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Macrophage activation syndrome (MAS) exemplifies a severe cytokine storm disorder with liver inflammation. In the liver, classical natural killer (cNK) cells and liver-resident type 1 innate lymphoid cells (ILC1s) dominate the ILC population. Thus far, research has primarily focused on the corresponding role of cNK cells. Considering the liver inflammation and cytokine storm in MAS, liver-resident ILC1s represent an interesting population to explore due to their rapid cytokine production upon environmental triggers. By utilizing a Toll-like receptor (TLR)9- and TLR3:4-triggered MAS model, we showed that ILC1s highly produce IFN-γ and TNF-α. However, activated ILC1s undergo apoptosis and are strongly reduced in numbers, while cNK cells resist inflammation-induced apoptosis. Signs of mitochondrial stress suggest that this ILC1 apoptosis may be driven by inflammation-induced mitochondrial impairment. To study whether early induction of highly cytokine-producing ILC1s influences MAS development, we used Hobit KO mice due to their paucity of liver ILC1s but unaffected cNK cell numbers. Nevertheless, neither the severity of MAS features nor the total inflammatory cytokine levels were affected in these Hobit KO mice, indicating that ILC1s are dispensable for MAS pathogenesis. Collectively, our data demonstrate that ILC1s undergo apoptosis during TLR-triggering and are dispensable for MAS pathogenesis., (© 2024 Wiley‐VCH GmbH.)

In both humans and mice, natural killer (NK) cells are important lymphocytes of the innate immune system. They are often considered pro-inflammatory effector cells but may also have a regulatory or pro-resolving function by switching their cytokine profile towards the production of anti-inflammatory cytokines, including interleukin-10 (IL-10) and transforming growth factor-β, and by killing pro-inflammatory immune cells. Here, the role of NK cells in the resolution of malaria lung pathology was studied. Malaria complications, such as malaria-associated acute respiratory distress syndrome (MA-ARDS), are often lethal despite the rapid and efficient killing of Plasmodium parasites with antimalarial drugs. Hence, studying the resolution and healing mechanisms involved in the recovery from these complications could be useful to develop adjunctive treatments. Treatment of Plasmodium berghei NK65 -infected C57BL/6 mice with a combination of artesunate and chloroquine starting at the appearance of symptoms was used as a model to study the resolution of MA-ARDS. The role of NK cells was studied using anti-NK1.1 depletion antibodies and NK cell-deficient mice. Using both methods, NK cells were found to be dispensable in the development of MA-ARDS, as shown previously. In contrast, NK cells were crucial in the initiation of resolution upon antimalarial treatment, as survival was significantly decreased in the absence of NK cells. Considerably increased IL-10 expression by NK cells suggested an anti-inflammatory and pro-resolving phenotype. Despite the increase in Il10 expression in the NK cells, inhibition of the IL-10/IL-10R axis using anti-IL10R antibodies had no effect on the resolution for MA-ARDS, suggesting that the pro-resolving effect of NK cells cannot solely be attributed to their IL-10 production. In conclusion, NK cells contribute to the resolution of experimental MA-ARDS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pollenus, Possemiers, Knoops, Prenen, Vandermosten, Pham, Buysrogge, Matthys and Van den Steen.)

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., (© 2024. The Author(s).)

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.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pollenus et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vandermosten, Prenen, Fogang, Dagneau de Richecour, Knoops, Donkeu, Nguefack, Taguebue, Ndombo, Ghesquière, Ayong and Van den Steen.)

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.

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)-β and C-C chemokine ligand (CCL)22 production, decreased inducible nitric oxide synthase (Nos2) , interleukin (IL)-1β 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.

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 ( Pb NK65) 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 Pb NK65 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Possemiers, Pollenus, Prenen, Knoops, Koshy and Van den Steen.)

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., (© 2022 John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pollenus, Pham, Vandermosten, Possemiers, Knoops, Opdenakker and Van den Steen.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2020 International Society on Thrombosis and Haemostasis.)

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.

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 α4β1 integrin, Src kinase family activity and the STAT3 transcription factor. Regulation of angiogenic genes is a novel contribution of MMP-9 to CLL pathology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© 2019 International Society on Thrombosis and Haemostasis.)

We previously showed that MMP-9 contributes to CLL pathology by regulating cell survival and migration and that, when present at high levels, MMP-9 induces cell arrest. To further explore the latter function, we studied whether MMP-9 influences the gene-expression profile in CLL. Microarray analyses rendered 131 differentially expressed genes in MEC-1 cells stably transfected with MMP-9 (MMP-9-cells) versus cells transfected with empty vector (Mock-cells). Ten out of twelve selected genes were also differentially expressed in MEC-1 cells expressing the catalytically inactive MMP-9MutE mutant (MMP-9MutE-cells). Incubation of primary CLL cells with MMP-9 or MMP-9MutE also regulated gene and protein expression, including CD99, CD226, CD52, and CD274. Because CD99 is involved in leukocyte transendothelial migration, we selected CD99 for functional and mechanistic studies. The link between MMP-9 and CD99 was reinforced with MMP-9 gene silencing studies, which resulted in CD99 upregulation. CD99 gene silencing significantly reduced CLL cell adhesion, chemotaxis and transendothelial migration, while CD99 overexpression increased cell migration. Mechanistic analyses indicated that MMP-9 downregulated CD99 via binding to α4β1 integrin and subsequent inactivation of the Sp1 transcription factor. This MMP-9-induced mechanism is active in CLL lymphoid tissues, since CD99 expression and Sp1 phosphorylation was lower in bone marrow-derived CLL cells than in their peripheral blood counterparts. Our study establishes a new gene regulatory function for MMP-9 in CLL. It also identifies CD99 as an MMP-9 target and a novel contributor to CLL cell adhesion, migration and arrest. CD99 thus constitutes a new therapeutic target in CLL, complementary to MMP-9.

Malaria reduces host fitness and survival by pathogen-mediated damage and inflammation. Disease tolerance mechanisms counter these negative effects without decreasing pathogen load. Here, we demonstrate that in four different mouse models of malaria, adrenal hormones confer disease tolerance and protect against early death, independently of parasitemia. Surprisingly, adrenalectomy differentially affects malaria-induced inflammation by increasing circulating cytokines and inflammation in the brain but not in the liver or lung. Furthermore, without affecting the transcription of hepatic gluconeogenic enzymes, adrenalectomy causes exhaustion of hepatic glycogen and insulin-independent lethal hypoglycemia upon infection. This hypoglycemia is not prevented by glucose administration or TNF-α neutralization. In contrast, treatment with a synthetic glucocorticoid (dexamethasone) prevents the hypoglycemia, lowers cerebral cytokine expression and increases survival rates. Overall, we conclude that in malaria, adrenal hormones do not protect against lung and liver inflammation. Instead, they prevent excessive systemic and brain inflammation and severe hypoglycemia, thereby contributing to tolerance.

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.

Background: Cerebral malaria and severe anaemia are the most common deadly complications of malaria, and are often associated, both in paediatric and adult patients, with hepatopathy, whose pathogenesis is not well characterized, and sometimes also with acute respiratory distress syndrome (ARDS). Here, two species of murine malaria, the lethal Plasmodium berghei strain NK65 and self-healing Plasmodium chabaudi strain AS which differ in their ability to cause hepatopathy and/or ARDS were used to investigate the lipid alterations, oxidative damage and host immune response during the infection in relation to parasite load and accumulation of parasite products, such as haemozoin., Methods: Plasma and livers of C57BL/6J mice injected with PbNK65 or PcAS infected erythrocytes were collected at different times and tested for parasitaemia, content of haemozoin and expression of tumour necrosis factor (TNF). Hepatic enzymes, antioxidant defenses and lipids content and composition were also evaluated., Results: In the livers of P. berghei NK65 infected mice both parasites and haemozoin accumulated to a greater extent than in livers of P. chabaudi AS infected mice although in the latter hepatomegaly was more prominent. Hepatic enzymes and TNF were increased in both models. Moreover, in P. berghei NK65 infected mice, increased lipid peroxidation, accumulation of triglycerides, impairment of anti-oxidant enzymes and higher collagen deposition were detected. On the contrary, in P. chabaudi AS infected mice the antioxidant enzymes and the lipid content and composition were normal or even lower than uninfected controls., Conclusions: This study demonstrates that in C57BL/6J mice, depending on the parasite species, malaria-induced liver pathology results in different manifestations, which may contribute to the different outcomes. In P. berghei NK65 infected mice, which concomitantly develop lethal acute respiratory distress syndrome, the liver tissue is characterized by an excess oxidative stress response and reduced antioxidant defenses while in P. chabaudi AS infected mice hepatopathy does not lead to lipid alterations or reduction of antioxidant enzymes, but rather to inflammation and cytokine burst, as shown earlier, that may favour parasite killing and clearance of the infection. These results may help understanding the different clinical profiles described in human malaria hepatopathy.

We previously showed that MMP-9 overexpression impairs migration of primary CLL cells and MEC-1 cells transfected with MMP-9. To determine the contribution of non-proteolytic activities to this effect we generated MEC-1 transfectants stably expressing catalytically inactive MMP-9MutE (MMP-9MutE-cells). In xenograft models in mice, MMP-9MutE-cells showed impaired homing to spleen and bone marrow, compared to cells transfected with empty vector (Mock-cells). In vitro transendothelial and random migration of MMP-9MutE-cells were also reduced. Biochemical analyses indicated that RhoAGTPase and p-Akt were not downregulated by MMP-9MutE, at difference with MMP-9. However, MMP-9MutE-cells or primary cells incubated with MMP-9MutE had significantly reduced p-ERK and increased PTEN, accounting for the impaired migration. Our results emphasize the role of non-proteolytic MMP-9 functions contributing to CLL progression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Astemizole is a H 1 -antagonist endowed with antimalarial activity, but has hERG liabilities. Systematic structural modifications of astemizole led to the discovery of analogues that display very potent activity as inhibitors of the growth of the Plasmodium parasite, but show a decreased hERG inhibition, when compared to astemizole. These compounds can be used as starting point for the development of a new class of antimalarials., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Malaria is a global disease associated with considerable mortality and morbidity. An appropriately balanced immune response is crucial in determining the outcome of malarial infection. The glucocorticoid (GC) metabolising enzyme, 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1) converts intrinsically inert GCs into active GCs. 11β-HSD1 shapes endogenous GC action and is immunomodulatory. We investigated the role of 11β-HSD1 in two mouse models of malaria. 11β-HSD1 deficiency did not affect survival after malaria infection, but it increased disease severity and parasitemia in mice infected with Plasmodium chabaudi AS. In contrast, 11β-HSD1 deficiency rather decreased parasitemia in mice infected with the reticulocyte-restricted parasite Plasmodium berghei NK65 1556Cl1. Malaria-induced antibody production and pathology were unaltered by 11β-HSD1 deficiency though plasma levels of IL-4, IL-6 and TNF-α were slightly affected by 11β-HSD1 deficiency, dependent on the infecting parasite. These data suggest that 11β-HSD1 is not crucial for survival of experimental malaria, but alters its progression in a parasite strain-specific manner.

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is an often lethal complication of malaria. Currently, no adequate therapy for this syndrome exists. Although glucocorticoids (GCs) have been used to improve clinical outcome of ARDS, their therapeutic benefits remain unclear. We previously developed a mouse model of MA-ARDS, in which dexamethasone treatment revealed GC resistance. In the present study, we investigated GC sensitivity of mouse microvascular lung endothelial cells stimulated with interferon-γ (IFN-γ) and Plasmodium berghei NK65 ( Pb NK65). Upon challenge with IFN-γ alone, dexamethasone inhibited the expression of CCL5 (RANTES) by 90% and both CCL2 (MCP-1) and CXCL10 (IP-10) by 50%. Accordingly, whole transcriptome analysis revealed that dexamethasone differentially affected several gene clusters and in particular inhibited a large cluster of IFN-γ-induced genes, including chemokines. In contrast, combined stimulation with IFN-γ and Pb NK65 extract impaired inhibitory actions of GCs on chemokine release, without affecting the capacity of the GC receptor to accumulate in the nucleus. Subsequently, we investigated the effects of GCs on two signaling pathways activated by IFN-γ. Dexamethasone left phosphorylation and protein levels of signal transducer and activator of transcription 1 (STAT1) unhampered. In contrast, dexamethasone inhibited the IFN-γ-induced activation of two mitogen-activated protein kinases (MAPK), JNK, and p38. However, Pb NK65 extract abolished the inhibitory effects of GCs on MAPK signaling, inducing GC resistance. These data provide novel insights into the mechanisms of GC actions in endothelial cells and show how malaria may impair the beneficial effects of GCs.

Malaria is a severe disease and kills over 400,000 people each year. Malarial complications are the main cause of death and include cerebral malaria and malaria-associated acute respiratory distress syndrome (MA-ARDS). Despite antimalarial treatment, lethality rates of MA-ARDS are still between 20 and 80%. Patients develop pulmonary edema with hemorrhages and leukocyte extravasation in the lungs. The vascular endothelial growth factor-A (VEGF-A) and the placental growth factor (PlGF) are vascular permeability factors and may be involved in the disruption of the alveolar-capillary membrane, leading to alveolar edema. We demonstrated increased pulmonary VEGF-A and PlGF levels in lungs of mice with experimental MA-ARDS. Depletion of pathogenic CD8 + T cells blocked pulmonary edema and abolished the increase of VEGF-A and PlGF. However, neutralization of VEGF receptor-2 (VEGFR-2) with the monoclonal antibody clone DC101 did not decrease pulmonary pathology. The broader spectrum receptor tyrosine kinase inhibitor sunitinib even increased lung pathology. These data suggest that the increase in alveolar VEGF-A and PlGF is not a cause but rather a consequence of the pulmonary pathology in experimental MA-ARDS and that therapeutic inhibition of VEGF receptors is not effective and even contra-indicated.

The endothelium plays a crucial role in inflammation. A balanced control of inflammation requires the action of glucocorticoids (GCs), steroidal hormones with potent cell-specific anti-inflammatory properties. Besides the classic anti-inflammatory effects of GCs on leukocytes, recent studies confirm that endothelial cells also represent an important target for GCs. GCs regulate different aspects of endothelial physiology including expression of adhesion molecules, production of pro-inflammatory cytokines and chemokines, and maintenance of endothelial barrier integrity. However, the regulation of endothelial GC sensitivity remains incompletely understood. In this review, we specifically examine the endothelial response to GCs in various inflammatory diseases ranging from multiple sclerosis, stroke, sepsis, and vasculitis to atherosclerosis. Shedding more light on the cross talk between GCs and endothelium will help to improve existing therapeutic strategies and develop new therapies better tailored to the needs of patients.

Coevolution of humans and malaria parasites has generated an intricate balance between the immune system of the host and virulence factors of the parasite, equilibrating maximal parasite transmission with limited host damage. Focusing on the blood stage of the disease, we discuss how the balance between anti-parasite immunity versus immunomodulatory and evasion mechanisms of the parasite may result in parasite clearance or chronic infection without major symptoms, whereas imbalances characterized by excessive parasite growth, exaggerated immune reactions or a combination of both cause severe pathology and death, which is detrimental for both parasite and host. A thorough understanding of the immunological balance of malaria and its relation to other physiological balances in the body is of crucial importance for developing effective interventions to reduce malaria-related morbidity and to diminish fatal outcomes due to severe complications. Therefore, we discuss in this review the detailed mechanisms of anti-malarial immunity, parasite virulence factors including immune evasion mechanisms and pathogenesis. Furthermore, we propose a comprehensive classification of malaria complications according to the different types of imbalances., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Malaria-associated acute lung injury (MA-ALI) and its more severe form malaria-associated acute respiratory distress syndrome (MA-ARDS) are common, often fatal complications of severe malaria infections. However, little is known about their pathogenesis. In this study, biochemical alterations of the lipid composition of the lungs were investigated as possible contributing factors to the severity of murine MA-ALI/ARDS. C57BL/6J mice were infected with Plasmodium berghei NK65 to induce lethal MA-ARDS, or with Plasmodium chabaudi AS, a parasite strain that does not induce lung pathology. The lipid profile of the lung tissue from mice infected with Plasmodium berghei NK65 developing MA-ALI/ARDS, but not that from mice without lung pathology or controls, was characterized by high levels of phospholipids -mainly phosphatidylcholine- and esterified cholesterol. The high levels of polyunsaturated fatty acids and the linoleic/oleic fatty acid ratio of the latter reflect the fatty acid composition of plasma cholesterol esters. In spite of the increased total polyunsaturated fatty acid pool, which augments the relative oxidability of the lung membranes, and the presence of hemozoin, a known pro-oxidant, no excess oxidative stress was detected in the lungs of Plasmodium berghei NK65 infected mice. The bronchoalveolar lavage (BAL) fluid of Plasmodium berghei NK65 infected mice was characterized by high levels of plasma proteins. The phospholipid profile of BAL large and small aggregate fractions was also different from uninfected controls, with a significant increase in the amounts of sphingomyelin and lysophosphatidylcholine and the decrease in phosphatidylglycerol. Both the increase of proteins and lysophosphatidylcholine are known to decrease the intrinsic surface activity of surfactant. Together, these data indicate that an altered lipid composition of lung tissue and BAL fluid, partially ascribed to oedema and lipoprotein infiltration, is a characteristic feature of murine MA-ALI/ARDS and possibly contribute to lung dysfunction.

Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes., (© 2015 Lin et al.)

Chemokines, binding their various G protein-coupled receptors, lead the way for leukocytes in health and inflammation. Yet chemokine receptor expression is not limited to leukocytes. Accordingly, chemokines are remarkably pleiotropic molecules involved in a range of physiological as well as pathological processes. For example, the CXCR3 chemokine receptor is expressed on activated T lymphocytes, dendritic cells and natural killer cells, but also fibroblasts and smooth muscle, epithelial and endothelial cells. In men, these cells express either CXCR3A, its splice variant CXCR3B or a balanced combination of both. The CXCR3 ligands, activating both receptor variants, include CXCL4, CXCL4L1, CXCL9, CXCL10 and CXCL11. Upon CXCR3A activation these ELR-negative CXC chemokines mediate chemotactic and proliferative responses, for example in leukocytes. In contrast, CXCR3B induces anti-proliferative and anti-migratory effects, as exemplified by angiostatic effects on endothelial cells. Taken together, the unusual and versatile characteristics of CXCR3 and its ligands form the basis for their pertinent involvement in a myriad of diseases. In this review, we discuss the presence and function of all CXCR3 ligands in various malignant, angiogenic, infectious, inflammatory and other disorders. By extension, we have also elaborated on the potential therapeutic applicability of CXCR3 ligand administration or blockade, as well as their additional value as predictive or prognostic biomarkers. This review illustrates the multifunctional, intriguing character of the various CXCR3-binding chemokines., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Gelatinase B/matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) cleaves many substrates and is produced by most cell types as a zymogen, proMMP-9, in complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 occurs as monomers, homomultimers and heterocomplexes, but our knowledge about the overall structure of proMMP-9 monomers and multimers is limited. We investigated biochemical, biophysical and functional characteristics of zymogen and activated forms of MMP-9 monomers and multimers. In contrast with a conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate that these are reduction-sensitive trimers. Based on the information from electrophoresis, AFM and TEM, we generated a 3D structure model of the proMMP-9 trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for TIMP-1 than the monomers. In vivo, this finding was reflected in a higher extent of TIMP-1 inhibition of angiogenesis induced by trimers compared with monomers. Our results show that proMMP-9 trimers constitute a novel structural and functional entity that is differentially regulated by TIMP-1.

Malaria is a global disease that clinically affects more than two hundred million people annually. Despite the availability of effective antimalarials, mortality rates associated with severe complications are high. Hepatopathy is frequently observed in patients with severe malarial disease and its pathogenesis is poorly understood. Previously, we observed high amounts of hemozoin or malaria pigment in livers from infected mice. In this study, we investigated whether hemozoin is associated with liver injury in different mouse malaria models. C57BL/6J mice infected with the rodent parasites Plasmodium berghei ANKA, P. berghei NK65 or P. chabaudi AS had elevated serum liver enzymes without severe histological changes in the liver, in line with the observations in most patients. Furthermore, liver enzymes were significantly higher in serum of P. chabaudi AS-infected mice compared to mice infected with the P. berghei parasite strains and a strong positive correlation was found between hepatic hemozoin levels, hepatocyte damage and inflammation in the liver with P. chabaudi AS. The observed liver injury was only marginally influenced by the genetic background of the host, since similar serum liver enzyme levels were measured in infected C57BL/6J and BALB/c mice. Intravenous injection of P. falciparum-derived hemozoin in malaria-free C57BL/6J mice induced inflammatory gene transcription in the liver, suggesting that hemozoin may be involved in the pathogenesis of malaria hepatopathy by inducing inflammation.

The Malaria Immunology Database (MalarImDB, www.malarimdb.org) is a novel literature-based database of host mediators in blood-stage malaria. We designed this open-access online tool because intensive malaria research has resulted in a dazzling complexity of host mediators with pathogenic or protective functions. MalarImDB allows comparisons between expression levels in humans, expression levels in murine models, and functional data from experimental treatments in mice. The database is equipped with multiple search engines to retrieve information from many published studies. The search output is visualized schematically in tables, thereby revealing similarities and disparities. Thus, the primary aim of this database is to present a clear overview of the currently available data about malaria and to simplify literature searches., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

By connection of Regasepin2, a heptapeptide inhibitor of matrix metalloproteinases (MMPs), to the N- or C-terminus of ES-2, an anti-angiogenic peptide of 11 residues, two designed peptides CPU1 and CPU2 were generated. Unexpectedly, CPU2 inhibited MMP-8 and MMP-9 activity in the nanomolar range, whereas CPU1 displayed a weaker inhibitory profile than Regasepin2 against TACE, MMP-8 and MMP-9. CPU1 showed a higher affinity than CPU2 with integrin α5β1 in a HUVEC adhesion assay. In an in vitro angiogenesis model of HUVEC migration, CPU1 showed higher inhibition potency than CPU2 (85% inhibition for CPU1 at a concentration of 0.8 μM versus 17% inhibition for CPU2 at the same concentration). In an in vivo angiogenesis model in chicken egg chorioallantoic membranes, 1.37 μM CPU1 showed a significant inhibition in the formation of new blood vessels, whereas CPU2 and Regasepin2 had no effect on angiogenesis. Furthermore, CPU1 significantly inhibited B16F10 melanoma growth in a syngeneic mouse model (inhibition rate of 56.91% by tumor weight analysis at a dose of 20 mg/kg/d), whereas CPU2 and Regasepin2 did not show any inhibitory effect. In view of recent findings that MMP-9 is pro-angiogenic, our results indicated that the combination of MMP inhibitors and anti-angiogenic peptides may generate novel molecules with potent in vivo anti-tumor effects.

Natural killer (NK) cells have different roles in the host response against Plasmodium-induced malaria depending on the stage of infection. Liver NK cells have a protective role during the initial hepatic stage of infection by production of the TH1-type cytokines IFN-γ and TNF-α. In the subsequent erythrocytic stage of infection, NK cells also induce protection through Th1-type cytokines but, in addition, may also promote development of cerebral malaria via CXCR3-induction on CD8(+) T cells resulting in migration of these cells to the brain. We have recently shown that the regulatory Ly49E NK receptor is expressed on liver NK cells in particular. The main objective of this study was therefore to examine the role of Ly49E expression in the immune response upon Plasmodium berghei ANKA infection, for which we compared wild type (WT) to Ly49E knockout (KO) mice. We show that the parasitemia was higher at the early stage, i.e. at days 6-7 of Plasmodium berghei ANKA infection in Ly49E KO mice, which correlated with lower induction of CD69, IFN-γ and TNF-α in DX5(-) liver NK cells at day 5 post-infection. At later stages, these differences faded. There was also no difference in the kinetics and the percentage of cerebral malaria development and in lymphocyte CXCR3 expression in WT versus Ly49E KO mice. Collectively, we show that the immune response against Plasmodium berghei ANKA infection is not drastically affected in Ly49E KO mice. Although NK cells play a crucial role in Plasmodium infection and Ly49E is highly expressed on liver NK cells, the Ly49E NK receptor only has a temporarily role in the immune control of this parasite.

Blood-stage malaria parasites produce insoluble hemozoin (Hz) crystals that are released in the blood circulation upon schizont rupture. In general, endogenous crystal formation or inhalation of crystalline materials is often associated with pathology. As the immune system responds differently to crystalline particles than to soluble molecules, in this review, the properties, immunological recognition, and pathogenic responses of Hz are discussed, and compared with two other major pathogenic crystals, monosodium urate (MSU) and asbestos. Because of the size and shape of MSU crystals and asbestos fibers, phagolysosomal formation is inefficient and often results in leakage of lysosomal content in the cell cytoplasm and/or in the extracellular environment with subsequent cell damage and cell death. Phagolysosomal formation after Hz ingestion is normal, but Hz remains stored inside these cells for months or even longer without any detectable degradation. Nonetheless, the different types of crystals are recognized by similar immune receptors, involving Toll-like receptors, the inflammasome, antibodies, and/or complement factors, and through similar signaling cascades, they activate both proinflammatory and anti-inflammatory immune responses that contribute to inflammation-associated pathology., (© 2013 International Union of Biochemistry and Molecular Biology.)

Endotoxin shock is a life-threatening disorder, associated with the rapid release of neutrophil enzymes, including neutrophil collagenase/matrix metalloproteinase-8 (MMP-8) and gelatinase B/matrix metalloproteinase-9 (MMP-9). After activation, these enzymes cleave extracellular matrix components and cytokines and thus may contribute to shock syndrome development. MMP inhibitors have been suggested as immunotherapy of endotoxin shock. However, little is known about the therapeutic time window of MMP inhibition. Here, a sublethal endotoxin shock mouse model was used to evaluate the effect of an MMP inhibiting peptide (P2) after intravenous or intraperitoneal injection and to study the time window between LPS and inhibitor injections. With the use of a specific ELISA the plasma P2 concentrations were monitored. Whereas we corroborated the treatment strategy of MMP targeting in endotoxin shock with a new inhibitor, we also demonstrated that the time window, within which effective MMP inhibition increased the survival rates, is rather limited.

Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is an increasingly reported, often lethal, and incompletely understood complication of malaria. We discuss and compare the pathogenesis of MA-ARDS in patients and in different murine models, including recent models without cerebral involvement, and summarize the roles of different leukocyte subclasses, adhesion molecules, cytokines, and chemokines. In patients as well as in mice, severe edema and impaired gas exchange are associated with abundant inflammatory infiltrates consisting of mainly mononuclear cells and parasite sequestration, and the pathogenesis appears different from cerebral malaria (CM). Experimental anti-inflammatory interventions are successful in mice and remain to be validated in patients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Research on matrix metalloproteinases (MMPs) and in particular on gelatinase B, alias MMP-9, has grown exponentially in the decade 2003-2012. Structural details about flexibility of MMP-9 monomers, together with glycosylation, oligomerization, heterogeneity and instability of the wildtype enzyme explain why crystallography experiments have not yet been successful for the intact enzyme. MMP-9 may be viewed as a multidomain enzyme in which the hemopexin, the O-glycosylated and the catalytic domains yield support for attachment, articulation and catalysis, respectively. The stepwise proteolytic activation of the inactive zymogen into a catalytically active form becomes gradually better understood. Priming of activation by MMP-3 may be executed by meprins that destabilize the interaction of the aminoterminus with the third fibronectin repeat. Alternatively, autocatalytic activation may occur in the presence of molecules that tightly bind to the catalytic site and that push the cystein residue in the prodomain away from the catalytic zinc ion. Thanks to the development of degradomics technologies, substrate repertoires of MMP-9 have been defined, but it remains a challenge to determine and prove which substrates are biologically relevant. The substrate repertoire has been enlarged from extracellular to membrane-bound and efficient intracellular substrates, such as crystallins, tubulins and actins. Biological studies of MMP-9 have tuned the field from being primarily cancer-oriented towards vascular and inflammatory research. In tumor biology, it has been increasingly appreciated that MMP-9 from inflammatory cells, particularly neutrophils, co-determines prognosis and outcome. Aside from the catalytic functions executed by aminoterminal domains of MMP-9, the carboxyterminal hemopexin (PEX) domain of gelatinase B exerts non-catalytic anti-apoptotic signaling effects. The recognition that gelatinase B is induced by many pro-inflammatory cytokines, whereas its inhibitors are increased by anti-inflammatory cytokines, has generated interest to target MMP-9 in acute lethal conditions, such as bacterial meningitis, sepsis and endotoxin shock, and in acute exacerbations of chronic diseases. Previously described transcriptional regulation of MMP-9 is complemented by epigenetic checkpoints, including histone modifications and microRNAs. Because activation of proMMP-9 may be executed by other MMPs, the therapeutic dogma that MMP inhibitors need to be highly selective may be keyed down for the treatment of life-threatening conditions. When inflammation and MMP-9 fulfill beneficial functions to clear damaging protein complexes, such as in systemic autoimmune diseases, therapeutic MMP inhibition has to be avoided. In Mmp9 gene knockout mice, specific spontaneous phenotypes emerged with effects on the skeletal, reproductive and nervous systems. These findings not only have clinical correlates in bone growth and fertility, but also stimulate research on the roles of MMPs and MMP-9 in endocrinology, immunology and the neurosciences. Mmp9-deficient mice are valuable tools to define MMP-9 substrates in vivo and to study the role of this enzyme in animal models of inflammatory, vascular, neoplastic and degenerative diseases. Future challenges include solving the crystal structure, definition of the functions of covalent oligomers and heteromers in biology and pathology, life-imaging of MMP-9 activity, substrate determination in situ and the study of inhibitor effects on fertility, cancer and inflammation and in neurobiology and regenerative medicine. Such studies will better define conditions in which inhibition of MMP-9 is beneficial or has to be avoided.

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial growth factor. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand 2, and keratinocyte-derived chemokine/CXCL1), cytokines (IL-1β, IL-6, IL-10, TNF, and transforming growth factor-β), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-2, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.

Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful, but often misinterpreted, tool yielding information on potential hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tissue sections with in situ zymography. In vivo zymography can pinpoint proteolytic activity to sites in an intact organism. Future development of novel substrate probes and improvement in detection and imaging methods will increase the applicability of zymography for (reverse) degradomics studies.

Meprin α and β, members of the astacin family of zinc metalloproteinases, are unique plasma membrane and secreted proteases known to cleave a wide range of biological substrates involved in inflammation, cancer and fibrosis. In this study, we identified proMMP-9 as a novel substrate and show that aminoterminal meprin-mediated clipping improves the activation kinetics of proMMP-9 by MMP-3, an efficient activator of proMMP-9. Interestingly, the NH(2)-terminus LVLFPGDL, generated by incubation with meprin α, is identical to the form produced in conditioned media from human neutrophils and monocytes. Hence, this meprin-mediated processing and enhancement of MMP-9 activation kinetics may have biological relevance in the context of in vivo inflammatory processes., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)