T Helper Plasticity Is Orchestrated by STAT3, Bcl6, and Blimp-1 Balancing Pathology and Protection in Malaria.

Hybrid Th1/Tfh cells (IFN-γ+IL-21+CXCR5+) predominate in response to several persistent infections. In Plasmodium chabaudi infection, IFN-γ+ T cells control parasitemia, whereas antibody and IL-21+Bcl6+ T cells effect final clearance, suggesting an evolutionary driver for the hybrid population. We found that CD4-intrinsic Bcl6, Blimp-1, and STAT3 coordinately regulate expression of the Th1 master regulator T-bet, supporting plasticity of CD4 T cells. Bcl6 and Blimp-1 regulate CXCR5 levels, and T-bet, IL-27Rα, and STAT3 modulate cytokines in hybrid Th1/Tfh cells. Infected mice with STAT3 knockout (KO) T cells produced less antibody and more Th1-like IFN-γ+IL-21-CXCR5lo effector and memory cells and were protected from re-infection. Conversely, T-bet KO mice had reduced Th1-bias upon re-infection and prolonged secondary parasitemia. Therefore, each feature of the CD4 T cell population phenotype is uniquely regulated in this persistent infection, and the cytokine profile of memory T cells can be modified to enhance the effectiveness of the secondary response.

Dissemination of non-typhoidal Salmonella during Plasmodium chabaudi infection affects anti-malarial immunity.

Malaria-associated bacteremia accounts for up to one-third of deaths from severe malaria, and non-typhoidal Salmonella (NTS) has been reported as a major complication of severe malarial infection. Patients who develop NTS bacteremia during Plasmodium infection show higher mortality rates than individuals with malaria alone. Systemic bacteremia can be caused by a wound or translocation from epithelial or endothelial sites. NTS is an intestinal pathogen, however the contribution of bacterial translocation from the intestinal tract during Plasmodium infection is not well studied. Here, we investigated the integrity of the intestinal barrier function of P. chabaudi-infected mice using large molecules and Salmonella infection. Intestinal histology and the adaptive immune response to malaria were also studied using light microscopy and flow cytometry. P. chabaudi infection compromised intestinal barrier function, which led to increased intestinal cellular infiltration. In addition, we observed increased serum lipopolysaccharide binding protein and leakage of soluble molecules from the intestine into the blood in infected mice. Plasmodium infection also increased intestinal translocation and dissemination of NTS to the liver. The adaptive immune response to P. chabaudi infection was also significantly impacted by NTS translocation. Reduced B and T cell activation were observed in co-infected animals, suggesting interference in the malaria-specific immune responses by bacteremia. These studies demonstrate that P. chabaudi infection induces failure of the barrier function of the intestinal wall and enhanced intestinal bacterial translocation, affecting anti-malarial immunity.

Elimination of intravascular thrombi prevents early mortality and reduces gliosis in hyper-inflammatory experimental cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is the most lethal outcome of Plasmodium infection. There are clear correlations between expression of inflammatory cytokines, severe coagulopathies, and mortality in human CM. However, the mechanisms intertwining the coagulation and inflammation pathways, and their roles in CM, are only beginning to be understood. In mice with T cells deficient in the regulatory cytokine IL-10 (IL-10 KO), infection with Plasmodium chabaudi leads to a hyper-inflammatory response and lethal outcome that can be prevented by anti-TNF treatment. However, inflammatory T cells are adherent within the vasculature and not present in the brain parenchyma, suggesting a novel form of cerebral inflammation. We have previously documented behavioral dysfunction and microglial activation in infected IL-10 KO animals suggestive of neurological involvement driven by inflammation. In order to understand the relationship of intravascular inflammation to parenchymal dysfunction, we studied the congestion of vessels with leukocytes and fibrin(ogen) and the relationship of glial cell activation to congested vessels in the brains of P. chabaudi-infected IL-10 KO mice. METHODS: Using immunofluorescence microscopy, we describe severe thrombotic congestion in these animals. We stained for immune cell surface markers (CD45, CD11b, CD4), fibrin(ogen), microglia (Iba-1), and astrocytes (GFAP) in the brain at the peak of behavioral symptoms. Finally, we investigated the roles of inflammatory cytokine tumor necrosis factor (TNF) and coagulation on the pathology observed using neutralizing antibodies and low-molecular weight heparin to inhibit both inflammation and coagulation, respectively. RESULTS: Many blood vessels in the brain were congested with thrombi containing adherent leukocytes, including CD4 T cells and monocytes. Despite containment of the pathogen and leukocytes within the vasculature, activated microglia and astrocytes were prevalent in the parenchyma, particularly clustered near vessels with thrombi. Neutralization of TNF, or the coagulation cascade, significantly reduced both thrombus formation and gliosis in P. chabaudi-infected IL-10 KO mice. CONCLUSIONS: These findings support the contribution of cytokines, coagulation, and leukocytes within the brain vasculature to neuropathology in malaria infection. Strikingly, localization of inflammatory leukocytes within intravascular clots suggests a mechanism for interaction between the two cascades by which cytokines drive local inflammation without considerable cellular infiltration into the brain parenchyma.

Protection by and maintenance of CD4 effector memory and effector T cell subsets in persistent malaria infection.

Protection at the peak of Plasmodium chabaudi blood-stage malaria infection is provided by CD4 T cells. We have shown that an increase in Th1 cells also correlates with protection during the persistent phase of malaria; however, it is unclear how these T cells are maintained. Persistent malaria infection promotes protection and generates both effector T cells (Teff), and effector memory T cells (Tem). We have previously defined new CD4 Teff (IL-7Rα-) subsets from Early (TeffEarly, CD62LhiCD27+) to Late (TeffLate, CD62LloCD27-) activation states. Here, we tested these effector and memory T cell subsets for their ability to survive and protect in vivo. We found that both polyclonal and P. chabaudi Merozoite Surface Protein-1 (MSP-1)-specific B5 TCR transgenic Tem survive better than Teff. Surprisingly, as Tem are associated with antigen persistence, Tem survive well even after clearance of infection. As previously shown during T cell contraction, TeffEarly, which can generate Tem, also survive better than other Teff subsets in uninfected recipients. Two other Tem survival mechanisms identified here are that low-level chronic infection promotes Tem both by driving their proliferation, and by programming production of Tem from Tcm. Protective CD4 T cell phenotypes have not been precisely determined in malaria, or other persistent infections. Therefore, we tested purified memory (Tmem) and Teff subsets in protection from peak pathology and parasitemia in immunocompromised recipient mice. Strikingly, among Tmem (IL-7Rαhi) subsets, only TemLate (CD62LloCD27-) reduced peak parasitemia (19%), though the dominant memory subset is TemEarly, which is not protective. In contrast, all Teff subsets reduced peak parasitemia by more than half, and mature Teff can generate Tem, though less. In summary, we have elucidated four mechanisms of Tem maintenance, and identified two long-lived T cell subsets (TemLate, TeffEarly) that may represent correlates of protection or a target for longer-lived vaccine-induced protection against malaria blood-stages.

Behavioural and neurological symptoms accompanied by cellular neuroinflammation in IL-10-deficient mice infected with Plasmodium chabaudi.

BACKGROUND: Cerebral malaria is one of the most severe complications of Plasmodium falciparum infection and occurs mostly in young African children. This syndrome results from a combination of high levels of parasitaemia and inflammation. Although parasite sequestration in the brain is a feature of the human syndrome, sequestering strains do not uniformly cause severe malaria, suggesting interplay with other factors. Host genetic factors such as mutations in the promoters of the cytokines IL-10 and TNF are also clearly linked to severe disease. Plasmodium chabaudi, a rodent malaria parasite, leads to mild illness in wildtype animals. However, IL-10(-/-) mice respond to parasite with increased levels of pro-inflammatory cytokines IFN-γ and TNF, leading to lethal disease in the absence of sequestration in the brain. These mice also exhibit cerebral symptoms including gross cerebral oedema and haemorrhage, allowing study of these critical features of disease without the influence of sequestration. METHODS: The neurological consequences of P. chabaudi infection were investigated by performing a general behavioural screen (SHIRPA). The immune cell populations found in the brain during infection were also analysed using flow cytometry and confocal microscopy. RESULTS: IL-10(-/-) mice suffer significant declines in behavioural and physical capacities during infection compared to wildtype. In addition, grip strength and pain sensitivity were affected, suggestive of neurological involvement. Several immune cell populations were identified in the perfused brain on day 7 post-infection, suggesting that they are tightly adherent to the vascular endothelium, or potentially located within the brain parenchyma. There was an increase in both inflammatory monocyte and resident macrophage (CD11b(hi), CD45(+), MHCII(+), Ly6C(+/-)) numbers in IL-10(-/-) compared to wildtype animals. In addition, the activation state of all monocytes and microglia (CD11b(int), CD45(-), MHC-II(+)) were increased. T cells making IFN-γ were also identified in the brain, but were localized within the vasculature, and not the parenchyma. CONCLUSIONS: These studies demonstrate exacerbated neuroinflammation concurrent with development of behavioural symptoms in P. chabaudi infection of IL-10(-/-) animals.

Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2 signaling in dictyostelium.

The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O(2) regulation of development, suggesting the existence of an ancient O(2)-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O(2) signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. (1)H NMR demonstrated formation of 4(trans)-hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFα by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3(SCF)ubiquitin ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily related E3(VBC)ubiquitin ligases.