Synthetic Plasmodium-Like Hemozoin Activates the Immune Response: A Morphology - Function Study

Increasing evidence points to an important role for hemozoin (HZ), the malaria pigment, in the immunopathology related to this infection. However, there is no consensus as to whether HZ exerts its immunostimulatory activity in absence of other parasite or host components. Contamination of native HZ preparations and the lack of a unified protocol to produce crystals that mimic those of Plasmodium HZ (PHZ) are major technical limitants when performing functional studies with HZ. In fact, the most commonly used methods generate a heterogeneous nanocrystalline material. Thus, it is likely that such aggregates do not resemble to PHZ and differ in their inflammatory properties. To address this issue, the present study was designed to establish whether synthetic HZ (sHZ) crystals produced by different methods vary in their morphology and in their ability to activate immune responses. We report a new method of HZ synthesis (the precise aqueous acid-catalyzed method) that yields homogeneous sHZ crystals (Plasmodium-like HZ) which are very similar to PHZ in their size and physicochemical properties. Importantly, these crystals are devoid of protein and DNA contamination. Of interest, structure-function studies revealed that the size and shape of the synthetic crystals influences their ability to activate inflammatory responses (e.g. nitric oxide, chemokine and cytokine mRNA) in vitro and in vivo. In summary, our data confirm that sHZ possesses immunostimulatory properties and underline the importance of verifying by electron microscopy both the morphology and homogeneity of the synthetic crystals to ensure that they closely resemble those of the parasite. Periodic quality control experiments and unification of the method of HZ synthesis are key steps to unravel the role of HZ in malaria immunopathology

Leishmania-Induced IRAK-1 Inactivation Is Mediated by SHP-1 Interacting with an Evolutionarily Conserved KTIM Motif

Parasites of the Leishmania genus can rapidly alter several macrophage (MØ) signalling pathways in order to tame down the innate immune response and inflammation, therefore favouring their survival and propagation within their mammalian host. Having recently reported that Leishmania and bacterial LPS generate a significantly stronger inflammatory response in animals and phagocytes functionally deficient for the Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), we hypothesized that Leishmania could exploit SHP-1 to inactivate key kinases involved in Toll-like receptor (TLR) signalling and innate immunity such as IL-1 receptor-associated kinase 1 (IRAK-1). Here we show that upon infection, SHP-1 rapidly binds to IRAK-1, completely inactivating its intrinsic kinase activity and any further LPS-mediated activation as well as MØ functions. We also demonstrate that the SHP-1/IRAK-1 interaction occurs via an evolutionarily conserved ITIM-like motif found in the kinase domain of IRAK-1, which we named KTIM (Kinase Tyrosyl-based Inhibitory Motif). This regulatory motif appeared in early vertebrates and is not found in any other IRAK family member. Our study additionally reveals that several other kinases (e.g. Erk1/2, IKKα/β) involved in downstream TLR signalling also bear KTIMs in their kinase domains and interact with SHP-1. We thus provide the first demonstration that a pathogen can exploit a host protein tyrosine phosphatase, namely SHP-1, to directly inactivate IRAK-1 through a generally conserved KTIM motif

Malarial Hemozoin Activates the NLRP3 Inflammasome through Lyn and Syk Kinases

The intraerythrocytic parasite Plasmodium—the causative agent of malaria—produces an inorganic crystal called hemozoin (Hz) during the heme detoxification process, which is released into the circulation during erythrocyte lysis. Hz is rapidly ingested by phagocytes and induces the production of several pro-inflammatory mediators such as interleukin-1β (IL-1β). However, the mechanism regulating Hz recognition and IL-1β maturation has not been identified. Here, we show that Hz induces IL-1β production. Using knockout mice, we showed that Hz-induced IL-1β and inflammation are dependent on NOD-like receptor containing pyrin domain 3 (NLRP3), ASC and caspase-1, but not NLRC4 (NLR containing CARD domain). Furthermore, the absence of NLRP3 or IL-1β augmented survival to malaria caused by P. chabaudi adami DS. Although much has been discovered regarding the NLRP3 inflammasome induction, the mechanism whereby this intracellular multimolecular complex is activated remains unclear. We further demonstrate, using pharmacological and genetic intervention, that the tyrosine kinases Syk and Lyn play a critical role in activation of this inflammasome. These findings not only identify one way by which the immune system is alerted to malarial infection but also are one of the first to suggest a role for tyrosine kinase signaling pathways in regulation of the NLRP3 inflammasome

Iron Prevents the Development of Experimental Cerebral Malaria by Attenuating CXCR3-Mediated T Cell Chemotaxis

<div><p>Cerebral malaria is a severe neurological complication of <i>Plasmodium falciparum</i> infection. Previous studies have suggested that iron overload can suppress the generation of a cytotoxic immune response; however, the effect of iron on experimental cerebral malaria (ECM) is yet unknown. Here we determined that the incidence of ECM was markedly reduced in mice treated with iron dextran. Protection was concomitant with a significant decrease in the sequestration of CD4⁺ and CD8⁺ T cells within the brain. CD4⁺ T cells demonstrated markedly decreased CXCR3 expression and had reduced IFNγ-responsiveness, as indicated by mitigated expression of IFNγR2 and T-bet. Additional analysis of the splenic cell populations indicated that parenteral iron supplementation was also associated with a decrease in NK cells and increase in regulatory T cells. Altogether, these results suggest that iron is able to inhibit ECM pathology by attenuating the capacity of T cells to migrate to the brain.</p></div

The Expression of CXCR3 on Splenic CD4⁺ T Cells is Decreased by Parenteral Iron Supplementation.

<p>Representative flow cytometric dot plots of CXCR3⁺ CD4⁺ and CD8⁺ T cells (<b>a</b>) and the percentage of CXCR3⁺ cells after gating on CD4⁺ or CD8⁺ T cells (<b>b</b>). Representative flow cytometric histograms of CXCR3 (<b>c</b>) and the MFI of CXCR3 (<b>d</b>) after gating on CD4⁺CD44^hi or CD8⁺CD44^hi T cells. All experiments were performed on day 7 post-infection. The numbers shown on the dot plots indicate the mean percentage of cells inside the gate ± S.E.M. <i>n</i> = 13 for all groups. The average of two individual experiments is shown. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (** <i>P</i> < 0.01 and *** <i>P</i> < 0.001), were determined by unpaired Student’s t-test.</p

Systemic Inflammation is Augmented in FeD Mice.

<p>Concentration of IFNγ (<b>a</b>), TNFα (<b>b</b>), IL-10 (<b>c</b>), IL-1β (<b>d</b>) and IL-6 (<b>e</b>) in the serum on day 7 post-infection. <i>n</i> = 5 for the control, uninfected group mice and <i>n</i> = 6 for all other groups. Levels of TNFα are below the limit of detection in the uninfected groups. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (* <i>P</i> < 0.05 and ** <i>P</i> < 0.01), were determined by unpaired Student’s t-test.</p

Sequestration of CD4⁺ and CD8⁺ T Cells in the Brain is Reduced in FeD Mice.

<p>The total number of cells recovered after isolation (<b>a</b>), the total number (<b>b</b>) and percentage (<b>c</b>) of CD8⁺ T cells after gating on infiltrating leukocytes (CD45⁺CD11b^lo-hi), representative flow cytometric dot plots of CD4⁺ and CD8⁺ T cells after gating on infiltrating leukocytes (<b>d</b>), and the total number (<b>e</b>) and percentage (<b>f</b>) of CD4⁺ T cells after gating on infiltrating leukocytes, on day 7 post-infection. The numbers shown on the dot plots indicate the mean percentage of cells inside the gate ± S.E.M. <i>n</i> = 5 mice were used for each group. UI = uninfected, I = infected, FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (* <i>P</i> < 0.05 and *** <i>P</i> < 0.001), were determined by unpaired Student’s t-test.</p

The Expression of Genes Involved in T Cell Chemotaxis are Attenuated by Parenteral Iron Supplementation.

<p>The expression of genes involved in T cell chemotaxis is shown for the brain (<b>a</b>) and the spleen (<b>b</b>) on day 7 post-infection. mRNA levels were normalized to <i>Gusb</i>. 2 samples pooled from 6 mice (3 mice per sample) were used for each group. UI = uninfected, I = infected, FeD = iron dextran, PBS = control.</p

FeD Mice have Modulated Frequencies of Splenic NK Cells and Tregs Early During the Infection.

<p>Representative flow cytometric dot plots of NK cells (<b>a</b>) and the percentage of NK cells (<b>b</b>) on day 3 and day 7 post-infection. Representative flow cytometric dot plots of Tregs (<b>c</b>) and the percentage of Tregs after gating on CD4⁺ T cells (<b>d</b>) on day 3 and day 7 post-infection. The numbers shown on the dot plots indicate the mean percentage of cells inside the gate ± S.E.M. On day 3 post-infection, <i>n</i> = 6 for control mice and <i>n</i> = 5 for FeD mice, except for Tregs, where <i>n</i> = 5 for the control mice. On day 7 post-infection, <i>n</i> = 6 for control mice and <i>n</i> = 6 for FeD mice. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (* <i>P</i> < 0.05 ** <i>P</i> < 0.01), were determined by unpaired Student’s t-test.</p

Tissue parasite sequestration is inhibited by parenteral iron supplementation.

<p>Parasite levels in the brain (<b>a</b>), spleen (<b>b</b>) and liver (<b>c</b>) on day 7 post-infection. Luciferase activity is shown as the total RLU per organ normalized to the total RLU in the control mice before symptoms. <i>n</i> = 8 for the control mice before symptoms, <i>n</i> = 8 for the control, symptomatic mice and <i>n</i> = 12 for the FeD mice. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (** <i>P</i> < 0.01 and *** <i>P</i> < 0.001), were determined by unpaired Student’s t-test.</p