CD4 T Cell Responses to Theileria parva in Immune Cattle Recognize a Diverse Set of Parasite Antigens Presented on the Surface of Infected Lymphoblasts.

Parasite-specific CD8 T cell responses play a key role in mediating immunity against Theileria parva in cattle (Bos taurus), and there is evidence that efficient induction of these responses requires CD4 T cell responses. However, information on the antigenic specificity of the CD4 T cell response is lacking. The current study used a high-throughput system for Ag identification using CD4 T cells from immune animals to screen a library of ∼40,000 synthetic peptides representing 499 T. parva gene products. Use of CD4 T cells from 12 immune cattle, representing 12 MHC class II types, identified 26 Ags. Unlike CD8 T cell responses, which are focused on a few dominant Ags, multiple Ags were recognized by CD4 T cell responses of individual animals. The Ags had diverse properties, but included proteins encoded by two multimember gene families: five haloacid dehalogenases and five subtelomere-encoded variable secreted proteins. Most Ags had predicted signal peptides and/or were encoded by abundantly transcribed genes, but neither parameter on their own was reliable for predicting antigenicity. Mapping of the epitopes confirmed presentation by DR or DQ class II alleles and comparison of available T. parva genome sequences demonstrated that they included both conserved and polymorphic epitopes. Immunization of animals with vaccine vectors expressing two of the Ags demonstrated induction of CD4 T cell responses capable of recognizing parasitized cells. The results of this study provide detailed insight into the CD4 T cell responses induced by T. parva and identify Ags suitable for use in vaccine development.

Tight blood glycaemic and blood pressure control in experimental diabetic nephropathy reduces extracellular matrix production without regression of fibrosis.

AIMS: Regression of albuminuria and renal fibrosis occurs in patients with diabetic nephropathy (DN) following tight control of blood glucose and blood pressure, however the pathways that promote regression remain poorly understood and we wished to characterize these using a rodent model. METHODS: Diabetes was induced with streptozotocin in Cyp1a1mRen2 rats and hypertension was generated by inducing renin transgene expression with dietary indole-3-carbinol (I-3-C) for 28 weeks. At this point an 'injury cohort' was culled, while in a 'reversal cohort' glycaemia was tightly controlled using insulin implants and blood pressure normalized by withdrawing dietary I-3-C for a further 8 weeks. Pathways activated during and following reversal of diabetes and hypertension were assessed by microarray profiling. RESULTS: Tight control of blood glucose and blood pressure reduced albuminuria and renal hypertrophy, but had no impact on renal fibrosis. 85 genes were up-regulated specifically during the injury phase, including genes encoding multiple myofibroblast and extracellular matrix (ECM) proteins. Conversely, 314 genes remained persistently elevated during reversal including genes linked to innate/adaptive immunity, phagocytosis, lysosomal processing and degradative metalloproteinases (MMPs). Despite increased MMP gene expression, MMP activity was suppressed during both injury and reversal, in association with up-regulation of tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Physical separation of the TIMP-1/MMP complexes during zymography of tissue homogenate restored MMP activity. CONCLUSION: Normalization of blood glucose and pressure ameliorates albuminuria and inhibits excess ECM production, however persistent TIMP-1 expression hinders attempts at ECM remodelling. Therapies which counteract the action of TIMPs may accelerate scar resolution.

Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis.

Apoptosis is essential for clearance of potentially injurious inflammatory cells and subsequent efficient resolution of inflammation. Here we report that human neutrophils contain functionally active cyclin-dependent kinases (CDKs), and that structurally diverse CDK inhibitors induce caspase-dependent apoptosis and override powerful anti-apoptosis signals from survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that the CDK inhibitor R-roscovitine (Seliciclib or CYC202) markedly enhances resolution of established neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice. In the pleurisy model, the caspase inhibitor zVAD-fmk prevents R-roscovitine-enhanced resolution of inflammation, indicating that this CDK inhibitor augments inflammatory cell apoptosis. We also provide evidence that R-roscovitine promotes apoptosis by reducing concentrations of the anti-apoptotic protein Mcl-1. Thus, CDK inhibitors enhance the resolution of established inflammation by promoting apoptosis of inflammatory cells, thereby demonstrating a hitherto unrecognized potential for the treatment of inflammatory disorders.

Macrophage/monocyte depletion by clodronate, but not diphtheria toxin, improves renal ischemia/reperfusion injury in mice.

The role of resident renal mononuclear phagocytes in acute kidney injury is controversial with experimental data suggesting both deleterious and protective functions. To help resolve this, we used mice transgenic for the human diphtheria toxin receptor under the control of the CD11b promoter and treated them with diphtheria toxin, or liposomal clodronate, or both to deplete monocyte/mononuclear phagocytes prior to renal ischemia/reperfusion injury. Although either system effectively depleted circulating monocytes and resident mononuclear phagocytes, depletion was most marked in diphtheria toxin-treated mice. Despite this, diphtheria toxin treatment did not protect from renal ischemia. In contrast, mice treated with clodronate exhibited reduced renal failure and acute tubular necrosis, suggesting key differences between these depletion strategies. Clodronate did not deplete CD206-positive renal macrophages and, unlike diphtheria toxin, left resident CD11c-positive cells unscathed while inducing dramatic apoptosis in hepatic and splenic mononuclear phagocyte populations. Abolition of the protected phenotype by administration of diphtheria toxin to clodronate-treated mice suggested that the protective effect of clodronate resulted from the presence of a cytoprotective intrarenal population of mononuclear phagocytes sensitive to diphtheria toxin-mediated ablation.

The cyclin-dependent kinase inhibitor R-roscovitine down-regulates Mcl-1 to override pro-inflammatory signalling and drive neutrophil apoptosis.

Successful resolution of inflammation requires inflammatory cells such as neutrophils to undergo apoptosis prior to non-inflammatory phagocytosis by professional phagocytes. Recently, cyclin-dependent kinase (CDK) inhibitors (e.g. R-roscovitine) have been shown to induce neutrophil apoptosis and enhance the resolution of inflammation. Interestingly, NF-kappaB and MAPK pathways and key endogenous survival proteins (typified by Mcl-1) are involved in the regulation of neutrophil apoptosis and, in cancer-cell lines, have been implicated as possible targets of CDK inhibitors. Here, we demonstrate that R-roscovitine over-rides TNF-alpha and LPS-induced survival (determined by morphological examination and binding of fluorescently labelled annexin-V) of isolated peripheral blood neutrophils. This effect did not appear to be mediated via effects on early markers of neutrophil activation (e.g. surface marker expression, shape change, aggregation and superoxide anion generation), by direct inhibition of NF-kappaB activation (assessed by cytoplasmic IkappaBalpha proteolysis and NF-kappaB p65 subunit translocation) and ERK activation (determined by specific ERK phosphorylation) but due to down-regulation (at protein and mRNA level) of the survival protein Mcl-1 but not the pro-apoptotic bcl-2 homologue Bim. These findings suggest that key endogenous survival proteins may be the targets of CDK inhibitors and consequently may be of critical importance in the resolution of inflammation.

Granulocyte macrophage-colony stimulating factor: A key modulator of renal mononuclear phagocyte plasticity.

Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF) play key roles in the differentiation of macrophages and dendritic cells (DCs). We examined the effect of treatment with M-CSF-containing macrophage medium or GM-CSF-containing DC medium upon the phenotype of murine bone marrow-derived macrophages and DCs. Culture of macrophages for 5 days in DC medium reduced F4/80 expression and increased CD11c expression with cells effectively stimulating T cell proliferation in a mixed lymphocyte reaction. DC medium treatment of macrophages significantly reduced phagocytosis of both apoptotic cells and latex beads and strongly induced the expression of the chemokine receptor CCR7 known to be involved in DC trafficking to lymph nodes. Lysates of obstructed murine kidneys expressed both M-CSF and GM-CSF though M-CSF expression was dominant (M-CSF:GM-CSF ratio ∼30:1). However, combination treatment with both M-CSF and GM-CSF (ratio 30:1) indicated that small amounts of GM-CSF skewed macrophages towards a DC-like phenotype. To determine whether macrophage phenotype might be modulated in vivo we tracked CD45.1+ bone marrow-derived macrophages intravenously administered to CD45.2+ mice with unilateral ureteric obstruction. Flow cytometry of enzyme dissociated kidneys harvested 3 days later indicated CD11c and MHC Class II upregulation by adoptively transferred CD45.1+ cells with CD45.1+ cells evident in draining renal lymph nodes. Our data suggests that GM-CSF modulates mononuclear phagocyte plasticity, which likely promotes resolution of injury and healing in the injured kidney.

Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype.

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

Isolation and characterization of equine native MSC populations.

BACKGROUND: In contrast to humans in which mesenchymal stem/stromal cell (MSC) therapies are still largely in the clinical trial phase, MSCs have been used therapeutically in horses for over 15 years, thus constituting a valuable preclinical model for humans. In human tissues, MSCs have been shown to originate from perivascular cells, namely pericytes and adventitial cells, which are identified by the presence of the cell surface markers CD146 and CD34, respectively. In contrast, the origin of MSCs in equine tissues has not been established, preventing the isolation and culture of defined cell populations in that species. Moreover, a comparison between perivascular CD146+ and CD34+ cell populations has not been performed in any species. METHODS: Immunohistochemistry was used to identify adventitial cells (CD34+) and pericytes (CD146+) and to determine their localization in relation to MSCs in equine tissues. Isolation of CD34+ (CD34+/CD146-/CD144-/CD45-) and CD146+ (CD146+/CD34-/CD144-/CD45-) cell fractions from equine adipose tissue was achieved by fluorescence-activated cell sorting. The isolated cell fractions were cultured and analyzed for the expression of MSC markers, using qPCR and flow cytometry, and for the ability to undergo trilineage differentiation. Angiogenic properties were analyzed in vivo using a chorioallantoic membrane (CAM) assay. RESULTS: Both CD34+ and CD146+ cells displayed typical MSC features, namely growth in uncoated tissue culture dishes, clonal growth when seeded at low density, expression of typical MSC markers, and multipotency shown by the capacity for trilineage differentiation. Of note, CD146+ cells were distinctly angiogenic compared with CD34+ and non-sorted cells (conventional MSCs), demonstrated by the induction of blood vessels in a CAM assay, expression of elevated levels of VEGFA and ANGPT1, and association with vascular networks in cocultures with endothelial cells, indicating that CD146+ cells maintain a pericyte phenotype in culture. CONCLUSION: This study reports for the first time the successful isolation and culture of CD146+ and CD34+ cell populations from equine tissues. Characterization of these cells evidenced their distinct properties and MSC-like phenotype, and identified CD146+ cells as distinctly angiogenic, which may provide a novel source for enhanced regenerative therapies.

Oxygen levels determine the ability of glucocorticoids to influence neutrophil survival in inflammatory environments.

GCs are highly effective in treating a wide range of inflammatory diseases but are limited in their ability to control neutrophilic lung inflammation in conditions such as COPD. Neutrophil apoptosis, a central feature of inflammation resolution, is delayed in response to microenvironmental cues, such as hypoxia and inflammatory cytokines, present at inflamed sites. GCs delay neutrophil apoptosis in vitro, and this may therefore limit the ability of GCs to control neutrophilic inflammation. This study assesses the effect GCs have on hypoxia- and inflammatory cytokine-induced neutrophil survival. Human neutrophils were treated with GCs in the presence or absence of GM-CSF or inflammatory macrophage-CM at a range of oxygen concentrations (21-1% oxygen). Neutrophil apoptosis and survival were assessed by flow cytometry and morphological analysis and neutrophil function, by stimulus-induced shape change and respiratory burst. Dexamethasone promoted neutrophil survival at 21%, 10%, and 5% oxygen but not at 1% oxygen. Interestingly, GM-CSF and inflammatory CM increased neutrophil survival significantly, even at 1% oxygen, with cells remaining functionally active at 96 h. Dexamethasone was able to reduce the prosurvival effect of GM-CSF and inflammatory CM in a hypoxic environment. In conclusion, we found that GCs do not augment neutrophil survival in the presence of severe hypoxia or proinflammatory mediators. This suggests that GCs would not promote neutrophil survival at sites of inflammation under these conditions.

Induction of eosinophil apoptosis by the cyclin-dependent kinase inhibitor AT7519 promotes the resolution of eosinophil-dominant allergic inflammation.

BACKGROUND: Eosinophils not only defend the body against parasitic infection but are also involved in pathological inflammatory allergic diseases such as asthma, allergic rhinitis and contact dermatitis. Clearance of apoptotic eosinophils by macrophages is a key process responsible for driving the resolution of eosinophilic inflammation and can be defective in allergic diseases. However, enhanced resolution of eosinophilic inflammation by deliberate induction of eosinophil apoptosis using pharmacological agents has not been previously demonstrated. Here we investigated the effect of a novel cyclin-dependent kinase inhibitor drug, AT7519, on human and mouse eosinophil apoptosis and examined whether it could enhance the resolution of a murine model of eosinophil-dominant inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Eosinophils from blood of healthy donors were treated with AT7519 and apoptosis assessed morphologically and by flow-cytometric detection of annexin-V/propidium iodide staining. AT7519 induced eosinophil apoptosis in a concentration dependent manner. Therapeutic administration of AT7519 in eosinophil-dominant allergic inflammation was investigated using an established ovalbumin-sensitised mouse model of allergic pleurisy. Following ovalbumin challenge AT7519 was administered systemically at the peak of pleural inflammation and inflammatory cell infiltrate, apoptosis and evidence of macrophage phagocytosis of apoptotic eosinophils assessed at appropriate time points. Administration of AT7519 dramatically enhanced the resolution of allergic pleurisy via direct induction of eosinophil apoptosis without detriment to macrophage clearance of these cells. This enhanced resolution of inflammation was shown to be caspase-dependent as the effects of AT7519 were reduced by treatment with a broad spectrum caspase inhibitor (z-vad-fmk). CONCLUSIONS: Our data show that AT7519 induces human eosinophil apoptosis and enhances the resolution of a murine model of allergic pleurisy by inducing caspase-dependent eosinophil apoptosis and enhancing macrophage ingestion of apoptotic eosinophils. These findings demonstrate the utility of cyclin-dependent kinase inhibitors such as AT7519 as potential therapeutic agents for the treatment of eosinophil dominant allergic disorders.

The CDK inhibitor, R-roscovitine, promotes eosinophil apoptosis by down-regulation of Mcl-1.

Eosinophils are major players in inflammatory allergic diseases such as asthma, hay fever and eczema. Here we show that the cyclin-dependent kinase inhibitor (CDKi) R-roscovitine efficiently and rapidly induces human eosinophil apoptosis using flow cytometric analysis of annexin-V/propidium iodide staining, morphological analysis by light microscopy, transmission electron microscopy and Western immunoblotting for caspase-3 cleavage. We further dissect these observations by demonstrating that eosinophils treated with R-roscovitine lose mitochondrial membrane potential and the key survival protein Mcl-1 is down-regulated. This novel finding of efficacious induction of eosinophil apoptosis by CDKi drugs has potential as a strategy for driving resolution of eosinophilic inflammation.

A novel hybrid aspirin-NO-releasing compound inhibits TNFalpha release from LPS-activated human monocytes and macrophages.

BACKGROUND: The cytoprotective nature of nitric oxide (NO) led to development of NO-aspirins in the hope of overcoming the gastric side-effects of aspirin. However, the NO moiety gives these hybrids potential for actions further to their aspirin-mediated anti-platelet and anti-inflammatory effects. Having previously shown that novel NO-aspirin hybrids containing a furoxan NO-releasing group have potent anti-platelet effects, here we investigate their anti-inflammatory properties. Here we examine their effects upon TNFalpha release from lipopolysaccharide (LPS)-stimulated human monocytes and monocyte-derived macrophages and investigate a potential mechanism of action through effects on LPS-stimulated nuclear factor-kappa B (NF-kappaB) activation. METHODS: Peripheral venous blood was drawn from the antecubital fossa of human volunteers. Mononuclear cells were isolated and cultured. The resultant differentiated macrophages were treated with pharmacologically relevant concentrations of either a furoxan-aspirin (B8, B7; 10 muM), their respective furazan NO-free counterparts (B16, B15; 10 muM), aspirin (10 muM), existing nitroaspirin (NCX4016; 10 muM), an NO donor (DEA/NO; 10 muM) or dexamethasone (1 muM), in the presence and absence of LPS (10 ng/ml; 4 h). Parallel experiments were conducted on undifferentiated fresh monocytes. Supernatants were assessed by specific ELISA for TNFalpha release and by lactate dehydrogenase (LDH) assay for cell necrosis. To assess NF-kappaB activation, the effects of the compounds on the loss of cytoplasmic inhibitor of NF-kappaB, IkappaBalpha (assessed by western blotting) and nuclear localisation (assessed by immunofluorescence) of the p65 subunit of NF-kappaB were determined. RESULTS: B8 significantly reduced TNFalpha release from LPS-treated macrophages to 36 +/- 10% of the LPS control. B8 and B16 significantly inhibited monocyte TNFalpha release to 28 +/- 5, and 49 +/- 9% of control, respectively. The B8 effect was equivalent in magnitude to that of dexamethasone, but was not shared by 10 muM DEA/NO, B7, the furazans, aspirin or NCX4016. LDH assessment revealed none of the treatments caused significant cell lysis. LPS stimulated loss of cytoplasmic IkappaBalpha and nuclear translocation of the p65 NF-kappaB subunit was inhibited by the active NO-furoxans. CONCLUSION: Here we show that furoxan-aspirin, B8, significantly reduces TNFalpha release from both monocytes and macrophages and suggest that inhibition of NF-kappaB activation is a likely mechanism for the effect. This anti-inflammatory action highlights a further therapeutic potential of drugs of this class.

The antimicrobial/elastase inhibitor elafin regulates lung dendritic cells and adaptive immunity.

Infections with bacteria and viruses such as adenovirus are a feature of chronic lung diseases such as chronic obstructive pulmonary diseases (COPD), and may be instrumental in the generation of disease exacerbations. We have previously shown in acute models that elafin (a lung natural chemotactic molecule for macrophages and neutrophils, with potent antimicrobial and neutrophil elastase inhibitor activity) is upregulated in infection and modulates innate immunity. Here we present data using two independent systems of elafin overexpression in vivo (recombinant adenovirus [Ad-elafin] and an elafin transgenic mouse line) to examine the function of elafin in adaptive immunity. We show that elafin increases the number (immunofluorescence) and activation status (flow cytometric measurement) of CD11c+/MHCII+ lung dendritic cells in vivo. Analysis of cytokines produced by spleen and lung cells, and of antibodies measured in serum and bronchoalveolar lavage fluid, shows that the immunity induced is biased toward a type 1 response (production of IL-12, IFN-gamma, and IgG2a). Furthermore, elafin overexpression protected mice against further challenge with Ad-LacZ, as assessed by antibody levels and neutralization titer, as well as LacZ expression in lung tissue. Thus, the pleiotropic molecule elafin has significant potential in modulating antigen-presenting cell numbers and activity, and could be beneficial in mucosal protective strategies.

Golgi fragmentation during Fas-mediated apoptosis is associated with the rapid loss of GM130.

During apoptosis, the Golgi complex becomes fragmented and key proteins (e.g., GRASP65 and p115) are targets for caspase cleavage. GM130, an integral membrane protein, contributes to the maintenance of Golgi structure and facilitates membrane fusion with secretory vesicles. We show that GM130 levels decrease during Fas-induced apoptosis but not during staurosporine-induced apoptosis while in both models p115 levels remain unaffected. We conclude that GM130 is rapidly diminished during Fas-mediated apoptosis associated with Golgi fragmentation in contrast to previous studies which have suggested that loss of GM130 during apoptosis is a late event.