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1.  Alternaria-derived serine protease activity drives IL-33–mediated asthma exacerbations 
Background
The fungal allergen Alternaria alternata is implicated in severe asthma and rapid onset life-threatening exacerbations of disease. However, the mechanisms that underlie this severe pathogenicity remain unclear.
Objective
We sought to investigate the mechanism whereby Alternaria was capable of initiating severe, rapid onset allergic inflammation.
Methods
IL-33 levels were quantified in wild-type and ST2−/− mice that lacked the IL-33 receptor given inhaled house dust mite, cat dander, or Alternaria, and the effect of inhibiting allergen-specific protease activities on IL-33 levels was assessed. An exacerbation model of allergic airway disease was established whereby mice were sensitized with house dust mite before subsequently being challenged with Alternaria (with or without serine protease activity), and inflammation, remodeling, and lung function assessed 24 hours later.
Results
Alternaria, but not other common aeroallergens, possessed intrinsic serine protease activity that elicited the rapid release of IL-33 into the airways of mice through a mechanism that was dependent upon the activation of protease activated receptor-2 and adenosine triphosphate signaling. The unique capacity of Alternaria to drive this early IL-33 release resulted in a greater pulmonary inflammation by 24 hours after challenge relative to the common aeroallergen house dust mite. Furthermore, this Alternaria serine protease–IL-33 axis triggered a rapid, augmented inflammation, mucus release, and loss of lung function in our exacerbation model.
Conclusion
Alternaria-specific serine protease activity causes rapid IL-33 release, which underlies the development of a robust TH2 inflammation and exacerbation of allergic airway disease.
doi:10.1016/j.jaci.2014.02.002
PMCID: PMC4152000  PMID: 24636086
Alternaria alternata; allergic airway disease; asthma exacerbation; protease; IL-33; AEBSF, 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride; ALT, Alternaria; BALF, BAL fluid; CAT, Cat dander; HDM, House dust mite; MCPT-1, Mast cell protease-1; MMP-9, Matrix metalloproteinase-9; PAP, Papain; PAR-2, Protease activated receptor 2; RAG, Ragweed; TRYP, Trypsin
2.  Respiratory Infections Cause the Release of Extracellular Vesicles: Implications in Exacerbation of Asthma/COPD 
PLoS ONE  2014;9(6):e101087.
Background
Infection-related exacerbations of respiratory diseases are a major health concern; thus understanding the mechanisms driving them is of paramount importance. Despite distinct inflammatory profiles and pathological differences, asthma and COPD share a common clinical facet: raised airway ATP levels. Furthermore, evidence is growing to suggest that infective agents can cause the release of extracellular vesicle (EVs) in vitro and in bodily fluids. ATP can evoke the P2X7/caspase 1 dependent release of IL-1β/IL-18 from EVs; these cytokines are associated with neutrophilia and are increased during exacerbations. Thus we hypothesized that respiratory infections causes the release of EVs in the airway and that the raised ATP levels, present in respiratory disease, triggers the release of IL-1β/IL-18, neutrophilia and subsequent disease exacerbations.
Methods
To begin to test this hypothesis we utilised human cell-based assays, ex vivo murine BALF, in vivo pre-clinical models and human samples to test this hypothesis.
Results
Data showed that in a murine model of COPD, known to have increased airway ATP levels, infective challenge causes exacerbated inflammation. Using cell-based systems, murine models and samples collected from challenged healthy subjects, we showed that infection can trigger the release of EVs. When exposed to ATP the EVs release IL-1β/IL-18 via a P2X7/caspase-dependent mechanism. Furthermore ATP challenge can cause a P2X7 dependent increase in LPS-driven neutrophilia.
Conclusions
This preliminary data suggests a possible mechanism for how infections could exacerbate respiratory diseases and may highlight a possible signalling pathway for drug discovery efforts in this area.
doi:10.1371/journal.pone.0101087
PMCID: PMC4074193  PMID: 24972036
3.  Neutrophils Recruited by IL-22 in Peripheral Tissues Function as TRAIL-Dependent Antiviral Effectors against MCMV 
Cell Host & Microbe  2014;15(4):471-483.
Summary
During primary infection, murine cytomegalovirus (MCMV) spreads systemically, resulting in virus replication and pathology in multiple organs. This disseminated infection is ultimately controlled, but the underlying immune defense mechanisms are unclear. Investigating the role of the cytokine IL-22 in MCMV infection, we discovered an unanticipated function for neutrophils as potent antiviral effector cells that restrict viral replication and associated pathogenesis in peripheral organs. NK-, NKT-, and T cell-secreted IL-22 orchestrated antiviral neutrophil-mediated responses via induction in stromal nonhematopoietic tissue of the neutrophil-recruiting chemokine CXCL1. The antiviral effector properties of infiltrating neutrophils were directly linked to the expression of TNF-related apoptosis-inducing ligand (TRAIL). Our data identify a role for neutrophils in antiviral defense, and establish a functional link between IL-22 and the control of antiviral neutrophil responses that prevents pathogenic herpesvirus infection in peripheral organs.
Graphical Abstract
Highlights
•Neutrophils are critical antiviral effector cells during MCMV virus infection•Neutrophils directly inhibit virus replication in a TRAIL-dependent manner•IL-22 inhibits virus replication in peripheral but not secondary lymphoid tissues•IL-22 orchestrates CXCL1-dependent neutrophil recruitment
Murine cytomegalovirus (MCMV) targets multiple peripheral organs during infection. Stacey et al. report that in response to MCMV infection, NK, NKT, and T cells secrete the cytokine IL-22, which recruits antiviral neutrophils to infected peripheral organs in a CXCL1-dependent manner. Neutrophils exert antiviral effector functions via proapoptotic TRAIL expression.
doi:10.1016/j.chom.2014.03.003
PMCID: PMC3989063  PMID: 24721575
4.  OX40 Ligand Fusion Protein Delivered Simultaneously With the BCG Vaccine Provides Superior Protection Against Murine Mycobacterium tuberculosis Infection 
The Journal of Infectious Diseases  2012;205(6):975-983.
Mycobacterium tuberculosis infection claims approximately 2 million lives per year, and improved efficacy of the BCG vaccine remains a World Health Organization priority. Successful vaccination against M. tuberculosis requires the induction and maintenance of T cells. Targeting molecules that promote T-cell survival may therefore provide an alternative strategy to classic adjuvants. We show that the interaction between T-cell–expressed OX40 and OX40L on antigen-presenting cells is critical for effective immunity to BCG. However, because OX40L is lost rapidly from antigen-presenting cells following BCG vaccination, maintenance of OX40-expressing vaccine-activated T cells may not be optimal. Delivering an OX40L:Ig fusion protein simultaneously with BCG provided superior immunity to intravenous and aerosol M. tuberculosis challenge even 6 months after vaccination, an effect that depends on natural killer 1.1+ cells. Attenuated vaccines may therefore lack sufficient innate stimulation to maintain vaccine-specific T cells, which can be replaced by reagents binding inducible T-cell costimulators.
doi:10.1093/infdis/jir868
PMCID: PMC3282567  PMID: 22315280
5.  Effect of the leukotriene A4 hydrolase aminopeptidase augmentor 4-methoxydiphenylmethane in a pre-clinical model of pulmonary emphysema 
The leukotriene A4 hydrolase enzyme is a dual functioning enzyme with the following two catalytic activities: an epoxide hydrolase function that transforms the lipid metabolite leukotriene A4 to leukotriene B4 and an aminopeptidase function that hydrolyzes short peptides. To date, all drug discovery efforts have focused on the epoxide hydrolase activity of the enzyme, because of extensive biological characterization of the pro- inflammatory properties of its metabolite, leukotriene B4. Herein, we have designed a small molecule, 4-methoxydiphenylmethane, as a pharmacological agent that is bioavailable and augments the aminopeptidase activity of the leukotriene A4 hydrolase enzyme. Pre-clinical evaluation of our drug showed protection against intranasal elastase-induced pulmonary emphysema in murine models.
doi:10.1016/j.bmcl.2011.09.048
PMCID: PMC3209762  PMID: 21983441
Emphysema; COPD; LTB4; aminopeptidase; leukotriene A4 hydrolase; murine model
6.  Lowering the Threshold of Lung Innate Immune Cell Activation Alters Susceptibility to Secondary Bacterial Superinfection 
The Journal of Infectious Diseases  2011;204(7):1086-1094.
Background. Previous studies have shown that the interaction of CD200R, a myeloid inhibitory receptor, with its ligand, CD200, is critical in the control of innate immune activation in the lung.
Methods and Results. Using a mouse model of bacterial superinfection following influenza, we show that an absence of CD200R (a negative regulator highly expressed by macrophages and dendritic cells), restricts commensal and exogenous bacterial invasiveness and completely prevents the mortality observed in wild-type mice. This benefit is due to a heightened innate immune response to influenza virus in cd200r knockout mice that limits immune pathogenesis and viral load. In wild-type mice, apoptotic cells expressing CD200 that we believe contribute to the suppressed innate immune response to bacteria dominate during the resolution phase of influenza-induced inflammation. We also show for the first time the presence of a variety of previously unidentified bacterial species in the lower airways that are significantly adjusted by influenza virus infection and may contribute to the pathophysiology of disease.
Conclusions. The interaction of CD200 with CD200R therefore contributes to the hyporesponsive innate immune state following influenza virus infection that predisposes to secondary bacterial infection, a phenomenon that has the potential for immune modulation.
doi:10.1093/infdis/jir467
PMCID: PMC3164429  PMID: 21881124
7.  MMP-9 Cleaves SP-D and Abrogates Its Innate Immune Functions In Vitro 
PLoS ONE  2012;7(7):e41881.
Possession of a properly functioning innate immune system in the lung is vital to prevent infections due to the ongoing exposure of the lung to pathogens. While mechanisms of pulmonary innate immunity have been well studied, our knowledge of how these systems are altered in disease states, leading to increased susceptibility to infections, is limited. One innate immune protein in the lung, the pulmonary collectin SP-D, has been shown to be important in innate immune defense, as well as clearance of allergens and apoptotic cells. MMP-9 is a protease with a wide variety of substrates, and has been found to be dysregulated in a myriad of lung diseases ranging from asthma to cystic fibrosis; in many of these conditions, there are decreased levels of SP-D. Our results indicate that MMP-9 is able to cleave SP-D in vitro and this cleavage leads to loss of its innate immune functions, including its abilities to aggregate bacteria and increase phagocytosis by mouse alveolar macrophages. However, MMP-9-cleaved SP-D was still detected in a solid-phase E. coli LPS-binding assay, while NE-cleaved SP-D was not. In addition, MMP-9 seems to cleave SP-D much more efficiently than NE at physiological levels of calcium. Previous studies have shown that in several diseases, including cystic fibrosis and asthma, patients have increased expression of MMP-9 in the lungs as well as decreased levels of intact SP-D. As patients suffering from many of the diseases in which MMP-9 is over-expressed can be more susceptible to pulmonary infections, it is possible that MMP-9 cleavage of SP-D may contribute to this phenotype.
doi:10.1371/journal.pone.0041881
PMCID: PMC3408449  PMID: 22860023
8.  Predicting patch occupancy in fragmented landscapes at the rangewide scale for an endangered species: an example of an American warbler 
Diversity & distributions  2012;18(2):158-167.
Aim
Our objective was to identify the distribution of the endangered golden-cheeked warbler (Setophaga chrysoparia) in fragmented oak–juniper woodlands by applying a geoadditive semiparametric occupancy model to better assist decision-makers in identifying suitable habitat across the species breeding range on which conservation or mitigation activities can be focused and thus prioritize management and conservation planning.
Location
Texas, USA.
Methods
We used repeated double-observer detection/non-detection surveys of randomly selected (n = 287) patches of potential habitat to evaluate warbler patch-scale presence across the species breeding range. We used a geoadditive semiparametric occupancy model with remotely sensed habitat metrics (patch size and landscape composition) to predict patch-scale occupancy of golden-cheeked warblers in the fragmented oak–juniper woodlands of central Texas, USA.
Results
Our spatially explicit model indicated that golden-cheeked warbler patch occupancy declined from south to north within the breeding range concomitant with reductions in the availability of large habitat patches. We found that 59% of woodland patches, primarily in the northern and central portions of the warbler’s range, were predicted to have occupancy probabilities ≤0.10 with only 3% of patches predicted to have occupancy probabilities >0.90. Our model exhibited high prediction accuracy (area under curve = 0.91) when validated using independently collected warbler occurrence data.
Main conclusions
We have identified a distinct spatial occurrence gradient for golden-cheeked warblers as well as a relationship between two measurable landscape characteristics. Because habitat-occupancy relationships were key drivers of our model, our results can be used to identify potential areas where conservation actions supporting habitat mitigation can occur and identify areas where conservation of future potential habitat is possible. Additionally, our results can be used to focus resources on maintenance and creation of patches that are more likely to harbour viable local warbler populations.
doi:10.1111/j.1472-4642.2011.00831.x
PMCID: PMC3298116  PMID: 22408381
Bayesian inference; golden-cheeked warbler; habitat conservation; occupancy; semiparametric regression; Setophaga chrysoparia
9.  A critical role for LTA4H in limiting chronic pulmonary neutrophilic inflammation 
Science (New York, N.Y.)  2010;330(6000):90-94.
Leukotriene A4 Hydrolase (LTA4H) is a pro-inflammatory enzyme which generates the inflammatory mediator leukotriene B4 (LTB4). LTA4H also possesses aminopeptidase activity with unknown substrate and physiological significance. We identified the neutrophil chemoattractant, Pro-Gly-Pro (PGP), as this physiological substrate. PGP is a biomarker for chronic obstructive pulmonary disease (COPD), and is implicated in neutrophil persistence in the lung. In acute neutrophil driven inflammation, PGP was degraded by LTA4H, which facilitated the resolution of inflammation. In contrast, cigarette smoke, a major risk factor for the development of COPD, selectively inhibited LTA4H aminopeptidase activity, which led to the accumulation of PGP and neutrophils. These studies imply that therapeutic strategies that inhibit LTA4H to prevent LTB4 generation may not reduce neutrophil recruitment because of elevated PGP.
doi:10.1126/science.1190594
PMCID: PMC3072752  PMID: 20813919
10.  NEUTROPHILS CONTAIN PROLYL ENDOPEPTIDASE AND GENERATE THE CHEMOTACTIC PEPTIDE, PGP, FROM COLLAGEN 
Journal of neuroimmunology  2009;217(1-2):51-54.
Prolyl endopeptidase (PE), a protease that cleaves after proline residues in oligopeptides, is highly active in brain and degrades neuropeptides in vitro. We have recently demonstrated that PE, in concert with MMP's, can generate PGP (prolineglycine-proline), a novel, neutrophil chemoattractant, from collagen. In this study, we demonstrate that human peripheral blood neutrophils contain PE, which is constitutively active, and can generate PGP de novo from collagen after activation with LPS. This novel, pro-inflammatory role for PE raises the possibility of a self-sustaining pathway of neutrophilic inflammation and may provide biomarkers and therapeutic targets for diseases caused by chronic, neutrophilic inflammation.
doi:10.1016/j.jneuroim.2009.09.020
PMCID: PMC2787998  PMID: 19875179
neutrophil; prolyl endopeptidase; PGP; collagen; inflammation
11.  Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production 
PLoS ONE  2010;5(7):e11465.
Background
Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 (Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages.
Methodology/Principal Findings
Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly I∶C). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-κB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase (P25911), the Rho guanine nucleotide exchange factor, Vav (P27870), and the adaptor protein Grb2 (Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo.
Conclusions/Significance
We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.
doi:10.1371/journal.pone.0011465
PMCID: PMC2903609  PMID: 20644716
12.  Respiratory Infections 
Although the outcome of respiratory infection alters with age, nutritional status, and immunologic competence, there is a growing body of evidence that we all develop a unique but subtle inflammatory profile. This uniqueness is determined by the sequence of infections or antigenic insults encountered that permanently mold our lungs through experience. This experience and learning process forms the basis of immunologic memory that is attributed to the acquired immune system. But what happens if the pathogen is not homologous to any preceding it? In the absence of cross-specific acquired immunity, one would expect a response similar to that of a subject who had never been infected with anything before. It is now clear that this is not the case. Prior inflammation in the respiratory tract alters immunity and pathology to subsequent infections even when they are antigenically distinct. Furthermore, the influence of the first infection is long lasting, not dependent on the presence of T and B cells, and effective against disparate pathogen combinations. We have used the term “innate imprinting” to explain this phenomenon, although innate education may be a closer description. This educational process, by sequential waves of infection, may be beneficial, as shown for successive viral infections, or significantly worse, as illustrated by the increased susceptibly to life-threatening bacterial pneumonia in patients infected with seasonal and pandemic influenza. We now examine what these long-term changes involve, the likely cell populations affected, and what this means to those studying inflammatory disorders in the lung.
doi:10.1513/pats.200706-066TH
PMCID: PMC2647650  PMID: 18073393
lung inflammation; heterologous immunity; respiratory tract; influenza; innate immunity
13.  Sustained desensitization to bacterial Toll-like receptor ligands after resolutionof respiratory influenza infection 
The World Health Organization estimates that lower respiratory tract infections (excluding tuberculosis) account for ∼35% of all deaths caused by infectious diseases. In many cases, the cause of death may be caused by multiple pathogens, e.g., the life-threatening bacterial pneumonia observed in patients infected with influenza virus. The ability to evolve more efficient immunity on each successive encounter with antigen is the hallmark of the adaptive immune response. However, in the absence of cross-reactive T and B cell epitopes, one lung infection can modify immunity and pathology to the next for extended periods of time. We now report for the first time that this phenomenon is mediated by a sustained desensitization of lung sentinel cells to Toll-like receptor (TLR) ligands; this is an effect that lasts for several months after resolution of influenza or respiratory syncytial virus infection and is associated with reduced chemokine production and NF-κB activation in alveolar macrophages. Although such desensitization may be beneficial in alleviating overall immunopathology, the reduced neutrophil recruitment correlates with heightened bacterial load during secondary respiratory infection. Our data therefore suggests that post-viral desensitization to TLR signals may be one possible contributor to the common secondary bacterial pneumonia associated with pandemic and seasonal influenza infection.
doi:10.1084/jem.20070891
PMCID: PMC2271005  PMID: 18227219

Results 1-13 (13)