William J. Janssen

Muc5b is required for airway defence

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b−/− mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α–responsive, heparan sulfate–specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

Lovastatin Enhances Clearance of Apoptotic Cells (Efferocytosis) with Implications for Chronic Obstructive Pulmonary Disease

Statins are potent, cholesterol-lowering agents with newly appreciated, broad anti-inflammatory properties, largely based upon their ability to block the prenylation of Rho GTPases, including RhoA. Because phagocytosis of apoptotic cells (efferocytosis) is a pivotal regulator of inflammation, which is inhibited by RhoA, we sought to determine whether statins enhanced efferocytosis. The effect of lovastatin on efferocytosis was investigated in primary human macrophages, in the murine lung, and in human alveolar macrophages taken from patients with chronic obstructive pulmonary disease. In this study, we show that lovastatin increased efferocytosis in vitro in an 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase-dependent manner. Lovastatin acted by inhibiting both geranylgeranylation and farnesylation, and not by altering expression of key uptake receptors or by increasing binding of apoptotic cells to phagocytes. Lovastatin appeared to exert its positive effect on efferocytosis by inhibiting RhoA, because it 1) decreased membrane localization of RhoA, to a greater extent than Rac-1, and 2) prevented impaired efferocytosis by lysophosphatidic acid, a potent inducer of RhoA. Finally, lovastatin increased efferocytosis in the naive murine lung and ex vivo in chronic obstructive pulmonary disease alveolar macrophages in an HMG-CoA reductase-dependent manner. These findings indicate that statins enhance efferocytosis in vitro and in vivo, and suggest that they may play an important therapeutic role in diseases where efferocytosis is impaired and inflammation is dysregulated.

Fas Determines Differential Fates of Resident and Recruited Macrophages during Resolution of Acute Lung Injury

RATIONALE During acute lung injury (ALI) the macrophage pool expands markedly as inflammatory monocytes migrate from the circulation to the airspaces. As inflammation resolves, macrophage numbers return to preinjury levels and normal tissue structure and function are restored. OBJECTIVES To determine the fate of resident and recruited macrophages during the resolution of ALI in mice and to elucidate the mechanisms responsible for macrophage removal. METHODS ALI was induced in mice using influenza A (H1N1; PR8) infection and LPS instillation. Dye labeling techniques, bone marrow transplantation, and surface immunophenotyping were used to distinguish resident and recruited macrophages during inflammation and to study the role of Fas in determining macrophage fate during resolving ALI. MEASUREMENTS AND MAIN RESULTS During acute and resolving lung injury from influenza A and LPS, a high proportion of the original resident alveolar macrophages persisted. In contrast, recruited macrophages exhibited robust accumulation in early inflammation, followed by a progressive decline in their number. This decline was mediated by apoptosis with local phagocytic clearance. Recruited macrophages expressed high levels of the death receptor Fas and were rapidly depleted from the airspaces by Fas-activating antibodies. In contrast, macrophage depletion was inhibited in mice treated with Fas-blocking antibodies and in chimeras with Fas-deficient bone marrow. Caspase-8 inhibition prevented macrophage apoptosis and delayed the resolution of ALI. CONCLUSIONS These findings indicate that Fas-induced apoptosis of recruited macrophages is essential for complete resolution of ALI.

Lung environment determines unique phenotype of alveolar macrophages

Alveolar macrophages (AM) are the most abundant antigen-presenting cells in the lungs, and they play a critical role in regulating pulmonary immune responses to inhaled pathogens and to allergens. However, compared with macrophages in other body sites, AM have an unusual phenotype that, in many respects, resembles the phenotype of dendritic cells (DC). Therefore, to more fully define the unique nature of AM, we compared the phenotype and function of AM with the phenotype and function of resident peritoneal lavage-derived macrophages (PLM). We found striking phenotypic differences between AM and PLM, particularly with regard to CD11c expression, and we also observed that AM had a significantly better antigen-presenting capability than PLM. Therefore, we investigated the role of the local airway environment in generation of the unusual phenotype of AM. We carried out cell transfer experiments to compare macrophage differentiation in the airways with that in the peritoneal cavity. We observed significant upregulation of CD11c expression on bone marrow macrophages and peritoneal macrophages when they were adoptively transferred into the airways. In contrast, CD11c expression was not upregulated after cell transfer into the peritoneal cavity, whereas CD11b expression was significantly increased. In vitro, culture of bone marrow-adherent cells with surfactant protein D (SP-D) or granulocyte/macrophage colony-stimulating factor (GM-CSF) induced significant upregulation of CD11c expression, and in vivo GM-CSF concentrations were significantly higher in bronchoalveolar than in peritoneal lavage fluid. Finally, GM-CSF(-/-) mice failed to develop CD11c(+) AM, but CD11c(+) AM were present in SP-D(-/-) mice. However, macrophages from GM-CSF(-/-) bone marrow could upregulate CD11c expression when transferred to the airways of wild-type mice. These results suggest that the airway environment promotes development of macrophages with unique DC-like characteristics and that this unusual phenotype is determined, to a large degree, by locally high concentrations of GM-CSF and, possibly, SP-D.

Surfactant Proteins A and D Suppress Alveolar Macrophage Phagocytosis via Interaction with SIRPα

RATIONALE Efficient removal of apoptotic cells is essential for the resolution of acute pulmonary inflammation. Alveolar macrophages ingest apoptotic cells less avidly than other professional phagocytes at rest but overcome this defect during acute inflammation. Surfactant protein (SP)-A and SP-D are potent modulators of macrophage function and may suppress clearance of apoptotic cells through activation of the transmembrane receptor signal inhibitory regulatory protein alpha (SIRP alpha). OBJECTIVES To investigate whether binding of SP-A and SP-D to SIRP alpha on alveolar macrophages suppresses apoptotic cell clearance. METHODS Phagocytosis of apoptotic cells was assessed using macrophages pretreated with SP-A, SP-D, or the collectin-like molecule C1q. Binding of SP-A and SP-D to SIRP alpha was confirmed in vitro using blocking antibodies and fibroblasts transfected with active and mutant SIRP alpha. The effects of downstream molecules SHP-1 and RhoA on phagocytosis were studied using SHP-1-deficient mice, sodium stibogluconate, and a Rho kinase inhibitor. Lipopolysaccharide was given to chimeric mice to study the effects of SP-A and SP-D binding on inflammatory macrophages. MEASUREMENTS AND MAIN RESULTS Preincubation of macrophages with SP-A or SP-D suppressed apoptotic cell clearance. Surfactant suppression of macrophage phagocytosis was reversed by blocking SIRP alpha and inhibiting downstream molecules SHP-1 and RhoA. Macrophages from inflamed lungs ingested apoptotic cells more efficiently than resting alveolar macrophages. Recruited mononuclear phagocytes with low levels of SP-A and SP-D mediated this effect. CONCLUSIONS SP-A and SP-D tonically inhibit alveolar macrophage phagocytosis by binding SIRP alpha. During acute pulmonary inflammation, defects in apoptotic cell clearance are overcome by recruited mononuclear phagocytes.

Cigarette Smoke Impairs Clearance of Apoptotic Cells through Oxidant-dependent Activation of RhoA

RATIONALE Cigarette smoke (CS) is the primary cause of chronic obstructive pulmonary disease (COPD), an effect that is, in part, due to intense oxidant stress. Clearance of apoptotic cells (efferocytosis) is a critical regulator of lung homeostasis, which is defective in smokers and in patients with COPD, suggesting a role in disease pathogenesis. OBJECTIVES We hypothesized that CS would impair efferocytosis through oxidant-dependent activation of RhoA, a known inhibitor of this process. METHODS We investigated the effect of CS on efferocytosis in vivo and ex vivo, using acute, subacute, and long-term mouse exposure models. MEASUREMENTS AND MAIN RESULTS Acute and subacute CS exposure suppressed efferocytosis by alveolar macrophages in a dose-dependent, reversible, and cell type-independent manner, whereas more intense CS exposure had an irreversible effect. In contrast, CS did not alter ingestion through the Fc gamma receptor. The inhibitory effect of CS on apoptotic cell clearance depended on oxidants, because the effect was blunted in oxidant-resistant ICR mice, and was prevented by either genetic or pharmacologic antioxidant strategies in vivo and ex vivo. CS inhibited efferocytosis through oxidant-dependent activation of the RhoA-Rho kinase pathway because (1) CS activated RhoA, (2) antioxidants prevented RhoA activation by CS, and (3) inhibitors of the RhoA-Rho kinase pathway reversed the suppressive effect of CS on apoptotic cell clearance in vivo and ex vivo. CONCLUSIONS These findings advance the hypothesis that impaired efferocytosis may contribute to the pathogenesis of COPD and suggest the therapeutic potential of drugs targeting the RhoA-Rho kinase pathway.

TNF-α Inhibits Macrophage Clearance of Apoptotic Cells via Cytosolic Phospholipase A2 and Oxidant-Dependent Mechanisms

Removal of apoptotic cells from inflammatory sites is an important step in the resolution of inflammation. Both murine and human macrophages stimulated with TNF-α or directly administered arachidonic acid showed an impaired ability to ingest apoptotic cells (efferocytosis). The inhibition was shown to be due to generation of reactive oxygen species, was blocked with a superoxide dismutase mimetic, MnTBAP, and was mimicked by direct addition of H2O2. To determine the mechanism of TNF-α-stimulated oxidant production, bone marrow-derived macrophages from gp91phox-deficient mice were examined but shown to still produce oxidants and exhibit defective apoptotic cell uptake. In contrast, a specific cytosolic phospholipase A2 inhibitor blocked the oxidant production and reversed the inhibited uptake. The suppressive effect of endogenous or exogenous oxidants on efferocytosis was mediated through activation of the GTPase, Rho. It was reversed in macrophages pretreated with C3 transferase to inactivate Rho or with an inhibitor of Rho kinase. During maturation of human monocyte-derived macrophages, only mature cells exhibited TNF-α-induced suppression of apoptotic cell clearance. The resistance of immature macrophages to such inhibition was shown to result not from defective generation of oxidants, but rather, from lack of response of these cells to the oxidants. Overall, the data suggest that macrophages in a TNF-α- and oxidant-rich inflammatory environment are less able to remove apoptotic cells and, thereby, may contribute to the local intensity of the inflammatory response.

The polymeric mucin Muc5ac is required for allergic airway hyperreactivity

In asthma, airflow obstruction is thought to result primarily from inflammation-triggered airway smooth muscle (ASM) contraction. However, anti-inflammatory and smooth muscle-relaxing treatments are often temporary or ineffective. Overproduction of the mucin MUC5AC is an additional disease feature that, while strongly associated pathologically, is poorly understood functionally. Here we show that Muc5ac is a central effector of allergic inflammation that is required for airway hyperreactivity (AHR) to methacholine (MCh). In mice bred on two well-characterized strain backgrounds (C57BL/6 and BALB/c) and exposed to two separate allergic stimuli (ovalbumin and Aspergillus extract), genetic removal of Muc5ac abolishes AHR. Residual MCh responses are identical to unchallenged controls, and although inflammation remains intact, heterogeneous mucus occlusion decreases by 74%. Thus, whereas inflammatory effects on ASM alone are insufficient for AHR, Muc5ac-mediated plugging is an essential mechanism. Inhibiting MUC5AC may be effective for treating asthma and other lung diseases where it is also overproduced.

A detailed evaluation of acute respiratory decline in patients with fibrotic lung disease: aetiology and outcomes.

Background and objective:  A comprehensive diagnostic evaluation is recommended for all patients with fibrotic lung disease and acute respiratory decompensation. However, the effect on clinical outcomes of this evaluation remains unknown.