Role of cyclin-dependent kinase 9 in the resolution of innate inflammation in a zebrafish tailfin injury model

Neutrophils are an important cell in host defence and migrate rapidly to sites of inflammation when the host is compromised (e.g., in infection or wounding). There, they produce and/or release inflammatory mediators (e.g., LTB4, TNF, IL-8) and ingest and degrade pathogens (e.g., by release of granule proteins and reactive oxygen species). Neutrophils then undergo apoptosis and are cleared by phagocytes such as macrophages, to allow efficient resolution of inflammation. Inducing neutrophil apoptosis by pharmacological means could be a therapeutic strategy to dampen inflammation in diseases where neutrophils are prevalent, e.g., acute respiratory distress syndrome (ARDS) and rheumatoid arthritis (RA). Inhibition of cyclin-dependent kinases (CDKs) using CDK inhibitor (CDKi) compounds induces mammalian neutrophil apoptosis in vitro, and can drive resolution of inflammation in vivo in mouse models. Evidence indicated that this is due to inhibition of CDK9 and CDK7-mediated transcription of the anti-apoptotic protein Mcl-1. The hypothesis of this project was that CDK9, CDK7 and Mcl-1 are pivotal regulators of resolution of inflammation in vivo. The model selected to test this hypothesis was tailfin injury of embryonic zebrafish (Danio rerio). Zebrafish are optically transparent and reporter transgenic lines with neutrophils labelled by enhanced GFP (EGFP - Tg[mpx:EGFP]i114) and macrophages (Tg[MPEG1:mCherry]) have been created, permitting the imaging of the behaviour of these cells in vivo. The model of tailfin transection was chosen to cause an inflammatory response in these animals, with neutrophil and macrophage recruitment to the tailfin. This response was manipulated using CDKi compounds and specific gene knockdowns (using morpholino and CRISPR/cas9 technologies). It was shown that CDKi compounds could reduce neutrophil numbers at 24 h post-injury at the transected tailfin, but did not affect macrophage numbers. The CDKi AT7519 increased neutrophil apoptosis at 12 h post-injury. Specific CDK9 knockdown using morpholinos or CRISPR/cas9 also reduced neutrophilic inflammation at the tailfin 24 h after transection, accompanied by increased apoptosis levels at 8 h in the morpholino-treated group. Inhibition of an endogenous CDK9 inhibitor, LaRP7, had the opposite effect and increased neutrophil numbers; and could oppose the neutrophil- reducing effect of AT7519 and CDK9 morpholino knockdown. Preliminary genetic knockdown studies into the roles of CDK7 and Mcl-1 have been carried out. Taken together, the results demonstrate CDK9 is important in the resolution of neutrophilic inflammation, indicating that manipulation of CDK9 activity could be a good target for therapeutic intervention in inflammatory disease

Oligodendrocyte precursor cells sculpt the visual system by regulating axonal remodeling

Many oligodendrocyte precursor cells (OPCs) do not differentiate to form myelin, suggesting additional roles of this cell population. The zebrafish optic tectum contains OPCs in regions devoid of myelin. Elimination of these OPCs impaired precise control of retinal ganglion cell axon arbor size during formation and maturation of retinotectal connectivity and degraded functional processing of visual stimuli. Therefore, OPCs fine-tune neural circuits independently of their canonical role to make myelin

Proteome Profile of Myelin in the Zebrafish Brain

The velocity of nerve conduction along vertebrate axons depends on their ensheathment with myelin. Myelin membranes comprise specialized proteins well characterized in mice. Much less is known about the protein composition of myelin in non-mammalian species. Here, we assess the proteome of myelin biochemically purified from the brains of adult zebrafish (Danio rerio), considering its increasing popularity as model organism for myelin biology. Combining gel-based and gel-free proteomic approaches, we identified > 1,000 proteins in purified zebrafish myelin, including all known constituents. By mass spectrometric quantification, the predominant Ig-CAM myelin protein zero (MPZ/P0), myelin basic protein (MBP), and the short-chain dehydrogenase 36K constitute 12%, 8%, and 6% of the total myelin protein, respectively. Comparison with previously established mRNA-abundance profiles shows that expression of many myelin-related transcripts coincides with the maturation of zebrafish oligodendrocytes. Zebrafish myelin comprises several proteins that are not present in mice, including 36K, CLDNK, and ZWI. However, a surprisingly large number of ortholog proteins is present in myelin of both species, indicating partial evolutionary preservation of its constituents. Yet, the relative abundance of CNS myelin proteins can differ markedly as exemplified by the complement inhibitor CD59 that constitutes 5% of the total zebrafish myelin protein but is a low-abundant myelin component in mice. Using novel transgenic reporter constructs and cryo-immuno electron microscopy, we confirm the incorporation of CD59 into myelin sheaths. These data provide the first proteome resource of zebrafish CNS myelin and demonstrate both similarities and heterogeneity of myelin composition between teleost fish and rodents

Flavones induce neutrophil apoptosis by down-regulation of Mcl-1 via a proteasomal-dependent pathway

Neutrophil apoptosis and subsequent nonphlogistic clearance by surrounding phagocytes are key to the successful resolution of neutrophilic inflammation, with dysregulated apoptosis reported in multiple human inflammatory diseases. Enhancing neutrophil apoptosis has proresolution and anti-inflammatory effects in preclinical models of inflammation. Here we investigate the ability of the flavones apigenin, luteolin, and wogonin to induce neutrophil apoptosis in vitro and resolve neutrophilic inflammation in vivo. Human neutrophil apoptosis was assessed morphologically and by flow cytometry following incubation with apigenin, luteolin, and wogonin. All three flavones induced time- and concentration-dependent neutrophil apoptosis (apigenin, EC(50)=12.2 μM; luteolin, EC(50)=14.6 μM; and wogonin, EC(50)=28.9 μM). Induction of apoptosis was caspase dependent, as it was blocked by the broad-spectrum caspase inhibitor Q-VD-OPh and was associated with both caspase-3 and caspase-9 activation. Flavone-induced apoptosis was preceded by down-regulation of the prosurvival protein Mcl-1, with proteasomal inhibition preventing flavone-induced Mcl-1 down-regulation and apoptosis. The flavones abrogated the survival effects of mediators that prolong neutrophil life span, including lipoteichoic acid, peptidoglycan, dexamethasone, and granulocyte-macrophage colony stimulating factor, by driving apoptosis. Furthermore, wogonin enhanced resolution of established neutrophilic inflammation in a zebrafish model of sterile tissue injury. Wogonin-induced resolution was dependent on apoptosis in vivo as it was blocked by caspase inhibition. Our data show that the flavones induce neutrophil apoptosis and have potential as neutrophil apoptosis-inducing anti-inflammatory, proresolution agents.—Lucas, C. D., Allen, K. C., Dorward, D. A., Hoodless, L. J., Melrose, L. A., Marwick, J. A., Tucker, C. S., Haslett, C., Duffin, R., Rossi, A. G. Flavones induce neutrophil apoptosis by down-regulation of Mcl-1 via a proteasomal-dependent pathway

The Neurotrophic Receptor Ntrk2 Directs Lymphoid Tissue Neovascularization during Leishmania donovani Infection

The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB) and its ligands brain derived neurotrophic factor (Bdnf), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) are known primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80hiCD11bloCD11c+ macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis

Increased Cycling Cell Numbers and Stem Cell Associated Proteins as Potential Biomarkers for High Grade Human Papillomavirus+ve Pre-Neoplastic Cervical Disease

High risk (oncogenic) human papillomavirus (HPV) infection causes cervical cancer. Infections are common but most clear naturally. Persistent infection can progress to cancer. Pre-neoplastic disease (cervical intraepithelial neoplasia/CIN) is classified by histology (CIN1-3) according to severity. Cervical abnormalities are screened for by cytology and/or detection of high risk HPV but both methods are imperfect for prediction of which women need treatment. There is a need to understand the host virus interactions that lead to different disease outcomes and to develop biomarker tests for accurate triage of infected women. As cancer is increasingly presumed to develop from proliferative, tumour initiating, cancer stem cells (CSCs), and as other oncogenic viruses induce stem cell associated gene expression, we evaluated whether presence of mRNA (detected by qRT-PCR) or proteins (detected by flow cytometry and antibody based proteomic microarray) from stem cell associated genes and/or increased cell proliferation (detected by flow cytometry) could be detected in well-characterised, routinely collected cervical samples from high risk HPV+ve women. Both cytology and histology results were available for most samples with moderate to high grade abnormality. We found that stem cell associated proteins including human chorionic gonadotropin, the oncogene TP63 and the transcription factor SOX2 were upregulated in samples from women with CIN3 and that the stem cell related, cell surface, protein podocalyxin was detectable on cells in samples from a subset of women with CIN3. SOX2, TP63 and human gonadotrophin mRNAs were upregulated in high grade disease. Immunohistochemistry showed that SOX2 and TP63 proteins clearly delineated tumour cells in invasive squamous cervical cancer. Samples from women with CIN3 showed increased proliferating cells. We believe that these markers may be of use to develop triage tests for women with high grade cervical abnormality to distinguish those who may progress to cancer from those who may be treated more conservatively

The role of the innate immune response regulatory gene ABCF1 in mammalian embryogenesis and development.

ABCF1 is an ABC transporter family protein that has been shown to regulate innate immune response and is a risk gene for autoimmune pancreatitis and arthritis. Unlike other members of ABC transporter family, ABCF1 lacks trans-membrane domains and is thought to function in translation initiation through an interaction with eukaryotic translation initiation factor 2 (eIF2). To study ABCF1 expression and function in development and disease, we used a single gene trap insertion in the Abcf1 gene in murine embryonic stem cells (ES cells) that allowed lineage tracing of the endogenous Abcf1 promoter by following the expression of a β-galactosidase reporter gene. From the ES cells, heterozygous mice (Abcf1+/-) were produced. No live born Abcf1-/- progeny were ever generated, and the lethality was not mouse strain-specific. Thus, we have determined that Abcf1 is an essential gene in development. Abcf1-/- mice were found to be embryonic lethal at 3.5 days post coitum (dpc), while Abcf1+/- mice appeared developmentally normal. Abcf1+/- mice were fertile and showed no significant differences in their anatomy when compared with their wild type littermates. The Abcf1 promoter was found to be active in all organs in adult mice, but varies in levels of expression in specific cell types within tissues. Furthermore, we observed high promoter activity in the blastocysts and embryos. Overall, Abcf1 expression in embryos is required for development and its expression in adults was highly correlated with actively proliferating and differentiating cell types

Comparison of growth factor mRNA abundance in F4/80^hiCD11b^lo cells compared to other MP populations isolated from <i>L. donovani</i>-infected spleens.

<p>Comparison of growth factor mRNA abundance in F4/80^hiCD11b^lo cells compared to other MP populations isolated from <i>L. donovani</i>-infected spleens.</p

Altered gene expression in F4/80^hiCD11b^lo cells compared to control macrophages.

<p>Altered gene expression in F4/80^hiCD11b^lo cells compared to control macrophages.</p

Infection-induced expression of Bdnf and Ntrk2 in spleen.

<p>Bdnf (black line) was expressed at significantly higher levels by F4/80^hiCD11b^lo cells compared to the other MP populations tested, as revealed by delta median fluorescence intensity levels (ΔMFI) using intracellular flow cytometry (A). Isotype control staining is highlighted by the solid grey histogram. Ntrk2 (red) expression in naïve mouse spleen is found in a subset of splenic red pulp macrophages (F4/80⁺, white) in naïve mouse spleen (B). Scale bar = 200microns. In <i>L. donovani</i> infected mice splenic white pulp vessels expressed Ntrk2 (C: red). F4/80⁺ (white) CD11c⁺ (green) MPs are found in close association with Nrtk2⁺ vessels (C: merge and inset). Endothelial cells (Meca-32, green) but not smooth muscle actin-positive cells (SMA, red) in white pulp express Nrtk2 (white, D and inset). All sections were stained with DAPI (blue). Scale bars = 100 microns and 50 microns in high magnification merge image.</p