NOD2/RICK-dependent β-defensin 2 regulation is protective for nontypeable Haemophilus influenzae-induced middle ear infection

PLoS One. 2014 Mar 13;9(3):e90933. doi: 10.1371/journal.pone.0090933. eCollection 2014.


Middle ear infection, otitis media (OM), is clinically important due to the high incidence in children and its impact on the development of language and motor coordination. Previously, we have demonstrated that the human middle ear epithelial cells up-regulate β-defensin 2, a model innate immune molecule, in response to nontypeable Haemophilus influenzae (NTHi), the most common OM pathogen, via TLR2 signaling. NTHi does internalize into the epithelial cells, but its intracellular trafficking and host responses to the internalized NTHi are poorly understood. Here we aimed to determine a role of cytoplasmic pathogen recognition receptors in NTHi-induced β-defensin 2 regulation and NTHi clearance from the middle ear. Notably, we observed that the internalized NTHi is able to exist freely in the cytoplasm of the human epithelial cells after rupturing the surrounding membrane. The human middle ear epithelial cells inhibited NTHi-induced β-defensin 2 production by NOD2 silencing but augmented it by NOD2 over-expression. NTHi-induced β-defensin 2 up-regulation was attenuated by cytochalasin D, an inhibitor of actin polymerization and was enhanced by α-hemolysin, a pore-forming toxin. NOD2 silencing was found to block α-hemolysin-mediated enhancement of NTHi-induced β-defensin 2 up-regulation. NOD2 deficiency appeared to reduce inflammatory reactions in response to intratympanic inoculation of NTHi and inhibit NTHi clearance from the middle ear. Taken together, our findings suggest that a cytoplasmic release of internalized NTHi is involved in the pathogenesis of NTHi infections, and NOD2-mediated β-defensin 2 regulation contributes to the protection against NTHi-induced otitis media.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line
  • Cytoplasm / metabolism
  • Ear, Middle / metabolism
  • Endocytosis
  • Gene Expression Regulation
  • Gene Silencing
  • Gentamicins / chemistry
  • Haemophilus Infections / immunology*
  • Haemophilus Infections / microbiology
  • Humans
  • Immunity, Innate
  • Inflammation / immunology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Electron, Transmission
  • Nod2 Signaling Adaptor Protein / metabolism*
  • Otitis Media / immunology*
  • Otitis Media / microbiology
  • Receptor-Interacting Protein Serine-Threonine Kinase 2
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism
  • Signal Transduction
  • Toll-Like Receptor 2 / metabolism
  • beta-Defensins / metabolism*


  • Defb2 protein, mouse
  • Gentamicins
  • Nod2 Signaling Adaptor Protein
  • Nod2 protein, mouse
  • Tlr2 protein, mouse
  • Toll-Like Receptor 2
  • beta-Defensins
  • Receptor-Interacting Protein Serine-Threonine Kinase 2
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ripk2 protein, mouse

Grant support

This work was supported in part by National Institutes of Health National Institute on Deafness and Other Communication Disorders (NIH-NIDCD) grants RO1 DC005025 and R21 DC011862. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.