title: Expression of cytokine and chemokine genes by human middle ear epithelial cells induced by formalin-killed Haemophilus influenzae or its lipooligosaccharide htrB and rfaD mutants.
abstract: To define the role of nontypeable Haemophilus influenzae (NTHI) lipooligosaccharide (LOS) in the induction of proinflammatory cytokine gene expression during otitis media, we compared the abilities of formalin-killed NTHI strain 2019 and its LOS htrB and rfaD mutants to stimulate human middle ear epithelial (HMEE) cell cytokine and chemokine gene expression and production in vitro. Strain DK-1, an rfaD gene mutant, expresses a truncated LOS consisting of only three deoxy-D-manno-octulosonic acid residues, a single heptose, and lipid A. Strain B29, an isogenic htrB mutant, possesses an altered oligosaccharide core and an altered lipid A. HMEE cells were incubated with formalin-killed NTHI 2019, B29, or DK-1. The supernatants and the cells were collected at 2, 4, 8, and 24 h after stimulation. Expression of genes for the cytokines tumor necrosis factor alpha (TNF-alpha), interleukin lbeta (IL-1beta), and IL-6 and for the chemokines macrophage inflammatory protein 1beta (MIP-1beta), monocyte chemotactic peptide 1 (MCP-1), and IL-8 was quantitated by real-time PCR. NTHI B29 did not significantly stimulate any cytokine or chemokine mRNA expression in HMEE cells. In striking contrast, NTHI 2019 induced up to 105-, 139-, and 187-fold increases in HMEE cell expression of IL-1beta, TNF-alpha, and MIP-1beta, respectively (P < 0.01 [2019 versus B29]). NTHI 2019 also induced upregulation of IL-8, IL-6, and MCP-1 mRNA expression (by 26-, 44-, and 14-fold, respectively [P < 0.05 (2019 versus B29)]). The significant induction of cytokine genes was confirmed by quantitating the secretion of cytokines in culture supernatants with an enzyme-linked immunosorbent assay. There were no significant differences in mRNA expression of IL-8, IL-6, and MCP-1 between the 2019- and DK-1-treated groups. The low levels of gene transcripts observed after incubation of HMEE cells with B29 indicate that products of the disrupted NTHI htrB LOS gene may play a major role in induction of these particular inflammatory mediators.
authors.affiliation: Department of Otolaryngology, College of Medicine and Public Health, The Ohio State University, Columbus 43210, USA.
authors.forename: H H
authors.initials: HH
authors.lastname: Tong
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: M
authors.initials: M
authors.lastname: James
authors.forename: J
authors.initials: J
authors.lastname: Van Deusen
authors.forename: D B
authors.initials: DB
authors.lastname: Welling
authors.forename: T F
authors.initials: TF
authors.lastname: DeMaria
mesh_terms: D000328:Adult; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D018925:Chemokines / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D004432:Ear, Middle / Q000166:cytology* / Q000276:immunology / Q000382:microbiology; D004847:Epithelial Cells / Q000276:immunology* / Q000382:microbiology; D005557:Formaldehyde / Q000494:pharmacology; D006193:Haemophilus influenzae / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D007700:Kinetics; D008070:Lipopolysaccharides / Q000276:immunology*; D009154:Mutation
chemical_list: D018925:Chemokines; D016207:Cytokines; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides; D005557:Formaldehyde; C037213:ADP-L-glycero-D-mannoheptose-6-epimerase; D002238:Carbohydrate Epimerases