Lethal Influenza Infection: Is A Macrophage To Blame?

Abstract

Alveolar macrophages (AMs) are critical for immunity against influenza A virus (IAV) infection. Depletion, hypo-reactivity, and disruption of AM development and differentiation are all associated with lethal IAV infection. AMs drive the innate immune response that limits IAV infection. AMs are crucial for steady-state homeostasis of pulmonary surfactant, and in turn surfactant proteins regulate AMs and participate in host defense against IAV. Known factors that are necessary for AM function and differentiation in vivo include surfactant proteins, the growth factor GM-CSF, the hormone receptor PPARγ, and the transcription factors PU.1 and Bach2. Although PU.1 and PPARγ are downstream effectors of GM-CSF, Bach2 works independently. GM-CSF and Bach2-deficient AMs have phenotypes with immature or alternatively activated states of differentiation, respectively, and both extremes are unsuitable for surfactant homeostasis. The activation state of AMs and the local microenvironment may determine the development of symptomatic vs. asymptomatic IAV infection in different individuals.

Alveolar macrophages are critical for host resistance to influenza

The indispensable role of alveolar macrophages (AMs) against influenza A virus (IAV) infection is evidenced in several lines of investigation. Depletion of AMs using clodronate liposomes prior to IAV infection led to uncontrolled viral replication and death in mice, pigs, and ferrets [1–3]. Conditional ablation of AMs by diptheria toxin (DT) administration in CD169(Siglec1)-DTR mice resulted in massive immunopathology and death, supporting an essential role of AMs [4]. Lack of AMs in GM-CSF or GM-CSF receptor-deficient mice impaired control of IAV replication and disrupted respiratory gas exchange causing 100% mortality [5]. Expression of GM-CSF under constitutive or inducible promoters in the lungs of GM-CSF-deficient mice conferred full or partial protection against IAV commensurate with activation [6] and local differentiation [7] of AMs. Exogenous delivery of GM-CSF to the lung protected against lethal IAV infection in wild type mice [8, 9]. Disruption of AM function by conditional deletion of PPARγ, a downstream effector of GM-CSF signaling, impaired clearance of apoptotic cells leading to severe pneumonia with high morbidity and mortality, despite adequate induction of humoral and cell-mediated immunity [5]. The role of Bach2 [10], which regulates differentiation of AMs independent of GM-CSF, in pathogenesis of IAV infection has not been determined. Assessing the role of genetics, it was found that the high susceptibility of DBA/2J mice to IAV infection compared to C57BL/6 mice stemmed from hypo-reactivity in innate responsiveness of AMs to IAV infection [11]. Restoration of the intrinsic antiviral Mx1 factor, which is deleted or mutated in most inbred mouse strains, failed to enhance protection in the susceptible DBA/2J mice [12], suggesting dysfunction in upstream mechanisms. A transformative finding in establishing the essential role of AMs is that neonatal transfer of wild type AM precursors to the lungs of GM-CSF βc receptor subunit-deficient mice protected adult mice from lethal IAV infection [5], indicating a direct and primary role of AMs in protection against IAV infection. The local environment in which IAV infection encounters AMs requires further analysis.

Alveolar Macrophages: at the interface of pulmonary homeostasis and host defense

Upon entry into the respiratory tract, IAV encounters a highly regulated immune environment designed to eliminate infection and avoid overt inflammation. Early studies demonstrated that AMs maintain a high threshold of immune activation avoiding inflammatory disease from exposure to innocuous antigens being highly efficient in phagocytosis and clearance of airborne agents and by secretion of mediators that suppress adaptive immunity as reviewed earlier [13]. AMs were subsequently found to be critical for the maintenance of surfactant levels through catabolism of excess surfactant proteins and lipids [14]. Excessive accumulation of surfactant during IAV infection in GM-CSF and PPARγ-deficient mice leads to respiratory insufficiency [5]. Epithelial cell derived GM-CSF drives differentiation of AM precursors that seed the lung at early stages of gestation. In postnatal life, AMs are maintained by local proliferation or differentiate from bone marrow precursors that travel to the lung in response to infection or inflammation [5]. Surfactant proteins contribute to the phenotype of AMs [15]. The ability of mature AMs to orchestrate immune homeostasis in the resting state involves intercellular communication, cell-cell contact of AMs with respiratory epithelial cells, interaction with pulmonary surfactant proteins A (SP-A) and D (SP-D), and possibly other surfactant components as reviewed previously [16, 17]. Significantly, genetic polymorphisms in SP-A were associated with increased susceptibility to acute inflammatory injury in patients infected with the 2009 pandemic H1N1 influenza [18], indicating that SP-A shapes the host response of AMs to IAV infection in humans. A question that has not been clearly addressed is whether GM-CSF modifies these homeostatic mechanisms in the course of an immune response. Increased levels of GM-CSF, in the context of IAV infection, may decrease the threshold of immune activation resulting in increased AM responsiveness and protection against infection or result in chronic inflammation [7]. A recent study demonstrated that low levels of IAV induce minimal expression of inflammatory genes but, depending on viral strain, once the virus titer exceeds a certain level, expression of inflammatory genes is strongly induced [19]. At this stage, IAV could disrupt the lung’s homeostatic circuit by depleting AMs; GM-CSF can prevent depletion of AMs by IAV [20]. It can be envisaged that provision of GM-CSF at a time frame when the virus titer is low reduces threshold of virus recognition, facilitating beneficial activation of AMs to eliminate IAV infection before it proliferates to dangerous levels. Furthermore, by rescuing AMs, GM-CSF may promote activation and differentiation of AM precursors encompassing a range of alternative activation states towards resolution of IAV-induced inflammation. In support for this notion, infection with an influenza A virus strain of intermediate virulence was characterized by the appearance of a heterogeneous population of activated alveolar macrophages and blood derived lung monocytes with both populations having a mixed M1/M2 phenotype at early stages of infection [21]. On the other hand, it is also reasonable to speculate that at high IAV titer, persistent activation of GM-CSF as induced by highly pathogenic pH1N1 and H5N1 IAV strains [19], may contribute to injurious inflammation and fibrosis by AMs. In this regard, the scavenger receptor MARCO, a known downstream effector of GM-CSF [7] drives macrophage polarization to the M2 alternative macrophage phenotype extreme leading to pulmonary fibrosis in response to injury [22]. Consistent with a pathogenic role, MARCO-deficient mice are resistant to IAV infection [7, 23], suggesting MARCO as a potential target for therapy against influenza-induced injury. Virus-induced GM-CSF could also sensitize AM pattern recognition receptors to tissue and virus-derived ligands enhancing inflammation. For example, activation of TLR-7 may result in oxidative injury through activation of the NADPH oxidase Nox2 in alveolar macrophages [24]; inhibition of Nox2 has been shown to attenuate influenza A virus-induced lung inflammation [25]. In this context, a decoy peptide that blocked activation of multiple toll-like receptors rescued mice from lethal influenza infection [26]. The interaction of these pathways with the Nox2 subunit rac1 downstream of SP-A receptor isoforms in response to IAV infection remains to be investigated [15]. These studies highlight that IAV evades multiple tasks of AMs in initiating, balancing, and resolution of the inflammatory response.

Abortive infection may direct host outcomes

The fate of infected AMs resulting in pathogenesis or resistance to IAV infection has not been established. Infection of AMs with IAV consists of a robust replication cycle of the viral genome and synthesis of viral proteins without packaging and release of new virus, which is termed abortive or non-productive infection. Given the importance of redox state in influenza pathogenesis [25, 27] with the myeloid Nox2 as a key mediator of oxidative injury [25], a question for further study is whether Nox2 facilitates abortive replication of the virus in AMs compared to Nox4 which was shown to mediate replication of IAV in epithelial cells [28]. Effective antiviral immunity by AMs rests on induction of adequate levels of type I interferon and inflammatory mediators, which suppress IAV infection in respiratory epithelial cells and promote adaptive and cell-mediated immunity [29]. It appears that IAV infection results in depletion of AMs [20], although other studies suggest that the AM population remains stable during IAV infection [30]. On the other hand, premature AM apoptosis as induced by the IAV polymerase gene product PB1-F2 [31], or timely apoptosis following production of type I interferon [29] may lead to pathogenesis or host resistance to IAV infection, respectively. The Type I interferon response by AMs is subject to inhibition by prostaglandin PGE2 produced by alveolar epithelial cells and macrophages during infection [29]. In this context, GM-CSF may be pivotal for suppression of PGE2 production activating the antiviral response of AMs [32]. One consideration that to our knowledge has not been determined is whether abortive IAV infection drives AMs towards alternative states of differentiation in vivo as has been shown in vitro [33]. In this case, Bach2 deficiency results in AMs with an M2 alternative activation state of differentiation independent of GM-CSF/PPARγ signaling pathways [10], suggesting Bach2 as a termination checkpoint for AM differentiation in the lung. The inability of bach2-deficient M2 AMs to catabolize surfactant suggests that additional factors in the local micro-environment modulate differentiation of AMs through Bach2. A similar phenotype was reported earlier in mice with constitutive overexpression of the M2 macrophage differentiation factor IL-4 [34], at a time when the M1/M2 macrophage differentiation model was not widely studied. The cross-talk between GM-CSF, Bach2, and IAV infection in AMs may thus determine progression of the infection towards disease resolution or disease pathogenesis in symptomatic vs asymptomatic IAV infection, respectively.

Concluding remarks

Influenza A virus causes highly contagious respiratory infections that spread from the upper to the lower airway and can lead to fatal viral pneumonia. New strains of IAV that easily spread between individuals arise frequently. Alveolar macrophages are essential for host resistance to IAV infection. We propose that AMs also serve as an essential portal for IAV infection, pathogenesis, and transmission. It is evident that IAV induces inflammatory/oxidative pathways to establish infection; such capacity would increase the risk for development of detrimental IAV-induced pneumonia by AMs. Controlled IAV infections of human volunteer cohorts revealed molecular signatures that can differentiate symptomatic from asymptomatic individuals during seasonal influenza [35]; asymptomatic individuals are not restrained by hospitalization and can freely shed infectious virus in the community. That GM-CSF, a key homeostatic factor for AMs, enhances host resistance to influenza supports the notion that IAV exploits the local homeostatic circuitry to establish infection and transmission. Through abortive infection of AMs, IAV is in a position to “polarize” the immune response towards tolerance or pathogenesis depending on inter-individual genetic differences distributed randomly in the population. We propose that understanding the mechanisms of IAV entry and persistence in AMs is of paramount importance for discovering how to prevent spread of deadly IAV infections. The set point that enables disposal of the offending virus and restoration of the lung microenvironment by AMs, as exemplified by treatment with GM-CSF or innate immune activation antagonists, is amenable to therapeutic intervention.