GPR84 was recently isolated and characterized from human B cells (Wittenberger et al., 2001, J Mol Biol, 307, 799-813) as the result of an expressed sequence tag data mining strategy, and also using a degenerate primer reverse transcriptase-polymerase chain reaction (RT-PCR) approach aimed to identify novel chemokine receptors expressed in neutrophils (Yousefi 2001).
GPR84 (also known as EX33) remained an orphan GPCR until the identification of medium-chain fatty acids (MCFA) with carbon chain lengths of 9-14 as ligands for this receptor (Wang 2006). GPR84 was described to be activated by capric acid, undecanoic acid and lauric acid with potencies of 5 μM, 9 μM and 11 μM, respectively. Two small molecules were also described to have some GPR84 agonist activity: 3,3′di indolylmethane (DIM) (Wang 2006) and Embelin (WO 2007/027661).
GPR84 expression has been shown to be expressed in immune cells including, but not limited to, polymorphonuclear leukocytes (PMN), neutrophils, monocytes, T cells, and B cells. (Wang 2006, Yousefi 2001, Venkataraman 2005, WO2007/027661). Higher levels of GPR84 were measured in neutrophils and eosinophils than in T-cells and B-cells. GPR84 expression was demonstrated in tissues that may play a role in the propagation of the inflammatory response such as lung, spleen, bone marrow.
For example, in a recent review, Du Bois reported the current status of therapies for lung interstitial diseases, such as idiopathic pulmonary fibrosis (IPF). There are almost 300 distinct injurious or inflammatory causes of interstitial lung disease that can result in diffuse lung scarring, and the initial stages of the IPF pathology are very likely to involve inflammation (Du Bois 2010), and combination therapies involving anti-inflammatory treatment could be advantageously used.
The expression of GPR84 was highly up-regulated in monocytes/macrophages upon LPS stimulation (Wang 2006).
GPR84 knock-out (KO) mice are viable and indistinguishable from wild-type littermate controls (Venkataraman 2005). The proliferation of T and B cells in response to various mitogens is reported to be normal in GPR84-deficient mice (Venkataraman 2005). T helper 2 (Th2) differentiated T cells from GPR84 KO secreted higher levels of IL4, IL5, IL13, the 3 major Th2 cytokines, compared to wild-type littermate controls. In contrast, the production of the Th1 cytokine, INFγ, was similar in Th1 differentiated T cells from GPR84 KO and wild-type littermate (Venkataraman 2005).
In addition, Capric acid, undecanoic acid and lauric acid dose-dependently increased the secretion of interleukin-12 p40 subunit (IL-12 p40) from RAW264.7 murine macrophage-like cells stimulated with LPS. The pro-inflammatory cytokine IL-12 plays a pivotal role in promoting cell-mediated immunity to eradicate pathogens by inducing and maintaining T helper 1 (Th1) responses and inhibiting T helper 2 (Th2) responses. MCFAs, through their direct actions on GPR84, may affect Th1/Th2 balance.
Berry et al. identified a whole-blood 393-gene transcriptional signature for active tuberculosis (TB) (Berry 2010). GPR84 was part of this whole-blood 393-gene transcriptional signature for active TB indicating a potential role for GPR84 in infectious diseases.
GPR84 expression was also described in the microglia, primary immune effector cells of the central nervous system (CNS) from myeloid-monocytic origin (Bouchard 2007). As observed in peripheral immune cells, GPR84 expression in microglia was highly inducible under inflammatory conditions such as TNFα and IL1 treatment but also notably endotoxemia and experimental autoimmune encephalomyelitis (EAE), suggesting a role in neuro-inflammatory processes. Those results suggest that GPR84 would be up-regulated in CNS not only during endotoxemia and multiple sclerosis, but also in all neurological conditions in which TNFα or IL1b pro-inflammatory cytokines are produced, including brain injury, infection, Alzheimer's disease (AD), Parkinson's disease (PD).
Neutrophil chemotaxis to sites of infection is crucial for the host's innate immune defense (Kubes 2002). Unregulated directional motility can lead to disorders such as chronic inflammation (Woolhouse 2002) and other disorders. Chemotaxis, migration directed by gradients of chemotactic agents, is a complex process that involves extracellular and intracellular signaling, regulation of the cytoskeleton, and interactions between cells and the extracellular matrix (Chung 2001). The migration of neutrophils is guided by a number of common chemotactic agents such as the bacterial product formyl-Met-Leu-Phe (fMLP) and host derived products like interleukin 8 (IL-8) and leukotriene B4 (LTB4) (Foxman 1997; Foxmann 1999; Baggiolini 1998).
Neutrophils sense the presence of chemotactic agents through the use of G-protein coupled receptors. This class of receptors is huge, representing a significant portion of the genome. The main classes of chemotaxis receptors are triggered by formyl peptides—formyl peptide receptors (FPR), chemokines—chemokine receptors (CCR or CXCR) and leukotrienes—leukotriene receptors (BLT).
It was suggested that GPR84 might be involved in the regulation of chemotaxis (Yousefi 2001). However, the authors proposed that GPR84 plays role in the classical chemotaxis pathway driven by IL8 and fMLP. As is demonstrated further herein and exemplified by the present invention, the role of GPR84 in neutrophil chemotaxis is highly specific and is not linked to the classical chemotaxis pathway, in fact GPR84 antagonists have no effect on the chemotaxis stimulated by the agents proposed in the publication by Yousefi.
The present invention provides evidence that GPR84 is involved in chemotaxis of immune cells, but not via one of the classic chemotaxis pathways described above. The present invention provides a novel assay for the identification of compounds that modulate GPR84 activity, in particular inhibit GPR84-agonist stimulated chemotaxis, and further a method for the identification of compounds useful in the prevention and/or treatment of inflammatory conditions (for example inflammatory bowel diseases (IBD), rheumatoid arthritis, vasculitis, lung diseases (e.g. chronic obstructive pulmonary disease (COPD) and lung interstitial diseases (e.g. idiopathic pulmonary fibrosis (IPF)), neuroinflammatory conditions, infectious diseases, autoimmune diseases and/or diseases involving impairment of immune cell functions.
USPTO Backgrounds
USPTO Backgrounds