Single cell RNA sequencing reveals endothelial cell killing and resolution pathways in experimental malaria-associated acute respiratory distress syndrome.

Plasmodium parasites cause malaria, a global health disease that is responsible for more than 200 million clinical cases and 600 000 deaths each year. Most deaths are caused by various complications, including malaria-associated acute respiratory distress syndrome (MA-ARDS). Despite the very rapid and efficient killing of parasites with antimalarial drugs, 15% of patients with complicated malaria succumb. This stresses the importance of investigating resolution mechanisms that are involved in the recovery from these complications once the parasite is killed. To study the resolution of MA-ARDS, P. berghei NK65-infected C57BL/6 mice were treated with antimalarial drugs after onset of symptoms, resulting in 80% survival. Micro-computed tomography revealed alterations of the lungs upon infection, with an increase in total and non-aerated lung volume due to edema. Whole body plethysmography confirmed a drastically altered lung ventilation, which was restored during resolution. Single-cell RNA sequencing indicated an increased inflammatory state in the lungs upon infection, which was accompanied by a drastic decrease in endothelial cells, consistent with CD8+ T cell-mediated killing. During resolution, anti-inflammatory pathways were upregulated and proliferation of endothelial cells was observed. MultiNicheNet interactome analysis identified important changes in the ligand-receptor interactions during disease resolution that warrant further exploration in order to develop new therapeutic strategies. In conclusion, our study provides insights in pro-resolving pathways that limit inflammation and promote endothelial cell proliferation in experimental MA-ARDS. This information may be useful for the design of adjunctive treatments to enhance resolution after Plasmodium parasite killing by antimalarial drugs.

Aspecific binding of anti-NK1.1 antibodies on myeloid cells in an experimental model for malaria-associated acute respiratory distress syndrome.

BACKGROUND: Conventional natural killer (cNK) cells play an important role in the innate immune response by directly killing infected and malignant cells and by producing pro- and anti-inflammatory cytokines. Studies on their role in malaria and its complications have resulted in conflicting results. METHODS: Using the commonly used anti-NK1.1 depletion antibodies (PK136) in an in-house optimized experimental model for malaria-associated acute respiratory distress syndrome (MA-ARDS), the role of cNK cells was investigated. Moreover, flow cytometry was performed to characterize different NK cell populations. RESULTS: While cNK cells were found to be dispensable in the development of MA-ARDS, the appearance of a NK1.1+ cell population was observed in the lungs upon infection despite depletion with anti-NK1.1. Detailed characterization of the unknown population revealed that this population consisted of a mixture of monocytes and macrophages that bind the anti-NK1.1 antibody in an aspecific way. This aspecific binding may occur via Fcγ receptors, such as FcγR4. In contrast, in vivo depletion using anti-NK1.1 antibodies was proved to be specific for cNK cells. CONCLUSION: cNK cells are dispensable in the development of experimental MA-ARDS. Moreover, careful flow cytometric analysis, with a critical mindset in relation to potential aspecific binding despite the use of commercially available Fc blocking reagents, is critical to avoid misinterpretation of the results.

Etiology of lactic acidosis in malaria.

Lactic acidosis and hyperlactatemia are common metabolic disturbances in patients with severe malaria. Lactic acidosis causes physiological adverse effects, which can aggravate the outcome of malaria. Despite its clear association with mortality in malaria patients, the etiology of lactic acidosis is not completely understood. In this review, the possible contributors to lactic acidosis and hyperlactatemia in patients with malaria are discussed. Both increased lactate production and impaired lactate clearance may play a role in the pathogenesis of lactic acidosis. The increased lactate production is caused by several factors, including the metabolism of intraerythrocytic Plasmodium parasites, aerobic glycolysis by activated immune cells, and an increase in anaerobic glycolysis in hypoxic cells and tissues as a consequence of parasite sequestration and anemia. Impaired hepatic and renal lactate clearance, caused by underlying liver and kidney disease, might further aggravate hyperlactatemia. Multiple factors thus participate in the etiology of lactic acidosis in malaria, and further investigations are required to fully understand their relative contributions and the consequences of this major metabolic disturbance.

Skeleton binding protein-1-mediated parasite sequestration inhibits spontaneous resolution of malaria-associated acute respiratory distress syndrome.

Malaria is a hazardous disease caused by Plasmodium parasites and often results in lethal complications, including malaria-associated acute respiratory distress syndrome (MA-ARDS). Parasite sequestration in the microvasculature is often observed, but its role in malaria pathogenesis and complications is still incompletely understood. We used skeleton binding protein-1 (SBP-1) KO parasites to study the role of sequestration in experimental MA-ARDS. The sequestration-deficiency of these SBP-1 KO parasites was confirmed with bioluminescence imaging and by measuring parasite accumulation in the lungs with RT-qPCR. The SBP-1 KO parasites induced similar lung pathology in the early stage of experimental MA-ARDS compared to wildtype (WT) parasites. Strikingly, the lung pathology resolved subsequently in more than 60% of the SBP-1 KO infected mice, resulting in prolonged survival despite the continuous presence of the parasite. This spontaneous disease resolution was associated with decreased inflammatory cytokine expression measured by RT-qPCR and lower expression of cytotoxic markers in pathogenic CD8+ T cells in the lungs of SBP-1 KO infected mice. These data suggest that SBP-1-mediated parasite sequestration and subsequent high parasite load are not essential for the development of experimental MA-ARDS but inhibit the resolution of the disease.

Experimental malaria-associated acute respiratory distress syndrome is dependent on the parasite-host combination and coincides with normocyte invasion.

BACKGROUND: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a complication of malaria with a lethality rate of up to 80% despite anti-malarial treatment. It is characterized by a vast infiltration of leukocytes, microhaemorrhages and vasogenic oedema in the lungs. Previously, a mouse model for MA-ARDS was developed by infection of C57BL/6 mice with the Edinburgh line NK65-E of Plasmodium berghei. RESULTS: Here, both host and parasite factors were demonstrated to play crucial roles in the development and severity of lung pathology. In particular, the genetic constitution of the host was an important determinant in the development of MA-ARDS. Both male and female C57BL/6, but not BALB/c, mice developed MA-ARDS when infected with P. berghei NK65-E. However, the New York line of P. berghei NK65 (NK65-NY) did not induce demonstrable MA-ARDS, despite its accumulation in the lungs and fat tissue to a similar or even higher extent as P. berghei NK65-E. These two commonly used lines of P. berghei differ in their red blood cell preference. P. berghei NK65-NY showed a stronger predilection for reticulocytes than P. berghei NK65-E and this appeared to be associated with a lower pathogenicity in the lungs. The pulmonary pathology in the C57BL/6/P. berghei NK65-E model was more pronounced than in the model with infection of DBA/2 mice with P. berghei strain ANKA. The transient lung pathology in DBA/2 mice infected with P. berghei ANKA coincided with the infection phase in which parasites mainly infected normocytes. This phase was followed by a less pathogenic phase in which P. berghei ANKA mainly infected reticulocytes. CONCLUSIONS: The propensity of mice to develop MA-ARDS during P. berghei infection depends on both host and parasite factors and appears to correlate with RBC preference. These data provide insights in induction of MA-ARDS and may guide the choice of different mouse-parasite combinations to study lung pathology.

Experimental malaria-associated acute kidney injury is independent of parasite sequestration and resolves upon antimalarial treatment.

Malaria remains a important global disease with more than 200 million cases and 600 000 deaths each year. Malaria-associated acute kidney injury (MAKI) may occur in up to 40% of patients with severe malaria and is associated with increased mortality. Histopathological characteristics of AKI in malaria are acute tubular injury, interstitial nephritis, focal segmental glomerulosclerosis, collapsing glomerulopathy and glomerulonephritis. We observed that C57BL/6 mice infected with Plasmodium berghei NK65 (PbNK65) develop MAKI in parallel with malaria-associated acute respiratory distress syndrome (MA-ARDS). MAKI pathology was associated with proteinuria, acute tubular injury and collapse of glomerular capillary tufts, which resolved rapidly after treatment with antimalarial drugs. Importantly, parasite sequestration was not detected in the kidneys in this model. Furthermore, with the use of skeleton binding protein-1 (SBP-1) KO PbNK65 parasites, we found that parasite sequestration in other organs and its subsequent high parasite load are not required for the development of experimental MAKI. Similar proteinuria, histopathological features, and increases in kidney expression of interferon-γ, TNF-α, kidney injury molecule-1 (KIM-1) and heme oxygenase-1 (HO-1) was observed in both infected groups despite a significant difference in parasite load. Taken together, we introduce a model of experimental AKI in malaria with important similarities to AKI in malaria patients. Therefore, this mouse model might be important to further study the pathogenesis of AKI in malaria.

Corrigendum: CCR2 Is Dispensable for Disease Resolution but Required for the Restoration of Leukocyte Homeostasis Upon Experimental Malaria-Associated Acute Respiratory Distress Syndrome.

[This corrects the article DOI: 10.3389/fimmu.2020.628643.].

CCR2 Is Dispensable for Disease Resolution but Required for the Restoration of Leukocyte Homeostasis Upon Experimental Malaria-Associated Acute Respiratory Distress Syndrome.

Malaria complications are often lethal, despite efficient killing of Plasmodium parasites with antimalarial drugs. This indicates the need to study the resolution and healing mechanisms involved in the recovery from these complications. Plasmodium berghei NK65-infected C57BL/6 mice develop malaria-associated acute respiratory distress syndrome (MA-ARDS) at 8 days post infection. Antimalarial treatment was started on this day and resulted in the recovery, as measured by the disappearance of the signs of pathology, in >80% of the mice. Therefore, this optimized model represents an asset in the study of mechanisms and leukocyte populations involved in the resolution of MA-ARDS. C-C chemokine receptor type 2 (CCR2) knock-out mice were used to investigate the role of monocytes and macrophages, since these cells are described to play an important role during the resolution of other inflammatory diseases. CCR2 deficiency was associated with significantly lower numbers of inflammatory monocytes in the lungs during infection and resolution and abolished the increase in non-classical monocytes during resolution. Surprisingly, CCR2 was dispensable for the development and the resolution of MA-ARDS, since no effect of the CCR2 knock-out was observed on any of the disease parameters. In contrast, the reappearance of eosinophils and interstitial macrophages during resolution was mitigated in the lungs of CCR2 knock-out mice. In conclusion, CCR2 is required for re-establishing the homeostasis of pulmonary leukocytes during recovery. Furthermore, the resolution of malaria-induced lung pathology is mediated by unknown CCR2-independent mechanisms.

Limitations of neutrophil depletion by anti-Ly6G antibodies in two heterogenic immunological models.

Neutrophil-depleting antibodies, such as anti-GR1 (RB6-8C5) and anti-Ly6G (1A8), are commonly used to study the in vivo function of neutrophils in murine disease models. Anti-Ly6G antibodies became the standard, because in contrast to anti-GR1, these do not bind Ly6C. The efficiency of the depletion needs to be carefully analysed as flow cytometry plots may be misinterpreted. For example, the staining intensity of GR1 on neutrophils (CD11b+ GR1hi) drops upon anti-Ly6G administration. We show that this drop is due to competition between anti-GR1 and anti-Ly6G antibodies. Neutrophil depletion with anti-Ly6G in naive mice was organ- and strain-specific. Furthermore, an incomplete anti-Ly6G-dependent neutrophil depletion was obtained in two immune-mediated mouse models, i.e. in malaria-infected C57BL/6 mice and in complete Freund's adjuvant (CFA)-challenged BALB/c mice. BrdU-incorporation studies show a slight increase in proliferating bone marrow neutrophils upon depletion in naive mice. Strikingly, depletion with anti-Ly6G in CFA-challenged BALB/c mice resulted in a significant increase in proliferating splenic neutrophils, causing a fast rebound of new immature neutrophils. In conclusion, our results emphasize the importance of careful panel design, gating strategies and duration of neutrophil depletion and highlight the context-dependent Ly6G depletion efficiency. It furthermore underlines the need for new tools to understand the in vivo role of neutrophils in immunological models.