Myeloid cells expressing VEGF and arginase-1 following uptake of damaged retinal pigment epithelium suggests potential mechanism that drives the onset of choroidal angiogenesis in mice

Whilst data recognise both myeloid cell accumulation during choroidal neovascularisation (CNV) as well as complement activation, none of the data has presented a clear explanation for the angiogenic drive that promotes pathological angiogenesis. One possibility that is a pre-eminent drive is a specific and early conditioning and activation of the myeloid cell infiltrate. Using a laser-induced CNV murine model, we have identified that disruption of retinal pigment epithelium (RPE) and Bruch's membrane resulted in an early recruitment of macrophages derived from monocytes and microglia, prior to angiogenesis and contemporaneous with lesional complement activation. Early recruited CD11b(+) cells expressed a definitive gene signature of selective inflammatory mediators particularly a pronounced Arg-1 expression. Accumulating macrophages from retina and peripheral blood were activated at the site of injury, displaying enhanced VEGF expression, and notably prior to exaggerated VEGF expression from RPE, or earliest stages of angiogenesis. All of these initial events, including distinct VEGF (+) Arg-1(+) myeloid cells, subsided when CNV was established and at the time RPE-VEGF expression was maximal. Depletion of inflammatory CCR2-positive monocytes confirmed origin of infiltrating monocyte Arg-1 expression, as following depletion Arg-1 signal was lost and CNV suppressed. Furthermore, our in vitro data supported a myeloid cell uptake of damaged RPE or its derivatives as a mechanism generating VEGF (+) Arg-1(+) phenotype in vivo. Our results reveal a potential early driver initiating angiogenesis via myeloid-derived VEGF drive following uptake of damaged RPE and deliver an explanation of why CNV develops during any of the stages of macular degeneration and can be explored further for therapeutic gain.

Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3

It is well-known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a vascular endothelial growth factor receptor (VEGFR) and PI3K/Akt dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Over-expression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 Protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3 and XBP1u occurred in the cytosol. Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.

The role of immune-related myeloid cells in angiogenesis

Macrophage function is not restricted to the innate and adaptive immune responses, but also includes host defence, wound healing, angiogenesis and homeostatic processes. Within the spectrum of macrophage activation there are two extremes: M1 classically activated macrophages which have a pro-inflammatory phenotype, and M2 alternatively activated macrophages which are pro-angiogenic and anti-inflammatory. An important property of macrophages is their plasticity to switch from one phenotype to the other and they can be defined in their polarisation state at any point between the two extremes. In order to determine what stage of activation macrophages are in, it is essential to profile various phenotypic markers for their identification. This review describes the angiogenic role for myeloid cells: circulating monocytes, Tie-2 expressing monocytes (TEMs), myeloid-derived suppressor cells (MDSCs), tumour associated macrophages (TAMs), and neutrophils. Each cell type is discussed by phenotype, roles within angiogenesis and possible targets as a cell therapy. In addition, we also refer to our own research on myeloid angiogenic cells (MACs), outlining their ability to induce angiogenesis and their similarities to alternatively activated M2 macrophages. MACs significantly contribute to vascular repair through paracrine mechanisms as they lack the capacity to differentiate into endothelial cells. Since MACs also retain plasticity, phenotypic changes can occur according to disease states and the surrounding microenvironment. This pro-angiogenic potential of MACs could be harnessed as a novel cellular therapy for the treatment of ischaemic diseases, such as diabetic retinopathy, hind limb ischaemia and myocardial infarction; however, caution needs to be taken when MACs are delivered into an inflammatory milieu.

A Functional Variant of Elafin With Improved Anti-inflammatory Activity for Pulmonary Inflammation

Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of elafin (GG- and QQ-elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) elafin. Similar to WT-elafin, GG- and QQ-elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-elafin, GG- and QQ-elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-elafin showed increased functionality when compared to WT-elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden.

A role for whey acidic protein four-disulfide-core 12 (WFDC12) in the regulation of the inflammatory response in the lung

Introduction: Secretory leucocyte protease inhibitor and elafin are members of the whey acidic protein (WAP), or WAP four disulfide-core (WFDC), family of proteins and have multiple contributions to innate defence including inhibition of neutrophil serine proteases and inhibition of the inflammatory response to lipopolysaccharide (LPS). This study aimed to explore potential activities of WFDC12, a previously uncharacterised WFDC protein expressed in the lung. Methods: Recombinant expression and purification of WFDC12 were optimised in Escherichia coli. Antiprotease, antibacterial and immunomodulatory activities of recombinant WFDC12 were evaluated and levels of endogenous WFDC12 protein were characterised by immunostaining and ELISA. Results: Recombinant WFDC12 inhibited cathepsin G, but not elastase or proteinase-3 activity. Monocytic cells pretreated with recombinant WFDC12 before LPS stimulation produced significantly lower levels of the pro-inflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared with cells stimulated with LPS alone. Recombinant WFDC12 became conjugated to fibronectin in a transglutaminase-mediated reaction and retained antiprotease activity. In vivo WFDC12 expression was confirmed by immunostaining of human lung tissue sections. WFDC12 levels in human bronchoalveolar lavage fluid from healthy and lung-injured patients were quantitatively compared, showing WFDC12 to be elevated in both patients with acute respiratory distress syndrome and healthy subjects treated with LPS, relative to healthy controls. Conclusions: Together, these results suggest a role for this lesser known WFDC protein in the regulation of lung inflammation.

Mesenchymal stromal cells for treatment of the acute respiratory distress syndrome: The beginning of the story

In spite of decades of research, the acute respiratory distress syndrome (ARDS) continues to have an unacceptably high mortality and morbidity. Mesenchymal stromal cells (MSCs) present a promising candidate for the treatment of this condition and have demonstrated benefit in preclinical models. MSCs, which are a topic of growing interest in many inflammatory disorders, have already progressed to early phase clinical trials in ARDS. While a number of their mechanisms of effect have been elucidated, a better understanding of the complex actions of these cells may pave the way for MSC modifications, which might enable more effective translation into clinical practice.

Intracellular survival of Burkholderia cepacia complex in phagocytic cells

Burkholderia cepacia complex (Bcc) species are a group of Gram-negative opportunistic pathogens that infect the airways of cystic fibrosis patients, and occasionally they infect other immunocompromised patients. Bcc bacteria display high-level multidrug resistance, and chronically persist in the infected host while eliciting robust inflammatory responses. Studies using macrophages, neutrophils and dendritic cells, combined with advances to genetically manipulate these bacteria have increased our understanding of the molecular mechanisms of virulence in these pathogens and the molecular details of cell-host responses triggering inflammation. This article discusses our current view of the intracellular survival of B. cenocepacia within macrophages.

Cigarette Smoke, Airway Epithelial Cells and Host Defence

In COPD inflammation driven by exposure to tobacco smoke results in impaired innate immunity in the airway and ultimately to lung injury and remodeling. To understand the biological processes involved in host interactions with cigarette derived toxins submerged epithelial cell culture is widely accepted as a model for primary human airway epithelial cell culture research. Primary nasal and bronchial epithelial cells can also be cultured in air-liquid interface (ALI) models. ALI and submerged culture models have their individual merits, and the decision to use either technique should primarily be determined primarily by the research hypothesis.

Cigarette smoke has gaseous and particulate matter, the latter constituent primarily represented in cigarette smoke extract (CSE). Although not ideal in order to facilitate our understanding of the responses of epithelial cells to cigarette smoke, CSE still has scientific merit in airway cell biology research. Using this model, it has been possible to demonstrate differences in levels of tight junction disruption after CSE exposure along with varied vulnerability to the toxic effects of CSE in cell cultures derived from COPD and control study groups.

Primary nasal epithelial cells (PNECs) have been used as an alternative to bronchial epithelial cells (PBECs). However, at least in subjects with COPD, PNECs cannot consistently substitute for PBECs. Although airway epithelial cells from patients with COPD exhibit a constitutional pro-inflammatory phenotype, these cells have a diminished inflammatory response to CSE exposure. COPD epithelial cells have an increased susceptibility to undergo apoptosis, and have reduced levels of Toll-like receptor-4 expression after CSE exposure, both of which may account for the reduced inflammatory response observed in this group.

The use of CSE in both submerged and ALI epithelial cultures has extended our understanding of the cellular mechanisms that are important in COPD, and helped to unravel important pathways which may be of relevance in its pathogenesis.

Molecular mechanisms of virulence of Burkholderia cepacia complex bacteria: Survival in Phagocytic Cells

Burkholderia cepacia complex (Bcc) species are a group of Gram-negative opportunistic pathogens that chronically infect the airways of cystic fibrosis patients, but they can also infect patients with various types of immunosuppressive disorders. Bcc members are multidrug resistant bacteria that have the ability to persist in the infected host and also elicit robust inflammatory responses. Studies using macrophages, neutrophils and dendritic cells, combined with dramatic advances in the ability to genetically manipulate these microorganisms have contributed to increase our understanding of the molecular mechanisms of virulence in these pathogens and the molecular details of the cell host responses triggering inflammation. This chapter reviews our understanding of the pathogenic mechanisms used by Bcc to establish an intracellular niche in phagocytic cells and modulate host cell responses that ultimately end up in cell death and a proinflammatory response.

Glucocorticoid receptor and Klf4 co-regulate anti-inflammatory genes in keratinocytes

The glucocorticoid (GC) receptor (GR) and Kruppel-like factor Klf4 are transcription factors that play major roles in skin homeostasis. However, whether these transcription factors cooperate in binding genomic regulatory regions in epidermal keratinocytes was not known. Here, we show that in dexamethasone-treated keratinocytes GR and Klf4 are recruited to genomic regions containing adjacent GR and KLF binding motifs to control transcription of the anti-inflammatory genes Tsc22d3 and Zfp36. GR- and Klf4 loss of function experiments showed total GR but partial Klf4 requirement for full gene induction in response to dexamethasone. In wild type keratinocytes induced to differentiate, GR and Klf4 protein expression increased concomitant with Tsc22d3 and Zfp36 up-regulation. In contrast, GR-deficient cells failed to differentiate or fully induce Klf4, Tsc22d3 and Zfp36 correlating with increased expression of the epithelium-specific Trp63, a known transcriptional repressor of Klf4. The identified transcriptional cooperation between GR and Klf4 may determine cell-type specific regulation and have implications for developing therapies for skin diseases.

TNF alpha-Induced p38MAPK Activation Regulates TRPA1 and TRPV4 Activity in Odontoblast-Like Cells

The transient receptor potential (TRP) channels are unique cellular sensors that are widely expressed in many neuronal and nonneuronal cells. Among the TRP family members, TRPA1 and TRPV4 are emerging as candidate mechanosensitive channels that play a pivotal role in inflammatory pain and mechanical hyperalgesia. Odontoblasts are nonneuronal cells that possess many of the features of mechanosensitive cells and mediate important defense and sensory functions. However, the effect of inflammation on the activity of the odontoblast's mechanosensitive channels remains unknown. By using immunohistochemistry and calcium microfluorimetry, we showed that odontoblast-like cells express TRPA1 and TRPV4 and that these channels were activated by hypotonicity-induced membrane stretch. Short treatment of odontoblast-like cells with tumor necrosis factor (TNF)-α enhanced TRPA1 and TRPV4 responses to their chemical agonists and membrane stretch. This enhanced channel activity was accompanied by phospho-p38 mitogen-activated protein kinase (MAPK) expression. Treatment of cells with the p38 inhibitor SB202190 reduced TNF-α effects, suggesting modulation of channel activity via p38 MAPK. In addition, TNF-α treatment also resulted in an up-regulation of TRPA1 expression but down-regulation of TRPV4. Unlike TRPV4, enhanced TRPA1 expression was also evident in dental pulp of carious compared with noncarious teeth. SB202190 treatment significantly reduced TNF-α-induced TRPA1 expression, suggesting a role for p38 MAPK signaling in modulating both the transcriptional and non-transcriptional regulation of TRP channels in odontoblasts.

Innate Lymphoid Cells are the Predominant Source of Interleukin-17A During the Early Pathogenesis of Acute Respiratory Distress Syndrome

Rationale: IL-17A is purported to help drive early pathogenesis in acute respiratory distress syndrome (ARDS) by enhancing neutrophil recruitment. Whilst IL-17A is the archetypal cytokine of T helper (Th)17 cells, it is produced by a number of lymphocytes, the source during ARDS being unknown.

Objectives: To identify the cellular source and the role of IL17A in the early phase of lung injury

Methods: Lung injury was induced in WT (C57BL/6) and IL-17 KO mice with aerosolised LPS (100 µg) or Pseudomonas aeruginosa infection. Detailed phenotyping of the cells expressing RORγt, the transcriptional regulator of IL-17 production, in the mouse lung at 24 hours was carried out by flow cytometry.

Measurement and Main Results: A 100-fold reduction in neutrophil infiltration was observed in the lungs of the IL-17A KO compared to wild type (WT) mice. The majority of RORγt+ cells in the mouse lung were the recently identified type 3 innate lymphoid cells (ILC3). Detailed characterisation revealed these pulmonary ILC3s (pILC3s) to be discrete from those described in the gut. The critical role of these cells was verified by inducing injury in Rag2 KO mice which lack T cells but retain ILCs. No amelioration of pathology was observed in the Rag2 KO mice.

Conclusions: IL-17 is rapidly produced during lung injury and significantly contributes to early immunopathogenesis. This is orchestrated largely by a distinct population of pILC3 cells. Modulation of pILC3s’ activity may potentiate early control of the inflammatory dysregulation seen in ARDS, opening up new therapeutic targets.

Alterations in Circulating Immune Cells in Neovascular Age-Related Macular Degeneration

Neovascular age-related macular degeneration (nAMD) is the leading cause of irreversible blindness in developed countries. Recent advances have highlighted the essential role of inflammation in the development of the disease. In addition to local retinal chronic inflammatory response, systemic immune alterations have also been observed in AMD patients. In this study we investigated the association between the frequency of circulating leukocyte populations and the prevalence as well as clinical presentations of nAMD. Leukocyte subsets of 103 nAMD patients (most of them were receiving anti-VEGF therapy prior to enrolment) and 26 controls were analysed by flow cytometry by relative cell size, granularity and surface markers. Circulating CD11b(+) cells and CD16(hi)HLA-DR(-) neutrophils were significantly increased (P = 0.015 and 0.009 respectively) in nAMD when compared to controls. The percentage of circulating CD4(+) T-cells was reduced in nAMD patients without subretinal fibrosis (P = 0.026) compared to patients with subretinal fibrosis. There was no correlation between the percentage of circulating leukocytes and the responsiveness to anti-VEGF therapy in nAMD patients. Our results suggest that higher levels of circulating CD11b(+) cells and neutrophils are associated with nAMD and that reduced levels of CD4(+) T-cells are associated with the absence of subretinal fibrosis in nAMD.

TH2 and TH17 inflammatory pathways are reciprocally regulated in asthma

Increasing evidence suggests that asthma is a heterogeneous disorder regulated by distinct molecular mechanisms. In a cross-sectional study of asthmatics of varying severity (n = 51), endobronchial tissue gene expression analysis revealed three major patient clusters: TH2-high, TH17-high, and TH2/17-low. TH2-high and TH17-high patterns were mutually exclusive in individual patient samples, and their gene signatures were inversely correlated and differentially regulated by interleukin-13 (IL-13) and IL-17A. To understand this dichotomous pattern of T helper 2 (TH2) and TH17 signatures, we investigated the potential of type 2 cytokine suppression in promoting TH17 responses in a preclinical model of allergen-induced asthma. Neutralization of IL-4 and/or IL-13 resulted in increased TH17 cells and neutrophilic inflammation in the lung. However, neutralization of IL-13 and IL-17 protected mice from eosinophilia, mucus hyperplasia, and airway hyperreactivity and abolished the neutrophilic inflammation, suggesting that combination therapies targeting both pathways may maximize therapeutic efficacy across a patient population comprising both TH2 and TH17 endotypes.

Targeting the complement system for the management of retinal inflammatory and degenerative diseases

The retina, an immune privileged tissue, has specialized immune defense mechanisms against noxious insults that may exist in diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), uveoretinitis and glaucoma. The defense system consists of retinal innate immune cells (including microglia, perivascular macrophages, and a small population of dendritic cells) and the complement system. Under normal aging conditions, retinal innate immune cells and the complement system undergo a low-grade activation (parainflammation) which is important for retinal homeostasis. In disease states such as AMD and DR, the parainflammatory response is dysregulated and develops into detrimental chronic inflammation. Complement activation in the retina is an important part of chronic inflammation and may contribute to retinal pathology in these disease states. Here, we review the evidence that supports the role of uncontrolled or dysregulated complement activation in various retinal degenerative and angiogenic conditions. We also discuss current strategies that are used to develop complement-based therapies for retinal diseases such as AMD. The potential benefits of complement inhibition in DR, uveoretinitis and glaucoma are also discussed, as well as the need for further research to better understand the mechanisms of complement-mediated retinal damage in these disease states.