Simon Phipps

Anti-IL-5 treatment reduces deposition of ECM proteins in the bronchial subepithelial basement membrane of mild atopic asthmatics

Eosinophil-derived TGF-beta has been implicated in remodeling events in asthma. We hypothesized that reduction of bronchial mucosal eosinophils with anti-IL-5 would reduce markers of airway remodeling. Bronchial biopsies were obtained before and after three infusions of a humanized, anti-IL-5 monoclonal antibody (mepolizumab) in 24 atopic asthmatics in a randomized, double-blind, placebo-controlled study. The thickness and density of tenascin, lumican, and procollagen III in the reticular basement membrane (RBM) were quantified immunohistochemically by confocal microscopy. Expression of TGF-beta1 mRNA by airway eosinophils was assessed by in situ hybridization, and TGF-beta1 protein was measured in bronchoalveolar lavage (BAL) fluid by ELISA. At baseline, airway eosinophil infiltration and ECM protein deposition was increased in the RBM of asthmatics compared with nonasthmatic controls. Treating asthmatics with anti-IL-5 antibody, which specifically decreased airway eosinophil numbers, significantly reduced the expression of tenascin, lumican, and procollagen III in the bronchial mucosal RBM when compared with placebo. In addition, anti-IL-5 treatment was associated with a significant reduction in the numbers and percentage of airway eosinophils expressing mRNA for TGF-beta1 and the concentration of TGF-beta1 in BAL fluid. Therefore eosinophils may contribute to tissue remodeling processes in asthma by regulating the deposition of ECM proteins.

Eosinophils: Biological Properties and Role in Health and Disease

Eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of innate and adaptive immunity. In this review, the biology of eosinophils is summarized, focusing on transcriptional regulation of eosinophil differentiation, characterization of the growing properties of eosinophil granule proteins, surface proteins and pleiotropic mediators, and molecular mechanisms of eosinophil degranulation. New views on the role of eosinophils in homeostatic function are examined, including developmental biology and innate and adaptive immunity (as well as their interaction with mast cells and T cells) and their proposed role in disease processes including infections, asthma, and gastrointestinal disorders. Finally, strategies for targeted therapeutic intervention in eosinophil‐mediated mucosal diseases are conceptualized.

Antagonism of microRNA-126 suppresses the effector function of TH2 cells and the development of allergic airways disease

Allergic asthma is an inflammatory disease of the lung characterized by abnormal T helper-2 (TH2) lymphocyte responses to inhaled antigens. The molecular mechanisms leading to the generation of TH2 responses remain unclear, although toll-like receptors (TLRs) present on innate immune cells play a pivotal role in sensing molecular patterns and in programming adaptive T cell responses. Here we show that in vivo activation of TLR4 by house dust mite antigens leads to the induction of allergic disease, a process that is associated with expression of a unique subset of small, noncoding microRNAs. Selective blockade of microRNA (miR)-126 suppressed the asthmatic phenotype, resulting in diminished TH2 responses, inflammation, airways hyperresponsiveness, eosinophil recruitment, and mucus hypersecretion. miR-126 blockade resulted in augmented expression of POU domain class 2 associating factor 1, which activates the transcription factor PU.1 that alters TH2 cell function via negative regulation of GATA3 expression. In summary, this study presents a functional connection between miRNA expression and asthma pathogenesis, and our data suggest that targeting miRNA in the airways may lead to anti-inflammatory treatments for allergic asthma.

Evidence that opioids may have toll-like receptor 4 and MD-2 effects

Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll-like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically-employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.

Toll/IL-1 Signaling Is Critical for House Dust Mite―specific Th1 and Th2 Responses

RATIONALE One of the immunopathological features of allergic inflammation is the infiltration of helper T type 2 (Th2) cells to the site of disease. Activation of innate pattern recognition receptors such as Toll-like receptors (TLRs) plays a critical role in helper T type 1 cell differentiation, yet their contribution to the generation of Th2 responses to clinically relevant aeroallergens remains poorly defined. OBJECTIVES To determine the requirement for TLR2, TLR4, and the Toll/IL-1 receptor domain adaptor protein MyD88 in a murine model of allergic asthma. METHODS Wild-type and factor-deficient ((-/-)) mice were sensitized intranasally to the common allergen house dust mite (HDM) and challenged 2 weeks later on four consecutive days. Measurements of allergic airway inflammation, T-cell cytokine production, and airway hyperreactivity were performed 24 hours later. MEASUREMENTS AND MAIN RESULTS Mice deficient in MyD88 were protected from the cardinal features of allergic asthma, including granulocytic inflammation, Th2 cytokine production and airway hyperreactivity. Although HDM activated NF-kappaB in TLR2- or TLR4-expressing HEK cells, only in TLR4(-/-) mice was the magnitude of allergic airway inflammation and hyperreactivity attenuated. The diminished Th2 response present in MyD88(-/-) and TLR4(-/-) mice was associated with fewer OX40 ligand-expressing myeloid dendritic cells in the draining lymph nodes during allergic sensitization. Finally, HDM-specific IL-17 production and airway neutrophilia were attenuated in MyD88(-/-) but not TLR4(-/-) mice. CONCLUSIONS Together, these data suggest that Th2- and Th17-mediated inflammation generated on inhalational HDM exposure is differentially regulated by the presence of microbial products and the activation of distinct MyD88-dependent pattern recognition receptors.

Toll-Interleukin-1 signalling is critical for house mite-specific th2 and th17 responses

RATIONALE One of the immunopathological features of allergic inflammation is the infiltration of helper T type 2 (Th2) cells to the site of disease. Activation of innate pattern recognition receptors such as Toll-like receptors (TLRs) plays a critical role in helper T type 1 cell differentiation, yet their contribution to the generation of Th2 responses to clinically relevant aeroallergens remains poorly defined. OBJECTIVES To determine the requirement for TLR2, TLR4, and the Toll/IL-1 receptor domain adaptor protein MyD88 in a murine model of allergic asthma. METHODS Wild-type and factor-deficient ((-/-)) mice were sensitized intranasally to the common allergen house dust mite (HDM) and challenged 2 weeks later on four consecutive days. Measurements of allergic airway inflammation, T-cell cytokine production, and airway hyperreactivity were performed 24 hours later. MEASUREMENTS AND MAIN RESULTS Mice deficient in MyD88 were protected from the cardinal features of allergic asthma, including granulocytic inflammation, Th2 cytokine production and airway hyperreactivity. Although HDM activated NF-kappaB in TLR2- or TLR4-expressing HEK cells, only in TLR4(-/-) mice was the magnitude of allergic airway inflammation and hyperreactivity attenuated. The diminished Th2 response present in MyD88(-/-) and TLR4(-/-) mice was associated with fewer OX40 ligand-expressing myeloid dendritic cells in the draining lymph nodes during allergic sensitization. Finally, HDM-specific IL-17 production and airway neutrophilia were attenuated in MyD88(-/-) but not TLR4(-/-) mice. CONCLUSIONS Together, these data suggest that Th2- and Th17-mediated inflammation generated on inhalational HDM exposure is differentially regulated by the presence of microbial products and the activation of distinct MyD88-dependent pattern recognition receptors.

The Relationship Between Allergen-Induced Tissue Eosinophilia and Markers of Repair and Remodeling in Human Atopic Skin

Several in vitro studies suggest that eosinophils may play a role in fibrosis, remodeling, and repair processes associated with IgE-mediated hypersensitivity. However, the relationship in vivo, between allergen-induced tissue eosinophilia and markers of repair has yet to be established in human atopic subjects. Using the allergen-induced cutaneous late-phase reaction as a model of allergic inflammation, we have tested the hypothesis that eosinophil-derived TGF-β1 and IL-13 are temporarily associated with myofibroblast formation and deposition of tenascin and procollagen I. Biopsies were taken from atopic volunteers at 1, 3, 6, 24, 48, and 72 h after intradermal allergen challenge and were examined by immunohistochemistry. Following the peak of the late-phase reaction (6 h) there were persisting TGF-β1+ eosinophils, α-smooth muscle actin+ myofibroblasts, tenascin immunoreactivity, and procollagen-I+ cells 24–48 h postchallenge. Direct evidence of generation of repair markers was obtained by coculture of eosinophils and fibroblasts. This resulted in α-smooth muscle actin immunoreactivity that was inhibitable by neutralizing Abs to TGF-β as well as production of tenascin transcripts and protein product. TGF-β1 and IL-13 also induced tenascin expression. We conclude that TGF-β1 and IL-13, provided partially by eosinophils, contribute to repair and remodeling events in allergic inflammation in human atopic skin.

RAGE and TLRs: relatives, friends or neighbours?

The innate immune system forms the first line of protection against infectious and non-infectious tissue injury. Cells of the innate immune system detect pathogen-associated molecular patterns or endogenous molecules released as a result of tissue injury or inflammation through various innate immune receptors, collectively termed pattern-recognition receptors. Members of the Toll-like receptor (TLR) family of pattern-recognition receptors have well established roles in the host immune response to infection, while the receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor predominantly involved in the recognition of endogenous molecules released in the context of infection, physiological stress or chronic inflammation. RAGE and TLRs share common ligands and signaling pathways, and accumulating evidence points towards their co-operative interaction in the host immune response. At present however, little is known about the mechanisms that result in TLR versus RAGE signalling or RAGE-TLR cross-talk in response to their shared ligands. Here we review what is known in relation to the physicochemical basis of ligand interactions between TLRs and RAGE, focusing on three shared ligands of these receptors: HMGB1, S100A8/A9 and LPS. Our aim is to discuss what is known about differential ligand interactions with RAGE and TLRs and to highlight important areas for further investigation so that we may better understand the role of these receptors and their relationship in host defense.

Differential Regulation of Human Eosinophil IL-3, IL-5, and GM-CSF Receptor α-Chain Expression by Cytokines: IL-3, IL-5, and GM-CSF Down-Regulate IL-5 Receptor α Expression with Loss of IL-5 Responsiveness, but Up-Regulate IL-3 Receptor α Expression

Our recent data suggested that tissue eosinophils may be relatively insensitive to anti-IL-5 treatment. We examined cross-regulation and functional consequences of modulation of eosinophil cytokine receptor expression by IL-3, IL-5 GM-CSF, and eotaxin. Incubation of eosinophils with IL-3, IL-5, or GM-CSF led to reduced expression of IL-5Rα, which was sustained for up to 5 days. Eosinophils incubated with IL-5 or IL-3 showed diminished respiratory burst and mitogen-activated protein kinase kinase phosphorylation in response to further IL-5 stimulation. In contrast to these findings, eosinophil expression of IL-3Rα was increased by IL-3, IL-5, and GM-CSF, whereas GM-CSF receptor α was down-regulated by GM-CSF, but was not affected by IL-3 or IL-5. CCR3 expression was down-regulated by IL-3 and was transiently reduced by IL-5 and GM-CSF, but rapidly returned toward baseline. Eotaxin had no effect on receptor expression for IL-3, IL-5, or GM-CSF. Up-regulation of IL-3Rα by cytokines was prevented by a phosphoinositol 3-kinase inhibitor, whereas this and other signaling inhibitors had no effect on IL-5Rα down-regulation. These data suggest dynamic and differential regulation of eosinophil receptors for IL-3, IL-5, and GM-CSF by the cytokine ligands. Since these cytokines are thought to be involved in eosinophil development and mobilization from the bone marrow and are present at sites of allergic inflammation, tissue eosinophils may have reduced IL-5R expression and responsiveness, and this may explain the disappointing effect of anti-IL-5 therapy in reducing airway eosinophilia in asthma.

NK cell deficiency predisposes to viral-induced Th2-type allergic inflammation via epithelial-derived IL-25

Severe respiratory syncytial virus (RSV) infection has long been associated with an increased risk for the development of childhood asthma and exacerbations of this disorder. Despite much research into the induction of Th2 responses by allergens and helminths, the factors associated with viral infection that predispose to Th2-regulated asthma remain unknown. Recently, clinical studies have shown reduced numbers of NK cells in infants suffering from a severe RSV infection. Here we demonstrate that NK cell deficiency during primary RSV infection of BALB/c mice results in the suppression of IFN-γ production and the development of an RSV-specific Th2 response and subsequent allergic lung disease. The outgrowth of the Th2 responses was dependent on airway epithelial cell-derived IL-25, which induced the upregulation of the notch ligand Jagged1 on dendritic cells. This study identifies a novel pathway underlying viral-driven Th2 responses that may have functional relevance to viral-associated asthma.