Marine Polyether Phycotoxin Palytoxin Induces Apoptotic Cell Death via Mcl-1 and Bcl-2 Downregulation.

Palytoxin is considered one of the most potent biotoxins. As palytoxin-induced cancer cell death mechanisms remain to be elucidated, we investigated this effect on various leukemia and solid tumor cell lines at low picomolar concentrations. As palytoxin did not affect the viability of peripheral blood mononuclear cells (PBMC) from healthy donors and did not create systemic toxicity in zebrafish, we confirmed excellent differential toxicity. Cell death was characterized by a multi-parametric approach involving the detection of nuclear condensation and caspase activation assays. zVAD-sensitive apoptotic cell death was concomitant with a dose-dependent downregulation of antiapoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL. Proteasome inhibitor MG-132 prevented the proteolysis of Mcl-1, whereas the three major proteasomal enzymatic activities were upregulated by palytoxin. Palytoxin-induced dephosphorylation of Bcl-2 further exacerbated the proapoptotic effect of Mcl-1 and Bcl-xL degradation in a range of leukemia cell lines. As okadaic acid rescued cell death triggered by palytoxin, protein phosphatase (PP)2A was involved in Bcl-2 dephosphorylation and induction of apoptosis by palytoxin. At a translational level, palytoxin abrogated the colony formation capacity of leukemia cell types. Moreover, palytoxin abrogated tumor formation in a zebrafish xenograft assay at concentrations between 10 and 30 pM. Altogether, we provide evidence of the role of palytoxin as a very potent and promising anti-leukemic agent, acting at low picomolar concentrations in cellulo and in vivo.

Correction: The scavenger receptor repertoire in six cnidarian species and its putative role in cnidarian-dinoflagellate symbiosis.

[This corrects the article DOI: 10.7717/peerj.2692.].

A diverse host thrombospondin-type-1 repeat protein repertoire promotes symbiont colonization during establishment of cnidarian-dinoflagellate symbiosis.

The mutualistic endosymbiosis between cnidarians and dinoflagellates is mediated by complex inter-partner signaling events, where the host cnidarian innate immune system plays a crucial role in recognition and regulation of symbionts. To date, little is known about the diversity of thrombospondin-type-1 repeat (TSR) domain proteins in basal metazoans or their potential role in regulation of cnidarian-dinoflagellate mutualisms. We reveal a large and diverse repertoire of TSR proteins in seven anthozoan species, and show that in the model sea anemone Aiptasia pallida the TSR domain promotes colonization of the host by the symbiotic dinoflagellate Symbiodinium minutum. Blocking TSR domains led to decreased colonization success, while adding exogenous TSRs resulted in a 'super colonization'. Furthermore, gene expression of TSR proteins was highest at early time-points during symbiosis establishment. Our work characterizes the diversity of cnidarian TSR proteins and provides evidence that these proteins play an important role in the establishment of cnidarian-dinoflagellate symbiosis.

Establishment of stably transfected cells constitutively expressing the full-length and truncated antigenic proteins of two genetically distinct mink astroviruses.

Astroviruses are becoming a growing concern in veterinary and public health. To date there are no registered vaccines against astrovirus-induced disease, mostly due to the difficulty to cultivate astroviruses to high titer for vaccine development using conventional techniques. As means to circumvent this drawback, we have developed stably transfected mink fetal cells and BHK21 cells constitutively expressing the full-length and truncated capsid proteins of two distinct genotypes of mink astrovirus. Protein expression in these stably transfected cells was demonstrated by strong signals as evaluated by in-situ PLA and IFA, and confirmed by Western blotting. The recombinant full-length and truncated proteins induced a high level of antibodies in mink, evaluated by ELISA, demonstrating their immunogenicity. In a challenge experiment in mink, a reduction in presentation clinical signs and virus shedding was observed in mink kits born from immunized females. The gene integration and protein expression were sustained through cell passage, showing that the used approach is robust and reliable for expression of functional capsid proteins for vaccine and diagnostic applications.

Genomic analysis and mRNA expression of equine type I interferon genes.

This study aimed at identifying all of the type I interferon (IFN) genes of the horse and at monitoring their expression in equine cells on in vitro induction. We identified 32 putative type I IFN loci on horse chromosome 23 and an unplaced genomic scaffold. A phylogentic analysis characterized these into 8 different type I IFN classes, that is, putative functional genes for 6 IFN-α, 4 IFN-ß, 8 IFN-ω (plus 4 pseudogenes), 3 IFN-δ (plus 1 pseudogene), 1 IFN-κ and 1 IFN-ε, plus 1 IFN-ν pseudogene, and 3 loci belonging to what has previously been called IFN-αω. Our analyses indicate that the IFN-αω genes are quite distinct from both IFN-α and IFN-ω, and we refer to this type I IFN as IFN-µ. Results from cell cultures showed that leukocytes readily expressed IFN-α, IFN-ß, IFN-δ, IFN-µ, and IFN-ω mRNA on induction with, for example, live virus; while fibroblasts only expressed IFN-ß mRNA on stimulation. IFN-κ or IFN-ε expression was not consistently induced in these cell cultures. Thus, the equine type I IFN family comprised 8 classes, 7 of which had putative functional genes, and mRNA expression of 5 was induced in vitro. Moreover, a relatively low number of IFN-α subtypes was found in the horse compared with other eutherian mammals.

Regulation of cnidarian-dinoflagellate mutualisms: Evidence that activation of a host TGFß innate immune pathway promotes tolerance of the symbiont.

Animals must manage interactions with beneficial as well as detrimental microbes. Immunity therefore includes strategies for both resistance to and tolerance of microbial invaders. Transforming growth factor beta (TGFß) cytokines have many functions in animals including a tolerance-promoting (tolerogenic) role in immunity in vertebrates. TGFß pathways are present in basal metazoans such as cnidarians but their potential role in immunity has never been explored. This study takes a two-part approach to examining an immune function for TGFß in cnidarians. First bioinformatic analyses of the model anemone Aiptasia pallida were used to identify TGFß pathway components and explore the hypothesis that an immune function for TGFßs existed prior to the evolution of vertebrates. A TGFß ligand from A. pallida was identified as one that groups closely with vertebrate TGFßs that have an immune function. Second, cellular analyses of A. pallida were used to examine a role for a TGFß pathway in the regulation of cnidarian-dinoflagellate mutualisms. These interactions are stable under ambient conditions but collapse under elevated temperature, a phenomenon called cnidarian bleaching. Addition of exogenous human TGFß suppressed an immune response measured as LPS-induced nitric oxide (NO) production by the host. Addition of anti-TGFß to block a putative TGFß pathway resulted in immune stimulation and a failure of the symbionts to successfully colonize the host. Finally, addition of exogenous TGFß suppressed immune stimulation in heat-stressed animals and partially abolished a bleaching response. These findings suggest that the dinoflagellate symbionts somehow promote host tolerance through activation of tolerogenic host immune pathways, a strategy employed by some intracellular protozoan parasites during their invasion of vertebrates. Insight into the ancient, conserved nature of host-microbe interactions gained from this cnidarian-dinoflagellate model is valuable to understanding the evolution of immunity and its role in the regulation of both beneficial and detrimental associations.

Role of the sphingosine rheostat in the regulation of cnidarian-dinoflagellate symbioses.

The symbiosis between host cnidarians, such as corals and anemones, and their dinoflagellate symbionts is regulated by largely undescribed mechanisms that stabilize the symbiosis during normal conditions but lead to symbiosis breakdown, or cnidarian bleaching, during stress. Previous transcriptomic studies identified the sphingosine rheostat as a putative symbiosis regulatory pathway. The sphingosine rheostat, which includes the sphingolipids sphingosine (Sph) and sphingosine 1-phosphate (S1P), is a key homeostatic cell regulatory pathway known to function in cell fate and immunity in animals. This study explores the role of sphingosine rheostat components in the stability of the symbiotic partnership. The anemone Aiptasia pallida, host to the dinoflagellate Symbiodinium sp., was used to test the hypothesis that S1P promotes symbiosis stability whereas Sph increases bleaching induced by heat stress. Anemones pre-incubated in exogenous S1P and FTY720, a synthetic S1P analog, were partially rescued from heat-stress-induced bleaching. In addition, they displayed a decrease in caspase activity, a measure of apoptosis, compared to controls. In contrast, when anemones were pre-incubated with Sph, both bleaching and caspase activity increased compared to untreated, heat-stressed controls. These data suggest that the sphingosine rheostat may play a role in the balance between stability and dysfunction in cnidarian-dinoflagellate symbioses.

IL-6 produced by type I IFN DC controls IFN-gamma production by regulating the suppressive effect of CD4+ CD25+ regulatory T cells.

The dendritic cell family is composed of different subsets differentially governing the immune response. Type I interferon (IFN) dendritic cells (DC) are endowed with the ability to trigger both Th1 and Th2 type responses. In view of the pivotal role of regulatory T cells in limiting the effectiveness of effector cells, we analyzed the interactions between these cells and type I IFN DC. DC were generated from monocytes in the presence of IFN-beta and interleukin (IL)-3 (DCI3) or granulocyte macrophage-colony-stimulating factor and IL-4 (DCG4) and activated by poly(I:C). Despite the release of lower amounts of IL-12 after maturation, DCI3 were able to induce a higher IFN-gamma production by T lymphocytes during the mixed leucocyte reaction (MLR) as compared with DCG4. mRNA analysis disclosed that DCI3 overtranscribed the IL-6 gene and secreted high amounts of the protein. Neutralization of IL-6 revealed that this cytokine specifically contributed to the IFN-gamma release induced by DCI3. Finally, depletion of CD25+ T cells before the MLR identified these cells as a target for IL-6. We conclude that DCI3 are endowed with the property of regulating the suppressive effect of regulatory T cells through high IL-6 production. This novel mechanism of T cell control is relevant for the use of DCI3 in vaccination strategies.

Immunostimulatory properties of human dendritic cells generated using IFN-beta associated either with IL-3 or GM-CSF.

Despite limited clinical efficacy in large trials, dendritic cells (DC)-based immunization has yielded impressive responses in some patients. Key questions remain to be solved in order to optimize this therapeutic vaccine. Among them, the nature of the DC type used and its state of maturation are pivotal. Besides myeloid DC which are mostly used in clinical trials, a new DC type has been recently described resulting from the differentiation of monocytes in the presence of type I IFNs. In the present study, we analyze the features of type I IFNs DC generated in the presence of either IL-3 (IL-3-DC) or GM-CSF (GM-CSF-DC) and compare their capacity to respond to poly(I:C) and to subsequently trigger T-cell activation. The two DC types disclose a similar immunophenotype characterized by high levels of chemokines receptors, co-stimulatory and HLA molecules expression. After poly(I:C) maturation, both DC types display a marked upregulation of CD80, CD83 and CD86 and the same pattern of gene expression. In addition, poly(I:C) stimulated them to secrete IFN-alpha and IL-12p70. Both DC types elicit potent allogeneic reactions. Priming of autologous T cells by IL-3-DC or GM-CSF-DC pulsed with an HLA-A2 restricted melan-A derived peptide, lead to the expansion of peptide specific CTL secreting high amounts of IFN-gamma. We conclude that poly(I:C) matured IL-3-DC and GM-CSF-DC share similar phenotype and functional properties including the capacity to prime tumor-associated antigen specific CTL.

Systemic and placental productions of tumor necrosis factor contribute to induce fetal mortality in mice acutely infected with Trypanosoma cruzi.

Blood levels and placental productions of IFN-gamma and TNF, known to be harmful for pregnancy, were determined in pregnant mice acutely infected with Trypanosoma cruzi and suffering massive fetal losses without congenital infection. INF-gamma was detected mainly at day 9 and TNF at days 17 and 19 of pregnancy in plasma of infected mice. TNF levels were significantly correlated to the percentages of dead fetuses. Placental cells produced TNF but not IFN-gamma, and addition of T. cruzi lysate to such cells strongly stimulated TNF production. Treatment of infected mice with pentoxifylline, known to decrease IFN-gamma production and to inhibit the TNF-alpha gene transcription, reduced the placental production of TNF, and the fetal mortality in comparison to control animals. Altogether these result suggest that TNF produced at systemic and placental levels plays a role in the fetal mortality induced in mice acutely infected with T. cruzi.

Acute Trypanosoma cruzi infection in mouse induces infertility or placental parasite invasion and ischemic necrosis associated with massive fetal loss.

Pathogens may impair reproduction in association or not with congenital infections. We have investigated the effect of acute infection with Trypanosoma cruzi, the protozoan agent of Chagas' disease in Latin America, on reproduction of mice. Although mating of infected mice occurred at a normal rate, 80% of them did not become gravid. In the few gravid infected mice, implantation numbers were as in uninfected control mice, but 28% of fetuses resorbed. Such infertility and early fetal losses were significantly associated with high maternal parasitemia. The remaining fetuses presented with reduced weights and all died later in gestation or within 48 hours after birth. Several organs of these fetuses were infiltrated by polynuclear cells and presented ischemic necrosis but did not harbor T. cruzi parasites, discarding congenital infection as the cause of mortality. However, surprisingly, the deciduas were massively invaded by T. cruzi parasites, harboring 125-fold more amastigotes than the maternal heart or other placental tissues. Parasites were significantly more numerous in the placentas of dead fetuses. In addition, placentas contained inflammatory infiltrates and displayed ischemic necrosis, fibrin deposits, and vascular thromboses. These results show that acute T. cruzi infection totally impairs reproduction in mice through inducing infertility or fetal-neonatal losses in association with placental parasite invasion and ischemic necrosis.