The effect of BPIFA1/SPLUNC1 genetic variation on its expression and function in asthmatic airway epithelium.

Bacterial permeability family member A1 (BPIFA1), also known as short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is a protein involved in the antiinflammatory response. The goal of this study was to determine whether BPIFA1 expression in asthmatic airways is regulated by genetic variations, altering epithelial responses to type 2 cytokines (e.g., IL-13). Nasal epithelial cells from patients with mild to severe asthma were collected from the National Heart, Lung, and Blood Institute Severe Asthma Research Program centers, genotyped for rs750064, and measured for BPIFA1. To determine the function of rs750064, cells were cultured at air-liquid interface and treated with IL-13 with or without recombinant human BPIFA1 (rhBPIFA1). Noncultured nasal cells with the rs750064 CC genotype had significantly less BPIFA1 mRNA expression than the CT and TT genotypes. Cultured CC versus CT and TT cells without stimulation maintained less BPIFA1 expression. With IL-13 treatment, CC genotype cells secreted more eotaxin-3 than CT and TT genotype cells. Also, rhBPIFA1 reduced IL-13-mediated eotaxin-3. BPIFA1 mRNA levels negatively correlated with serum IgE and fractional exhaled nitric oxide. Baseline FEV1% levels were lower in the asthma patients with the CC genotype (n = 1,016). Our data suggest that less BPIFA1 in asthma patients with the CC allele may predispose them to greater eosinophilic inflammation, which could be attenuated by rhBPIFA1 protein therapy.

Immunoproteasomes as a novel antiviral mechanism in rhinovirus-infected airways.

Rhinovirus (RV) infection is involved in acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). RV primarily infects upper and lower airway epithelium. Immunoproteasomes (IP) are proteolytic machineries with multiple functions including the regulation of MHC class I antigen processing during viral infection. However, the role of IP in RV infection has not been explored. We sought to investigate the expression and function of IP during airway RV infection. Primary human tracheobronchial epithelial (HTBE) cells were cultured at air-liquid interface (ALI) and treated with RV16, RV1B, or interferon (IFN)-λ in the absence or presence of an IP inhibitor (ONX-0914). IP gene (i.e. LMP2) deficient mouse tracheal epithelial cells (mTECs) were cultured for the mechanistic studies. LMP2-deficient mouse model was used to define the in vivo role of IP in RV infection. IP subunits LMP2 and LMP7, antiviral genes MX1 and OAS1 and viral load were measured. Both RV16 and RV1B significantly increased the expression of LMP2 and LMP7 mRNA and proteins, and IFN-λ mRNA in HTBE cells. ONX-0914 down-regulated MX1 and OAS1, and increased RV16 load in HTBE cells. LMP2-deficient mTECs showed a significant increase in RV1B load compared with the wild-type (WT) cells. LMP2-deficient (compared with WT) mice increased viral load and neutrophils in bronchoalveolar lavage (BAL) fluid after 24 h of RV1B infection. Mechanistically, IFN-λ induction by RV infection contributed to LMP2 and LMP7 up-regulation in HTBE cells. Our data suggest that IP are induced during airway RV infection, which in turn may serve as an antiviral and anti-inflammatory mechanism.

Air-Liquid Interface Culture of Human and Mouse Airway Epithelial Cells.

Air-liquid interface culture enables airway epithelial cells to differentiate into a pseudostratified cell layer, consisting of ciliated cells, goblet/secretory cells, and basal cells (Ghio et al., Part Fibre Toxicol 10:25, 2013). This technique is critically important for in vitro studies of lung diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, since differentiated airway epithelial cells are more representative of the in vivo lung environment than non-differentiated cells (Derichs et al., FASEB J 25:2325-2332, 2011; Hackett et al., Am J Respir Cell Mol Biol 45:1090-1100, 2011;Schneider et al., Am J Respir Crit Care Med 182: 332-340, 2010). Here we describe the process of isolating and expanding human and mouse airway epithelial cells, as well as differentiation of airway epithelial cells by air-liquid interface culture.

Toll-Interacting Protein, Tollip, Inhibits IL-13-Mediated Pulmonary Eosinophilic Inflammation in Mice.

Toll-interacting protein (Tollip) is a key negative regulator of innate immunity by preventing excessive proinflammatory responses. Tollip genetic variation has been associated with airflow limitation in asthma subjects and Tollip expression. Whether Tollip regulates lung inflammation in a type 2 cytokine milieu (e.g., IL-13) is unclear. Our goal was to determine the in vivo role of Tollip in IL-13-mediated lung eosinophilic inflammation and the underlying mechanisms. Tollip-knockout (KO) and wild-type (WT) mice were inoculated intranasally with recombinant mouse IL-13 protein to examine lung inflammation. To determine how Tollip regulates inflammation, alveolar macrophages and bone marrow-derived macrophages from Tollip KO and WT mice were cultured with or without IL-13 and/or IL-33. IL-13-treated Tollip KO mice significantly increased lung eosinophilic inflammation and eotaxin-2 (CCL24) levels compared with the WT mice. IL-13- treated Tollip KO (vs. WT) macrophages, in the absence and particularly in the presence of IL-33, increased expression of the IL-33 receptor ST2L and CCL24, which was in part dependent on enhanced activation of interleukin (IL)-1 receptor-associated kinase 1 (IRAK1) and signal transducer and activator of transcription 6 (STAT6). Our results suggest that Tollip downregulates IL-13-mediated pulmonary eosinophilia in part through inhibiting the activity of the ST2L/IL-33/IRAK1 axis and STAT6.

Overproduction of growth differentiation factor 15 promotes human rhinovirus infection and virus-induced inflammation in the lung.

Human rhinovirus (HRV) is the most common virus contributing to acute exacerbations of chronic obstructive pulmonary disease (COPD) nearly year round, but the mechanisms have not been well elucidated. Recent clinical studies suggest that high levels of growth differentiation factor 15 (GDF15) protein in the blood are associated with an increased yearly rate of all-cause COPD exacerbations. Therefore, in the current study, we investigated whether GDF15 promotes HRV infection and virus-induced lung inflammation. We first examined the role of GDF15 in regulating host defense and HRV-induced inflammation using human GDF15 transgenic mice and cultured human GDF15 transgenic mouse tracheal epithelial cells. Next, we determined the effect of GDF15 on viral replication, antiviral responses, and inflammation in human airway epithelial cells with GDF15 knockdown and HRV infection. Finally, we explored the signaling pathways involved in airway epithelial responses to HRV infection in the context of GDF15. Human GDF15 protein overexpression in mice led to exaggerated inflammatory responses to HRV, increased infectious particle release, and decreased IFN-λ2/3 (IL-28A/B) mRNA expression in the lung. Moreover, GDF15 facilitated HRV replication and inflammation via inhibiting IFN-λ1/IL-29 protein production in human airway epithelial cells. Lastly, Smad1 cooperated with interferon regulatory factor 7 (IRF7) to regulate airway epithelial responses to HRV infection partly via GDF15 signaling. Our results reveal a novel function of GDF15 in promoting lung HRV infection and virus-induced inflammation, which may be a new mechanism for the increased susceptibility and severity of respiratory viral (i.e., HRV) infection in cigarette smoke-exposed airways with GDF15 overproduction.

Oncostatin M and TNF-α Induce Alpha-1 Antitrypsin Production in Undifferentiated Adipose Stromal Cells.

Alpha-1 antitrypsin (A1AT), a circulating acute-phase reactant antiprotease, is produced and secreted by cells of endodermal epithelial origin, primarily hepatocytes, and by immune cells. Deficiency of A1AT is associated with increased risk of excessive lung inflammation and injury, especially following chronic cigarette smoke (CS) exposure. Exogenous administration of mesenchymal progenitor cells, including adipose tissue-derived stromal/stem cells (ASC), alleviates CS-induced lung injury through paracrine effectors such as growth factors. It is unknown, however, if mesodermal ASC can secrete functional A1AT and if CS exposure affects their A1AT production. Human ASC collected via liposuction from nonsmoking or smoking donors were stimulated by inflammatory cytokines tumor necrosis alpha (TNFα), oncostatin M (OSM), and/or dexamethasone (DEX) or were exposed to sublethal concentrations of ambient air control or CS extract (0.5%-2%). We detected minimal expression and secretion of A1AT by cultured ASC during unstimulated conditions, which significantly increased following stimulation with TNFα or OSM. Furthermore, TNFα and OSM synergistically enhanced A1AT expression and secretion, which were further increased by DEX. The A1AT transcript variant produced by stimulated ASC resembled that produced by bronchial epithelial cells rather than the variant produced by monocytes/macrophages. While the cigarette smoking status of the ASC donor had no measurable effect on the ability of ASC to induce A1AT expression, active exposure to CS extract markedly reduced A1AT expression and secretion by cultured ASC, as well as human tracheobronchial epithelial cells. ASC-secreted A1AT covalently complexed with neutrophil elastase in control ASC, but not in cells transfected with A1AT siRNA. Undifferentiated ASC may require priming to secrete functional A1AT, a potent antiprotease that may be relevant to stem cell therapeutic effects.

MUC18 regulates IL-13-mediated airway inflammatory response.

OBJECTIVE: To evaluate the effects of MUC18 on IL-13-mediated airway inflammatory responses in human airway epithelial cells and in mice. MATERIALS: Primary normal human tracheobronchial epithelial (HTBE) cells, wild-type (WT) and Muc18 knockout (KO) mice, and mouse tracheal epithelial cells (mTECs) were utilized. TREATMENT: Cultured HTBE cells treated with MUC18 siRNA or MUC18 expressing lentivirus were incubated with IL-13 (10 ng/mL) for 24 h. Mice were intranasally instilled with 500 ng of IL-13 for 3 days. mTECs were treated with IL-13 (10 ng/mL) for 3 days. METHODS: PCR was used to measure mRNA expression. Western Blot and ELISAs were used to quantify protein expression. Cytospins of bronchoalveolar lavage (BAL) cells were used to obtain leukocyte differentials. RESULTS: MUC18 siRNA reduced IL-13-mediated eotaxin-3 (183 ± 44 vs. 380 ± 59 pg/mL, p < 0.05), while MUC18 overexpression increased IL-13-mediated eotaxin-3 (95 ± 3 vs. 58 ± 3 pg/mL, p < 0.05) in HTBE cells. IL-13-treated Muc18 KO mice had a lower percentage of neutrophils in BAL than WT mice (25 ± 3 vs. 35 ± 3%, p = 0.0565). CONCLUSIONS: These results implicate MUC18 as a potential enhancer of airway inflammation in a type 2 cytokine (e.g., IL-13) milieu.

A novel mouse model of conditional IRAK-M deficiency in myeloid cells: application in lung Pseudomonas aeruginosa infection.

Myeloid cells such as macrophages are critical to innate defense against infection. IL-1 receptor-associated kinase M (IRAK-M) is a negative regulator of TLR signaling during bacterial infection, but the role of myeloid cell IRAK-M in bacterial infection is unclear. Our goal was to generate a novel conditional knockout mouse model to define the role of myeloid cell IRAK-M during bacterial infection. Myeloid cell-specific IRAK-M knockout mice were generated by crossing IRAK-M floxed mice with LysM-Cre knock-in mice. The resulting LysM-Cre+/IRAK-Mfl/wt and control (LysM-Cre-/IRAK-Mfl/wt) mice were intranasally infected with Pseudomonas aeruginosa (PA). IRAK-M deletion, inflammation, myeloperoxidase (MPO) activity and PA load were measured in leukocytes, bronchoalveolar lavage (BAL) fluid and lungs. PA killing assay with BAL fluid was performed to determine mechanisms of IRAK-M-mediated host defense. IRAK-M mRNA and protein levels in alveolar and lung macrophages were significantly reduced in LysM-Cre+/IRAK-Mfl/wt mice compared with control mice. Following PA infection, LysM-Cre+/IRAK-Mfl/wt mice have enhanced lung neutrophilic inflammation, including MPO activity, but reduced PA load. The increased lung MPO activity in LysM-Cre+/IRAK-Mfl/wt mouse BAL fluid reduced PA load. Generation of IRAK-M conditional knockout mice will enable investigators to determine precisely the function of IRAK-M in myeloid cells and other types of cells during infection and inflammation.

MUC18 Regulates Lung Rhinovirus Infection and Inflammation.

BACKGROUND: MUC18 is upregulated in the lungs of asthma and COPD patients. It has been shown to have pro-inflammatory functions in cultured human airway epithelial cells during viral infections and in mice during lung bacterial infections. However, the in vivo role of MUC18 in the context of viral infections remains poorly understood. The goal of this study is to define the in vivo function of MUC18 during respiratory rhinovirus infection. METHODS: Muc18 wild-type (WT) and knockout (KO) mice were infected with human rhinovirus 1B (HRV-1B) and sacrificed after 1 day to determine the inflammatory and antiviral responses. To examine the direct effects of Muc18 on viral infection, tracheal epithelial cells isolated from WT and KO mice were grown under air-liquid interface and infected with HRV-1B. Finally, siRNA mediated knockdown of MUC18 was performed in human airway epithelial cells (AECs) to define the impact of MUC18 on human airway response to HRV-1B. RESULTS: Both viral load and neutrophilic inflammation were significantly decreased in Muc18 KO mice compared to WT mice. In the in vitro setting, viral load was significantly lower and antiviral gene expression was higher in airway epithelial cells of Muc18 KO mice than the WT mice. Furthermore, in MUC18 knockdown human AECs, viral load was decreased and antiviral gene expression was increased compared to controls. CONCLUSIONS: Our study is the first to demonstrate MUC18's pro-inflammatory and pro-viral function in an in vivo mouse model of rhinovirus infection.