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Inflammatory bowel disease (IBD) is a group of chronic inflammatory conditions of the gastrointestinal (GI) tract. IBD is also associated with a number of co-morbidities that can include inflammation in the lung (Black et al. Chest 2007). IBD-induced pulmonary inflammation is characterized by polymorphonuclear neutrophil (PMN) and lymphocyte inflammation. We have demonstrated neutrophilia and an increase in PMN in the pulmonary system in the DSS model of colitis. We hypothesized that colitis induces PMN-mediated inflammation in the lung, which attracts GI-primed lymphocytes. Employing the DSS model of colitis, we observed a significant correlation between PMN in the circulatory and pulmonary system (R2=0.845, N=12). Extravasation of PMN into lung tissue was observed and was associated with a 2-fold increase in integrin β2 expression and an increase in IL-1β and CCL2 protein levels. To examine whether DSS pulmonary inflammation resulted in lymphocyte recruitment to the lungs, an adoptive transfer experimen...

Infants born very preterm are usually exposed to high oxygen concentrations but this may impair lung function in survivors in later life. However, the precise changes involved are poorly understood. We determined how neonatal hyperoxia alters lung function at mid-adulthood in mice. Neonatal C57BL/6J mice inhaled 65% oxygen (HE group) from birth for 7 days. They then breathed room air until 11 months of age (P11mo); these mice experienced growth restriction. Controls breathed only room air. To exclude the effects of growth restriction, a group of dams was rotated between hyperoxia and normoxia during the exposure period (HE + DR group). Lung function was measured at P11mo. HE mice had increased inspiratory capacity, work of breathing and tissue damping. HE + DR mice had further increases in inspiratory capacity and work of breathing, and reduced FEV 100 /FVC. Total lung capacity was increased in HE+DR males. HE males had elevated responses to methacholine. Neonatal hyperoxia alters lung function at mid-adulthood, especially in males.

Background : The zoonotic bacterium Campylobacter jejuni has a broad host range but is especially associated with birds, both domestic and wild. Earlier studies have indicated thrushes of the genus Turdus in Europe to be frequently colonized with C. jejuni , and predominately with host-associated specific genotypes. The European Blackbird Turdus merula has a large distribution in Europe, including some oceanic islands, and was also introduced to Australia by European immigrants in the 1850s. Methods : The host specificity and temporal stability of European Blackbird C. jejuni was investigated with multilocus sequence typing in a set of isolates collected from Sweden, Australia, and The Azores. Results : Remarkably, we found that the Swedish, Australian, and Azorean isolates were genetically highly similar, despite extensive spatial and temporal isolation. This indicates adaptation, exquisite specificity, and stability in time for European Blackbirds, which is in sharp contrast with the high levels of recombination and mutation found in poultry-related C. jejuni genotypes. Conclusion : The maintenance of host-specific signals in spatially and temporally separated C. jejuni populations suggests the existence of strong purifying selection for this bacterium in European Blackbirds. Keywords: gastrointestinal pathogen ; Campylobacter jejuni; Blackbird; MLST; population structure (Published: 2 December 2015) Citation: Infection Ecology and Epidemiology 2015, 5: 28383 - http://dx.doi.org/10.3402/iee.v5.28383

© 2015 King et al. Nontypeable Haemophilus influenzae (NTHi) is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1), oxidative stress and 2), protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS) production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT) in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps.

Viral exacerbations of chronic obstructive pulmonary disease (COPD), commonly caused by rhinovirus (RV) infections, are poorly controlled by current therapies. This is due to a lack of understanding of the underlying immunopathological mechanisms. Human studies have identified a number of key immune responses that are associated with RV-induced exacerbations including neutrophilic inflammation, expression of inflammatory cytokines and deficiencies in innate anti-viral interferon. Animal models of COPD exacerbation are required to determine the contribution of these responses to disease pathogenesis. We aimed to develop a short-term mouse model that reproduced the hallmark features of RV-induced exacerbation of COPD. Evaluation of complex protocols involving multiple dose elastase and lipopolysaccharide (LPS) administration combined with RV1B infection showed suppression rather than enhancement of inflammatory parameters compared with control mice infected with RV1B alone. Therefore, these approaches did not accurately model the enhanced inflammation associated with RV infection in patients with COPD compared with healthy subjects. In contrast, a single elastase treatment followed by RV infection led to heightened airway neutrophilic and lymphocytic inflammation, increased expression of tumour necrosis factor (TNF)-α, C-X-C motif chemokine 10 (CXCL10)/IP-10 (interferon γ-induced protein 10) and CCL5 [chemokine (C-C motif) ligand 5]/RANTES (regulated on activation, normal T-cell expressed and secreted), mucus hypersecretion and preliminary evidence for increased airway hyper-responsiveness compared with mice treated with elastase or RV infection alone. In summary, we have developed a new mouse model of RV-induced COPD exacerbation that mimics many of the inflammatory features of human disease. This model, in conjunction with human models of disease, will provide an essential tool for studying disease mechanisms and allow testing of novel therapies with potential to be translated into clinical practice., The present study describes a new short-term mouse model of rhinovirus (RV)-induced exacerbation of COPD (chronic obstructive pulmonary disease) which will facilitate insight into disease mechanisms and could provide a more efficient tool to test novel therapies with potential to be translated into clinical practice.

The complex etiology and heterogeneity of respiratory diseases have made it difficult to design pharmacological reagents that show good efficacy across a diseased population. The discovery of small ‘non-coding’ RNA molecules (small interfering (si)- and micro-RNAs) that regulate the function of a gene or clusters of genes and the design of synthetic analogues of these molecules may provide new directions for the treatment and characterization of respiratory disorders.

Chlamydia pneumoniae causes a range of respiratory infections including bronchitis, pharyngitis and pneumonia. Infection has also been implicated in exacerbation/initiation of asthma and chronic obstructive pulmonary disease (COPD) and may play a role in atherosclerosis and Alzheimer's disease. We have used a mouse model of Chlamydia respiratory infection to determine the effectiveness of intranasal (IN) and transcutaneous immunization (TCI) to prevent Chlamydia lung infection. Female BALB/c mice were immunized with chlamydial major outer membrane protein (MOMP) mixed with cholera toxin and CpG oligodeoxynucleotide adjuvants by either the IN or TCI routes. Serum and bronchoalveolar lavage (BAL) were collected for antibody analysis. Mononuclear cells from lung-draining lymph nodes were stimulated in vitro with MOMP and cytokine mRNA production determined by real time PCR. Animals were challenged with live Chlamydia and weighed daily following challenge. At day 10 (the peak of infection) animals were sacrificed and the numbers of recoverable Chlamydia in lungs determined by real time PCR. MOMP-specific antibody-secreting cells in lung tissues were also determined at day 10 post-infection. Both IN and TCI protected animals against weight loss compared to non-immunized controls with both immunized groups gaining weight by day 10-post challenge while controls had lost 6% of body weight. Both immunization protocols induced MOMP-specific IgG in serum and BAL while only IN immunization induced MOMP-specific IgA in BAL. Both immunization routes resulted in high numbers of MOMP-specific antibody-secreting cells in lung tissues (IN>TCI). Following in vitro re-stimulation of lung-draining lymph node cells with MOMP; IFNgamma mRNA increased 20-fold in cells from IN immunized animals (compared to non-immunized controls) while IFNgamma levels increased 6- to 7-fold in TCI animals. Ten days post challenge non-immunized animals had >7,000 IFU in their lungs, IN immunized animals

Asthma affects 10% of the population in Westernized countries, being most common in children. It is a heterogeneous condition characterized by chronic allergic airway inflammation, mucus hypersecretion, and airway hyperresponsiveness (AHR) to normally innocuous antigens. Combination therapies with inhaled corticosteroids and bronchodilators effectively manage mild to moderate asthma, but there are no cures, and patients with severe asthma do not respond to these treatments. The inception of asthma is linked to respiratory viral (respiratory syncytial virus, rhinovirus) and bacterial (Chlamydia, Mycoplasma) infections. The examination of mouse models of early-life infections and allergic airway disease (AAD) provides valuable insights into the mechanisms of disease inception that may lead to the development of more effective therapeutics. For example, early-life, but not adult, Chlamydia respiratory infections in mice permanently modify immunity and lung physiology. This increases the severity of AAD by promoting IL-13 expression, mucus hypersecretion, and AHR. We have identified novel roles for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and IL-13 in promoting infection-induced pathology in early life and subsequent chronic lung disease. Genetic deletion of TRAIL or IL-13 variously protected against neonatal infection-induced inflammation, mucus hypersecretion, altered lung structure, AHR, and impaired lung function. Therapeutic neutralization of these factors prevented infection-induced severe AAD. Other novel mechanisms and avenues for intervention are also being explored. Such studies indicate the immunological mechanisms that may underpin the association between early-life respiratory infections and the development of more severe asthma and may facilitate the development of tailored preventions and treatments.

Background: Avian influenza viruses (AIVs) are found worldwide in numerous bird species, causing significant disease in gallinaceous poultry and occasionally other species. Surveillance of wild bird reservoirs provides an opportunity to add to the understanding of the epidemiology of AIVs. Methods: This study examined key findings from the National Avian Influenza Wild Bird Surveillance Program over a 5-year period (July 2007-June 2012), the main source of information on AIVs circulating in Australia. Results: The overall proportion of birds that tested positive for influenza A via PCR was 1.90.1%, with evidence of widespread exposure of Australian wild birds to most low pathogenic avian influenza (LPAI) subtypes (H1-13, H16). LPAI H5 subtypes were found to be dominant and widespread during this 5-year period. Conclusion: Given Australia's isolation, both geographically and ecologically, it is important for Australia not to assume that the epidemiology of AIV from other geographic regions applies here. Despite all previous highly pathogenic avian influenza outbreaks in Australian poultry being attributed to H7 subtypes, widespread detection of H5 subtypes in wild birds may represent an ongoing risk to the Australian poultry industry.

The McDonald Islands (53°S, 73°E) originally comprised three small islands that lie on the Kerguelen Plateau, 44 km west of Heard Island. No volcanic activity was observed since their discovery in 1874 until 1997, when two passing ships recorded major changes and eruptive behaviour. A 2001 satellite image showed that the main island had doubled its area. This paper reports observations made from a cruise ship in November 2002, supplemented by a high-resolution satellite image acquired in March 2003. A new volcanic complex comprises lava domes, spines and flows, all assumed to be phonolitic, similar to the older volcanic rocks. The complex shows dormant volcanic activity, with numerous fumaroles, recent spine evolution and lava flows. Changes in relative sea level have connected Flat and McDonald Islands. A spit about 1km long with extensive shoals beyond, now extends eastward from McDonald Island and presents new hazards to shipping. Biological changes include colonization by king penguins (Aptenodytes patagonica), previously absent, and a large reduction in numbers of formerly widespread macaroni penguins (Eudyptes chrysolophus chrysolophus).

Asthma is a common inflammatory disease of the airways that results in airway narrowing and wheezing. Allergic asthma is characterised by a T-helper cell-type (Th) 2 response, immunoglobulin (Ig) E production, and eosinophilic influx into the airways. Recently, many clinical studies have implicated Mycoplasma pneumoniae and Chlamydia pneumoniae in the development and exacerbation of both chronic and acute asthma. It is widely accepted that M. pneumoniae and C. pneumoniae infections require Th1 immunity for clearance; therefore, according to the hygiene hypothesis, these infections should be protective against asthma. Here, we review the clinical evidence for the association and mechanisms of predisposition to and protection against asthma by these infections. We will examine the following question: Is it the absence of infection or the age of the individual on infection that confers susceptibility or resistance to asthma and does this vary between normal and predisposed individuals? We put forward a hypothesis of the effects of these infections on the development and prevention of asthma and how novel preventative and treatment strategies involving these microbes may be targeted against asthma.

Chronic obstructive pulmonary disease (COPD) patients are at increased risk of developing nonsmall cell lung carcinoma, irrespective of their smoking history. Although the mechanisms behind this observation are not clear, established drivers of carcinogenesis in COPD include oxidative stress and sustained chronic inflammation. Mitochondria are critical in these two processes and recent evidence links increased oxidative stress in COPD patients to mitochondrial damage. We therefore postulate that mitochondrial damage in COPD patients leads to increased oxidative stress and chronic inflammation, thereby increasing the risk of carcinogenesis.The functional state of the mitochondrion is dependent on the balance between its biogenesis and degradation (mitophagy). Dysfunctional mitochondria are a source of oxidative stress and inflammasome activation. In COPD, there is impaired translocation of the ubiquitin-related degradation molecule Parkin following activation of the Pink1 mitophagy pathway, resulting in excessive dysfunctional mitochondria. We hypothesise that deranged pathways in mitochondrial biogenesis and mitophagy in COPD can account for the increased risk in carcinogenesis. To test this hypothesis, animal models exposed to cigarette smoke and developing emphysema and lung cancer should be developed. In the future, the use of mitochondria-based antioxidants should be studied as an adjunct with the aim of reducing the risk of COPD-associated cancer.

© 2018 American Physiological Society. All rights reserved. Chronic obstructive pulmonary disease (COPD) is the third leading cause of morbidity and death and imposes major socioeconomic burdens globally. It is a progressive and disabling condition that severely impairs breathing and lung function. There is a lack of effective treatments for COPD, which is a direct consequence of the poor understanding of the underlying mechanisms involved in driving the pathogenesis of the disease. Toll-like receptor (TLR)2 and TLR4 are implicated in chronic respiratory diseases, including COPD, asthma and pulmonary fibrosis. However, their roles in the pathogenesis of COPD are controversial and conflicting evidence exists. In the current study, we investigated the role of TLR2 and TLR4 using a model of cigarette smoke (CS)-induced experimental COPD that recapitulates the hallmark features of human disease. TLR2, TLR4, and associated coreceptor mRNA expression was increased in the airways in both experimental and human COPD. Compared with wild-type (WT) mice, CS-induced pulmonary inflammation was unaltered in TLR2-deficient (Tlr2-/-) and TLR4-deficient (Tlr4-/-) mice. CS-induced airway fibrosis, characterized by increased collagen deposition around small airways, was not altered in Tlr2-/- mice but was attenuated in Tlr4-/- mice compared with CS-exposed WT controls. However, Tlr2-/- mice had increased CS-induced emphy-sema-like alveolar enlargement, apoptosis, and impaired lung function, while these features were reduced in Tlr4-/- mice compared with CS-exposed WT controls. Taken together, these data highlight the complex roles of TLRs in the pathogenesis of COPD and suggest that activation of TLR2 and/or inhibition of TLR4 may be novel therapeutic strategies for the treatment of COPD.

Bronchoalveolar lavage (BAL) is a simple, yet informative tool in understanding the immunopathology of various lung diseases via quantifying various inflammatory cells, cytokines and growth factors. At present, this traditional method is often blended with several robust and sophisticated molecular and biological techniques sustaining the significance and longevity of this technique. Crucially, the existence of slightly distinct approaches and variables employed at different laboratories around the globe in performing BAL aspiration indeed demands an utmost need to optimize and develop an effective, cost-effective and a reproducible technique. This mini review will be of importance to the biological translational scientist, particularly respiratory researchers in understanding the fundamentals and approaches to apply and consider with BAL aspiration techniques. This will ensure generating a meaningful and clinically relevant data which in turn accelerate the development of new and effective therapeutic moieties for major respiratory conditions.

The immune processes involved in the clearance and immunopathology of Chlamydia infection in the female reproductive tract (FRT) are not well understood. In previous ground-breaking studies we showed that IFN-ɛ, a novel type I IFN that is exclusively and constitutively expressed in the FRT, plays an important role in protecting against Chlamydia infections. Here, we examined the effects of IFN-ɛ on innate immune processes in the FRT in order to elucidate the mechanisms by which IFN-ɛ protects against Chlamydia infections. Female WT and IFN-ɛ−/− C57BL/6 mice were pre-treated with progesterone and infected intra-vaginally with Chlamydia muridarum or sham-infected. Uterine horns were harvested and the effects of IFN-ɛ deficiency on immune factor expression and cellular infiltration were assessed using microarray/bioinformatics analyses, real-time qPCR and flow cytometry. IFN-ɛ uniquely regulated the expression of 744 genes at baseline and 802 genes during Chlamydia infection, and universally regulated the transcription of 61 genes, regardless of infection status. Of note, pathways associated with protective innate responses, such as IFN regulatory factor activation and pattern recognition receptor signalling were down-regulated in Chlamydia-infected IFN-ɛ−/− mice compared to WT controls. These changes correlate with a decrease in the expression of many important IFN-γ signalling molecules and number of IFN-γ-producing NK cells in the FRT during infection. These findings suggest that IFN-ɛ may protect against Chlamydia FRT infections by potentiating innate immune processes important for the clearance of infection, particularly pathways associated with IFN-γ signalling and the activation of NK cell responses.

CO2 produced by systemic cellular respiration is hydrated into carbonic acid (H2CO3) that dissociates into H+ and HCO3−. These are transported in the plasma to the lungs where HCO3− is converted back into H2CO3 and CO2 that are expelled through breathing. Evidence suggests that dysfunction of the mechanisms that govern these processes may result in the development of respiratory acidosis (RA) and the cardinal features of severe, steroid-resistant (SSR) asthma. Reduced lung function, which occurs in SSR asthma, impairs removal of volatile H2CO3 and CO2, resulting in acid accumulation and increased arterial PaCO2. Patients with severe asthma often develop complications from increased PaCO2, which skews the PaCO2/HCO3− ratio resulting in increased H+ concentration and reduced pH. We developed three mouse models of respiratory infection and ovalbumin-induced SSR allergic airways disease (SSRAAD) that are highly representative of SSR asthma in humans. We used these models to show a role for impaired homeostatic acid-base balance in SSR asthma. All three infections suppress the induction of the expression of the chloride (Cl−)/HCO− pump, Slc26a4, in the airway mucosa in AAD. Importantly, SSRAAD is associated with increased levels of free H+ ions in bronchoalveolar lavage fluid. Administration of Slc26a4-specific siRNA in steroid-sensitive AAD, which mimics the effect of decreased Slc26a4 responses in SSRAAD, induced RA and steroid-resistant airway inflammation and AHR. Importantly, treatment of RA with NaHCO3 during infection-induced SSRAAD suppressed steroid-resistant AHR. Thus, we have identified a previously unrecognised role for deficient Slc26a4 responses that result in the development of RA and the pathogenesis of SSRAAD.

Altered iron levels and/or dysregulated iron homeostasis have been associated with a number of lung diseases, however, the mechanisms that underpin these associations, and whether iron plays a role in the pathogenesis of disease, are yet to be fully elucidated. In this study, systemic and pulmonary iron and lung structure and function were assessed in transferrin receptor (TFR)2 mutant and wild-type (WT) BALB/c mice fed a high-iron diet (containing 2% carbonyl iron) compared to normal diet controls, respectively. The effects of increased iron loading on murine models of ovalbumin- and house dust mite-induced allergic airways disease (AAD) were also assessed. Excess iron accumulation was observed in the lungs in both the genetic and diet-induced models of iron overloading. Increased iron levels in the lung were associated with emphysema-like alveolar enlargement, small airways collagen deposition, alterations in baseline lung function and increased airways hyper-responsiveness (AHR). Increased iron loading also resulted in altered type 1, 2 and 17 cytokine production, increased eosinophilic inflammation and severe, steroid-resistant AHR in AAD. Interestingly, AAD also results in altered systemic and pulmonary iron levels and iron regulatory molecule expression. These data show that increased iron levels in the lung results in emphysema and airways fibrosis that corresponds with reduced lung function. We also show that lung disease may be closely associated with changes in iron homeostasis. These models will be used to characterize the interplay between iron and immunity in the pathogenesis of lung disease and determine the therapeutic effectiveness of correcting dysregulated iron homeostasis for the treatment of lung disease.

There is a critical window in early-life where the lung is still maturing and susceptible to infection. Indeed, severe respiratory infections in early-life are a risk factor for the development of chronic lung diseases. The aim of this study was to identify the mechanisms involved. Neonatal wild-type (WT), TLR2 deficient (−/−), IL-13−/−, MyD88−/− and STAT6−/− mice were infected with the natural mouse bacterial pathogen Chlamydia muridarum, as a model of severe respiratory tract infection in early-life. In some experiments WT mice were treated with miR-21 antagomirs, PI3K inhibitors, or relevant controls during early-life infection. The impact of targeting these specific immune molecules during early-life on infection-induced impairment of lung function and structure were assessed. Neonatal Chlamydia respiratory infection increased TLR2, IL-13-receptor, miR-21 and PI3K expression and/or activity in the lung. TLR2 signalling induced IL-13-receptor expression, IL-13 signalling induced miR-21 expression and miR-21 increased PI3K activity. TLR2 signalling also increased IL-13+ ILC2s in the lung. TLR2−/− and IL-13−/− mice were protected against infection-induced airway hyperresponsiveness (AHR), but not emphysema-like alveolar enlargement. This TLR2/IL-13 mediated phenotype was independent of MyD88, but dependent on STAT6. Specific targeting of miR-21 prevented AHR but not emphysema. Pan-PI3K inhibition did not affect AHR, but protected against emphysema. Interestingly, early-life infection-induced AHR was steroid insensitive. This study identifies a novel signalling network that may be targeted for the prevention of the long-term deleterious effects of early-life infection on lung function and structure.

Chronic respiratory diseases, including asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), are among the leading causes of mortality and morbidity worldwide. In the past decade, the interest in the role of microbiome in maintaining lung health and in respiratory diseases has grown exponentially. The advent of sophisticated multiomics techniques has enabled the identification and characterisation of microbiota and their roles in respiratory health and disease. Furthermore, associations between the microbiome of the lung and gut, as well as the immune cells and mediators that may link these two mucosal sites, appear to be important in the pathogenesis of lung conditions. Here we review the recent evidence of the role of normal gastrointestinal and respiratory microbiome in health and how dysbiosis affects chronic pulmonary diseases. The potential implications of host and environmental factors such as age, gender, diet and use of antibiotics on the composition and overall functionality of microbiome are also discussed. We summarise how microbiota may mediate the dynamic process of immune development and/or regulation focusing on recent data from both clinical human studies and translational animal studies. This furthers the understanding of the pathogenesis of chronic pulmonary diseases and may yield novel avenues for the utilisation of microbiota as potential therapeutic interventions.

BackgroundThe underlying mechanisms of idiopathic pulmonary fibrosis (IPF) are unknown. This progressive disease has high mortality rates, and current models for prediction of mortality have limited value in identifying which patients will progress. We previously showed that the glycoprotein fibulin-1 is involved in enhanced proliferation and wound repair by mesenchymal cells and, thus, may contribute to lung fibrosis in IPF.MethodsSerum, lung tissue, and lung function values were obtained from four independent locations (Sydney, NSW, and Perth, WA, Australia; San Francisco, CA; and Modena, Italy). Patients with IPF were followed for a minimum of 1 year and progression was defined as a significant decline in lung function or death. Primary parenchymal lung fibroblasts of 15 patients with and without IPF were cultured under nonstimulatory conditions. Fibulin-1 levels in serum, and secreted or deposited by fibroblasts, were measured by western blot and in lung tissue by immunohistochemistry.ResultsSerum fibulin-1 levels were increased in patients with IPF compared with subjects without lung disease (P = .006). Furthermore, tissue fibulin-1 levels were increased in patients with IPF (P = .02) and correlated negatively with lung function (r = -0.9, P < .05). Primary parenchymal fibroblasts from patients with IPF produced more fibulin-1 than those from subjects without IPF (P < .05). Finally, serum fibulin-1 levels at first blood draw predicted disease progression in IPF within 1 year (area under the curve , 0.71; 95% CI, 0.57-0.86; P = .012).ConclusionsFibulin-1 is a novel potential biomarker for disease progression in IPF and raises the possibility that it could be used as a target for the development of new treatments.

Background Steroid-insensitive endotypes of asthma are an important clinical problem and effective therapies are required. They are associated with bacterial infection and non-eosinophilic inflammatory responses in the asthmatic lung. Macrolide therapy is effective in steroid-insensitive endotypes, such as non-eosinophilic asthma. However, whether the effects of macrolides are due to antimicrobial or anti-inflammatory mechanisms is not known. Objective To determine and assess the efficacy of macrolide (ie, clarithromycin) and non-macrolide (ie, amoxicillin) antibiotic treatments in experimental models of infection-induced, severe, steroid-insensitive neutrophilic allergic airways disease (SSIAAD), compared with steroid-sensitive AAD and to delineate the antimicrobial and anti-inflammatory effects of macrolide therapy. Methods We developed and used novel mouse models of Chlamydia and Haemophilus lung infection-induced SSIAAD. We used these models to investigate the effects of clarithromycin and amoxicillin treatment on immune responses and airways hyper-responsiveness (AHR) in Ova-induced, T helper lymphocyte (Th) 2 -associated steroid-sensitive AAD and infection-induced Th1/Th17-associated SSIAAD compared with dexamethasone treatment. Results Clarithromycin and amoxicillin had similar antimicrobial effects on infection. Amoxicillin did attenuate some features, but did not broadly suppress either form of AAD. It did restore steroid sensitivity in SSIAAD by reducing infection. In contrast, clarithromycin alone widely suppressed inflammation and AHR in both steroid-sensitive AAD and SSIAAD. This occurred through reductions in Th2 responses that drive steroid-sensitive eosinophilic AAD and tumour necrosis factor α and interleukin 17 responses that induce SSIAAD. Conclusions Macrolides have broad anti-inflammatory effects in AAD that are likely independent of their antimicrobial effects. The specific responses that are suppressed are dependent upon the responses that dominate during AAD.

The pathogenesis of chronic obstructive pulmonary disease (COPD) and its exacerbations, are intricately linked to colonisation and infection with bacteria and other microbes. Despite their undeniable importance, we have a poor understanding of the complex relationships between COPD phenotypes, physiology, cellular and molecular biology and the roles of colonising microbe or infecting pathogens. The management algorithms for the care of patients with COPD that include microbial influences, have almost exclusively been developed using microbial methods that were entirely dependent on the ability to grow bacteria on suitable media. The shortcomings of this approach are becoming clear now that it is possible to completely and accurately define the microbial ecology of ecosystems using genomic methods, which do not rely on the ability to cultivate the organisms present. Whilst our appreciation of the relationships between some bacterial ecosystems and the organ in which they reside in humans is now relatively advanced, this is not true for lung. This perspective serves to highlight the growing importance of including an accurate description of bacterial ecology in any attempt to decipher the pathobiology of COPD. While this field is in its infancy, there is significant potential to gain new insights which will translate into more rational and effective treatment algorithms for patients with COPD.

Protease serine member S31 (Prss31)/transmembrane tryptase/tryptase-γ is a mast cell (MC)-restricted protease of unknown function that is retained on the outer leaflet of the plasma membrane when MCs are activated. We determined the nucleotide sequences of the Prss31 gene in different mouse strains and then used a Cre/loxP homologous recombination approach to create a novel Prss31(-/-) C57BL/6 mouse line. The resulting animals exhibited no obvious developmental abnormality, contained normal numbers of granulated MCs in their tissues, and did not compensate for their loss of the membrane tryptase by increasing their expression of other granule proteases. When Prss31-null MCs were activated with a calcium ionophore or by their high affinity IgE receptors, they degranulated in a pattern similar to that of WT MCs. Prss31-null mice had increased baseline airway reactivity to methacholine but markedly reduced experimental chronic obstructive pulmonary disease and colitis, thereby indicating both beneficial and adverse functional roles for the tryptase. In a cigarette smoke-induced model of chronic obstructive pulmonary disease, WT mice had more pulmonary macrophages, higher histopathology scores, and more fibrosis in their small airways than similarly treated Prss31-null mice. In a dextran sodium sulfate-induced acute colitis model, WT mice lost more weight, had higher histopathology scores, and contained more Cxcl-2 and IL-6 mRNA in their colons than similarly treated Prss31-null mice. The accumulated data raise the possibility that inhibitors of this membrane tryptase may provide additional therapeutic benefit in the treatment of humans with these MC-dependent inflammatory diseases.

Pregnancy provides a unique challenge for maternal immunity, requiring the ability to tolerate the presence of a semi-allogeneic foetus, and yet still being capable of inducing an immune response against invading pathogens. To achieve this, numerous changes must occur in the activity and function of maternal immune cells throughout the course of pregnancy. Respiratory viruses take advantage of these changes, altering the sensitive balance of maternal immunity, leaving the mother with increased susceptibility to viral infections and increased disease severity. Influenza virus is one of the most common respiratory virus infections during pregnancy, leading to an increased risk of ICU hospitalisations, pneumonia, acute respiratory distress syndrome and even death. Whilst much research has been performed to understand the changes that must take place in maternal immunity during pregnancy, considerable work is still needed to fully comprehend this tremendous feat. To date, few studies have focused on the alterations that occur in maternal immunity during respiratory virus infections. This review highlights the role of dendritic cells (DCs) and CD8 T cells during pregnancy, and the changes that occur in these antiviral cells following influenza virus infections.

Tetramer-forming tryptase (hTryptase-β) was recently discovered to have a prominent role in preventing the internal accumulation of life-threatening fibrin deposits and fibrin-platelet clots. The anticoagulant activity of hTryptase-β is an explanation for the presence of hemorrhagic disorders in some patients with anaphylaxis or mastocytosis. The fragments of hFibrinogen formed by the proteolysis of this prominent protein by hTryptase-β could be used as biomarkers in the blood and/or urine for the identification and monitoring of patients with mast cell-dependent disorders. Recombinant hTryptase-β has potential to be used in clinical settings where it is desirable to inhibit blood coagulation.

International audience; Objective The spondyloarthritides share genetic susceptibility, interleukin-23 (IL-23) dependence, and the involvement of microbiota. The aim of the current study was to elucidate how host genetics influence gut microbiota and the relationship between microbiota and organ inflammation in spondyloarthritides. Methods BALB/c ZAP-70(W163C)-mutant (SKG) mice, Toll-like receptor 4 (TLR-4)-deficient SKG mice, and wild-type BALB/c mice were housed under specific pathogen-free conditions. SKG and wild-type BALB/c mice were maintained under germ-free conditions, and some of these mice were recolonized with altered Schaedler flora. All of the mice were injected intraperitoneally with microbial -1,3-glucan (curdlan). Arthritis, spondylitis, and ileitis were assessed histologically. Microbiome composition was analyzed in serial fecal samples obtained from mice that were co-housed beginning at the time of weaning, using 454 pyrosequencing. Infiltrating cells and cytokines in the peritoneal cavity were measured by flow cytometry and enzyme-linked immunosorbent assay. Cytokine, endoplasmic reticulum (ER) stress marker, and tight junction protein transcription was measured by quantitative real-time polymerase chain reaction. Results Microbiota content and response to curdlan varied according to whether T cell receptor signal strength was normal or was impaired due to the ZAP-70(W163C) mutation. Curdlan triggered acute inflammation regardless of the presence of the SKG allele or microbiota. However, no or limited microbiota content attenuated the severity of arthritis. In contrast, ileal IL-23 expression, ER stress, lymph node IL-17A production, goblet cell loss, and ileitis development were microbiota-dependent. Ileitis but not arthritis was suppressed by microbiota transfer upon co-housing SKG mice with wild-type BALB/c mice, as well as by TLR-4 deficiency. Conclusion The interaction between immunogenetic background and host microbiota leads to an IL-23-dependent loss of mucosal function, triggering ileitis in response to curdlan.

Characterisation of 35 Kluyveromyces lactis strains lacking mitochondrial DNA has shown that mutations suppressing rho(0)-lethality are limited to the ATP1, 2 and 3 genes coding for the alpha-, beta- and gamma- subunits of mitochondrial F(1)-ATPase. All atp mutations reduce growth on glucose and three alleles, atp1-2, 1-3 and atp3-1, produce a respiratory deficient phenotype that indicates a drop in efficiency of the F(1)F(0)-ATP synthase complex. ATPase activity is needed for suppression as a double mutant containing an atp allele, together with a mutation abolishing catalytic activity, does not suppress rho(0)-lethality. Positioning of the seven amino acids subject to mutation on the bovine F(1)-ATPase structure shows that two residues are found in a membrane proximal region while five amino acids occur at a region suggested to be a molecular bearing. The intriguing juxtaposition of mutable amino acids to other residues subject to change suggests that mutations affect subunit interactions and alter the properties of F(1) in a manner yet to be determined. An explanation for suppressor activity of atp mutations is discussed in the context of a possible role for F(1)-ATPase in the maintenance of mitochondrial inner membrane potential.

Kluyveromyces lactis is a petite-negative yeast that does not form viable mitochondrial genome-deletion mutants (petites) when treated with DNA-targeting drugs. Loss of mtDNA is lethal for this yeast but mutations at three loci termed MGI, for mitochondrial genome integrity, can suppress this lethality. The three loci encode the alpha-, beta- and gamma-subunits of mitochondrial F1-ATPase. In this study we report the isolation and characterization of the KlATPdelta gene encoding the delta-subunit of F1-ATPase. The deduced protein contains 158 amino acids showing 72% identity to the protein from Saccharomyces cerevisiae and a putative mitochondrial targeting sequence of 23 amino acids. Disruption of the gene causes cells to become respiratory deficient while the introduction of ATPdelta from S. cerevisiae restores growth on glycerol. Cells with a disrupted ATPdelta gene, like strains with disruptions of alpha-, beta- and gamma-F1-subunits, do not produce petite mutants when treated with ethidium bromide. However, unlike strains with disruptions in the three largest F1-subunits, disruption of ATPdelta in the presence of some mgi alleles does not abolish the Mgi- phenotype. By contrast, elimination of ATPdelta in other mgi strains removes resistance to ethidium bromide and rho0 mutants are not formed. Hence the ATPdelta subunit of F1-ATPase, while not mandatory for a Mgi- phenotype, aids some mgi alleles in suppressing rho0 lethality.

The induction of regulatory T cells (Tregs) to suppress aberrant inflammation and immunity has potential as a therapeutic strategy for asthma. Recently, we identified key immunoregulatory components of Streptococcus pneumoniae, type 3 polysaccharide and pneumolysoid (T+P), which suppress allergic airways disease (AAD) in mouse models of asthma. To elucidate the mechanisms of suppression, we have now performed a thorough examination of the role of Tregs. BALB/c mice were sensitized to OVA (day 0) i.p. and challenged intranasal (12–15 d later) to induce AAD. T+P was administered intratracheally at the time of sensitization in three doses (0, 12, and 24 h). T+P treatment induced an early (36 h–4 d) expansion of Tregs in the mediastinal lymph nodes, and later (12–16 d) increases in these cells in the lungs, compared with untreated allergic controls. Anti-CD25 treatment showed that Treg-priming events involving CD25, CCR7, IL-2, and TGF-β were required for the suppression of AAD. During AAD, T+P-induced Tregs in the lungs displayed a highly suppressive phenotype and had an increased functional capacity. T+P also blocked the induction of IL-6 to prevent the Th17 response, attenuated the expression of the costimulatory molecule CD86 on myeloid dendritic cells (DCs), and reduced the number of DCs carrying OVA in the lung and mediastinal lymph nodes. Therefore, bacterial components (T+P) drive the differentiation of highly suppressive Tregs, which suppress the Th2 response, prevent the Th17 response and disable the DC response resulting in the effective suppression of AAD.

BackgroundTumstatin is a segment of the collagen-IV protein that is markedly reduced in the airways of asthmatics. Tumstatin can play an important role in the development of airway remodelling associated with asthma due to its anti-angiogenic properties. This study assessed the anti-angiogenic properties of smaller peptides derived from tumstatin, which contain the interface tumstatin uses to interact with the αVβ3 integrin.MethodsPrimary human lung endothelial cells were exposed to the LF-15, T3 and T7 tumstatin-derived peptides and assessed for cell viability and tube formation in vitro. The impact of the anti-angiogenic properties on airways hyperresponsiveness (AHR) was then examined using a murine model of chronic OVA-induced allergic airways disease.ResultsThe LF-15 and T7 peptides significantly reduced endothelial cell viability and attenuated tube formation in vitro. Mice exposed to OVA+ LF-15 or OVA+T7 also had reduced total lung vascularity and AHR was attenuated compared to mice exposed to OVA alone. T3 peptides reduced cell viability but had no effect on any other parameters.ConclusionThe LF-15 and T7 peptides may be appropriate candidates for use as novel pharmacotherapies due to their small size and anti-angiogenic properties observed in vitro and in vivo.

Preterm infants who receive supplemental oxygen for prolonged periods are at increased risk of impaired lung function later in life. This suggests that neonatal hyperoxia induces persistent changes in small conducting airways (bronchioles). Although the effects of neonatal hyperoxia on alveolarization are well documented, little is known about its effects on developing bronchioles. We hypothesized that neonatal hyperoxia would remodel the bronchiolar walls, contributing to altered lung function in adulthood. We studied three groups of mice (C57BL/6J) to postnatal day 56 (P56; adulthood) when they either underwent lung function testing or necropsy for histological analysis of the bronchiolar wall. One group inhaled 65% O2 from birth until P7, after which they breathed room air; this group experienced growth restriction (HE+GR group). We also used a group in which hyperoxia-induced GR was prevented by dam rotation (HE group). A control group inhaled room air from birth. At P56, the bronchiolar epithelium of HE mice contained fewer Clara cells and more ciliated cells, and the bronchiolar wall contained ∼25% less collagen than controls; in HE+GR mice the bronchiolar walls had ∼13% more collagen than controls. Male HE and HE+GR mice had significantly thicker bronchiolar epithelium than control males and altered lung function (HE males: greater dynamic compliance; HE+GR males: lower dynamic compliance). We conclude that neonatal hyperoxia remodels the bronchiolar wall and, in adult males, affects lung function, but effects are altered by concomitant growth restriction. Our findings may partly explain the reports of poor lung function in ex-preterm children and adults.

Chronic inflammatory diseases of the lung are leading causes of morbidity and mortality worldwide. Many of these disorders can be attributed to abnormal immune responses to environmental stimuli and infections. As such, understanding the innate host defense pathways and their regulatory systems will be critical to developing new approaches to treatment. In this regard, there is increasing interest in the role of microRNAs (miRNAs) in the regulation of pulmonary innate host defense responses and the inflammatory sequelae in respiratory disease. In this review, we discuss recent findings that indicate an important role for miRNAs in the regulation in mouse models of various respiratory diseases and in host defense against bacterial and viral infection. We also discuss the potential utility and limitations of targeting these molecules as anti-inflammatory strategies and also as a means to improve pathogen clearance from the lung.

A Role for IFN-ɛ Type I interferons (IFNs) are critical cytokines involved in host defense against pathogens, particularly viruses. IFN-ɛ is an IFN-like gene encoded within the type I IFN locus in mice and humans whose function has not been characterized. Fung et al. (p. 1088 ) created mice with a genetic deletion in Ifn -ɛ and found that, like other type I IFNs, IFN-ɛ signals through the IFN-α receptors 1 and 2. However, unlike these other cytokines, which are primarily expressed by immune cells and are induced upon immune cell triggering, IFN-ɛ was expressed exclusively by epithelial cells of the female reproductive tract in both mice and humans and its expression was hormonally regulated. IFN-ɛ–deficient mice were more susceptible to infection with herpes simplex virus 2 and Chlamydia muridarum , two common sexually transmitted pathogens.

Chronic obstructive pulmonary disease (COPD) is a common and serious respiratory disease, particularly in older individuals, characterised by fixed airway obstruction and persistent airway neutrophilia. The mechanisms that lead to these features are not well established. We investigated the contribution of age, prior smoking, and fixed airflow obstruction on sputum neutrophils, TLR2 expression, and markers of neutrophilic inflammation. Induced sputum from adults with COPD (n = 69) and healthy controls (n = 51) was examined. A sputum portion was dispersed, total, differential cell count and viability recorded, and supernatant assayed for CXCL8, matrix metalloproteinase- (MMP-) 9, neutrophil elastase, and soluble TLR2. Peripheral blood cells (n = 7) were stimulated and TLR2 activation examined. TLR2 levels were increased with ageing, while sputum neutrophils and total sputum MMP-9 levels increased with age, previous smoking, and COPD. In multivariate regression, TLR2 gene expression and MMP-9 levels were significant independent contributors to the proportion of sputum neutrophils after adjustment for age, prior smoking, and the presence of airflow obstruction. TLR2 stimulation led to enhanced release of MMP-9 from peripheral blood granulocytes. TLR2 stimulation activates neutrophils for MMP-9 release. Efforts to understand the mechanisms of TLR2 signalling and subsequent MMP-9 production in COPD may assist in understanding neutrophilic inflammation in COPD. © 2013 Jodie L. Simpson et al.

Background: Emerging evidence suggests that non-olfactory tissues and cells can express olfactory receptors (ORs), however, the exact function of ectopic OR expression remains unknown. We have previously shown in mouse models that a unique cooperation between interferon-γ (IFN-γ) and lipopolysaccharide (LPS) drives the activation of pulmonary macrophages and leads to the induction of pathogenic responses in the respiratory tract. Further, through gene array studies, we have shown that activation of macrophages by these molecules results in the selective expression of a number of ORs. In this study, we validated the expression of these ORs in mouse airway and pulmonary macrophages in response to IFN-γ and LPS (γ/LPS) stimulation, and further explored the effect of odorant stimulation on macrophage function. Methodology/Principal Findings: OR expression in airway and pulmonary macrophages in response to IFN-γ, LPS or γ/LPS treatments was assessed by microarray and validated by q-PCR. OR expression (e.g. OR622) on macrophages was confirmed by visualization in immunofluoresence assays. Functional responses to odorants were assessed by quantifying inflammatory cytokine and chemokine expression using q-PCR and cell migration was assessed by a modified Boyden chamber migration assay. Our results demonstrate that eight ORs are expressed at basal levels in both airway and pulmonary macrophages, and that γ/LPS stimulation cooperatively increased this expression. Pulmonary macrophages exposed to the combined treatment of γ/LPS+octanal (an odorant) exhibited a 3-fold increase in MCP-1 protein production, compared to cells treated with γ/LPS alone. Supernatants from γ/LPS+octanal exposed macrophages also increased macrophage migration in vitro. Conclusions/Significance: Eight different ORs are expressed at basal levels in pulmonary macrophages and expression is upregulated by the synergistic action of γ/LPS. Octanal stimulation further increased MCP-1 production and the motility of macrophages. Our results suggest that ORs may mediate macrophage function by regulating MCP-1 production and cell migration. © 2013 Li et al.

Background: Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short timeframe. Objectives: We sought to create an early-onset mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short time-frame. We also sought to use this model to better understand pathogenesis and the roles of macrophages and mast cells (MCs) in patients with COPD. Methods: Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathologic features of COPD was assessed. The roles of macrophages and MC tryptase in pathogenesis were evaluated by using depletion and in vitro studies and MC protease 6-deficient mice. Results: After just 8 weeks of smoke exposure, wild-type mice had chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart and increased susceptibility to respiratory tract infections also were observed. Macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced proinflammatory responses from cultured macrophages. Conclusion: A short-term mouse model of cigarette smoke-induced COPD was developed in which the characteristic features of the disease were induced more rapidly than in existing models. The model can be used to better understand COPD pathogenesis, and we show a requirement for macrophages and tryptase-expressing MCs. © 2013 American Academy of Allergy, Asthma & Immunology.

Corticosteroids are broadly active and potent anti-inflammatory agents that, despite the introduction of biologics, remain as the mainstay therapy for many chronic inflammatory diseases, including inflammatory bowel diseases, nephrotic syndrome, rheumatoid arthritis, chronic obstructive pulmonary disease and asthma. Significantly, there are cohorts of these patients with poor sensitivity to steroid treatment even with high doses, which can lead to many iatrogenic side effects. The dose-limiting toxicity of corticosteroids, and the lack of effective therapeutic alternatives, leads to substantial excess morbidity and healthcare expenditure. We have developed novel murine models of respiratory infection-induced, severe, steroid-resistant asthma that recapitulate the hallmark features of the human disease. These models can be used to elucidate novel disease mechanisms and identify new therapeutic targets in severe asthma. Hypothesis-driven studies can elucidate the roles of specific factors and pathways. Alternatively, 'Omics approaches can be used to rapidly generate new targets. Similar approaches can be used in other diseases.

Chlamydia pneumoniae commonly causes respiratory tract infections in children, and epidemiological investigations strongly link infection to the pathogenesis of asthma. The immune system in early life is immature and may not respond appropriately to pathogens. Toll-like receptor (TLR)2 and 4 are regarded as the primary pattern recognition receptors that sense bacteria, however their contribution to innate and adaptive immunity in early life remains poorly defined. We investigated the role of TLR2 and 4 in the induction of immune responses to Chlamydia muridarum respiratory infection, in neonatal wild-type (Wt) or TLR2-deficient ((-/-)), 4(-/-) or 2/4(-/-) BALB/c mice. Wt mice had moderate disease and infection. TLR2(-/-) mice had more severe disease and more intense and prolonged infection compared to other groups. TLR4(-/-) mice were asymptomatic. TLR2/4(-/-) mice had severe early disease and persistent infection, which resolved thereafter consistent with the absence of symptoms in TLR4(-/-) mice. Wt mice mounted robust innate and adaptive responses with an influx of natural killer (NK) cells, neutrophils, myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells, and activated CD4(+) and CD8(+) T-cells into the lungs. Wt mice also had effective production of interferon (IFN)γ in the lymph nodes and lung, and proliferation of lymph node T-cells. TLR2(-/-) mice had more intense and persistent innate (particularly neutrophil) and adaptive cell responses and IL-17 expression in the lung, however IFNγ responses and T-cell proliferation were reduced. TLR2/4(-/-) mice had reduced innate and adaptive responses. Most importantly, neutrophil phagocytosis was impaired in the absence of TLR2. Thus, TLR2 expression, particularly on neutrophils, is required for effective control of Chlamydia respiratory infection in early life. Loss of control of infection leads to enhanced but ineffective TLR4-mediated inflammatory responses that prolong disease symptoms. This indicates that TLR2 agonists may be beneficial in the treatment of early life Chlamydia infections and associated diseases.

Background 20–30% of patients with asthma have neutrophilic airway inflammation and reduced responsiveness to steroid therapy. They often have chronic airway bacterial colonisation and Haemophilus influenzae is one of the most commonly isolated bacteria. The relationship between chronic airway colonisation and the development of steroid-resistant neutrophilic asthma is unclear. Objectives To investigate the relationship between H influenzae respiratory infection and neutrophilic asthma using mouse models of infection and ovalbumin (OVA)-induced allergic airways disease. Methods BALB/c mice were intratracheally infected with H influenzae (day 10), intraperitoneally sensitised (day 0) and intranasally challenged (day 12–15) with OVA. Treatment groups were administered dexamethasone intranasally during OVA challenge. Infection, allergic airways disease, steroid sensitivity and immune responses were assessed (days 11, 16 and 21). Results The combination of H influenzae infection and allergic airways disease resulted in chronic lung infection that was detected on days 11, 16 and 21 (21, 26 and 31 days after infection). Neutrophilic allergic airways disease and T helper 17 cell development were induced, which did not require active infection. Importantly, all features of neutrophilic allergic airways disease were steroid resistant. Toll-like receptor 4 expression and activation of phagocytes was reduced, but most significantly the influx and/or development of phagocytosing neutrophils and macrophages into the airways was inhibited. Conclusions The combination of infection and allergic airways disease promotes bacterial persistence, leading to the development of a phenotype similar to steroid-resistant neutrophilic asthma and which may result from dysfunction in innate immune cells. This indicates that targeting bacterial infection in steroid-resistant asthma may have therapeutic benefit.

The mouse and human TPSB2 and TPSAB1 genes encode tetramer-forming tryptases stored in the secretory granules of mast cells (MCs) ionically bound to heparin-containing serglycin proteoglycans. In mice these genes encode mouse MC protease-6 (mMCP-6) and mMCP-7. The corresponding human genes encode a family of serine proteases that collectively are called hTryptase-β. We previously showed that the α chain of fibrinogen is a preferred substrate of mMCP-7. We now show that this plasma protein also is highly susceptible to degradation by hTryptase-β· and mMCP-6·heparin complexes and that Lys(575) is a preferred cleavage site in the protein α chain. Because cutaneous mouse MCs store substantial amounts of mMCP-6·heparin complexes in their secretory granules, the passive cutaneous anaphylaxis reaction was induced in the skin of mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(-) C57BL/6 mice. In support of the in vitro data, fibrin deposits were markedly increased in the skin of the double-deficient mice 6 h after IgE-sensitized animals were given the relevant antigen. Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Our discovery that mouse and human tetramer-forming tryptases destroy fibrinogen before this circulating protein can be converted to fibrin changes the paradigm of how MCs hinder fibrin deposition and blood coagulation internally. Because of the adverse consequences of fibrin deposits in tissues, our data explain why mice and humans lack a circulating protease inhibitor that rapidly inactivates MC tryptases and why mammals have two genes that encode tetramer-forming serine proteases that preferentially degrade fibrinogen.