Activation of A ₁ -Adenosine Receptors Promotes Leukocyte Recruitment to the Lung and Attenuates Acute Lung Injury in Mice Infected with Influenza A/WSN/33 (H1N1) Virus

Abstract

We have shown that bronchoalveolar epithelial A 1 -adenosine receptors (A 1 -AdoR) are activated in influenza A virus-infected mice. Alveolar macrophages and neutrophils also express A 1 -AdoRs, and we hypothesized that activation of A 1 -AdoRs on these cells will promote macrophage and neutrophil chemotaxis and activation and thereby play a role in the pathogenesis of influenza virus-induced acute lung injury. Wild-type (WT) C57BL/6 mice, congenic A 1 -AdoR knockout (A 1 -KO) mice, and mice that had undergone reciprocal bone marrow transfer were inoculated intranasally with 10,000 PFU/mouse influenza A/WSN/33 (H1N1) virus. Alternatively, WT mice underwent daily treatment with the A 1 -AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) from 1 day prior to inoculation. Infection increased bronchoalveolar lining fluid (BALF) adenosine comparably in WT and A 1 -KO mice. Infection of WT mice resulted in reduced carotid arterial O 2 saturation (hypoxemia), lung pathology, pulmonary edema, reduced lung compliance, increased basal airway resistance, and hyperresponsiveness to methacholine. These effects were absent or significantly attenuated in A 1 -KO mice. Levels of BALF leukocytes, gamma interferon (IFN-␥), and interleukin 10 (IL-10) were significantly reduced in infected A 1 -KO mice, but levels of KC, IP-10, and MCP-1 were increased. Reciprocal bone marrow transfer resulted in WT-like lung injury severity, but BALF leukocyte levels increased only in WT and A 1 -KO mice with WT bone barrow. Hypoxemia, pulmonary edema, and levels of BALF alveolar macrophages, neutrophils, IFN-␥, and IL-10 were reduced in DPCPX-treated WT mice. Levels of viral replication did not differ between mouse strains or treatment groups. These findings indicate that adenosine activation of leukocyte A 1 -AdoRs plays a significant role in their recruitment to the infected lung and contributes to influenza pathogenesis. A 1 -AdoR inhibitor therapy may therefore be beneficial in patients with influenza virus-induced lung injury. IMPORTANCEBecause antiviral drugs are of limited efficacy in patients hospitalized for influenza virus-induced respiratory failure, there is an urgent need for new therapeutics that can limit the progression of lung injury and reduce influenza death rates. We show that influenza A virus infection results in increased production of the nucleoside adenosine in the mouse lung and that activation of A 1 -subtype adenosine receptors by adenosine contributes significantly to both recruitment of innate immune cells to the lung and development of acute lung injury following influenza virus infection. We also show that treatment with an A 1 -adenosine receptor antagonist reduces the severity of lung injury in influenza virus-infected mice. Our findings indicate that adenosine plays an important and previously unrecognized role in the innate immune response to influenza virus infection and suggest that drugs which can inhibit either generation of adenosine or activation of A 1 -adenosine receptors may be beneficia...

Mouse Models of Acute Respiratory Distress Syndrome

“…While not included in this list, induction of acute lung injury using influenza virus instillation also has been well established T. Xu et al 2006;Traylor, Aeffner, and Davis 2013;Zhang et al 2012;Aeffner, Woods, and Davis 2014;Dai et al 2014). These models may be used in tandem.…”

“…Later, the remaining cells or a portion of them can be processed as cytospin preparations (by centrifugation at 1,000 rpm for 5 min on a Cytospin™ 4 unit; Thermo Fisher Scientific, Waltham, MA) and dyed with Wright-Giemsa stain or a similar method. Differential cell counts typically are performed at 10 (ocular) Â 100 (objective) magnification via oil-immersion microscopy by counting a minimum of 200 cells (optimal: 300 or more cells; Naito et al 2013;McGarry, Protherone, and Lee 2009;Aeffner, Woods, and Davis 2014).…”

“…For the latter, lungs are harvested as described earlier and stored at À80 C until enough samples are collected. Lungs are then homogenized by hand or mechanically (e.g., FastPrep-24 instrument; MP Biomedical, Santa Ana, CA), after which enzyme activity is measured using a commercially available MPO kit or a colorimetric assay according to the manufacturer's instructions (e.g., Mouse Myeloperoxidase ELISA Kit, Cell Sciences, Canton, MA; Lomas et al 2003;Hirsh et al 2004;Neff et al 2005;Hoke et al 2007;Aeffner, Woods, and Davis 2014;E. Zhou et al 2014).…”

Means of accelerating tendon graft healing after anterior cruciate reconstruction

“…Matsumoto et al revealed the presence of abundant vascular stem cells with a characteristic expression of CD34+ in the injured ACL tissues, which displayed higher expansion and multiple lineage differentiation potential than CD34-cells [39]. In a further study, ACL-derived CD34+ cells were injected into nude mice articular cavities after ACL reconstruction, where more fibrocartilage cells, as well as enhanced angiogenesis and osteogenesis, were suggested in a CD34+ sorted group [38]. Additionally, CD34+ cells were previously isolated from peripheral blood or bone marrow and recognised as a rich population of haematopoietic/endothelial progenitor cells, which have been already used in clinical settings for the repair of various damaged tissues [40].…”

“…ACL-derived CD34+ cells have been shown to enhance tendon-bone healing in ACL reconstruction animal models, mostly by enhancing angiogenesis and osteogenesis [37,38]. Matsumoto et al revealed the presence of abundant vascular stem cells with a characteristic expression of CD34+ in the injured ACL tissues, which displayed higher expansion and multiple lineage differentiation potential than CD34-cells [39].…”