ORs are classically regarded as smell sense receptors that are predominantly expressed in the olfactory epithelium within the nasal cavity 
. However, various studies have reported that ORs may also be expressed in non-olfactory tissues, where the roles of these ectopically expressed receptors remain unclear 
. In the present study, we demonstrate that a group of ORs (OR65, OR272, OR352, OR446, OR568, OR622, OR657 and OR1014) are expressed in mouse airway tissue and pulmonary macrophages ( and ). We further demonstrate that IFN-γ and LPS stimulation act synergistically to enhance the expression of this group of ORs (). We further demonstrate the presence of OR622 protein in pulmonary macrophages by immunofluorescence (). Our data suggest that innate immune signals linked to the control of infection (IFN-γ and LPS) may regulate OR expression and suggest that these receptors may play a role in host defence responses, regulating macrophage function.
Although there are a limited number of studies on the role of ORs in immunity, a previous study demonstrated a role for odorants in activation of pulmonary immune response. Intranasal application of odorants derived from female mice stimulated IL-1β-independent activation of lung immunity and leukocyte mobilization in the lungs of male mice 
. In our study, stimulation of ORs with the odorant agonists, octanal, amyl acetate and diaminopimelic acid, increased MCP-1 production in pulmonary macrophages ( and ). The effect appears to be selective for MCP-1, as no changes were observed in other major macrophage inflammatory mediators including NOS2, ARG1, FIZZ1, YM1, IL-10, MIP-1, MCP-2, MCP-3, KC, TNF-α, IFN-γ and IL-12 (Figure S2
. Furthermore, supernatants from octanal-stimulated macrophages (with exposure to γ/LPS) significantly enhanced the migration of peritoneal-derived macrophages, at levels that were comparable to the levels observed by the addition of 10 ng/ml MCP-1 protein (). As MCP-1 predominantly acts on macrophages, these data collectively suggest that the activation of ORs in pulmonary macrophages leads to increased production of MCP-1, which further amplifies macrophage migration during immune responses 
Pulmonary macrophages are the most abundant innate immune cells in the lung and they are critical for maintaining pulmonary homeostasis, contributing to the clearance of foreign substances, elimination of infectious agents through phagocytosis and regulating aspects of innate and adaptive immune responses 
. Broadly, macrophages can be classified into M1 and M2 subtypes based on their expression of specific markers 
. M1 macrophages are associated with T helper type 1 (Th1) immune responses and contribute to the clearance of intracellular pathogens, killing of tumour cells and removal of tissue debris 
. By contrast, M2 macrophages are associated with T helper type 2 (Th2) immune responses that underpin parasite eradication, suppression of inflammation and tissue remodelling 
. We investigated whether octanal stimulation of ORs influences polarization of macrophages to specific phenotypes and phagocytosis of NTHi bacteria. Interestingly, octanal stimulation of bone marrow derived macrophages did not alter their M1/M2 polarisation phenotype. Furthermore, exposure of pulmonary macrophages to octanal did not alter the rate of phagocytosis or clearance of NTHi by pulmonary macrophages. These results imply that the roles of ORs expressed on macrophages are to facilitate the recruitment of these cells through the production of MCP-1, rather than directly altering their proinflammatory or innate host defence function.
Interestingly, many bacterial metabolites can act as OR agonists. For example, a recently identified odorant, diaminopimelic acid, is an intermediate product of the peptidoglycan and lysine biosynthetic pathways of bacteria 
. This in conjunction with our data suggests that pulmonary macrophages have the potential to detect bacteria in their surroundings by sensing bacterial metabolites. Similarly, this notion is supported by the findings that P. aeruginosa
-generated acyl-homoserine lactones (quorum-sensing molecules) activate taste receptors in human upper respiratory epithelium 
. This pathogenic bacterium is commonly found in the lungs of patients with cystic fibrosis 
. Although it is not clear at present if these quorum-sensing molecules can stimulate ORs, studies in C. elegans
have shown that nematodes can respond to acyl-homoserine lactones through ORs expressed on their neuron cells 
. These studies suggest that bacterial quorum-sensing molecules not only directly stimulate taste receptors but also have the potential to activate the ORs of mammalian cells. Highly volatile compounds such as acetic acid, ethanol and 4-methylphenol are well-known odorant molecules that have also been identified in the culture of P. aeruginosa
. Interestingly, Fukuda and colleagues have recently shown that acetate (an acetic acid molecule lacking H+
) released by Bifidobacteria
protects the host from pathogenic infection through the activation of intestinal epithelial cells in a mouse model of E. coli
O157 infection 
Our findings demonstrate that stimulation of macrophages with the bacterial bio-product LPS in conjunction with the host defence cytokine IFN-γ synergistically upregulates the expression of ORs. Activation of these ORs with the odorant octanal results in the increased expression and production of MCP-1 (but not other proinflammatory or host defence molecules). Although the fundamental roles of ORs on pulmonary macrophages needs to be further explored, we speculate that the ectopic expression of ORs on macrophages may serve as another pathogen-recognition pathway for the sensing of odorants linked to the metabolic activity of pathogenic bacteria. Thus, OR activation may act to promote the migration and accumulation of macrophages at the site of bacterial infection, where they can be further activated by specific pathogen recognition pathways (e.g. Toll like receptors).