Role of formyl peptide receptor variants in mucosal innate immune defense

描述(申请人提供)：这项研究的长期目标是确定新的治疗目标和策略，以最大限度地减少粘膜的急性和慢性炎症。在美国，每年有数百万人受到这些疾病的影响。具体地说，我们计划测试这一新想法，即甲酰肽受体(FPR)与感染的早期阶段有关，并且FPR的不充分激活可能会导致细菌增殖和炎症。粘膜炎症的病因可能涉及环境、遗传和免疫调节因素之间的复杂相互作用。其中一些因素已经被确定，例如参与识别某些细菌成分的蛋白质：例如，NOD2和TLR-4的突变已经被证明与一些克罗恩病病例有关，这些突变影响了它们的配体(细菌胞壁二肽和脂多糖)的结合。我们认为在健康和疾病中影响粘膜免疫的另一个可能的候选因素是FPR。FPR以高亲和力结合细菌肽，通过激活NADPH氧化酶系统，导致中性粒细胞趋化和细菌杀灭。FPR1基因的编码区包含6个非同义单核苷酸多态(SNPs)和4个同义SNPs，导致人类40种不同的单倍型。我们已经证明，一种fpr单倍型与检测和响应来自禽分枝杆菌SSP的多肽的能力降低有关。副结核。由于人的粘膜被500-1000种细菌定植，FPR功能的差异可能会影响中性粒细胞对某些细菌肽的反应能力，从而损害先天免疫反应，导致炎症。为了验证我们的假设，即某些人可能由于对某些细菌物种的反应缺陷而更容易受到细菌感染，我们将检查在95%的高加索人口中发现的12种常见的FPR变体的功能。这些fpr变异体将在异源表达系统中表达，并将通过检测细胞信号和趋化作用来检测fpr对来自不同共生和致病黏膜细菌的细菌肽的反应。此外，我们还提出了一种假设，即FPR编码区的SNPs影响蛋白质翻译，从而影响每个细胞中的受体数量。我们将测量不同的FPR单倍型在与萤火虫荧光素酶的融合构建中的翻译效率。将被测试的FPR单倍型是根据基于SNP的密码子偏差和密码子对偏差计算的结果选择的，这些计算表明某些单倍型可能被低估或被过度表达。表达水平的差异将为密码子和密码子对的使用在蛋白质翻译中的作用提供额外的证据，并可能在一定程度上解释人类FPR表达的个体差异。如果发现某些fpr变异体对细菌肽的反应发生改变或缺陷，未来的基因筛查可能对疾病预防以及诊断和治疗有利。 相关性：中性粒细胞甲酰肽受体在粘膜天然免疫防御中的作用尚未确定。我们将验证我们的假设，即某些甲酰肽受体变体在对某些细菌物种的反应中存在缺陷，从而导致免疫反应延迟。我们还将测试假设，即甲酰肽受体中某些单核苷酸多态的组合包含代表不足的密码子和密码子对，导致翻译效率较低和受体表达水平降低，这可能会影响中性粒细胞对细菌感染的快速反应能力。

DESCRIPTION (provided by applicant): 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. RELEVANCE: 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.