Induction of proinflammatory cytokines and caspase-1 by leptin in monocyte/macrophages from holstein cows

The proliferation of peripheral blood mononuclear cells (PBMC) containing both monocyte/macrophages and T lymphocytes increased after treatment with T-cell mitogen (concanavalin A: Con A). PBMC treated with either leptin alone or combination of leptin and ConA showed enhanced proliferative activity by 10-40%, compared with those treated with ConA alone. In contrast, isolated T lymphocytes treated with leptin and ConA showed lowered proliferative activity than the ConA-treated alone, indicating that leptin induced production of some cytokines from monocyte/macrophages, that subsequently resulted in enhancement of T lymphocytes proliferation in PBMC. Among the cytokines examined, monocyte/monocytes constitutively expressed interleukin (IL)-1beta, IL-12p35, IL-18 mRNA, and faintly expressed tumor necrosis factor (TNF)-alpha and IL-12p40 mRNA. Leptin treatment augmented the monocyte/macrophages mRNA expression of only TNF-alpha and IL-12p40 to comparable levels of cells treated with lipopolysaccharide (LPS). However, leptin treatment increased monocyte/macrophages production of IL-1beta as well as TNF-alpha, and induced the mRNA expression of caspase-1, which is shown to mediate the conversion of latent pro-IL-1beta and pro-IL-18 to active forms. These results suggest that leptin directly acts on monocyte/macrophages to produce factors that induce T lymphocytes proliferation such as IL-12p35/p40 complex through IL-12p40 induction and IL-1beta/IL-18 production through caspase-1 induction.     

Development of a microarray for studying porcine cytokine production in blood mononuclear cells and intestinal biopsies

A microarray for demonstration of a limited number of porcine cytokines was initiated. Polymerase chain reaction (PCR) products were synthesized for four house-keeping genes, cyclophilin, beta-actin, hypoxanthine phosphoribosyltransferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the following cytokines: interleukin (IL)-1beta, IL-4, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, IL-18, interferon (IFN)-alpha, IFN-beta, IFN-gamma, tumour necrosis factor (TNF)-alpha, macrophage inhibition factor (MIF) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Cytokine production was induced by incubation of porcine peripheral blood mononuclear cells (PBMC) with Concanavalin A (ConA) or oligodeoxynucleotide (ODN) 2216. RNA was isolated after 6 or 24 h from stimulated cells or unstimulated control cells and from intestinal biopsies. Cytokine expression was analysed using a 3-DNA Array 350(TM) labelling kit from Genisphere. Data were normalized using external control genes and analysed with the genepix pro 5.0 software. All the cytokines could be induced in PBMC and expressed on the array and the cytokines IL-6 and IFN-alpha were also analysed at protein level. All but one cytokine were expressed in samples from intestinal biopsies. Densitometric analyses of PCR products of the house-keeping genes were performed to validate the results from the microarray. Thus, this microarray will enable analyses of the cytokine profile during local and systemic infections in the pig.     

Differential production of proinflammatory cytokines: in vitro PRRSV and Mycoplasma hyopneumoniae co-infection model

An in vitro culture system was developed to investigate the induction of proinflammatory cytokines by Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV). M. hyopneumoniae infected porcine tracheal ring explants were co-cultured with PRRSV infected pulmonary alveolar macrophages (PAMs) for 24h to assess the cytokine production of each pathogen alone and the interaction between the two pathogens in vitro. Semiquantitative RT-PCR was used to measure interleukin (IL) 1alpha, IL1beta, IL6, IL8, IL10, IL12 and tumor necrosis factor (TNF) alpha mRNA in PAMs. Commercial ELISAs were used to measure soluble IL1beta, IL8, IL10 and TNF in the culture supernatant. In the dual infected group, mRNA expression of IL1alpha, IL1beta, IL8 and TNF was increased. Both the M. hyopneumoniae- and PRRSV-infected only groups tended to have increased expression of IL1alpha, IL1beta and IL8 mRNA, although no statistical difference was observed. Increased levels of IL1beta, IL8 and IL10 were present in the supernatant of the dual infected group as measured by ELISA. No increase in soluble TNF was observed in any of the groups. IL8 levels appeared high in all groups independent of infection status. The cause of the elevated IL8 was unknown, however, it may have been a non-specific response by the cells to tissue damage during the harvesting of the tracheal rings. Correlation between mRNA expression and the soluble cytokine levels were similar in the dual infected groups with the exception of IL10 and TNF. Levels of mRNA and soluble protein levels in the single pathogen infected groups were not as consistent. The increased production of proinflammatory cytokines IL1alpha, IL1beta, IL8 and TNF in the group infected with both M. hyopneumoniae and PRRSV suggests that cytokine induced inflammation may play an important role in the severe, chronic pneumonia induced by the concurrent infection of the two pathogens.     

Molecular cloning and phylogenetic analysis of inflammatory cytokines of the ferret (Mustela putorius furo)

The present study determined the cDNA and deduced amino acid sequences of ferret (Mustela putorius furo) inflammatory cytokines, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor (TNF)-alpha. The homologies of the nucleotide sequences of IFN-gamma, IL-1beta, IL-6, IL-8 and TNF-alpha of the ferret to those from other mammalian species ranged from 64.3-92.9%, 73.0-83.9, 58.1-84.8%, 58.1-89.7% and 79.0-95.0%, respectively. As distinctive amino acid residues constituting various motifs and ligand-binding sites and cysteine residues were highly conserved in ferret inflammatory cytokine proteins, ferret cytokines may have fundamentally similar functions to those of other mammals. Phylogenetic analyses based on the deduced amino acid sequences revealed that all ferret inflammatory cytokines were more closely related to those of the Carnivora order, specifically dog and cat, than to other species.     

The Effects of Selenium Source on Measures of Selenium Status of Mares and Selenium Status and Immune Function of Their Foals

The effect of organic and inorganic sources of selenium (Se) on measures of Se status of mares and their foals, Se concentrations of colostrum and milk, and indices of immune function of foals was studied. Twenty late-gestation Standardbred mares were randomly assigned to one of two groups. All mares received an identical balanced ration, except for the source of supplementary Se: one group received organic Se (Se yeast) and the other group received inorganic Se (sodium selenite), fed to deliver 0.3 mg/kg supplementary Se based on total ration dry matter. Mares received the experimental diet from 2 months before estimated due date until 1 month after foaling. Source of Se did not affect Se concentrations in maternal plasma, red blood cells, colostrum, or milk. At 1 month of age, foals from mares fed organic Se had higher red blood cell Se concentration than foals from mares fed inorganic Se (P < .05). Measures of immunity included serum immunoglobulin G concentration, lymphocyte proliferation in response to concanavalin A, and relative cytokine gene expression of stimulated lymphocytes (interferon gamma [IFNγ], interleukin [IL]-2, IL-5, IL-10, tumor necrosis factor alpha [TNFα]) and neutrophils (IL-1, IL-6, IL-8, IL-12, TNFα). Relative gene expression of IL-2, TNFα, and IFNγ by foal lymphocytes was associated with the source of Se supplementation provided to the mares. We conclude that the source of dietary Se provided to mares may influence the immune function of foals at 1 month of age through changes in relative gene expression of certain lymphocyte cytokines.     

Cytokines and acute phase proteins associated with acute swine influenza infection in pigs

This study set out to investigate the cytokines and acute phase proteins (APPs) associated with the acute stages of experimentally-induced swine influenza virus (SIV) infection in 3-week-old, colostrum-deprived, caesarean-derived piglets. The piglets were inoculated intratracheally with 10^7.5 50% egg infective dose [EID₅₀] Swine/Belgium/1/98 (H1N1) SIV and were euthanased at time-points between 0 and 120 h post-inoculation (PI). Broncho-alveolar lavage fluid (BALF), lung homogenates and sera were examined for inflammatory mediators by bioassay or ELISA. Interferon (IFN)-α, interleukin (IL)-6, IL-1 and tumour necrosis factor (TNF)-α peaked in BALF 24–30 h PI, when virus titres and the severity of clinical signs were maximal.Whereas IFN-γ and IL-12, but not IL-18, increased in tandem in BALF, serum cytokine concentrations were either undetectable or were up to 100-fold lower. The APP C-reactive protein (CRP) and haptoglobin peaked 24 h later than the cytokines and reached higher levels in serum than in BALF. In contrast, lipopolysaccharide (LPS)-binding protein (LBP) only increased in BALF. Lung virus titres tightly correlated with BALF IFN-α, IL-6, IL-1, TNF-α, IFN-γ and IL-12, as well as with serum IL-6, IFN-α and IFN-γ. Signs of disease correlated with the same cytokines in BALF and serum, as well as with BALF LBP and serum CRP. The findings suggest that IFN-γ and IL-12 play a role in the pathogenesis of SIV and that APPs are induced by cytokines. This influenza infection model may have value in assessing the therapeutic potential of cytokine antagonists.     

Proinflammatory cytokine responses of cultured equine keratinocytes to bacterial pathogen‐associated molecular pattern motifs

Reasons for performing study: Further knowledge of equine keratinocyte physiology and keratinocyte response to various stimuli is important in developing a better understanding of disease states involving the epidermis. Objectives: To assess the inflammatory cytokine response of cultured equine keratinocytes to various pathogen‐associated molecular pattern molecules (PAMPs) from both Gram‐negative and positive bacteria likely to be present in equine sepsis. Methods: Keratinocytes were isolated from skin of 2 horses and primary cultures performed. Keratinocytes were harvested for RNA extraction after exposure to lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), bacterial DNA (CpG), flagellin or maintained in medium (controls) for 4 or 24 h. Real time‐quantitative PCR was used to quantify interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and CXCL8 mRNA concentrations. Results: Increases (P<0.05) in IL‐1β, IL‐6 and CXCL8 mRNA concentrations were induced by LPS exposure compared to controls. Increased mRNA concentrations of both IL‐6 and CXCL8 were also noted (vs. controls) upon exposure to flagellin. Overall, responses were greater at 4 h. No increases (P>0.05) in cytokine expression by keratinocytes were present after LTA, PGN or CpG exposure. Conclusions: Increased proinflammatory cytokine expression in response to LPS and flagellin indicate that equine keratinocytes have functional TLR4 and TLR5 receptor signalling. However, the lack of keratinocyte stimulation by PGN, LTA or CpG provides no evidence for functional TLR2, TLR9 or NOD receptor signalling. These results suggest that equine keratinocytes are more responsive to PAMPs usually associated with Gram‐negative sepsis and unresponsive to PAMPs most commonly associated with Gram‐positive sepsis. Potential relevance: The increased incidence of injury of epidermal structures in clinical cases of Gram‐negative (vs. Gram‐positive) sepsis in the horse may be due to a lack of functional TLR signalling for Gram‐positive PAMPs in the equine keratinocyte.     

The impact of weight loss on circulating cytokines in Beagle dogs

Chronic low-grade inflammation in obesity is characterized by an increased production of pro-inflammatory and chemotactic cytokines that are contributing to insulin resistance and related co-morbidities. Cytokines act in networks and exhibit pleiotropic effects so we investigated the circulating levels of a wide array of cytokines (pro and anti-inflammatory, chemotactic and growth factors) in a canine model of weight loss. The dogs served as their own control in order to study the impact of weight loss independent of potential confounding factors, such as history of excess weight or gender. While low-grade inflammation had been previously investigated in obese dogs by measuring changes in adipokines, acute phase proteins and key pro-inflammatory cytokines, to the best of our knowledge this is the first study to evaluate how weight loss impacts a wide array of circulating cytokines.Eighteen overweight Beagle dogs were recruited (six spayed females and 12 neutered males), and none of them were grossly obese according to the body condition score (BCS). All the dogs reached an ideal weight by the end of the program. Parameters were assessed before (baseline), at mid-point (month 3) and at end-point (month 6). Plasma GM-CSF, IL-2, Il-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, IFNγ, IP-10, TNFα, monocyte chemotactic protein 1 (MCP-1), keratinocyte chemokine (KC) were measured with canine multiplex immunoassays. Fat mass was assessed by dual energy X-ray absorption (DEXA).Several cytokines decreased throughout the weight loss program (p < 0.01) and were correlated with the percentage of fat measured by DEXA (p < 0.05): chemotactic (MCP-1), growth factors (GM-CSF, IL-7 and IL-2), and pro-inflammatory (KC and IL-18). We could not show trends for several cytokines, possibly because their level may be lower than the assay sensitivity: anti-inflammatory (IL-4 and IL-10), and pro-inflammatory (IL-6 and TNFα).In conclusion, while our findings for several pro-inflammatory and chemotactic cytokines are in accordance with human and rodent studies, we may have identified additional cytokines, such as growth factors, related to obesity-induced low-grade inflammation. Considering the weight loss was enabled by an adjusted diet, the role of this association of cytokines in insulin resistance and related co-morbidities needs to be clarified. Our results could help better understand the cytokine biology in dogs, and as such are relevant for further elucidating the relationship between immune function and metabolism/nutrition.     

Characterization of cytokine expression in milk somatic cells during intramammary infections with Escherichia coli or Staphylococcus aureus by real-time PCR

The expression of inflammatory cytokines, including interleukin (IL)-6, IL-8, IL-12, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma, by milk somatic cells was characterized by real-time polymerase chain reaction (PCR) in dairy cows experimentally challenged with either E. coli (n = 8) or S. aureus (n = 8). The mRNA abundance of a target gene was calibrated with that of a reference gene (beta-actin) and expressed as fold of induction over the control quarter at each time point. At no single time point did all eight quarters challenged with the same type of bacteria demonstrated increased expression of a target gene and there was large variation among animals at each given time. As a consequence, most tested comparisons were not statistically significant except the peak time points of IL-8 expression (75- and 29- fold in glands challenged with E. coli and S. aureus, respectively). However, the average fold induction of all targeted cytokines was increased in response to both bacterial challenges with the exception of IFN-gamma. The expression of IFN-gamma was only increased in milk somatic cells isolated from E. coli, but not S. aureus, challenged mammary glands. Moreover, upregulated expression of cytokine genes had higher magnitudes and/or faster responses in glands challenged with E. coli in comparison with those challenged with S. aureus. We propose that the compromised upregulation of inflammatory cytokines in S. aureus infected glands may, at least partially, contribute to the chronic course of infection caused by this pathogen. Further research on identifying factors responsible for the differentially expressed cytokine profiles may be fundamental to developing strategies that mitigate the outcome of bovine mastitis.     

Cytokine induction in pulmonary airways of horses with heaves and effect of therapy with inhaled fluticasone propionate

Work in humans and laboratory animals has identified a central role for cytokines and chemokines in development and persistence of lower airway inflammation. The objectives of this study were to determine interleukin (IL)-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha induction in bronchoalveolar lavage (BAL) of control horses and horses with heaves both during remission and exacerbation of the disease, and to determine the effect of therapy with inhaled fluticasone propionate on the cytokine profile of horses with heaves. IL-1 beta and TNF-alpha mRNA expression was significantly higher in horses with heaves after exposure to moldy hay compared to either values obtained during clinical remission or to healthy controls. IL-8 mRNA expression and protein concentrations were significantly higher in horses with heaves than in controls. Both IL-4 and IFN-gamma mRNA expression was increased at various times in heaves-susceptible horses compared to controls. IL-2, IL-5 and IL-10 mRNA expression was not detected in BAL cells of either group. Therapy with inhaled fluticasone propionate after induction of a severe heaves exacerbation resulted in complete resolution of clinical signs, normalization of pulmonary function tests, and significant decrease in BAL neutrophilia. This was associated with a significant decrease in IL-4 mRNA expression and increase in IFN-gamma/IL-4 ratio in horses with heaves. These results demonstrate the clinical efficacy of inhaled fluticasone propionate for the treatment of heaves and suggest a role for cytokines in the development of lower airway inflammation in heaves-susceptible horses.     

Autologous conditioned serum: the comparative cytokine profiles of two commercial methods (IRAP and IRAP II) using equine blood

Reasons for performing study: Osteoarthritis (OA) is one of the most prevalent and debilitating conditions affecting the horse. Autologous conditioned serum (ACS), commercially available as IRAP and IRAP II, is a recently developed treatment for OA in which plasma is prepared from venous blood by incubation with glass beads for 24 h. This product has been shown to increase anti‐inflammatory cytokines and growth factors in human blood. However, data for equine ACS preparations are lacking. Objectives: To characterise the protein profiles produced by commercially available ACS systems in equine blood. Methods: Blood was drawn from 5 horses into 6 groups: red top vacutainer (control), IRAP and IRAP II, with and without heparin. Samples were collected 1 or 24 h post draw and analysed for IL‐1ra, IL‐10, IGF‐1, TGF‐β, TNF‐α and IL‐1β using ELISAs. Results: Twenty‐four hour IRAP and IRAP II samples contained significantly higher levels of all cytokines relative to 1 h serum controls. At 24 h, IRAP II contained significantly higher levels of IL‐1ra and IRAP contained significantly higher levels of TNF‐α, compared to 24 h controls. In addition, TGF‐β, IL‐10 and IL‐1β in IRAP and IRAP II sera were similar to 24 h serum controls. The addition of heparin significantly reduced levels of IGF‐1, TNF‐α and TGF‐β, and significantly elevated levels of IL‐1ra. Conclusions: The cytokine profile that IRAP II produced is modestly better than IRAP. Incubation of whole blood in glass tubes stimulated cytokine synthesis, although not as efficiently as IRAP II. Potential relevance: Although high levels of IL‐1ra were found in ACS, elevation of other factors suggests these cytokines play a previously understated role in clinical improvements. Because ACS has been shown to alleviate clinical symptoms of OA, the present study suggests that factors other than IL‐1ra alone might be involved in its clinical efficacy. Species‐dependent elevations of cytokines warrant further investigation and optimisation of the systems appears to be necessary based on the differences between human and equine blood.     

猪IL-2、IL-12p40和IFN-γ SYBR Green I real-time PCR检测方法的建立

针对猪Th1型细胞因子IL-2、IL-12p40、IFN-γ以及管家基因β-actin的基因序列分别设计一对特异性引物,构建含有各自引物扩增序列的重组质粒作为阳性标准品,建立了检测IL-2、IL-12p40、IFN-γ及β-actin的SYBR Green I real-time PCR方法。该方法线性关系好,标准曲线的相关系数均达到0.997以上;敏感性高,初始模板的检出下限均达到100拷贝/μL;特异性强,扩增产物形成单一的特异性熔解峰;重复性好,组内与组间的变异系数均小于3.5%。应用所建立的方法对猪繁殖与呼吸综合征病毒感染仔猪外周血单个核细胞中IL-2、IL-12p40和IFN-γ mRNA的表达水平进行了检测。结果表明,所建立的real-time PCR检测方法灵敏度高、特异性强、重复性好。     

Interleukin-6 and tumour necrosis factor in synovial fluid from horses with carpal joint pathology

The carpal joints are common sites of traumatic arthritis and osteoarthritis (OA) in athletic horses. The pro-inflammatory cytokines interleukin (IL)-6 and tumour necrosis factor (TNF) may be of great importance in the development of intra-articular lesions. The aim of the present study was to investigate possible associations between synovial fluid levels of bioactive IL-6 and TNF and different types of joint lesions seen in traumatic arthritis and OA. Synovial fluid was collected from horses with carpal lameness immediately before arthroscopic surgery. Articular cartilage, synovial membranes and intra-articular ligaments were assessed macroscopically at arthroscopy. Synovial fluid levels of IL-6 and TNF were determined by bioassays, and the cytokine levels between different grades of morphologic changes in each type of assessed tissue were compared. The highest levels of IL-6 were detected in joints with chip fractures. All joints with chip fractures also showed some degree of synovitis. Tumour necrosis factor bioactivity was low and not associated with any joint lesion. Hence, TNF is not useful as a biomarker indicating a specific joint lesion in equine traumatic arthritis or OA. We conclude that a dramatic increase of IL-6 in synovial fluid indicates the presence of osteochondral fragmentation, although low or undetectable levels of IL-6 do not exclude chip fractures. The role of IL-6 in the disease process of osteochondral fragmentation needs further investigation.     

Establishment of a multiplex RT-PCR assay for the rapid detection of fish cytokines

To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1β, IL-6, IL-17A/F3, IL-18, TNF-α, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-β1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-γ), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1β, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-α, TNF-N, I-IFN-1 and IFN-γ genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish.