghei ANKA infection. Combined, the data indicate that Pht analogs should be further explored, which could offer novel value to the antimalarial drug development pipeline.

The monoterpene 1,8-cineole prevents cerebral edema in a murine model of severe malaria.

1,8-Cineole is a naturally occurring compound found in essential oils of different plants and has well-known anti-inflammatory and antimicrobial activities. In the present work, we aimed to investigate its potential antimalarial effect, using the following experimental models: (1) the erythrocytic cycle of Plasmodium falciparum; (2) an adhesion assay using brain microvascular endothelial cells; and (3) an experimental cerebral malaria animal model induced by Plasmodium berghei ANKA infection in susceptible mice. Using the erythrocytic cycle of Plasmodium falciparum, we characterized the schizonticidal effect of 1,8-cineole. This compound decreased parasitemia in a dose-dependent manner with a half maximal inhibitory concentration of 1045.53 ± 63.30 μM. The inhibitory effect of 972 μM 1,8-cineole was irreversible and independent of parasitemia. Moreover, 1,8-cineole reduced the progression of intracellular development of the parasite over 2 cycles, inducing important morphological changes. Ultrastructure analysis revealed a massive loss of integrity of endomembranes and hemozoin crystals in infected erythrocytes treated with 1,8-cineole. The monoterpene reduced the adhesion index of infected erythrocytes to brain microvascular endothelial cells by 60%. Using the experimental cerebral malaria model, treatment of infected mice for 6 consecutive days with 100 mg/kg/day 1,8-cineole reduced cerebral edema with a 50% reduction in parasitemia. Our data suggest a potential antimalarial effect of 1,8-cineole with an impact on the parasite erythrocytic cycle and severe disease.

NK cell-derived IL-10 enhances Plasmodium-specific CD4+ Tfh differentiation and IL-27 production in mice

Abstract Malaria, which results from infection with Plasmodium parasites, remains a major public health problem with over 200 million cases and nearly 500,000 deaths annually. Although long-lived, sterilizing immunity does not develop in humans following repeated exposure to Plasmodium parasites, protection against symptomatic disease correlates with the acquisition of humoral immunity. Despite the established role antibodies play in protection to malaria disease, excessive inflammation is thought to contribute to the ineffective immune response to Plasmodium infection. Plasmodium berghei ANKA (PbA) infection results in a fatal severe malaria disease in mice. We previously showed that treatment of mice with IL-15 complex (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) induces IL-10 expression in NK cells, which protects mice from PbA-induced death by dampening the fatal inflammatory response. Interestingly, IL-15C treatment promotes Tfh differentiation at 7 days post-PbA infection (dpi), and genetic deletion of NK cell-derived IL-10 or antibody blockade of IL-10R prevents IL-15C-induced Tfh differentiation. Additionally, IL-15C treatment enhances CD4 T cell-derived IL-27p28 production at 7 dpi and results in increased anti-PbA IgG antibody levels at 42 dpi. These data suggest that IL-15C treatment, via its induction of IL-10 from NK cells, modulates the inflammatory milieu to promote Tfh differentiation, IL-27 production, and PbA-specific antibody generation. These findings will facilitate improved control of malaria infection and protection from disease by informing therapeutic strategies and vaccine design. Supported by grants from NIH (K22 AI143969-01A1, K08 AI141761-01)