No abstract

Macrophage phagocytosis of particles and pathogens is an essential aspect of innate host defense. Phagocytic function requires cytoskeletal rearrangements that depend on the interaction between macrophage surface receptors, particulates/pathogens and the extracellular matrix. In the present report we determine the role of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), in integrating the lipopolysaccharide (LPS) and matrix stiffness signals to control macrophage phenotypic change for host defense and resolution from lung injury. We demonstrate that active TRPV4 mediates LPS-stimulated murine macrophage phagocytosis of non-opsonized particles (E. coli) in vitro and opsonized particles (IgG-coated latex beads) in vitro and in vivo in intact mice. Intriguingly, matrix stiffness in the range seen in inflamed or fibrotic lung is required to sensitize the TRPV4 channel to mediate the LPS-induced increment in macrophage phagocytosis. Furthermore, TRPV4 is required for the LPS induction of anti-inflammatory/pro-resolution cytokines. These findings suggest that signaling through TRPV4, triggered by changes in extracellular matrix stiffness, cooperates with LPS-induced signals to mediate macrophage phagocytic function and lung injury resolution. These mechanisms are likely to be important in regulating macrophage function in the context of pulmonary infection and fibrosis.

The microenvironment of the lung in asthma is acidic, yet the effect of acidity on inflammatory cells has not been well established. We now demonstrate that acidity inhibits eosinophil apoptosis and increases cellular viability in a dose-dependent manner between pH 7.5 and 6.0. Notably, acidity induced eosinophil cyclic adenosine 5-monophosphate (cAMP) production and enhanced cellular viability in an adenylate cyclasedependent manner. Furthermore, we identify G protein-coupled receptor 65 (GPR65) as the chief acid-sensing receptor expressed by eosinophils, as GPR65-deficient eosinophils were resistant to acid-induced eosinophil cAMP production and enhanced viability. Notably, GPR65 ؊/؊ mice had attenuated airway eosinophilia and increased apoptosis in 2 distinct models of allergic airway disease. We conclude that eosinophil viability is increased in acidic microenvironments in a cAMP-and GPR65-dependent manner. IntroductionAsthma is a chronic lung disease characterized by episodes of inflammation and narrowing of the airways in response to diverse stimuli. 1 Over the past 2 decades, the incidence of asthma has increased worldwide and is now one of the chief diagnoses responsible for pediatric hospitalization. Extracellular acidosis is commonly observed in inflammatory diseases such as asthma. Several studies have shown that the pH of exhaled breath condensate (EBC) in patients with acute asthma is significantly lower than that of normal subjects. The pH of EBC is mildly alkaline in control persons (7.65 Ϯ 0.20), reaching 5.23 plus or minus 0.21 during asthma exacerbations. 2 Furthermore, the EBC pH of pediatric and adult patients with chronic asthma is also significantly decreased compared with healthy people and correlates with asthma severity. 3,4 Whereas EBC is not a direct measurement of airway lining fluid, airway acidification in asthma is supported by alternative approaches, including direct measurements of bronchial secretions. 2 Although EBC pH is not an asthma-specific biomarker, early clinical studies have suggested that acidification contributes to the disease process as inhalation of buffers that increase airway pH alleviates asthma symptoms. 5 G protein-coupled receptor 65 (GPR65), also known as T-cell death-associated gene 8, belongs to a group of acid-sensing receptors in the G2A G protein-coupled receptor (GPCR) family. [6][7][8] This group includes G2A, GPR65, ovarian cancer GPCR1 (OGR1), and GPR4. Some members of the G2A family, including GPR65, have been shown to be proton sensors in lymphocytes. Acid-sensing capacity is mediated by proton transfer to histidines in the first extracellular loop of GPR65, presumably causing a conformational change in GPR65 that activates G␣ s . This acid-sensing activity has been demonstrated by measuring intracellular cyclic adenosine 5Ј-monophosphate (cAMP) in GPR65-deficient lymphocytes incubated at several proton concentrations. 7 To better understand the functional implications of acidic microenvironments on eosinophils, hallmark cells of asthmatic inflam...

The effect of moderate alcohol consumption on liver fibrosis is not well understood, but evidence suggests that adenosine may play a role in mediating the effects of moderate ethanol on tissue injury. Ethanol increases the concentration of adenosine in the liver. Adenosine 2A receptor (A2AR) activation is known to enhance hepatic stellate cell (HSC) activation and A2AR deficient mice are protected from fibrosis in mice. Making use of a novel mouse model of moderate ethanol consumption in which female C57BL/6J mice were allowed continued access to 2% (vol/vol) ethanol (11% calories) or pair-fed control diets for 2 days, 2 weeks or 5 weeks and superimposed with exposure to CCl4, we tested the hypothesis that moderate ethanol consumption increases fibrosis in response to carbon tetrachloride (CCl4) and that treatment of mice with an A2AR antagonist prevents and/or reverses this ethanol-induced increase in liver fibrosis. Neither the expression or activity of CYP2E1, required for bio-activation of CCl4, nor AST and ALT activity in the plasma were affected by ethanol, indicating that moderate ethanol did not increase the direct hepatotoxicity of CCl4. However, ethanol feeding enhanced HSC activation and exacerbated liver fibrosis upon exposure to CCl4. This was associated with an increased sinusoidal angiogenic response in the liver. Treatment with A2AR antagonist both prevented and reversed the ability of ethanol to exacerbate liver fibrosis.ConclusionModerate ethanol consumption exacerbates hepatic fibrosis upon exposure to CCl4. A2AR antagonism may be a potential pharmaceutical intervention to decrease hepatic fibrosis in response to ethanol.

Pro-fibrotic mesenchymal cells are known to be the key effector cells of fibroproliferative disease, but the specific matrix signals and the induced cellular responses that drive the fibrogenic phenotype remain to be elucidated. The key mediators of the fibroblast fibrogenic phenotype were characterized using a novel assay system that measures fibroblast behavior in response to actual normal and fibrotic lung tissue. Using this system, we demonstrate that normal lung promotes fibroblast motility and polarization, while fibrotic lung immobilizes the fibroblast and promotes myofibroblast differentiation. These context-specific phenotypes are surprisingly both mediated by myosin II. The role of myosin II is supported by the observation of an increase in myosin phosphorylation and a change in intracellular distribution in fibroblasts on fibrotic lung, as compared with normal lung. Moreover, loss of myosin II activity has opposing effects on protrusive activity in fibroblasts on normal and fibrotic lung. Loss of myosin II also selectively inhibits myofibroblast differentiation in fibroblasts on fibrotic lung. Importantly, these findings are recapitulated by varying the matrix stiffness of polyacrylamide gels in the range of normal and fibrotic lung tissue. Comparison of the effects of myosin inhibition on lung tissue with that of polyacrylamide gels suggests that matrix fiber organization drives the fibroblast phenotype under conditions of normal/soft lung, while matrix stiffness drives the phenotype under conditions of fibrotic/stiff lung. This work defines novel roles for myosin II as a key regulatory effector molecule of the pro-fibrotic phenotype, in response to biophysical properties of the matrix.