Cytokine induced 3-D organotypic psoriasis skin model demonstrates distinct roles for NF-κB and JAK pathways in disease pathophysiology.

Psoriasis vulgaris is an inflammatory skin disease that affects 2%-3% of the population worldwide. One of the major challenges in discovering novel therapies is the poor translatability of animal models to human disease. Therefore, it is imperative to develop human preclinical models of psoriasis that are amenable to pharmacological intervention. Here, we report a 3-D reconstituted human epidermis (RHE) culture system treated with cytokines commonly associated with psoriasis (TNFα, IL-17A and IL-22) that reproduced some key features of the human disease. The effects on epidermal morphology, gene transcription and cytokine production, which are dysregulated in psoriasis were assessed. Certain morphological features of psoriatic epidermis were evident in cytokine-stimulated RHEs, including hypogranulosis and parakeratosis. In addition, RHEs responded to a cytokine mix in a dose-dependent manner by expressing genes and proteins associated with impaired keratinocyte differentiation (keratin 10/K10, loricrin), innate immune responses (S100A7, DEFB4, elafin) and inflammation (IL-1α, IL-6, IL-8, IL-10, IL-12/23p40, IL-36γ, GM-CSF and IFNγ) typical of psoriasis. These disease-relevant changes in morphology, gene transcription and cytokine production were robustly attenuated by pharmacologically blocking TNFα/IL-17A-induced NF-κB activation with IKK-2 inhibitor IV. Conversely, inhibition of IL-22-induced JAK1 signalling with ABT-317 strongly attenuated morphological features of the disease but had no effect on NFκB-dependent cytokine production, suggesting distinct mechanisms of action by the cytokines driving psoriasis. These data support the use of cytokine-induced RHE models for identifying and targeting keratinocyte signalling pathways important for disease progression and may provide translational insights into novel keratinocyte mechanisms for novel psoriasis therapies.

Intercellular Adhesion Molecule 1 Functions as an Efferocytosis Receptor in Inflammatory Macrophages.

Intercellular adhesion molecule-1 (ICAM-1) is up-regulated during inflammation by several cell types. ICAM-1 is best known for its role in mediating leukocyte adhesion to endothelial cells and guiding leukocytes across the vascular wall. Recently, macrophages have been shown to express ICAM-1, however, their role in macrophage function is unclear. We found that ICAM-1 expression was induced during inflammatory macrophage polarization and high numbers of ICAM-1-expressing macrophages were noted in inflamed colon tissue in a murine colitis model and in human inflammatory bowel disease. Because tissue macrophages play a critical role in removing apoptotic/necrotic cells in inflammation and injury, a process termed efferocytosis, it was examined whether ICAM-1 contributes to this process. Genetic deletion (ICAM-1 knockout mice) or siRNA-mediated knockdown of ICAM-1 in isolated murine and human macrophages significantly impaired apoptotic cell (AC) engulfment. Impairment in the engulfment of Jurkat T cells, neutrophils, and epithelial cells was confirmed ex vivo by inflammatory macrophages and in vivo by thioglycolate-recruited peritoneal macrophages. Decreased efferocytosis was also seen in vitro and in vivo with inhibition of ICAM-1 adhesive interactions, using a function blocking anti-ICAM-1 antibody. Mechanistically, it was found that ICAM-1 actively redistributes to cluster around engulfed ACs to facilitate macrophage-AC binding. Our findings define a new role for ICAM-1 in promoting macrophage efferocytosis, a critical process in the resolution of inflammation and restoration of tissue homeostasis.

Neutrophil Microparticles Deliver Active Myeloperoxidase to Injured Mucosa To Inhibit Epithelial Wound Healing.

Neutrophil (PMN) infiltration of the intestinal mucosa often leads to severe epithelial injury; however, how this process occurs is unclear. This article describes a novel mechanism whereby membrane-derived microparticles released by tissue infiltrating PMNs (PMN-MPs) serve as shuttles to protect and deliver active mediators to locally modulate cellular function during inflammation. Specifically, myeloperoxidase (MPO), which is abundantly expressed in PMN azurophilic granules and is used for microbial killing, was found to be mobilized to the PMN surface and subsequently released in association with PMN-MPs upon PMN activation and binding to intestinal epithelial cells (IECs). The enzymatic activity of PMN-MP-associated MPO was enhanced compared with soluble protein, leading to potent inhibition of wound closure following PMN-MP binding to IECs. Importantly, localized microinjection of PMN-MPs into wounded colonic mucosa was sufficient to impair epithelial wound healing in vivo. PMN-MP/MPO-dependent inhibition of IEC wound healing was due to impaired IEC migration and proliferation, resulting from impeded actin dynamics, cell spreading, and cell cycle arrest. Thus, our findings provide new insight into mechanisms governing PMN-induced tissue injury and implicate PMN-MPs and MPO as important regulators of cellular function.

Altering PI3K-Akt signalling in zebrafish embryos affects PTEN phosphorylation and gastrulation.

BACKGROUND INFORMATION: PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a negative regulator of the PI3K (phosphoinositide 3-kinase)-Akt (also called protein kinase B) signalling pathway and is essential for embryogenesis, but its function in early vertebrate embryos is unclear. RESULTS: To address how PTEN functions in early embryos, we overexpressed one of the four zebrafish PTEN isoforms at the 1-2-cell stage. Overexpression of Ptena454 alters phospho-Akt levels and impairs cell movements associated with gastrulation. Heat shocking embryos increases phospho-Akt levels and lowers phospho-Ptena454 levels. Inhibiting CK2 (protein kinase CK2) activity reduces phospho-Pten levels and augments the effects due to Ptena454 overexpression. Low phospho-Akt and corresponding low phospho-GSK-3 (glycogen synthase kinase-3) and high phospho-Pten levels accompany wortmannin or LY294002 treatment, which inhibit PI3K activity. CONCLUSIONS: These results suggest that Ptena454 regulation is correlated to changes in phospho-Akt levels. We propose a model in which homoeostasis in rapidly dividing and migrating embryonic cells depends on a counterbalance between pro-survival signalling employing CK2 and GSK-3 and the pro-apoptotic activity of Ptena454.

Assessing neuraxial microstructural changes in a transgenic mouse model of early stage Amyotrophic Lateral Sclerosis by ultra-high field MRI and diffusion tensor metrics.

OBJECTIVE: Cell structural changes are one of the main features observed during the development of amyotrophic lateral sclerosis (ALS). In this work, we propose the use of diffusion tensor imaging (DTI) metrics to assess specific ultrastructural changes in the central nervous system during the early neurodegenerative stages of ALS. METHODS: Ultra-high field MRI and DTI data at 17.6T were obtained from fixed, excised mouse brains, and spinal cords from ALS (G93A-SOD1) mice. RESULTS: Changes in fractional anisotropy (FA) and linear, planar, and spherical anisotropy ratios (CL, CP, and CS, respectively) of the diffusion eigenvalues were measured in white matter (WM) and gray matter (GM) areas associated with early axonal degenerative processes (in both the brain and the spinal cord). Specifically, in WM structures (corpus callosum, corticospinal tract, and spinal cord funiculi) as the disease progressed, FA, CL, and CP values decreased, whereas CS values increased. In GM structures (prefrontal cortex, hippocampus, and central spinal cord) FA and CP decreased, whereas the CL and CS values were unchanged or slightly smaller. Histological studies of a fluorescent mice model (YFP, G93A-SOD1 mouse) corroborated the early alterations in neuronal morphology and axonal connectivity measured by DTI. CONCLUSIONS: Changes in diffusion tensor shape were observed in this animal model at the early, nonsymptomatic stages of ALS. Further studies of CL, CP, and CS as imaging biomarkers should be undertaken to refine this neuroimaging tool for future clinical use in the detection of the early stages of ALS.

Mouse G-protein gamma3 expression in the developing CNS and neural crest cell derivatives.

Heterotrimeric G-protein signaling, involving alpha, beta and gamma subunits, plays a number of roles in differentiation and development. Individual gamma subunits interact with a beta subunit and as a heterodimer, is responsible for modulating many G protein-mediated cellular responses. The 12 gamma subunits in mammals have highly variable distribution and expression patterns in adult tissues. gamma3 is abundantly and widely expressed in the brain and when its expression is knocked-out, the mice show increased susceptibility to seizures, reduced body weights and decreased adiposity compared to the wild-type littermates (Schwindinger et al., 2004). Recent evidence has shown the Gng3 gene being strongly induced in activated CD4+ T-cells (Dubeykovskiy et al., 2006) and its involvement in the developing mammalian enteric nervous system (Heanue and Pachnis, 2006). Given this diversity in expression and interest in finding models of human disease, and to extend our previous investigation with zebrafish gamma3 (Kelly et al., 2001), we undertook an analysis to report the temporal and spatial expression patterns of gamma3 mRNA during mouse embryogenesis. Analysis reveals that gamma3 transcripts were first expressed in mid-late embryonic stages. Specifically, signals were predominant in the CNS and in neural crest cell derivatives including but not limited to the trigeminal and dorsal root (spinal) ganglia, and in cells of the adrenal medulla. These data indicate that G protein coupled signaling involving gamma3 participates in a number of physiological roles, not only in the CNS, but also in numerous cells derived from the neural crest.

Unveiling early cortical and subcortical neuronal degeneration in ALS mice by ultra-high field diffusion MRI.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease primarily characterized by the progressive impairment of motor functions. However, a significant portion of affected patients develops severe cognitive dysfunction, developing a widespread white (WM) and gray matter (GM) microstructural impairment. The objective of this study is to determine if Gaussian and non-Gaussian diffusion models gathered by ultra-high field diffusion MRI (UHFD-MRI) are an appropriate tool to detect early structural changes in brain white and gray matter in a preclinical model of ALS. ALS brains (G93A-SOD1mice) were scanned in a 16.7 T magnet. Diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI) have shown presymptomatic decrease in axonal organization by Fractional Anisotropy (FA) and neurite content by Intracellular Volume Fraction (ICVF) across deep WM (corpus callosum) as well as superficial (cortex) and deep (hippocampus) GM. Additional diffusion kurtosis imaging (DKI) analysis demonstrated broader and earlier GM reductions in mean kurtosis (MK), possibly related to the decrease in neuronal complexity. Histological validation was obtained by an ALS fluorescent mice reporter (YFP, G93A-SOD1 mice). The combination of DTI, NODDI, and DKI models have proved to provide a more complete assessment of the early microstructural changes in the ALS brain, particularly in areas associated with high cognitive functions. This comprehensive approach should be considered as a valuable tool for the early detection of neuroimaging markers.

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies.

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50-1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

Constitutive p38HOG mitogen-activated protein kinase activation induces permanent cell cycle arrest and senescence.

Cellular senescence, initially observed during subculturing of normal diploid fibroblasts, can also be induced by chronic exposure to cellular stress, such as UV light, oxidative stress, or DNA damaging agents. Here we demonstrate that stable expression of an activated form of MKK6 (MKK6EE), a direct activator of the stress-induced p38(HOG) mitogen-activated protein kinase pathway, is sufficient for inducing features of senescence including a flattened, vacuolated, and irregular morphology, staining for acidic beta-galactosidase, and accumulation of age-associated pigments. Consistent with the senescent phenotype, p38(HOG) activation induces a G(1) cell cycle arrest, which is permanent and irreversible after 4 days. MKK6EE also induces biochemical features of senescence in a p38-dependent manner, including enhanced expression of p21(CIP), a cyclin-dependent kinase inhibitor. Microarray analysis of MKK6EE cells showed a pattern of gene expression noted previously in Werner Syndrome and senescent fibroblasts. These results define p38(HOG) as an intracellular pathway that activates a senescence checkpoint in tumor cells and may play a role in Ras- or stress-induced senescence.

Human megakaryocyte progenitors derived from hematopoietic stem cells of normal individuals are MHC class II-expressing professional APC that enhance Th17 and Th1/Th17 responses.

Platelets, like stromal cells, present antigen only via MHC class I, but the immune potential of their progenitors has not been explored in humans. We derived CD34(+)CD117(+)CD41(+)CD151(+) megakaryocyte progenitors (MKp) in vitro from mobilized peripheral blood hematopoietic stem and progenitor cells (HSPC) of normal subjects using culture conditions akin to bone marrow niche, or organs that support extramedullary hematopoiesis. The MKp expressed MHC Class II in contrast to platelets and functioned as professional APC before they matured further. Moreover, MKp constitutively expressed mRNA encoding mediators for human Th17 expansion, including IL-1, IL-18, IL-6, TGFß, IL-23, BAFF, and COX2. MKp also expressed high levels of type I interferon and IRF5 mRNA. In contrast to platelets, MKp augmented the expansion of Th17, Th1, and potent Th17/Th1 double-positive cells in normal PBMC and CD4 line T cells from normal subjects or lupus patients. The Th cell augmentation involved pre-committed memory cells, and was significant although modest, because only non-cognate MKp-T cell interactions could be studied, under non-polarizing conditions. Importantly, the MKp-mediated expansion was observed in the presence or absence of direct MKp-T cell contact. Furthermore, MKp augmented Th17 responses against Candida albicans, a serious opportunistic pathogen. These results indicate an immunologic role of MKp in situations associated with extramedullary hematopoiesis and mobilization of HSPC.

Hephaestus encodes a polypyrimidine tract binding protein that regulates Notch signalling during wing development in Drosophila melanogaster.

We describe the role of the Drosophila melanogaster hephaestus gene in wing development. We have identified several hephaestus mutations that map to a gene encoding a predicted RNA-binding protein highly related to human polypyrimidine tract binding protein and Xenopus laevis 60 kDa Vg1 mRNA-binding protein. Polypyrimidine tract binding proteins play diverse roles in RNA processing including the subcellular localization of mRNAs, translational control, internal ribosome entry site use, and the regulation of alternate exon selection. The analysis of gene expression in imaginal discs and adult cuticle of genetic mosaic animals supports a role for hephaestus in Notch signalling. Somatic clones lacking hephaestus express the Notch target genes wingless and cut, induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of DELTA: These results lead to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta. This is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in the Notch signalling pathway.