Sensing of apoptotic cells through Axl causes lung basal cell proliferation in inflammatory diseases.

Epithelial cell proliferation, division, and differentiation are critical for barrier repair following inflammation, but the initial trigger for this process is unknown. Here we define that sensing of apoptotic cells by the TAM receptor tyrosine kinase Axl is a critical indicator for tracheal basal cell expansion, cell cycle reentry, and symmetrical cell division. Furthermore, once the pool of tracheal basal cells has expanded, silencing of Axl is required for their differentiation. Genetic depletion of Axl triggers asymmetrical cell division, leading to epithelial differentiation and ciliated cell regeneration. This discovery has implications for conditions associated with epithelial barrier dysfunction, basal cell hyperplasia, and continued turnover of dying cells in patients with chronic inflammatory pulmonary diseases.

Defective lung function following influenza virus is due to prolonged, reversible hyaluronan synthesis.

Little is known about the impact of viral infections on lung matrix despite its important contribution to mechanical stability and structural support. The composition of matrix also indirectly controls inflammation by influencing cell adhesion, migration, survival, proliferation and differentiation. Hyaluronan is a significant component of the lung extracellular matrix and production and degradation must be carefully balanced. We have discovered an imbalance in hyaluronan production following resolution of a severe lung influenza virus infection, driven by hyaluronan synthase 2 from epithelial cells, endothelial cells and fibroblasts. Furthermore hyaluronan is complexed with inter-α-inhibitor heavy chains due to elevated TNF-stimulated gene 6 expression and sequesters CD44-expressing macrophages. We show that intranasal administration of exogenous hyaluronidase is sufficient to release inter-α-inhibitor heavy chains, reduce lung hyaluronan content and restore lung function. Hyaluronidase is already used to facilitate dispersion of co-injected materials in the clinic. It is therefore feasible that fibrotic changes following severe lung infection and inflammation could be overcome by targeting abnormal matrix production.

Axl and MerTK receptor tyrosine kinases maintain human macrophage efferocytic capacity in the presence of viral triggers.

The requirement to remove apoptotic cells is equally important in homeostasis and inflammatory disease. In particular, during viral infections large quantities of infected cells undergo apoptosis and need to be efficiently cleared by phagocytes to prevent secondary necrosis. Although specific roles of several apoptotic cell sensors, such as the TAM (Tyro3, Axl, MerTK) receptor family, have been characterized in mouse models, little is known about their regulation and involvement in apoptotic cell uptake (efferocytosis) by human macrophages under inflammatory conditions. We show that whereas pro-inflammatory stimuli consistently downregulated MerTK expression in human monocyte-derived macrophages (MDMs), stimuli indicative of a viral infection, interferon-α (IFN-α) and the TLR3 ligand poly(I:C), specifically induced Axl expression and promoted binding of the bridging molecule Gas6. Axl induction by IFN-α and poly(I:C) was associated with higher MDM efferocytic capacity compared to cells treated with other pro-inflammatory stimuli, such as LPS and IFN-γ. While MerTK blocking antibody uniformly suppressed apoptotic cell uptake by MDMs, Axl blocking antibody significantly reduced efferocytosis by poly(I:C)-stimulated MDMs, but not by resting MDMs. Our observations demonstrate that Axl induction during viral infections contributes to maintaining macrophage capacity to engulf apoptotic cells, which may have important consequences for resolution of anti-viral immune responses.

The Axl receptor tyrosine kinase is a discriminator of macrophage function in the inflamed lung.

Much of the biology surrounding macrophage functional specificity has arisen through examining inflammation-induced polarizing signals, but this also occurs in homeostasis, requiring tissue-specific environmental triggers that influence macrophage phenotype and function. The TAM receptor family of receptor tyrosine kinases (Tyro3, Axl and MerTK) mediates the non-inflammatory removal of apoptotic cells by phagocytes through the bridging phosphatidylserine-binding molecules growth arrest-specific 6 (Gas6) or Protein S. We show that one such TAM receptor (Axl) is exclusively expressed on mouse airway macrophages, but not interstitial macrophages and other lung leukocytes, under homeostatic conditions and is constitutively ligated to Gas6. Axl expression is potently induced by granulocyte-macrophage colony-stimulating factor expressed in the healthy and inflamed airway, and by type I interferon or Toll-like receptor-3 stimulation on human and mouse macrophages, indicating potential involvement of Axl in apoptotic cell removal under inflammatory conditions. Indeed, an absence of Axl does not cause sterile inflammation in health, but leads to exaggerated lung inflammatory disease upon influenza infection. These data imply that Axl allows specific identification of airway macrophages, and that its expression is critical for macrophage functional compartmentalization in the airspaces or lung interstitium. We propose that this may be a critical feature to prevent excessive inflammation because of secondary necrosis of apoptotic cells that have not been cleared by efferocytosis.

Benzodiazepine augmented γ-amino-butyric acid signaling increases mortality from pneumonia in mice.

OBJECTIVES: Benzodiazepines are used for treating anxiety, epilepsy, muscle spasm, alcohol withdrawal, palliation, insomnia, and sedation as they allosterically modulate γ-amino-butyric acid type A (GABAA) receptors. Despite widespread use, the importance and mechanism of their immune side-effects are poorly understood. Herein we sought to elucidate the impact and mechanism of benzodiazepine-induced susceptibility to infection at anxiolytic doses in mice. DESIGN: Animal randomized controlled trial. SETTING: Laboratory. SUBJECTS: Adult female C57BL/6 and BALB/c mice. INTERVENTIONS: The effect of a subsedative, anxiolytic dose of diazepam (2 mg kg intraperitoneal) was investigated in a murine Streptococcus pneumoniae pneumonia model. MEASUREMENT AND MAIN RESULTS: Mortality, bacterial and cytokine load, cell recruitment, and intracellular pH were measured. Diazepam treatment did not affect immune homeostasis in the lung. However, diazepam increased mortality and bacterial load from S. pneumoniae pneumonia. The increases in mortality and bacterial load were reversed by a GABAA antagonist, bicuculline, indicating dependence on GABAA receptor signaling. While cell recruitment was unaltered by diazepam, the cytokine response to infection was affected, suggesting that local responses to the pathogen were perturbed. Macrophage and monocytes expressed benzodiazepine sensitive (α1-γ2) GABAA receptors. Interestingly macrophage GABAA receptor expression was regulated by bacterial toll-like receptor agonists and cytokines indicating an endogenous role in the immune response. Functionally diazepam appeared to counteract the endogenous down-regulation of GABAA signaling during infection. Consistent with augmented GABAA signaling, diazepam provoked intracellular acidosis in macrophage, leading to impaired cytokine production, bacterial phagocytosis and killing. In contrast, selective benzodiazepines that do not target the α1 GABAA subunit did not affect macrophage function ex vivo or increase susceptibility to pneumonia in vivo. CONCLUSIONS: Our data highlight the regulation of macrophage function by GABAA receptor signaling and the potential harm of benzodiazepine exposure during pneumonia. Therapeutically, selective drugs may improve the safety profile of benzodiazepines.