IRGM3 contributes to immunopathology and is required for differentiation of antigen-specific effector CD8+ T cells in experimental cerebral malaria.

Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection with Plasmodium species. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used the Plasmodium berghei ANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA for Irgm1 and Irgm3 was found in the brains and spleens of infected mice at times of peak IFN-γ production. Irgm3-/- but not Irgm1-/- mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8+ T cells within the brain. Although antigen-specific proliferation of transferred CD8+ T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred into Irgm3-/- recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens of Irgm3-/- mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology in Irgm3-/- mice occurs due to impaired generation of CD8+ effector function. This defect is nonintrinsic to CD8+ T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.

Mechanisms of murine cerebral malaria: Multimodal imaging of altered cerebral metabolism and protein oxidation at hemorrhage sites.

Using a multimodal biospectroscopic approach, we settle several long-standing controversies over the molecular mechanisms that lead to brain damage in cerebral malaria, which is a major health concern in developing countries because of high levels of mortality and permanent brain damage. Our results provide the first conclusive evidence that important components of the pathology of cerebral malaria include peroxidative stress and protein oxidation within cerebellar gray matter, which are colocalized with elevated nonheme iron at the site of microhemorrhage. Such information could not be obtained previously from routine imaging methods, such as electron microscopy, fluorescence, and optical microscopy in combination with immunocytochemistry, or from bulk assays, where the level of spatial information is restricted to the minimum size of tissue that can be dissected. We describe the novel combination of chemical probe-free, multimodal imaging to quantify molecular markers of disturbed energy metabolism and peroxidative stress, which were used to provide new insights into understanding the pathogenesis of cerebral malaria. In addition to these mechanistic insights, the approach described acts as a template for the future use of multimodal biospectroscopy for understanding the molecular processes involved in a range of clinically important acute and chronic (neurodegenerative) brain diseases to improve treatment strategies.

The kynurenine pathway contributes to long-term neuropsychological changes in experimental pneumococcal meningitis.

Pneumococcal meningitis is a lethal form of bacterial infection in the central nervous system that often causes lifelong neurological sequelae, despite therapeutic advances. The contemporary view is that the inflammatory response to infection contributes to the functional disabilities among survivors of this disease. We previously have established a mouse model of neurobehavioural deficits, using an automated IntelliCage™ system that revealed long-term behavioural and cognitive deficits in C57BL/6J female mice cured of meningitis by ceftriaxone treatment. We now have investigated the roles of two kynurenine pathway enzymes, indoleamine dioxygenase-1 (IDO1) and tryptophan dioxygenase-2 (TDO2), in the pathomechanisms of pneumococcal meningitis. Since tryptophan metabolism has long been implicated in behavioural and cognitive modulation through the production of neuroactive compounds, we hypothesised that preventing the actions of these enzymes through gene knockout would be beneficial in mice subjected to pneumococcal infection. We found no significant effect of IDO1 or TDO2 on mortality. Post-meningitic wild-type mice showed long-term diurnal hypoactivity and nocturnal hyperactivity when they were exposed to an Intellicage adaptation test throughout both the light and dark phases. These changes were not apparent in IDO1(-/-) survivors, but were present in the TDO2(-/-) survivors. Both IDO1(-/-) and TDO2(-/-) survivors were not protected against developing long-term cognitive deficits as measured in IntelliCage-based patrolling or reversal tasks. Collectively, these observations suggest (i) involvement of the kynurenine pathway in causing some behavioural sequelae of pneumococcal meningitis and (ii) that this pathway might operate synergistically with, or independently of, other pathways to cause other aspects of neurological sequelae.

FTIR imaging of brain tissue reveals crystalline creatine deposits are an ex vivo marker of localized ischemia during murine cerebral malaria: general implications for disease neurochemistry.

Phosphocreatine is a major cellular source of high energy phosphates, which is crucial to maintain cell viability under conditions of impaired metabolic states, such as decreased oxygen and energy availability (i.e., ischemia). Many methods exist for the bulk analysis of phosphocreatine and its dephosphorylated product creatine; however, no method exists to image the distribution of creatine or phosphocreatine at the cellular level. In this study, Fourier transform infrared (FTIR) spectroscopic imaging has revealed the ex vivo development of creatine microdeposits in situ in the brain region most affected by the disease, the cerebellum of cerebral malaria (CM) diseased mice; however, such deposits were also observed at significantly lower levels in the brains of control mice and mice with severe malaria. In addition, the number of deposits was observed to increase in a time-dependent manner during dehydration post tissue cutting. This challenges the hypotheses in recent reports of FTIR spectroscopic imaging where creatine microdeposits found in situ within thin sections from epileptic, Alzheimer's (AD), and amlyoid lateral sclerosis (ALS) diseased brains were proposed to be disease specific markers and/or postulated to contribute to the brain pathogenesis. As such, a detailed investigation was undertaken, which has established that the creatine microdeposits exist as the highly soluble HCl salt or zwitterion and are an ex-vivo tissue processing artifact and, hence, have no effect on disease pathogenesis. They occur as a result of creatine crystallization during dehydration (i.e., air-drying) of thin sections of brain tissue. As ischemia and decreased aerobic (oxidative metabolism) are common to many brain disorders, regions of elevated creatine-to-phosphocreatine ratio are likely to promote crystal formation during tissue dehydration (due to the lower water solubility of creatine relative to phosphocreatine). The results of this study have demonstrated that although the deposits do not occur in vivo, and do not directly play any role in disease pathogenesis, increased levels of creatine deposits within air-dried tissue sections serve as a highly valuable marker for the identification of tissue regions with an altered metabolic status. In this study, the location of crystalline creatine deposits were used to identify whether an altered metabolic state exists within the molecular and granular layers of the cerebellum during CM, which complements the recent discovery of decreased oxygen availability in the brain during this disease.

Inflammasome-dependent IFN-γ drives pathogenesis in Streptococcus pneumoniae meningitis.

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ(-/-) mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ(-/-) mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ(-/-) mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ(-/-) mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ(-/-) mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.

Chemical alterations to murine brain tissue induced by formalin fixation: implications for biospectroscopic imaging and mapping studies of disease pathogenesis.

Understanding biochemical mechanisms and changes associated with disease conditions and, therefore, development of improved clinical treatments, is relying increasingly on various biochemical mapping and imaging techniques on tissue sections. However, it is essential to be able to ascertain whether the sampling used provides the full biochemical information relevant to the disease and is free from artefacts. A multi-modal micro-spectroscopic approach, including FTIR imaging and PIXE elemental mapping, has been used to study the molecular and elemental profile within cryofixed and formalin-fixed murine brain tissue sections. The results provide strong evidence that amino acids, carbohydrates, lipids, phosphates, proteins and ions, such as Cl(-) and K(+), leach from tissue sections into the aqueous fixative medium during formalin fixation of the sections. Large changes in the concentrations and distributions of most of these components are also observed by washing in PBS even for short periods. The most likely source of the chemical species lost during fixation is the extra-cellular and intra-cellular fluid of tissues. The results highlight that, at best, analysis of formalin-fixed tissues gives only part of the complete biochemical "picture" of a tissue sample. Further, this investigation has highlighted that significant lipid peroxidation/oxidation may occur during formalin fixation and that the use of standard histological fixation reagents can result in significant and differential metal contamination of different regions of tissue sections. While a consistent and reproducible fixation method may be suitable for diagnostic purposes, the findings of this study strongly question the use of formalin fixation prior to spectroscopic studies of the molecular and elemental composition of biological samples, if the primary purpose is mechanistic studies of disease pathogenesis.

Coincident parasite and CD8 T cell sequestration is required for development of experimental cerebral malaria.

Cerebral malaria (CM) is a fatal complication of Plasmodium falciparum infection. Using a well defined murine model, we observed the effect on disease outcome of temporarily reducing parasite burden by anti-malarial drug treatment. The anti-malarial treatment regime chosen decreased parasitaemia but did not cure the mice, allowing recrudescence of parasites. These mice were protected against CM, despite their parasitaemia having increased, following treatment cessation, to levels surpassing that associated with CM in mice not treated with the drug. The protection was associated with reduced levels of cytokines, chemokines, CD8(+) T cells and parasites in the brain. The results suggest that the development of the immunopathological response that causes CM depends on a continuous stimulus provided by parasitised red blood cells, either circulating or sequestered in small vessels.

Artemisone effective against murine cerebral malaria.

BACKGROUND: Artemisinins are the newest class of drug approved for malaria treatment. Due to their unique mechanism of action, rapid effect on Plasmodium, and high efficacy in vivo, artemisinins have become essential components of malaria treatment. Administration of artemisinin derivatives in combination with other anti-plasmodials has become the first-line treatment for uncomplicated falciparum malaria. However, their efficiency in cases of cerebral malaria (CM) remains to be determined. METHODS: The efficacy of several artemisinin derivatives for treatment of experimental CM was evaluated in ICR or C57BL/6 mice infected by Plasmodium berghei ANKA. Both mouse strains serve as murine models for CM. RESULTS: Artemisone was the most efficient drug tested, and could prevent death even when administered at relatively late stages of cerebral pathogenesis. No parasite resistance to artemisone was detected in recrudescence. Co-administration of artemisone together with chloroquine was more effective than monotherapy with either drug, and led to complete cure. Artemiside was even more effective than artemisone, but this substance has yet to be submitted to preclinical toxicological evaluation. CONCLUSIONS: Altogether, the results support the use of artemisone for combined therapy of CM.

Modulation of cerebral malaria by fasudil and other immune-modifying compounds.

Malaria continues to cause millions of deaths annually. No specific effective treatment has yet been found for cerebral malaria, one of the most severe complications of the disease. The pathology of cerebral malaria is considered to be primarily immunological. We examined a number of compounds with known effects on the immune system, in a murine model of cerebral malaria. Of the compounds tested, only fasudil and curcumin had significant effects on the progression of the disease. Although neither drug caused a reduction in parasitemia, survival of the treated mice was significantly increased, and the development of cerebral malaria was either delayed or prevented. Our results support the hypothesis that an immunomodulator efficient in preventing CM should be administered together with anti-plasmodial drugs to prevent severe malaria disease; curcumin and fasudil should be further investigated to determine efficiency and feasibility of treatment.

Chemokine gene expression during fatal murine cerebral malaria and protection due to CXCR3 deficiency.

Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. Using murine models of malaria, we found much greater up-regulation of a number of chemokine mRNAs, including those for CXCR3 and its ligands, in the brain during fatal murine CM (FMCM) than in a model of non-CM. Expression of CXCL9 and CXCL10 RNA was localized predominantly to the cerebral microvessels and in adjacent glial cells, while expression of CCL5 was restricted mainly to infiltrating lymphocytes. The majority of mice deficient in CXCR3 were found to be protected from FMCM, and this protection was associated with a reduction in the number of CD8+ T cells in brain vessels as well as reduced expression of perforin and FasL mRNA. Adoptive transfer of CD8+ cells from C57BL/6 mice with FMCM abrogated this protection in CXCR3-/- mice. Moreover, there were decreased mRNA levels for the proinflammatory cytokines IFN-gamma and lymphotoxin-alpha in the brains of mice protected from FMCM. These data suggest a role for CXCR3 in the pathogenesis of FMCM through the recruitment and activation of pathogenic CD8+ T cells.

Characterization of an indoleamine 2,3-dioxygenase-like protein found in humans and mice.

Indoleamine 2,3-dioxygenase (INDO) and tryptophan 2,3-dioxygenase (TDO) each catalyze the first step in the kynurenine pathway of tryptophan metabolism. We describe the discovery of another enzyme with this activity, indoleamine 2,3-dioxygenase-like protein (INDOL1), which is closely related to INDO and is expressed in mice and humans. The corresponding genes have a similar genomic structure and are situated adjacent to each other on human and mouse chromosome 8. They are likely to have arisen by gene duplication before the origin of the tetrapods. The expression of INDOL1 is highest in the mouse kidney, followed by epididymis, and liver. Expression of mouse INDOL1 was further localized to the tubular cells in the kidney and the spermatozoa. INDOL1 was assigned its name because of its structural similarity to INDO. We demonstrate that INDOL1 catalyses the conversion of tryptophan to kynurenine therefore a more appropriate nomenclature for the enzymes might be INDO-1 and INDO-2, or the more commonly-used abbreviations, IDO-1 and IDO-2. Although the two proteins have similar enzymatic activities, their different expression patterns within tissues and during malaria infection, suggests a distinct role for each protein. This identification of INDOL1 may help to explain the regulation of the diversity of physiological and patho-physiological processes in which the kynurenine pathway is involved.

Brain gene expression, metabolism, and bioenergetics: interrelationships in murine models of cerebral and noncerebral malaria.

Malaria infection can cause cerebral symptoms without parasite invasion of brain tissue. We examined the relationships between brain biochemistry, bioenergetics, and gene expression in murine models of cerebral (Plasmodium berghei ANKA) and noncerebral (P. berghei K173) malaria using multinuclear NMR spectroscopy, neuropharmacological approaches, and real-time RT-PCR. In cerebral malaria caused by P. berghei ANKA infection, we found biochemical changes consistent with increased glutamatergic activity and decreased flux through the Krebs cycle, followed by increased production of the hypoxia markers lactate and alanine. This was accompanied by compromised brain bioenergetics. There were few significant changes in expression of mRNA for metabolic enzymes or transporters or in the rate of transport of glutamate or glucose. However, in keeping with a role for endogenous cytokines in malaria cerebral pathology, there was significant up-regulation of mRNAs for TNF-alpha, interferon-gamma, and lymphotoxin. These changes are consistent with a state of cytopathic hypoxia. By contrast, in P. berghei K173 infection the brain showed increased metabolic rate, with no deleterious effect on bioenergetics. This was accompanied by mild up-regulation of expression of metabolic enzymes. These changes are consistent with benign hypermetabolism whose cause remains a subject of speculation.

For want of a nail. ramifications of a single gene deletion, dystrophin, in the brain of the mouse.

Lack of expression of a single gene, dystrophin, causes the severe, progressive muscle wasting and mental deficits characteristic of Duchenne muscular dystrophy. In this work, we investigated the impact of dystrophin deletion on expression of other genes in the brain cortex, hippocampus and cerebellum using the murine homologue, the mdx mouse, and RT-PCR. Expression of the brain glucose transporters GLUT1 and GLUT2 was found to be decreased, as were some subunits of the GABAA and nicotinic acetylcholine receptors. Genes involved in bioenergetic homeostasis, such as the mitochondrial creatine kinase and the gamma subunit of ATP synthase were also found to be abnormally expressed, while expression of the structural proteins beta-dystrobrevin and rapsyn was also significantly affected. We relate these findings to known functional deficits and discuss the possible mechanisms behind the altered gene expression.

Inhibition of glutamine transport depletes glutamate and GABA neurotransmitter pools: further evidence for metabolic compartmentation.

The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mm) glutamine to displace 13C label supplied as [3-13C]pyruvate, [2-13C]acetate, l-[3-13C]lactate, or d-[1-13C]glucose was investigated using NMR spectroscopy. Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. This uptake was inhibited by MeAIB. When [1-13C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are discussed.