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.

The Immunopathology of Pulmonary Rejection after Murine Lung Transplantation.

To improve outcomes following lung transplantation, it is essential to understand the immunological mechanisms that result in chronic graft failure. The associated clinical syndrome is termed chronic lung allograft dysfunction (CLAD), which is known to be induced by alloimmune-dependent (i.e., rejection) and alloimmune-independent factors (e.g., infections, reflux and environmental factors). We aimed to explore the alloimmune-related mechanism, i.e., pulmonary rejection. In this study, we use a murine orthotopic left lung transplant model using isografts and allografts (C57BL/6 or BALB/c as donors to C57BL/6 recipients), with daily immunosuppression (10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone). Serial sacrifice was performed at days 1, 7 and 35 post-transplantation (n = 6 at each time point for each group). Left transplanted lungs were harvested, a single-cell suspension was made and absolute numbers of immune cells were quantified using multicolor flow cytometry. The rejection process followed the principles of a classic immune response, including innate but mainly adaptive immune cells. At day 7 following transplantation, the numbers of interstitial macrophages, monocytes, dendritic cells, NK cells, NKT cells, CD4+ T cells and CD8+ T and B cells were increased in allografts compared with isografts. Only dendritic cells and CD4+ T cells remained elevated at day 35 in allografts. Our study provides insights into the immunological mechanisms of true pulmonary rejection after murine lung transplantation. These results might be important in further research on diagnostic evaluation and treatment for CLAD.

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.

Glucocorticoid dysfunction in children with severe malaria.

Introduction: Malaria remains a widespread health problem with a huge burden. Severe or complicated malaria is highly lethal and encompasses a variety of pathological processes, including immune activation, inflammation, and dysmetabolism. Previously, we showed that adrenal hormones, in particular glucocorticoids (GCs), play critical roles to maintain disease tolerance during Plasmodium infection in mice. Here, GC responses were studied in Cameroon in children with uncomplicated malaria (UM), severe malaria (SM) and asymptomatic controls (AC). Methods: To determine the sensitivity of leukocytes to GC signaling on a transcriptional level, we measured the ex vivo induction of glucocorticoid induced leucine zipper (GILZ) and FK506-binding protein 5 (FKBP5) by GCs in human and murine leukocytes. Targeted tracer metabolomics on peripheral blood mononuclear cells (PBMCs) was performed to detect metabolic changes induced by GCs. Results: Total cortisol levels increased in patients with clinical malaria compared to AC and were higher in the SM versus UM group, while cortisol binding globulin levels were unchanged and adrenocorticotropic hormone (ACTH) levels were heterogeneous. Induction of both GILZ and FKBP5 by GCs was significantly reduced in patients with clinical malaria compared to AC and in malaria-infected mice compared to uninfected controls. Increased activity in the pentose phosphate pathway was found in the patients, but this was not affected by ex vivo stimulation with physiological levels of hydrocortisone. Interestingly, hydrocortisone induced increased levels of cAMP in AC, but not in clinical malaria patients. Discussion: Altogether, this study shows that patients with SM have increased cortisol levels, but also a decreased sensitivity to GCs, which may clearly contribute to the severity of disease.

A Multimodal Imaging-Supported Down Syndrome Mouse Model of RSV Infection.

Individuals with Down syndrome (DS) are more prone to develop severe respiratory tract infections. Although a RSV infection has a high clinical impact and severe outcome in individuals with DS, no vaccine nor effective therapeutics are available. Any research into infection pathophysiology or prophylactic and therapeutic antiviral strategies in the specific context of DS would greatly benefit this patient population, but currently such relevant animal models are lacking. This study aimed to develop and characterize the first mouse model of RSV infection in a DS-specific context. Ts65Dn mice and wild type littermates were inoculated with a bioluminescence imaging-enabled recombinant human RSV to longitudinally track viral replication in host cells throughout infection progression. This resulted in an active infection in the upper airways and lungs with similar viral load in Ts65Dn mice and euploid mice. Flow cytometric analysis of leukocytes in lungs and spleen demonstrated immune alterations with lower CD8+ T cells and B-cells in Ts65Dn mice. Overall, our study presents a novel DS-specific mouse model of hRSV infection and shows that potential in using the Ts65Dn preclinical model to study immune-specific responses of RSV in the context of DS and supports the need for models representing the pathological development.

Absence of CCR2 Promotes Proliferation of Alveolar Macrophages That Control Lung Inflammation in Acute Respiratory Distress Syndrome in Mice.

Acute respiratory distress syndrome (ARDS) consists of uncontrolled inflammation that causes hypoxemia and reduced lung compliance. Since it is a complex process, not all details have been elucidated yet. In a well-controlled experimental murine model of lipopolysaccharide (LPS)-induced ARDS, the activity and viability of macrophages and neutrophils dictate the beginning and end phases of lung inflammation. C-C chemokine receptor type 2 (CCR2) is a critical chemokine receptor that mediates monocyte/macrophage activation and recruitment to the tissues. Here, we used CCR2-deficient mice to explore mechanisms that control lung inflammation in LPS-induced ARDS. CCR2-/- mice presented higher total numbers of pulmonary leukocytes at the peak of inflammation as compared to CCR2+/+ mice, mainly by enhanced influx of neutrophils, whereas we observed two to six-fold lower monocyte or interstitial macrophage numbers in the CCR2-/-. Nevertheless, the time needed to control the inflammation was comparable between CCR2+/+ and CCR2-/-. Interestingly, CCR2-/- mice presented higher numbers and increased proliferative rates of alveolar macrophages from day 3, with a more pronounced M2 profile, associated with transforming growth factor (TGF)-ß and C-C chemokine ligand (CCL)22 production, decreased inducible nitric oxide synthase (Nos2), interleukin (IL)-1ß and IL-12b mRNA expression and increased mannose receptor type 1 (Mrc1) mRNA and CD206 protein expression. Depletion of alveolar macrophages significantly delayed recovery from the inflammatory insult. Thus, our work shows that the lower number of infiltrating monocytes in CCR2-/- is partially compensated by increased proliferation of resident alveolar macrophages during the inflammation control of experimental ARDS.

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.].

Neutrophils in malaria: The good, the bad or the ugly?

Neutrophils are the most abundant circulating leukocytes in human peripheral blood. They are often the first cells to respond to an invading pathogen and might therefore play an important role in malaria. Malaria is a globally important disease caused by Plasmodium parasites, responsible for more than 400,000 deaths each year. Most of these deaths are caused by complications, including cerebral malaria, severe malarial anaemia, placental malaria, renal injury, metabolic problems and malaria-associated acute respiratory distress syndrome. Neutrophils contribute in the immune defence against malaria, through clearance of parasites via phagocytosis, production of reactive oxygen species and release of neutrophil extracellular traps (NETs). However, Plasmodium parasites diminish antibacterial functions of neutrophils, making patients more susceptible to other infections. Neutrophils might also be involved in the development of malaria complications, for example via the release of toxic granules and NETs. However, technical pitfalls in the determination of the roles of neutrophils have caused contradicting results. Further investigations need to consider these pitfalls, in order to elucidate the role of neutrophils in malaria complications.

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.

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.

Hemozoin in Malarial Complications: More Questions Than Answers.

Plasmodium parasites contain various virulence factors that modulate the host immune response. Malarial pigment, or hemozoin (Hz), is an undegradable crystalline product of the hemoglobin degradation pathway in the parasite and possesses immunomodulatory properties. An association has been found between Hz accumulation and severe malaria, suggesting that the effects of Hz on the host immune response may contribute to the development of malarial complications. Although the immunomodulatory roles of Hz have been widely investigated, many conflicting data exist, likely due to the variability between experimental set-ups and technical limitations of Hz generation and isolation methods. Here, we critically assess the potential immunomodulatory effects of Hz, its role in malarial complications, and its potential effects after parasite clearance.

von Willebrand factor increases in experimental cerebral malaria but is not essential for late-stage pathogenesis in mice.

BACKGROUND: Cerebral malaria (CM) is the most severe complication of malaria. Endothelial activation, cytokine release, and vascular obstruction are essential hallmarks of CM. Clinical studies have suggested a link between von Willebrand factor (VWF) and malaria pathology. OBJECTIVES: To investigate the contribution of VWF in the pathogenesis of experimental cerebral malaria (ECM). METHODS: Both Vwf+/+ and Vwf-/- mice were infected with Plasmodium berghei ANKA (PbANKA) to induce ECM. Alterations of plasma VWF and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), platelet count, neurological features, and accumulation of platelets and leukocytes in the brain were examined following infection. RESULTS: Plasma VWF levels significantly increased upon PbANKA infection in Vwf+/+ animals. While ADAMTS13 activity was not affected, high molecular weight VWF multimers disappeared at the end-stage ECM, possibly due to an ongoing hypercoagulability. Although the number of reticulocytes, a preferential target for the parasites, was increased in Vwf-/- mice compared to Vwf+/+ mice early after infection, parasitemia levels did not markedly differ between the two groups. Interestingly, Vwf-/- mice manifested overall clinical ECM features similar to those observed in Vwf+/+ animals. At day 8.5 post-infection, however, clinical ECM features in Vwf-/- mice were slightly more beneficial than in Vwf+/+ animals. Despite these minor differences, overall survival was not different between Vwf-/- and Vwf+/+ mice. Similarly, PbANKA-induced thrombocytopenia, leukocyte, and platelet accumulations in the brain were not altered by the absence of VWF. CONCLUSIONS: Our study suggests that increased VWF concentration is a hallmark of ECM. However, VWF does not have a major influence in modulating late-stage ECM pathogenesis.

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.

Release of endothelial activation markers in lungs of patients with malaria-associated acute respiratory distress syndrome.

BACKGROUND: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is an understudied complication of malaria and is characterized by pulmonary inflammation and disruption of the alveolar-capillary membrane. Its pathogenesis remains poorly understood. Since endothelial activation plays an important role in other malarial complications, the expression of two endothelial activation markers, von Willebrand factor (VWF) and angiopoietin-2 (ANG-2), was investigated in the lungs of patients with MA-ARDS. METHODS: Post-mortem lung sections of Plasmodium falciparum-infected patients without alveolar oedema (NA), P. falciparum-infected patients with alveolar oedema (MA-ARDS), and uninfected people who died accidentally with no pathological changes to the lungs (CON) were immunohistochemically stained for VWF and ANG-2, and were evaluated with semi-quantitative analysis. RESULTS: Alveolar oedematous VWF and ANG-2 and intravascular VWF staining were significantly increased in patients with MA-ARDS versus infected and uninfected control groups. The levels of VWF in the alveolar septa and endothelial lining of large blood vessels of patients with MA-ARDS was significantly decreased compared to controls. ANG-2 expression was increased in the alveolar septa of malaria patients without alveolar oedema versus control patients, while ANG-2+ leukocytes were increased in the alveoli in both infected patient groups. CONCLUSIONS: This study documents a high level of VWF and ANG-2, two endothelial activation markers in the oedematous alveoli of post-mortem lung sections of Thai patients with MA-ARDS. Decreased detection of VWF in the endothelial lining of blood vessels, in parallel with an increased presence of intravascular VWF staining suggests marked endothelial activation and Weibel-Palade body release in the lungs of patients with MA-ARDS.

Matrix metalloproteinase-9 induces a pro-angiogenic profile in chronic lymphocytic leukemia cells.

Increased angiogenesis is commonly observed in chronic lymphocytic leukemia (CLL) tissues in correlation with advanced disease. CLL cells express pro- and anti-angiogenic genes and acquire a pro-angiogenic pattern upon interaction with the microenvironment. Because MMP-9 (a microenvironment component) plays important roles in solid tumor angiogenesis, we have studied whether MMP-9 influenced the angiogenic pattern in CLL cells. Immunofluorescence analyses confirmed the presence of MMP-9 in CLL tissues. MMP-9 interaction with CLL cells increased their MMP-9 expression and secretion into the medium. Accordingly, the conditioned media of MMP-9-primed CLL cells significantly enhanced endothelial cell proliferation, compared to control cells. MMP-9 also increased VEGF and decreased TSP-1 and Ang-2 expression, all at the gene and protein level, inducing a pro-angiogenic pattern in CLL cells. Mechanistic analyses demonstrated that downregulation of the selected gene TSP-1 by MMP-9 involved α4ß1 integrin, Src kinase family activity and the STAT3 transcription factor. Regulation of angiogenic genes is a novel contribution of MMP-9 to CLL pathology.

Critical Roles of Endogenous Glucocorticoids for Disease Tolerance in Malaria.

During malaria, the hypothalamic-pituitary-adrenal (HPA) axis is activated and glucocorticoid (GC) levels are increased, but their essential roles have been largely overlooked. GCs are decisive for systemic regulation of vital processes such as immune responses, vascular function, and metabolism, which are crucial in malaria. Here, we introduce GCs in general, followed by their versatile roles for disease tolerance in malaria. A complementary comparison is provided with their role in sepsis. Finally, potential translational implications are considered. The failed clinical trials of dexamethasone against cerebral malaria in the past have diminished the interest in GCs in malaria. However, the issue of relative corticosteroid insufficiency has barely been explored in malaria patients, but may hold promise for a better understanding and treatment of specific malaria complications.

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.

von Willebrand factor in experimental malaria-associated acute respiratory distress syndrome.

BACKGROUND: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a lethal complication of severe malaria, characterized by marked pulmonary inflammation. Patient studies have suggested a link between von Willebrand factor (VWF) and malaria severity. OBJECTIVES: To investigate the role of VWF in the pathogenesis of experimental MA-ARDS. METHODS: Plasmodium berghei NK65-E (PbNK65) parasites were injected in Vwf+/+ and Vwf-/- mice. Pathological parameters were assessed following infection. RESULTS: In accordance with patients with severe malaria, plasma VWF levels were increased and ADAMTS13 activity levels were reduced in experimental MA-ARDS. ADAMTS13- and plasmin-independent reductions of high molecular weight VWF multimers were observed at the end stage of disease. Thrombocytopenia was VWF-independent because it was observed in both Vwf+/+ and Vwf-/- mice. Interestingly, Vwf-/- mice had a shorter survival time compared with Vwf+/+ controls following PbNK65 infection. Lung edema could not explain this shortened survival because alveolar protein levels in Vwf-/- mice were approximately two times lower than in Vwf+/+ controls. Parasite load, on the other hand, was significantly increased in Vwf-/- mice compared with Vwf+/+ mice in both peripheral blood and lung tissue. In addition, anemia was only observed in PbNK65-infected Vwf-/- mice. Of note, Vwf-/- mice presented with two times more reticulocytes, a preferential target of the parasites. CONCLUSIONS: This study suggests that parasite load together with malarial anemia, rather than alveolar leakage, might contribute to shortened survival in PbNK65-infected Vwf-/- mice. VWF deficiency is associated with early reticulocytosis following PbNK65 infection, which potentially explains the increase in parasite load.