Induction of experimental cerebral malaria is independent of TLR2/4/9

The contribution of the Toll-like receptor (TLR) cascade to the pathogenesis of cerebral malaria (CM) is controversially discussed. TLR2 and TLR9 were reported to be involved in the induction of CM in a study while recently TLR signaling was shown to be dispensable for the development of CM. Using Plasmodium berghei ANKA (PbA) infection of mice as a model of CM, we demonstrate here that the induction of CM is independent of TLR2, 4 and 9. Using triple TLR2/4/9-deficient mice, we exclude synergistic effects between the single TLRs that have been previously implicated with malaria pathology. In conclusion, this study shows that the activation of the innate immune response and the development of CM is not dependent on the engagement of TLR2/4/9.     

Pathological role of Toll-like receptor signaling in cerebral malaria

Toll-like receptors (TLRs) recognize malaria parasites or their metabolites; however, their physiological roles in malaria infection in vivo are not fully understood. Here, we show that myeloid differentiation primary response gene 88 (MyD88)-dependent TLR signaling mediates brain pathogenesis of severe malaria infection, namely cerebral malaria (CM). A significant number of MyD88-, but not TIR domain containing adaptor-inducing IFN-beta (TRIF)-deficient or wild-type (WT) mice survived CM caused by Plasmodium berghei ANKA (PbA) infection. Although systemic parasitemia was comparable, sequestration of parasite and hemozoin load in the brain blood vessels was significantly lower in MyD88-deficient mice compared with those in TRIF-deficient or WT mice. Moreover, brain-specific pathological changes were associated with MyD88-dependent infiltration of CD8+, CCR5+ T cells and CD11c+ dendritic cells, including CD11c+, NK1.1+ and B220+ cells, and up-regulation of genes such as Granzyme B, Lipocalin 2, Ccl3 and Ccr5. Further studies using mice lacking various TLRs suggest that TLR2 and TLR9, but not TLR4, 5 and 7, were involved in CM. These results strongly suggest that TLR2- and/or TLR9-mediated, MyD88-dependent brain pathogenesis may play a critical role in CM, the lethal complication during PbA infection.     

Protein kinase C-theta is required for development of experimental cerebral malaria

Cerebral malaria is the most severe neurologic complication in children and young adults infected with Plasmodium falciparum. T-cell activation is required for development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (CM). To characterize the T-cell activation pathway involved, the role of protein kinase C-theta (PKC-θ) in experimental CM development was examined. PKC-θ-deficient mice are resistant to CM development. In the absence of PKC-θ, no neurologic sign of CM developed after blood stage PbA infection. Resistance of PKC-θ-deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and magnetic resonance angiography, whereas wild-type mice developed distinct microvascular pathology. Recruitment and activation of CD8(+) T cells, and ICAM-1 and CD69 expression were reduced in the brain of resistant mice; however, the pulmonary inflammation and edema associated with PbA infection were still present in the absence of functional PKC-θ. Resistant PKC-θ-deficient mice developed high parasitemia, and died at 3 weeks with severe anemia. Therefore, PKC-θ signaling is crucial for recruitment of CD8(+) T cells and development of brain microvascular pathology resulting in fatal experimental CM, and may represent a novel therapeutic target of CM.     

Resistance to cerebral malaria in tumor necrosis factor-alpha/beta-deficient mice is associated with a reduction of intercellular adhesion molecule-1 up-regulation and T helper type 1 response.

Tumor necrosis factor (TNF) induced by Plasmodium berghei ANKA (PbA) infection was suggested to play an important role in the development of cerebral malaria (CM). We asked whether TNF-alpha/beta double-deficient mice, which have a complete disruption of the TNF-signaling pathways, are protected from CM and what might be the possible mechanisms of protection. PbA infection induces fatal CM in wild-type mice, which die within 5 to 8 days with severe neurological signs. In contrast, TNF-alpha/beta-deficient mice are completely resistant to PbA-induced CM. As PbA-induced up-regulation of endothelial intercellular adhesion molecule (ICAM)-1 expression as well as the systemic release of nitric oxide is found only in wild-type mice, TNF is apparently central for the recruitment of mononuclear cells and microvascular damage. Mononuclear cell adhesion to the endothelium, vascular leak and, perivascular hemorrhage are found only in the brain of wild-type mice. By contrast, the development of parasitemia and anemia is independent of TNF. Resistance to CM in TNF-alpha/beta-deficient mice is associated with reduced interferon-gamma and interleukin-12 expression in the brain, in the absence of increased T helper type 2 cytokines. In conclusion, TNF apparently is required for PbA-induced endothelial ICAM-1 up-regulation and subsequent microvascular pathology resulting in fatal CM. In the absence of TNF, ICAM-1 and nitric oxide up-regulation are reduced, and PbA infection fails to cause fatal CM.     

Interferon-γ is essential for the development of cerebral malaria

Infection with Plasmodium berghei ANKA (PbA) causes fatal cerebral malaria (CM). While a pathogenic role for tumor necrosis factor (TNF) has been established, we asked whether a disruption of interferon-γ (IFN-γ) signaling would modulate CM. We demonstrate here that IFN-γR-deficient mice are completely protected from CM. PbA-induced release of TNF and up-regulation of endothelial intercellular adhesion molecule (ICAM)-1 expression, recruitment of mononuclear cells, and cerebral microvascular damage with vascular leakage occur only in wild-type mice. Protected mice die at a later time of severe anemia and overwhelming parasitemia. Resistance to CM in IFN-γR-deficient mice is associated with reduced serum TNF levels, reduced interleukin-12 expression in the brain and increased T-helper 2 cytokines. In conclusion, IFN-γ is apparently required for PbA-induced endothelial ICAM-1 up-regulation and subsequent microvascular pathology, resulting in fatal CM. In the absence of IFN-γ signaling, ICAM-1 and TNF up-regulation is reduced; hence, PbA infection fails to cause fatal CM.     

Mast cell-mediated immune responses through IgE antibody and Toll-like receptor 4 by malarial peroxiredoxin

In this study, 2-Cys Plasmodium berghei ANKA (PbA) peroxiredoxin (Prx) was identified as an antigenic protein recognized by an anti-PbA IgE antibody using two-dimensional polyacrylamide gel electrophoresis and proteomic analysis. Innate immune responses to PbAPrx were examined using cells from mice deficient in Toll-like receptors (TLR) or related molecules, and it was demonstrated that responses were severely impaired in TLR4(-/-), MyD88(-/-) and MD-2(-/-) mice, but not in Toll/IL-1 receptor domain-containing adaptor inducing IFN-gamma (TRIF)(-/-), TLR2(-/-) or radioprotective 105 (RP105)(-/-) mice. An association between PbAPrx and TLR4 was observed following immunoprecipitation and immunoblotting, suggesting that PbAPrx was associated with TLR4/MD-2. Interactions between Prx and TLR4/MD-2 were also examined by flow cytometry using TLR4/MD-2- or TLR2-expressing cells. NFkappaB/GFP activity was observed in TLR4/MD-2- but not in TLR2-expressing cells following stimulation with Prx. However, this effect was not observed after treatment with proteinase K, suggesting that PbAPrx is a protein ligand for TLR4 and that the PbAPrx activity observed in this study is not due to contamination with LPS. These findings indicate that malarial Prx induces IgE-mediated protection through FcepsilonRI on mast cells and innate immunity through TLR4 with MyD88 and MD-2, suggesting a novel function for malarial Prx in innate and acquired immune responses in malaria.     

Experimental cerebral malaria develops independently of caspase recruitment domain-containing protein 9 signaling

The outcome of infection depends on multiple layers of immune regulation, with innate immunity playing a decisive role in shaping protection or pathogenic sequelae of acquired immunity. The contribution of pattern recognition receptors and adaptor molecules in immunity to malaria remains poorly understood. Here, we interrogate the role of the caspase recruitment domain-containing protein 9 (CARD9) signaling pathway in the development of experimental cerebral malaria (ECM) using the murine Plasmodium berghei ANKA infection model. CARD9 expression was upregulated in the brains of infected wild-type (WT) mice, suggesting a potential role for this pathway in ECM pathogenesis. However, P. berghei ANKA-infected Card9(-/-) mice succumbed to neurological signs and presented with disrupted blood-brain barriers similar to WT mice. Furthermore, consistent with the immunological features associated with ECM in WT mice, Card9(-/-) mice revealed (i) elevated levels of proinflammatory responses, (ii) high frequencies of activated T cells, and (iii) CD8(+) T cell arrest in the cerebral microvasculature. We conclude that ECM develops independently of the CARD9 signaling pathway.     

TLR3 and TLR4 are innate antiviral immune receptors in human microglia: Role of IRF3 in modulating antiviral and inflammatory response in the CNS

In the CNS, microglia are the primary targets of HIV infection. In this study, we investigated the effect of activation of the innate antiviral receptors TLR3 and TLR4 on HIV infection of primary human microglia, as well as microglial cell signaling and gene expression. Ligands for both TLR3 and TLR4 potently inhibited HIV replication in microglia through a pathway requiring IRF3. Surprisingly, a remarkably similar pattern of cell signaling and gene expression was observed in TLR3- and TLR4-activated microglia, suggesting a relatively minor role for MyD88 following TLR4 activation in these cells. HIV did not activate IRF3 but rather decreased IRF3 protein, indicating that HIV does not activate TLR3 or RIG-like helicases in microglia. Taken together, these results indicate that activation of TLR3 or TLR4 will elicit antiviral immunity, in addition to inducing proinflammatory responses. We suggest that a balanced expression between inflammatory and innate immune genes might be achieved by IRF3 over-expression.     

Interleukin-10 modulates susceptibility in experimental cerebral malaria.

In this study, we examined the effects of interleukin-10 (IL-10) on the outcome of experimental cerebral malaria (CM), a lethal neurological syndrome that occurs in susceptible strains of mice after infection with Plasmodium berghei ANKA (PbA). Constitutive IL-10 mRNA levels were significantly higher in the spleen and brain of resistant animals. In vivo neutralization of endogenous IL-10 in CM-resistant mice induced the neurological syndrome in 35.7% of these mice, as opposed to 7.7% in controls. IL-10 inhibited PbA antigen-specific interferon-gamma (IFN-gamma) production in vitro but not tumour necrosis factor (TNF) serum levels in vivo. Susceptible mice, on the other hand, were significantly protected against CM when injected with recombinant IL-10. Overall, our findings suggest that IL-10 plays a protective role against experimental cerebral malaria.     

T cell response in malaria pathogenesis: selective increase in T cells carrying the TCR V(beta)8 during experimental cerebral malaria.

To characterize the T cells involved in the pathogenesis of cerebral malaria (CM) induced by infection with Plasmodium berghei ANKA clone 1.49L (PbA 1.49L), the occurrence of the disease was assessed in mice lacking T cells of either the alphabeta or gammadelta lineage (TCRalphabeta(-/-) or TCRgammadelta(-/-)). TCRgammadelta(-/-) mice were susceptible to CM, whereas all TCRalphabeta(-/-) mice were resistant, suggesting that T cells of the alphabeta lineage are important in the genesis of CM. The repertoire of TCR V(beta) segment gene expression was examined by flow cytometry in B10.D2 mice, a strain highly susceptible to CM induced by infection with PbA 1.49L. In these mice, CM was associated with an increase of T cells bearing the V(beta)8.1, 2 segments in the peripheral blood lymphocytes. Most V(beta)8.1, 2(+) T cells from peripheral blood lymphocytes of the mice that developed CM belonged to the CD8 subset, and exhibited the CD69(+), CD44(high) and CD62L(low) phenotype surface markers. The link between the increase in V(beta)8.1, 2(+) T cells and the neuropathological consequences of PbA infection was strengthened by the observation that the occurrence of CM was significantly reduced in mice treated with KJ16 antibodies against the V(beta)8.1 and V(beta)8.2 chains, and in mice rendered deficient in V(beta)8.1(+) T cells by a mouse mammary tumor virus superantigen.     

Nucleosome-induced neutrophil activation occurs independently of TLR9 and endosomal acidification: implications for systemic lupus erythematosus

The nucleosome is a major autoantigen known to activate PMN in systemic lupus erythematosus (SLE). TLR9 recognizes bacterial and even mammalian DNA under certain circumstances. Nevertheless, the role of TLR9 in SLE development is still unclear. Since nucleosomes are composed of DNA, we investigated whether TLR9 is required for nucleosome-induced PMN activation. Isolated neutrophils were cultured with nucleosomes, plasma from lupus patients and other stimuli in the presence/absence of various inhibitors. Cells were analyzed by flow cytometry, ELISA and confocal microscopy. We found that nucleosomes circulating in lupus plasma induce the secretion of pro-inflammatory cytokines by PMN. Nucleosomes activate human PMN independently of unmethylated CpG sequences in nucleosomal DNA, leading to IL-8/IL-6/TNF secretion and CD11b up-regulation. Nucleosomes accumulate in the cytoplasm of PMN upon endocytosis, induce TLR9 up-regulation and act synergistically with TLR9 ligands. Nucleosome-induced activation was not inhibited by polymyxin B (PB), chloroquine (CQ), ammonium chloride (AC) or a TLR9 antagonist. Moreover, both PMN isolated from WT and TLR9-KO mice were activated by nucleosomes, as detected by MIP-2 secretion and CD11b up-regulation. Activation occurred therefore independently of endotoxins, endosomal acidification, TLR9 and CpG motifs. TLR9 may thus be differently required in the triggering of nucleosome-induced innate immunity and anti-nucleosome B-cell autoimmunity.     

Characterization of cerebral malaria in the outbred Swiss Webster mouse infected by Plasmodium berghei ANKA

Plasmodium berghei ANKA (PbA) infection in susceptible inbred mouse strains is the most commonly used experimental model to study pathogenesis of cerebral malaria (CM). Indeed, many concepts on mechanisms related to this complication have arisen from works using this model. Although inbred strains present several advantages and are indicated for most studies, the use of outbred models can show unique usefulness in a number of approaches such as fine post-quantitative trait loci mapping and discovery of genes relevant to CM susceptibility or resistance, as well as pharmacological and vaccine studies. Here we describe the features of PbA infection and CM incidence, and characterize the associated multiorgan pathology in the outbred Swiss Webster mouse. This model showed a sizeable (62.7%) and reproducible incidence of CM demonstrated by clinical signs and histopathological changes in brain (microhaemorrhages, oedema and vessel plugging by mononuclear cells). Major pathological changes were also observed in lungs, liver, thymus and spleen, analogous to those observed in inbred strains. Parasitaemia levels were associated with the risk of CM development, the risk being significantly higher in mice showing higher values of parasitaemia on days 6–7 of infection. This outbred CM model is then suitable for genetic, vaccine and drug studies targeting this malaria complication.     

Alterations of splenic architecture in malaria are induced independently of Toll-like receptors 2, 4, and 9 or MyD88 and may affect antibody affinity

Splenic microarchitecture is substantially altered during acute malaria infections, which may affect the development and regulation of immune responses. Here we investigated whether engagement of host Toll-like receptor 2 (TLR2), TLR4, TLR9, and the adaptor protein MyD88 is required for induction of the changes and whether antibody responses are modified when immunization takes place during the period of splenic disruption. The alterations in splenic microarchitecture were maximal shortly after the peak of parasitemia and were not dependent on engagement of TLR2, TLR4, or TLR9, and they were only minimally affected by the absence of the MyD88 adaptor molecule. Although germinal centers were formed in infected mice, they did not contain the usual light and dark zones. Immunization of mice with chicken gamma globulin 2 weeks prior to acute Plasmodium chabaudi infection did not affect the quantity or avidity of the immunoglobulin G antibody response to this antigen. However, immunization at the same time as the primary P. chabaudi infection resulted in a clear transient reduction in antibody avidity in the month following immunization. These data suggest that the alterations in splenic structure, particularly the germinal centers, may affect the quality of an antibody response during a malaria infection and could impact the development of immunity to malaria or to other infections or immunizations given during a malaria infection.     

Roles of the small intestine for induction of toll-like receptor 4-mediated innate resistance in naturally acquired murine toxoplasmosis

Peroral infection of Toxoplasma gondii is thought to reflect the typical infection route of naturally acquired toxoplasmosis in humans. We have investigated possible differential roles of toll-like receptor 2 (TLR2) and TLR4 in host defense against naturally acquired murine toxoplasmosis. After peroral inoculation of T. gondii ME49 cysts, TLR4-deficient C3H/HeJ mice were more susceptible to infection than wild-type (WT) C3H/HeN mice, as shown by increased cyst number and low production of cytokines, which are the key factors in protective immunity. When mice were inoculated by intra-peritoneal inoculation of T. gondii, there were no significant differences in the number of brain cysts and cytokine productions between C3H/HeJ and C3H/HeN mice. Histopathologic examination revealed severe inflammation in the small intestine of C3H/HeJ (TLR4-deficient) mice, while an increased number of TLR4-positive mononuclear cells was found in C3H/HeN (WT) mice. To confirm these phenomena, TLR2(-/-) or TLR4(-/-) mice were infected perorally with T. gondii cysts. TLR4(-/-) mice were more susceptible to infection compared with TLR2(-/-) and C57BL/6 mice. Nuclear factor-kappa B activation through TLR4 agonistic activity of T. gondii ME49 was demonstrated by luciferase assay using stably expressing mouse (m) TLR2 or mTLR4/mMD-2 transfectants. We demonstrate here for the first time that innate immune recognition by TLR4 is involved in protective mechanisms against peroral infection with T. gondii ME49. These results suggest that the small intestine plays an important role in the induction of innate immunity in naturally acquired toxoplasmosis.     

Is Ischemia Involved in the Pathogenesis of Murine Cerebral Malaria?

Sequestration of parasitized erythrocytes in the central nervous system microcirculation and increased cerebrospinal fluid lactate are prominent features of cerebral malaria (CM), suggesting that sequestration causes mechanical obstruction and ischemia. To examine the potential role of ischemia in the pathogenesis of CM, Plasmodium berghei ANKA (PbA) infection in CBA mice was compared to infection with P. berghei K173 (PbK) which does not cause CM (the non-CM model, NCM). Cerebral metabolite pools were measured by (1)H nuclear magnetic resonance spectroscopy during PbA and PbK infections. Lactate and alanine concentrations increased significantly at the terminal stage of CM, but not in NCM mice at any stage. These changes did not correlate with parasitemia. Brain NAD/NADH ratio was unchanged in CM and NCM mice at any time studied, but the total NAD pool size decreased significantly in the CM mice on day 7 after inoculation. Brain levels of glutamine and several essential amino acids were increased significantly in CM mice. There was a significant linear correlation between the time elapsed after infection and small, progressive decreases in the cell density/cell viability markers glycerophosphocholine and N-acetylaspartate in CM, indicative of gradual loss of cell viability. The metabolite changes followed a different pattern, with a sudden significant alteration in the levels of lactate, alanine, and glutamine at the time of terminal CM. In NCM, there were significant decreases with time of glutamate, the osmolyte myo-inositol, and glycerophosphocholine. These results are consistent with an ischemic change in the metabolic pattern of the brain in CM mice, whereas in NCM mice the changes were more consistent with hypoxia without vascular obstruction. Mild obstructive ischemia is a likely cause of the metabolic changes during CM, but a role for immune cell effector molecules cannot be ruled out.     

A novel negative regulator for IL-1 receptor and Toll-like receptor 4

The Toll-IL-1 receptor (TIR) superfamily, defined by the presence of an intracellular TIR domain, initiates innate immunity via NF-kappaB activation, leading to production of proinflammatory cytokines. ST2 is a member of the TIR family that does not activate NF-kappaB and has been suggested as an important effector molecule of type 2 T helper cell responses. We have recently demonstrated that the membrane bound form of ST2 (ST2L) negatively regulated IL-1RI and TLR4 but not TLR3 signaling by sequestrating the adaptors MyD88 and Mal. In contrast to wild-type mice, ST2 deficient mice failed to develop endotoxin tolerance. Thus, ST2 suppresses IL-1R and TLR4 signaling via MyD88- and Mal-dependent pathways and modulates innate immunity. The results provide a molecular explanation for the role of ST2 in T(H)2 responses since inhibition of TLRs will promote a T(H)2 response and also identify ST2 as a key regulator of endotoxin tolerance.     

Toll-like receptor 9 regulates the lung macrophage phenotype and host immunity in murine pneumonia caused by Legionella pneumophila

Experiments were performed to determine the contribution of TLR9 to the generation of protective immunity against the intracellular respiratory bacterial pathogen Legionella pneumophila. In initial studies, we found that the intratracheal (i.t.) administration of L. pneumophila to mice deficient in TLR9 (TLR9(-/-)) resulted in significantly increased mortality, which was associated with an approximately 10-fold increase in the number of lung CFU compared to that of wild-type BALB/c mice. Intrapulmonary bacterial challenge in TLR9(-/-) mice resulted in the reduced accumulation of myeloid dendritic cells (DC) and activated CD4(+) T cells. Lung macrophages isolated from Legionella-infected TLR9(-/-) mice displayed the impaired internalization of bacteria and evidence of alternative rather than classical activation, as manifested by the markedly reduced expression of nitric oxide and type 1 cytokines, whereas the expression of Fizz-1 and arginase-1 was enhanced. The adoptive transfer of bone marrow-derived DC from syngeneic wild-type, but not TLR9(-/-), mice administered i.t. reconstituted anti-legionella immunity and restored the macrophage phenotype in TLR9(-/-) mice. Finally, the i.t., but not intraperitoneal, administration of the TLR9 agonist molecule CpG oligodeoxynucleotide stimulated protective immunity in Legionella-infected mice. In total, our findings indicate that TLR9 is required for effective innate immune responses against the intracellular bacterial pathogen L. pneumophila, and approaches to maximize TLR9-mediated responses may serve as a means to augment antibacterial immunity in pneumonia.     

Induction of adaptive immunity by flagellin does not require robust activation of innate immunity

The ability of TLR agonists to promote adaptive immune responses is attributed to their ability to robustly activate innate immunity. However, it has been observed that, for adjuvants in actual use in research and vaccination, TLR signaling is dispensable for generating humoral immunity. Here, we examined the role of TLR5 and MyD88 in promoting innate and humoral immunity to flagellin using a prime/boost immunization regimen. We observed that eliminating TLR5 greatly reduced flagellin‐induced cytokine production, except for IL‐18, and ablated DC maturation but did not significantly impact flagellin's ability to promote humoral immunity. Elimination of MyD88, which will ablate signaling through TLR and IL‐1β/IL‐18 generated by Nod‐like receptors, reduced, but did not eliminate flagellin's promotion of humoral immunity. In contrast, loss of the innate immune receptor for profilin‐like protein (PLP), TLR11, greatly reduced the ability of PLP to elicit humoral immunity. Together, these results indicate that, firstly, the degree of innate immune activation induced by TLR agonists may be in great excess of that needed to promote humoral immunity and, secondly, there is considerable redundancy in mechanisms that promote the humoral immune response upon innate immune recognition of flagellin. Thus, it should be possible to design innate immune activators that are highly effective vaccine adjuvants yet avoid the adverse events associated with systemic TLR activation.