The roles of phosphocholine expressed on NTHi bacteria and host CRP in acute otitis media

Thesis

Otitis Media (OM) is inflammation of the middle ear (ME). Non-typeable Haemophilus influenzae (NTHi) is one of the leading otopathogens causing OM. Phosphocholine (PCho) on the NTHi lipopolysaccharide influences host-pathogen interaction. C-Reactive Protein (CRP), an acute phase protein, recognizes PCho and can mediate bacterial killing. However, some strains of NTHi survive well in the presence of CRP. We aimed to study the interaction of CRP with NTHi to understand its role in bacterial survival in acute OM (AOM). NTHi can efficiently infect the Junbo mouse, a characterised model of chronic and AOM. We developed a CRP ELISA that showed PCho off heptose III on NTHi surface enhanced CRP binding compared to PCho off heptose I. This increased interaction made the NTHi more susceptible to serum bactericidal killing. Gene expression analysis of macrophages infected with NTHi 375 strains confirmed that PCho influenced inflammatory cytokine gene expression whereby having no PCho or PCho off heptose III drove a M1 macrophage response. We inoculated the Junbo mouse with NTHi 375 strains and studied infection and immune responses at days 1-, 3- and 7- post NTHi inoculation. The highest levels of acute response were measured at 1-day post infection. A greater than 90% infection rate of the Junbo mouse was measured for NTHi bacteria expressing PCho compared to the PCho mutant, which had an infection rate of approximately 50%. Immune cell profiling of the innate and adaptive cells in the middle ear fluid (MEF), blood and nasal associated lymphoid tissue showed interesting cellular changes in response to PCho presence and position. NTHi promoted anti-inflammatory immune cell types in the MEF by activating M2-like macrophages and regulatory T cells. NTHi expressing PCho off heptose I survive in the MEF of the Junbo mouse even in the presence of a T cell cytokine/chemokine response. Together, these data confirm that the presence of CRP in the Junbo mouse MEF may not contribute to NTHi killing because it does not bind well to PCho off specific heptoses on the bacterial surface. Thus, NTHi bacteria can survive in the presence of CRP dependent upon the presence and position of PCho on their surface-expressed LPS.

Authors: Bharj, GK

• DOI: 10.5287/ora-2arvzaj5e
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: regulatory T cells; acute otitis media; innate; non-typeable haemophilus influenzae; middle ear; macrophages
• Subjects: Acute otitis media; Bacteria; Immunology