A prevailing assumption is that CS is a toxic irritant that broadly inhibits leukocyte function (23
). However, a growing body of literature suggests that this perspective is too narrow and that chronic CS exposure can augment specific innate and adaptive immune functions in COPD patients (7
). In the course of infection, NK cells play an important dual role, the early release of cytokines and directed cytotoxicity. Previously, we have shown that NK cells from CS-exposed mice have enhanced cytokine production in response to stimulating cytokines or to TLR ligands that mimic pathogen associated molecular patterns (PAMPS) (13
). In the current study, we show that this enhanced function is dependent on the presence of NKG2D. NK cells from CS-exposed, NKG2D-deficient do not exhibit enhanced IFNγ production when stimulated with IL-12 and/or IL-18. Excessive NK cell activation after infection could worsen the outcomes associated with CS exposure. In the context of COPD, the presence of NK cells hyperresponsive to viral provocation could increase the inflammatory response and contribute to the progression of COPD.
Although there have been a number of studies in humans investigating the effect of CS exposure on cytotoxic lymphocyte effector functions, primarily cytotoxicity, the outcomes of these studies are equivocal. Several studies reported increased cytotoxic activity (28
) while others show decreased activity in smokers with and without COPD (27
). Analyses of NK cell function in mice exposed to CS show a similar variability in results (13
). The recent reports from Urbanowicz et al. indicate that compartmental differences in cytotoxic lymphocyte function may account for these discordant results. Those investigators found that NK cells collected from the peripheral blood of long term smokers showed reduced cytotoxicity (27
), whereas NK cells collected from the sputum of similar patient populations showed increased cytotoxicity (32
). Ultimately, the reasons for the disparities are unclear but the inconsistencies highlight the need for mechanistic studies to understand the effects of CS exposure on NK cell function and the role of NK cells in COPD pathogenesis.
NK cells interact with cells through an extensive array of stimulatory and inhibitory receptor-ligand interactions including NKG2D (8
). In this study, we report increased NKG2D-mediated cytotoxicity in a mouse model of COPD. Previously, we demonstrated that inducible expression of RAET1 on mouse pulmonary epithelial cells, and subsequent activation of NKG2D, causes an emphysematous phenotype that was associated with increased epithelial cell apoptosis (12
). Moreover, these mice exhibited enhanced cytokine release in response to TLR stimulation (13
) and enhanced clearance of pulmonary bacterial infection (39
). Our findings appear contradictory to reports that repeated activation of NKG2D in transgenic mice caused receptor downregulation and loss of function (8
). However, these studies examined the effects of constitutive expression of NKG2D ligands. In contrast, our mouse model conditionally expresses Raet1α
in pulmonary epithelial cells, an exposure more comparable to the CS model of COPD, and did not exhibit downregulation of NKG2D expression (12
). Together with the current results demonstrating enhanced NKG2D function in our CS model of COPD, our findings suggest that a hyperresponsive NK cell phenotype is achieved through chronic, lung-specific NKG2D stimulation.
Cigarette smoke exposure increases pulmonary inflammation and lung remodeling in response to viral infection. Earlier studies did not identify specific mechanisms that may contribute to CS-induced viral exacerbations (43
), whereas Kang et al. identified multiple soluble mediators (IL-18, IFNγ) and signaling pathways (e.g., TLR3) that contribute to viral-induced pathologies in CS-exposed mice (47
). However, the specific cell populations and pathways affected by CS exposure that, in turn contribute to the heightened response to influenza, have not been identified. This distinction is important because neither cytokine nor receptor pathways were altered by CS exposure in the absence of infection in the previous studies (43
). The integrity of these pathways may reflect their importance for responses to pulmonary viral pathogens with established tobacco smoke induced injury. Here, we have identified significant cell-specific alterations in response to CS exposure that directly contribute to the increased pathology after viral infection. In the context of influenza infection of CS-exposed mice, we demonstrate the requirement for NKG2D+ NK cells in the exacerbation of COPD pathologies. The CS-exposed NKG2D- deficient mice could not mount the prototypic injury response of CS exposed wild-type mice that included the influx of inflammatory cells, the loss/destruction of an intact respiratory epithelium and diminished expression of the CCSP, a phenotypic marker of the protective Clara cell. However, reconstitution with NKG2D+ NK cells from CS-exposed wild-type mice in the NKG2D-deficient mice reestablishes the prototypic injury response. Of note, there were no differences in viral load between any groups of influenza-infected mice. This finding is important because it suggests that the exaggerated immune response to virus and the subsequent increased pathology in COPD, which is dependent on NKG2D, is not a function of immunosuppression and increased susceptibility to infection. Rather, the reported increased incidence and severity of symptoms in smokers is likely due to the increased inflammation and airway obstruction as a consequence of enhanced NK cell function in response to the viral infection.
Although not directly examined in this study, it is tempting to speculate that the diminished pathologies in CS-exposed, influenza-infected, NKG2D-deficient mice was due to the failure of these NK cells to develop a hyperresponsive phenotype in the COPD model. An alternative explanation for these observations is that there is a fundamental requirement for NKG2D in response to pulmonary influenza infection. If so, the FA-exposed Klrk1+/+ or Klrk1-/- mice would reveal this requirement for homeostatic responses to influenza infection in terms of inflammation, injury and clearance. However, the lack of effect of NKG2D deficiency on these basal responses strongly supports the concept that NKG2D-dependent, CS-induced, NK cell hyperresponsiveness is an important mechanism in disease pathogenesis.
Together with our previous report demonstrating CS-induced Raet1 expression in the lung (12
), our new findings suggest a model whereby CS induces the chronic local expression of NKG2D ligands on resident pulmonary cells which primes NK cells to independent stimulation by cytokines, TLR ligands, and NKG2D ligands. These NK activating mediators are known to increase during viral infection and lead to enhanced inflammation and cytotoxicity. Important to the exacerbation of COPD, the enhanced NK cell activation leads to increased pulmonary injury and airways obstruction. Although we demonstrate a clear role for NKG2D in the priming of NK cell function in the context of long-term CS exposure, the mechanisms whereby NKG2D mediates NK cell priming and the subsequent response to influenza remain to be fully elucidated.
In the current study, we present evidence that NK cells regulate inflammation in a model of viral infection of COPD and that NKG2D mediates enhanced NK cell effector function and influenza-induced pathologies in a mouse model of COPD. These findings indicate that alterations in NK cell function will have significant consequences in COPD patients infected with virus. Although NK cells are by far the predominant cell type expressing NKG2D in mice, NKG2D is also expressed almost ubiquitously on human CD8 T cells (8
). However, studies suggest that NKG2D plays a co-stimulatory role based on the relative inability to stimulate T cell responses with NKG2D ligands alone (48
). This difference in NKG2D expression and function between mice and humans coupled with evidence for a role of CD8 T cells in COPD pathogenesis (51
) necessitates further examination of the contribution of NKG2D-bearing lymphocyte subpopulations in COPD pathogenesis.