RhoA- and Actin-Dependent Functions of Macrophages from the Rodent Cardiac Transplantation Model Perspective -Timing Is the Essence

Abstract

The small GTPase RhoA, and its down-stream effector ROCK kinase, and the interacting Rac1and and mTORC2 pathways, are the principal regulators of the actin cytoskeleton and actin-related functions in all eukaryotic cells, including the immune cells. As such, they also regulate the phenotypes and functions of macrophages in the immune response and beyond. Here, we review the results of our and other's studies on the role of the actin and RhoA pathway in shaping the macrophage functions in general and macrophage immune response during the development of chronic (long term) rejection of allografts in the rodent cardiac transplantation model. We focus on the importance of timing of the macrophage functions in chronic rejection and how the circadian rhythm may affect the anti-chronic rejection therapies.

Keywords: ROCK; Rac1; RhoA; actin; chronic rejection; circadian rhythm; macrophage; mouse; rat; timing; transplantation.

Figure 1

Actin in receptor recycling and Golgi. (A) An “used” ligand-bound receptor is internalized by endocytosis. The ligand is released in the acidic interior of the late endosome, and sorted into the lysosome-fusing vesicles. The receptors are sorted into the recycling vesicles and return to the cell membrane. The endocytic vesicles may move using actin comets, which are blocked by the GTPase inhibitors. (B) Some surface receptors are also recycled through the Golgi complex, which can exchange components with the endosomal pathway. The Golgi cisternae and budding vesicles are supported and anchored by the actin filaments.

Figure 2

Two modes of tunneling nanotubes (TNTs) formation. The distant cells extend filopodia, which fuse and elongate to form the TNT (upper left panel). The closely apposing cells attach and move outward extending the membrane connection into a TNT (upper right panel). The bottom panel shows two cells connected by TNT, which transports organelles, such as mitochondria and vesicles, between cells.

Figure 3

Podosome structure and function. Microscope image of mouse macrophage stained for actin (red color) shows podosomes arranged into a rosette (left panel). The diagram of the longitudinal section through the podosome (right panel) depicting the actin filament core surrounded by the adhesion molecules such as vinculin, which anchor actin filaments at the membrane. Podosome delivers matrix metalloproteinase enzymes (MMPs) that digest extracellular matrix components such as collagen.

Figure 4

Nuclear actin. The diagram depicts some of the known functions of nuclear actin. The cell nucleus is anchored in q specific place within the cytoplasm by the cellular cytoskeleton containing actin filaments (F actin). The entry of the globular actin (G actin) to the nucleus is facilitated by the karyopherin protein Importin, and the exit is facilitated by the karyoprotein Exportin. Nuclear G actin plays a role in chromatin remodeling and chromatin movement. By regulating the activity of RNA polymerases I, II, III, and transcription factors, it regulates gene expression. It also participates in mRNA processing and DNA repair, and inhibits histone deacetylation. Additionally, nuclear matrix-associated actin plays a structural role in the overall nuclear organization.

Figure 5

Timeline of transplant rejection and therapeutic intervention in the mouse cardiac transplantation model. The acute rejection, which occurs a few days after transplantation depends mainly on the T cells. The immunosuppressive drugs targeting T cells such as CTLA4Ig (in the mouse model) inhibit acute rejection but not the chronic rejection, which relies mainly on the macrophages. Administration of the RhoA pathway inhibitors within the first week of post-transplantation inhibits macrophage recruitment into the graft and inhibits chronic rejection.

Figure 6

Effect of the RhoA pathway inhibition on macrophages and chronic rejection. RhoA is regulated by GEFs. It is also affected by the mTORC2 pathway and reciprocally interacts with Rac1, which in turn affects the mTORC2 pathway. All these pathways regulate actin polymerization and actin-dependent functions of macrophages. After transplantation, the macrophages infiltrate the graft and cause vessel occlusion, fibrosis, and chronic rejection (left panel). Inhibition of RhoA pathway (or co-inhibition of RhoA and mTORC2), or macrophage specific deletion of RhoA, disrupts actin polymerization and actin-dependent functions, causes extreme elongation (hummingbird phenotype), dispersion of the Golgi, and relocation of the nucleus (nu) and mitochondria toward the tail. It also inhibits the expression of CX3CR1 receptors and aggregates the focal adhesions (FA) at the tip of the tail. All these changes prevent macrophage movement into the graft and prevent vessel occlusion and chronic rejection (right panel).