PROS1/AXL signaling protects mice from lethal influenza infection by inducing M2 macrophage polarization

Abstract

AXL, a member of the TAM (Tyro3, AXL, and Mertk) subfamily of RTKs, is abundantly expressed in lung tissue and has been implicated in viral infections and lung injury. PROS1, one of the ligands known to activate AXL, functions as an immunomodulator in many diseases. However, the role of PROS1/AXL signaling in influenza A virus (IAV) infection and infection-induced lung injury is largely unknown. In this study, we find that the exogenous administration of PROS1 mitigates lung injury and protects mice from lethal infection by IAVs through the activation of AXL. PROS1 induces the phosphorylation of AXL, which in turn recruits Gab1 and p85, a regulatory subunit of PI3K, to form a complex that activates Gab1 and its downstream PI3K/AKT/mTOR in alveolar macrophages. Gab1 knockdown in vivo, or LY294002 (a PI3K inhibitor), abolishes the PROS1/AXL-induced protective activity against lethal influenza infection in mice. We also show that PROS1/AXL signaling induces M2 polarization of alveolar macrophages through Gab1 activation both in vitro and in vivo. Gab1 knockdown inhibits M2 macrophage accumulation in IAV-infected lungs and attenuates the protective effect of PROS1. These results indicate that PROS1/AXL signaling can activate Gab1 in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype, thereby eliciting protective activity against lethal infection with IAVs. These data also highlight the PROS1/AXL signal as a novel therapeutic target for IAV infection.

1. Introduction

Influenza A viruses (IAVs) are zoonotic, negative-sense, single-stranded RNA viruses that are closely associated with worldwide pandemics [1]. Annual outbreaks of IAV cause considerable morbidity and mortality in communities [2]. The deadliest of these outbreaks, the 1918 pandemic “Spanish Flu”, caused by an unusually virulent H1N1 subtype of IAV, resulted in 500 million infections and 50 million deaths [3]. In recent years, avian influenza variants, including H5N1 and H7N9, have been able to cross-infect humans and have high rates of infection and lethality compared with other IAV strains, with pandemic potential risk [4]. Severe influenza infections are often lethal, manifesting as cytokine storms, inflammation, flooding of alveolar spaces, and even acute respiratory distress syndrome and respiratory failure. However, the factors that control IAV replication and limit lethal lung inflammation remain largely unknown. Vaccines and anti-influenza medications, such as neuraminidase inhibitors, are available but may be much less effective in immunocompromised populations or those infected with drug-resistant IAVs [5,6]. Therefore, the identification of novel antiinfluenza targets and agents is urgently needed.

Tyro3, AXL, and Mertk (TAM) receptors are a subfamily of receptor tyrosine kinases (RTKs) that are expressed in various cells and tissues. In the lung, TAM receptors are expressed in alveolar type II epithelial cells (ATII cells), alveolar macrophages and neutrophils [7–10]. Vitamin K-dependent protein growth arrest specific protein 6 (Gas6) and protein S (PROS1) are two ligands that bind to and activate the TAM receptor [11]. TAM receptors and their ligands have potent immunomodulatory functions. Currently, the most studied TAM receptors (Tyro3/AXL/Mertk) recognize phosphatidylserine on apoptotic cells via the bridging molecules Gas6 and PROS1, thus mediating the clearance of apoptotic cells [12,13]. TAM receptor activation-mediated efferocytosis and inhibition of inflammatory pathways protect the brain from ischemic stroke and viral infections [14,15]. However, the role of the immunomodulatory functions of TAM receptors and their ligands in combating IAV infection and infection-induced lung pathology is unclear. Exogenous administration of PROS1 or Gas6 alleviated acute lung injury in mice by inhibiting inflammatory cytokine secretion through the activation of AXL [16,17]. AXL plays a crucial role in inducing protective antiviral adaptive immunity by limiting the immunosuppressive effects of type I IFNs [18]. Alveolar macrophages (AMs) are critical for immunity to influenza A virus (IAV) infection [19], and PROS1 has been reported to promote an immunosuppressive microenvironment in gliomas by polarizing tumor-associated macrophages toward the M2 phenotype [20], suggesting that PROS1 may also affect alveolar macrophage polarization and function. All of the above studies suggest an important and beneficial role for the TAM receptor and its ligands in modulating immune responses during IAV infection.