G蛋白偶联甲酰基肽受体在抗细菌感染和恶性肿瘤宿主防御中的作用
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摘要
细菌感染和恶性肿瘤与以白细胞浸润为特征的宿主保护性炎症反应，即宿主防御密切相关。炎性细胞的浸润受G蛋白偶联受体（G protein-coupled receptors, GPCRs），包括甲酰基肽受体（Formylpeptide receptors, FPRs）和趋化因子类GPCRs介导。FPRs（小鼠FPRs称为Fprs）具有病原模式识别受体（pathogen pattern recognizing receptor,PRR）特性，能够识别多种病原和宿主来源的趋化因子，而趋化因子类GPCRs通过感受多种内源性趋化因子而介导白细胞浸润。活化GPCRs能够触发一系列信号放大效应并最终促使中性粒细胞迁移，增强其对病原体和损伤组织的吞噬作用，同时能够增加杀菌性活性氧（reactive oxygen species, ROS）的产生和基因转录。本课题通过系列实验证实Fprs（包括Fpr1和Fpr2）能够与Listeria菌释放的趋化性成分结合而介导中性粒细胞在Listeria感染的小鼠肝内快速聚集，从而对宿主抗Listeria感染的防御反应起到决定性作用。另外，Fpr2具有维持肿瘤相关巨噬细胞向具有抗肿瘤效应的M1方向分化的作用而利于机体免疫系统限制肿瘤发展。
第一部分：FPRs在抗细菌感染宿主防御中的作用。
材料与方法
1.建立系统性Listeria感染模型。野生型（wild type, WT）和Fpr基因敲除（knockout, KO；包括Fpr1-/-, Fpr2-/-和Fpr1/2-/-）小鼠分别尾静脉注射对数分裂期Listeria细菌建立系统性感染模型，观察WT和KO小鼠死亡率；检测小鼠肝脏内细菌数量、病灶数量和大小；确定病灶区中性粒细胞分布规律。
2.肝内趋化因子检测。在不同时间点，ELISA检测小鼠肝脏内趋化因子CXCL1和CXCL2表达。
3.趋化实验。使用合成Listeria肽（fMIVIL）和Listeria细菌裂解液检测其对HEK293细胞（human embryonic kidney293cells）和转染了Fpr1或Fpr2的HEK293细胞（HEK293/Fpr1和HEK293/Fpr2）细胞的趋化能力，证实Listeria含有FPR激动性配体（agonist）；使用WT和Fpr KO（包括Fpr1-/-, Fpr2-/-, Fpr1/2-/-）小鼠中性粒细胞证实Listeria所致的中性粒细胞趋化由FPRs介导。
4.中性粒细胞对Listeria的吞噬和杀伤能力。中性粒细胞和热灭活Listeria共培养（1:100）后流式细胞术和免疫荧光检测中性粒细胞对Listeria的吞噬能力；中性粒细胞和Listeria活菌共培养（1:100）后接种于琼脂糖平板（Agar plate）计数细菌菌落形成单位（colony forming unit, CFU）；检测中性粒细胞对H2O2（hydrogen superoxide，过氧化氢）的表达能力。
5. Listeria对中性粒细胞内信号分子磷酸化的影响。Listeria裂解液刺激中性粒细胞后裂解细胞提取总蛋白，检测细胞外信号调节激酶（extracellular signal-regulatedkinase, Erk1/2）磷酸化水平。
6.骨髓移植和骨髓细胞竞争性重分布。使用致死剂量放射性铯照射Fpr2-/-和Fpr1/2-/-小鼠并移植WT小鼠骨髓细胞，6周后建立感染模型，观察嵌合体小鼠（chimera）死亡率；使用不同荧光染料预标记WT和Fpr1/2-/-小鼠骨髓细胞，并等量注射入Fpr1/2-/-小鼠尾静脉，建立感染模型，观察WT和Fpr1/2-/-小鼠骨髓细胞在感染区的数量。
结果
1. Fpr基因敲除导致小鼠抗Listeria感染的宿主防御障碍。成功建立Listeria系统性感染模型，WT小鼠半数致死量为2×104个细菌，亚致死量为1×104个细菌。和WT小鼠相比（死亡率50%），Fpr1-/-和Fpr2-/-小鼠死亡率显著增高（10天内死亡率分别为100%和90%）；而Fpr1/2-/-小鼠在3天内死亡率达到100%。Listeria感染的Fpr1-/-、Fpr2-/-和Fpr1/2-/-小鼠肝内CFUs均显著增加（较WT小鼠分别增高50、40和80倍）。Fpr1-/-、Fpr2-/-和Fpr1/2-/-小鼠肝内脓肿数量增加、直径变大，而中性粒细胞浸润减少。
2. Fprs介导感染小鼠肝内中性粒细胞快速聚集。在WT小鼠，肝内中性粒细胞在感染后30min内即快速增加并于2h内达到高峰；在Fpr基因敲除小鼠肝内中性粒细胞趋化延迟，峰值数量显著降低。骨髓细胞竞争性重分布显示感染区WT小鼠骨髓细胞显著多于Fpr1/2-/-小鼠骨髓细胞；移植WT小鼠骨髓细胞后能够显著降低Listeria感染的Fpr2-/-和Fpr1/2-/-小鼠死亡率。
3.肝内趋化因子表达晚于早期中性粒细胞趋化聚集。肝内CXCL1和CXCL2表达上升分别开始于感染后8h和24h，显著晚于中性粒细胞趋化、聚集。
4. Fpr1和Fpr2是Listeria趋化中性粒细胞的独有受体。Listeria裂解液能够趋化HEK293/Fpr1和HEK293/Fpr2细胞而不能趋化HEK293细胞，表明Listeria裂解液含有FPR agonist。Listeria裂解液对Fpr1-/-和Fpr2-/-小鼠中性粒细胞的趋化作用显著低于WT小鼠中性粒细胞，而对Fpr1/2-/-小鼠中性粒细胞完全不具有趋化作用，说明Listeria裂解液所导致的中性粒细胞趋化作用系通过Fpr1和Fpr2所实现，而Fpr1和Fpr2是介导该趋化反应的独有受体。
5. Fpr1和Fpr2介导中性粒细胞H2O2分泌和杀菌作用。和WT小鼠中性粒细胞相比，Fpr基因敲除小鼠中性粒细胞对Listeria的吞噬作用未见改变，但是杀菌作用显著减弱。Fpr基因敲除小鼠中性粒细胞对H2O2的分泌能力显著降低；Listeria所致WT小鼠中性粒细胞对H2O2的分泌能够被Fpr特异性抑制性配体（antagonists）所抑制。
6.上述作用主要由信号分子Erk1/2介导。Listeria裂解液能够显著上调中性粒细胞中Erk1/2的磷酸化，该作用能被Frp antagonists选择性阻断。
第二部分：Fpr2在抗Lewis肺癌（Lewis lung carcinoma, LLC）宿主防御中的作用。
材料与方法
1.肿瘤模型。5×105LLC细胞接种于小鼠右侧胁腹部建立肿瘤模型，测量肿瘤体积和小鼠死亡率。在转移试验中，4×105LLC细胞经尾静脉注射，15天后计数肺部转移灶数量。
2.免疫荧光染色。肿瘤组织冰冻切片，anti-CD11b，anti-F4/80或anti-CD31标记，免疫荧光和FACS检测肿瘤内骨髓来源白细胞、巨噬细胞数量，免疫荧光检测血管生成情况。
3.趋化实验。检测LLC上清液对WT, Fpr2-/-和Fpr转基因（transgenic, Tg）小鼠巨噬细胞的趋化能力，并使用Fpr2antagonist、CCL2中和性抗体、CCR2抗体及CCR4antagonist选择性抑制趋化反应，检测LLC上清液对巨噬细胞趋化的特异性。
4. Western blotting。骨髓来源巨噬细胞以LLC上清液，LPS+IFNγ或IL4+IL13刺激，提取蛋白检测Arg1表达和STAT1、STAT3及STAT6磷酸化水平。
5.肿瘤组织浸润白细胞的检测。Percoll法纯化肿瘤组织内浸润白细胞。纯化的白细胞经FACS检测肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）、CD45+CD11b+F4/80+CD206+和CD45+CD11b+F4/80+CD11c+细胞数量及比例。
6. RT-qPCR （reverse transcription quantitative PCR，反转录定量PCR）。RT-qPCR检测Arg1, iNOS and TNFα mRNA表达。
结果
1. Fpr2基因敲除导致LLC肿瘤生长加速，荷瘤小鼠死亡率增加，肺部转移灶增多。
2. Fpr2-/-小鼠LLC肿瘤内浸润白细胞（CD11b+）增加，巨噬细胞（F4/80+）增加，血管生成增多（CD31+细胞增多）。
3. Fpr2基因敲除导致巨噬细胞对LLC上清液趋化反应增强，该反应能够被CCL2抗体完全阻断，而被CCR2中和性抗体部分抑制。
4. Fpr2-/-小鼠巨噬细胞表达CCR4增加。FACS、RT-qPCR检测显示Fpr2-/-小鼠骨髓来源巨噬细胞和TAM表达CCR4蛋白和mRNA增加。
5. Fpr2基因敲除导致LLC内巨噬细胞向M2转化。Fpr2-/-小鼠骨髓来源巨噬细胞和TAM表达M2标记物iNOS，TNFα和phospho-STAT1减少，而表达M2标记物Arg1,phospho-STAT3和phospho-STAT6增加。
结论
1. Fpr1和Fpr2介导Listeria系统性感染时小鼠肝内中性粒细胞快速趋化和聚集以及H2O2依赖性的杀菌作用，从而参与宿主对细菌感染的早期防御反应；
2. Fpr1和Fpr2是介导Listeria所致中性粒细胞趋化的独有受体；
3. Fpr2通过限制肿瘤相关巨噬细胞向M2转化而促进宿主抗肿瘤防御；
4. Fprs通过直接感知细菌趋化分子而介导中性粒细胞向细菌感染部位聚集，同时Fpr2通过调控肿瘤相关巨噬细胞浸润和分化而参与宿主抗肿瘤防御。
综上所述，本文通过小鼠Listeria菌系统性感染模型，发现Fpr1和Fpr2介导细菌感染性疾病中中性粒细胞的早期、快速趋化和聚集，并通过调节H2O2的释放而参与中性粒细胞的杀菌作用，证实Fprs在感染性疾病中以早期中性粒细胞趋化聚集为主的宿主保护性免疫反应中起到重要作用；使用小鼠LLC模型，证实FPR2（Fpr2）能够限制肿瘤相关巨噬细胞向M2转化而促进宿主抗肿瘤防御。本研究为感染性疾病和肿瘤治疗提供了良好的理论基础和潜在的治疗靶点。
Bacterial infection and cancer are associated with inflammation characterized byinfiltration of leukocytes, which plays pivotal roles in host defense against pathogenicmicroorganisms and tumor progression. Infiltration of leukocyte is mediated by Gprotein-coupled chemoattractant receptors (GPCRs), including formylpeptide receptors(FPRs) and chemokine GPCRs. FPRs in human (Fprs in mice) exhibit pathogen patternrecognition receptor (PRR) properties by recognizing pathogen-and host-derivedchemotactic agonists, while chemokine GPCRs mediate leukocyte response to a plethora ofendogenous chemotactic cytokines. Activation of chemoattractant GPCRs triggers acascade of signaling events in leukocytes culminating in cell migration, increasedphagocytosis of pathogens and damaged tissues, the release of bactericidal reactive oxygenspecies (ROS) and gene transcription. The critical role of chemoattractant GPCRs inbacterial infection and cancer was exemplified by our studies of mice deficient in Fprs, inwhich, two subtypes of Fprs, Fpr1and Fpr2, mediate the rapid first wave neutrophilaccumulation in Listeria-infected liver in response to bacteria produced Fpr chemotacticagonists. The early neutrophil mobilization into infected liver by Fprs in Listeria-infectedmice is critical for mouse resistance to pathogen. In addition to Listeria infection, we alsohave shown that one of the Fprs, Fpr2, is capable of sustaining the polarization ofmacrophages into an anti-tumor M1macrophage phenotype, which is critical for the hostimmune system to limit tumor growth. Our observations demonstrate that Fprs are not onlyimportant for directly mediating neutrophil migration and activation by bacterialchemoattractants, but one of the subtype Fpr2is also capable of orchestrating hostanti-tumor defense.
Part1. Fprs in host defense against Listeria infection
Materials and methods
1. Systemic Listeria infection. Wild type (WT) and Fpr knock out (KO) C57/Bl6micewere i.v. injected with Listeria to establish systemic infection. Survival rate of infected mice and colony forming units (CFUs) of Listeria from mouse livers were determined.Microabscesses and the kinetics of neutrophil infiltration in the livers were analyzed withH&E, immunofluorescence staining and fluorescence-activated cell sorting (FACS).
2. Measurement of chemokines in the liver. The production of neutrophil specificchemokines CXCL1and CXCL2in the liver of na ve and infected mice was detected withenzyme-linked immunosorbence assay (ELISA).
3. Chemotaxis assay. To test the production of Fpr agonists by Listeria, Listeria lysatewas used to induce migration of Fpr1or Fpr2transfected HEK293(HEK293/Fpr1andHEK293/Fpr2) cells. Listeria lysate was tested for chemotactic activity for neutrophils fromWT and Fpr-deficient (Fpr1-/-, Fpr2-/-and Fpr1/2-/-) mice.
4. Phagocytosis and killing of Listeria by neutrophils. Neutrophils were incubated withheat inactivated Listeria and the efficacy of phagocytosis was measured with FACS andimmunofluorescence staining. Neutrophils were also incubated with live bacteria then toCFUs after the cells were lysed to measure the bactericidal capabilities. Listeria-inducedH2O2production by neutrophils in the presence or absence of Fpr specific antagonists or aTLR2neutralizing antibody was determined to clarify the specificity of Fpr inListeria-triggered H2O2production by neutrophils.
5. Phosphorylation of signaling molecules down-stream of Fpr activation. Mouseneutrophils were stimulated with Listeria lysate and the phosphorylation of extracellularsignal-regulated kinase1and2(Erk1/2) in cell lysate was detected with Western blotting.
6. Competitive repopulation and transplantation of bone marrow cells. For competitiverepopulation of bone marrow cells, bone marrow cells from WT and Fpr1/2-/-mice werepre-labeled with different colors and i.v. injected immediately after infection. Color spotsrepresenting repopulated cells in the livers were counted. For bone marrow transfer, Fpr2-/-and Fpr1/2-/-mice were irradiated and i.v. injected with bone marrow cells from WT mice4h later. All recipient mice were infected with1×104Listeria6weeks after bone marrowtransfer and survival rate was measured to test the rescue effect of transferred bone marrowfrom WT mice.
Results
1. Fpr-deficiency impairs host defense against Listeria infection. Systemic Listeriainfection was established with a50%lethal dose of2×104Listeria and a sublethal dose of 1×104in WT mice. Fpr-deficiency markedly increased the susceptibility of mice toListeria infection (50%WT mice died in10days,90%Fpr1-/-in10days,100%Fpr2-/-in7days and100%Fpr1/2-/-in3days). The Listeria load in the liver was50-,40-and80-foldhigher in Fpr1-/-, Fpr2-/-and Fpr1/2-/-mice than in WT mice, respectively.
2. Fprs are responsible for rapid neutrophil infiltration in infected organs. A rapid waveof neutrophil accumulation in WT mouse liver was detected, initiating at30min andpeaking at4h post infection. In contrast, in the liver of Fpr single-or double-deficient mice,neutrophil accumulation was markedly delayed. Histological examination revealedincreased abscess formation in the liver of Fpr-deficient mice with substantially reducedneutrophils surrounding the core of injured hepatocytes. Competitive repopulation ofneutrophils in Listeria-infected Fpr1/2-/-mice showed greatly increased WT cellsinfiltrating the infected liver. In addition, transplantation of bone marrow cells from WTmice significantly reduced the mortality of Fpr-deficient mice after Listeria infection.Compared with mice without WT mouse bone marrow transfer, the survival rates of Fpr2-/-and Fpr1/2-/-mice receiving WT mouse bone marrow transfer markedly increased.
3. The infiltration of neutrophils antecedes the production of chemokines. In the liverof WT mice, despite the rapid infiltration of neutrophils, the production of neutrophilspecific chemokines CXCL1and CXCL2was not detectable at8h and24h post infection.There was no difference in CXCL1and CXCL2levels in the infected livers of Fpr-deficientmice and WT mice, indicating chemokines are not involved in the rapid infiltration ofneutrophil in the infected liver.
4. Fprs are sole receptors for Listeria-derived chemotactic signals. Compared withneutrophils from WT mice, Fpr1-/-or Fpr2-/-mouse neutrophils exhibited decreasedchemotaxis to a synthetic Listeria peptide, fMIVIL. Fpr1/2-/-mouse neutrophils failed torespond to the peptide. However, Fpr-deficient mouse neutrophils retained normalchemotaxis induced by ligands using other GPCRs. In addition, Listeria lysate induced themigration of HEK293cells transfected to express Fprs, but not the parental HEK293cells.WT mouse neutrophils also migrated potently to Listeria lysate. In contrast, Fpr1-/-orFpr2-/-mouse cells showed reduced chemotaxis to Listeria lysate, with complete absence ofresponse of Fpr1/2-/-mouse cells.
5. Fprs mediate H2O2-dependent Listeria killing by neutrophils. There was no difference in phagocytosis of both live and heat-inactivated bacteria by neutrophils fromWT and Fpr-deficient mice. However, the killing of Listeria by Fpr1-/-and Fpr2-/-neutrophils was considerably reduced with an even greater reduction in killing by Fpr1/2-/-neutrophils. The Listeria killing capacity of neutrophils was correlated with their H2O2production in response to heat-inactivated bacteria. In WT neutrophils, Listeria-inducedH2O2production was partially inhibited by selective Fpr1or Fpr2antagonists, with furtherreduction by combination of two antagonists. In Fpr-deficient mice, absence of a single Fprsubstantially reduced neutrophil H2O2production induced by Listeria, with complete lossof production by Fpr1/2-/-cells. In support of the specificity of Fprs in Listeria-stimulatedH2O2production, WT and Fpr-deficient neutrophils responded equally well to phorbol ester(PMA), which was not inhibited by Fpr antagonists.
6. Erk1/2are involved in Listeria-induced neutrophil activation. Listeria lysateincreased the phosphorylation of Erk1/2in WT mouse neutrophils, which was selectivelyinhibited by Fpr1or Fpr2antagonist.
Part2. Fpr2in host defense against transplanted Lewis lung carcinoma (LLC)
Materials and methods
1. Tumor inoculation.5×105LLC cells were subcutaneously injected into the rightflank of mice and the size of tumors were measured twice per week. The survival rate ofmice was monitored. For tumor metastasis,4×105LLC cells in250μL PBS were injectedinto the mouse tail vein. On Day15, the lungs were harvested and tumor nodules werecounted.
2. Immunofluorescence staining. Cryosections of LLC tumors were stained withanti-CD11b, anti-F4/80, or anti-CD31antibody respectively to detect tumor-infiltratingmyeloid cells, vasculature and macrophage differentiation markers.
3. Chemotaxis assay. WT, Fpr2-/-and Fpr-transgenic (Tg) mouse macrophages weremeasured for chemotaxis in response to LLC supernatant and the chemokine CCL2.
4. Western blotting to detect the expression of specific macrophage subtype markers.BM-derived macrophages treated with LLC Sup, LPS plus IFNγ or IL4plus IL13fordifferent times were lysed and detected for the expression of Arg1and the phosphorylationof STAT1, STAT3and STAT6.
5. Isolation of tumor-infiltrating leukocytes. Tumors were homogenized and total cells were centrifuged with discontinuous Percoll gradient centrifugation. Leukocytes werecollected from the interface for further analysis.
6. FACS. BM cells and BM-derived macrophages were stained with antibodies againstCD11b-PECy5, F4/80-FITC, and CCR4-PE. Tumor-associated macrophages (TAMs) werelabeled with CD45-FITC, CD11b-PE, F4/80-PerCP-Cy5.5and CD206-APC or CD11c-APCantibodies for FACS analyses.
7. RT-qPCR (reverse transcription quantitative PCR). RT-qPCR was used to detect theexpression of M1and M2macrophage marker genes, such as Arg1, iNOS and TNFαmRNA.
Results
1. Fpr2deficiency increases tumor growth and mouse death. The volume ofsubcutaneous (s.c.) tumors, survival rate and lung metastasis in Fpr2-/-mice weresignificantly increased compared to WT mice.
2. Increased myeloid cell infiltration and angiogenesis in LLC tumors in Fpr2-/-mice.Immunofluorescence staining showed significantly increased CD11b+, F4/80+and CD31+cells in tumor in Fpr2-/-mice.
3. Increased chemotactic response of Fpr2-/-mouse macrophages to LLC tumor cellsupernatant and the chemokine CCL2. Chemotaxis assay revealed a more potent responseof Fpr2-/-mouse macrophages to LLC supernatant and CCL2, with complete inhibition by aCCL2antibody. But macrophage chemotaxis was only partially inhibited by an antibodyagainst CCR2, a major GPCR for CCL2, suggesting another CCL2receptor onmacrophages may play a role.
4. Increased expression of CCR4by Fpr2-/-mouse macrophages. FACS and RT-qPCRdetected an increase in CCR4protein and mRNA expression by LLCsupernatant-stimulated Fpr2-/-mouse macrophages. CCR4partially accounted for Fpr2-/-macrophage chemotaxis in response to LLC supernatant and CCL2.
5. Increased expression of M2markers by Fpr2-/-mouse macrophages in response toLLC Sup. The expression of M1markers, iNOS, TNFα and phosphor-STAT1, wasdownregulated in Fpr2-/-mouse macrophages; while M2markers, Arg1, phosphor-STAT3and phosphor-STAT6were upregulated.
Conclusions
1. Fpr1and Fpr2mediate host defense against systemic Listeria infection in micethrough mobilization of rapid neutrophil infiltration and H2O2-dependent Listeria killing;
2. Frp1and Fpr2are sole receptors for Listeria-derived chemotactic signals;
3. Fpr2promotes anti-tumor host defense by limiting M2polarization of macrophages;
4. Fprs control leukocyte infiltration by directly mediating neutrophil response tobacterial chemoattractant in Listeria infection, and by regulating the polarization andinfiltration of macrophages in tumor.
第一部分：FPRs在抗细菌感染宿主防御中的作用。
材料与方法
1.建立系统性Listeria感染模型。野生型（wild type, WT）和Fpr基因敲除（knockout, KO；包括Fpr1-/-, Fpr2-/-和Fpr1/2-/-）小鼠分别尾静脉注射对数分裂期Listeria细菌建立系统性感染模型，观察WT和KO小鼠死亡率；检测小鼠肝脏内细菌数量、病灶数量和大小；确定病灶区中性粒细胞分布规律。
2.肝内趋化因子检测。在不同时间点，ELISA检测小鼠肝脏内趋化因子CXCL1和CXCL2表达。
3.趋化实验。使用合成Listeria肽（fMIVIL）和Listeria细菌裂解液检测其对HEK293细胞（human embryonic kidney293cells）和转染了Fpr1或Fpr2的HEK293细胞（HEK293/Fpr1和HEK293/Fpr2）细胞的趋化能力，证实Listeria含有FPR激动性配体（agonist）；使用WT和Fpr KO（包括Fpr1-/-, Fpr2-/-, Fpr1/2-/-）小鼠中性粒细胞证实Listeria所致的中性粒细胞趋化由FPRs介导。
4.中性粒细胞对Listeria的吞噬和杀伤能力。中性粒细胞和热灭活Listeria共培养（1:100）后流式细胞术和免疫荧光检测中性粒细胞对Listeria的吞噬能力；中性粒细胞和Listeria活菌共培养（1:100）后接种于琼脂糖平板（Agar plate）计数细菌菌落形成单位（colony forming unit, CFU）；检测中性粒细胞对H2O2（hydrogen superoxide，过氧化氢）的表达能力。
5. Listeria对中性粒细胞内信号分子磷酸化的影响。Listeria裂解液刺激中性粒细胞后裂解细胞提取总蛋白，检测细胞外信号调节激酶（extracellular signal-regulatedkinase, Erk1/2）磷酸化水平。
6.骨髓移植和骨髓细胞竞争性重分布。使用致死剂量放射性铯照射Fpr2-/-和Fpr1/2-/-小鼠并移植WT小鼠骨髓细胞，6周后建立感染模型，观察嵌合体小鼠（chimera）死亡率；使用不同荧光染料预标记WT和Fpr1/2-/-小鼠骨髓细胞，并等量注射入Fpr1/2-/-小鼠尾静脉，建立感染模型，观察WT和Fpr1/2-/-小鼠骨髓细胞在感染区的数量。
结果
1. Fpr基因敲除导致小鼠抗Listeria感染的宿主防御障碍。成功建立Listeria系统性感染模型，WT小鼠半数致死量为2×104个细菌，亚致死量为1×104个细菌。和WT小鼠相比（死亡率50%），Fpr1-/-和Fpr2-/-小鼠死亡率显著增高（10天内死亡率分别为100%和90%）；而Fpr1/2-/-小鼠在3天内死亡率达到100%。Listeria感染的Fpr1-/-、Fpr2-/-和Fpr1/2-/-小鼠肝内CFUs均显著增加（较WT小鼠分别增高50、40和80倍）。Fpr1-/-、Fpr2-/-和Fpr1/2-/-小鼠肝内脓肿数量增加、直径变大，而中性粒细胞浸润减少。
2. Fprs介导感染小鼠肝内中性粒细胞快速聚集。在WT小鼠，肝内中性粒细胞在感染后30min内即快速增加并于2h内达到高峰；在Fpr基因敲除小鼠肝内中性粒细胞趋化延迟，峰值数量显著降低。骨髓细胞竞争性重分布显示感染区WT小鼠骨髓细胞显著多于Fpr1/2-/-小鼠骨髓细胞；移植WT小鼠骨髓细胞后能够显著降低Listeria感染的Fpr2-/-和Fpr1/2-/-小鼠死亡率。
3.肝内趋化因子表达晚于早期中性粒细胞趋化聚集。肝内CXCL1和CXCL2表达上升分别开始于感染后8h和24h，显著晚于中性粒细胞趋化、聚集。
4. Fpr1和Fpr2是Listeria趋化中性粒细胞的独有受体。Listeria裂解液能够趋化HEK293/Fpr1和HEK293/Fpr2细胞而不能趋化HEK293细胞，表明Listeria裂解液含有FPR agonist。Listeria裂解液对Fpr1-/-和Fpr2-/-小鼠中性粒细胞的趋化作用显著低于WT小鼠中性粒细胞，而对Fpr1/2-/-小鼠中性粒细胞完全不具有趋化作用，说明Listeria裂解液所导致的中性粒细胞趋化作用系通过Fpr1和Fpr2所实现，而Fpr1和Fpr2是介导该趋化反应的独有受体。
5. Fpr1和Fpr2介导中性粒细胞H2O2分泌和杀菌作用。和WT小鼠中性粒细胞相比，Fpr基因敲除小鼠中性粒细胞对Listeria的吞噬作用未见改变，但是杀菌作用显著减弱。Fpr基因敲除小鼠中性粒细胞对H2O2的分泌能力显著降低；Listeria所致WT小鼠中性粒细胞对H2O2的分泌能够被Fpr特异性抑制性配体（antagonists）所抑制。
6.上述作用主要由信号分子Erk1/2介导。Listeria裂解液能够显著上调中性粒细胞中Erk1/2的磷酸化，该作用能被Frp antagonists选择性阻断。
第二部分：Fpr2在抗Lewis肺癌（Lewis lung carcinoma, LLC）宿主防御中的作用。
材料与方法
1.肿瘤模型。5×105LLC细胞接种于小鼠右侧胁腹部建立肿瘤模型，测量肿瘤体积和小鼠死亡率。在转移试验中，4×105LLC细胞经尾静脉注射，15天后计数肺部转移灶数量。
2.免疫荧光染色。肿瘤组织冰冻切片，anti-CD11b，anti-F4/80或anti-CD31标记，免疫荧光和FACS检测肿瘤内骨髓来源白细胞、巨噬细胞数量，免疫荧光检测血管生成情况。
3.趋化实验。检测LLC上清液对WT, Fpr2-/-和Fpr转基因（transgenic, Tg）小鼠巨噬细胞的趋化能力，并使用Fpr2antagonist、CCL2中和性抗体、CCR2抗体及CCR4antagonist选择性抑制趋化反应，检测LLC上清液对巨噬细胞趋化的特异性。
4. Western blotting。骨髓来源巨噬细胞以LLC上清液，LPS+IFNγ或IL4+IL13刺激，提取蛋白检测Arg1表达和STAT1、STAT3及STAT6磷酸化水平。
5.肿瘤组织浸润白细胞的检测。Percoll法纯化肿瘤组织内浸润白细胞。纯化的白细胞经FACS检测肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）、CD45+CD11b+F4/80+CD206+和CD45+CD11b+F4/80+CD11c+细胞数量及比例。
6. RT-qPCR （reverse transcription quantitative PCR，反转录定量PCR）。RT-qPCR检测Arg1, iNOS and TNFα mRNA表达。
结果
1. Fpr2基因敲除导致LLC肿瘤生长加速，荷瘤小鼠死亡率增加，肺部转移灶增多。
2. Fpr2-/-小鼠LLC肿瘤内浸润白细胞（CD11b+）增加，巨噬细胞（F4/80+）增加，血管生成增多（CD31+细胞增多）。
3. Fpr2基因敲除导致巨噬细胞对LLC上清液趋化反应增强，该反应能够被CCL2抗体完全阻断，而被CCR2中和性抗体部分抑制。
4. Fpr2-/-小鼠巨噬细胞表达CCR4增加。FACS、RT-qPCR检测显示Fpr2-/-小鼠骨髓来源巨噬细胞和TAM表达CCR4蛋白和mRNA增加。
5. Fpr2基因敲除导致LLC内巨噬细胞向M2转化。Fpr2-/-小鼠骨髓来源巨噬细胞和TAM表达M2标记物iNOS，TNFα和phospho-STAT1减少，而表达M2标记物Arg1,phospho-STAT3和phospho-STAT6增加。
结论
1. Fpr1和Fpr2介导Listeria系统性感染时小鼠肝内中性粒细胞快速趋化和聚集以及H2O2依赖性的杀菌作用，从而参与宿主对细菌感染的早期防御反应；
2. Fpr1和Fpr2是介导Listeria所致中性粒细胞趋化的独有受体；
3. Fpr2通过限制肿瘤相关巨噬细胞向M2转化而促进宿主抗肿瘤防御；
4. Fprs通过直接感知细菌趋化分子而介导中性粒细胞向细菌感染部位聚集，同时Fpr2通过调控肿瘤相关巨噬细胞浸润和分化而参与宿主抗肿瘤防御。
综上所述，本文通过小鼠Listeria菌系统性感染模型，发现Fpr1和Fpr2介导细菌感染性疾病中中性粒细胞的早期、快速趋化和聚集，并通过调节H2O2的释放而参与中性粒细胞的杀菌作用，证实Fprs在感染性疾病中以早期中性粒细胞趋化聚集为主的宿主保护性免疫反应中起到重要作用；使用小鼠LLC模型，证实FPR2（Fpr2）能够限制肿瘤相关巨噬细胞向M2转化而促进宿主抗肿瘤防御。本研究为感染性疾病和肿瘤治疗提供了良好的理论基础和潜在的治疗靶点。
Bacterial infection and cancer are associated with inflammation characterized byinfiltration of leukocytes, which plays pivotal roles in host defense against pathogenicmicroorganisms and tumor progression. Infiltration of leukocyte is mediated by Gprotein-coupled chemoattractant receptors (GPCRs), including formylpeptide receptors(FPRs) and chemokine GPCRs. FPRs in human (Fprs in mice) exhibit pathogen patternrecognition receptor (PRR) properties by recognizing pathogen-and host-derivedchemotactic agonists, while chemokine GPCRs mediate leukocyte response to a plethora ofendogenous chemotactic cytokines. Activation of chemoattractant GPCRs triggers acascade of signaling events in leukocytes culminating in cell migration, increasedphagocytosis of pathogens and damaged tissues, the release of bactericidal reactive oxygenspecies (ROS) and gene transcription. The critical role of chemoattractant GPCRs inbacterial infection and cancer was exemplified by our studies of mice deficient in Fprs, inwhich, two subtypes of Fprs, Fpr1and Fpr2, mediate the rapid first wave neutrophilaccumulation in Listeria-infected liver in response to bacteria produced Fpr chemotacticagonists. The early neutrophil mobilization into infected liver by Fprs in Listeria-infectedmice is critical for mouse resistance to pathogen. In addition to Listeria infection, we alsohave shown that one of the Fprs, Fpr2, is capable of sustaining the polarization ofmacrophages into an anti-tumor M1macrophage phenotype, which is critical for the hostimmune system to limit tumor growth. Our observations demonstrate that Fprs are not onlyimportant for directly mediating neutrophil migration and activation by bacterialchemoattractants, but one of the subtype Fpr2is also capable of orchestrating hostanti-tumor defense.
Part1. Fprs in host defense against Listeria infection
Materials and methods
1. Systemic Listeria infection. Wild type (WT) and Fpr knock out (KO) C57/Bl6micewere i.v. injected with Listeria to establish systemic infection. Survival rate of infected mice and colony forming units (CFUs) of Listeria from mouse livers were determined.Microabscesses and the kinetics of neutrophil infiltration in the livers were analyzed withH&E, immunofluorescence staining and fluorescence-activated cell sorting (FACS).
2. Measurement of chemokines in the liver. The production of neutrophil specificchemokines CXCL1and CXCL2in the liver of na ve and infected mice was detected withenzyme-linked immunosorbence assay (ELISA).
3. Chemotaxis assay. To test the production of Fpr agonists by Listeria, Listeria lysatewas used to induce migration of Fpr1or Fpr2transfected HEK293(HEK293/Fpr1andHEK293/Fpr2) cells. Listeria lysate was tested for chemotactic activity for neutrophils fromWT and Fpr-deficient (Fpr1-/-, Fpr2-/-and Fpr1/2-/-) mice.
4. Phagocytosis and killing of Listeria by neutrophils. Neutrophils were incubated withheat inactivated Listeria and the efficacy of phagocytosis was measured with FACS andimmunofluorescence staining. Neutrophils were also incubated with live bacteria then toCFUs after the cells were lysed to measure the bactericidal capabilities. Listeria-inducedH2O2production by neutrophils in the presence or absence of Fpr specific antagonists or aTLR2neutralizing antibody was determined to clarify the specificity of Fpr inListeria-triggered H2O2production by neutrophils.
5. Phosphorylation of signaling molecules down-stream of Fpr activation. Mouseneutrophils were stimulated with Listeria lysate and the phosphorylation of extracellularsignal-regulated kinase1and2(Erk1/2) in cell lysate was detected with Western blotting.
6. Competitive repopulation and transplantation of bone marrow cells. For competitiverepopulation of bone marrow cells, bone marrow cells from WT and Fpr1/2-/-mice werepre-labeled with different colors and i.v. injected immediately after infection. Color spotsrepresenting repopulated cells in the livers were counted. For bone marrow transfer, Fpr2-/-and Fpr1/2-/-mice were irradiated and i.v. injected with bone marrow cells from WT mice4h later. All recipient mice were infected with1×104Listeria6weeks after bone marrowtransfer and survival rate was measured to test the rescue effect of transferred bone marrowfrom WT mice.
Results
1. Fpr-deficiency impairs host defense against Listeria infection. Systemic Listeriainfection was established with a50%lethal dose of2×104Listeria and a sublethal dose of 1×104in WT mice. Fpr-deficiency markedly increased the susceptibility of mice toListeria infection (50%WT mice died in10days,90%Fpr1-/-in10days,100%Fpr2-/-in7days and100%Fpr1/2-/-in3days). The Listeria load in the liver was50-,40-and80-foldhigher in Fpr1-/-, Fpr2-/-and Fpr1/2-/-mice than in WT mice, respectively.
2. Fprs are responsible for rapid neutrophil infiltration in infected organs. A rapid waveof neutrophil accumulation in WT mouse liver was detected, initiating at30min andpeaking at4h post infection. In contrast, in the liver of Fpr single-or double-deficient mice,neutrophil accumulation was markedly delayed. Histological examination revealedincreased abscess formation in the liver of Fpr-deficient mice with substantially reducedneutrophils surrounding the core of injured hepatocytes. Competitive repopulation ofneutrophils in Listeria-infected Fpr1/2-/-mice showed greatly increased WT cellsinfiltrating the infected liver. In addition, transplantation of bone marrow cells from WTmice significantly reduced the mortality of Fpr-deficient mice after Listeria infection.Compared with mice without WT mouse bone marrow transfer, the survival rates of Fpr2-/-and Fpr1/2-/-mice receiving WT mouse bone marrow transfer markedly increased.
3. The infiltration of neutrophils antecedes the production of chemokines. In the liverof WT mice, despite the rapid infiltration of neutrophils, the production of neutrophilspecific chemokines CXCL1and CXCL2was not detectable at8h and24h post infection.There was no difference in CXCL1and CXCL2levels in the infected livers of Fpr-deficientmice and WT mice, indicating chemokines are not involved in the rapid infiltration ofneutrophil in the infected liver.
4. Fprs are sole receptors for Listeria-derived chemotactic signals. Compared withneutrophils from WT mice, Fpr1-/-or Fpr2-/-mouse neutrophils exhibited decreasedchemotaxis to a synthetic Listeria peptide, fMIVIL. Fpr1/2-/-mouse neutrophils failed torespond to the peptide. However, Fpr-deficient mouse neutrophils retained normalchemotaxis induced by ligands using other GPCRs. In addition, Listeria lysate induced themigration of HEK293cells transfected to express Fprs, but not the parental HEK293cells.WT mouse neutrophils also migrated potently to Listeria lysate. In contrast, Fpr1-/-orFpr2-/-mouse cells showed reduced chemotaxis to Listeria lysate, with complete absence ofresponse of Fpr1/2-/-mouse cells.
5. Fprs mediate H2O2-dependent Listeria killing by neutrophils. There was no difference in phagocytosis of both live and heat-inactivated bacteria by neutrophils fromWT and Fpr-deficient mice. However, the killing of Listeria by Fpr1-/-and Fpr2-/-neutrophils was considerably reduced with an even greater reduction in killing by Fpr1/2-/-neutrophils. The Listeria killing capacity of neutrophils was correlated with their H2O2production in response to heat-inactivated bacteria. In WT neutrophils, Listeria-inducedH2O2production was partially inhibited by selective Fpr1or Fpr2antagonists, with furtherreduction by combination of two antagonists. In Fpr-deficient mice, absence of a single Fprsubstantially reduced neutrophil H2O2production induced by Listeria, with complete lossof production by Fpr1/2-/-cells. In support of the specificity of Fprs in Listeria-stimulatedH2O2production, WT and Fpr-deficient neutrophils responded equally well to phorbol ester(PMA), which was not inhibited by Fpr antagonists.
6. Erk1/2are involved in Listeria-induced neutrophil activation. Listeria lysateincreased the phosphorylation of Erk1/2in WT mouse neutrophils, which was selectivelyinhibited by Fpr1or Fpr2antagonist.
Part2. Fpr2in host defense against transplanted Lewis lung carcinoma (LLC)
Materials and methods
1. Tumor inoculation.5×105LLC cells were subcutaneously injected into the rightflank of mice and the size of tumors were measured twice per week. The survival rate ofmice was monitored. For tumor metastasis,4×105LLC cells in250μL PBS were injectedinto the mouse tail vein. On Day15, the lungs were harvested and tumor nodules werecounted.
2. Immunofluorescence staining. Cryosections of LLC tumors were stained withanti-CD11b, anti-F4/80, or anti-CD31antibody respectively to detect tumor-infiltratingmyeloid cells, vasculature and macrophage differentiation markers.
3. Chemotaxis assay. WT, Fpr2-/-and Fpr-transgenic (Tg) mouse macrophages weremeasured for chemotaxis in response to LLC supernatant and the chemokine CCL2.
4. Western blotting to detect the expression of specific macrophage subtype markers.BM-derived macrophages treated with LLC Sup, LPS plus IFNγ or IL4plus IL13fordifferent times were lysed and detected for the expression of Arg1and the phosphorylationof STAT1, STAT3and STAT6.
5. Isolation of tumor-infiltrating leukocytes. Tumors were homogenized and total cells were centrifuged with discontinuous Percoll gradient centrifugation. Leukocytes werecollected from the interface for further analysis.
6. FACS. BM cells and BM-derived macrophages were stained with antibodies againstCD11b-PECy5, F4/80-FITC, and CCR4-PE. Tumor-associated macrophages (TAMs) werelabeled with CD45-FITC, CD11b-PE, F4/80-PerCP-Cy5.5and CD206-APC or CD11c-APCantibodies for FACS analyses.
7. RT-qPCR (reverse transcription quantitative PCR). RT-qPCR was used to detect theexpression of M1and M2macrophage marker genes, such as Arg1, iNOS and TNFαmRNA.
Results
1. Fpr2deficiency increases tumor growth and mouse death. The volume ofsubcutaneous (s.c.) tumors, survival rate and lung metastasis in Fpr2-/-mice weresignificantly increased compared to WT mice.
2. Increased myeloid cell infiltration and angiogenesis in LLC tumors in Fpr2-/-mice.Immunofluorescence staining showed significantly increased CD11b+, F4/80+and CD31+cells in tumor in Fpr2-/-mice.
3. Increased chemotactic response of Fpr2-/-mouse macrophages to LLC tumor cellsupernatant and the chemokine CCL2. Chemotaxis assay revealed a more potent responseof Fpr2-/-mouse macrophages to LLC supernatant and CCL2, with complete inhibition by aCCL2antibody. But macrophage chemotaxis was only partially inhibited by an antibodyagainst CCR2, a major GPCR for CCL2, suggesting another CCL2receptor onmacrophages may play a role.
4. Increased expression of CCR4by Fpr2-/-mouse macrophages. FACS and RT-qPCRdetected an increase in CCR4protein and mRNA expression by LLCsupernatant-stimulated Fpr2-/-mouse macrophages. CCR4partially accounted for Fpr2-/-macrophage chemotaxis in response to LLC supernatant and CCL2.
5. Increased expression of M2markers by Fpr2-/-mouse macrophages in response toLLC Sup. The expression of M1markers, iNOS, TNFα and phosphor-STAT1, wasdownregulated in Fpr2-/-mouse macrophages; while M2markers, Arg1, phosphor-STAT3and phosphor-STAT6were upregulated.
Conclusions
1. Fpr1and Fpr2mediate host defense against systemic Listeria infection in micethrough mobilization of rapid neutrophil infiltration and H2O2-dependent Listeria killing;
2. Frp1and Fpr2are sole receptors for Listeria-derived chemotactic signals;
3. Fpr2promotes anti-tumor host defense by limiting M2polarization of macrophages;
4. Fprs control leukocyte infiltration by directly mediating neutrophil response tobacterial chemoattractant in Listeria infection, and by regulating the polarization andinfiltration of macrophages in tumor.
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