Plasmodium-Induced Inflammation by Uric Acid

Infection of erythrocytes with the Plasmodium parasite causes the pathologies associated with malaria, which result in at least one million deaths annually. The rupture of infected erythrocytes triggers an inflammatory response, which is induced by parasite-derived factors that still are not fully characterized. Induced secretion of inflammatory cytokines by these factors is considered a major cause of malaria pathogenesis. In particular, the inflammatory cytokine tumor necrosis factor (TNF) is thought to mediate most of the life-threatening pathologies of the disease. Here we describe the molecular characterization of a novel pathway that results in the secretion of TNF by host cells. We found that erythrocytes infected by Plasmodium accumulate high concentrations of hypoxanthine and xanthine. Degradation of Plasmodium-derived hypoxanthine/xanthine results in the formation of uric acid, which triggers the secretion of TNF. Since uric acid is considered a “danger signal” released by dying cells to alert the immune system, Plasmodium appears to have co-evolved to exploit this warning system. Identifying the mechanisms used by the parasite to induce the host inflammatory response is essential to develop urgently needed therapies against this disease

Correction: Population Genomics of the Immune Evasion (var) Genes of Plasmodium falciparum

Var genes encode the major surface antigen (PfEMP1) of the blood stages of the human malaria parasite Plasmodium falciparum. Differential expression of up to 60 diverse var genes in each parasite genome underlies immune evasion. We compared the diversity of the DBLalpha domain of var genes sampled from 30 parasite isolates from a malaria endemic area of Papua New Guinea (PNG) and 59 from widespread geographic origins (global). Overall, we obtained over 8,000 quality-controlled DBLalpha sequences. Within our sampling frame, the global population had a total of 895 distinct DBLalpha "types" and negligible overlap among repertoires. This indicated that var gene diversity on a global scale is so immense that many genomes would need to be sequenced to capture its true extent. In contrast, we found a much lower diversity in PNG of 185 DBLalpha types, with an average of approximately 7% overlap among repertoires. While we identify marked geographic structuring, nearly 40% of types identified in PNG were also found in samples from different countries showing a cosmopolitan distribution for much of the diversity. We also present evidence to suggest that recombination plays a key role in maintaining the unprecedented levels of polymorphism found in these immune evasion genes. This population genomic framework provides a cost effective molecular epidemiological tool to rapidly explore the geographic diversity of var genes

Population Genomics of the Immune Evasion (var) Genes of Plasmodium falciparum

Var genes encode the major surface antigen (PfEMP1) of the blood stages of the human malaria parasite Plasmodium falciparum. Differential expression of up to 60 diverse var genes in each parasite genome underlies immune evasion. We compared the diversity of the DBLα domain of var genes sampled from 30 parasite isolates from a malaria endemic area of Papua New Guinea (PNG) and 59 from widespread geographic origins (global). Overall, we obtained over 8,000 quality-controlled DBLα sequences. Within our sampling frame, the global population had a total of 895 distinct DBLα “types” and negligible overlap among repertoires. This indicated that var gene diversity on a global scale is so immense that many genomes would need to be sequenced to capture its true extent. In contrast, we found a much lower diversity in PNG of 185 DBLα types, with an average of approximately 7% overlap among repertoires. While we identify marked geographic structuring, nearly 40% of types identified in PNG were also found in samples from different countries showing a cosmopolitan distribution for much of the diversity. We also present evidence to suggest that recombination plays a key role in maintaining the unprecedented levels of polymorphism found in these immune evasion genes. This population genomic framework provides a cost effective molecular epidemiological tool to rapidly explore the geographic diversity of var genes

Uric Acid Is a Mediator of the Plasmodium falciparum-Induced Inflammatory Response

Malaria triggers a high inflammatory response in the host that mediates most of the associated pathologies and contributes to death. The identification of pro-inflammatory molecules derived from Plasmodium is essential to understand the mechanisms of pathogenesis and to develop targeted interventions. Uric acid derived from hypoxanthine accumulated in infected erythrocytes has been recently proposed as a mediator of inflammation in rodent malaria.We found that human erythrocytes infected with Plasmodium falciparum gradually accumulate hypoxanthine in their late stages of development. To analyze the role of hypoxanthine-derived uric acid induced by P. falciparum on the inflammatory cytokine response from human blood mononuclear cells, cultures were treated with allopurinol, to inhibit uric acid formation from hypoxanthine, or with uricase, to degrade uric acid. Both treatments significantly reduce the secretion of TNF, IL-6, IL-1beta and IL-10 from human cells.Uric acid is a major contributor of the inflammatory response triggered by P. falciparum in human peripheral blood mononuclear cells. Since the inflammatory reaction induced by P. falciparum is considered a major cause of malaria pathogenesis, identifying the mechanisms used by the parasite to induce the host inflammatory response is essential to develop urgently needed therapies against this disease

A Molecular Epidemiological Study of var Gene Diversity to Characterize the Reservoir of Plasmodium falciparum in Humans in Africa

BACKGROUND: The reservoir of Plasmodium infection in humans has traditionally been defined by blood slide positivity. This study was designed to characterize the local reservoir of infection in relation to the diverse var genes that encode the major surface antigen of Plasmodium falciparum blood stages and underlie the parasite's ability to establish chronic infection and transmit from human to mosquito. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the molecular epidemiology of the var multigene family at local sites in Gabon, Senegal and Kenya which differ in parasite prevalence and transmission intensity. 1839 distinct var gene types were defined by sequencing DBLα domains in the three sites. Only 76 (4.1%) var types were found in more than one population indicating spatial heterogeneity in var types across the African continent. The majority of var types appeared only once in the population sample. Non-parametric statistical estimators predict in each population at minimum five to seven thousand distinct var types. Similar diversity of var types was seen in sites with different parasite prevalences. CONCLUSIONS/SIGNIFICANCE: Var population genomics provides new insights into the epidemiology of P. falciparum in Africa where malaria has never been conquered. In particular, we have described the extensive reservoir of infection in local African sites and discovered a unique var population structure that can facilitate superinfection through minimal overlap in var repertoires among parasite genomes. Our findings show that var typing as a molecular surveillance system defines the extent of genetic complexity in the reservoir of infection to complement measures of malaria prevalence. The observed small scale spatial diversity of var genes suggests that var genetics could greatly inform current malaria mapping approaches and predict complex malaria population dynamics due to the import of var types to areas where no widespread pre-existing immunity in the population exists

<i>P. falciparum</i>-derived uric acid induces TNF, IL-6 and IL-1β release from PBMCs.

<p>PBMCs were incubated with media alone (control), uninfected erythrocytes (RBCs) or <i>P. falciparum</i>-infected erythrocytes (iRBCs) at a ratio of (5∶1; erythrocyte∶PBMC) for 6 h (24 h for IL-10) in the absence (black bars) or presence (white bars) of 2 mM allopurinol (A–D) or 0.1 mg/ml uricase (E–H). Incubation media were collected and TNF (A,E), IL-6 (B,F), IL-1β (C,G) and IL-10 (D,H) concentrations were determined by flow cytometry using cytometric bead array. Data represent the average of triplicated samples with standard deviations. *, indicates significant differences (<i>p</i><0.05) in cytokine release by PBMCs incubated with iRBCs when compared to IRBCs in the presence of allopurinol or uricase.</p

Mature <i>P. falciparum</i> infected erythrocytes accumulate high levels of hypoxanthine.

<p>Hypoxanthine was analyzed in the soluble fraction of lysates of human erythrocytes infected with <i>P. falciparum</i> at different times after infection in a synchronized culture. Lysates of uninfected erythrocytes cultured for the same times were used as controls. Shown are GC - selected reaction monitoring MS ion plots using the m/z 365.2 to m/z 251.2 product ion MS/MS transition. Hypoxanthine accumulated in cultured uninfected erythrocytes (red lines) and <i>P. falciparum</i> infected erythrocytes (black lines) is shown. Infected erythrocytes at 33 h (mature trophozoites, thick lines) and 40 h (schizonts, think lines) were purified from synchronized cultures of 2% parasitemia.</p

Mature <i>P. falciparum</i> infected erythrocytes induce TNF, IL-6 and IL-1β from PBMCs.

<p>(A–C) PBMCs were incubated with mature <i>P. falciparum</i> infected erythrocytes (squares) or uninfected erythrocytes (circles) at the indicated ratio of erythrocyte to PBMC for 6 h. Data represent the average of triplicated samples with standard deviations. Incubation media were collected and TNF (A), IL-6 (B), or IL-1β (C) concentrations were determined by flow cytometry using cytometric bead array. (D) <i>P. falciparum</i> infected erythrocytes were cultivated alone or in the presence of 2 mM allopurinol. Synchronized cultures were seeded at 0.5% rings and the culture media was changed daily. The percentage of infected erythrocytes was calculated after 0 h (black bars), 24 h (white bars) and 48 h (grey bars) of culture.</p