Enhanced GITR/GITRL interactions augment IL-27 expression and induce IL-10-producing Tr-1 like cells.

The glucocorticoid-induced TNFR-related (GITR) protein is a coactivating receptor that is constitutively expressed on Treg cells and induced on activated T cells. To better under-stand the role of long-term GITR signaling, we generated a mouse that constitutively expresses GITR ligand (GITRL) on APCs that mimics the physiological distribution of GITRL in vivo. Despite a five-fold expansion of the Treg-cell pool, there is increased activation and depletion of naive T cells in the transgenic (Tg) mice, suggesting that the increased number of Treg cells cannot fully suppress T-cell activation. Interestingly, GITRL Tg mice have multiorgan lymphocytic infiltrates yet display no overt autoimmunity, indicating the existence of a compensatory immunoregulatory mechanism(s). In the spleens and tissue infiltrates ofGITRL Tg mice, we found increased numbers of Foxp3(-) IL-10-producing type 1 regulatory T (Tr-1)-like cells that suppress naïve T-cell proliferation in an IL-10-dependent fashion. Increased IL-27 production from Tg APCs and activation of c-Maf in the Tr1-like cells suggest a possible mechanism for their induction. Our results demonstrate that enhanced GITR/GITRL interactions have a pleiotropic role on the regulation of T-cell responses, which includes promoting the differentiation of Tr-1-like cells, which contribute to the maintenance of peripheral T-cell tolerance.

Imidazotriazines: Spleen Tyrosine Kinase (Syk) Inhibitors Identified by Free-Energy Perturbation (FEP).

There has been significant interest in spleen tyrosine kinase (Syk) owing to its role in a number of disease states, including autoimmunity, inflammation, and cancer. Ongoing therapeutic programs have resulted in several compounds that are now in clinical use. Herein we report our optimization of the imidazopyrazine core scaffold of Syk inhibitors through the use of empirical and computational approaches. Free-energy perturbation (FEP) methods with MCPRO+ were undertaken to calculate the relative binding free energies for several alternate scaffolds. FEP was first applied retrospectively to determine if there is any predictive value; this resulted in 12 of 13 transformations being predicted in a directionally correct manner. FEP was then applied in a prospective manner to evaluate 17 potential targets, resulting in the realization of imidazotriazine 17 (3-(4-(3,4-dimethoxyphenylamino)imidazo[1,2-f][1,2,4]triazin-2-yl)benzamide), which shows a tenfold improvement in activity relative to the parent compound and no increase in atom count. Optimization of 17 led to compounds with nanomolar cellular activity.

Serine protease HTRA1 antagonizes transforming growth factor-ß signaling by cleaving its receptors and loss of HTRA1 in vivo enhances bone formation.

HTRA1 is a member of the High Temperature Requirement (HTRA1) family of serine proteases, which play a role in several biological and pathological processes. In part, HTRA1 regulation occurs by inhibiting the TGF-ß signaling pathway, however the mechanism of inhibition has not been fully defined. Previous studies have shown that HTRA1 is expressed in a variety of tissues, including sites of skeletal development. HTRA1 has also been implicated in the process of bone formation, although the precise manner of regulation is still unknown. This study investigated how HTRA1 regulates TGF-ß signaling and examined the in vivo effects of the loss of HTRA1. We demonstrated that recombinant HTRA1 was capable of cleaving both type II and type III TGF-ß receptors (TßRII and TßRIII) in vitro in a dose-dependent manner, but it did not affect the integrity of TßRI or TGF-ß. Overexpression of HTRA1 led to decreased levels of both TßRII and III on the cell surface but had no effect on TßRI. Silencing HTRA1 expression significantly increased TGF-ß binding to the cell surface and TGF-ß responsiveness within the cell. To examine the role of HTRA1 in vivo, we generated mice with a targeted gene deletion of HTRA1. Embryonic fibroblasts isolated from these mice displayed an increase in TGF-ß-induced expression of several genes known to promote bone formation. Importantly, the loss of HTRA1 in the knockout mice resulted in a marked increase in trabecular bone mass. This study has identified a novel regulatory mechanism by which HTRA1 antagonizes TGF-ß signaling, and has shown that HTRA1 plays a key role in the regulation of bone formation.