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Liver inflammation is a common extraintestinal manifestation in inflammatory bowel disease (IBD), yet, the mechanisms driving gut-liver axis inflammation remain poorly understood. IBD leads to a breakdown in the integrity of the intestinal barrier causing an increase in portal and systemic gut-derived antigens, which challenge the liver. Here, we examined the role of platelet activating factor receptor (PAFR) in colitis-associated liver damage using dextran sulfate sodium (DSS) and anti-CD40-induced colitis models. Both DSS and anti-CD40 models exhibited liver inflammation associated with colitis. Colitis reduced global PAFR protein expression in mouse livers causing an exclusive re-localization of PAFR to the portal triad. The global decrease in liver PAFR was associated with increased sirtuin 1 while relocalized PAFR expression was limited to Kupffer cells (KCs) and co-localized with toll-like receptor 4. DSS activated the NLRP3-inflammasome and increased interleukin (IL)-1β in the liver. Antagonism of PAFR amplified the inflammasome response by increasing NLRP3, caspase-1, and IL-1β protein levels in the liver. LPS also increased NLRP3 response in human hepatocytes, however, overexpression of PAFR restored the levels of NLPR3 and caspase-1 proteins. Interestingly, KCs depletion also increased IL-1β protein in mouse liver after DSS challenge. These data suggest a protective role for PAFR-expressing KCs during colitis and that regulation of PAFR is important for gut-liver axis homeostasis.

Th22 cells are a major source of IL-22 and have been found at sites of infection and in a range of inflammatory diseases. However, their molecular characteristics and functional roles remain largely unknown because of our inability to generate and isolate pure populations. We developed a novel Th22 differentiation assay and generated dual IL-22/IL-17A reporter mice to isolate and compare pure populations of cultured Th22 and Th17 cells. Il17a fate-mapping and transcriptional profiling provide evidence that these Th22 cells have never expressed IL-17A, suggesting that they are potentially a distinct cell lineage from Th17 cells under in vitro culture conditions. Interestingly, Th22 cells also expressed granzymes, IL-13, and increased levels of Tbet. Using transcription factor–deficient cells, we demonstrate that RORγt and Tbet act as positive and negative regulators of Th22 differentiation, respectively. Furthermore, under Th1 culture conditions in vitro, as well as in an IFN-γ–rich inflammatory environment in vivo, Th22 cells displayed marked plasticity toward IFN-γ production. Th22 cells also displayed plasticity under Th2 conditions in vitro by upregulating IL-13 expression. Our work has identified conditions to generate and characterize Th22 cells in vitro. Further, it provides evidence that Th22 cells develop independently of the Th17 lineage, while demonstrating plasticity toward both Th1- and Th2-type cells.

MicroRNAs (miRNAs) are short noncoding RNAs that regulate a broad spectrum of biological processes, including immune responses. Although the contributions of miRNAs to the function of immune cells are beginning to emerge, their specific roles remain largely unknown. IL-33 plays an important role in macrophage activation for innate host defense and proinflammatory responses. In this study, we report that miR-487b can suppress the levels of mRNA and protein for IL-33 during the differentiation of bone marrow–derived macrophages (BMDMs). This results in inhibition of IL-33–induced expression of Ag-presenting and costimulatory molecules and proinflammatory mediators. A luciferase assay showed that miR-487b binds to the IL-33 3′-untranslated region. We also confirmed that IL-33 directly promotes the activation of BMDMs by increasing the expression of MHC class I, MHC class II, CD80/CD86, and inducible NO synthase (iNOS) in a dose-dependent manner. Exposure of BMDMs to the TLR4 ligand, LPS, decreased miR-487b expression, increased IL-33 transcript levels, and induced the production of proinflammatory mediators (e.g., iNOS, IL-1β, IL-6, and TNF-α). Treatment with a specific inhibitor of miR-487b function also resulted in increased levels of IL-33 mRNA, which augmented LPS-induced expression of these inflammatory mediators in macrophages. Collectively, our results indicate that miR-487b plays a negative regulatory role in macrophages by controlling the levels of IL-33 transcript and protein to fine-tune innate immune host defense and proinflammatory responses of these cells. Thus, miR-487b plays an important role in the regulation of macrophage homeostasis and activation by targeting IL-33 transcripts.

// Hong Zhou 1,2 , Jie Zhang 1 , Fiona Eyers 2 , Yang Xiang 3 , Cristan Herbert 4 , Hock L. Tay 2 , Paul S. Foster 2,* and Ming Yang 2,* 1 Department of Respiratory Medicine, The Second Hospital, Jilin University, ChangChun, Jilin, People’s Republic of China 2 Priority Research Centre for Asthma and Respiratory Diseases, School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, The University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW, Australia 3 Department of Physiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, People’s Republic of China 4 Inflammation and Infection Research Centre, School of Medical Sciences, UNSW Australia, Sydney, Australia * These authors have contributed equally to this wok Correspondence to: Ming Yang, email: // Paul S. Foster, email: // Keywords : microRNA, macrophage, differentiation, transcriptional regulation, Immunology and Microbiology Section, Immune response, Immunity Received : December 23, 2015 Accepted : April 13, 2016 Published : April 22, 2016 Abstract Limited evidence is available about the specific miRNA networks that regulate differentiation of specific immune cells. In this study, we characterized miRNA expression and associated alterations in expression with putative mRNA targets that are critical during differentiation of macrophages. In an effort to map the dynamic changes in the bone marrow (BM), we profiled whole BM cultures during differentiation into macrophages. We identified 112 miRNAs with expression patterns that were differentially regulated 5-fold or more during BMDM development. With TargetScan and MeSH databases, we identified 1267 transcripts involved in 30 canonical pathways linked to macrophage biology as potentially regulated by these specific 112 miRNAs. Furthermore, by employing miRanda and Ingenuity Pathways Analysis (IPA) analysis systems, we identified 18 miRNAs that are temporally linked to the expression of CSF1R, CD36, MSR1 and SCARB1; 7 miRNAs linked to the regulation of the transcription factors RUNX1 and PU.1, and 14 miRNAs target the nuclear receptor PPARα and PPARγ. This novel information provides an important reference resource for further study of the functional links between miRNAs and their target mRNAs for the regulation of differentiation and function of macrophages.

Ceriumdioxide (NM212) and bariumsulphate (NM220) nanoparticles were tested according to the OECD test guideline no. 453; additions were made to the standard protocol to find evidence of inflammation and potential lung tumours with high sensitivity. Aerosol concentrations were concentrations 0.1, 0.3, 1 and 3 mg/m3 and 50 mg/m³ was tested, respectively. Levels of cerium measured in the organs increased with higher exposure concentrations and over time. However, the accumulation only reached a very low level. Lung burdens of barium were unexpectedly low during the first three months of exposure, due to fast clearance most probably by dissolution in vivo. Barium lung burdens increased thereafter. Animals in all exposure groups showed chronic inflammation of the lungs, with stronger inflammation at higher exposure concentrations. The level of particles present in the lungs was higher for bariumsulphate than for the highest level of ceriumdioxide. The nature of the inflammatory tissue changes varied between the two nanoparticles. Ceriumdioxide already triggered a chronic inflammation effect at the lowest dose, which was unexpected for insoluble nanoparticles without inherent toxicity. Despite chronic inflammation at all dose levels, no lung tumours were found which could be attributed to ceriumdioxide or bariumsulphate exposure. This may indicate that a particle-related, low-level chronic inflammation is not sufficient to cause tumour formation. Low-level accumulation in extrapulmonary organs did not lead to any pathological changes. No evidence of further health impacts was found for ceriumdioxide and bariumsulphate beyond those already known for granular particles. Acknowledgement: The project was carried out and funded jointly by the German Federal Environment Ministry, the Federal Environment Agency, the Federal Institute for Occupational Safety and Health and BASF SE as well as the EU project NanoREG and Cefic LRI. Histopathological examinations were performed by Fraunhofer Institute for Toxicology and Experimental Medicine and the biodistribution was analysed by the Federal Institute for Risk Assessment.

Accumulating evidence highlights links between iron regulation and respiratory disease. Here, we assessed the relationship between iron levels and regulatory responses in clinical and experimental asthma.We show that cell-free iron levels are reduced in the bronchoalveolar lavage (BAL) supernatant of severe or mild–moderate asthma patients and correlate with lower forced expiratory volume in 1 s (FEV1). Conversely, iron-loaded cell numbers were increased in BAL in these patients and with lower FEV1/forced vital capacity (FVC) ratio. The airway tissue expression of the iron sequestration molecules divalent metal transporter 1 (DMT1) and transferrin receptor 1 (TFR1) are increased in asthma, with TFR1 expression correlating with reduced lung function and increased Type-2 (T2) inflammatory responses in the airways. Furthermore, pulmonary iron levels are increased in a house dust mite (HDM)-induced model of experimental asthma in association with augmented Tfr1 expression in airway tissue, similar to human disease. We show that macrophages are the predominant source of increased Tfr1 and Tfr1+ macrophages have increased Il13 expression. We also show that increased iron levels induce increased pro-inflammatory cytokine and/or extracellular matrix (ECM) responses in human airway smooth muscle (ASM) cells and fibroblasts ex vivo and induce key features of asthma in vivo, including airway hyper-responsiveness (AHR) and fibrosis, and T2 inflammatory responses.Together these complementary clinical and experimental data highlight the importance of altered pulmonary iron levels and regulation in asthma, and the need for a greater focus on the role and potential therapeutic targeting of iron in the pathogenesis and severity of disease.

In this review, we highlight experiments conducted in our laboratories that have elucidated functional roles for CD4+ T-helper type-2 lymphocytes (TH2 cells), their associated cytokines and eosinophils in the regulation of hallmark features of allergic asthma. Notably, we consider the complexity of Type-2 responses and studies that have explored integrated signaling among classical TH2 cytokines (IL-4, IL-5 and IL-13), which together with CCL11 (eotaxin-1) regulate critical aspects of eosinophil recruitment, allergic inflammation and airways hyperresponsiveness (AHR). Among our most important findings, we have provided evidence that the initiation of TH2 responses is regulated by airway epithelial cell-derived factors, including TRAIL and MID1, which promote TH2 cell development via STAT6-dependent pathways. Further, we highlight studies demonstrating that microRNA are key regulators of allergic inflammation and potential targets for anti-inflammatory therapy. On the background of TH2 inflammation, we have demonstrated that innate immune cells (notably, airway macrophages) play essential roles in the generation of steroid-resistant inflammation and AHR secondary to allergen and pathogen-induced exacerbations. Our work clearly indicates that understanding the diversity and spatiotemporal role of the inflammatory response and its interactions with resident airway cells is critical to advancing knowledge on asthma pathogenesis and the development of new therapeutic approaches.

Background and aims: Symptomatic carotid stenosis is associated with an increased risk of early stroke recurrence. Severity of the stenosis, current basis of revascularization, explains only the cerebral ischemic mechanism of regional hypoperfusion. Plaque inflammation, the initiating event for plaque rupture and thromboembolism (artery-to-artery embolism), is not evaluated routinely. Using 18F-fluorodeoxyglucose positron-emission tomography (FDG-PET)/computed tomography (CT) and high resolution magnetic resonance imaging (HR-MRI), we investigated the role of plaque imaging and stroke recurrence in our cohort of stroke patients with recently symptomatic carotid stenosis. Methods: This ongoing prospective study included consecutive patients within 30-days of recent stroke and ipsilateral carotid stenosis (≥50%). FDG uptake was quantified as mean standardized uptake values (SUV, g/ml). The ratio of T1 hyperintensity of carotid plaque to the ipsilateral sternocleidomastoid muscle (SCM) was recorded on T1-weighted fat suppressed images. Patients were followed prospectively for stroke recurrence within 90-days. Embolic potential of carotid plaque is estimated by presence of spontaneous microembolic signals (MES) on extended transcranial Doppler monitoring of ipsilateral middle cerebral artery. Results: Of the 33 patients included in the study, 6 (18%) suffered from recurrent cerebral ischemic event in the same vascular territory within 90-days. Compared to patients without subsequent cerebral ischemic events, patients with recurrent cerebral ischemia showed higher mean T1 carotid-SCM ratio (2.49 versus 1.53; p Conclusions: FDG-PET/CT and HR-MRI imaging of carotid stenosis helps in identification of patients at higher risk of subsequent cerebral ischemic events and may aid in better therapeutic decision-making.

Respiratory syncytial virus (RSV) infection induces asthma exacerbations, which leads to worsening of clinical symptoms and may result in a sustained decline in lung function. Exacerbations are the main cause of morbidity and mortality associated with asthma, and significantly contribute to asthma-associated healthcare costs. Although glucocorticoids are used to manage exacerbations, some patients respond to them poorly. The underlying mechanisms associated with steroid-resistant exacerbations remain largely unknown. We have previously established a mouse model of RSV-induced exacerbation of allergic airways disease, which mimics hallmark clinical features of asthma. In this study, we have identified key roles for macrophage IFN-γ and IL-27 in the regulation of RSV-induced exacerbation of allergic airways disease. Production of IFN-γ and IL-27 was steroid-resistant, and neutralization of IFN-γ or IL-27 significantly suppressed RSV-induced steroid-resistant airway hyperresponsiveness and airway inflammation. We have previously implicated activation of pulmonary macrophage by TNF-α and/or MCP-1 in the mechanisms of RSV-induced exacerbation. Stimulation of pulmonary macrophages with TNF-α and/or MCP-1 induced expression of both IFN-γ and IL-27. Our findings highlight critical roles for IFN-γ and IL-27, downstream of TNF-α and MCP-1, in the mechanism of RSV-induced exacerbation. Thus, targeting the pathways that these factors activate may be a potential therapeutic approach for virus-induced asthma exacerbations.

Viral exacerbations continue to represent the major burden in terms of morbidity, mortality and health care costs associated with asthma. Those at greatest risk for acute asthma are those with more severe airways disease and poor asthma control. It is this group with established asthma in whom acute exacerbations triggered by virus infections remain a serious cause of increased morbidity. A range of novel therapies are emerging to treat asthma and in particular target this group with poor disease control, and in most cases their efficacy is now being judged by their ability to reduce the frequency of acute exacerbations. Critical for the development of new treatment approaches is an improved understanding of virus-host interaction in the context of the asthmatic airway. This requires research into the virology of the disease in physiological models in conjunction with detailed phenotypic characterisation of asthma patients to identify targets amenable to therapeutic intervention.

©2015 Plank et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source arecredited. MicroRNAs are post-transcriptional regulators of gene expression that are differentially regulated during development and in inflammatory diseases. A role for miRNAs in allergic asthma is emerging and further investigation is required to determine whether they may serve as potential therapeutic targets. We profiled miRNA expression in murine lungs from an ovalbumin-induced allergic airways disease model, and compared expression to animals receiving dexamethasone treatment and non-allergic controls. Our analysis identified 29 miRNAs that were significantly altered during allergic inflammation. Target prediction analysis revealed novel genes with altered expression in allergic airways disease and suggests synergistic miRNA regulation of target mRNAs. To assess the impacts of one induced miRNA on pathology, we targeted miR-155-5p using a specific antagomir. Antagomir administration successfully reduced miR-155-5p expression with high specificity, but failed to alter the disease phenotype. Interestingly, further investigation revealed that antagomir delivery has variable efficacy across different immune cell types, effectively targeting myeloid cell populations, but exhibiting poor uptake in lymphocytes. Our findings demonstrate that antagomir-based targeting of miRNA function in the lung is highly specific, but highlights cell-specificity as a key limitation to be considered for antagomir-based strategies as therapeutics.

Interleukin-22 (IL-22) belongs to the IL-10 cytokine family and is expressed in the lung during infection with Chlamydia muridarum (Cmu) and Pneumonia Virus of Mice (PVM). IL-22 can have both pro-inflammatory and tissue protective roles depending on the inflammatory context, tissue tropisms, and local cytokine milieu. Several cell types can produce IL-22, including innate lymphoid cells, neutrophils, γδ T cells, and CD4+ Th cells. While IL-22 has previously been associated with Th17 cells, recently a novel lineage of CD4+ T helper cells (known as Th22 cells) have been identified that predominantly produce IL-22 in the absence of IL-17a. Current knowledge of the cellular source of IL-22 in inflammatory lung infections is unclear and our understanding of a role for IL-22 in the pathogenesis of these diseases remains limited. We have mapped, for the first time, the cellular source of IL-22 in both Cmu and PVM lung infections using novel transgenic dual reporter mice for IL-17a and IL-22. We show that IL-22 expression only partially overlaps with IL-17a expression and identify Th22 cells as the major source of IL-22 in the lung. Using IL-22 deficient mice, we show a functional role for IL-22 in Cmu infection, but fail to identify a role in PVM infection. Our study provides novel insight into the cellular source of IL-22 in the context of lung infections and identifies a role for IL-22 in Cmu lung infection.

Purpose: To present our experience with minimally invasive surgery for central and left-sided diaphragmatic defects in the neonate and infants.Methods: For congenital diaphragmatic hernia (CDH), the laparoscopic technique is helped by a port position well out in the left flank, a hitch stitch to hold the posterior muscle shelf forward during the repair, and the patient positioned with the head up and the left side rotated well forwards. For hemi-diaphragmatic palsy, in addition, two rows of plication could be achieved only after the left lobe of the liver had been detached from the diaphragm. For Morgagni hernia, the defect was closed with muscle-to-muscle sutures, with the patient supine.Results: Four patients, ranging in age from a neonate to 18 months, underwent successful primary laparoscopic repair of one Morgagni hernia, one hemi-diaphragmatic palsy, and two diaphragmatic hernias. Presenting symptoms ranged from minor, to one neonate requiring ventilatory support. The maximum length of stay after su...

MicroRNAs are small non-coding RNAs that bind to multiple target mRNAs to control gene expression post-transcriptionally by inhibiting translation. In mammalian cells, microRNAs play important roles in a diverse array of cellular processes (e.g. cell proliferation and differentiation). However, alterations in their levels may compromise cellular function, predisposing to disease. In this review, we discuss microRNAs that have been linked with pathogenesis of asthma and propose functional roles in the regulation of disease. MicroRNAs have the potential to be biomarkers for asthma and provide the platform for the development of new classes of therapeutic compounds.

Respiratory virus infections are often pathogenic, driving severe inflammatory responses. Most research has focused on localized effects of virus infection and inflammation. However, infection can induce broad-reaching, systemic changes that are only beginning to be characterized. In this study, we assessed the impact of acute pneumovirus infection in C57BL/6 mice on bone marrow hematopoiesis. We hypothesized that inflammatory cytokine production in the lung upregulates myeloid cell production in response to infection. We demonstrate a dramatic increase in the percentages of circulating myeloid cells, which is associated with pronounced elevations in inflammatory cytokines in serum (IFN-γ, IL-6, CCL2), bone (TNF-α), and lung tissue (TNF-α, IFN-γ, IL-6, CCL2, CCL3, G-CSF, osteopontin). Increased hematopoietic stem/progenitor cell percentages (Lineage−Sca-I+c-kit+) were also detected in the bone marrow. This increase was accompanied by an increase in the proportions of committed myeloid progenitors, as determined by colony-forming unit assays. However, no functional changes in hematopoietic stem cells occurred, as assessed by competitive bone marrow reconstitution. Systemic administration of neutralizing Abs to either TNF-α or IFN-γ blocked expansion of myeloid progenitors in the bone marrow and also limited virus clearance from the lung. These findings suggest that acute inflammatory cytokines drive production and differentiation of myeloid cells in the bone marrow by inducing differentiation of committed myeloid progenitors. Our findings provide insight into the mechanisms via which innate immune responses regulate myeloid cell progenitor numbers in response to acute respiratory virus infection.