Chronic obstructive pulmonary disease (COPD) is a debilitating, progressive lung disease punctuated by exacerbations of symptoms. COPD exacerbations are most often associated with viral infections, and exposure to cigarette smoke (CS) followed by viral infection has been shown experimentally to enhance lung inflammation, tissue destruction, and airway fibrosis. Despite this, however, the cellular mechanisms responsible for this effect are unknown. In this study, we examined NK cell function in a mouse model of COPD given the vital role of NK cells following viral infection. Ex vivo stimulation of lung leukocytes with poly(I:C), ssRNA40, or ODN1826 enhanced production of NK cell-derived IFN-γ in CS-exposed mice. NK cells from CS-exposed mice exhibited a novel form of priming; highly purified NK cells from CS-exposed mice, relative to NK cells from filtered air-exposed mice, produced more IFN-γ following stimulation with IL-12, IL-18, or both. Further, NK cell priming was lost following smoking cessation. NKG2D stimulation through overexpression of Raet1 on the lung epithelium primed NK cell responsiveness to poly(I:C), ssRNA40, or ODN1826 stimulation, but not cytokine stimulation. In addition, NK cells from CS-exposed mice expressed more cell surface CD107a upon stimulation, demonstrating that the NK cell degranulation response was also primed. Together, these results reveal a novel mechanism of activation of the innate immune system and highlight NK cells as important cellular targets in controlling COPD exacerbations.

Rationale: Pathogenic T cells drive, or sustain, a number of inflammatory diseases. Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with the accumulation of activated T cells. We previously demonstrated that chronic cigarette smoke (CS) exposure causes oligoclonal expansion of lung CD4 1 T cells and CD8 1 T cells in a mouse model of COPD, thus implicating these cells in disease pathogenesis. Objectives: To determine whether T cells are pathogenic in a CSinduced mouse model of COPD. Methods: We transferred lung CD3 1 T cells from filtered air (FA)-and CS-exposed mice into Rag2 2/2 recipients. Endpoints associated with the COPD phenotype were then measured. Measurements and Main Results: Here, we demonstrate that chronic CS exposure generates pathogenic T cells. Transfer of CD3 1 T cells from the lungs of CS-exposed mice into Rag2 2/2 recipients led to substantial pulmonary changes pathognomonic of COPD. These changes included monocyte/macrophage and neutrophil accumulation, increased expression of cytokines and chemokines, activation of proteases, apoptosis of alveolar epithelial cells, matrix degradation, and airspace enlargement reminiscent of emphysema. Conclusions: These data formally demonstrate, for the first time, that chronic CS exposure leads to the generation of pathogenic T cells capable of inducing COPD-like disease in Rag2 2/2 mice. This report provides novel insights into COPD pathogenesis.

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 toll-like receptor ligands. Here, we show that the activating receptor NKG2D is a key mediator for CS stimulated NK cell hyperresponsiveness as 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 towards cells that express the NKG2D ligand RAET1ε. We also demonstrate that NKG2D-deficent mice exhibit diminished airway damage and reduced inflammation in a model of viral COPD exacerbation without affecting 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 WT 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.

Cigarette smoke (CS) exposure is the primary risk factor for the development of chronic obstructive pulmonary disease (COPD). COPD is characterized by chronic peribronchial, perivascular and alveolar inflammation. The inflammatory cells consist primarily of macrophage, neutrophils and lymphocytes. Although myeloid cells are well studied, the role of lymphocyte populations in pathogenesis of COPD remains unclear. Using a mouse model of CS-induced emphysema our laboratory has previously demonstrated that CS exposure causes changes in the T cell receptor repertoire suggestive of an antigen specific response and triggers a pathogenic T cell response sufficient to cause alveolar destruction and inflammation. We extend these findings to demonstrate that T cells from CS-exposed mice of Balb/cJ or C57B6 strain are sufficient to transfer pulmonary pathology to CS-naïve, immunosufficient mice. CS exposure causes a proinflammatory phenotype among pulmonary T cells consistent with from COPD patients. We provide evidence that donor T cells from CS-exposed mice depend on antigen recognition to transfer alveolar destruction using MHC class I deficient recipient mice. Neither CD4+ nor CD8+ T cells from donor mice exposed to CS are alone sufficient to cause inflammation or pathology in recipient mice. We found no evidence of impaired suppression of T cell proliferation among regulatory T cells from CS-exposed mice. These results suggest that CS exposure initiates an antigen specific response that leads to pulmonary destruction and inflammation that involves both CD8+ and CD4+ T cells. These results are direct evidence for an autoimmune response initiated by CS exposure.

Chronic Obstructive Pulmonary Disease (COPD) is a devastating disease with no effective therapies. We investigated the role of the C-type lectin receptor, CLEC5A, in macrophage activation and pulmonary pathogenesis in a mouse model of COPD. We demonstrate that CLEC5A is expressed on alveolar macrophages in mice exposed long-term to cigarette smoke (CS) and in human smokers. We also show that CLEC5A-mediated activation of macrophages enhanced cytokine elaboration alone, and in combination with LPS or GM-CSF in CS-exposed mice. Furthermore, using Clec5a-deficient mice, we demonstrate that CS-induced macrophage responsiveness is mediated by CLEC5A and CLEC5A is required for the development of inflammation, proinflammatory cytokine expression and airspace enlargement. These findings suggest a novel mechanism that promotes airway inflammation and pathologies in response to CS exposure and identifies CLEC5A as a novel target for the therapeutic control of COPD pathogenesis.

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CCR7 is a chemokine receptor expressed on the surfaces of T cells, B cells, and mature dendritic cells that controls cell migration in response to the cognate ligands CCL19 and CCL21. CCR7 is critical for the generation of an adaptive T cell response. However, the roles of CCR7 in the host defense against pulmonary infection and innate immunity are not well understood. We investigated the role of CCR7 in the host defense against acute pulmonary infection with Pseudomonas aeruginosa. We intranasally infected C57BL/6 mice with P. aeruginosa and characterized the expression of CCR7 ligands and the surface expression of CCR7 on pulmonary leukocytes. In response to infection, expression of CCL19 and expression of CCL21 were oppositely regulated, and myeloid dendritic cells upregulated CCR7 expression. We further examined the effects of CCR7 deficiency on the inflammatory response to P. aeruginosa infection. We infected Ccr7 ؊/؊ and wild-type mice with P. aeruginosa and characterized the accumulation of pulmonary leukocytes, production of proinflammatory mediators, neutrophil activation, and bacterial clearance. CCR7 deficiency led to an accumulation of myeloid dendritic cells and T cells in the lung in response to infection. CCR7 deficiency resulted in higher expression of CD80 and CD86 on dendritic cells; increased production of interleukin-12/23p40 (IL-12/23p40), gamma interferon (IFN-␥), and IL-1␣; increased neutrophil respiratory burst; and, ultimately, increased clearance of acute P. aeruginosa infection. In conclusion, our results suggest that CCR7 deficiency results in a heightened proinflammatory environment in response to acute pulmonary P. aeruginosa infection and contributes to more efficient clearance.

Long-term exposure to cigarette smoke (CS) can have deleterious effects on lung epithelial cells including cell death and the initiation of inflammatory responses. CS-induced cell injury can elaborate cell surface signals and cellular byproducts that stimulate immune system surveillance. Our previous work has shown that the expression of ligands for the cytotoxic lymphocyte activating receptor NKG2D is enhanced in patients with COPD and that the induction of these ligands in a mouse model can replicate COPD pathologies. Here, we extend these findings to demonstrate a role for the NKG2D receptor in CS-induced pathophysiology and provide evidence linking nucleic acid-sensing endosomal toll-like receptor (TLR) signaling to COPD pathology through NKG2D activation. Specifically, we show that mice deficient in NKG2D exhibit attenuated pulmonary inflammation and airspace enlargement in a model of CS-induced emphysema. Additionally, we show that CS exposure induces the release of free nucleic acids in the bronchoalveolar lavage and that direct exposure of mouse lung epithelial cells to cigarette smoke extract similarly induces functional nucleic acids as assessed by TLR3, 7, and 9 reporter cell lines. We demonstrate that exposure of mouse lung epithelial cells to TLR ligands stimulates the surface expression of RAET1, a ligand for NKG2D, and that mice deficient in TLR3/7/9 receptor signaling do not exhibit CS-induced NK cell hyperresponsiveness and airspace enlargement. The findings indicate that CS-induced airway injury stimulates TLR signaling by endogenous nucleic acids leading to elevated NKG2D ligand expression. Activation of these pathways plays a major role in the altered NK cell function, pulmonary inflammation and remodeling related to long-term CS exposure.