CX3CR1 deficiency exacerbates neuronal loss and impairs early regenerative responses in the target-ablated olfactory epithelium

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Abstract

The olfactory epithelium is a site of sustained adult neurogenesis where olfactory sensory neurons are continuously replaced from endogenous stem/progenitor cells. Epithelial macrophages have been implicated in the phagocytosis of degenerating cells but the molecular mechanisms allowing for their recruitment and activation while maintaining a neurogenic microenvironment are poorly understood. We have previously shown that the chemokine fractalkine (CX3CL1) is expressed by olfactory sensory neurons and ensheathing cells in the olfactory epithelium. In turn, the fractalkine receptor, CX3CR1, is expressed on macrophages and dendritic cells within the olfactory epithelium. We report that a selective cell death of olfactory sensory neurons in the epithelium of CX3CR1-deficient mice via target ablation (i.e. olfactory bulbectomy) results in an exacerbated loss of olfactory sensory neurons compared to wild-type mice. In addition, reduced proliferation of intraepithelial stem/progenitor cells was observed in lesioned CX3CR1-deficient mice, suggesting an impaired regenerative response. Importantly, a lack of CX3CL1-signaling caused increased recruitment of macrophages into the olfactory epithelium, which in turn contained higher levels of pro-inflammatory cytokines (e.g. TNF-α and IL-6) as determined by qPCR. We also present novel data showing that, relative to wild-type, CX3CR1-deficient macrophages have diminished phagocytic activity following stimulation with CX3CL1. Collectively, our data indicate that signaling through the CX3CR1 receptor modulates macrophage activity, resulting in an environment conducive to olfactory sensory neuron clearance and targeted replacement from endogenous stem/progenitor cells.

Introduction

Olfactory sensory neurons (OSNs) in the nasal neuroepithelium are bipolar sensory neurons with ciliated dendritic nerve endings that are in direct contact with the external environment (Farbman and Buchholz, 1992). This cytoarchitectural feature allows for the immediate sampling of olfactory cues from the environment but also creates a vulnerability against pathogens and olfactory toxicants that may enter the nasal cavity. Indeed, OSN lifespan appears to depend on environmental conditions and the death of these cells is a relatively common event in the neuroepithelium (Moulton, 1974, Hinds et al., 1984, Mackay-Sim and Kittel, 1991). To counter continuous OSN degeneration, and thus maintain a receptive field for the sense of smell, the olfactory neuroepithelium has sustained the ability to effectively replace its neurons throughout adult life from a basal stem/progenitor cell compartment (Harding et al., 1977, Graziadei et al., 1979, Leung et al., 2007). Adult-born OSNs successfully regrow an axon into their central nervous system (CNS) target, the olfactory bulb, and restore appropriate connectivity with second-order neurons. Because of its accessibility, ease of manipulation and unsurpassed regenerative potential, the olfactory pathway provides an attractive model to study various aspects of neuronal injury and regeneration.

Tissue macrophages play an important role during injury and subsequent regenerative events in the olfactory neuroepithelium. Macrophage numbers rapidly increase following OSN degeneration and the influx of these phagocytes coincides with the time course of neuronal death (Graziadei et al., 1979, Suzuki et al., 1995, Cowan et al., 2001). In addition to removing degenerating OSNs from the neuroepithelium, macrophage activation and infiltration is also thought to be important for the induction and regulation of regenerative responses from the basal stem/progenitor cell compartment. Appropriately activated macrophages are a rich source of factors that influence neuronal survival and stem/progenitor cell proliferation (e.g. Nan et al., 2001, Borders et al., 2007a). In line with this, macrophage depletion exacerbates neuronal death and impairs regenerative tissue responses (Borders et al., 2007b). However, the molecular mechanisms involved in the cross-talk between neurons and macrophages as well as the regulation of macrophage activation profiles remain largely unclear.

We recently reported that CX3CL1/fractalkine is expressed on OSNs while its receptor, CX3CR1, is expressed on intraepithelial macrophages and putative dendritic cells (Ruitenberg et al., 2008, Vukovic et al., 2010); this allows for direct communication between epithelial neurons and these phagocytic immune cells. An important feature of CX3CL1 is that it is produced in a membrane-bound form (Bazan et al., 1997), from which the chemokine domain can be released through shedding by TACE/ADAM17 and ADAM10 (Garton et al., 2001, Tsou et al., 2001, Hundhausen et al., 2003). ADAM10 is thought to contribute to constitutive cleavage whereas TACE seems to mediate inducible shedding following injury or stress (Hundhausen et al., 2003). Together, these findings suggest that the shedding of CX3CL1 and release of the chemokine module may function as an early neuronal distress signal. Here, we used comparative histological and molecular analysis between wild-type and CX3CR1-deficient mice to test this hypothesis and further elucidate the physiological importance of CX3CL1-signaling in the olfactory neuroepithelium.

Section snippets

OSN death in response to bulbectomy

Immunofluorescent staining for OMP was performed to visualize mature OSN numbers in tissue sections from Cx3cr1+/+ and Cx3cr1gfp/gfp mice at 48 h after unilateral olfactory bulbectomy (OB-X; Figs. 1A–D). As expected, numerous OMP+ cells were found in the intermediate (neuronal) layer of the OE on the uninjured side, i.e. contralateral to olfactory bulbectomy, for both Cx3cr1+/+ and Cx3cr1gfp/gfp mice (Figs. 1A and C). Ipsilateral to the ablated olfactory bulb, a dramatic decline in OMP+ neurons

Discussion

The present study shows that the chemokine receptor CX3CR1 plays an important role in the macrophage response to injury within the primary olfactory pathway by maintaining an environment that reduces secondary cell death and allows for neuronal replacement from endogenous stem/progenitor cells. After bulbectomy, the vast majority of the mature (OMP+) olfactory sensory neurons in the olfactory epithelium dies via retrograde apoptosis (Cowan et al., 2001), resulting in macrophage recruitment and

Experimental animals

A total of 74 C57BL/6J mice (♂/♀; 2–4 months of age) were used in this study. Wild-type (Cx3cr1+/+), heterozygous (Cx3cr1gfp/+) and homozygous knock-in (Cx3cr1gfp/gfp) mice were generated from heterozygous Cx3cr1gfp breeding pairs (Jung et al., 2000). In Cx3cr1gfp mice, the coding sequence for enhanced green fluorescent protein (eGFP) was inserted into the Cx3cr1 coding region via targeted deletion, i.e. rendering the CX3CL1/fractalkine receptor non-functional and placing eGFP under Cx3cr1

Acknowledgments

The authors are grateful to Dr. Peter Mark and Mr. Greg Cozens (UWA) for assistance with quantitative PCR experiments. Work in the laboratory of M.J.R. was financially supported by the Australian Research Council (Discovery Grant DP0774113 to M.J.R.), The School of Biomedical Sciences at The University of Queensland and an Enabling Grant from the office of the DVC(R) of The University of Queensland (to M.J.R.). M.J.R. was additional supported by an ARC Postdoctoral Fellowship (2007–2009;

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