Malarial hemozoin is a Nalp3 inflammasome activating danger signal

Abstract

Background: Characteristic symptoms of malaria include recurrent fever attacks and neurodegeneration, signs that are also found in patients with a hyperactive Nalp3 inflammasome. Plasmodium species produce a crystal called hemozoin that is generated by detoxification of heme after hemoglobin degradation in infected red blood cells. Thus, we hypothesized that hemozoin could activate the Nalp3 inflammasome, due to its particulate nature reminiscent of other inflammasome-activating agents.

Methodology/principal findings: We found that hemozoin acts as a proinflammatory danger signal that activates the Nalp3 inflammasome, causing the release of IL-1beta. Similar to other Nalp3-activating particles, hemozoin activity is blocked by inhibiting phagocytosis, K(+) efflux and NADPH oxidase. In vivo, intraperitoneal injection of hemozoin results in acute peritonitis, which is impaired in Nalp3-, caspase-1- and IL-1R-deficient mice. Likewise, the pathogenesis of cerebral malaria is dampened in Nalp3-deficient mice infected with Plasmodium berghei sporozoites, while parasitemia remains unchanged.

Significance/conclusions: The potent pro-inflammatory effect of hemozoin through inflammasome activation may possibly be implicated in plasmodium-associated pathologies such as cerebral malaria.

Figure 1. Hemozoin activates IL-1β secretion in murine and human macrophages and dendritic cells.

(a,b) Bone marrow derived macrophages (BMDMs) were stimulated for 6 h with 150 µg/ml hemozoin and 2.5 µg/ml CpG. Cytokines and chemokines were measured by CBA. (c) THP1 cells were stimulated for 1, 3 or 6 h with hemozoin, MSU or Nigericin and analysed by western blot. (d) Bone marrow derived dendritic cells (BMDCs) were stimulated for 6 h with hemozoin, MSU, Nigericin or CpG. Cell extracts and supernatants were analysed by western blot. Data shown are representative of three independent experiments.

Figure 2. Hemozoin induced IL-1β secretion is NALP3 inflammasome-dependent.

(a,b) BMDMs from wild-type (WT), ASC-, Nalp3- or Ipaf-deficient mice were stimulated for 6 h with hemozoin (150 µg/ml) or MSU (150 µg/ml ) in the presence of 20 µM z-VAD or 130 mM KCl where indicated (* nonspecific band). (c) BMDMs were primed or not with LPS before stimulation with hemozoin or MSU. Data shown are representative of three independent experiments.

Figure 3. Hemozoin induced IL-1β production is independent from P2X7 activation.

(a) BMDMs were stimulated for 45 min with 5 mM ATP, or for 6 h with 100 µg/ml hemozoin and 100 µg/ml MSU. (b) Uricase treatment (0,1 U/ml) of THP1 cells does not affect IL-1β production by hemozoin. Cells were stimulated with 150 µg/ml hemozoin, 150 µg/ml asbestos and 50 µg/ml MSU. (c) Heme does not activate caspase-1 cleavage in BMDMs as compared to hemozoin (150 µg/ml) or MSU (150 µg/ml), but leads to PARP cleavage, indicating its toxicity. Cell supernatants and extracts were analysed by Western blot.

Figure 4. Hemozoin induced IL-1β production is independent from MyD88-mediated signaling pathways.

(a) Caspase-1 activation can still be observed in MyD88-deficient BMDMs, albeit to a lesser extent. (b) BMDMs were stimulated with hemozoin, MSU and Nigericin in the presence or absence of DNaseI (100 U/ml). Cells were stimulated for 6 h with 150 µg/ml hemozoin, 150 µg/ml MSU and 1.34 µM Nigericin. Cell supernatants and extracts were analysed by Western blot. (c) BMDMs were stimulated with hemozoin, MSU, Nigericin or CpG (2,5 µg/ml) in the presence or absence of 10 µM chloroquine. (d,e) BMDMs (d) and THP1 cells (e) were stimulated with hemozoin, MSU, nigericin and CpG in the presence or absence of bafilomycinA1 (250 nM in (d) and 50 and 250 nM in (e)). IL-1β secretion and caspase-1 cleavage were analysed by western blot; IL-6 was analysed by CBA.

Figure 5. Phagocytosis, K⁺ efflux and activation of a NADPH oxidase are essential for hemozoin-mediated inflammasome activation.

(a) Crystal phagocytosis is essential for hemozoin induced IL-1β production as evidenced by the use of cytochalasin D (2 µM) to block the actin cytoskeleton. (b) Hemozoin induced IL-1β production can be reduced by blocking the K⁺ efflux from the cells by adding high extracellular K⁺ concentration to the culture medium (130 mM KCl) or by using glybenclamide, an ATP-sensitive potassium channel inhibitor (50 µM). (c) Hemozoin induced IL-1β production can be reduced by the use of ROS inhibitors, such as APDC (50 µM) and DPI (20 µM). (d) NADPH oxidase subunit p22phox is essential for inflammasome activation by hemozoin. (e) gp91phox(NOX2)-deficient BMDMs were stimulated with different crystals and nigericin for 6 h. (f) Cathepsin B-deficient BMDMs were stimulated with the indicated inflammasome activators for 6 h. THP1 cells were stimulated for 6 h with 150 µg/ml hemozoin, 100 µg/ml MSU, 100 µg/ml asbestos and 1,34 µM Nigericin. Salmonella were added to the cells at an MOI of 10. BMDMs were stimulated with 150 µg/ml hemozoin, 150 µg/ml MSU, 250 µg/ml silica, 150 µg/ml alum, 15 µg/ml R837 and 1,34 µM Nigericin Cell supernatants and extracts were analysed by Western blot.

Figure 6. Role of the inflammasome in a mouse model of hemozoin-induced peritonitis.

(a-f) The indicated WT or mutant mice were injected intraperitoneally with 0.25 mg of hemozoin, 0.2 mg of zymosan or PBS as a control. Neutrophil influx (CD11b⁺ Ly-6C⁺ Ly-6G^high F4/80⁻) was quantified 6 h later by FACS (values are mean±s.e.m and n = 4–6 mice per group). Differences between groups were calculated using the unpaired t test and were considered significant (*) when p≤0.05. Data shown are representative of three independent experiments.

Figure 7. Role of Nalp3 in a mouse model of cerebral malaria.

(a-d) Nalp3-deficient mice have increased resistance to cerebral malaria (CM). (a, b) Survival of Nalp3^+/+ (solid line) and Nalp3^−/− (dotted line) mice was monitored daily (n = 18–22 for each group, (*) log-rank p = 0.0263) and parasitemia was assessed by blood smears (mean parasitemia±s.e.m., n = 6 for each group, N3^+/+ = black square, N3^−/− = open circles). (c) Nalp3^−/−mice are protected from CM compared to Nalp3^+/+ mice (n = 15 for each group). (d) Histological analysis of brain sections of Nalp3^+/+ (panel i and iii) and Nalp3^−/− (panel ii and iv) mice 9 days after Plasmodium infection. Haematoxylin & Eosin staining is shown in panels i and ii, CD45 staining is shown in panels iii and iv. Infected Nalp3^+/+ mice brains show pathological endothelial cells (open triangles) and lymphomonocytic infiltrates (closed triangles) whereas in the brains from infected Nalp3-deficient mice these pathological signs are markedly reduced (panels i and ii).