Carcinoembryonic antigen (CEA)-related cell adhesion molecules are co-expressed in the human lung and their expression can be modulated in bronchial epithelial cells by non-typable Haemophilus influenzae, Moraxella catarrhalis, TLR3, and type I and II interferons

Klaile, Esther; Klassert, Tilman E; Scheffrahn, Inka; Müller, Mario M; Heinrich, Annina; Heyl, Kerstin A; Dienemann, Hendrik; Grünewald, Christiane; Bals, Robert; Singer, Bernhard B; Slevogt, Hortense

Primary normal human bronchial epithelial (NHBE) cells (Lonza) were propagated as suggested by the supplier in collagen I-coated flasks or plates (BD Biosciences) in Bronchial Epithelial Cell Basal Medium (BEBM, Lonza) supplemented with bovine pitituary extract (BPE), Hydrocortisone, human Epidermal Growth Factor (hEGF) Epinephrine, Transferrin, Insulin, Retinoic acid and Triiodothyronine (from the BEGM bullet Kit, Lonza). Prior to stimulation, cells in passage 4-6 were grown to confluence. NHBE cells were treated for the indicated durations with 100 ng/ml IFNα (interferon alpha 1a, ImmunoTools), 100 ng/ml IFNβ (interferon beta 1a, ImmunoTools), 100 ng/ml IFNγ (recombinant human interferon gamma, Promokine), 100 ng/ml TNFα (recombinant human tumor necrosis factor alpha, R&D Systems), 100 ng/ml MALP-2 (Enzo Life Sciences GmbH), 100 ng/ml poly I:C (high molecular weight, InvivoGene), 100 ng/ml flagellin (Invivogene Biotech), or 4% CSE (see above). The concentrations chosen for the treatments described above are standard concentrations used for lung epithelial cells [37] [38] [39] [40] [41] [42] [43] . Infections werde done using the following strains: M. catarrhalis: 25238 (wild type, American Type Culture Collection); BBH18 (wild type) and BBH18.1 (adhesin UspA1 deletion mutant unable to bind CEACAM1), both kindly provided by Kristian Riesbeck, Malmö Lund University, Skåne University Hospital, Malmö, Sweden; nontypable Haemophilus influenzae (NTHi): 2019 (wild type), kindly provided by Edward Swords, University of Iowa, USA; 1128 (wild type) and 1128f-(adhesin P5 deletion mutant unable to bind to CEACAM1), both kindly provided by Lauren O. Bakaletz, The Research Institute at Nationwide Children's Hospital and The Ohio State University College of Medicine, USA. For infection, bacteria were freshly grown over night at 37°C, 5% CO 2 on Columbia Agar or Chocolate Agar, respectively (both BD Biosciences). Bacteria were incubated to exponential growth (mid-log phase) in liquid Brain Heart Infusion (BHI) broth at 210 rpm, 37°C (BHI, BD Biosciences; for NTHi supplemented with 10 μg/ml hemin, Sigma Aldrich, and 10 μg/ml NAD, MP Biomedicals). Bacteria were harvested by centrifugation and re-suspended in PBS. Colony forming units (cfu) were determined by measurement of optical densities (M. catarrhalis: OD 405 nm = 0.3 correlates with 5×10 7 cfu/ml; NTHi: OD 600 nm = 0.1 correlates with 10 8 cfu/ml). Cells were infected for 24 h at a multiplicity of infection (MOI) of 5 (Moraxella catarrhalis) or 100 (NTHi). Optimal MOIs were determined in pilot experiments by analysis of maximal induction of interleukin 8 secretion in the absence of cytotoxic effects (data not shown).