CpG-Induced Stimulation of Cytokine Expression by Peripheral Blood Mononuclear Cells of Foals and Their Dams

A relative immunodeficiency of young foals is considered to account for the increased susceptibility of foals to infectious diseases, including pneumonia caused by Rhodococcus equi. In this report, peripheral blood mononuclear cells (PBMCs) from healthy foals at 14 and 56 days of age, or from their dams, were incubated with three stimulatory and one nonstimulatory (control) synthetic cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODNs), and mRNA expression of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin (IL) -6, IL-8, IL12-p35, and IL-12p40 were determined. Results indicated that synthetic CpG-ODNs can induce strong, rapid cytokine responses in healthy foals and adult horses. B-class CpG-ODNs 2135 and 2142 induced greater messenger RNA (mRNA) expression of IFN-γ, IL-6, and IL-12p40 than the C-class CpG-ODN 2395 in foal PBMCs. In foals, B-class CpG-ODNs induced IFN-γ, IL-6, and IL-12P40 mRNA expression that was similar to or higher in magnitude than that observed in adult horses. These observations indicate that CpG-ODNs might be useful as immunomodulators or as potential adjuvants for vaccines to aid in preventing R. equi pneumonia and other bacterial diseases of foals.     

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.     

The in vitro effects of CpG oligodeoxynucleotides on the expression of cytokine genes in the common carp (Cyprinus carpio L.) head kidney cells

Synthetic oligodeoxynucleotides with CpG motifs (CpG-ODNs) have gained attention because it elicits strong innate immune responses. CpG-ODNs promote the production of T-helper 1(T(H)1) and pro-inflammatory cytokines. In fish, knowledge of the effects of CpG-ODNs on the expression of cytokine genes is scarce. In this study, we report that CpG-ODNs induce the expression of pro-inflammatory cytokines in common carp head kidney leucocytes in vitro. Evidence of a T(H)1 type immune stimulation was observed as the fish homologs of IP-10 (interferon gamma-inducible protein 10; CXCL10) and MCP (monocyte chemotactic protein a CC-chemokine) were induced by CpG. Interleukin 10, a cytokine inhibitory factor, failed to be induced by CpG. These results suggest a robust immune stimulation can be achieved by CpGs and has a potential as an immunostimulant in aquaculture.     

Immunostimulation of bronchoalveolar lavage cells from recurrent airway obstruction-affected horses by different CpG-classes bound to gelatin nanoparticles

Recurrent airway obstruction (RAO) in horses has become a common problem in stabled horses in industrialized countries and deserves new therapeutic strategies. CpG-oligodeoxynucleotides (CpG-ODNs) were developed as effective immunostimulating agents to induce a Th2/Th1 shift. These agents showed a beneficial therapeutic effect in allergic diseases with predominant Th2 immunoresponse. CpG-ODN delivery by gelatin nanoparticles (GNPs) resulted in enhanced cellular uptake in murine and human in vitro studies and was a starting point for the present trial. The aim of this study was to identify an optimal stimulating CpG motif in horses with regard to species specificity on equine bronchoalveolar lavage (BAL) cells, in terms of a possible specific immunomodulation effect (Th2/Th1 shift) by used CpG-ODN. Accordingly, GNPs were evaluated as a delivery system to improve CpG-ODN immunostimulation in equine BAL cells. BAL fluid (BALF) was obtained from seven horses with moderate RAO and from four healthy horses and was subsequently incubated with five different CpG-ODN sequences (from A-, B- and C-class) and one ODN without any CpG motif. Release of three key cytokines (IL-4, IL-10 and IFN-γ) was quantified by ELISA to detect an allergy mediated Th2 immunoresponse (IL-4) as well as a proinflammatory Th1 response (IFN-γ). Due to its specific anti-inflammatory and anti-allergic effects, IL-10 was considered as a beneficial agent in pathophysiology of RAO. Results showed a significant upregulation of IL-10 and IFN-γ on the one hand and a downregulation of IL-4 on the other hand in RAO affected horses. Cell cultures from healthy horses had a significantly stronger response in cytokine release to all the applied stimuli in contrast to RAO derived cells. Comparing all five CpG sequences, A-class 2216 significantly showed the highest immunomodulatory effects on equine BALF cells and, hence, was chosen for follow-up preliminary clinical studies.     

Evaluation of cytokine messenger RNA expression in peripheral blood mononuclear cells from dogs with canine demodicosis

Using RT-PCR and semi-quantitative PCR, mRNA expression for canine interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5, IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta in peripheral blood mononuclear cells (PBMCs) was examined in dogs with or without demodicosis. mRNA expression for IFN-gamma as well as TNF-alpha in dogs with demodicosis (localized (LD) and generalized (GD)) was slightly lower than those in dogs without demodicosis (healthy controls). Expression of IL-5 mRNA in dogs with demodicosis was higher than that in control dogs, but there were no significant differences in IL-4 and IL-10 mRNA expression levels among the three groups. On the other hand, expression levels of TGF-beta mRNA in dogs with GD were higher than those in control dogs and dogs with LD. The expression levels of IL-5 and TGF-beta mRNA decreased in all three dogs with GD which showed resolution of the clinical signs. Taken together, these results suggest that the Th2-like response in PBMCs from dogs with demodicosis is up-regulated, and that subsequent increased expression of IL-5 and TGF-beta mRNA in dogs with GD is reversible after treatment. Therefore, these cytokines, particularly IL-5, might be a useful clinical index of the clinical course in demodicosis. Also, increased TGF-beta mRNA expression might be a key factor for revealing the difference in the mechanism of onset between LD and GD.     

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.     

Semi-quantitative analysis of cytokine expression in asymptomatic canine leishmaniasis

The dog is the main reservoir of Leishmania infantum, the parasite responsible for visceral leishmaniasis in Mediterranean countries. The infection in dogs shows different clinical presentations, from subclinical/asymptomatic to a fully developed disease, depending on the host's immune responses. The Th1/Th2 dichotomy is not clear in the different forms of canine leishmaniasis, since the data available from studies of immunity response in canine leishmaniasis are scarce and fragmented. The present work describes the cytokine expression in peripheral blood mononuclear cells (PBMC) obtained from asymptomatic dogs experimentally infected with L. infantum that present a cellular protective immune response. The results obtained from freshly isolated PBMC showed expressions of TNF-alpha, IL-2, IFN-gamma, IL-10 and IL-18 mRNA, similar to those from non-infected dogs. However, there was almost no expression of IL-4 mRNA detected in the asymptomatic infected dogs compared to the control dogs. Unspecific stimulation with ConA promoted the expression in a greater or lower degree of all the cytokines studied. In vitro stimulation of PBMC with soluble leishmanial antigen (SLA) promoted the expression of IL-2, IFN-gamma, TNF-alpha, IL-18, IL-4, IL-6 and IL-10 mRNA, with the two first being specifically induced. Although both Th1 and Th2 cytokines are produced, cell mediated immunity observed in these L. infantum-infected asymptomatic dogs depended on the preferential expression of Th1 cytokines.     

Porcine circovirus type 2 (PCV2) viral components immunomodulate recall antigen responses

Porcine circovirus type 2 (PCV2) is a single-stranded circular DNA virus infecting domestic pigs worldwide. Interaction of this virus with the immune system apparently modulates the immune response of the host. In the present study, the implication of different components of PCV2 in the modulation of the immune response of the host were investigated by using PCV2 viral-like particles (VLPs) and 16 novel oligodeoxyribonucleotides containing CpG motifs (CpG-ODNs) based on the PCV2 genomic sequence. The role of these viral components was studied by evaluating the cytokine profiles (IFN-alpha, IFN-gamma, IL-10, IL-2 and IL-12) on porcine peripheral mononuclear cell (PBMC) and bone marrow-derived dendritic cell (BMDC) cultures. Also, the effect of PCV2 and its elements were examined in recall antigen (pseudorabies virus, PRV) responses. While PCV2 was a potent inducer of IL-10 by PBMCs, such effect was not observed using CpG-ODNs or VLPs. However, IFN-gamma and IL-2 production by recall antigen was repressed in presence of PCV2 and most of the studied CpG-ODNs. VLPs did not have such repressive effect. In BMDC cultures, PCV2 and most of CpG-ODNs were able to inhibit IFN-alpha secretion induced by PRV. Interestingly, CpG-ODNs with inhibitory effect were located within the PCV2 Rep gene. Additionally, PCV2 virus was a very strong IL-12 inducer in BMDC cultures. Whereas, IFN-alpha modulation on BMDC after PCV2 VLP treatment was neglectable, PCV2 VLPs were potent IL-12 inducers. Our data shows that PCV2 viral elements can distinctly regulate cytokine production depending on the cell population studied. Thus, the final immune response upon PCV2 infection seems to depend on the fine balance between the regulatory elements present in viral DNA and structural protein within the host immune system.     

Immunological findings in 3 dogs clinically diagnosed with allergic rhinitis

Three dogs clinically diagnosed with allergic rhinitis (AR) were examined for their immunological findings. House dust mites (HDM) such as Dermatophagoides farinae (DF) and D. pteronyssinus (DP) were identified as positive allergens in the 3 dogs with both intradermal skin test and serum antigen-specific IgE test. Lymphocyte blastogenic response of peripheral blood mononuclear cells (PBMCs) under stimulation with DF antigen in dogs with AR was higher than that in 4 healthy control dogs. Expression level of IL-4 mRNA in PBMCs obtained from the 3 AR dogs was higher than that in PBMCs obtained from 4 healthy control dogs before and after stimulation with DF antigen. Expression level of IFN-gamma mRNA in PBMCs was not different between the AR and control dogs before and after stimulation with DF antigen. These results suggested that allergic reaction to HDM antigen and T(H)2-type immune response were associated with the development of AR in 3 dogs examined in this study.     

Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs within certain flanking base pairs are recognized as a danger signal by the innate immune system of vertebrates. Using lymphocyte proliferative response (LPR) and IFN-gamma secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on bovine peripheral blood mononuclear cells. ODN composed of a nuclease resistant phosphorothioate backbone and a leading 5'-TCGTCGTT-3' motif with two 5'-GTCGTT-3' motifs were highly stimulatory in both assays. Flow cytometric analysis and cell-specific surface marker labeling determined that B-cells (surface IgM(+)) were the primary cell population responding in the LPR assay. Depletion of T cells (CD3(+)) from the PBMC population did not affect IFN-gamma secretion or B-cell proliferation when cultured with CpG-ODN. However, depletion of monocytes (DH59B(+)) completely abrogated the ability of CpG-ODN to stimulate IFN-gamma secretion, and significantly reduced the B-cell proliferative response. These data establish the identity of an optimal immunostimulatory CpG motif for cattle and demonstrate that monocytes play a pivotal role in the ability of cell populations to respond to CpG-ODN. These data provide insight for future studies investigating the mechanism of CpG-ODN bioactivity and its application in novel vaccine formulations and immunotherapy.     

Antigen-specific regulatory T cells in bovine paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) is an intracellular pathogen that survives in the host's intestinal macrophages and causes chronic enteritis in ruminants. The subclinical stage of MAP infection is accompanied by loss of pro-inflammatory T(H)1 response, and a predominant, but ineffective, antibody-mediated T(H)2 response. How MAP interacts with the bovine immune system and suppresses T(H)1 responses is unclear. Studies carried out in our lab and others indicate that when peripheral blood mononuclear cells (PBMCs) from subclinical MAP-infected cattle are stimulated with MAP-antigen, IL-10 is up-regulated and leads to suppression of IFN-gamma expression in MAP-antigen-reactive effector T cells. IL-10 up-regulation and reduction in IFN-gamma would favor MAP survival and proliferation in macrophages. Depletion studies in PBMCs from MAP-infected cattle also revealed that the MAP responsive T-cell population that produces IL-10 is CD4(+) and CD25(+). Therefore, we hypothesize that cattle infected with MAP develop regulatory T (Treg) cells capable of producing IL-10 that in turn limits peripheral and tissue-specific T(H)1 immune responses. The aim of this review is to summarize current thinking regarding Treg cells and provide preliminary evidence that infection of cattle with MAP may lead to development of Treg cells.     

Quantitative analysis of inflammatory and immune responses in dogs with gastritis and their relationship to Helicobacter spp. infection

The present study sought to quantitatively examine mucosal inflammatory and immune responses in dogs with gastritis and the relationship of these responses to infection with Helicobacter. Gastric biopsies from 30 dogs were evaluated for B- and T-lymphocytes, neutrophils, eosinophils, macrophages, and mast cells. Mucosal atrophy, fibrosis, cellularity, and severity of gastritis were graded qualitatively. Messenger-RNA (mRNA) for actin, interleukin-1beta (IL-1beta), IL-4, IL-8, and IL-10, transforming growth factor beta (TGF-beta), and interferon gamma (IFN-gamma) was quantified by polymerase chain reaction (PCR). The presence of Helicobacter spp. was determined by urease activity, histology, PCR, and enzyme-linked immunosorbent assay. mRNA for IL-1beta, IL-8, IL-10, TGF-beta, and IFN-gamma was detected in most dogs. IL-4 mRNA was detected in only 1 dog. Correlations were observed for IL-1beta versus IL-8 and IL-10; IL-8 versus IL-10, IFN-gamma, and TGF-beta; and IL-10 versus IFN-y. Mucosal pathology was related to cytokine mRNA expression (neutrophils to IL-8 and IFN-gamma, macrophages and lymphocytes to IFN-gamma, and fibrosis to IL-1beta). Gastritis was categorized as lymphoplasmacytic in all dogs, and its histologic severity correlated with atrophy, infiltration with lymphocytes and macrophages, and expression of IL-10 and IFN-gamma. Of the dogs examined, 76.7% were infected with Helicobacter spp. Infection was associated with increased expression of TGF-beta and fibrosis. Circulating anti-Helicobacter immunoglobulin G titers were higher in uninfected than infected dogs. We conclude that lymphoplasmacytic gastritis in dogs is characterized by concurrent activation of proinflammatory and immunomodulatory cytokines, with increased mRNA expression related to mucosal pathology. No significant associations between Helicobacter infection and proinflammatory cytokine expression, severity of gastritis, or differences in the pathogenicity of different Helicobacter spp. were found.     

Cyclosporine A inhibits the mRNA expressions of IL-2, IL-4 and IFN-gamma, but not TNF-alpha, in canine mononuclear cells

The effects of the calcineurin inhibitors cyclosporine A (CsA) and FK506 on the mRNA expressions of various cytokines were evaluated in dogs to determine whether the effects of CsA and FK506 in dogs were similar to those in humans. The mRNA expression levels of the cytokines IL-2, IL-4, IFN-gamma and TNF-alpha were measured in PHA-stimulated canine PBMC using real-time RT-PCR after incubation with CsA or FK506 for 5 hr. Both reagents inhibited IL-2, IL-4 and IFN-gamma mRNA expressions in a dose-dependent manner. However, CsA hardly inhibited the mRNA expression of TNF-alpha. These findings are important for assessing the indications of CsA treatment in dogs.     

Increased expression of fractalkine and its receptor CX(3)CR1 in canine inflammatory bowel disease and their possible role in recruitment of intraepithelial lymphocytes

The interaction between fractalkine/CX(3)CL1 and its receptor CX(3)CR1 has been reported to play an important role in various human inflammatory diseases, including inflammatory bowel disease (IBD) mediated by lymphocyte chemoattraction. The objective of this study was to investigate the role of fractalkine and CX(3)CR1 in lymphocyte migration in canine IBD. IBD was diagnosed in 34 dogs, and 19 healthy beagles were used as normal controls. We quantified intestinal mRNA and protein expression of fractalkine and CX(3)CR1 by real-time RT-PCR and ELISA, respectively, and examined the localization of fractalkine in canine intestine by immunohistochemistry. The expression of CX(3)CR1 and surface antigens on peripheral blood mononuclear cells (PBMCs) and intraepithelial lymphocytes (IELs) was analyzed by flow cytometry. Intestinal fractalkine and CX(3)CR1 mRNA was significantly up-regulated in IBD dogs compared with the healthy control dogs. In addition, fractalkine expression on intestinal epithelial cells was significantly increased in the intestinal mucosa of IBD dogs compared with the healthy dogs. CX(3)CR1(+) PBMCs were significantly elevated in IBD dogs and positively correlated with the histopathological severity of IELs infiltration. These CX(3)CR1(+) PBMCs predominantly expressed markers for cytotoxic T cells. Almost all IELs expressed CD3, and the majority of cells expressed CD8 rather than CD4, which was analogous to the CX(3)CR1(+) PBMCs. These results suggest that the fractalkine-CX(3)CR1 interaction may contribute to the pathogenesis of canine IBD through migration of IELs.     

Expression of interleukin 4, interleukin 4 splice variants and interferon gamma mRNA in calves experimentally infected with Fasciola hepatica

Interleukin 4 (IL-4) is expected to play a dominant role in the development of T helper (Th) 2 cells. Th2 immune responses with expression of relatively large amounts of interleukin 4 (IL-4) but little interferon gamma (IFN-gamma) are characteristic for chronic helminth infections. But no information is available about IL4 expression during early Fasciola hepatica (F. hepatica) infections in cattle. Therefore, we investigated F. hepatica specific IL-4 and IFN-gamma mRNA expression in peripheral blood mononuclear cells (PBMCs) from calves experimentally infected with F. hepatica. Cells were collected prior to infection and on post-inoculation days (PIDs) 10, 28 and 70. Interestingly, PBMCs responded to stimulation with F. hepatica secretory-excretory products (FhSEP) already on PID 10 and expressed high amounts of IL-4 but not of IFN-gamma mRNA suggesting that F. hepatica induced a Th2 biased early immune response which was not restricted to the site of infection. Later in infection IL-4 mRNA expression decreased whereas IFN-gamma mRNA expression increased slightly. Isolated lymph node cells (LNCs) stimulated with FhSEP and, even more importantly, non-stimulated LN tissue samples indicated highly polarized Th2 type immune responses in the draining (hepatic) lymph node, but not in the retropharyngeal lymph node. During preliminary experiments, two splice variants of bovine IL-4 mRNA, boIL-4delta2 and boIL-4delta3, were detected. Since a human IL-4delta2 was assumed to act as competitive inhibitor of IL-4, it was important to know whether expression of these splice variants of bovine IL-4 have a regulatory function during an immune response to infection with F. hepatica. Indeed, IL-4 splice variants could be detected in a number of samples, but quantitative analysis did not yield any clue to their function. Therefore, the significance of bovine IL-4 splice variants remains to be determined.     

CpG oligodeoxynucleotides augment the immune responses of piglets to swine Pasteurella multocida living vaccine in vivo

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) prevent development of T-helper type 2 (Th2) immune response and reverse established allergic responses in mouse models. However, little work on immune responses in piglets has been conducted in vivo. In this report, the ability of a porcine-specific CpG ODN to act as an immunostimulant and enhance immune responses of piglets to swine Pasteurella multocida living vaccine (SPML vaccine) was determined. The titre of IgG and IgG1/IgG2 isotype to SPML vaccine in serum, the proliferation of lymphocytes, SPML-specific interferon-gamma (IFN-gamma) and IL-6, TNF-alpha, IL-4 production of PBMCs in vitro and IFN-gamma, IL-6, TNF-alpha, IL-4, IL-10 in piglets serum were examined to identify the immune responses of the piglets. Immune responses of the piglets vaccinated with SPML and CpG ODN were significantly stronger than responses of piglets vaccinated with SPML alone. All these data summarized that immunostimulatory CpG ODN could modulate the immune response towards a Th1-like response when co-administered to piglets during SPML vaccination, which suggested that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.