Mechansims of Macrophage Mechanosensation in Collagen Matrix

Pardis Pakshir 1, 2, 3 ; Moien Alizadehgiashi 4 ; Boaz Wong 5 ; Nuno Miranda Coelho 2 ; Christopher McCulloch 2 ; Boris Hinz 1, 2, 3;

1.  Laboratory of Tissue Repair and Regeneration; 2.  Matrix Dynamics Group, Faculty of Dentistry; 3.  Institute of Biomaterials and Biomedical Engineering; 4.  Department of Chemistry, University of Toronto, Toronto, ; 5.  Department of Physiology, University of Western Ontario, London, Ontario, Canada;

Background: Intimate communication between macrophages (Mϕ) and fibroblasts is important for tissue repair after injury and miscommunication can lead to pathological healing and fibrosis. Our previous results show that single contracting fibroblasts generate deformation fields in fibrillar collagen extracellular matrix (ECM) that provide far-reaching physical cues for Mϕ. We found that Mϕ detect and follow local strain rate changes in their substrate but the mechanisms of Mϕ mechanosensing remain elusive.

Hypothesis: Mϕ use collagen binding receptors and stress-activated membrane channels to sense and respond to local deformations in their ECM.

Objective: To elucidate the molecular mechanism and receptors of Mϕ mechanosensation in response to dynamic mechanical cues transmitted through collagen ECM.

Methods: Mouse bone marrow-derived Mϕ were seeded onto fibrillar collagen ECM. To identify potential mechanosensors, we assessed fibrillar collagen receptors: discoidin domain receptors (DDRs), integrins α1β1 and α21β1, using Western blotting and flow cytometry. To study the involvement of these receptors in mechanosensing, Mϕ were incorporated into 3D collagen ECM and subjected to cyclic uniaxial strain. Mϕ were then fixed and stained for cytoskeletal markers of cell migration polarization to identify Mϕ mechano-responses to ECM strain. To mimic force fields produced by single contractile fibroblasts, Mϕ migration was tracked on collagen ECM that was gradually deformed using micromanipulator-controlled microneedles. Both experimental series were performed in presence and absence of collagen receptor and ion channel inhibitors.

Results: Recruitment of α2β1 integrin was upregulated in Mϕ exposed to fibrillar collagen versus non-coated substrates. Upon periodic stretching, Mϕ oriented within collagen gels, indicating mechanosensing. Collagen receptor function-blocking antibodies, most prominently β1 integrin inhibitors, altered Mϕ alignment indicating their functional involvement in Mϕ mechanosensation. Mϕ migrated towards actuated microneedles, which was impaired in the presence of selective ion channel blockers indicating a functional role of specific membrane channels in Mϕ mechano-responses to ECM strain.

Conclusions: Mϕ mechanosensing of strain in collagen ECM is dependent on membrane ion channels and β1 integrins.