Activation of ILC2s through constitutive IFNγ signaling reduction leads to spontaneous pulmonary fibrosis.

Pulmonary fibrosis (PF), a condition characterized by inflammation and collagen deposition in the alveolar interstitium, causes dyspnea and fatal outcomes. Although the bleomycin-induced PF mouse model has improved our understanding of exogenous factor-induced fibrosis, the mechanism governing endogenous factor-induced fibrosis remains unknown. Here, we find that Ifngr1-/-Rag2-/- mice, which lack the critical suppression factor for group 2 innate lymphoid cells (ILC2), develop PF spontaneously. The onset phase of fibrosis includes ILC2 subpopulations with a high Il1rl1 (IL-33 receptor) expression, and fibrosis does not develop in ILC-deficient or IL-33-deficient mice. Although ILC2s are normally localized near bronchioles and blood vessels, ILC2s are increased in fibrotic areas along with IL-33 positive fibroblasts during fibrosis. Co-culture analysis shows that activated-ILC2s directly induce collagen production from fibroblasts. Furthermore, increased IL1RL1 and decreased IFNGR1 expressions are confirmed in ILC2s from individuals with idiopathic PF, highlighting the applicability of Ifngr1-/-Rag2-/- mice as a mouse model for fibrosis research.

ASP7266, a Novel Antibody against Human Thymic Stromal Lymphopoietin Receptor for the Treatment of Allergic Diseases.

Thymic stromal lymphopoietin (TSLP), positioned at the top of the inflammatory cascade, is a key regulator that enhances allergic inflammatory responses by activating T helper type 2 cells, Group 2 innate lymphoid cells (ILC2), and myeloid dendritic cells (mDCs) via the TSLP receptor (TSLPR). We evaluated the inhibitory effects of ASP7266, a novel recombinant fully human IgG1 monoclonal antibody against TSLPR, on TSLP signaling and inflammation. The inhibitory effects of ASP7266 and the control antibody tezepelumab on TSLP and TSLPR interactions were investigated using a proliferation assay with TSLP stimulation and a chemokine production assay. The pharmacological effects of ASP7266 were investigated by examining differentiation of naive CD4+ T cells, ILC2 cytokine production, and ascaris extract-induced skin allergic reaction in cynomolgus monkeys. ASP7266 potently inhibited TSLP-induced cell proliferation and C-C motif chemokine ligand 17 production. Furthermore, ASP7266 inhibited TSLP-stimulated mDC-mediated naive CD4+ T-cell differentiation and interleukin 5 production by lineage-negative peripheral blood mononuclear cells, which can be considered ILC2 in vitro. In sensitized monkeys, ASP7266 completely suppressed ascaris extract-induced allergic skin reactions. Based on these results, ASP7266, a novel human therapeutic antibody against TSLPR, is a potential therapy for patients with allergic diseases. SIGNIFICANCE STATEMENT: TSLP, positioned at the top of the inflammatory cascade, plays a key role in various allergic diseases, including asthma, chronic rhinosinusitis with nasal polyposis, and atopic dermatitis. Here we show that the anti-TSLPR antibody ASP7266 exhibited excellent pharmacological activity in preclinical studies. Therefore, ASP7266 has the potential to be a promising treatment option for patients with allergic disorders.

Mechanism analysis of long-term graft survival by monocarboxylate transporter-1 inhibition.

BACKGROUND: Monocarboxylate transporter (MCT)-1, a member of a family of molecules, transports monocarboxylates such as lactate. Inhibiting MCT-1 leads to long-term graft survival in rodent heart transplantation and induces tolerance. We evaluated an MCT-1 inhibitor, AS2495674, in a rat heart transplant model and analyzed its underlying mechanism. METHODS: AS2495674 was tested on rat lymphocytes to determine its effect on lactate accumulation, proliferation, and immunoglobulin production. The effect of AS2495674 on graft survival was tested on the Brown Norway to Lewis rat strain combination with a second heart transplantation to test donor-specific suppression. Histology and ex vivo analyses were done to examine the AS2495674 effects on the immune response. RESULTS: In vitro, AS2495674 resulted in lactate accumulation, inhibited lymphocyte proliferation, and prevented immunoglobulin production. AS2495674 induced long-term allograft survival with little evidence of chronic rejection and induced donor-specific suppression. Evaluation of the allograft and peripheral T lymphocytes from the AS2495674 group compared with that of vehicle showed (1) decreased donor-specific T lymphocyte response, (2) more forkhead box P3+ (Foxp3+) and CD45RA+ cells in the allograft, (3) higher gene expression of chemokines and chemokine receptors in the allograft, and (4) preferential inhibition of Foxp3(-) cells with little or no effect on Foxp3+ cells. CONCLUSIONS: AS2495674 prevents acute rejection, reduces features of chronic rejection, and induces tolerance. Our data suggest that the mechanism of AS2495674 involves generating a tolerogenic graft environment by preferentially targeting T effector cells while sparing the generation of T regulatory cells.

Species selectivity of small-molecular antagonists for the CCR5 chemokine receptor.

The species selectivity of four structurally different compounds, SCH-351125, E-913, TAK-779 and UK-427857 has been examined using cloned human, rhesus, and mouse CCR5 receptors. SCH-351125 and E-913 potently inhibited the binding of [125I]-CCL3 to human CCR5, but their inhibitory activities against rhesus CCR5 were more than 10-fold weaker. In contrast, TAK-779 and UK-427857 inhibited binding to human and rhesus CCR5 with similar potency. The inhibitory activities of all four compounds against mice CCR5 receptors were weak. The inhibitory activities of the CCR5 antagonists in the [125I]-CCL3 binding assay agreed well with those induced by CCL3 in the intracellular calcium ([Ca(2+)]i) elevation assay. Mutational analysis of the human CCR5 receptor showed that its Ile198 component plays a critical role in the inhibitory activities of both SCH-351125 and E-913, but not that of TAK-779 or UK-427857. These results provide a structural basis for understanding how specific antagonists interact with CCR5, and will aid the process of creating new, improved CCR5 antagonists.

Structural basis for the interaction of CCR5 with a small molecule, functionally selective CCR5 agonist.

The chemokine receptor CCR5 is an attractive target for HIV-1 drug development, as individuals whose cells lack surface CCR5 expression are highly resistant to HIV-1 infection. CCR5 ligands, such as CCL5/RANTES, effectively inhibit HIV-1 infection by competing for binding opportunities to the CCR5 and inducing its internalization. However, the inherent proinflammatory activity of the chemotactic response of CCR5 ligands has limited their clinical use. In this study, we found that a novel small molecule, functionally selective CCR5 agonist, 2,2-dichloro-1-(triphenylphosphonio)vinyl formamide perchlorate (YM-370749), down-modulates CCR5 from the cell surface without inducing a chemotactic response and inhibits HIV-1 replication. In molecular docking studies of YM-370749 and a three-dimensional model of CCR5 based on the rhodopsin crystal structure as well as binding and functional studies using various CCR5 mutants, the amino acid residues necessary for interaction with YM-370749 were marked. These results provide a structural basis for understanding the activation mechanism of CCR5 and for designing functionally selective agonists as a novel class of anti-HIV-1 agents.

Transgenic mouse expressing human CCR5 as a model for in vivo assessments of human selective CCR5 antagonists.

The species selectivity of receptor antagonists often hinders their preclinical assessment in vivo. In order to evaluate human selective CC chemokine receptor type 5 (CCR5) antagonists in vivo, we generated human CCR5 transgenic mice that expressed the transgene on both peripheral blood leukocytes as well as thymocytes. The selective CCR5 ligand CC chemokine ligand 4 (CCL4)/macrophage inflammatory protein (MIP)-1beta induced the chemotaxis of thymocytes that had been derived from the transgenic mice, but not from littermate mice, suggesting that the human CCR5 expressed in the transgenic mice were functional. The binding of the human CCR5 specific antibody 45531 to peripheral blood granulocytes from the transgenic mice was inhibited by human selective CCR5 antagonist SCH-351125. Using this antibody, we developed an ex vivo assay system that is suitable for the evaluation of a test compound's ability to occupy the human CCR5 receptor on mouse peripheral blood leukocytes. This transgenic mouse model is useful for estimating the pharmacodynamics of human selective CCR5 antagonists in vivo.