Aryl Hydrocarbon Receptor Targets Pathways Extrinsic to Bone Marrow Cells to Enhance Neutrophil Recruitment during Influenza Virus Infection

Although vaccination and improved drug therapy have dramatically reduced mortality from infectious diseases, respiratory viral infections remain among the leading causes of mortality worldwide (World Health Organization, 2005). With the continued emergence of new viral strains, influenza viruses in particular pose a threat to human health and the global economy. The immune response to influenza virus relies on the activation of cells from the innate and adaptive arms of the immune system, leading ultimately to the creation of virus-specific CD8+ cytotoxic T lymphocytes (CTL), and antibodies (Gerhard, 2001; Woodland et al., 2001). During a primary infection, the generation of CTL and virus-specific antibodies takes 7–10 days, during which time cells of the innate immune system emigrate to the lung and presumably keep the infection at bay. In contrast to the well-characterized role of lymphocytes in antiviral immune responses, less is known about the precise role that cells of the innate immune system, and neutrophils in particular, play during respiratory viral infections. It is becoming increasingly clear that the recruitment of neutrophils to the lung requires exquisite control. Evidence for this includes studies demonstrating that the accumulation of excess neutrophils is associated with host tissue damage and increased mortality (Fernandez et al., 2001; Patel et al., 1999; Shimizu et al., 1999; Teske et al., 2005). On the other hand, depletion of neutrophils can diminish survival following infection (Bliss et al., 2001; Sayles and Johnson, 1996; Stephens-Romero et al., 2005; Tumpey et al., 2005). Thus, there is growing evidence that the magnitude of neutrophil influx to the lung probably plays a very important role in the host’s ability to survive viral infection. Therefore, it is important to understand what factors influence the differential recruitment of neutrophils to the lung upon infection with different subtypes of influenza A virus or among different individuals. We have recently reported that activation of the aryl hydrocarbon receptor (AhR) markedly increases the number of neutrophils in lungs of mice infected with influenza A virus (Teske et al., 2005). This AhR-mediated increase in the number of neutrophils peaks on the seventh day of infection, was observed in both the airways and lung interstium, but was not observed in the absence of infection. Furthermore, by depleting neutrophils in vivo, we were able to improve the survival of infected mice, suggesting that AhR-mediated recruitment of excess neutrophils to the lung contributes to the decreased survival from influenza virus (Teske et al., 2005). The AhR is a member of the Per-Arnt-Sim family of transcriptional regulators and plays a role in xenobiotic metabolism and development (Gu et al., 2000). The AhR is activated by a diverse spectrum of ligands, including plant-derived natural compounds, tryptophan metabolites, and environmental contaminants (Denison and Nagy, 2003). Of the AhR agonists characterized thus far, the pollutant 2,3,7,8-tetrachlorodi-benzo-p-dioxin (TCDD or “dioxin”) binds to it with the highest affinity, and is often used as a prototypical AhR agonist. In addition to TCDD, other pollutants that bind and activate the AhR include coplanar polychlorinated biphenyls and polyaromatic hydrocarbons (PAH), such as benzo[a]pyrene and 7,12-dimethylbenzanthracene, which are found in cigarette smoke and diesel exhaust (Behnisch et al., 2003; Denison and Nagy, 2003). In short, humans are exposed to AhR ligands daily through ingestion and inhalation (Charnley and Doull, 2005; Schecter et al., 2001). Moreover, diminished host resistance and altered immune function following exposure to PAH-containing pollutants correlates with an increased incidence of influenza and other respiratory infections (Burchiel and Luster, 2001; Sopori and Kozak, 1998). In rodents, AhR activation impairs survival following infection with influenza virus (Burleson et al., 1996; Luebke et al., 2002; Teske et al., 2005; Warren et al., 2000), further illustrating the relationship between exposure to AhR ligands and altered host resistance to infection. The AhR is broadly expressed in mammalian tissues, and cells of the immune system, including neutrophils, have been reported to express it (Ackermann et al., 1989; Lang et al., 1998; Lawrence et al., 1996; Williams et al., 1996; Yamamoto et al., 2004,). AhR is also found in both the human and rodent lung (Dolwick et al., 1993; Lang et al., 1998; Thatcher et al., 2007; Yamamoto et al., 2004). Furthermore, in humans and rodents exposure to AhR agonists has been linked to enhanced pulmonary inflammation, including increased neutrophil influx to the lung (Diaz-Sanchez et al., 2000; Harrod et al., 2003; Luebke et al., 2002; Teske et al., 2005; Warren et al., 2000). However, the mechanism by which AhR activation enhances the directional migration of neutrophils has proved difficult to determine. The immune system is a very well known and sensitive target organ for the toxicity of dioxins and related compounds, and studies using AhR-deficient mice demonstrate that their toxicity is AhR dependent (Kerkvliet et al., 2002; Neff-LaFord et al., 2007; Teske et al., 2005; Vorderstrasse et al., 2001; and our unpublished observations). In particular, we have previously reported that increased neutrophilic inflammation in lungs of TCDD-treated mice infected with influenza virus is AhR dependent (Teske et al., 2005). Therefore, much effort to delineate the mechanism by which AhR activation enhances neutrophil recruitment has focused on an immune-mediated mechanism. However, neither the infection-induced increase in soluble neutrophil chemoattractants nor the upregulation of adhesion molecules on neutrophils is perturbed when the AhR is activated by TCDD (Teske et al., 2005). Likewise, the functional activity of neutrophils from infected, TCDD-treated mice was equivalent to neutrophils from vehicle control-treated mice (Teske et al., 2005). In addition to affecting known soluble neutrophil chemo-attractants, it is possible that the increase in the number of neutrophils results from nonspecific leakage of leukocytes from the blood into the lung. However, the following pieces of evidence do not support this idea: (1) the number of macrophages in lungs of infected mice is the same, regardless of TCDD treatment (Warren et al., 2000); (2) AhR activation by TCDD reduces the number of lymphocytes in lungs of infected mice (Mitchell and Lawrence, 2003; Warren et al., 2000); and (3) compared with infected controls, there is no change in the levels of protein or LDH in lavage fluid from infected mice treated with TCDD (Bohn et al., 2005). Collectively, these observations suggest that the directional migration of excess neutrophils to the lung during influenza virus infection is likely the result of AhR-dependent deregulation of a neutrophil-specific recruitment processes. To characterize how activation of the AhR deregulates neutrophil-specific processes, we first determined whether AhR activation enhances the number of neutrophils systemically or only at the site of antigen challenge (i.e., the lung). We then determined whether AhR-mediated events within or extrinsic to the immune system drive the recruitment of excess neutrophils to the lungs of TCDD-treated, infected mice. The findings from these studies provide novel information regarding how AhR activation alters neutrophil recruitment to the lung, and emphasizes that environmental exposure to AhR ligands may have a profound effect on disease outcome during infection with a common respiratory virus.