Resonance Raman spectroscopy in malaria research

In recent years, the field of Raman spectroscopy has witnessed a surge in technological development, with the incorporation of ultrasensitive, charge-coupled devices, improved laser sources and precision Rayleigh-filter systems. This has led to the development of sensitive confocal micro-Raman spectrometers and imaging spectrometers that are capable of obtaining high spatial-resolution spectra and images of subcellular components within single living cells. This review reports on the application of resonance micro-Raman spectroscopy to the study of malaria pigment (hemozoin), a by-product of hemoglobin catabolization by the malaria parasite, which is an important target site for antimalarial drugs. The review aims to briefly describe recent studies on the application of this technology, elucidate molecular and electronic properties of the malaria pigment and its synthetic analog β-hematin, provide insight into the mechanism of hemozoin formation within the food vacuole of the parasite, and comment on developing strategies for using this technology in drug-screening protocols.     

Evaluation of naphthoquinones identified the acetylated isolapachol as a potent and selective antiplasmodium agent

Abstract

This study reports on the design, synthesis and antiparasitic activity of three new semi-synthetic naphthoquinones structurally related to the naturally-occurring lapachol and lapachone. Of the compounds tested, 3-(3-methylbut-1-en-1-yl)-1,4-dioxo-1,4-dihydronaphthalen-2-yl acetate (1) was the most active against Plasmodium falciparum among both natural and semi-synthetic naphthoquinones, showing potent and selective activity. Compound 1 was able to reduce the in vitro parasite burden, in vitro parasite cell cycle, as well as the blood parasitemia in Plasmodium berghei-infected mice. More importantly, infection reduction under compound 1-treatment was achieved without exhibiting mouse genotoxicity. Regarding the molecular mechanism of action, this compound inhibited the hemozoin crystal formation in P. falciparum treated cells, and this was further confirmed by observing that it inhibits the β-hematin polymerization process similarly to chloroquine. Interestingly, this compound did not affect either mitochondria structure or cause DNA fragmentation in parasite treated cells. In conclusion, we identified a semi-synthetic antimalarial naphthoquinone closely related to isolapachol, which had stronger antimalarial activity than lapachol.     

Intracellular pathogen sensor NOD2 programs macrophages to trigger Notch1 activation

Intracellular pathogen sensor, NOD2, has been implicated in regulation of wide range of anti-inflammatory responses critical during development of a diverse array of inflammatory diseases; however, underlying molecular details are still imprecisely understood. In this study, we demonstrate that NOD2 programs macrophages to trigger Notch1 signaling. Signaling perturbations or genetic approaches suggest signaling integration through cross-talk between Notch1-PI3K during the NOD2-triggered expression of a multitude of immunological parameters including COX-2/PGE(2) and IL-10. NOD2 stimulation enhanced active recruitment of CSL/RBP-Jk on the COX-2 promoter in vivo. Intriguingly, nitric oxide assumes critical importance in NOD2-mediated activation of Notch1 signaling as iNOS(-/-) macrophages exhibited compromised ability to execute NOD2-triggered Notch1 signaling responses. Correlative evidence demonstrates that this mechanism operates in vivo in brain and splenocytes derived from wild type, but not from iNOS(-/-) mice. Importantly, NOD2-driven activation of the Notch1-PI3K signaling axis contributes to its capacity to impart survival of macrophages against TNF-α or IFN-γ-mediated apoptosis and resolution of inflammation. Current investigation identifies Notch1-PI3K as signaling cohorts involved in the NOD2-triggered expression of a battery of genes associated with anti-inflammatory functions. These findings serve as a paradigm to understand the pathogenesis of NOD2-associated inflammatory diseases and clearly pave a way toward development of novel therapeutics.     

IL-13 pre-treatment of murine peritoneal macrophages increases their anti-Toxoplasma gondii activity induced by lipopolysaccharides

Th1 cytokines and microbial lipopolysaccharides (LPS) activate macrophages to produce inflammatory mediators and effector molecules. Althrough Th2 cytokines often have an opposite action to Th1 cytokines and down-modulate the inflammatory response of macrophages, they can induce a distinct alternative activation that is beneficial in host defence. In this study, we report that IL-13 enhances the anti-Toxoplasma activity of LPS-activated murine macrophages. The inhibition of parasite proliferation was not related to reduced Toxoplasma gondii penetration into the cells, nor to the conversion of tachyzoites into bradyzoites. Used alone, IL-13 triggers the polarisation of macrophages towards type 2. However, in LPS-activated macrophages, we show the priming capacity of this cytokine to enhance the expression of inducible nitric oxide synthase (iNOS), a major marker of type 1 macrophages. This effect of IL-13 was not dependent on the activation state of macrophages (resident versus thioglycolate-elicited) or the timing of pre-treatment. We demonstrate a correlation between the enhancement of NO production and upgrading of the microbicidal effectiveness of the macrophages. Thus, both Th2 and Th1 cytokines could activate macrophages to control infections.     

Lancemaside A inhibits lipopolysaccharide‐induced inflammation by targeting LPS/TLR4 complex

In our previous study, lancemaside A isolated from Codonopsis lanceolata (family Campanulaceae) ameliorated colitis in mice. In this study, the anti‐inflammatory effects of lancemaside A was investigated in lipopolysaccharide (LPS)‐stimulated mice and their peritoneal macrophage cells. Lancemaside A suppressed the production of pro‐inflammatory cytokines, TNF‐α and IL‐1β, in vitro and in vivo. Lancemaside A also down‐regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2), as well as the inflammatory mediators, nitric oxide (NO), and PGE₂. Lancemaside A also inhibited the expression of IL‐1 receptor‐associated kinase‐4 (IRAK‐4), the phosphorylation of IKK‐β and IκB‐α, the nuclear translocation of NF‐κB and the activation of mitogen‐activated protein kinases in LPS‐stimulated peritoneal macrophages. Furthermore, lancemaisde A inhibited the interaction between LPS and TLR4, as well as IRAK‐4 expression in peritoneal macrophages. Based on these findings, lancemaside A expressed anti‐inflammatory effects by regulating both the binding of LPS to TLR4 on macrophages. J. Cell. Biochem. 111: 865–871, 2010. © 2010 Wiley‐Liss, Inc.     

A patatin-like protein protects Toxoplasma gondii from degradation in activated macrophages

The apicomplexan parasite Toxoplasma gondii is able to suppress nitric oxide production in activated macrophages. A screen of over 6000 T. gondii insertional mutants identified two clones, which were consistently unable to suppress nitric oxide production from activated macrophages. One strain, called 89B7, grew at the same rate as wild‐type parasites in naïve macrophages, but unlike wild type, the mutant was degraded in activated macrophages. This degradation was marked by a reduction in the number of parasites within vacuoles over time, the loss of GRA4 and SAG1 protein staining by immunofluorescence assay, and the vesiculation and breakdown of the internal parasite ultrastructure by electron microscopy. The mutagenesis plasmid in the 89B7 clone disrupts the promoter of a 3.4 kb mRNA that encodes a predicted 68 kDa protein with a cleavable signal peptide and a patatin‐like phospholipase domain. Genetic complementation with the genomic locus of this patatin‐like protein restores the parasites ability to suppress nitric oxide and replicate in activated macrophages. A haemagglutinin‐tagged version of this patatin‐like protein shows punctate localization into atypical T. gondii structures within the parasite. This is the first study that defines a specific gene product that is needed for parasite survival in activated but not naïve macrophages.     

Recent advances in understanding the mechanism of hemozoin (malaria pigment) formation

The recent literature on hemozoin/β-hematin formation is reviewed, with an emphasis on the mechanism of its formation. Recent findings from unrelated organisms that produce hemozoin, namely the malaria parasite Plasmodium falciparum, the worm Schistosoma mansoni and the kissing bug Rhodnius prolixus all of which consume human hemoglobin show that the formation of this crystalline substance occurs within or at the surface of lipids. Biomimetic experimental models of the lipid–water interface as well as computational studies indicate that these lipid environments are probably extraordinarily efficient at producing hemozoin. A rethink is now needed, with a new emphasis on Fe(III)PPIX in non-aqueous environments that mimic lipids and indeed within the lipid environment itself. These findings are explored and discussed in the context of earlier studies on β-hematin formation.     

Induction of protective immunity by primed B-1 cells in Toxoplasma gondii -infected B cell-deficient mice

We examined the role of B‐1 cells in protection against Toxoplasma gondii infection using B cell‐deficient mice (μMT mice). We found that primed but not naïve B‐1 cells from wild‐type C57BL/6 mice protected B cell‐deficient recipients from challenge infection. All μMT mice transferred with primed B‐1 cells survived more than 5 months after T. gondii infection, whereas 100% of μMT mice transferred with naïve B‐1 cells succumbed by 18 days after infection. Additionally, high expression of both T help (Th) 1‐ and Th2‐type cytokines and a high level of nitric oxide production were observed in T. gondii‐infected μMT mice transferred with primed B‐1 cells. Thus, it was clearly demonstrated that B‐1 cells play an important role in host protection against T. gondii infection in μMT mice.     

TLR2 mediates immunity to experimental cysticercosis

Information concerning TLR-mediated antigen recognition and regulation of immune responses during helminth infections is scarce. TLR2 is a key molecule required for innate immunity and is involved in the recognition of a wide range of viruses, bacteria, fungi and parasites. Here, we evaluated the role of TLR2 in a Taenia crassiceps cysticercosis model. We compared the course of T. crassiceps infection in C57BL/6 TLR2 knockout mice (TLR2^-/-) with that in wild type C57BL/6 (TLR2^+/+) mice. In addition, we assessed serum antibody and cytokine profiles, splenic cellular responses and cytokine profiles and the recruitment of alternatively activated macrophages (AAMφs) to the site of the infection. Unlike wild type mice, TLR2^-/- mice failed to produce significant levels of inflammatory cytokines in either the serum or the spleen during the first two weeks of Taenia infection. TLR2^-/- mice developed a Th2-dominant immune response, whereas TLR2^+/+ mice developed a Th1-dominant immune response after Taenia infection. The insufficient production of inflammatory cytokines at early time points and the lack of Th1-dominant adaptive immunity in TLR2^-/- mice were associated with significantly elevated parasite burdens; in contrast, TLR2^+/+ mice were resistant to infection. Furthermore, increased recruitment of AAMφs expressing PD-L1, PD-L2, OX40L and mannose receptor was observed in TLR2^-/- mice. Collectively, these findings indicate that TLR2-dependent signaling pathways are involved in the recognition of T. crassiceps and in the subsequent activation of the innate immune system and production of inflammatory cytokines, which appear to be essential to limit infection during experimental cysticercosis.     

Role of Nitric Oxide in the Progression of Pneumoconiosis

Conflicting evidence has been reported as to whether nitric oxide (NO) possesses anti-inflammatory or inflammatory properties. Data are presented indicating that in vitro or in vivo exposure to selected occupational dusts, i.e., crystalline silica, organic dust contaminated with endotoxin, or asbestos, results in upregulation of inducible nitric oxide synthase (iNOS) and the production of NO by alveolar macrophages and pulmonary epithelial cells. Nitric oxide production is associated temporally and anatomically with pulmonary damage, inflammation, and disease progression in response to occupational dusts. Blockage of inducible nitric oxide synthase by administration of NOS inhibitors or in iNOS knockout mice decreases the magnitude of injury and inflammation following in vivo exposure to silica, endotoxin, or asbestos. Therefore, NO may play an important role in the initiation and progression of pneumoconiosis.     

Innate immune responses to endosymbiotic Wolbachia bacteria in Brugia malayi and Onchocerca volvulus are dependent on TLR2, TLR6, MyD88, and Mal, but not TLR4, TRIF, or TRAM

The discovery that endosymbiotic Wolbachia bacteria play an important role in the pathophysiology of diseases caused by filarial nematodes, including lymphatic filariasis and onchocerciasis (river blindness) has transformed our approach to these disabling diseases. Because these parasites infect hundreds of millions of individuals worldwide, understanding host factors involved in the pathogenesis of filarial-induced diseases is paramount. However, the role of early innate responses to filarial and Wolbachia ligands in the development of filarial diseases has not been fully elucidated. To determine the role of TLRs, we used cell lines transfected with human TLRs and macrophages from TLR and adaptor molecule-deficient mice and evaluated macrophage recruitment in vivo. Extracts of Brugia malayi and Onchocerca volvulus, which contain Wolbachia, directly stimulated human embryonic kidney cells expressing TLR2, but not TLR3 or TLR4. Wolbachia containing filarial extracts stimulated cytokine production in macrophages from C57BL/6 and TLR4(-/-) mice, but not from TLR2(-/-) or TLR6(-/-) mice. Similarly, macrophages from mice deficient in adaptor molecules Toll/IL-1R domain-containing adaptor-inducing IFN-beta and Toll/IL-1R domain-containing adaptor-inducing IFN-beta-related adaptor molecule produced equivalent cytokines as wild-type cells, whereas responses were absent in macrophages from MyD88(-/-) and Toll/IL-1R domain-containing adaptor protein (TIRAP)/MyD88 adaptor-like (Mal) deficient mice. Isolated Wolbachia bacteria demonstrated similar TLR and adaptor molecule requirements. In vivo, macrophage migration to the cornea in response to filarial extracts containing Wolbachia was dependent on TLR2 but not TLR4. These results establish that the innate inflammatory pathways activated by endosymbiotic Wolbachia in B. malayi and O. volvulus filaria are dependent on TLR2-TLR6 interactions and are mediated by adaptor molecules MyD88 and TIRAP/Mal.     

Organic dust augments nucleotide-binding oligomerization domain expression via an NF-{kappa}B pathway to negatively regulate inflammatory responses

Nucleotide-binding oligomerization domain 2 (NOD2) is involved in innate immune responses to peptidoglycan degradation products. Peptidoglycans are important mediators of organic dust-induced airway diseases in exposed agriculture workers; however, the role of NOD2 in response to complex organic dust is unknown. Monocytes/macrophages were exposed to swine facility organic dust extract (ODE), whereupon NOD2 expression was evaluated by real-time PCR and Western blot. ODE induced significant NOD2 mRNA and protein expression at 24 and 48 h, respectively, which was mediated via a NF-κB signaling pathway as opposed to a TNF-α autocrine/paracrine mechanism. Specifically, NF-κB translocation increased rapidly following ODE stimulation as demonstrated by EMSA, and inhibition of the NF-κB pathway significantly reduced ODE-induced NOD2 expression. However, there was no significant reduction in ODE-induced NOD2 gene expression when TNF-α was inhibited or absent. Next, it was determined whether NOD2 regulated ODE-induced inflammatory cytokine production. Knockdown of NOD2 expression by small interfering RNA resulted in increased CXCL8 and IL-6, but not TNF-α production in response to ODE. Similarly, primary lung macrophages from NOD2 knockout mice demonstrated increased IL-6, CXCL1, and CXCL1, but not TNF-α, expression. Lastly, a higher degree of airway inflammation occurred in the absence of NOD2 following acute (single) and repetitive (3 wk) ODE exposure in an established in vivo murine model. In summary, ODE-induced NOD2 expression is directly dependent on NF-κB signaling, and NOD2 is a negative regulator of complex, organic dust-induced inflammatory cytokine/chemokine production in mononuclear phagocytes.     

P2X7 Receptor Modulates Inflammatory and Functional Pulmonary Changes Induced by Silica

Silicosis is an occupational lung disease, characterized by irreversible and progressive fibrosis. Silica exposure leads to intense lung inflammation, reactive oxygen production, and extracellular ATP (eATP) release by macrophages. The P2X7 purinergic receptor is thought to be an important immunomodulator that responds to eATP in sites of inflammation and tissue damage. The present study investigates the role of P2X7 receptor in a murine model of silicosis. To that end wild-type (C57BL/6) and P2X7 receptor knockout mice received intratracheal injection of saline or silica particles. After 14 days, changes in lung mechanics were determined by the end-inflation occlusion method. Bronchoalveolar lavage and flow cytometry analyzes were performed. Lungs were harvested for histological and immunochemistry analysis of fibers content, inflammatory infiltration, apoptosis, as well as cytokine and oxidative stress expression. Silica particle effects on lung alveolar macrophages and fibroblasts were also evaluated in cell line cultures. Phagocytosis assay was performed in peritoneal macrophages. Silica exposure increased lung mechanical parameters in wild-type but not in P2X7 knockout mice. Inflammatory cell infiltration and collagen deposition in lung parenchyma, apoptosis, TGF-β and NF-κB activation, as well as nitric oxide, reactive oxygen species (ROS) and IL-1β secretion were higher in wild-type than knockout silica-exposed mice. In vitro studies suggested that P2X7 receptor participates in silica particle phagocytosis, IL-1β secretion, as well as reactive oxygen species and nitric oxide production. In conclusion, our data showed a significant role for P2X7 receptor in silica-induced lung changes, modulating lung inflammatory, fibrotic, and functional changes.     

Role of phosphatidylinositol-3-kinase-gamma (PI3Kgamma)-mediated pathway in 17beta-estradiol-induced killing of L. mexicana in macrophages from C57BL/6 mice

We recently demonstrated that 17beta-estradiol (E2) enhances killing of Leishmania mexicana in macrophages from both male and female DBA/2 mouse by increasing nitric oxide (NO) production. Here, we analyzed the effect of E2 on leishmanicidal activity and cytokine production by bone marrow-derived macrophages (BMDMs) from male and female C57BL/6 mice in vitro, specifically examining the role of phosphatidylinositol-3-kinase-gamma (PI3Kgamma) in E2-induced parasite killing. Unlike its effect on macrophages from both male and female DBA/2 mice, E2 only increased leishmanicidal activity in macrophages from female C57BL/6 mice, which was evident by a significant reduction in both infection rates and infection levels compared to sham controls. E2-treated BMDMs from female C57BL/6 mice expressed higher levels of interferon-gammaRalpha, and also produced more interleukin (IL)-12, IL-6 and NO than both the sham controls and E2-treated male-derived macrophages. Sham-treated BMDMs from female PI3Kgamma-/- C57BL/6 mice displayed lower infection rates and infection levels compared to sham-treated wild-type (WT) macrophages. However E2, unlike its effect on macrophages from female WT C57BL/6 mice, failed to reduce infection rates and infection levels in BMDMs from female PI3Kgamma-/- mice. Interestingly, E2-treated BMDMs from female C57BL/6 mice produced significant amounts of inflammatory cytokines and NO in levels comparable to those observed in sham-treated PI3Kgamma-deficient macrophages as well as E2-treated macrophages from WT mice. These findings show that E2 exerts a distinct effect on leishmanicidal activity of macrophages from male versus female C57BL/6 mice. In addition, they suggest that PI3Kgamma is not required for E2-induced cytokine and NO production in L. mexicana-infected macrophages from female C57BL/6 mice but it may be involved in parasite clearance from these cells.     

Immunohistochemical expression of Bcl-2, p53 and caspase-9 in hypothalamus magnocellular centers of nNOS knockout mice following water deprivation

Abstract

We studied the interactions between apoptosis regulator proteins (Bcl-2, p53 and caspase-9) and neuronal nitric oxide in vasopressinergic magnocellular centers of the hypothalamus using neuronal nitric oxide synthase (nNOS) gene knockout mice. nNOS gene deletion resulted in accumulation of Bcl-2, p53 and caspase-9 in the paraventricular (PVN) and supraoptic (SON) nuclei in controls. Dehydration increased the levels of all three apoptosis regulator proteins studied in nuclei of wild type mice. In the hypothalamus magnocellular centers of nNOS knockout mice, however, expression of Bcl-2, p53 and caspase-9 was unchanged after dehydration. The number of magnocellular neurons did not change in the SON and PVN of nNOS deficient mice compared to wild type, and after dehydration, cell death was not observed in either nucleus of wild type or knockout mice despite activation of apoptosis regulator protein expression. Thus, we demonstrated that gene disruption of nNOS prevents activation of Bcl-2, p53 and caspase-9 expression during water deprivation, and that nNOS deficiency did not affect survival of magnocellular neurons of the hypothalamus.     

Induction of nitric oxide synthase by saponins of heat-processed ginseng

Total saponin of heat-processed ginseng (TSHG) stimulated the production of nitric oxide (NO) in interferon-gamma (IFN-gamma)-primed macrophages through the increased expression of inducible nitric oxide synthase (iNOS). However, TSHG by itself had a very weak effect on the NO synthesis without IFN-gamma priming. The saponins of white ginseng inhibited the NO production in lipopolysaccharide (LPS)/IFN-gamma activated macrophages rather than the stimulation of NO production found in IFN-gamma primed macrophages. The NO production by TSHG-stimulated macrophages was inhibited by the NOS inhibitor (N(G)-monomethyl-L-arginine (L-NMMA)) and nuclear factor-kappaB inhibitor (pyrrolidine dithiocarbamate (PDTC)). TSHG showed different serum-dependence from LPS on the activation of IFN-gamma primed macrophages. This property of TSHG may explain the intensified anti-tumor properties of heat-processed ginseng through its immunostimulating activity.     

The oral histone deacetylase inhibitor ITF2357 reduces cytokines and protects islet β cells in vivo and in vitro

In type 1 diabetes, inflammatory and immunocompetent cells enter the islet and produce proinflammatory cytokines such as interleukin-1β (IL-1β), IL-12, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); each contribute to β-cell destruction, mediated in part by nitric oxide. Inhibitors of histone deacetylases (HDAC) are used commonly in humans but also possess antiinflammatory and cytokine-suppressing properties. Here we show that oral administration of the HDAC inhibitor ITF2357 to mice normalized streptozotocin (STZ)-induced hyperglycemia at the clinically relevant doses of 1.25-2.5 mg/kg. Serum nitrite levels returned to nondiabetic values, islet function improved and glucose clearance increased from 14% (STZ) to 50% (STZ + ITF2357). In vitro, at 25 and 250 nmol/L, ITF2357 increased islet cell viability, enhanced insulin secretion, inhibited MIP-1α and MIP-2 release, reduced nitric oxide production and decreased apoptosis rates from 14.3% (vehicle) to 2.6% (ITF2357). Inducible nitric oxide synthase (iNOS) levels decreased in association with reduced islet-derived nitrite levels. In peritoneal macrophages and splenocytes, ITF2357 inhibited the production of nitrite, as well as that of TNFα and IFNγ at an IC(50) of 25-50 nmol/L. In the insulin-producing INS cells challenged with the combination of IL-1β plus IFNγ, apoptosis was reduced by 50% (P < 0.01). Thus at clinically relevant doses, the orally active HDAC inhibitor ITF2357 favors β-cell survival during inflammatory conditions.     

Radioprotective effect of E. coli endotoxin on hematopoietic stem cells is partially suppressed by inhibiting production of nitric oxide by administering N-omega-nitro-L-arginine

Lipopolysaccharide of E. coli (LPS) injected to mice one day before the total gamma-irradiation caused a substantial increase in the level of endogeneous colonies formed in the spleen. It is known that this type of endotoxin may result in considerable production of nitric oxide from macrophages in different tissues. Therefore it is possible that the activation of hemopoietic stem cells after LPS-treatment was completely or partially stimulated by nitric oxide, which is the most important physiological mediator, or by action some other mediators (cytokines and growth factors) produced by hemopoietic microenvironment elements in response to the action of nitric oxide. This assumption was checked in experience with combined treatment of mice by LPS and a nonspecific inhibitor of nitric oxide production--N omega-nitro-1-arginine (NA). NA used in high dose (250 mg/kg) reduced partially (approximately by 30%) the LPS-increased level of spleen endogeneous colonies. When LPS was injected to mice 15 minutes after gamma-irradiation, this led to a slight increase in level of spleen colonies. In case when LPS was used together with NA after gamma-irradiation, this increase was still found.