Lung Nk Cells During Respiratory Virus Infection

Natural killer (NK) cells are innate lymphocytes which are one of cells in firstline of host defence. NK cells pass activating and inhibitory signals through amultitude of germline-encoded receptors. Activating receptors include the naturalcytotoxicity receptors (NCRs), such as NKp46and NKp44, the Fc receptor CD16and NKG2D. Inhibitory receptors, such as killer immunoglobulin-like receptors(KIRs) and the NKG2A:CD94dimer. In addition, NK cells are activated bycytokines, including type I interferons, interleukin (IL)-12and IL-18. Once activated,NK cells can direct cytolytic granules towards the synapse to directly kill a targetcell. Our understanding of NK cells is evolving rapidly and their functions clearly gobeyond those of innate killer cells. Importantly, NK cells are a potent and earlysource of cytokines, particularly interferon (IFN)-γ, but they can also produce Thelper type2(Th2)-associated cytokines, such as IL-5and IL-13, and the regulatorycytokine IL-10. In addition, NK cells can be activated by interactions with dendriticcells and macrophages and profoundly influence the generation of the adaptiveresponse. The relationship between NK cells and viral infection is one of hot spot forimmunologist. Recently, researchers have found that NK cell can play a dual role inviral infection: protecting against virus or inducing injury.Lung, a very special organ, is commonly exposed to multiple non-self substanceand various pathogens from external environment continuously. Unique lungenvironment specialize the immune system of lung, for example, there are highthresholds when the immune response occurred in the lungs. NK cells also specializein their function at different tissue locations: recently, a novel IL-22-secreting subsetof NK cells has been described in the gut and tonsils. These cells also appear in thelungs during respiratory bacterial and viral infection. The airways are a major routeof entry of many important pathogens into the body and the ability of NK cells torespond rapidly to infection suggests an important role for these cells in acutepulmonary infection. However, what differences are there between lung NK cell andNK cells from other tissues on phenotype and functions? This is the question wewant to find out.The aim of our studies is to investigate the important role of lung NK cellsduring respiratory virus infection. We focus on the lung NK cells and Respiratory syncytial virus (RSV) infection. Meanwhile, we compare the different phenotype ofNK cells between lung and other tissues. We hope to provide a new sight forunderstanding the function of lung NK cells through these studies.In this study, we isolated the mononuclear cells from lymph node, bone marrow,spleen, blood, liver and lung in BALB/c and C57BL/6mice and detected thephenotype of NK cells using flow cytometry; We established the mouse model forRSV infection by intranasal infection of106PFU of RSV in female BALB/c mice.The histopathology of lung was assessed by H&E staining. ELISA was used toexamine the levels of cytokines in brochoalveolar lavage fluid (BALF). Theproportion and phenotype of lung NK cells were determined by flow cytometry.Intracellular staining was used to examine the secretion of cytokines and otherfunctional molecules. For NK cell depletion, Mice were treated intravenous (i.v.)with rabbit anti-mouse anti-asialo GM-1(AsGM-1) antibodies. The major results ofour studies are shown as follows:1. The frequency of NK cells is high in the lung in miceFirstly, we compared the frequency of NK cells in different tissues in normalmice. We found that NK cells widely distributed in lymphoid and non-lymphoidtissues, and the frequencies of NK cells in lymphocytes were inconformity indifferent tissues of C57BL/6mice and BALB/c mice. The frequency of NK cells inlymphocytes in the lung (10~12%) was the highest among all tissues, and the nextwas liver (6~8%), followed by blood (3~4%), spleen (2%), bone marrow (BM)(2%)and lymph node (LN)(0.5%). Together, these results implied that high NK cellsfrequency may has some special meanings for the lung.2. Lung NK cells maintain a more mature phenotype under steady-state conditionsNext, we detected the expression of CD27and CD11b and found more than70%of lung NK cells were CD11bhighCD27lowNK cell subset under steady-stateconditions, which was higher than CD11bhighCD27lowNK cell subset in blood(65.19%), spleen (46.20%), liver (31.15%), LN (25.25%), BM (17.85%),respectively. Almost all lung NK cells displayed the phenotype CD11bhigh,suggesting that lung NK cells are in a more mature status. Furthermore, the maturesurface markers DX5, CD122, Ly49s and CD43were also higher on lung NK cells,and the immature surface marker CD51was lower on lung NK cells than NK cells from any other tissue. Together, these date indicated that lung NK cells possess amore mature phenotype.3. Lung NK cells are quiescent under steady-state conditionsFurther, we compared the phenotype of NK cells among lung, spleen and bonemarrow. We found that the expressions of inhibitory receptors CD94-NKG2A on lungNK cells were higher than on spleen and BM NK cells. Moreover, the expression ofactivating receptors NKp46and NKG2D and activating marker CD69on lung NKcells were lower than on spleen and BM NK cells. The expression of migrationassociated molecules on lung NK cells were lower than on spleen and BM NK cells,such as, adhesion molecules (CD11a, CD18, CD244, CD48, CD62L, CD44andCD11c), chemokine receptors (CCR5and CXCR2). Meanwhile, lung NK cellsexpressed lower levels of co-stimulatory molecules CD86, B220, CD1d, PDL1andPD1than spleen or BM NK cells. Thus, these data indicated that lung NK cells arequiescent cells under normal conditions.4. Lung NK cells are activated and gain functions rapidly after respiratory infectionIn this part, we found lung NK cells could be activated rapidly after bacteria(Staphylococcus aureus or Klebsiella pneumoniae) and virus (A/PR8influenza virus)infection, and the time for lung NK cells activation was earlier than spleen NK cells.As time went on, the expression of CD69on lung NK cells steady increased afterrespiratory infection. Furthermore, accompanying with activation, the expressions offunctional molecules CD107a, TRAIL, FasL, IFN-γ and Granzyme B on lung NKcells were also increased on day2after K. pneumonia infection. Thus, although lungNK cells are quiescent cells under normal condition, they would be activated andexpressed functional molecules rapidly to eliminate harmful pathogens duringrespiratory infection.5. NK cells are involved in acute lung immune injury caused by RSV infectionHere, we found that RSV infection induced severe acute lung immune injuryand promoted the accumulation and activation of lung natural killer (NK) cells at theearly stage of infection in BALB/c mice. Activated lung NK cells highly expressedactivating receptors NKG2D and CD27and became functional NK cells byproducing a large amount of interferon-γ (IFN-γ), which might be responsible for acute lung immune injury. NK cell depletion significantly attenuated lung immuneinjury and reduced infiltration of total inflammatory cells and production of IFN-γ inBALF. These data show that NK cells are involved in exacerbating the lung immuneinjury at the early stage of RSV infection via IFN-γ secretion.Taken together, we found that there are high frequency of NK cells in lung inboth C57BL/6and BALB/c mice. And these NK cells maintain a more maturephenotype. Under steady-state conditions, lung NK cells are quiescent cells. LungNK cell activated and expressed functional molecules rapidly to eliminate harmfulpathogens during respiratory infection. NK cells are involved in acute lung immuneinjury at the early stage of RSV infection via IFN-γ secretion. We discussed thecrucial roles of NK cells in lung homeostasis and lung injury in this study andprovided a new sight for understanding the function of lung NK cells.