Toll-like receptor agonists and antagonists and methods of use thereof
View Patent ↗The present disclosure relates to compositions and methods of modulating inflammatory and immune responses through binding of SAA to TLR2 in a subject (e.g., human, non-human primate, rodent, etc.), and compositions and methods for screening TLR2 agonists and antagonists. In the studies described herein, a potential role of SAA in neutrophilia was investigated and the results demonstrated that SAA is a potent inducer for macrophage secretion of G-CSF, which leads to neutrophilia in mice. Using G-CSF −/− and TLR2 −/− mice, it was found that the SAA-induced neutrophilia is dependent on TLR2-mediated production of G-CSF. Based on direct binding assay and gain-of-function studies in TLR2-transfected cells, SAA was identified as a novel ligand for TLR2 and a link between increased SAA concentration and TLR2-mediated inflammatory responses such as neutrophilia was established. Additional embodiments are disclosed.
1. A method for identifying a TLR2 antagonist comprising:
providing a composition comprising a candidate TLR2 antagonist;
contacting a TLR2 polypeptide comprising a TLR2 extracellular domain with a serum amyloid A (SAA) polypeptide in the presence of the composition; and
determining binding of the TLR2 polypeptide to the SAA polypeptide, wherein a disruption in binding of TLR2 to SAA compared to TLR2 binding to SAA in the absence of the composition indicates the composition is a TLR2 antagonist.
2. The method of claim 1 , wherein the candidate TLR2 antagonist is selected from the group consisting of: a small molecule, a macromolecule, a peptide, and a nonpeptide, wherein the small molecule, macromolecule, peptide, and nonpeptide can be naturally occurring or synthetic.
3. The method of claim 1 , wherein the composition further comprises a pharmaceutically acceptable carrier.
4. The method of claim 1 , wherein the TLR2 polypeptide is expressed on the surface of a cell.
5. The method of claim 4 , wherein the TLR2 polypeptide is expressed from a cDNA encoding full-length TLR2 expressed in the cell.
6. The method of claim 5 , wherein the cell further comprises a nucleic acid encoding a plurality of NF-κB binding sites operably linked to at least one expression control sequence and a reporter gene.
7. The method of claim 6 , wherein the reporter gene is luciferase.
8. The method of claim 6 , wherein determining the binding of the recombinant TLR2 polypeptide to the SAA polypeptide is by detecting expression of the reporter gene.
9. The method of claim 5 , wherein determining the binding of the recombinant TLR2 polypeptide to the SAA polypeptide is by detecting the expression of a SAA-inducible protein produced by the cell.
10. The method of claim 9 , wherein a SAA-inducible protein produced by the cell is a proinflammatory cytokine in the cell.
11. The method of claim 10 , wherein the cytokine is G-CSF.
12. The method of claim 1 , wherein the recombinant polypeptide comprising a TLR2 extracellular domain is fused to an antibody Fc fragment.
13. The method of claim 12 , wherein determining the binding of the recombinant TLR2 polypeptide to the SAA polypeptide is by ELISA assay.
14. The method of claim 2 , wherein the candidate TLR2 antagonist is an antibody.
15. The method of claim 1 , wherein the SAA polypeptide is a recombinant SAA polypeptide.
16. The method of claim 15 , wherein the recombinant SAA polypeptide is fusion protein.