Unique Lipid Signatures of Extracellular Vesicles from the Airways of Asthmatics.

Asthma is a chronic inflammatory disease process involving the conductive airways of the human lung. The dysregulated inflammatory response in this disease process may involve multiple cell-cell interactions mediated by signaling molecules, including lipid mediators. Extracellular vesicles (EVs) are lipid membrane particles that are now recognized as critical mediators of cell-cell communication. Here, we compared the lipid composition and presence of specific lipid mediators in airway EVs purified from the bronchoalveolar lavage (BAL) fluid of healthy controls and asthmatic subjects with and without second-hand smoke (SHS) exposure. Airway exosome concentrations were increased in asthmatics, and correlated with blood eosinophilia and serum IgE levels. Frequencies of HLA-DR+ and CD54+ exosomes were also significantly higher in asthmatics. Lipidomics analysis revealed that phosphatidylglycerol, ceramide-phosphates, and ceramides were significantly reduced in exosomes from asthmatics compared to the non-exposed control groups. Sphingomyelin 34:1 was more abundant in exosomes of SHS-exposed asthmatics compared to healthy controls. Our results suggest that chronic airway inflammation may be driven by alterations in the composition of lipid mediators within airway EVs of human subjects with asthma.

Subsets of airway myeloid-derived regulatory cells distinguish mild asthma from chronic obstructive pulmonary disease.

BACKGROUND: Subsets of myeloid-derived regulatory cells (MDRCs), which are phenotypically similar to the myeloid-derived suppressor cells found in patients with cancer, have recently been appreciated as critical regulators of airway inflammation in mouse models of asthma. OBJECTIVE: We test the hypothesis that subsets of airway MDRCs contribute differentially to the inflammatory milieu in human asthma and chronic obstructive pulmonary disease (COPD). METHODS: We used bronchoalveolar lavage to identify and characterize human airway MDRCs from 10 healthy subjects, 9 patients with mild asthma, and 8 patients with COPD, none of whom were treated with inhaled or systemic corticosteroids. We defined subsets of airway MDRCs using flow cytometry, the molecular mediators they produce, and their abilities to regulate proliferation of polyclonally activated autologous T lymphocytes. RESULTS: We found substantial differences in the functional potential of MDRC subsets in healthy subjects, patients with asthma, and patients with COPD, with these differences regulated by the nitrosative and oxidative free radicals and cytokines they produced. Nitric oxide-producing MDRCs suppressed and superoxide-producing MDRCs enhanced proliferation of polyclonally activated autologous CD4 T cells. HLA-DR(+)CD11b(+)CD11c(+)CD163(-) superoxide-producing MDRCs, which stimulated proliferation of autologous T cells, comprised a high fraction of MDRCs in the airways of patients with mild asthma or COPD but not those of healthy control subjects. CD11b(+)CD14(+)CD16(-)HLA-DR(-) nitric oxide-producing MDRCs, which suppressed T-cell proliferation, were present in high numbers in airways of patients with mild asthma but not patients with COPD or healthy control subjects. CONCLUSION: Subsets of airway MDRCs conclusively discriminate patients with mild asthma, patients with COPD, and healthy subjects from each other. The distinctive activities of these MDRCs in patients with asthma or COPD might provide novel targets for new therapeutics for these common disorders. [Corrected]

IL-22-producing neutrophils contribute to antimicrobial defense and restitution of colonic epithelial integrity during colitis.

IL-22 plays an important role in mucosal epithelial cell homeostasis. Using a dextran sodium sulfate-induced mouse model of acute colitis, we observed an IL-23-dependent up-regulation of IL-22 in the middle and distal colon at the onset of epithelial cell damage. This heightened IL-22 correlated with an influx of innate immune cells, suggesting an important role in colonic epithelial protection. Freshly isolated colon-infiltrating neutrophils produced IL-22 contingent upon IL-23 signaling, and IL-22 production was augmented by TNF-α. Importantly, the depletion of neutrophils resulted in diminished IL-22 levels in the colon, and the transfer of IL-22-competent neutrophils to Il22a-deficient mice protected the colonic epithelium from dextran sodium sulfate-induced damage. In addition, IL-22-producing neutrophils targeted colonic epithelial cells to up-regulate the antimicrobial peptides, RegIIIß and S100A8. This study establishes a role for neutrophils in providing IL-22-dependent mucosal epithelial support that contributes to the resolution of colitis.

Toll-like receptor 4 engagement inhibits adenosine 5'-monophosphate-activated protein kinase activation through a high mobility group box 1 protein-dependent mechanism.

Despite the potent antiinflammatory effects of pharmacologically induced adenosine 5'-monophosphate kinase (AMPK) activation on Toll-like receptor 4 (TLR4)-induced cellular activation, there is little evidence that AMPK is activated during inflammatory conditions. In the present studies, we examined mechanisms by which TLR4 engagement may affect the ability of AMPK to become activated in neutrophils and macrophages under in vitro conditions and in the lungs during lipopolysaccharide (LPS)-induced acute lung injury. We found that incubation of neutrophils or macrophages with LPS diminished the ability of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) or hydrogen peroxide (H(2)O(2)) to activate AMPK. Although ratios of AMP to adenosine 5'-triphosphate (ATP) were increased in LPS-treated neutrophils and in the lungs of LPS exposed mice, a condition that should result in AMPK activation, no activation of AMPK was found. Immunocytochemistry and Western blot analysis revealed that nuclear to cytosolic translocation of the proinflammatory mediator high mobility group box 1 protein (HMGB1) correlated with inhibition of AMPK activation in LPS-stimulated macrophages. Moreover, while induced overexpression of HMGB1 resulted in inhibition of AMPK activation, Small interfering RNA (siRNA)-induced knockdown of HMGB1 was associated with enhanced activation of AMPK in macrophages incubated with AICAR. Increased interaction between liver kinase B1 (LKB1), an upstream activator of AMPK, and HMGB1 was found in LPS-stimulated macrophages and in the lungs of mice exposed to LPS. These results suggest that nuclear to cytoplasmic translocation of HMGB1 in TLR4-activated cells potentiates inflammatory responses by binding to LKB1, thereby inhibiting the antiinflammatory effects of AMPK activation.

Inflammasome-derived IL-1ß regulates the production of GM-CSF by CD4(+) T cells and γδ T cells.

Recent findings have demonstrated an indispensable role for GM-CSF in the pathogenesis of experimental autoimmune encephalomyelitis. However, the signaling pathways and cell populations that regulate GM-CSF production in vivo remain to be elucidated. Our work demonstrates that IL-1R is required for GM-CSF production after both TCR- and cytokine-induced stimulation of immune cells in vitro. Conventional αß and γδ T cells were both identified to be potent producers of GM-CSF. Moreover, secretion of GM-CSF was dependent on IL-1R under both IL-12- and IL-23-induced stimulatory conditions. Deficiency in IL-1R conferred significant protection from experimental autoimmune encephalomyelitis, and this correlated with reduced production of GM-CSF and attenuated infiltration of inflammatory cells into the CNS. We also find that GM-CSF production in vivo is not restricted to a defined CD4(+) T cell lineage but is rather heterogeneously expressed in the effector CD4(+) T cell population. In addition, inflammasome-derived IL-1ß upstream of IL-1R is a critical regulator of GM-CSF production by T cells during priming, and the adapter protein, MyD88, promotes GM-CSF production in both αß and γδ T cells. These findings highlight the importance of inflammasome-derived IL-1ß and the IL-1R/MyD88 signaling axis in the regulation of GM-CSF production.

AMP-activated protein kinase enhances the phagocytic ability of macrophages and neutrophils.

Although AMPK plays well-established roles in the modulation of energy balance, recent studies have shown that AMPK activation has potent anti-inflammatory effects. In the present experiments, we examined the role of AMPK in phagocytosis. We found that ingestion of Escherichia coli or apoptotic cells by macrophages increased AMPK activity. AMPK activation increased the ability of neutrophils or macrophages to ingest bacteria (by 46 ± 7.8 or 85 ± 26%, respectively, compared to control, P<0.05) and the ability of macrophages to ingest apoptotic cells (by 21 ± 1.4%, P<0.05 compared to control). AMPK activation resulted in cytoskeletal reorganization, including enhanced formation of actin and microtubule networks. Activation of PAK1/2 and WAVE2, which are downstream effectors of Rac1, accompanied AMPK activation. AMPK activation also induced phosphorylation of CLIP-170, a protein that participates in microtubule synthesis. The increase in phagocytosis was reversible by the specific AMPK inhibitor compound C, siRNA to AMPKα1, Rac1 inhibitors, or agents that disrupt actin or microtubule networks. In vivo, AMPK activation resulted in enhanced phagocytosis of bacteria in the lungs by 75 ± 5% vs. control (P<0.05). These results demonstrate a novel function for AMPK in enhancing the phagocytic activity of neutrophils and macrophages.

Inhibition of neutrophil apoptosis by PAI-1.

Increased circulating and tissue levels of plasminogen activator inhibitor 1 (PAI-1) are often present in severe inflammatory states associated with neutrophil activation and accumulation and correlate with poor clinical outcome from many of these conditions. The mechanisms by which PAI-1 contributes to inflammation have not been fully delineated. In the present experiments, we found that addition of PAI-1 to neutrophil cultures diminished the rate of spontaneous and TNF-related apoptosis-inducing ligand-induced apoptotic cell death. The effects of PAI-1 on cell viability were associated with activation of antiapoptotic signaling pathways, including upregulation of PKB/Akt, Mcl-1, and Bcl-x(L). Although urokinase-plasminogen activator receptor, lipoprotein receptor-related protein, and vitronectin are primary ligands for PAI-1, these molecules were not involved in mediating its antiapoptotic properties. In contrast, blocking pertussis toxin-sensitive G protein-coupled receptors and selective inhibition of phosphatidylinositide 3-kinase reversed the ability of PAI-1 to extend neutrophil viability. The antiapoptotic effects of PAI-1 were also evident under in vivo conditions during LPS-induced acute lung injury, where enhanced apoptosis was present among neutrophils accumulating in the lungs of PAI-1(-/-) compared with PAI-1(+/+) mice. These results demonstrate a novel antiapoptotic role for PAI-1 that may contribute to its participation in neutrophil-associated inflammatory responses.

Marginal zone precursor B cells as cellular agents for type I IFN-promoted antigen transport in autoimmunity.

The pathogenic connection of type I IFN and its role in regulating the migration response of Ag delivery by B cells into lymphoid follicles in an autoimmune condition has not been well-identified. Here, we show that there was a significantly larger population of marginal zone precursor (MZ-P) B cells, defined as being IgM(hi)CD1d(hi)CD21(hi)CD23(hi) in the spleens of autoimmune BXD2 mice compared with B6 mice. MZ-P B cells were highly proliferative compared with marginal zone (MZ) and follicular (FO) B cells. The intrafollicular accumulation of MZ-P B cells in proximity to germinal centers (GCs) in BXD2 mice facilitated rapid Ag delivery to the GC area, whereas Ag-carrying MZ B cells, residing predominantly in the periphery, had a lower ability to carry Ag into the GCs. IFN-alpha, generated by plasmacytoid dendritic cells, induced the expression of CD69 and suppressed the sphingosine-1-phosphate-induced chemotactic response, promoting FO-oriented Ag transport by MZ-P B cells. Knockout of type I IFN receptor in BXD2 (BXD2-Ifnalphar(-/-)) mice substantially diffused the intrafollicular MZ-P B cell conglomeration and shifted their location to the FO-MZ border near the marginal sinus, making Ag delivery to the FO interior less efficient. The development of spontaneous GCs was decreased in BXD2-Ifnalphar(-/-) mice. Together, our results suggest that the MZ-P B cells are major Ag-delivery B cells and that the FO entry of these B cells is highly regulated by type I IFN-producing plasmacytoid dendritic cells in the marginal sinus in the spleens of autoimmune BXD2 mice.

Critical role of macrophages and their activation via MyD88-NFκB signaling in lung innate immunity to Mycoplasma pneumoniae.

Mycoplasma pneumoniae (Mp), a common cause of pneumonia, is associated with asthma; however, the mechanisms underlying this association remain unclear. We investigated the cellular immune response to Mp in mice. Intranasal inoculation with Mp elicited infiltration of the lungs with neutrophils, monocytes and macrophages. Systemic depletion of macrophages, but not neutrophils, resulted in impaired clearance of Mp from the lungs. Accumulation and activation of macrophages were decreased in the lungs of MyD88(-/-) mice and clearance of Mp was impaired, indicating that MyD88 is a key signaling protein in the anti-Mp response. MyD88-dependent signaling was also required for the Mp-induced activation of NFκB, which was essential for macrophages to eliminate the microbe in vitro. Thus, MyD88-NFκB signaling in macrophages is essential for clearance of Mp from the lungs.

Antigen and lipopolysaccharide play synergistic roles in the effector phase of airway inflammation in mice.

T helper (Th) cells play major roles in orchestrating asthmatic airway inflammation, but the molecular mechanisms controlling Th-cell recruitment to the airways remain incompletely defined. Innate immunity contributes importantly to the recruitment of effector T cells into sites of inflammation. To understand better the role of innate immune signals in the development of airway inflammation, we used a murine model in which lipopolysaccharide (LPS) contaminating the antigen is thought to trigger Toll-like receptor 4 (TLR4). To investigate the importance of the TLR4-signaling pathway in induction of lung inflammation, we compared recruitment of adoptively transferred ovalbumin-specific Th1 and Th2 cells in wild-type and TLR4 mutant (TLR4m) mice after intranasal or aerosol challenge. Intranasal challenge of TLR4m mice with ovalbumin resulted in decreased recruitment of Th1 and Th2 cells compared with that of wild-type mice. The numbers of Th1 and Th2 cells recruited to the airways of TLR4m mice were less profoundly reduced after aerosol ovalbumin challenge. Comparing the effects of altering the dose of ovalbumin with that of LPS suggested that both contribute to the magnitude of the response in wild-type mice. Our findings demonstrate the importance of both antigen and endotoxin acting in a synergistic manner in the development of airway inflammation.

Taking control of follicular dendritic cells.

Follicular dendritic cells signal the formation of B cell follicles as well as B cell activation, proliferation, and maturation. However, the precise molecular pathways controlling these processes are just beginning to be unraveled.

Induction of a late asthmatic response associated with airway inflammation in mice.

To investigate mechanisms underlying the late asthmatic response, we developed a murine model using repetitive intratracheal antigen challenge. BALB/c mice sensitized by i.p. injection with ovalbumin+alum were challenged with ovalbumin intratracheally 4 times. The 1st challenge induced early airway obstruction peaking at 30 min but without a late response; however, the 4th challenge caused not only early but also late airway obstruction at 2-8 h. Eosinophils, and CD4+ and CD8+ T lymphocytes were increased in the airway before the 4th but not before the 1st-3rd challenges. The numbers of IgE+/CD117+ (mast) cells were also increased in the lung before the 4th challenge. Levels of Th2 cytokines were also increased in the airway. Daily administration of dexamethasone during the challenge period suppressed all these inflammatory events. Thus, this experimental late asthmatic response is associated with Th2 cytokine production from inflammatory cells recruited as a consequence of the 1st-3rd challenges.

Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function.

The gastrointestinal mucosa contains a complex network of lymphoid compartments that have evolved to efficiently protect the host from invading pathogens. Recently, an additional lymphoid structure resembling Peyer's patches (PP) in composition and architecture has been identified in the murine small intestine, the isolated lymphoid follicle (ILF). In this study we examine the nature and factors required for ILF formation. We observed a spectrum of structures fitting the previous descriptions of ILFs, ranging from clusters of B220(+) cells (which we have termed immature ILFs) to well-organized lymphoid nodules (which we have termed mature ILFs). Here we demonstrate that that similar to PP formation, ILF formation requires lymphotoxin (LT)- and LT beta receptor-dependent events. However unlike PP formation, the LT- and LT beta receptor-dependent events required for ILF formation can occur in adulthood and require LT-sufficient B lymphocytes. We demonstrate that mature ILF formation occurs in response to lumenal stimuli, including normal bacterial flora, and requires TNF receptor I function. These findings suggest that ILFs are organized intestinal lymphoid structures whose formation can be induced and whose mass can be expanded in response to mucosal challenges.

Cell cooperation in development of eosinophil-predominant inflammation in airways.

A large body of research supports a pathogenic role of Th2 cells in allergic diseases such as asthma. These disorders are characterized by recruitment to selected peripheral tissues of a mixed leukocyte inflammatory infiltrate including a predominant eosinophil component. The development of this inflammatory response is dependenton accumulation of Th2 cells in the affected tissues. Our studies aim to define the mechanisms that control the development of this tissue inflammatory response, focusing particularly on the mechanisms that recruit Th2 cells to the lung and airway. We have found that Th2 cells are on their own poorly competent for antigen-induced recruitment to the lung. By contrast, Th1 cells are avidly recruited to the lungs in response to airway antigen challenge. More important, recruitment of Th1 cells to the lung resulted in enhanced recruitment of Th2 cells to this tissue. The increased Th1 cell-induced recruitment of Th2 cells was associated with upregulation of endothelial vascular cell adhesion molecule-1 (VCAM-1) expression in airway-associated endothelial cells and could be largely blocked by systemic treatment with a monoclonal anti-VCAM-1 antibody. Systemic blocking of tumor necrosis factor (TNF) also blunted the airway inflammatory response. The prominent roles of TNF and VCAM-1 in recruitment of Th2 cells suggested that an inflammatory microenvironment was essential for the recruitment of Th2 cells. In fact, recruitment of Th2 cells to the airway could be induced in an antigen-independent fashion by proinflammatory stimuli such as intranasal instillation of endotoxin. This antigen nonspecificity of the Th2 cell recruitment suggested a model in which Th2 cell recruitment is in response to general inflammatory signals rather than to antigen itself. This model provides an explanation for the clinical observation that bacterial or viral respiratory tract infections are associated with disease exacerbations in allergic asthmatics. More generally, these data imply that Th2 cells, like other leukocytes, are recruited efficiently to sites of tissue inflammation, and that these nonspecifically recruited Th2 cells have substantial potential to modulate local inflammatory processes.

Nonhematopoietic expression of Janus kinase 3 is required for efficient recruitment of Th2 lymphocytes and eosinophils in OVA-induced airway inflammation.

Tyrosine kinases of the Janus kinase (Jak) family transduce signals from the type I and type II cytokine receptors. Jak3 is unique in this family because its expression must be induced and is predominantly limited to cells of the lymphoid and myeloid lineages. Deficient expression of Jak3 interferes with normal development and function of T, B, and NK cells. Using irradiated Jak3-deficient (Jak3-/-) mice reconstituted with normal bone marrow (Jak3-/-chimeric mice), we have investigated possible actions of Jak3 outside of the hematopoietic system. We show that efficient recruitment of inflammatory cells to the airways of OVA-sensitized mice challenged with aerosolized OVA requires the expression of Jak3 in radioresistant nonhematopoietic cells. Failure to develop eosinophil-predominant airway inflammation in Jak3-/- chimeric mice is not due to failure of T cell sensitization, because Jak3-/- chimeric mice showed delayed-type hypersensitivity responses indistinguishable from wild-type chimeric mice. Jak3-/- chimeric mice, however, express less endothelial-associated VCAM-1 after airway Ag challenge. Given the key role of VCAM-1 in recruitment of Th2 cells and eosinophils, our data suggest that Jak3 in airway-associated endothelial cells is required for the expression of eosinophilic airway inflammation. This requirement for nonhematopoietic expression of Jak3 represents the first demonstration of a physiological function of Jak3 outside of the lymphoid lineages.