Xian C. Li

IL-9 and Th9 cells: progress and challenges

T(h)9 cells are a new subset of helper T cells, and the signature cytokine for T(h)9 cells is IL-9. Both T(h)9 cells and T(h)9 products are implicated in multiple disease settings. Thus, a clear understanding of how T(h)9 cells are induced and controlled is an important and clinically relevant issue. There are different molecular pathways identified thus far in the induction of T(h)9 cells, and activation of such diverse pathways requires integration of signals from TGF-β and IL-4 cytokine receptors as well as costimulatory molecules. These signals converge on the induction of multiple transcription factors that collectively drive the development of T(h)9 cells.

ROCK inhibition impedes macrophage polarity and functions.

Macrophages play an important role in immune responses including allograft rejection and they are one of the potential targets of anti-rejection therapies in organ transplantation. Macrophage alloreactivity relies on their phenotype/polarity, motility, phagocytosis and matrix degradation, which in turn depend on proper functioning of actin cytoskeleton and its regulators, the small GTPase RhoA and its downstream effector the Rho-associated protein kinase (ROCK). Several laboratories showed that administration of ROCK inhibitor Y-27632 to the graft recipient inhibits chronic rejection or rodent cardiac allografts. Here we studied the effect of Y-27632 on mouse peritoneal macrophage structure, polarity and functions in in vitro assays. We show that Y-27632 inhibitor affects macrophage phenotype/polarity, phagocytosis, migration, and matrix degradation. These novel findings suggest that the impediment of macrophage structure and function via interference with the RhoA/ROCK pathway has a potential to be therapeutically effective in organ transplantation.

Mouse macrophage polarity and ROCK1 activity depend on RhoA and non-apoptotic Caspase 3.

The macrophages have different subtypes with different functions in immune response and disease. It has been generally accepted that M1 macrophages are responsible for stimulation of immune system and inflammation while M2 macrophages play a role in tissue repair. Irrespective of the type, macrophage functions depend on actin cytoskeleton, which is under the control of small GTPase RhoA pathway and its downstream effector ROCK1. We generated RhoA-deleted macrophages and compared the effect of RhoA deletion on M0, M1 and M2 macrophage phenotype. Our studies showed that, unexpectedly, the RhoA deletion did not eliminate macrophage ROCK1 expression and increased ROCK1 activity. The RhoA deletion effect on macrophage phenotype, structure and polarity was different for each subtype. Moreover, our study indicates that the up-regulation of ROCK1 activity in RhoA-deleted macrophages and macrophage phenotype/polarity are dependent on non-apoptotic Caspase-3 and are sensitive to Caspase-3 inhibition. These novel findings will revise/complement our understanding of RhoA pathway regulation of cell structure and polarity.

The newly found functions of MTOC in immunological response.

The MTOCs are present in all eukaryotic cells. In animal somatic cells, the MTOC function is played by a centrosome, which contains centrioles and PCM. The traditional view is that the MTOC is responsible for the organization of microtubular structures (the intracellular network, cilia, and flagella) in interphase cells, and the formation of the mitotic and meiotic spindle apparatus which is required for the partitioning of chromosomes in dividing cells. Recent evidence suggests that MTOC also plays a key role in the engagement of molecular motors, directional transport of granules, and polarization of subcellular structures and molecules. All of these functions are crucial for targeted cytotoxicity and the regulation of immune cells. In this review, we focus on the ultrastructural and molecular aspects of MTOCs in various aspects of immune cell functions, with specific emphasis on the formation of the IS and targeted cell killing.

Dissonant response of M0/M2 and M1 bone-marrow-derived macrophages to RhoA pathway interference

Macrophages have a multitude of functions in innate and adaptive immune response and organ and tissue homeostasis. Many experimental studies are performed on bone-marrow-derived macrophages differentiated in vitro into M1 (inflammatory) and M2 (anti-inflammatory) subtypes that express different molecular markers pertaining to their prospective functions. Macrophage phenotype, polarity and functions depend on the actin cytoskeleton, which is regulated by small GTPase RhoA, its downstream effector ROCK, and non-apoptotic Caspase-3. We generated transgenic mice with the macrophage-specific deletion of RhoA and compared the effect of Rho pathway interference (RhoA deletion and ROCK and Caspase-3 inhibition) on the phenotype, polarity and expression of subtype-specific molecular markers of bone-marrow-derived M0, M1 and M2 macrophages. We show that M0 and M2 macrophages have a radically different phenotype and polarity from M1 macrophages, and that this is mirrored in dissonant response to RhoA pathway interference. The RhoA pathway interference induces extreme elongation (hummingbird phenotype) of M0 and M2 but not M1 macrophages and inhibits the expression of M2-specific but not M1-specific molecular markers. These dramatic differences in the response of M0/M2 versus M1 macrophages to the same molecular cues ought to be important considerations in the interpretation of experimental data and therapeutic use of bone-marrow-derived macrophages.

Macrophage/monocyte-specific deletion of Ras homolog gene family member A (RhoA) downregulates fractalkine receptor and inhibits chronic rejection of mouse cardiac allografts

BACKGROUND The cellular and molecular mechanisms of chronic rejection of transplanted organs remain obscure; however, macrophages are known to play a critical role in the injury and repair of allografts. Among multiple factors influencing macrophage infiltration to allografts, the fractalkine chemokine (C-X3-C motif) ligand 1(CX3CL1)/chemokine (C-X3-C motif) receptor 1 (CX3CR1) signaling pathway and actin cytoskeleton, which is regulated by a small guanosine-5׳-triphosphatase Ras homolog gene family member A (RhoA), are of the utmost importance. To define the role of macrophage/RhoA pathway involvement in chronic rejection, we generated mice with monocyte/macrophage-specific deletion of RhoA. METHODS Hearts from BALB/c (H-2d) donors were transplanted into RhoAflox/flox (no Cre) and heterozygous Lyz2Cre+/-RhoAflox/flox recipients treated with cytotoxic T-lymphocyte-associated protein 4 immunoglobulin to inhibit early T-cell response. Allografts were assessed for chronic rejection and monocyte/macrophage functions. RESULTS The deletion of RhoA inhibited macrophage infiltration, neointimal hyperplasia of vasculature, and abrogated chronic rejection of the allografts. The RhoA deletion downregulated G protein-coupled fractalkine receptor CX3CR1, which activates the RhoA pathway and controls monocyte/macrophage trafficking into the vascular endothelium. This in turn promotes, through overproliferation and differentiation of smooth muscle cells in the arterial walls, neointimal hyperplasia. CONCLUSIONS Our finding of codependence of chronic rejection on monocyte/macrophage CX3CR1/CX3CL1 and RhoA signaling pathways may lead to the development of novel anti-chronic rejection therapies.

The phenotype of peritoneal mouse macrophages depends on the mitochondria and ATP/ ADP homeostasis

Different macrophage subtypes have different morphologies/shapes and functions. Naïve M0 macrophages are elongated. Pro-inflammatory M1 that produce the bactericidal molecule iNos are round. Anti-inflammatory M2 macrophages that produce the pro-healing enzyme Arg-1 are highly elongated. We showed previously that the morphologies of M0 and M2 but not M1 macrophages are RhoA-dependent. Macrophage-specific deletion of RhoA causes the extreme elongation (hummingbird phenotype) of M0 and M2 but not M1 macrophages. The M1 and M2 macrophages also differ in their metabolic status. Here, we studied the effect of the oxidative phosphorylation inhibitors, antimycin A and oligomycin A, at a suboptimal dose, which depolarizes mitochondria but does not eliminate mitochondrial functions, on the mitochondria/energy production and phenotype of wild-type and RhoA-deleted M0, M1 and M2 peritoneal mouse macrophages. We found that, while untreated M1 macrophages had the lowest and the M2 had the highest level of ATP the ATP/ADP ratio was nearly identical between M0, M1 and M2 macrophages. Inhibitor treatment resulted in approximately 60% increase in ATP level and ATP/ADP ratio in M0 and M2 macrophages, and decrease in the level of filamentous (F) actin, and these changes correlated with a drastic shortening/tail retraction of M0 and M2 macrophages, and decreased expression of Arg-1 in M2 macrophages. The treatment of M1 macrophages caused only a 30% increase in the ATP level and ATP/ADP ratio, and while it did not affect the shape of M1 macrophages, it increased the production of iNos. This indicates that the maintenance of mouse macrophage phenotypes depends on mitochondrial function and ATP/ADP homeostasis.

Rho-specific Guanine nucleotide exchange factors (Rho-GEFs) inhibition affects macrophage phenotype and disrupts Golgi complex

Macrophages play crucial role in tissue homeostasis and the innate and adaptive immune response. Depending on the state of activation macrophages acquire distinct phenotypes that depend on actin, which is regulated by small GTPase RhoA. The naive M0 macrophages are slightly elongated, pro-inflammatory M1 are round and M2 anti-inflammatory macrophages are elongated. We showed previously that interference with RhoA pathway (RhoA deletion or RhoA/ROCK kinase inhibition) disrupted actin, produced extremely elongated (hummingbird) macrophage phenotype and inhibited macrophage movement toward transplanted hearts. The RhoA function depends on the family of guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP and activate RhoA that reorganizes actin cytoskeleton. Using actin staining, immunostaining, Western blotting, flow cytometry and transmission electron microscopy we studied how a direct inhibition of Rho-GEFs with Rhosin (Rho GEF-binding domain blocker) and Y16 (Rho GEF DH-PH domain blocker) affects M0, M1 and M2 macrophage phenotypes. We also studied how Rho-GEFs inhibition and RhoA deletion affects organization of Golgi complex that is crucial for normal macrophage functions such as phagocytosis, antigen presentation and receptor recycling. We found that GEFs inhibition differently affected M0, M1 and M2 macrophages phenotype and that GEFs inhibition and RhoA deletion both caused changes in the ultrastructure of the Golgi complex. These results suggest that actin/RhoA- dependent shaping of macrophage phenotype has different requirements for activity of RhoA/GEFs pathway in M0, M1 and M2 macrophages, and that RhoA and Rho-GEFs functions are necessary for the maintenance of actin-dependent organization of Golgi complex.

Macrophages and RhoA Pathway in Transplanted Organs

RhoA is a small GTPase that, via its downstream effectors, regulates a variety of cell functions such as cytokinesis, cell migration, vesicular trafficking, and phagocytosis. As such the RhoA pathway is also pivotal for proper functioning of immune cells including macrophages. By controlling actin cytoskeleton organization, RhoA pathway modulates macrophages polarity and basic functions: phagocytosis, migration, and extracellular matrix degradation. Numerous studies indicate that macrophages are very important effectors contributing to acute and chronic rejection of transplanted organs. In this review we discuss the role of RhoA pathway in governance of macrophages functions in terms of transplanted organs.