The Chemoattractant Formylpeptide Receptors Promote Host Defense Against Bacterial Infection And Malignant Tumor

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 infectionMaterials and methods1. 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.Results1. 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 methods1. 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.Results1. 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. Conclusions1. 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.