Blockade of caspase cascade overcomes malaria-associated acute respiratory distress syndrome in mice.

Malaria is an enormous burden on global health that caused 409,000 deaths in 2019. Severe malaria can manifest in the lungs, an illness known as acute respiratory distress syndrome (ARDS). Not much is known about the development of malaria-associated ARDS (MA-ARDS), especially regarding cell death in the lungs. We had previously established a murine model that mimics various human ARDS aspects, such as pulmonary edema, hemorrhages, pleural effusion, and hypoxemia, using DBA/2 mice infected with Plasmodium berghei ANKA. Here, we explored the mechanisms and the involvement of apoptosis in this syndrome. We found that apoptosis contributes to the pathogenesis of MA-ARDS, primarily as facilitators of the alveolar-capillary barrier breakdown. The protection of pulmonary endothelium by inhibiting caspase activation could be a promising therapeutic strategy to prevent the pathogenicity of MA-ARDS. Therefore, intervention in the programmed death cell mechanism could help patients not to develop severe malaria.

Adverse pregnancy outcomes are associated with Plasmodium vivax malaria in a prospective cohort of women from the Brazilian Amazon.

BACKGROUND: Malaria in Brazil represents one of the highest percentages of Latin America cases, where approximately 84% of infections are attributed to Plasmodium (P.) vivax. Despite the high incidence, many aspects of gestational malaria resulting from P. vivax infections remain poorly studied. As such, we aimed to evaluate the consequences of P. vivax infections during gestation on the health of mothers and their neonates in an endemic area of the Amazon. METHODS AND FINDINGS: We have conducted an observational cohort study in Brazilian Amazon between January 2013 and April 2015. 600 pregnant women were enrolled and followed until delivery. After applying exclusion criteria, 329 mother-child pairs were included in the analysis. Clinical data regarding maternal infection, newborn's anthropometric measures, placental histopathological characteristics, and angiogenic and inflammatory factors were evaluated. The presence of plasma IgG against the P. vivax (Pv) MSP119 protein was used as marker of exposure and possible associations with pregnancy outcomes were analyzed. Multivariate logistic regression analysis revealed that P. vivax infections during the first trimester of pregnancy are associated with adverse gestational outcomes such as premature birth (adjusted odds ratio [aOR] 8.12, 95% confidence interval [95%CI] 2.69-24.54, p < 0.0001) and reduced head circumference (aOR 3.58, 95%CI 1.29-9.97, p = 0.01). Histopathology analysis showed marked differences between placentas from P. vivax-infected and non-infected pregnant women, especially regarding placental monocytes infiltrate. Placental levels of vasomodulatory factors such as angiopoietin-2 (ANG-2) and complement proteins such as C5a were also altered at delivery. Plasma levels of anti-PvMSP119 IgG in infected pregnant women were shown to be a reliable exposure marker; yet, with no association with improved pregnancy outcomes. CONCLUSIONS: This study indicates that P. vivax malaria during the first trimester of pregnancy represents a higher likelihood of subsequent poor pregnancy outcomes associated with marked placental histologic modification and angiogenic/inflammatory imbalance. Additionally, our findings support the idea that antibodies against PvMSP119 are not protective against poor pregnancy outcomes induced by P. vivax infections.

Plasmodium falciparum infection dysregulates placental autophagy.

Plasmodium (P.) falciparum malaria during pregnancy has been frequently associated with severe consequences such as maternal anemia, abortion, premature birth, and reduced birth weight. Placental damage promotes disruption of the local homeostasis; though, the mechanisms underlying these events are still to be elucidated. Autophagy is a fundamental homeostatic mechanism in the natural course of pregnancy by which cells self-recycle in order to survive in stressful environments. Placentas from non-infected and P. falciparum-infected women during pregnancy were selected from a previous prospective cohort study conducted in the Brazilian Amazon (Acre, Brazil). Newborns from infected women experienced reduced birth weight (P = 0.0098) and placental immunopathology markers such as monocyte infiltrate (P < 0.0001) and IL-10 production (P = 0.0122). The placentas were evaluated for autophagy-related molecules. As a result, we observed reduced mRNA levels of ULK1 (P = 0.0255), BECN1 (P = 0.0019), and MAP1LC3B (P = 0.0086) genes in placentas from P. falciparum-infected, which was more striking in those diagnosed with placental malaria. Despite the protein levels of these genes followed the same pattern, the observed reduction was not statistically significant in placentas from P. falciparum-infected women. Nevertheless, our data suggest that chronic placental immunopathology due to P. falciparum infection leads to autophagy dysregulation, which might impair local homeostasis during malaria in pregnancy that may result in poor pregnancy outcomes.

Association of Malaria Infection During Pregnancy With Head Circumference of Newborns in the Brazilian Amazon.

Importance: Malaria during pregnancy is associated with adverse events for the fetus and newborn, but the association of malaria during pregnancy with the head circumference of the newborn is unclear. Objective: To investigate the association of malaria during pregnancy with fetal head growth. Design, Setting, and Participants: Two cohort studies were conducted at the general maternity hospital of Cruzeiro do Sul (Acre, Brazil) in the Amazonian region. One cohort study prospectively enrolled noninfected and malaria-infected pregnant women who were followed up until delivery, between January 2013 and April 2015. The other cohort study was assembled retrospectively using clinical and malaria data from all deliveries that occurred between January 2012 and December 2013. Data analyses were conducted from January to August 2017 and revised in November 2018. Clinical data from pregnant women and anthropometric measures of their newborns were evaluated. A total of 600 pregnant women were enrolled through volunteer sampling (prospective cohort study), and 4697 pregnant women were selected by population-based sampling (retrospective cohort study). After application of exclusion criteria, data from 251 (prospective cohort study) and 232 (retrospective cohort study) malaria-infected and 158 (prospective cohort study) and 3650 (retrospective cohort study) noninfected women were evaluated. Exposure: Malaria during pregnancy. Main Outcomes and Measures: The primary end point was the incidence of altered head circumference in newborns delivered from malaria-infected mothers compared with that from noninfected mothers. Secondary end points included measures of placental pathology relative to newborn head circumference. Results: In total, 4291 maternal-child pairs were analyzed. Among 409 newborns in the prospective cohort study, the mothers of 251 newborns had malaria during pregnancy, infected with Plasmodium vivax, Plasmodium falciparum, or both. Among 3882 newborns in the retrospective cohort study, 232 were born from mothers that had malaria during pregnancy. The prevalence of newborns with a small head (19 [30.7%] in the prospective cohort study and 30 [36.6%] in the retrospective cohort study) and the prevalence of microcephaly among newborns (5 [8.1%] in the prospective cohort study and 6 [7.3%] in the retrospective cohort study) were higher among newborns from women infected with P falciparum during pregnancy. Multivariate logistic regression analyses revealed that P falciparum infection during pregnancy represented a significant risk factor for the occurrence of small head circumference in newborns (prospective cohort study: odds ratio, 3.15; 95% CI, 1.52-6.53; P = .002; retrospective cohort study: odds ratio, 1.91; 95% CI, 1.21-3.04; P = .006). Placental pathologic findings corroborated this association, with more syncytial nuclear aggregates and inflammatory infiltrates occurring in placentas of newborns born with decreased head circumference. Conclusions and Relevance: This study indicates that falciparum malaria during pregnancy is associated with decreased head circumference in newborns, which is in turn associated with evidence of placental malaria.

Fetal-Derived MyD88 Signaling Contributes to Poor Pregnancy Outcomes During Gestational Malaria.

Placental malaria (PM) remains a severe public health problem in areas of high malaria transmission. Despite the efforts to prevent infection poor outcomes in Plasmodium endemic areas, there is still a considerable number of preterm births and newborns with low birth weight resulting from PM. Although local inflammation triggered in response to malaria is considered crucial in inducing placental damage, little is known about the differential influence of maternal and fetal immune responses to the disease progression. Therefore, using a PM mouse model, we sought to determine the contribution of maternal and fetal innate immune responses to PM development. For this, we conducted a series of cross-breeding experiments between mice that had differential expression of the MyD88 adaptor protein to obtain mother and correspondent fetuses with distinct genetic backgrounds. By evaluating fetal weight and placental vascular spaces, we have shown that the expression of MyD88 in fetal tissue has a significant impact on PM outcomes. Our results highlighted the existence of a distinct contribution of maternal and fetal immune responses to PM onset. Thus, contributing to the understanding of how inflammatory processes lead to the dysregulation of placental homeostasis ultimately impairing fetal development.

Malaria during pregnancy and newborn outcome in an unstable transmission area in Brazil: A population-based record linkage study.

BACKGROUND: Malaria in pregnancy (MiP) is one of the major causes of mortality and morbidity in tropical regions, causing maternal anemia, intrauterine growth retardation, preterm birth, and low birth weight (LBW). The integration of the information systems on pregnancy and malaria could prove to be a useful method of improved decision making for better maternal-child health. METHODS: A population-based observational study acquired information retrospectively from all live births that occurred between 2006 and 2014 in Cruzeiro do Sul (Acre, Brazil). Social and clinical data of the mother and newborn was extracted from the Information System of Live Births. Malaria episodes information was obtained from the Brazilian Epidemiological Surveillance Information System Malaria. A deterministic record linkage was performed to assess malaria impact on pregnancy. RESULTS: The studied population presented a malaria incidence of 8.9% (1283 pregnant women infected), of which 63.9% infected by Plasmodium (P.) vivax. Reduction of newborn birth weight at term (small for gestational age (SGA) and LBW) has been found associated with P. vivax infection during pregnancy (SGA-OR 1.24, 95% CI 1.02-1.52, p = 0.035; term LBW-OR 1.39, 95% CI 1.03-1.88, p = 0.033). Additionally, P. falciparum infection during pregnancy has been found to be associated with preterm births (OR 1.54, 95% CI 1.09-2.18, p = 0.016), which is related with late preterm births (OR 1.59, 95% CI 1.11-2.27, p = 0.011). CONCLUSIONS: Despite the decrease of malaria cases during the evaluation period and regardless of Plasmodium species, we present evidence of the deleterious effects of MiP in a low transmission area in the Amazonian region.

Publisher Correction: TLR4-Mediated Placental Pathology and Pregnancy Outcome in Experimental Malaria.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

TLR4-Mediated Placental Pathology and Pregnancy Outcome in Experimental Malaria.

Malaria-associate pregnancy has a significant impact on infant morbidity and mortality. The detrimental effects of malaria infection during pregnancy have been shown to correlate with immune activation in the placental tissue. Herein we sought to evaluate the effect of Toll-like receptors (TLRs) activation on placental malaria (PM) development by using the Plasmodium berghei NK65GFP infection model. We observed that activation of the innate immune system by parasites leads to PM due to local inflammation. We identified TLR4 activation as the main pathway involved in the inflammatory process in the placental tissue since the absence of functional TLR4 in mice leads to a decrease in the pro-inflammatory responses, which resulted in an improved pregnancy outcome. Additionally, a similar result was obtained when infected pregnant mice were treated with IAXO-101, a TLR4/CD14 blocker. Together, this study illustrates the importance of TLR4 signalling for the generation of the severe inflammatory response involved in PM pathogenesis. Therefore, our results implicate that TLR4 blockage could be a potential candidate for therapeutic interventions to reduce malaria-induced pathology both in the mother and the fetus.

HGF Secreted by Activated Kupffer Cells Induces Apoptosis of Plasmodium-Infected Hepatocytes.

Malaria liver stage infection is an obligatory parasite development step and represents a population bottleneck in Plasmodium infections, providing an advantageous target for blocking parasite cycle progression. Parasite development inside hepatocytes implies a gross cellular insult evoking innate host responses to counteract intra-hepatocytic infection. Using primary hepatocyte cultures, we investigated the role of Kupffer cell-derived hepatocyte growth factor (HGF) in malaria liver stage infection. We found that Kupffer cells from Plasmodium-infected livers produced high levels of HGF, which trigger apoptosis of infected hepatocytes through a mitochondrial-independent apoptosis pathway. HGF action in infected hepatocyte primary cultures results in a potent reduction of parasite yield by specifically sensitizing hepatocytes carrying established parasite exo-erythrocytic forms to undergo apoptosis. This apoptosis mechanism is distinct from cell death that is spontaneously induced in infected cultures and is governed by Fas signaling modulation through a mitochondrial-dependent apoptosis pathway. This work indicates that HGF and Fas signaling pathways are part of an orchestrated host apoptosis response that occurs during malaria liver stage infection, decreasing the success of infection of individual hepatocytes. Our results raise the hypothesis that paracrine signals derived from Kupffer cell activation are implicated in directing death of hepatocytes infected with the malaria parasite.

Emerging Plasmodium vivax resistance to chloroquine in South America: an overview.

The global emergence of Plasmodium vivax strains resistant to chloroquine (CQ) since the late 1980s is complicating the current international efforts for malaria control and elimination. Furthermore, CQ-resistant vivax malaria has already reached an alarming prevalence in Indonesia, East Timor and Papua New Guinea. More recently, in vivo studies have documented CQ-resistant P. vivax infections in Guyana, Peru and Brazil. Here, we summarise the available data on CQ resistance across P. vivax-endemic areas of Latin America by combining published in vivo and in vitro studies. We also review the current knowledge regarding the molecular mechanisms of CQ resistance in P. vivax and the prospects for developing and standardising reliable molecular markers of drug resistance. Finally, we discuss how the Worldwide Antimalarial Resistance Network, an international collaborative effort involving malaria experts from all continents, might contribute to the current regional efforts to map CQ-resistant vivax malaria in South America.

Emerging Plasmodium vivax resistance to chloroquine in South America: an overview

The global emergence of Plasmodium vivax strains resistant to chloroquine (CQ) since the late 1980s is complicating the current international efforts for malaria control and elimination. Furthermore, CQ-resistant vivax malaria has already reached an alarming prevalence in Indonesia, East Timor and Papua New Guinea. More recently, in vivo studies have documented CQ-resistant P. vivax infections in Guyana, Peru and Brazil. Here, we summarise the available data on CQ resistance across P. vivax-endemic areas of Latin America by combining published in vivo and in vitro studies. We also review the current knowledge regarding the molecular mechanisms of CQ resistance in P. vivax and the prospects for developing and standardising reliable molecular markers of drug resistance. Finally, we discuss how the Worldwide Antimalarial Resistance Network, an international collaborative effort involving malaria experts from all continents, might contribute to the current regional efforts to map CQ-resistant vivax malaria in South America.

TREM2 governs Kupffer cell activation and explains belr1 genetic resistance to malaria liver stage infection.

Plasmodium liver stage infection is a target of interest for the treatment of and vaccination against malaria. Here we used forward genetics to search for mechanisms underlying natural host resistance to infection and identified triggering receptor expressed on myeloid cells 2 (TREM2) and MHC class II molecules as determinants of Plasmodium berghei liver stage infection in mice. Locus belr1 confers resistance to malaria liver stage infection. The use of newly derived subcongenic mouse lines allowed to map belr1 to a 4-Mb interval on mouse chromosome 17 that contains the Trem2 gene. We show that Trem2 expression in the nonparenchymal liver cells closely correlates with resistance to liver stage infection, implicating TREM2 as a mediator of the belr1 genetic effect. Trem2-deficient mice are more susceptible to liver stage infection than their WT counterparts. We found that Kupffer cells are the principle cells expressing TREM2 in the liver, and that Trem2(-/-) Kupffer cells display altered functional activation on exposure to P. berghei sporozoites. TREM2 expression in Kupffer cells contributes to the limitation of parasite expansion in isolated hepatocytes in vitro, potentially explaining the increased susceptibility of Trem2(-/-) mice to liver stage infection. The MHC locus was also found to control liver parasite burden, possibly owing to the expression of MHC class II molecules in hepatocytes. Our findings implicate unexpected Kupffer-hepatocyte cross-talk in the control Plasmodium liver stage infection and demonstrate that TREM2 is involved in host responses against the malaria parasite.

IL-12p40 deficiency leads to uncontrolled Trypanosoma cruzi dissemination in the spinal cord resulting in neuronal death and motor dysfunction.

Chagas' disease is a protozoosis caused by Trypanosoma cruzi that frequently shows severe chronic clinical complications of the heart or digestive system. Neurological disorders due to T. cruzi infection are also described in children and immunosuppressed hosts. We have previously reported that IL-12p40 knockout (KO) mice infected with the T. cruzi strain Sylvio X10/4 develop spinal cord neurodegenerative disease. Here, we further characterized neuropathology, parasite burden and inflammatory component associated to the fatal neurological disorder occurring in this mouse model. Forelimb paralysis in infected IL-12p40KO mice was associated with 60% (p<0.05) decrease in spinal cord neuronal density, glutamate accumulation (153%, p<0.05) and strong demyelization in lesion areas, mostly in those showing heavy protein nitrosylation, all denoting a neurotoxic degenerative profile. Quantification of T. cruzi 18S rRNA showed that parasite burden was controlled in the spinal cord of WT mice, decreasing from the fifth week after infection, but progressive parasite dissemination was observed in IL-12p40KO cords concurrent with significant accumulation of the astrocytic marker GFAP (317.0%, p<0.01) and 8-fold increase in macrophages/microglia (p<0.01), 36.3% (p<0.01) of which were infected. Similarly, mRNA levels for CD3, TNF-α, IFN-γ, iNOS, IL-10 and arginase I declined in WT spinal cords about the fourth or fifth week after infection, but kept increasing in IL-12p40KO mice. Interestingly, compared to WT tissue, lower mRNA levels for IFN-γ were observed in the IL-12p40KO spinal cords up to the fourth week of infection. Together the data suggest that impairments of parasite clearance mechanisms in IL-12p40KO mice elicit prolonged spinal cord inflammation that in turn leads to irreversible neurodegenerative lesions.

Malaria liver stage susceptibility locus identified on mouse chromosome 17 by congenic mapping.

Host genetic variants are known to confer resistance to Plasmodium blood stage infection and to control malaria severity both in humans and mice. This work describes the genetic mapping of a locus for resistance to liver stage parasite in the mouse. First, we show that decreased susceptibility to the liver stage of Plasmodium berghei in the BALB/c mouse strain is attributable to intra-hepatic factors and impacts on the initial phase of blood stage infection. We used QTL mapping techniques to identify a locus controlling this susceptibility phenotype (LOD score 4.2) on mouse chromosome 17 (belr1 locus). Furthermore, analysis of congenic mouse strains delimited the belr1 locus boundaries distally to the H2 region. Quantification of parasites in the liver of infected congenic mice strongly suggested that the belr1 locus represents a genetic factor controlling the expansion of P. berghei in the hepatic tissue. The mapping of belr1 locus raises the hypothesis that host gene variation is able to control the progression of Plasmodium liver stage infection and opens the possibility that the human genomic region orthologue to belr1 may contain genes that confer resistance to the human malaria liver stage.