The long-term goal of this research is to identify new therapeutic targets and strategies to minimize acute and chronic inflammation of the mucosa. These disorders affect millions of people in the United States every year. Specifically, we plan to test the novel idea that the formyl peptide receptor (FPR) is associated with early stages of infection, and that insufficient activation of FPR may allow bacterial proliferation and inflammation. The etiology of inflammation of the mucosa likely involves a complex interplay between environmental, genetic and immunoregulatory factors. Some of these factors have been identified, such as proteins involved in recognition of certain bacterial components: For example, mutations in NOD2 and TLR-4 that affect binding of their ligands (bacterial muramyl dipeptide and LPS) have been shown to be associated with some Crohn's disease cases. We believe that a possible additional candidate affecting mucosal immunity in health and disease is FPR. FPR binds bacterial peptides with high affinity and results in neutrophil chemotaxis and bacterial killing through activation of the NADPH oxidase system. The coding region of the FPR1 gene contains six non-synonymous single nucleotide polymorphisms (SNPs) and four synonymous SNPs resulting in >40 different haplotypes in humans. We have already shown that one FPR haplotype is associated with a reduced capacity to detect and respond to a peptide from Mycobacterium avium ssp. paratuberculosis. Since the human mucosa is colonized by 500-1000 bacterial species, differences in FPR function could affect the neutrophils'ability to respond to certain bacterial peptides and therefore impair the innate immune response, resulting in inflammation. To test our hypothesis that certain individuals may be more susceptible to bacterial infection due to a defect in their response to certain bacterial species, we will examine the function of 12 common FPR variants found in 95% of the Caucasian population. These FPR variants will be expressed in a heterologous expression system, and FPR function in response to bacterial peptides from different commensal and pathogenic mucosal bacteria will be examined by measuring cell signaling and chemotaxis. Furthermore, we propose to examine the hypothesis that the SNPs in the coding region of FPR affect protein translation and therefore quantity of receptor in each cell. We will measure the translational efficiency of different FPR haplotypes in a fusion construct with firefly luciferase. The FPR haplotypes to be tested have been selected on the basis of results from SNP-based codon bias and codon pair bias calculations that suggest that certain haplotypes may be either under- or overrepresented. Differences in expression levels will provide additional evidence for a role of codon and codon pair usage in protein translation, and may in part explain the individual variations in FPR expression in humans. If certain FPR variants are found to be altered or defective in their response to bacterial peptides, future genetic screening may be advantageous for disease prevention as well as diagnosis and treatment.
The role of neutrophil formyl peptide receptor in the innate immune defense at the mucosa has not been characterized. We will test our hypothesis that some formyl peptide receptor variants have a defect in their response to certain bacterial species resulting in a delayed immune response. We will also test out hypothesis that certain combinations of single nucleotide polymorphisms in the formyl peptide receptor contain underrepresented codons and codon pairs leading to lower translation efficiencies and reduced receptor expression levels, which may affect the neutrophils'ability to promptly respond to bacterial infection.
| Miettinen, Heini M (2011) Regulation of human formyl peptide receptor 1 synthesis: role of single nucleotide polymorphisms, transcription factors, and inflammatory mediators. PLoS One 6:e28712 |
