Engineering macrophages to control the inflammatory response and angiogenesis

• RAW264.7 cells were transduced with an engineered TLR4 construct (MФ-cTLR4 cells).
• The MФ-cTLR4 cells are activated by the addition of a cell permeable CID drug.
• CID-treated MФ-cTLR4 cells activated TLR4-specific pathways.
• CID-treated MФ-cTLR4 cells displayed M1-like MФ phenotype characteristics.
• MФ-cTLR4 cells could be used as a tool to determine ideal healing conditions.

Macrophage (MΦ) dysregulation is increasingly becoming recognized as a risk factor for a number of inflammatory complications including atherosclerosis, cancer, and the host response elicited by biomedical devices. It is still unclear what roles the pro-inflammatory (M1) MΦ and pro-healing (M2) MΦ phenotypes play during the healing process. However, it has been shown that a local overabundance of M1 MΦs can potentially lead to a chronically inflamed state of the tissue; while a local over-exuberant M2 MΦ response can lead to tissue fibrosis and even promote tumorigenesis. These notions strengthen the argument that the tight temporal regulation of this phenotype balance is necessary to promote inflammatory resolution that leads to tissue homeostasis. In this study, we have engineered pro-inflammatory MΦs, MΦ-cTLR4 cells, which can be activated to a M1-like MΦ phenotype with a small molecule, the chemical inducer of dimerization (CID) drug. The MΦ-cTLR4 cells when activated with the CID drug, express increased levels of TNFα, IL-6, and iNOS. Activated MΦ-cTLR4 cells stay stimulated for at least 48 h; once the CID drug is withdrawn, the MΦ-cTLR4 cells return to baseline state within 18 h. Further, in vitro CID-activated MΦ-cTLR4 cells induce upregulation of VCAM-1 and ICAM-1 on endothelial cells (EC) in a TNFα-dependent manner. With the ability to specifically modulate the MФ-cTLR4 cells with the presence or absence of a small molecule, we now have the tool necessary to observe a primarily M1 MФ response during inflammation. By isolating this phase of the wound healing response, it may be possible to determine conditions for ideal healing.