Chitin recognition via chitotriosidase promotes pathologic type-2 helper T cell responses to cryptococcal infection.

Pulmonary mycoses are often associated with type-2 helper T (Th2) cell responses. However, mechanisms of Th2 cell accumulation are multifactorial and incompletely known. To investigate Th2 cell responses to pulmonary fungal infection, we developed a peptide-MHCII tetramer to track antigen-specific CD4+ T cells produced in response to infection with the fungal pathogen Cryptococcus neoformans. We noted massive accruement of pathologic cryptococcal antigen-specific Th2 cells in the lungs following infection that was coordinated by lung-resident CD11b+ IRF4-dependent conventional dendritic cells. Other researchers have demonstrated that this dendritic cell subset is also capable of priming protective Th17 cell responses to another pulmonary fungal infection, Aspergillus fumigatus. Thus, higher order detection of specific features of fungal infection by these dendritic cells must direct Th2 cell lineage commitment. Since chitin-containing parasites commonly elicit Th2 responses, we hypothesized that recognition of fungal chitin is an important determinant of Th2 cell-mediated mycosis. Using C. neoformans mutants or purified chitin, we found that chitin abundance impacted Th2 cell accumulation and disease. Importantly, we determined Th2 cell induction depended on cleavage of chitin via the mammalian chitinase, chitotriosidase, an enzyme that was also prevalent in humans experiencing overt cryptococcosis. The data presented herein offers a new perspective on fungal disease susceptibility, whereby chitin recognition via chitotriosidase leads to the initiation of harmful Th2 cell differentiation by CD11b+ conventional dendritic cells in response to pulmonary fungal infection.

Chitinase 3-like-1 regulates both visceral fat accumulation and asthma-like Th2 inflammation.

RATIONALE: Obesity, especially truncal obesity, is a risk factor for asthma incidence, prevalence, and severity. Chitinase 3-like-1 (Chi3l1) is an evolutionarily conserved moiety that plays a critical role in antipathogen and Th2 responses. However, the mechanisms that underlie the association between asthma and obesity and the role(s) of Chi3l1 in fat accumulation have not been defined. OBJECTIVES: To determine whether Chi3l1 is regulated by a high-fat diet (HFD) and simultaneously plays an important role(s) in the pathogenesis of asthma and obesity. METHODS: We evaluated the regulation of Chi3l1 by an HFD and Th2 inflammation. We also used genetically modified mice to define the roles of Chi3l1 in white adipose tissue (WAT) accumulation and Th2 inflammation and blockers of sirtuin 1 (Sirt1) to define its roles in these responses. Finally, the human relevance of these findings was assessed with a case-control study involving obese and lean control subjects and those with asthma. MEASUREMENTS AND MAIN RESULTS: These studies demonstrate that an HFD and aeroallergen challenge augment the expression of WAT and pulmonary Chi3l1. Chi3l1 also played a critical role in WAT accumulation and lung Th2 inflammation. In addition, Chi3l1 inhibited Sirt1 expression, and the deficient visceral fat and Th2 responses in Chi3l1 null mice were reversed by Sirt1 inhibition. Finally, serum and sputum Chi3l1 were positively associated with truncal adiposity, and serum Chi3l1 was associated with persistent asthma and low lung function in obese subjects with asthma. CONCLUSIONS: Chi3l1 is induced by an HFD and Th2 inflammation, and simultaneously contributes to the genesis of obesity and asthma.

Fatty acid binding protein 4 regulates VEGF-induced airway angiogenesis and inflammation in a transgenic mouse model: implications for asthma.

Neovascularization of the airways occurs in several inflammatory lung diseases, including asthma. Vascular endothelial growth factor (VEGF) plays an important role in vascular remodeling in the asthmatic airways. Fatty acid binding protein 4 (FABP4 or aP2) is an intracellular lipid chaperone that is induced by VEGF in endothelial cells. FABP4 exhibits a proangiogenic function in vitro, but whether it plays a role in modulation of angiogenesis in vivo is not known. We hypothesized that FABP4 promotes VEGF-induced airway angiogenesis and investigated this hypothesis with the use of a transgenic mouse model with inducible overexpression of VEGF165 under a CC10 promoter [VEGF-TG (transgenic) mice]. We found a significant increase in FABP4 mRNA levels and density of FABP4-expressing vascular endothelial cells in mouse airways with VEGF overexpression. FABP4(-/-) mouse airways showed a significant decrease in neovessel formation and endothelial cell proliferation in response to VEGF overexpression. These alterations in airway vasculature were accompanied by attenuated expression of proinflammatory mediators. Furthermore, VEGF-TG/FABP4(-/-) mice showed markedly decreased expression of endothelial nitric oxide synthase, a well-known mediator of VEGF-induced responses, compared with VEGF-TG mice. Finally, the density of FABP4-immunoreactive vessels in endobronchial biopsy specimens was significantly higher in patients with asthma than in control subjects. Taken together, these data unravel FABP4 as a potential target of pathologic airway remodeling in asthma.

Hyperoxia causes angiopoietin 2-mediated acute lung injury and necrotic cell death.

The angiogenic growth factor angiopoietin 2 (Ang2) destabilizes blood vessels, enhances vascular leak and induces vascular regression and endothelial cell apoptosis. We considered that Ang2 might be important in hyperoxic acute lung injury (ALI). Here we have characterized the responses in lungs induced by hyperoxia in wild-type and Ang2-/- mice or those given either recombinant Ang2 or short interfering RNA (siRNA) targeted to Ang2. During hyperoxia Ang2 expression is induced in lung epithelial cells, while hyperoxia-induced oxidant injury, cell death, inflammation, permeability alterations and mortality are ameliorated in Ang2-/- and siRNA-treated mice. Hyperoxia induces and activates the extrinsic and mitochondrial cell death pathways and activates initiator and effector caspases through Ang2-dependent pathways in vivo. Ang2 increases inflammation and cell death during hyperoxia in vivo and stimulates epithelial necrosis in hyperoxia in vitro. Ang2 in plasma and alveolar edema fluid is increased in adults with ALI and pulmonary edema. Tracheal Ang2 is also increased in neonates that develop bronchopulmonary dysplasia. Ang2 is thus a mediator of epithelial necrosis with an important role in hyperoxic ALI and pulmonary edema.

Vascular endothelial growth factor enhances macrophage clearance of apoptotic cells.

Efficient clearance of apoptotic cells from the lung by alveolar macrophages is important for the maintenance of tissue structure and function. Lung tissue from humans with emphysema contains increased numbers of apoptotic cells and decreased levels of vascular endothelial growth factor (VEGF). Mice treated with VEGF receptor inhibitors have increased numbers of apoptotic cells and develop emphysema. We hypothesized that VEGF regulates apoptotic cell clearance by alveolar macrophages (AM) via its interaction with VEGF receptor 1 (VEGF R1). Our data show that the uptake of apoptotic cells by murine AMs and human monocyte-derived macrophages is inhibited by depletion of VEGF and that VEGF activates Rac1. Antibody blockade or pharmacological inhibition of VEGF R1 activity also decreased apoptotic cell uptake ex vivo. Conversely, overexpression of VEGF significantly enhanced apoptotic cell uptake by AMs in vivo. These results indicate that VEGF serves a positive regulatory role via its interaction with VEGF R1 to activate Rac1 and enhance AM apoptotic cell clearance.

Chitin is a size-dependent regulator of macrophage TNF and IL-10 production.

Chitin is a ubiquitous polysaccharide in fungi, insects, and parasites. We hypothesized that chitin is a size-dependent regulator of innate immunity. To test this hypothesis, we characterized the effects of chitins of different sizes on murine bronchoalveolar or peritoneal macrophages. In these studies, large chitin fragments were inert, while both intermediate-sized chitin (40-70 microm) and small chitin (SC; <40 microm, largely 2-10 microm) stimulated TNF elaboration. In contrast, only SC induced IL-10 elaboration. The effects of intermediate-sized chitin were mediated by pathways that involve TLR2, dectin-1, and NF-kappaB. In contrast, the effects of SC were mediated by TLR2-dependent and -independent, dectin-1-dependent pathways that involved the mannose receptor and spleen tyrosine kinase. Chitin contains size-dependent pathogen-associated molecular patterns that stimulate TLR2, dectin-1, and the mannose receptor, differentially activate NF-kappaB and spleen tyrosine kinase, and stimulate the production of pro- and anti-inflammatory cytokines.

TLR-2 and IL-17A in chitin-induced macrophage activation and acute inflammation.

Chitin is a ubiquitous polysaccharide in fungi, insects, and parasites. To test the hypothesis that chitin is an important immune modulator, we characterized the ability of chitin fragments to regulate murine macrophage cytokine production in vitro and induce acute inflammation in vivo. In this study, we show that chitin is a size-dependent stimulator of macrophage IL-17A production and IL-17AR expression and demonstrate that these responses are TLR-2 and MyD88-dependent. We further demonstrate that IL-17A pathway activation is an essential event in the stimulation of some but not all chitin-stimulated cytokines and that chitin uses a TLR-2, MyD88-, and IL-17A-dependent mechanism(s) to induce acute inflammation. These studies demonstrate that chitin is a size-dependent pathogen-associated molecular pattern that activates TLR-2 and MyD88 in a novel IL-17A/IL-17AR-based innate immunity pathway.

Developmental regulation of NO-mediated VEGF-induced effects in the lung.

Vascular endothelial growth factor (VEGF) is known to have a pivotal role in lung development and in a variety of pathologic conditions in the adult lung. Our earlier studies have shown that NO is a critical mediator of VEGF-induced vascular and extravascular effects in the adult murine lung. As significant differences have been reported in the cytokine responses in the adult versus the neonatal lung, we hypothesized that there may be significant differences in VEGF-induced alterations in the developing as opposed to the mature lung. Furthermore, nitric oxide (NO) mediation of these VEGF-induced effects may be developmentally regulated. Using a novel externally regulatable lung-targeted transgenic murine model, we found that VEGF-induced pulmonary hemorrhage was mediated by NO-dependent mechanisms in adults and newborns. VEGF enhanced surfactant production in adults as well as increased surfactant and lung development in newborns, via an NO-independent mechanism. While the enhanced survival in hyperoxia in the adult was partly NO-dependent, there was enhanced hyperoxia-induced lung injury in the newborn. In addition, human amniotic fluid VEGF levels correlated positively with surfactant phospholipids. Tracheal aspirate VEGF levels had an initial spike, followed by a decline, and then a subsequent rise, in human neonates with an outcome of bronchopulmonary dysplasia or death. Our data show that VEGF can have injurious as well as potentially beneficial developmental effects, of which some are NO dependent, others NO independent. This opens up the possibility of selective manipulation of any VEGF-based intervention using NO inhibitors for maximal potential clinical benefit.

Adenosine mediates IL-13-induced inflammation and remodeling in the lung and interacts in an IL-13-adenosine amplification pathway.

IL-13 is an important mediator of inflammation and remodeling. We hypothesized that adenosine accumulation, alterations in adenosine receptors, and adenosine-IL-13 autoinduction are critical events in IL-13-induced pathologies. To test this, we characterized the effects of IL-13 overexpression on the levels of adenosine, adenosine deaminase (ADA) activity, and adenosine receptors in the murine lung. We also determined whether adenosine induced IL-13 in lungs from ADA-null mice. IL-13 induced an inflammatory and remodeling response that caused respiratory failure and death. During this response, IL-13 caused a progressive increase in adenosine accumulation, inhibited ADA activity and mRNA accumulation, and augmented the expression of the A1, A2B, and A3 but not the A2A adenosine receptors. ADA enzyme therapy diminished the IL-13-induced increase in adenosine, inhibited IL-13-induced inflammation, chemokine elaboration, fibrosis, and alveolar destruction, and prolonged the survival of IL-13-transgenic animals. In addition, IL-13 was strongly induced by adenosine in ADA-null mice. These findings demonstrate that adenosine and adenosine signaling contribute to and influence the severity of IL-13-induced tissue responses. They also demonstrate that IL-13 and adenosine stimulate one another in an amplification pathway that may contribute to the nature, severity, progression, and/or chronicity of IL-13 and/or Th2-mediated disorders.

Antifibrotic role of vascular endothelial growth factor in pulmonary fibrosis.

The chronic progressive decline in lung function observed in idiopathic pulmonary fibrosis (IPF) appears to result from persistent nonresolving injury to the epithelium, impaired restitution of the epithelial barrier in the lung, and enhanced fibroblast activation. Thus, understanding these key mechanisms and pathways modulating both is essential to greater understanding of IPF pathogenesis. We examined the association of VEGF with the IPF disease state and preclinical models in vivo and in vitro. Tissue and circulating levels of VEGF were significantly reduced in patients with IPF, particularly in those with a rapidly progressive phenotype, compared with healthy controls. Lung-specific overexpression of VEGF significantly protected mice following intratracheal bleomycin challenge, with a decrease in fibrosis and bleomycin-induced cell death observed in the VEGF transgenic mice. In vitro, apoptotic endothelial cell-derived mediators enhanced epithelial cell injury and reduced epithelial wound closure. This process was rescued by VEGF pretreatment of the endothelial cells via a mechanism involving thrombospondin-1 (TSP1). Taken together, these data indicate beneficial roles for VEGF during lung fibrosis via modulating epithelial homeostasis through a previously unrecognized mechanism involving the endothelium.

Chitinase 3-like 1 synergistically activates IL6-mediated STAT3 phosphorylation in intestinal epithelial cells in murine models of infectious colitis.

BACKGROUND: Chitinase 3-like 1 (CHI3L1) is an inducible molecule on intestinal epithelial cells during the development of inflammatory bowel disease. METHODS: To investigate the role of CHI3L1 in bacterial infectious colitis, we orally inoculated pathogenic Salmonella typhimurium and potentially pathogenic adherent-invasive Escherichia coli (AIEC) LF82 virulent strain into C57Bl/6 wild-type mice or CHI3L1 knockout (KO) mice. RESULTS: Both S. typhimurium and AIEC LF82 were found to efficiently induce severe intestinal inflammation in wild-type mice but not in CHI3L1 KO mice. These bacteria-infected CHI3L1 KO mice exhibit decreased cellular infiltration, bacterial translocation, and production of interleukin (IL)-6 and IL-22, as compared with those of wild-type mice. More importantly, CHI3L1 KO mice displayed aberrant STAT3 activation after bacterial infections. Co-stimulation of CHI3L1 and IL-6, but not IL-22, synergistically activates STAT3 signaling pathway in intestinal epithelial cells in an NF-κB/MAPK-dependent manner. CONCLUSIONS: CHI3L1 promotes the onset of selected gram-negative bacterial infectious colitis through IL-6/STAT3 pathway.

Novel biomarkers in asthma: chemokines and chitinase-like proteins.

PURPOSE OF REVIEW: Allergic asthma is a frequent lung disease in Western civilizations and is characterized by airway inflammation and tissue remodeling. Without early diagnosis and specific treatment, asthma results in a loss of lung function, impaired quality of life and the risk to die from uncontrolled asthma attacks. Thus, there is a need for specific biomarkers to detect asthma as soon as possible and to initiate the correct clinical treatment. RECENT FINDINGS: Recent studies have highlighted the potential role of the chemokine (C-C motif) ligand 17 and the chitinase-like protein YKL-40 as novel biomarkers in asthma. Patient studies suggest that these proteins could be useful to identify asthmatics, to characterize disease severity or both in patients with asthma. Functional studies indicate that these molecules are more than correlated epiphenomena and instead contribute in significant ways to asthma pathogenesis. SUMMARY: Assessments of chemokine (C-C motif) ligand 17 and YKL-40 may allow physicians to more accurately diagnose and predict the course of asthma and thereby allow therapy to be appropriately tailored for a given patient.

Acidic mammalian chitinase is secreted via an ADAM17/epidermal growth factor receptor-dependent pathway and stimulates chemokine production by pulmonary epithelial cells.

Acidic mammalian chitinase (AMCase) is expressed in an exaggerated fashion in epithelial cells at sites of pulmonary T helper cell type 2 inflammation and plays important roles in the pathogenesis of anti-parasite and asthma-like responses. However, the mechanisms that control epithelial cell AMCase secretion and its effector responses have not been adequately defined. To address these issues, we used in vivo and in vitro experimental systems to define the pathways of epithelial AMCase secretion and its epithelial regulatory effects. Here we demonstrate that, in murine T helper cell type 2 modeling systems, AMCase colocalizes with the epidermal growth factor receptor (EGFR) and ADAM17 (a membrane disintegrin and metallopeptidase 17) in lung epithelial cells. In vitro cotransfection experiments in A549 cells demonstrated that AMCase and EGFR physically interact with each other. Cotransfection of AMCase and EGFR also increased, whereas EGFR inhibition decreased AMCase secretion. Interestingly, AMCase secretion was not significantly altered by treatment with EGF but was significantly decreased when the upstream EGFR transactivator ADAM17 was inhibited. AMCase secretion was also decreased when the EGFR-downstream Ras was blocked. Transfected and recombinant AMCase induced epithelial cell production of CCL2, CCL17, and CXCL8. These studies demonstrate that lung epithelial cells secrete AMCase via an EGFR-dependent pathway that is activated by ADAM17 and mediates its effects via Ras. They also demonstrate that the AMCase that is secreted feeds back in an autocrine and/or paracrine fashion to stimulate pulmonary epithelial cell chemokine production.

Role of 5-lipoxygenase in IL-13-induced pulmonary inflammation and remodeling.

Exaggerated levels of IL-13 and leukotriene (LT) pathway activation frequently coexist at sites of Th2 inflammation and in tissue fibrotic responses. However, the relationship(s) between the IL-13 and LTs in these responses have not been defined. We hypothesized that the 5-lipoxygenase (5-LO) pathway of LT metabolism plays an important role in the pathogenesis of IL-13-induced chronic inflammation and remodeling. To test this hypothesis, we evaluated the effects of IL-13 on components of the 5-LO metabolic and activation pathways. We also compared the effects of transgenic IL-13 in C57BL/6 mice with wild-type and null 5-LO genetic loci. These studies demonstrate that IL-13 increases the levels of mRNA encoding cytosolic phospholipase A(2), LTA(4) hydrolase, and 5-LO-activating protein without altering the expression of 5-LO, LTC(4) synthase, LTB(4) receptors 1 and 2, and cysteinyl-LT receptors 1 and 2. They also demonstrate that this activation is associated with the enhanced accumulation of LTB(4) but not of cysteinyl-LTs. Furthermore, they demonstrate that this stimulation plays a critical role in the pathogenesis of IL-13-induced inflammation, tissue fibrosis, and respiratory failure-induced death while inhibiting alveolar remodeling. Lastly, mechanistic insights are provided by demonstrating that IL-13-induced 5-LO activation is required for optimal stimulation and activation of TGF-beta(1) and the inhibition of matrix metalloproteinase-12. When viewed in combination, these studies demonstrate that 5-LO plays an important role in IL-13-induced inflammation and remodeling.

Partially adenosine deaminase-deficient mice develop pulmonary fibrosis in association with adenosine elevations.

Adenosine, a signaling nucleoside, exhibits tissue-protective and tissue-destructive effects. Adenosine levels in tissues are controlled in part by the enzyme adenosine deaminase (ADA). ADA-deficient mice accumulate adenosine levels in multiple tissues, including the lung, where adenosine contributes to the development of pulmonary inflammation and chronic airway remodeling. The present study describes the development of pulmonary fibrosis in mice that have been genetically engineered to possess partial ADA enzyme activity and, thus, accumulate adenosine over a prolonged period of time. These partially ADA-deficient mice live for up to 5 mo and die from apparent respiratory distress. Detailed investigations of the lung histopathology of partially ADA-deficient mice revealed progressive pulmonary fibrosis marked by an increase in the number of pulmonary myofibroblasts and an increase in collagen deposition. In addition, in regions of the distal airways that did not exhibit fibrosis, an increase in the number of large foamy macrophages and a substantial enlargement of the alveolar air spaces suggest emphysemic changes. Furthermore, important proinflammatory and profibrotic signaling pathways, including IL-13 and transforming growth factor-beta1, were activated. Increases in tissue fibrosis were also seen in the liver and kidneys of these mice. These changes occurred in association with pronounced elevations of lung adenosine concentrations and alterations in lung adenosine receptor levels, supporting the hypothesis that elevation of endogenous adenosine is a proinflammatory and profibrotic signal in this model.

Essential role of nitric oxide in VEGF-induced, asthma-like angiogenic, inflammatory, mucus, and physiologic responses in the lung.

VEGF, nitric oxide (NO), inflammation, and vascular- and extravascular remodeling coexist in asthma and other disorders. In these responses, VEGF regulates angiogenesis. VEGF also induces inflammation and remodeling. The mechanisms of the latter responses have not been defined, however. We hypothesized that VEGF-induces extravascular tissue responses via NO-dependent mechanisms. To evaluate this hypothesis, we compared the effects of transgenic VEGF165 in lungs from normal mice, mice treated with pan-NO synthase (NOS) or endothelial NOS (eNOS) inhibitors, and mice with null mutations of inducible NOS (iNOS) or eNOS. These studies demonstrate that VEGF selectively stimulates eNOS and iNOS. They also demonstrate that VEGF induces pulmonary alterations via NO-dependent and -independent mechanisms with angiogenesis, edema, mucus metaplasia, airway hyperresponsiveness, lymphocyte accumulation, dendritic cell hyperplasia and S-nitrosoglutathione reductase stimulation being NO-dependent and dendritic cell activation being NO-independent. Furthermore, they demonstrate that eNOS and iNOS both contribute to these responses. NO/NOS-based interventions may be therapeutic in VEGF-driven inflammation and remodeling.

Genetic control of a mouse serum lipoprotein factor that inactivates murine leukemia viruses: evaluation of apolipoprotein F as a candidate.

Mice contain a serum factor capable of inactivating some subgroups of murine leukemia viruses. This leukemia virus-inactivating factor (LVIF) is distinct from immunoglobulin and complement; it has been associated with lipoprotein serum fractions and may be an apolipoprotein. The present study demonstrates that some Swiss-derived inbred strains are LVIF negative. Genetic crosses show this factor to be under control of a single gene that maps to distal chromosome 10 at or near the gene encoding a minor serum apolipoprotein, apolipoprotein F (ApoF). To evaluate this gene as a potential candidate for LVIF, the mouse ApoF gene was cloned and sequenced and its expression was assessed in LVIF-positive and -negative mice; no obvious differences were detected, suggesting that LVIF is under the control of a distinct linked gene.

Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling.

Since the first tetracycline-controlled transcriptional activation system was designed nearly a decade ago, new variants, modifications, and improvements have been steadily added to this powerful set of tools for temporal control of transgene expression in mammalian systems. Tetracycline-based externally regulatable (Tet-based) systems have been successfully used to control the expression of numerous transgenes in cultured cells and in whole organisms, especially in mice. The application of these systems has provided invaluable insights into the function and regulation of a variety of genes under physiological and pathological conditions. Because of the favorable characteristics of the inducing agent doxycycline and the efficiency and effectiveness of the operating mechanism, the Tet-based systems have attracted substantial attention from the transgenic research community and are rapidly gaining popularity. The original tetracycline-controlled transcriptional activator (tTA) is a regulator with tight control of target gene expression and a broad range of inducibility. The reverse tetracycline-controlled transcriptional activator (rtTA) activates the responsive elements only in the presence of doxycycline, giving a convenient control over the target transgene. The recently developed tetracycline-controlled transcriptional silencer (tTS) has been successfully used in cultured cells and in transgenic mice. In combination with rtTA, tTS actively suppresses background expression or "leakiness" without impeding the inducibility of the target gene, providing a true "On/Off" transgenic switch. New variants of Tet-based regulators with improved features are still emerging and the utilities of these systems are constantly being tested.