Longitudinal analysis of cytokine gene expression and parasite load in PBMC in Leishmania infantum experimentally infected dogs

Canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, an intracellular protozoan parasite that causes a severe infectious disease. To evaluate the gene expression profile associated to CVL in vivo, we have measured monthly by real-time PCR over one year the IL-4, IL-10, IL-12, IL-13, IFN-gamma, TGF-beta and TNF-alpha mRNA levels in peripheral blood mononuclear cells in 6 experimentally infected dogs that exhibited different progressions of the illness. While in two dogs no parasite, or a very low number of parasites, was detected and the two dogs did not show any clinico-pathological abnormalities at the end of the study (L dogs), for the remaining dogs high parasite loads were detected and they developed clinical leishmaniasis (H dogs). The L dogs have null expression of both IL-4 and IL-13 for the first 4 months after the infection, whereas an early IL-4 and IL-13 expression occurs in this period of infection in most of the dogs that developed clinical leishmaniasis (H dogs). Furthermore, a higher IFN-gamma expression was associated with the increase of parasite load and clinical status in these dogs. Moreover, the high variability of expression at the pre-infection stage causes us to reject the possibility that the basal levels of these cytokines indicate the prognosis of the subsequent response against infection.     

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.     

A role for balance of interferon-gamma and interleukin-4 production in protective immunity against Neospora caninum infection

A suitable balance in the production of Th1/Th2-type cytokines has a crucial role in the control of microbial infections. We investigated cytokine production patterns and effects during Neospora caninum infection, based on two mouse models and an in vitro system. In the acute infection of N. caninum, BALB/c-background IFN-gamma-deficient mice that were sensitive to the N. caninum infection showed high levels of IL-10 production, whereas significant levels of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production were observed in resistant wild type mice. BALB/c mice vaccinated with recombinant vaccinia virus expressing N. caninum surface protein NcSRS2 resisted parasite spread throughout the body, low levels of IFN-gamma production and high levels of IL-4 production were observed compared to unvaccinated animals. The treatment of N. caninum-infected cells with IFN-gamma or IL-10 decreased the host-cell viability in an in vitro system using mouse macrophage J774A.1 cells. On the other hand, IL-4, but not IL-10 administration, increased the viability of N. caninum-infected and IFN-gamma-treated cells. In the light of the balance of Th1/Th2-type cytokine production, an IFN-gamma/IL-4 balance may have a crucial role for the control of cellular responses against the parasite invasion.     

Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs

The factors responsible for the clinical progress of visceral leishmaniasis (VL) in dogs have not been yet established. The starting hypothesis was the possibility of associating the changing level of a specific type of cytokines with the evolution of the infection towards infection-manifested disease or resistant behaviour. For this purpose the authors have established a connection between Leishmania load, cytokine mRNA accumulation, and the progression of the disease in naturally infected asymptomatic dogs. We made use of real-time (RT) PCR system to detect the expression of cytokine mRNA levels during all the phases of the infection. In particular, we measured the amount of parasites in samples such as blood, lymph nodes and skin, and the expression levels of IFN-gamma, IL-2, IL-4, IL-10, IL-12 and IL-18 cytokines in the blood. We employed different targeted real-time PCR assay on 40 naturally infected dogs, initially asymptomatic; 20 of these progressed to overt disease, and the 20 remaining dogs remained asymptomatic throughout the period of study (2 years). Two other groups included: 20 naturally infected dogs with clinical signs of VL, and 20 healthy dogs living in a non-endemic area. All these animals were employed as positive and negative controls, respectively. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels represents a reliable tool for predicting disease development, and thus for choosing the best treatment for the asymptomatic form of the disease.     

Development and validation of puma (Felis concolor) cytokine and lentivirus real-time PCR detection systems

Studies of immune correlates of disease outcome associate humoral immune response mediated by T-helper 2 cytokines (IL-4, IL-10) with more virulent disease relative to a cell-mediated response driven by T-helper 1 cytokines (IL-2, IFN-gamma), particularly in viral and other intra-cellular infections. Specifically, the kinetics of both human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV) infection are closely associated with Type 1 versus Type 2 cytokine profiles. Puma (Felis concolor) lentivirus (PLV) is closely related to FIV, but based on phylogenetic and clinical studies, is more ancient and less pathogenic. The aims of this study were to validate feline real-time PCR primer/probe systems for puma cytokines and PLV as sensitive, quantitative assays for use in investigations of PLV pathogenicity. We demonstrate that primer/probe systems for IL-4, IL-10, IFN-gamma, TNF-alpha, GAPDH, and the pol region of PLV-1695 amplify puma cytokines and PLV-1695 with high amplification efficiency and sensitivity. Detection of PLV-1695 provirus in experimentally inoculated domestic cats proved to be of equivalent sensitivity, specificity, and positive and negative predictive value to co-culture of one million peripheral blood mononuclear cells (PBMC). Evaluation of cytokine induction during naturally occurring PLV infection will allow insight into mechanisms of host control associated with apathogenic infection. In addition, determination of viral loads during different stages of PLV infection or in different tissues from domestic cats or pumas will further elucidate capacity of these viruses to replicate and establish infection.     

Early hepatic immune response in rats infected with Fasciola hepatica

We investigated the phenotype of the T cells (CD4+ and CD8+) that produced Th1 (IFN-gamma) and Th2 cytokines (IL-4 and IL-10) during the firsttwo weeks of experimental fasciolosis in rats. We also followed the kinetics of the cytokine and proliferative responses of hepatic mononuclear cells (HMNC) over the same period. We found that HMNC were more numerous in the infected animals than in the controls. The percentage of CD4+ cells increased significantly after infection, whereas the percentage of CD8+ cells did not change. Moreover, the frequency of the cells producing (CP) cytokine changed after infection. The frequency of CP IFN-gamma on 7 days postinfection (pi) was similar to that in control animals. However, the frequency of CP IFN-gamma was clearly lower on day 14 pi, whereas the frequency of CP IL-4 and CP IL-10 had increased. The CP IL-10-were mostly CD4+. Mitogenic stimulation (phorbol myristate acetate/ionomycin) of HMNC led to an increase in the amounts of the Th2 cytokines in the supernatant on days 7 and 14 pi, with the increase more pronounced on day 14. In contrast, IFN-gamma levels also increased by day 7 pi but then decreased to below control levels by day 14. In addition, HMNC proliferation in response to mitogen followed a similar pattern to IFN-gamma production. These findings suggested that, during the first 2 weeks of infection, F hepatica induced a transient ThO cytokine profile followed by downregulation of the cellular response and the induction of a Th2 cytokine profile.     

Immune response of gnotobiotic piglets against Mycoplasma hyopneumoniae

In this study, several cytokine responses were investigated during Mycoplasma hyopneumoniae (Mhp) infection using a gnotobiotic infection model. We found that several inflammatory cytokines (IL-1beta, IL-8, IL-18, and TNF-alpha) and an anti-inflammatory cytokine IL-10 were induced from peripheral blood mononuclear cells (PBMC) of germ-free (GF) piglets stimulated with heat killed Mhp whole antigens, but no IFN-gamma and IL-4 were induced by Mhp. After the intranasal infection of Mhp, IL-1beta, IL-8, IL-18, and IFN-gamma were also detected in the broncho-alveolar lavage fluids (BALF). The antigen-specific IFN-gamma and IL-10 responses after infection of Mhp were gradually suppressed during Mhp infection as well as non-specific immune response to concanavalin A (ConA) and lipopolysacchalide (LPS) at early stage of infection. These results suggested that Mhp infection modulates the immune response of pigs by inducing several cytokines, and causes immuno-suppression of pigs in a gnotobiotic condition.     

Toxocara canis infection induces antigen-specific IL-10 and IFNgamma production in pregnant dogs and their puppies

Toxocara canis (T. canis) is originally a parasite of canine bitches and their pups. The pathogenicity of T. canis infection is enhanced during pregnancy and puppyhood. The aim of this study was to investigate if modification of IFNgamma and IL-10 secretion occurs during infection in pregnant dogs and puppies. Analysis of cytokines secreted could let us hypothesize a role for IL-10 and/or IFNgamma in T. canis infection. We tested T. canis-specific production of IFNgamma and IL-10 by lymphocytes of pregnant dogs and their puppies after in vitro re-exposure to purified excretory/secretory antigen (ESAg) from T. canis. Blood mononuclear cells (BMC) isolated from pregnant dogs and their puppies were cultured in the presence of ESAg. Cultures' supernatants were tested for cytokine levels by ELISA. Results obtained showed that IL-10 concentrations increased during pregnancy in infected animals and in the meantime IFNgamma production decreased. In puppyhood, we observed that, IL-10 concentration decreased with the age of puppies mainly in infected animals while IFNgamma increased. In conclusion, our data suggests that BMC of infected dogs have a particular modification of IL-10 and IFNgamma synthesis. These data could be the basis to design immunotherapeutic approaches.     

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.     

Cytokine and antibody subclass responses in the intestinal lymph of sheep during repeated experimental infections with the nematode parasite Trichostrongylus colubriformis

The expression of interleukin (IL)-4, IL-5, IL-10, IL-13, TNF-alpha and IFN-gamma genes, and parasite-specific IgM, IgG1, IgG2, IgA and total IgE levels, were monitored daily in intestinal lymph of sheep infected repeatedly with the nematode parasite Trichostrongylus colubriformis. Host genotype had a significant influence on IL-13 gene activity, with resistant-line (R) sheep consistently expressing higher levels of mRNA than susceptible-line (S) sheep. Mean gene expression of IL-13, IL-4 and IFN-gamma did not differ significantly between the first and second nematode challenge. Field-primed R and S as well as field-primed R and na?ve S sheep had lower mean gene expression of IL-5 and IL-10, respectively, during the second when compared to primary challenge. Genes for IL-13 and IL-5 were transiently and strongly up-regulated after nematode infection, particularly in animals with previous exposure to nematodes. Genes for TNF-alpha and IFN-gamma were also transiently up-regulated, but to a lesser extent and more typically after primary challenge. Na?ve sheep of both genotypes produced relatively little antibody response after primary challenge. A second nematode challenge resulted in large increases in the lymphatic levels of all antibody sub-classes which were significant for adult antigen-specific IgA and larval antigen-specific IgG1. In na?ve S line sheep, the larval-specific IgA and IgG2 response appeared delayed when compared to the R line animals. Field-primed R and S line sheep had relatively high lymphatic IgG1 levels prior to experimental infection and these did not change significantly afterwards. These results demonstrate that during nematode infections, the intestinal micro-environment of sheep is transiently skewed towards Th2 cytokine dominance, although IFN-gamma gene expression continues. This response is accompanied by increases of nematode-specific IgG1, IgA, IgG2 and IgM, as well as of total IgE in lymph plasma.     

Immunological responses and cytokine gene expression analysis to Cooperia punctata infections in resistant and susceptible Nelore cattle

Cellular and humoral immune response, as well as cytokine gene expression, was assessed in Nelore cattle with different degrees of resistance to Cooperia punctata natural infection. One hundred cattle (male, weaned, 11-12 months old), kept together on pasture, were evaluated. Faecal and blood samples were collected for parasitological and immunological assays. Based on nematode faecal egg counts (FEC) and worm burden, the seven most resistant and the eight most susceptible animals were selected. Tissue samples of the small intestine were collected for histological quantification of inflammatory cells and analysis of cytokine gene expression (IL-2, IL-4, IL-8, IL-12p35, IL-13, TNF-alpha, IFN-gamma, MCP-1, MCP-2, and MUC-1) using real-time RT-PCR. Mucus samples were also collected for IgA levels determination. Serum IgG1 mean levels against C. punctata antigens were higher in the resistant group, but significant differences between groups were only observed 14 days after the beginning of the experiment against infective larvae (L3) and 14 and 84 days against adult antigens. The resistant group also presented higher IgA levels against C. punctata (L3 and adult) antigens with significant difference 14 days after the beginning of the trial (P<0.05). In the small-intestine mucosa, levels of IgA anti-L3 and anti-adult C. punctata were higher in the resistant group, compared with the susceptible group (P<0.05). Gene expression of both T(H)2 cytokines (IL-4 and IL-13) in the resistant group and T(H)1 cytokines (IL-2, IL-12p35, IFN-gamma and MCP-1) in the susceptible group was up-regulated. Such results suggested that immune response to C. punctata was probably mediated by T(H)2 cytokines in the resistant group and by T(H)1 cytokines in the susceptible group.     

Systemic cytokine profile in feeder pigs suffering from natural postweaning multisystemic wasting syndrome (PMWS) as determined by semiquantitative RT-PCR and flow cytometric intracellular cytokine detection

Postweaning multisystemic wasting syndrome (PMWS) is an economically important disease in pigs caused by porcine circovirus type 2 (PCV2). Development of this disease is presumably associated with an impairment of the immune system. We, therefore, investigated the systemic expression of relevant cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, TNF-alpha, IFN-gamma) and IL-2Ralpha at mRNA (semiquantitative RT-PCR) and at protein level (flow cytometric intracellular cytokine detection after short-time stimulation of peripheral blood mononuclear cells) in 10 feeder pigs aged 14 weeks suffering from natural PMWS and in 10 clinically healthy pen-mates. Hematological examination revealed a significant (p < 0.001) relative lymphopenia in the diseased animals when compared to reference pigs. IL-1alpha and IL-10 mRNA levels were notably increased in the affected pigs, whereas IL-2 and IL-2Ralpha (CD25) mRNA levels tended to be down-regulated. IL-8, TNF-alpha and IFN-gamma mRNA expressions appeared to be slightly increased. Intracellular cytokine levels as measured by flow cytometry revealed an increase of IL-1beta, IL-2, and IL-6, whereas IL-12 and TNF-alpha expressions were not affected. IFN-gamma was slightly decreased in the diseased animals. In conclusion, despite the assumption, that the cellular immune response to PMWS as a virus-induced disease should be characterized by either a Th1 driven cytokine profile or a cytokine profile indicative of T cell immunosuppression, our results did not support that hypothesis. Nevertheless, data from intracellular cytokine detection suggest an even increased percentage of the remaining lymphocytes capable to produce IL-2 upon in vitro stimulation, which is in contrast to the slightly diminished IL-2 mRNA levels reflecting the in vivo situation at least at the mRNA level.