Immunostimulatory capacity of DNA vaccine vectors in porcine PBMC: a specific role for CpG-motifs?

With the development of DNA vaccines in pigs, the possibility was investigated that the nature and the amount of certain CpG-motifs present on plasmid DNA might have an effect on their immunostimulatory capacity. A panel of three CpG-oligodeoxynucleotides (ODN) and three eukaryotic expression vectors currently used in experimental DNA vaccines in pigs (pcDNA1, pcDNA3.1 and pCI) were screened for their immunostimulatory activity on porcine PBMC by evaluating in vitro the lymphocyte proliferative responses and cytokine profiles (IL-1alpha, IL-2, IL-4, IL-6, IL-10, IFN-gamma, TGF-beta, TNF-alpha). The vectors were chosen so that they differed in number and nature of certain CpG-motifs present on their backbone. CpG-ODN A (5'ATCGAT3') and to a lesser extend CpG-ODN C (5'AACGTT3') significantly enhanced the proliferation of porcine PBMC in contrast to CpG-ODN B (5'GACGTT3') where no effect was observed. Furthermore, CpG-ODN A significantly induced IL-6 and TNF-alpha together with elevated levels of IFN-gamma and IL-2 mRNA expression even though considerable heterogeneity was observed in the response of individual pigs. Comparison of the three vectors showed significantly increased proliferative responses for both pcDNA3.1 and pCI combined with a significant increase in IL-6 mRNA levels for pCI. For pcDNA1, proliferation was absent together with significantly decreased levels of IL-6 and IFN-gamma. CpG-ODN and plasmids both suppressed the TGF-beta and IL-1alpha mRNA expression. Taken together, these data confirm the identity of an optimal immunostimulating CpG-motif in pigs (5'-ggTGCATCGATGCAG-3') and demonstrates that the choice of the vector or the insertion of immunostimulatory motifs can be important in the future design of DNA vaccines in pigs, although further research is necessary to explore the possible link between certain CpG-motifs and the immunogenicity of DNA vaccines.

Porcine-specific CpG-oligodeoxynucleotide activates B-cells and increases the expression of MHC-II molecules on lymphocytes.

Two CpG-oligodeoxynucleotide motifs, a mouse-specific one (CpG(mouse)) 5'-GCTAGACGTTAGCGT-3' and a porcine-specific one (CpG(pig)), 5'-TGCATCGATGCAG-3' were synthesized by two different companies and tested in vitro for their capacity to stimulate porcine peripheral blood monomorphonuclear cells (PBMC). The porcine-specific motif, consisting of a nuclease-resistant phosphorothioate guanosines at the 5' and at the 3'-end (CpG(pig)-S), enhanced significantly the proliferation of porcine PBMC in comparison with CpG(mouse). The latter motif did not induce any proliferation. Methylation of CpG(pig) diminished the proliferation. Four days of culture with CpG(pig)-S increased the percentage of B-cells as well as B-cell blasting. Moreover, CpG(pig)-S also enhanced the expression of class II MHC in most cultures while there were no changes in percentage of macrophages or in the degree of expression of the macrophage marker (monoclonal 74-22-15). In conclusion, in this study, it was confirmed that 5'-ggTGCATCGATGCAGggggg-3' is a swine-specific CpG-ODN, that activates porcine B-cells and deserves further evaluation in vivo as a potential immunostimulating adjuvant.

Comparative analysis of porcine cytokine production by mRNA and protein detection.

To analyze the correlation between cytokine mRNA transcription and secretion, interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) were quantified by enzyme-linked immunosorbent assay and IL-2 by bioassay and compared with their mRNA levels, determined by reverse transcription polymerase chain reaction (RT-PCR). For this purpose, peripheral blood monomorphonuclear cells (PBMC) were stimulated in vitro with the lectins pokeweed mitogen (PWM), phytohemagglutinin (PHA) and concanavalin A (ConA), respectively, and with F4 fimbriae in an antigen-specific assay. Analyses were performed 4, 8, 16, 24 and 48 h after stimulation on the stimulated PBMC for mRNA and on the respective culture supernatants for proteins. RT-PCR products were quantified by densitometric scanning of the electrophoresis bands and related to the band intensity of the housekeeping gene, cyclophilin. Low levels of IL-2, IL-4 and IFN-gamma mRNA were detected in unstimulated PBMC. Stimulation with all three mitogens (PWM, ConA, PHA) led to an increase in mRNA transcription. In contrast, substantial IL-10 mRNA levels were detected in both unstimulated and stimulated cells with practically no difference between the three mitogens used. IL-2 mRNA expression tended to peak after 8-16 h for all three mitogens. The cells stimulated with PWM and ConA showed higher levels of gene expression for IFN-gamma and lower for IL-4 then the cells stimulated with PHA, however, differences were not statistically significant. For cells stimulated with F4 fimbriae only the IFN-gamma mRNA expression increased with an early peak at 8h post-stimulation. The analysis of the culture supernatants for secreted cytokines revealed a correlation between the levels of mRNA transcription and the respective secreted cytokines during the first 24h of stimulation. After 24h of stimulation, however, a decrease in IFN-gamma and IL-2 mRNA levels was accompanied by an increase or a less pronounced decrease in cytokine concentration; only the ConA induced IL-2 mRNA and protein concentration slopes showed similar profiles. In conclusion, similar cytokine production profiles as defined by mRNA and protein, respectively are obtained only during the first 24h after stimulation of the cell cultures.

BNIP3 supports melanoma cell migration and vasculogenic mimicry by orchestrating the actin cytoskeleton.

BNIP3 is an atypical BH3-only member of the BCL-2 family of proteins with reported pro-death as well as pro-autophagic and cytoprotective functions, depending on the type of stress and cellular context. In line with this, the role of BNIP3 in cancer is highly controversial and increased BNIP3 levels in cancer patients have been linked with both good as well as poor prognosis. In this study, using small hairpin RNA (shRNA) lentiviral transduction to stably knockdown BNIP3 (BNIP3-shRNA) expression levels in melanoma cells, we show that BNIP3 supports cancer cell survival and long-term clonogenic growth. Although BNIP3-shRNA increased mitochondrial mass and baseline levels of reactive oxygen species production, which are features associated with aggressive cancer cell behavior, it also prevented cell migration and completely abolished the ability to form a tubular-like network on matrigel, a hallmark of vasculogenic mimicry (VM). We found that this attenuated aggressive behavior of these melanoma cells was underscored by severe changes in cell morphology and remodeling of the actin cytoskeleton associated with loss of BNIP3. Indeed, BNIP3-silenced melanoma cells displayed enhanced formation of actin stress fibers and membrane ruffles, while lamellopodial protrusions and filopodia, tight junctions and adherens junctions were reduced. Moreover, loss of BNIP3 resulted in re-organization of focal adhesion sites associated with increased levels of phosphorylated focal adhesion kinase. Remarkably, BNIP3 silencing led to a drop of the protein levels of the integrin-associated protein CD47 and its downstream signaling effectors Rac1 and Cdc42. These observations underscore that BNIP3 is required to maintain steady-state levels of intracellular complexes orchestrating the plasticity of the actin cytoskeleton, which is integral to cell migration and other vital processes stimulating cancer progression. All together these results unveil an unprecedented pro-tumorigenic role of BNIP3 driving melanoma cell's aggressive features, like migration and VM.

Perk-dependent repression of miR-106b-25 cluster is required for ER stress-induced apoptosis.

Activation of the unfolded protein response sensor PKR-like endoplasmic reticulum kinase (Perk) attenuates endoplasmic reticulum (ER) stress levels. Conversantly, if the damage is too severe and ER function cannot be restored, this signaling branch triggers apoptosis. Bcl-2 homology 3-only family member Bim is essential for ER stress-induced apoptosis. However, the regulatory mechanisms controlling Bim activation under ER stress conditions are not well understood. Here, we show that downregulation of the miR-106b-25 cluster contributes to ER stress-induced apoptosis and the upregulation of Bim. Hypericin-mediated photo-oxidative ER damage induced Perk-dependent cell death and led to a significant decrease in the levels of miRNAs belonging to miR-106b-25 cluster in wild-type (WT) but not in Perk⁻/⁻ MEFs. Further, we show that expression of miR-106b-25 and Mcm-7 (host gene of miR-106b-25) is co-regulated through the transcription factors Atf4 (activating transcription factor 4) and Nrf2 (nuclear factor-erythroid-2-related factor 2). ER stress increased the activity of WT Bim 3'UTR (untranslated region) construct but not the miR-106b-25 recognition site-mutated Bim 3'UTR construct. Overexpression of miR-106b-25 cluster inhibits ER stress-induced cell death in WT but did not confer any further protection in Bim-knockdown cells. Further, we show downregulation in the levels of miR-106b-25 cluster in the symptomatic SOD1(G86R) transgenic mice. Our results suggest a molecular mechanism whereby repression of miR-106b-25 cluster has an important role in ER stress-mediated increase in Bim and apoptosis.

PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress.

Endoplasmic reticulum stress is emerging as an important modulator of different pathologies and as a mechanism contributing to cancer cell death in response to therapeutic agents. In several instances, oxidative stress and the onset of endoplasmic reticulum (ER) stress occur together; yet, the molecular events linking reactive oxygen species (ROS) to ER stress-mediated apoptosis are currently unknown. Here, we show that PERK (RNA-dependent protein kinase (PKR)-like ER kinase), a key ER stress sensor of the unfolded protein response, is uniquely enriched at the mitochondria-associated ER membranes (MAMs). PERK(-/-) cells display disturbed ER morphology and Ca(2+) signaling as well as significantly weaker ER-mitochondria contact sites. Re-expression of a kinase-dead PERK mutant but not the cytoplasmic deletion mutant of PERK in PERK(-/-) cells re-establishes ER-mitochondria juxtapositions and mitochondrial sensitization to ROS-mediated stress. In contrast to the canonical ER stressor thapsigargin, during ROS-mediated ER stress, PERK contributes to apoptosis twofold by sustaining the levels of pro-apoptotic C/EBP homologous protein (CHOP) and by facilitating the propagation of ROS signals between the ER and mitochondria through its tethering function. Hence, this study reveals an unprecedented role of PERK as a MAMs component required to maintain the ER-mitochondria juxtapositions and propel ROS-mediated mitochondrial apoptosis. Furthermore, it suggests that loss of PERK may cause defects in cell death sensitivity in pathological conditions linked to ROS-mediated ER stress.

The jejunal Peyer's patches are the major inductive sites of the F4-specific immune response following intestinal immunisation of pigs with F4 (K88) fimbriae.

A recently developed oral immunisation model in pigs in which F4 (K88) fimbriae of enterotoxigenic Escherichia coli are administered to induce a protective intestinal immunity, was used to determine the optimal inductive sites of the F4-specific intestinal immune response. Hereto, pigs were immunised with F4 orally, in the lumen of the mid-jejunum, ileum or mid-colon. Throughout the small intestine, the highest number of ASC was found following jejunal immunisation, followed by ileal, oral and colonic immunisation. To determine the signifance of Peyer's patches in the induced immune response, F4 was injected into the jejunal Peyer's patches (JPP), lamina propria (LP) and ileal Peyer's patches (IPP). Immunisation in the JPP induced the highest number ASC in the small intestine, whereas immunisation in the LP and IPP resulted in lower intestinal antibody responses. In conclusion, we have shown that the JPP are the major inductive sites of the F4-specific intestinal antibody response. This knowledge could be important when using the pig as an animal model for vaccination studies.

1alpha,25-dihydroxyvitamin D3 increases IgA serum antibody responses and IgA antibody-secreting cell numbers in the Peyer's patches of pigs after intramuscular immunization.

Pigs were injected intramuscularly (i.m.) twice with human serum albumin (HSA) with or without 1alpha,25-dihydroxyvitamin D3[1alpha,25(OH)2D3] with a 5-week interval. The supplementation of 1alpha,25(OH)2D3 enhanced the HSA-specific IgA serum antibody response but decreased the IgM, IgG, IgG1 and IgG2 responses. Furthermore, higher numbers of HSA-specific IgA antibody-secreting cells were obtained in systemic lymphoid tissues (local draining lymph node, spleen and bone marrow) as well as in Peyer's patches and lamina propria of the gut (GALT). In addition, the in vivo mRNA expression for Th1 [interferon (IFN)-gamma, interleukin (IL-2)], Th2 (IL-4, IL-6 and IL-10) and Th3 [transforming growth factor (TGF)-beta] cytokines as well as the percentage of different cell subsets (CD2+, CD4+, CD8+, IgM+, MHC II+, CD25+) of monomorphonuclear cells from the local draining lymph node were determined at different time-points after the i.m. immunizations. Cytokine profiles did not resemble a typical Th-cytokine profile using 1alpha,25(OH)2D3: higher levels of IL-10 and significantly lower levels of IL-2 were observed the first day after the primary immunization. However, significantly higher levels of IL-2 and significantly lower levels of IFN-gamma were observed the first day after the second immunization. Furthermore, after the second immunization TGF-beta mRNA expression decreased more quickly in the 1alpha,25(OH)2D3 group. This difference became significant 7 days after the second immunization. One week later a significantly higher percentage of CD25+ cells was observed in this group, indicating more activated T and B cells using the steroid hormone. These results suggest that in pigs the addition of 1alpha,25(OH)2D3 to an intramuscularly injected antigen can enhance the antigen-specific IgA-response and prime GALT tissues, but the relation with cytokines and cell phenotype in the local draining lymph node needs further clarification.

Differentiation of EL4 lymphoma cells by tumoral environment is associated with inappropriate expression of the large chondroitin sulfate proteoglycan PG-M and the tumor-associated antigen HTgp-175.

Progression to malignancy of transformed cells involves complex genetic alterations and aberrant gene expression patterns. While aberrant gene expression is often caused by alterations in individual genes, the contribution of the tumoral environment to the triggering of this gene expression is less well established. The stable but heterogeneous expression in cultured EL4/13 cells of a novel tumor-associated antigen, designated as HTgp-175, was chosen for the investigation of gene expression during tumor formation. Homogeneously HTgp-175-negative EL4/13 cells, isolated by cell sorting or obtained by subcloning, acquired HTgp-175 expression as a result of tumor formation. The tumorigenicity of HTgp-175-negative vs. HTgp-175-positive EL4 variants was identical, indicating that induction but not selection accounted for the phenotypic switch from HTgp-175-negative to HTgp-175-positive. Although mutagenesis experiments showed that the protein was not essential for tumor establishment, tumor-derived cells showed increased malignancy, linking HTgp-175 expression with genetic changes accompanying tumor progression. This novel gene expression was not an isolated event, since it was accompanied by ectopic expression of the large chondroitin sulfate proteoglycan PG-M and of normal differentiation antigens. We conclude that signals derived from the tumoral microenvironment contribute significantly to the aberrant gene expression pattern of malignant cells, apparently by fortuitous activation of differentiation processes and cause expression of novel differentiation antigens as well as of inappropriate tumor-associated and ectopic antigens.

Priming of piglets against enterotoxigenic E. coli F4 fimbriae by immunisation with FAEG DNA.

Early vaccination is necessary to protect pigs against postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). However, at present no commercial vaccine allows successful vaccination. This is partly due to the presence of maternally derived antibodies. Since DNA vaccines are suggested to be superior to protein vaccines in young animals with maternal antibodies, we determined whether the fimbrial adhesin (FaeG) of F4ac(+) ETEC could be used as a plasmid DNA vaccine to prime piglets in a heterologous prime-boost approach. Hereto, pcDNA1/faeG19 was constructed and expression of rFaeG in Cos-7 cells was demonstrated. Thereafter, pigs were immunised (days 0, 21 and 42) intramuscularly by injection or intradermally by gene gun and humoral and cellular immune responses were analysed. Even though responses were low, results demonstrated that intramuscular injection was superior to gene gun delivery for priming the humoral immune response since higher antibody titres were raised, whereas gene gun delivery better induced a cellular response, evaluated by a lymphocyte proliferation assay. Effective priming of the humoral immune response was evidenced by high IgG titres 1 week after a protein boost with purified F4. The low responses to the pcDNA1/faeG19 DNA vaccination suggest that delivery of the DNA and/or the expression of the faeG gene should be improved.