A malaria protein factor induces IL-4 production by dendritic cells via PI3K-Akt-NF-κB signaling independent of MyD88/TRIF and promotes Th2 response.

Dendritic cells (DC) and cytokines produced by DC play crucial roles in inducing and regulating pro-/anti-inflammatory and Th1/Th2 responses. DC are known to produce a Th1-promoting cytokine, interleukin (IL)-12, in response to malaria and other pathogenic infections, but it is thought that DC do not produce Th2-promoting cytokine, IL-4. Here, we show that a protein factor of malaria parasites induces IL-4 responses by CD11chiMHCIIhiCD3ϵ-CD49b-CD19-FcϵRI- DC via PI3K-Akt-NF-κB signaling independent of TLR-MyD88/TRIF. Malaria parasite-activated DC induced IL-4 responses by T cells both in vitro and in vivo, favoring Th2, and il-4-deficient DC were unable to induce IL-4 expression by T cells. Interestingly, lethal parasites, Plasmodium falciparum and Plasmodium berghei ANKA, induced IL-4 response primarily by CD8α- DC, whereas nonlethal Plasmodium yoelii induced IL-4 by both CD8α+ and CD8α- DC. In both P. berghei ANKA- and P. yoelii-infected mice, IL-4-expressing CD8α- DC did not express IL-12, but a distinct CD8α- DC subset expressed IL-12. In P. berghei ANKA infection, CD8α+ DC expressed IL-12 but not IL-4, whereas in P. yoelii infection, CD8α+ DC expressed IL-4 but not IL-12. These differential IL-4 and IL-12 responses by DC subsets may contribute to different Th1/Th2 development and clinical outcomes in lethal and nonlethal malaria. Our results for the first time demonstrate that a malaria protein factor induces IL-4 production by DC via PI3K-Akt-NF-κB signaling, revealing signaling and molecular mechanisms that initiate and promote Th2 development.

CD36 receptor regulates malaria-induced immune responses primarily at early blood stage infection contributing to parasitemia control and resistance to mortality.

In malaria, CD36 plays several roles, including mediating parasite sequestration to host organs, phagocytic clearance of parasites, and regulation of immunity. Although the functions of CD36 in parasite sequestration and phagocytosis have been clearly defined, less is known about its role in malaria immunity. Here, to understand the function of CD36 in malaria immunity, we studied parasite growth, innate and adaptive immune responses, and host survival in WT and Cd36-/- mice infected with a non-lethal strain of Plasmodium yoelii Compared with Cd36-/- mice, WT mice had lower parasitemias and were resistant to death. At early but not at later stages of infection, WT mice had higher circulatory proinflammatory cytokines and lower anti-inflammatory cytokines than Cd36-/- mice. WT mice showed higher frequencies of proinflammatory cytokine-producing and lower frequencies of anti-inflammatory cytokine-producing dendritic cells (DCs) and natural killer cells than Cd36-/- mice. Cytokines produced by co-cultures of DCs from infected mice and ovalbumin-specific, MHC class II-restricted α/ß (OT-II) T cells reflected CD36-dependent DC function. WT mice also showed increased Th1 and reduced Th2 responses compared with Cd36-/- mice, mainly at early stages of infection. Furthermore, in infected WT mice, macrophages and neutrophils expressed higher levels of phagocytic receptors and showed enhanced phagocytosis of parasite-infected erythrocytes than those in Cd36-/- mice in an IFN-γ-dependent manner. However, there were no differences in malaria-induced humoral responses between WT and Cd36-/- mice. Overall, the results show that CD36 plays a significant role in controlling parasite burden by contributing to proinflammatory cytokine responses by DCs and natural killer cells, Th1 development, phagocytic receptor expression, and phagocytic activity.

The TORC1-activated Proteins, p70S6K and GRB10, Regulate IL-4 Signaling and M2 Macrophage Polarization by Modulating Phosphorylation of Insulin Receptor Substrate-2.

Lung M2 macrophages are regulators of airway inflammation, associated with poor lung function in allergic asthma. Previously, we demonstrated that IL-4-induced M2 gene expression correlated with tyrosine phosphorylation of the insulin receptor substrate-2 (IRS-2) in macrophages. We hypothesized that negative regulation of IRS-2 activity after IL-4 stimulation is dependent upon serine phosphorylation of IRS-2. Herein, we describe an inverse relationship between tyrosine phosphorylation (Tyr(P)) and serine phosphorylation (Ser(P)) of IRS-2 after IL-4 stimulation. Inhibiting serine phosphatase activity increased Ser(P)-IRS-2 and decreased Tyr(P)-IRS-2 leading to reduced M2 gene expression (CD200R, CCL22, MMP12, and TGM2). We found that inhibition of p70S6K, downstream of TORC1, resulted in diminished Ser(P)-IRS-2 and prolonged Tyr(P)-IRS-2 as well. Inhibition of p70S6K increased expression of CD200R and CCL22 indicating that p70S6K negatively regulates some, but not all, human M2 genes. Knocking down GRB10, another negative regulatory protein downstream of TORC1, enhanced both Tyr(P)-IRS-2 and increased expression of all four M2 genes. Furthermore, GRB10 associated with IRS-2, NEDD4.2 (an E3-ubiquitin ligase), IL-4Rα, and γC after IL-4 stimulation. Both IL-4Rα and γC were ubiquitinated after 30 min of IL-4 treatment, suggesting that GRB10 may regulate degradation of the IL-4 receptor-signaling complex through interactions with NEDD4.2. Taken together, these data highlight two novel regulatory proteins that could be therapeutically manipulated to limit IL-4-induced IRS-2 signaling and polarization of M2 macrophages in allergic inflammation.

Phagosomal Acidification Prevents Macrophage Inflammatory Cytokine Production to Malaria, and Dendritic Cells Are the Major Source at the Early Stages of Infection: IMPLICATION FOR MALARIA PROTECTIVE IMMUNITY DEVELOPMENT.

Inflammatory cytokines produced at the early stages of malaria infection contribute to shaping protective immunity and pathophysiology. To gain mechanistic insight into these processes, it is important to understand the cellular origin of cytokines because both cytokine input and cytokine-producing cells play key roles. Here, we determined cytokine responses by monocytes, macrophages, and dendritic cells (DCs) to purified Plasmodium falciparum and Plasmodium berghei ANKA, and by spleen macrophages and DCs from Plasmodium yoelii 17NXL-infected and P. berghei ANKA-infected mice. The results demonstrate that monocytes and macrophages do not produce inflammatory cytokines to malaria parasites and that DCs are the primary source early in infection, and DC subsets differentially produce cytokines. Importantly, blocking of phagosomal acidification by inhibiting vacuolar-type H(+)-ATPase enabled macrophages to elicit cytokine responses. Because cytokine responses to malaria parasites are mediated primarily through endosomal Toll-like receptors, our data indicate that the inability of macrophages to produce cytokines is due to the phagosomal acidification that disrupts endosomal ligand-receptor engagement. Macrophages efficiently produced cytokines to LPS upon simultaneously internalizing parasites and to heat-killed Escherichia coli, demonstrating that phagosomal acidification affects endosomal receptor-mediated, but not cell surface receptor-mediated, recognition of Toll-like receptor agonists. Enabling monocytes/macrophages to elicit immune responses to parasites by blocking endosomal acidification can be a novel strategy for the effective development of protective immunity to malaria. The results have important implications for enhancing the efficacy of a whole parasite-based malaria vaccine and for designing strategies for the development of protective immunity to pathogens that induce immune responses primarily through endosomal receptors.

CD36 contributes to malaria parasite-induced pro-inflammatory cytokine production and NK and T cell activation by dendritic cells.

The scavenger receptor CD36 plays important roles in malaria, including the sequestration of parasite-infected erythrocytes in microvascular capillaries, control of parasitemia through phagocytic clearance by macrophages, and immunity. Although the role of CD36 in the parasite sequestration and clearance has been extensively studied, how and to what extent CD36 contributes to malaria immunity remains poorly understood. In this study, to determine the role of CD36 in malaria immunity, we assessed the internalization of CD36-adherent and CD36-nonadherent Plasmodium falciparum-infected red blood cells (IRBCs) and production of pro-inflammatory cytokines by DCs, and the ability of DCs to activate NK, and T cells. Human DCs treated with anti-CD36 antibody and CD36 deficient murine DCs internalized lower levels of CD36-adherent IRBCs and produced significantly decreased levels of pro-inflammatory cytokines compared to untreated human DCs and wild type mouse DCs, respectively. Consistent with these results, wild type murine DCs internalized lower levels of CD36-nonadherent IRBCs and produced decreased levels of pro-inflammatory cytokines than wild type DCs treated with CD36-adherent IRBCs. Further, the cytokine production by NK and T cells activated by IRBC-internalized DCs was significantly dependent on CD36. Thus, our results demonstrate that CD36 contributes significantly to the uptake of IRBCs and pro-inflammatory cytokine responses by DCs, and the ability of DCs to activate NK and T cells to produce IFN-γ. Given that DCs respond to malaria parasites very early during infection and influence development of immunity, and that CD36 contributes substantially to the cytokine production by DCs, NK and T cells, our results suggest that CD36 plays an important role in immunity to malaria. Furthermore, since the contribution of CD36 is particularly evident at low doses of infected erythrocytes, the results imply that the effect of CD36 on malaria immunity is imprinted early during infection when parasite load is low.

Kinetics and thermodynamics of glycans and glycoproteins binding to Holothuria scabra lectin: a fluorescence and surface plasmon resonance spectroscopic study.

Holothuria scabra produces a monomeric lectin (HSL) of 182 kDa. HSL showed strong antibacterial activity and induced bacterial agglutination under in vitro conditions, indicating its role in animals' innate immune responses. Very few lectins have been reported from echinoderms and none of these lectins have been explored in detail for their sugar-binding kinetics. Affinity, kinetics and thermodynamic analysis of glycans and glycoproteins binding to HSL were studied by fluorescence and surface plasmon resonance spectroscopy. Lectin binds with higher affinity to O-linked than N-linked asialo glycans, and the affinities were relatively higher than that for sialated glycans and glycoproteins. T-antigen α-methyl glycoside was the most potent ligand having the highest affinity (Ka 8.32 ×10(7) M(-1)). Thermodynamic and kinetic analysis indicated that the binding of galactosyl Tn-antigen and asialo glycans is accompanied by an enthalpic contribution in addition to higher association rate coupled by low activation energy for the association process. Presence of sialic acid or protein matrix inhibits binding. Higher affinity of HSL for O-glycans than N-glycans had biological implications; since HSL specifically recognizes bacteria, which have mucin or O-glycan cognate on their cell surfaces and play a major role in animal innate immunity. Since, HSL had higher affinity to T-antigen, makes it a useful tool for cancer diagnostic purpose.

TLR9 and MyD88 are crucial for the development of protective immunity to malaria.

Effective resolution of malaria infection by avoiding pathogenesis requires regulated pro- to anti-inflammatory responses and the development of protective immunity. TLRs are known to be critical for initiating innate immune responses, but their roles in the regulation of immune responses and development of protective immunity to malaria remain poorly understood. In this study, using wild-type, TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) mice infected with Plasmodium yoelii, we show that TLR9 and MyD88 regulate pro/anti-inflammatory cytokines, Th1/Th2 development, and cellular and humoral responses. Dendritic cells from TLR9(-/-) and MyD88(-/-) mice produced significantly lower levels of proinflammatory cytokines and higher levels of anti-inflammatory cytokines than dendritic cells from wild-type mice. NK and CD8(+) T cells from TLR9(-/-) and MyD88(-/-) mice showed markedly impaired cytotoxic activity. Furthermore, mice deficient in TLR9 and MyD88 showed higher Th2-type and lower Th1-type IgGs. Consequently, TLR9(-/-) and MyD88(-/-) mice exhibited compromised ability to control parasitemia and were susceptible to death. Our data also show that TLR9 and MyD88 distinctively regulate immune responses to malaria infection. TLR9(-/-) but not MyD88(-/-) mice produced significant levels of both pro- and anti-inflammatory cytokines, including IL-1ß and IL-18, by other TLRs/inflammasome- and/or IL-1R/IL-18R-mediated signaling. Thus, whereas MyD88(-/-) mice completely lacked cell-mediated immunity, TLR9(-/-) mice showed low levels of cell-mediated immunity and were slightly more resistant to malaria infection than MyD88(-/-) mice. Overall, our findings demonstrate that TLR9 and MyD88 play central roles in the immune regulation and development of protective immunity to malaria, and have implications in understanding immune responses to other pathogens.

Iκb-ζ plays an important role in the ERK-dependent dysregulation of malaria parasite GPI-induced IL-12 expression.

Plasmodium falciparum glycosylphosphatidylinositols (GPIs) have been proposed as malaria pathogenic factors based on their ability to induce proinflammatory responses in macrophages and malaria-like symptoms in mice. Parasite GPIs induce the production of inflammatory cytokines by activating the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways. Importantly, inhibition of the extracellular-signal-regulated kinase (ERK) pathway upregulates a subset of cytokines, including IL-12. We investigated the role of nuclear transcription factor, IκB-ζ, in the GPI-induced dysregulated expression of IL-12 on inhibition of the ERK pathway. GPIs efficiently induced the expression of IκB-ζ in macrophages regardless of whether cells were pretreated or untreated with ERK inhibitors, indicating that ERK has no role in IκB-ζ expression. However, on ERK inhibition followed by stimulation with GPIs, NF-κB binding to Il12b promoter κB site was markedly increased, suggesting that the ERK pathway regulates Il12b transcription. Knockdown of IκB-ζ using siRNA markedly reduced the GPI-induced IL-12 production and abrogated the dysregulated IL-12 production in ERK inhibited cells. Together these results demonstrate that ERK modulates IL-12 expression by regulating IκB-ζ-dependent binding of NF-κB transcription factors to Il12b gene promoter. Additionally, our finding that IκB-ζ can be knocked down efficiently in primary macrophages is valuable for studies aimed at gaining further insights into IκB-ζ function.

The nucleosome (histone-DNA complex) is the TLR9-specific immunostimulatory component of Plasmodium falciparum that activates DCs.

The systemic clinical symptoms of Plasmodium falciparum infection such as fever and chills correspond to the proinflammatory cytokines produced in response to the parasite components released during the synchronized rupture of schizonts. We recently demonstrated that, among the schizont-released products, merozoites are the predominant components that activate dendritic cells (DCs) by TLR9-specific recognition to induce the maturation of cells and to produce proinflammatory cytokines. We also demonstrated that DNA is the active constituent and that formation of a DNA-protein complex is essential for the entry of parasite DNA into cells for recognition by TLR9. However, the nature of endogenous protein-DNA complex in the parasite is not known. In this study, we show that parasite nucleosome constitute the major protein-DNA complex involved in the activation of DCs by parasite nuclear material. The parasite components were fractionated into the nuclear and non-nuclear materials. The nuclear material was further fractionated into chromatin and the proteins loosely bound to chromatin. Polynucleosomes and oligonucleosomes were prepared from the chromatin. These were tested for their ability to activate DCs obtained by the FLT3 ligand differentiation of bone marrow cells from the wild type, and TLR2(-/-), TLR9(-/-) and MyD88(-/-) mice. DCs stimulated with the nuclear material and polynucleosomes as well as mono- and oligonucleosomes efficiently induced the production of proinflammatory cytokines in a TLR9-dependent manner, demonstrating that nucleosomes (histone-DNA complex) represent the major TLR9-specific DC-immunostimulatory component of the malaria parasite nuclear material. Thus, our data provide a significant insight into the activation of DCs by malaria parasites and have important implications for malaria vaccine development.

Plasmodium falciparum: differential merozoite dose requirements for maximal production of various inflammatory cytokines.

The ligand specificity of TLRs and the details of signaling pathways that are activated by ligand-receptor engagements have been studied extensively. However, it is not known whether the signaling events initiated by defined doses of ligand are uniformly effective in producing various cytokines. In this study, we investigated the dose requirement for the saturated production of representative inflammatory cytokines, TNF-α, IL-6 and IL-12, by DCs stimulated with Plasmodium falciparum merozoites/protein-DNA complex or a CpG ODN TLR9 ligand. The data demonstrate that the ligand doses required for the maximal expression of TNF-α and IL-6 are substantially higher than those required for the maximal production of IL-12. The data also demonstrate that the uptake capacity of malaria parasite by plasmacytoid DCs is markedly lower than that of myeloid DCs, and that, like myeloid DCs, plasmacytoid DCs produce significant levels of TNF-α and IL-12 when the uptake of malarial DNA is facilitated by carrier molecules such as polylysine or cationic lipids. These results have implications for enhancing the effectiveness of vaccine against malaria by modulating the innate immune responses of plasmacytoid DCs to malaria parasites.

Protein-DNA complex is the exclusive malaria parasite component that activates dendritic cells and triggers innate immune responses.

Dendritic cells (DCs) play a crucial role in the development of protective immunity to malaria. However, it remains unclear how malaria parasites trigger immune responses in DCs. In this study, we purified merozoites, food vacuoles, and parasite membrane fragments released during the Plasmodium falciparum schizont burst to homogeneity and tested for the activation of bone marrow-derived DCs from wild-type and TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) C57BL/6J mice. The results demonstrate that a protein-DNA complex is the exclusive parasite component that activates DCs by a TLR9-dependent pathway to produce inflammatory cytokines. Complex formation with proteins is essential for the entry of parasite DNA into DCs for TLR9 recognition and, thus, proteins convert inactive DNA into a potent immunostimulatory molecule. Exogenous cationic polymers, polylysine and chitosan, can impart stimulatory activity to parasite DNA, indicating that complex formation involves ionic interactions. Merozoites and DNA-protein complex could also induce inflammatory cytokine responses in human blood DCs. Hemozoin is neither a TLR9 ligand for DCs nor functions as a carrier of DNA into cells. Additionally, although TLR9 is critical for DCs to induce the production of IFN-gamma by NK cells, this receptor is not required for NK cells to secret IFN-gamma, and cell-cell contact among myeloid DCs, plasmacytoid DCs, and NK cells is required for IFN-gamma production. Together, these results contribute substantially toward the understanding of malaria parasite-recognition mechanisms. More importantly, our finding that proteins and carbohydrate polymers are able to confer stimulatory activity to an otherwise inactive parasite DNA have important implications for the development of a vaccine against malaria.

Rhodotorula aurantiaca penicillin V acylase: active site characterization and fluorometric studies.

Penicillin V acylase (PVA), a member of newly evolved Ntn-hydrolase superfamily, is a pharmaceutically important enzyme to produce 6-aminopenicillanic acid. Active site characterization of recently purified monomeric PVA from Rhodotorula aurantiaca (Ra-PVA), the yeast source, showed the involvement of serine and tryptophan in the enzyme activity. Modification of the protein with serine and tryptophan specific reagents such as PMSF and NBS showed partial loss of PVA activity and substrate protection. Ra-PVA was found to be a multi-tryptophan protein exhibiting one tryptophan, in native and, four in its denatured condition. Various solute quenchers and substrate were used to probe the microenvironment of the putative reactive tryptophan through fluorescence quenching. The results obtained indicate that the tryptophan residues of Ra-PVA were largely buried in hydrophobic core of the protein matrix. Quenching of the fluorescence by acrylamide was collisional. Acrylamide was the most effective quencher amongst all the used quenchers, which quenched 71.6% of the total intrinsic fluorescence of the protein, at a very less final concentration of 0.1M. Surface tryptophan residues were found to have predominantly more electropositively charged amino acids around them, however differentially accessible for ionic quenchers. Denaturation led to shift in lambda(max) from 336, in native state, to 357 nm and more exposed to the solvent, consequently increase in fluorescence quenching with all quenchers. This is an attempt towards the conformational studies of Ra-PVA.

MAPK-activated protein kinase 2 differentially regulates plasmodium falciparum glycosylphosphatidylinositol-induced production of tumor necrosis factor-{alpha} and interleukin-12 in macrophages.

Proinflammatory responses induced by Plasmodium falciparum glycosylphosphatidylinositols (GPIs) are thought to be involved in malaria pathogenesis. In this study, we investigated the role of MAPK-activated protein kinase 2 (MK2) in the regulation of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12, two of the major inflammatory cytokines produced by macrophages stimulated with GPIs. We show that MK2 differentially regulates the GPI-induced production of TNF-alpha and IL-12. Although TNF-alpha production was markedly decreased, IL-12 expression was increased by 2-3-fold in GPI-stimulated MK2(-/-) macrophages compared with wild type (WT) cells. MK2(-/-) macrophages produced markedly decreased levels of TNF-alpha than WT macrophages mainly because of lower mRNA stability and translation. In the case of IL-12, mRNA was substantially higher in MK2(-/-) macrophages than WT. This enhanced production is due to increased NF-kappaB binding to the gene promoter, a markedly lower level expression of the transcriptional repressor factor c-Maf, and a decreased binding of GAP-12 to the gene promoter in MK2(-/-) macrophages. Thus, our data demonstrate for the first time the role of MK2 in the transcriptional regulation of IL-12. Using the protein kinase inhibitors SB203580 and U0126, we also show that the ERK and p38 pathways regulate TNF-alpha and IL-12 production, and that both inhibitors can reduce phosphorylation of MK2 in response to GPIs and other toll-like receptor ligands. These results may have important implications for developing therapeutics for malaria and other infectious diseases.

T-antigen binding lectin with antibacterial activity from marine invertebrate, sea cucumber (Holothuria scabra): possible involvement in differential recognition of bacteria.

In invertebrates, cellular and humoral components are evolved to maintain their body immunity and integrity. Both these factors respond to different antigens such as microorganisms, vertebrate erythrocytes and foreign proteins. In this article, we report a study of a lectin (HSL) involved in immune response in the echinoderm, sea cucumber (Holothuria scabra). Correlative studies indicate that the expression of this defensive lectin is induced by bacterial challenge, wherein cell wall glycoconjugates of bacteria are involved in lectin induction. HSL showed strong broad spectrum antibacterial activity against both gram-positive and gram-negative bacteria. Under in vitro conditions, purified HSL mediate agglutination of the test bacteria, there by indicating a possible mode of action in physiological situation.

Purification and characterization of a T-antigen specific lectin from the coelomic fluid of a marine invertebrate, sea cucumber (Holothuria scabra).

A novel lectin was purified from the coelomic fluid of the sea cucumber Holothuria scabra (HSL), subjected to bacterial challenge. HSL is a monomeric glycoprotein of molecular mass 182 kDa. The lectin is highly thermostable as it retains full activity for 1 h at 80 degrees C. Further, the hemagglutination activity of HSL is unaffected by pH in the range 2-11. Unlike other lectins purified from marine invertebrates, the hemagglutination activity of HSL does not require any divalent metal ions. The affinity profile of HSL was studied by a combination of hemagglutination inhibition and fluorescence spectroscopy. HSL binds to desialylated glycoproteins, MealphaGal, T-antigen and T (alpha-ser)-antigen with a distinction between beta1-4 and beta1-3 linkages. Mealpha-T-antigen was a potent ligand having highest affinity (Ka 8.32 x 10(7)M(-1)). Monosaccharide binding is enthalphically driven while disaccharide binding involves both entropic and enthalpic contributions.