Olfactory axons are ensheathed by OECs that are unique in having a presence in both the PNS and the CNS. OECs, because of their close proximity to both olfactory neurons and to the outside environment in the nasal cavity are in a prime position to assist with host immunity against invading pathogens. Attention was drawn to their proposed role in host immunity by data from microarray experiments demonstrating that OECs expressed mRNA of proteins associated with the innate immune system including lysozyme and chemokines such as CXCL1 and MCP1 (Vincent et al. 2005a). Nuclear translocation of NF-κB with subsequent increased expression of Gro1 protein and TLR4 in OECs incubated with E. coli or LPS was also demonstrated (Vincent et al. 2007).
If OECs have an immune function it is possible that they behave as other immune cells such as macrophages. OECs appeared to associate with and internalise fluorescently labelled dead
Micrococcus luteus (Vincent et al. 2005b). This function was investigated further by others who demonstrated that cultured OECs extend pseudopodia towards E. coli (Leung et al. 2008). Using LysoTracker (a fluorescent marker for lysozymes) and transmission electron microscopy Leung and co-workers observed that OECs were capable of phagocytosing E. coli. Co-immunoprecipitation experiments confirmed that the LPS component of E. coli was recognised by TLR4 which was expressed on OECs (Leung et al. 2008). Confocal analysis showing phagocytosis of degenerating OSNs by OECs rather than by macrophages confirm their place as the primary immune cells in the olfactory mucosa (Su et al. 2013a). Selectivity for ability to phagocytose bacterial species was observed in a recent study involving human and mouse OECs and Schwann cells derived from mouse trigeminal nerves and dorsal root ganglia. It was found that the three cells types were able to phagocytose E. coli but that murine OECs and Schwann cells were unable to phagocytose Burkholderia thailandensis.
The authors proposed that this may have implications in the use of OECs in CNS repair therapies (Panni et al. 2013).
Thus, it has been firmly established that OECs can behave like other immune cells such as macrophages and microglia, and can phagocytose pathogens. Induction of chemokines are an important component of the immune response and the discovery that both OSNs and OECs express CX3CL1 provides further support for an immune role for OECs (Ruitenberg et al. 2008).
These data support the notion that OECs possess the cellular machinery to enable them to play a role in innate immunity. Infections via this route are relatively uncommon and powerful endogenous mechanisms for preventing microbial infection must exist but these remain poorly understood. Could theproperties of OECs be one of the reasons that microbial infection via olfactory neurons is uncommon? Much of the research into immune properties of OECs is currently based on cell culture models. No mechanism for killing of microbes has as yet been established and the hypothesised immune role of OECs has not been
demonstrated in vivo. Increased understanding of the functional plasticity of OECs may lead to multiple functions extending beyond their role in the olfactory pathway. Their future developments clinically can only be helped by a better understanding of their cell biology.
Hypothesis and aims of thesis
I hypothesise that OECs form an integral part of the innate immune system and that they work in concert with macrophages and microglia in the olfactory pathway to maintain an effective immune barrier to the brain.
This hypothesis leads to the following questions, which will be tested in the aims of this thesis.
1. Do OECs contribute to the innate immune defence barrier by production of the antimicrobial agent NO?
2. Do OECs constitute part of the additional defence barrier which exists to protect a compromised olfactory pathway?
3. What is the overall immune response to bacterial challenge in the compromised olfactory pathway?
Mitral cell dendrite Glomerular layer OSN axon terminal Cribriform plate Bowman’s gland
OSN cell body Mucous layer OSN cilia
OEC
Olfactory nerve layer OSN axon
Dura
Blood vessel Olfactory epithelium
Lamina propria
Columnar epithelial cell (supporting/ sustentacular cell)
Olfactory bulb
Glomerulus
Figure 1.1. Anatomy of the rat olfactory pathway.
Olfactory ensheathing cells (OECs) ensheath and accompany olfactory sensory neurons (OSNs) as they traverse from the nasal cavity via the lamina propria, through the cribriform plate of the skull and synapse with mitral cells within the glomerular layer of the olfactory bulb. The
dendrites of OSNs terminate in the nasal cavity within the mucus layer, and are in direct contact with the outside environment.
Adapted from Mathison et al. 1998.
Basal cell
Perineural cell Duct
FIGURE 1.2
Figure 1.2. Synthesis of NO.
The synthesis of NO is a 2-step process. L-arginine is converted to Nw-hydroxy-L-arginine, then to L-citrulline and NO (Bredt and Snyder 1989; Palmer et al. 1988a; Palmer and Moncada 1989; Palmer et al. 1988b). Nicotinamide adenine dinucleotide phosphate (NADPH) is an electron donor and several co factors are required including flavin adenine nucleotide (FAD) (Stuehr et al. 1989), flavin mononucleotide (FMN) (Forstermann et al. 1994), tetrahydrobiopterin (BH4) (Tayeh and Marletta 1989), NADPH (Palmer and Moncada
1989), molecular oxygen as a co-substrate (Kwon et al. 1990) and calmodulin.
NO has a short half life and rapidly degrades to NO2- (nitrite) and then NO3- (nitrate) which
can be S-nitrosylated. NO also reacts with O2 to form O2- (superoxide) and ONOO-
(peroxynitrite).
Adapted from Aktan et al, 2004
BH4 FAD FMN FAD FMN NO2-, NO3- S-nitrosylation +O2 O2- ONOO-
FIGURE 1.3
Figure 1.3. Nitric oxide production by macrophages.
Macrophages respond to pro inflammatory cytokines TNF-α, IFN-γ and IL-6 through cytokine receptors expressed on the cell surface. They also respond to immunoglobulin immune complexes and to bacterial cell wall components such as LPS via TLRs. Signalling pathways within the cell are activated leading to nuclear translocation of transcription factors e.g. NF-κB to the cell nucleus and binding to the promoter of genes such as iNOS. NADPH oxidase catalyses the respiratory burst to produce superoxide that then combines with NO forming peroxynitrite. iNOS catalyses the oxidation of L-arginine to L-citrulline and NO. NO participates in a number of reactions including diffusing into the phagosome to mediate antimicrobial killing or, as it is a soluble free radical, diffusing out of the cell into neighbouring cells and tissues to participate in antimicrobial killing, tissue injury inflammation and cytokine/chemokine induction.
Adapted from “Production of nitric oxide by phagocytes via iNOS” poster library Abcam. Cambridge. MA. USA.
IFNγ
Antimicrobial killing, inflammation, tissue injury, chemokine/cytokine induction Immunoglobulin LPS Pro inflammatory cytokines TNF-α, IL-6, IFNγ Cytokine receptor
PATHOGEN MODEL Region of infection and detection methods REFERENCES
S. aureus human OM- nasal swabs and blood-PCR (von Eiff et al. 2001)
S. pneumoniae mouse OM, perineural space-OB - microscopy (Rake 1937) Adeno virus rat OM, OSNs, GL of OB- immunofluorescence (Zhao et al. 1996) Borna Virus rat OM, OB - immunohistochemistry (Morales et al. 1988) Canine distemper virus ferret OM, OSNs, cribriform plate, GmL of OB - immunohistochemistry (Rudd et al. 2006) Herpes Simplex Virus mouse OE, perineural cells, OB - immunofluorescence (Esiri and Tomlinson 1984) Influenza A mouse OM, OSNs, OB - mRNA (Aronsson et al. 2003) Mouse hepatitus virus mouse
OM, periglomerular layer, mitral, GL of OB, cortices,
basal forebrain, amygdala - immunohistochemistry (Barnett et al. 1993a) Neisseria meningitidis mice OM, OSNs, OB, meninges -electron microscopy (Sjolinder and Jonsson 2010) Polio virus monkey OM,OSNs, OB, olfactory tracts – light microscopy (Howe and Bodian 1941) Rabies virus mouse Supporting cells of OE, OB, olfactory tracts - immunofluorescence (Astic et al. 1993) St Louis encephalitis syrian hamster OM,OSNs, OB, olfactory tracts - microscopic mapping (Monath et al. 1983) Sendai virus mouse OM, OSNs, GmL - PCR, immunohistochemistry (Mori et al. 1995)
Semliki forest virus mouse OM, OSNs, OB – light microscopy (Oliver and Fazakerley 1998) Venezuelan equine virus mouse OM, CNS - ultrastructural studies (Ryzhikov et al. 1995a) Yellow fever virus mouse, monkey OM, OSNs, CNS – light microscopy (Findlay GM and LP 1935) Vesicular stomatitis virus mouse OM, supporting cells, basal cells, OSNs, OB, olfactory tracts - immunofluorescence (Plakhov et al. 1995) Balamuthia mandrillaris-Amoebic
meningocephalitis mouse
OM, OE, LP, OSNs,cribriform plate, CNS -
immunohistochemistry (Kiderlen and Laube 2004)