Olfactory receptor 2 promotes recruitment of monocytes in abdominal aortic aneurysm

P. Schelemei (Köln)¹, F. S. R. Picard (Köln)¹, D. Mehrkens (Köln)², J. Wrobel (Köln)², H. Nemade (Köln)¹, Y. Park (Köln)¹, E. Wagner (Köln)¹, P. Arkenberg (Köln)¹, A. Hof (Köln)³, W. Ibing (Düsseldorf)⁴, H. Schelzig (Düsseldorf)⁴, M. Wagenhäuser (Düsseldorf)⁴, J. Roy (Solna)⁵, M. L. Liljeqvist (Solna)⁵, M. Orrechioni (Augusta)⁶, B. Levkau (Düsseldorf)⁷, N. Gerdes (Düsseldorf)⁸, R. T. Megens (München)⁹, C. Weber (München)⁹, S. Baldus (Köln)¹⁰, M. Mollenhauer (Köln)², H. Winkels (Köln)^2
1Universitätsklinikum Köln Klinik III für Innere Medizin - Experimentelle Kardiologie Köln, Deutschland; ²Herzzentrum der Universität zu Köln Klinik III für Innere Medizin Köln, Deutschland; ³Universitätsklinikum Köln Herzzentrum - Kardiologie Köln, Deutschland; ⁴Universitätsklinikum Düsseldorf Düsseldorf, Deutschland; ⁵Karolinska Institutet Solna, Schweden; ⁶Augusta University Immunology Center of Georgia Augusta, USA; ⁷Universitätsklinikum Düsseldorf Institut für Molekulare Medizin III Düsseldorf, Deutschland; ⁸Universitätsklinikum Düsseldorf Klinik für Kardiologie, Pneumologie und Angiologie Düsseldorf, Deutschland; ⁹LMU Klinikum der Universität München Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten München, Deutschland; ¹⁰Herzzentrum der Universität zu Köln Klinik für Kardiologie, Angiologie, Pneumologie und Internistische Intensivmedizin Köln, Deutschland

Background:

Abdominal aortic aneurysm is a chronic, inflammatory disease defined by localized dilation of the abdominal aorta driven by smooth muscle cell death and fibrosis. Macrophage mediated inflammation is crucial to the progression of AAA, yet their activation and underlying mechanisms are incompletely understood. The olfactory receptor 2 (Olfr2) is a G-protein coupled receptor involved in mediating the sense of smelling. Extra-nasal expression of Olfr2 has been identified on vascular macrophages, but its role in AAA is unknown.

Methods & Results:
Flow cytometric (FACS) analysis of peripheral blood leukocytes of patients (n=15 each) with small (<5cm) and large (>5cm) AAA revealed increased expression of OR6A2 (the human ortholog of Olfr2) on classical, intermediate and non-classical monocytes in patients with large AAA. Staining of human AAA tissue further confirmed an increased frequency of OR6A2 expressing macrophages in AAA compared to aortic control tissue. Additionally, in-silico micro array analysis of human abdominal aortic samples with and without thrombus formation further demonstrated significant upregulation of OR6A2 in AAA with thrombus formation compared to controls.  

We next investigated the dynamics of Olfr2 expression via spectral flow cytometry of aortic mouse leukocytes in WT mice that underwent porcine pancreatic elastase infusion (PPE) into the infrarenal aorta. Olfr2 was specifically upregulated by aortic MHCII⁺CCR2^low monocytes and macrophages at day 7 post AAA induction which was reduced to baseline levels at day 28.
To determine whether Olfr2 plays a functional role in AAA we induced AAA in Olfr2-deficient (KO) and WT control mice. Ultrasound and histological analysis at day 28 post PPE showed marked protection from aortic dilation as well as preserved elastin structure in KO mice. Immunofluorescence and flow cytometry further demonstrated a significant reduction of monocytes and macrophages in KO mice compared to WT. 
Concurrently, pharmacological activation by injection of the Olfr2 agonist octanal into WT mice increased aortic dilation, elastin degradation and macrophage content compared to vehicle treated mice whereas the Olfr2 antagonist citral ameliorated AAA development and aortic inflammation.
To investigate the recruitment of monocytes, expression of chemokine receptors was assessed via FACS. Ly6C^high monocytes of KO mice showed reduced expression of CX3CR1 during AAA development. Furthermore, injection of octanal induced CX3CR1 expression on circulating monocytes at day 7 post PPE compared to vehicle treated mice. Interestingly, OR6A2 expression positively correlated with increased CX3CR1 expression on human classical and non-classical monocytes in AAA patients.
In vitro, isolated bone marrow monocytes of KO mice showed impaired recruitment towards the CX3CR1 ligand CX3CL1 compared to WT monocytes in a chemotaxis assay. Adoptive transfer of labeled WT and KO monocytes into mice with a developing AAA (day 3) confirmed reduced recruitment of KO monocytes into the aorta as assayed by flow cytometry 24h after transfer.
Finally we assessed whether increased CX3CR1 expression might be causative for Olfr2/Octanal exacerbated AAA formation. Indeed, treatment of WT mice with both octanal and a CX3CR1 inhibitor ameliorated aortic dilation compared to mice treated only with octanal.  

Conclusion:
We highlight a novel mechanism by which Olfr2 drives AAA formation by promoting CX3CR1 mediated monocyte recruitment.