α2-3 Sialic acid glycoconjugate loss and its effect on infection with Toxoplasma parasites

Recognition of sialylated glycoconjugates is important for host cell invasion by Apicomplexan parasites. Toxoplasma gondii parasites penetrate host cells via interactions between their microneme proteins and sialylated glycoconjugates on the surface of host cells. However, the role played by sialic acids during infection with T. gondii is not well understood. Here, we focused on the role of α2-3 sialic acid linkages as they appear to be widely expressed in vertebrates. Removal of α2-3 sialic acid linkages on macrophages by neuraminidase treatment did not influence the rate of infection or growth of T. gondii, nor did it affect phagocytosis in vitro. Sialyltransferase ST3Gal-I deficient mice (ST3Gal-I^−/− mice) lost α2-3 sialic acid linkages in macrophages and spleen cells. The numbers of T. gondii-infected CD11b⁺ cells in peritoneal cavities of the infected ST3Gal-I^−/− mice were relatively lower than those of the infected wild type animals. In addition, CD8⁺ T cell populations and numbers in the spleens and peritoneal cavities of the ST3Gal-I^−/− mice were significantly lower than those in the wild type animals before and after the T. gondii infection. ST3Gal-I^−/− mice had severe liver damage and reduced survival rates following peritoneal infection with T. gondii. Furthermore, adoptive transfer of immune CD8⁺ cells from wild type mice to ST3Gal-I^−/− mice increased their survival during infection with T. gondii. Our data show that parasite invasion via α2-3 sialic acid linkages might not contribute on host survival and indicate the impact that loss of α2-3 sialic acid linkages has on CD8⁺ T cell populations, which are necessary for effective immune responses against infection with T. gondii.     

Natural killer cell intrinsic toll-like receptor MyD88 signaling contributes to IL-12-dependent IFN-γ production by mice during infection with Toxoplasma gondii

Myeloid differentiation factor 88 (MyD88)-dependent IL-12 secretion by dendritic cells is critical for natural killer cell-mediated IFN-γ production and innate resistance to Toxoplasma gondii. Although MyD88^−/− mice challenged with T. gondii have defective IL-12 responses and succumb to infection, administration of IL-12 to MyD88^−/− mice fails to prevent acute mortality, suggesting that MyD88 may mediate signals within natural killer cells important for IL-12-dependent IFN-γ production and innate resistance to this parasite. In this study, we found that T. gondii antigens and IL-12 could synergistically trigger IFN-γ secretion by natural killer cells, which was dependent on toll-like receptor-MyD88 signaling. Further analysis showed that p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, c-Jun N-terminal kinase and NF-κB multiple pathways downstream of MyD88 contributed to IFN-γ production by natural killer cells. Moreover, the well-established toll-like receptor agonists, T. gondii profilin (Tgprofilin) and T. gondii heat shock protein 70 (TgHSP70) could evoke a similar IFN-γ secretory response in natural killer cells to that evoked by T. gondii antigens. In vivo adoptive transfer experiments showed that, upon challenge with T. gondii, NOD/SCID-β2 microglobulin null (NOD/SCID-β2m^−/−) mice injected i.v. with MyD88^−/− natural killer cells had reduced serum IFN-γ levels and increased splenic tachyzoite burdens compared with those injected i.v. with wild-type natural killer cells. Taken together, these findings demonstrate a critical role for natural killer cell intrinsic toll-like receptor-MyD88 signaling in IL-12-dependent early IFN-γ production and innate resistance to T. gondii.     

Toxoplasma gondii: Impaired maturation and pro-inflammatory response of dendritic cells in MIF-deficient mice favors susceptibility to infection

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of MIF-dependent responses to Toxoplasma gondii, we investigated host resistance in MIF−/− mice (BALB/c background) during natural oral infection. We focused on the potential involvement of MIF in Dendritic Cell (DC) maturation and IL-12 production. Following oral T. gondii infection, wild type mice developed a strong IL-12 response with an adequate maturation of their draining mesenteric lymph node DC (MLNDC) population and were resistant to challenge with either 40 or 100 cysts (ME49 strain). In contrast, similarly infected MIF−/− mice mounted a weak IL-12 response, displayed immature MLNDCs in the early phases of infection and rapidly succumbed to both type of challenges. Lack of maturation and IL-12 production of DCs in response to T. gondii antigens was confirmed by in vitro studies, and these effects were reversed following treatment with recombinant MIF. These findings demonstrate that MIF-induced early DC maturation and IL-12 production mediate resistance to T. gondii infection.     

MAP Kinase Phosphatase-2 Plays a Key Role in the Control of Infection with Toxoplasma gondii by Modulating iNOS and Arginase-1 Activities in Mice

The dual specific phosphatase, MAP kinase phosphatase-2 (MKP-2) has recently been demonstrated to negatively regulate macrophage arginase-1 expression, while at the same time to positively regulate iNOS expression. Consequently, MKP-2 is likely to play a significant role in the host interplay with intracellular pathogens. Here we demonstrate that MKP-2^−/− mice on the C57BL/6 background have enhanced susceptibility compared with wild-type counterparts following infection with type-2 strains of Toxoplasma gondii as measured by increased parasite multiplication during acute infection, increased mortality from day 12 post-infection onwards and increased parasite burdens in the brain, day 30 post-infection. MKP-2^−/− mice did not, however, demonstrate defective type-1 responses compared with MKP-2^+/+ mice following infection although they did display significantly reduced serum nitrite levels and enhanced tissue arginase-1 expression. Early resistance to T. gondii in MKP-2^+/+, but not MKP-2^−/−, mice was nitric oxide (NO) dependent as infected MKP-2^+/+, but not MKP-2^−/− mice succumbed within 10 days post-infection with increased parasite burdens following treatment with the iNOS inhibitor L-NAME. Conversely, treatment of infected MKP-2^−/− but not MKP-2^+/+ mice with nor-NOHA increased parasite burdens indicating a protective role for arginase-1 in MKP-2^−/− mice. In vitro studies using tachyzoite-infected bone marrow derived macrophages and selective inhibition of arginase-1 and iNOS activities confirmed that both iNOS and arginase-1 contributed to inhibiting parasite replication. However, the effects of arginase-1 were transient and ultimately the role of iNOS was paramount in facilitating long-term inhibition of parasite multiplication within macrophages.     

T cell-derived interferon-γ is required for host defense to Toxoplasma gondii

Interferon-γ (IFN-γ) is important for host defense against various intracellular organisms including a protozoan pathogen Toxoplasma gondii. Various immune cells are recently shown to produce IFN-γ in T. gondii infection, however, it remains elusive which cell types are important for anti-T. gondii host defense so far. Here we generate a new IFN-γ reporter "GREVEN" mouse line in which a fusion protein of Venus and NanoLuc to analyze IFN-γ producing cells during T. gondii infection and find that CD4⁺, CD8⁺, γδ T cells and natural killer cells express Venus in a time dependent manner. Furthermore, Lck-Cre/Ifng^fl/fl mice are highly susceptible to T. gondii infection. Taken together, our results demonstrate that T cell-derived IFN-γ plays an important role in anti-T. gondii host defense.     

Toxoplasma gondii-induced foetal resorption in mice involves interferon-gamma-induced apoptosis and spiral artery dilation at the maternofoetal interface

The severity of congenital toxoplasmosis depends on the stage of the pregnancy at which infection takes place. Infection during the first trimester generally leads to miscarriage, through an unknown mechanism. Toxoplasma gondii infection is normally controlled by a strong Th1-type response with IFN-γ production. To investigate the mechanisms of foetal resorption induced by T. gondii, pregnant Swiss-Webster mice were infected 1 day post coïtum with the avirulent Me49 strain. Mated recipients were examined at mid-gestation. Few parasites and no cytolytic effects were detected 10 days post coïtum in implantation sites undergoing resorption. Resorption was accompanied by haemorrhage, spiral artery dilation, hypocellularity of the decidua basalis, apoptosis of placental cells, a decline in uterine mature natural killer cell numbers, increased indoleamine 2,3-dioxygenase mRNA levels and reduced IL-15 mRNA levels. Given the role of IFN-γR^−/− in non-infectious abortive processes, IFN-γR^−/− mice were used to investigate its local role in T. gondii-induced foetal resorption. IFN-γR^−/− mice showed 50% less foetal resorption than their wild-type counterparts, and spiral artery dilation and placental cell apoptosis were both abolished. These results strongly suggest that, at least in mice, T. gondii-induced abortion in early gestation is not due to a direct action of the parasite at the maternofoetal interface but rather to massive IFN-γ release.     

IL-7 and IL-15 do not synergize during CD8 T cell recall response against an obligate intracellular parasite

Long-term protection against Toxoplasma gondii is dependent on robust CD8⁺ T cell immunity. In the absence of this response, the host is unable to maintain chronicity, which results in recrudescence of infection and possible death. Factors needed for the persistence of protective CD8⁺ T cells against the parasite need to be evaluated. Previous studies from our laboratory have reported that synergism between γ chain cytokines like IL-7 and IL-15 is critical for the generation of CD8⁺ T cell response needed for protection during acute infection. In this study we report that the situation is different during the recall response where CD8⁺ T cell response is almost entirely dependent on IL-15, with IL-7 at best playing a minor role. In the absence of IL-15, CD8⁺ T cells fail to respond optimally to parasitic re-challenge and hosts are unable to control their replication, which leads to their death. Thus T. gondii infection may represent a unique situation where CD8⁺ T cell response during secondary challenge is primarily dependent on IL-15 with other γ chain cytokines having nominal effect. These findings provide important information regarding factors involved in the generation of protective immunity against T. gondii with strong implications in developing immunotherapeutic agents against the pathogen.     

Cryptosporidium parvum: the contribution of Th1-inducing pathways to the resolution of infection in mice

The contribution of cytokines IL-12, IL-18, IL-23, and IFN-γ, and Stat1 signaling molecules involved in Th1 responses associated with host resistance to Cryptosporidium parvum infection was investigated in adult IL-12p40^−/−mice. Host resistance to C. parvum infection was assessed in different mouse strains lacking IL-12, IL-18, and IL-23 genes. We found that as in IL-12p40^−/− mice (which lack both IL-12 and IL-23), IL-12p35^−/− mice (which lack IL-12) and IL-18 deficient mice were also susceptible to infection with C. parvum. Varied levels of resistance were observed when mice were treated with cytokines like IL-18, IL-23 and IFN-γ. Mice treated with IL-12, as expected, were completely resistant to infection until day 5 post infection, and had significantly decreased (85%) parasite loads at peak infection (day 7), whereas rIL-23 had a lesser effect, decreasing parasite load by approximately 45%. Interestingly, IL-18 appears to play a significant role in initial immune response, even in the absence of IL-12, since treatment with IL-18 in IL-12p40^−/− knockout mice decreased parasite load by approximately 70%. In addition, the establishment of C. parvum infection in mice lacking the Stat1 gene demonstrated the involvement of this pathway in resolution of infection. These observations indicate a strong requirement for Th1 response in the development of immunity to C. parvum in the adult IL-12p40^−/− mice, information that will be essential to further investigate the immune responses during infections and in the development of potential vaccine candidates.     

Mannose-Binding Lectin Regulates Host Resistance and Pathology during Experimental Infection with Trypanosoma cruzi

Mannose-binding lectin (MBL) is a humoral pattern-recognition molecule important for host defense. Although recent genetic studies suggest an involvement of MBL/MASP2-associated pathways in Chagas’ disease, it is currently unknown whether MBL plays a role in host resistance to the intracellular protozoan Trypanosoma cruzi, the causative agent of Chagas’ disease. In this study we employed MBL^−/− mice to assess the role of MBL in resistance to experimental infection with T. cruzi. T. cruzi infection enhanced tissue expression of MBL both at the mRNA and protein level. Similarly, symptomatic acute Chagas’ disease patients displayed increased serum concentrations of MBL compared to patients with indeterminate, asymptomatic forms of the disease. Furthermore, increased parasite loads in the blood and/or tissue were observed in MBL^−/− mice compared to WT controls. This was associated with reduced systemic levels of IL-12/23p40 in MBL^−/− mice. Importantly, MBL^−/− mice infected with a cardiotropic strain of T. cruzi displayed increased myocarditis and cardiac fibrosis compared to WT controls. The latter was accompanied by elevated hydroxyproline content and mRNA levels of collagen-1 and -6 in the heart. These observations point to a previously unappreciated role for MBL in regulating host resistance and cardiac inflammation during infection with a major human pathogen.     

Immunomodulation of dual specificity phosphatase 4 during visceral leishmaniasis

DUSP4, an inducible protein has a substrate specificity toward ERK1/2, a component of MAP kinase which is enhanced during Leishmania infection. The DUSP4^?/? mice show increased susceptibility towards the infection caused by Toxoplasma gondii and Leishmania mexicana. These observations emphatically established the fact that unlike DUSP1, DUSP4 has host protective role. In our study, it has been Leishmania donovani, the causative agent of visceral leishmaniasis (VL) significantly reduced the expression of DUSP4 during infection. In order to find out the host protective role of DUSP4 in macrophages during VL, we silenced DUSP4 prior to infection and the parasite number within macrophage was counted. Under DUSP4 knock-down condition, phosphorylation of p38 MAPK and generation of pro-inflammatory response like IL-12, TNF-α, and iNOS was decreased significantly. Silencing DUSP4 promoted the phosphorylation of ERK1/2 and the generation of anti-inflammatory response like- IL-10, TGF-β with increased Arginase-1 and Cox-2 activity. Glycyrrhizic Acid (GA), an immunomodulator, already known to suppress L. donovani infection, found to up-regulate DUSP4 expression during L. donovani infection. On the other hand, GA failed to increase Th1 cytokine production and decrease Th2 response during DUSP4 knock-down condition suggesting the key role of DUSP4 while providing protection during L. donovani infection.     

Toxoplasma gondii: The role of IFN-gamma, TNFRp55 and iNOS in inflammatory changes during infection

In order to examine the role of IFN-γ, TNFRp55 and iNOS in inflammatory reaction during toxoplasmosis, IFN-γ^−/−, TNFRp55^−/− and iNOS^−/− mice were experimentally infected with Toxoplasma gondii ME-49 strain. The organs of the mice were evaluated for histology and immunohistochemistry in detection of tissue parasitism and iNOS positive cells. IFN-γ^−/− mice presented mild inflammation in peripheral organs associated with a high parasitism and mortality in the acute phase of infection. In contrast, the peripheral organs of WT, TNFRp55^−/− and iNOS^−/− mice, presented a significant inflammatory reaction and low tissue parasitism in the same period of infection. The inflammatory lesions and tissue parasitism were increased and more severe in the Central Nervous System (CNS) of TNFRp55^−/− and iNOS^−/− with a progression of infection, when compared to WT mice. In these knockout animals, the inflammatory changes were associated with low levels or no expression of iNOS in TNFRp55^−/− and iNOS^−/− mice, respectively.     

The complexity of signaling in host-pathogen interactions revealed by the Toxoplasma gondii-dependent modulation of JNK phosphorylation

The inhibition of apoptosis by Toxoplasma gondii is governed by its modulation of several signaling cascades including the NFκappaB and JNK pathways. This is evident in the dysregulation of JNK activation following treatment with UV and TNFα, both apoptogenic stimuli. Infection-mediated interference with the JNK cascade was found to be highly reproducible in HeLa cells. In light of emerging evidence regarding cross talk between the JNK and NFκB cascades, we examined the impact of infection in wild type and RelA/p65−/− mouse embryonic fibroblasts (MEF). Remarkably, parasite infection failed to significantly impact both UV and TNFα-mediated JNK phosphorylation in both cell lines suggesting a cell type specific effect. Furthermore siRNA-mediated knockdown of RelA/p65 failed to impact the parasite mediated effects on stimulus dependent activation of JNK in HeLa cells. Finally, the infection mediated suppression of JNK phosphorylation in HeLa cells did not result in decreased JNK kinase activity. Rather, the reduced levels of phospho-JNK in infected cells correlated with increased phosphatase activity noted by the partial rescue of the phenotype following treatment with okadaic acid. Taken together the results indicate that manipulation of the JNK pathway does not involve NFκB and is furthermore not a central component of the parasite enforced block of apoptosis. It further highlights the complexity of these systems and the danger of extrapolating results both within and across pathogen-host cell systems based on limited studies.     

Toxoplasma,testosterone, and behavior manipulation: the role of parasite strain,host variations,and intensity of infection

Toxoplasma gondii is an intracellular parasite involved in the etiology of various behavioral and hormonal alterations in humans and rodents. Various mechanisms, including induction changes of testosterone production, have been proposed in the etiology of behavioral alterations during T. gondii infection. However, controversy remains about the effects of T. gondii infection on testosterone production; in some studies, increased levels of testosterone were reported, whereas other studies reported decreased levels. This is a significant point, because testosterone has been shown to play important roles in various processes, from reproduction to fear and behavior. This contradiction seems to indicate that different factors--primarily parasite strains and host variations--have diverse effects on the intensity of T. gondii infection, which consequently has diverse effects on testosterone production and behavioral alterations. This paper reviews the role of parasite strains, host variations, and intensity of T. gondii infection on behavioral alterations and testosterone production, as well as the role of testosterone in the etiology of these alterations during toxoplasmosis.     

Chlamydia pneumoniae harness host NLRP3 inflammasome-mediated caspase-1 activation for optimal intracellular growth in murine macrophages

Chlamydia pneumoniae is an obligate intracellular pathogen that replicates within a vacuole and acquires host cell nutrients. We show that C. pneumoniae utilizes host innate immune signaling NLRP3/ASC/caspase-1 inflammasome for intracellular growth. Bone marrow-derived macrophages (BMMs) secreted mature interleukin-1β upon infection with C. pneumoniae depending on the NLRP3 inflammasome activation. Intracellular growth of C. pneumoniae was severely impaired in BMMs from Nlrp3^−/−, Asc^−/−, and Casp1^−/− mice but not wild type or Nlrc4^−/− mice. Furthermore defective NLRP3 inflammasome components led to accumulation of lipid droplets inside the infected BMMs, suggesting that uptake and/or utilization of lipids is disturbed in the absence of NLRP3 inflammasome activation. These results suggest C. pneumoniae has evolved to harness both host innate immune response and NLRP3 inflammasome activation, for the acquisition of essential nutrients necessary for intracellular growth. This unique property of C. pneumoniae may shed a new light on how C. pneumoniae increase the risk of atherosclerosis and metabolic syndrome.     

Induction of IL-10-producing regulatory B cells following Toxoplasma gondii infection is important to the cyst formation

Toxoplasma gondii is a protozoan parasite that infects humans and animals via congenital or postnatal routes. During parasite infection, IL-10-producing Bregs are stimulated as part of the parasite-induced host immune responses that favor infection. In this study, we investigated whether T. gondii infection induces immune regulatory cells including IL-10-producing CD1d^highCD5⁺ regulatory B cells (Bregs) and whether Breg induction is critical for the development of chronic infection of T. gondii. Furthermore, B cell-deficient (μMT) mice revealed that the IL-10-producing B cells might be associated with the development of chronic T. gondii infection. To better understand the mechanism underlying the accumulation of IL-10-producing B cells upon T. gondii infection, we determined the effect of products released by T. gondii on the induction and differentiation of IL-10-producing B cells during the acute stage of infection using transgenic green fluorescent protein (GFP)-expressing T. gondii strain. We demonstrated that products secreted at the stage of cell lysis by fully replicated tachyzoites induced the differentiation of naive B cells to IL-10-producing Bregs. Our results indicated that the downregulation of the immune response via Bregs during T. gondii infection is related to cyst formation in the host brain and to the establishment of chronic infection.     

The P-glycoprotein Inhibitor GF120918 Modulates Ca2+-Dependent Processes and Lipid Metabolism in Toxoplasma Gondii

Up-regulation of the membrane-bound efflux pump P-glycoprotein (P-gp) is associated with the phenomenon of multidrug-resistance in pathogenic organisms, including protozoan parasites. In addition, P-gp plays a role in normal physiological processes, however our understanding of these P-gp functions remains limited. In this study we investigated the effects of the P-gp inhibitor GF120918 in Toxoplasma gondii, a model apicomplexan parasite and an important human pathogen. We found that GF120918 treatment severely inhibited parasite invasion and replication. Further analyses of the molecular mechanisms involved revealed that the P-gp inhibitor modulated parasite motility, microneme secretion and egress from the host cell, all cellular processes known to depend on Ca²⁺ signaling in the parasite. In support of a potential role of P-gp in Ca²⁺-mediated processes, immunoelectron and fluorescence microscopy showed that T. gondii P-gp was localized in acidocalcisomes, the major Ca²⁺ storage in the parasite, at the plasma membrane, and in the intravacuolar tubular network. In addition, metabolic labeling of extracellular parasites revealed that inhibition or down-regulation of T. gondii P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of T. gondii P-gp in essential processes of the parasite biology and further validate the potential of P-gp activity as a target for drug development.