NKG2D mediates NK cell hyperresponsiveness and influenza-induced pathologies in a mouse model of chronic obstructive pulmonary disease

Abstract

Chronic obstructive pulmonary disease (COPD) is characterized by peribronchial and perivascular inflammation and largely irreversible airflow obstruction. Acute disease exacerbations, due frequently to viral infections, lead to enhanced disease symptoms and contribute to long-term progression of COPD pathology. Previously, we demonstrated that NK cells from cigarette smoke (CS)-exposed mice exhibit enhanced effector functions in response to stimulating cytokines or TLR ligands. In this article, we show that the activating receptor NKG2D is a key mediator for CS-stimulated NK cell hyperresponsiveness, because CS-exposed NKG2D-deficient mice (Klrk1(-/-)) did not exhibit enhanced effector functions as assessed by cytokine responsiveness. NK cell cytotoxicity against MHC class I-deficient targets was not affected in a COPD model. However, NK cells from CS-exposed mice exhibit greater cytotoxic activity toward cells that express the NKG2D ligand RAET1ε. We also demonstrate that NKG2D-deficient mice exhibit diminished airway damage and reduced inflammation in a model of viral COPD exacerbation, which do not affect viral clearance. Furthermore, adoptive transfer of NKG2D(+) NK cells into CS-exposed, influenza-infected NKG2D-deficient mice recapitulated the phenotypes observed in CS-exposed, influenza-infected wild-type mice. Our findings indicate that NKG2D stimulation during long-term CS exposure is a central pathway in the development of NK cell hyperresponsiveness and influenza-mediated exacerbations of COPD.

Figure 1. Characterization of NK cell markers in a mouse model of COPD

NK cells were isolated from C57BL/6 mice exposed to FA or CS for 6 months and analyzed by flow cytometry. (A-B) Total NK cells enumerated by NKp46+ expression. (C-D) The percentage of NKp46+ cells expressing the indicated NK cell markers. (E-F) The geometric mean fluorescent intensity (MFI) of NK cell receptors. Values are presented as means ± SEM. n = 5-6 per group. All data representative of two independent experiments.

Figure 2. NK cell cytotoxicity against MHC class I-deficient and NKG2D ligand expressing targets in mouse model of COPD

(A) Representation of flow cytometry analysis comparing in vivo clearance. CFSE labeled C57BL/6 (CFSE low) cells and C57BL/6 B2m^-/- (CFSE high) cells were injected i.v .into receipt mice exposed to exposed to FA or CS. Mice were bled 16 hrs following injections and labeled target cells were analyzed by flow cytometry. (B) Time course of NK cell cytotoxicity against B2m^-/- target cells, as described in (A), in mice exposed to FA or CS for 3-10 months. Data representative of results from four independent experiments.

Figure 3. NK cells of CS exposed mice demonstrate increased cytotoxic activity towards RAE1 expressing cells

NK cells were purified from pooled splenocytes of mice exposed to FA or CS for 6 months and cytotoxicity towards RAE1ε-targets was assessed ex vivo. NK cells and CFSE-labeled RMA cells transfected to express RAE1ε (or mock transfected) were combined at increasing effector to target (E:T) ratios. Cells were incubated for 4 h, harvested and analyzed by flow cytometry as described in the methods. Figure is representative of four independent experiments each using NK cells pooled from two mice. Significant differences between groups at all E:T ratios are indicated.

Figure 4. Klrk1^-/- mice do not develop enhanced cellular responses in mouse model of COPD

NK cells from the pooled spleens of 5 mice were highly purified (>99% NKp46+) and stimulated overnight with cytokines as labeled. IFNγ was quantified by ELISA. The marked (*) axis is the scale representing levels of IFNγ released by IL-12/18 stimulation. Values are presented as means ± SEM . Relevant significant differences between groups are highlighted. Figure is representative of three independent experiments.

Figure 5. CS-exposed Klrk1^-/- mice lack enhanced cellular responses associated with influenza infection

Mice were infected with 2 × 10³ pfu influenza virus and endpoints were measured 4 days after infection. (A-B) H&E stained lung sections representing changes in lung inflammation and airways obstruction between groups. (C-D) Clara cell secretory protein (CCSP) immunohistochemistry staining of lung sections showing epithelial damage of large airways and small airways. (E-F) Total RNA was isolated from lung homogenates of FA- or CS-exposed Klrk1^+/+ and Klrk1^-/- mice with and without influenza infection. Raet1 and Mult1 transcripts were assayed by quantitative RT-PCR and normalized to Rpl32. (G) H&E stained lung sections of influenza-infected CS-exposed Klrk1^-/- mice which received NK cells from FA or CS-exposed Klrk1^+/+ mice. (H) Semi-quantitative assessment of inflammation severity and distribution. (I) Quantitation of the percentage of intrapulmonary airways exhibiting any degree of airways obstruction. (n = 4-8 mice per group)

Figure 6. Long-term CS exposure does not affect viral clearance in Klrk1^+/+ or Klrk1^-/- mice

Influenza titers in individual mice were determined by plaque assay as described in Methods. Data are presented as plaque-forming units (PFU) per gram of wet lung tissue. All data representative of two independent experiments (n = 4-8 mice per group).