RESUMO
Host-derived antimicrobial peptides play an important role in the defense against extracellular bacterial infections. However, the capacity of antimicrobial peptides derived from macrophages as potential antibacterial effectors against intracellular pathogens remains unknown. In this study, we report that normal (wild-type, WT) mouse macrophages increased their expression of cathelin-related antimicrobial peptide (CRAMP, encoded by Camp) after infection by viable E. coli or stimulation with inactivated E. coli and its product lipopolysaccharide (LPS), a process involving activation of NF-κB followed by protease-dependent conversion of CRAMP from an inactive precursor to an active form. The active CRAMP was required by WT macrophages for elimination of phagocytosed E. coli, with participation of autophagy-related proteins ATG5, LC3-II and LAMP-1, as well as for aggregation of the bacteria with p62 (also known as SQSTM1). This process was impaired in CRAMP-/- macrophages, resulting in retention of intracellular bacteria and fragmentation of macrophages. These results indicate that CRAMP is a critical component in autophagy-mediated clearance of intracellular E. coli by mouse macrophages.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Escherichia coli , Animais , Autofagia , Macrófagos , Camundongos , FagocitoseRESUMO
The cathelin-related antimicrobial peptide CRAMP protects the mouse colon from inflammation, inflammation-associated carcinogenesis, and disrupted microbiome balance, as shown in systemic Cnlp-/- mice (also known as Camp-/- mice). However, the mechanistic basis for the role and the cellular source of CRAMP in colon pathophysiology are ill defined. This study, using either epithelial or myeloid conditional Cnlp-/- mice, demonstrated that epithelial cell-derived CRAMP played a major role in supporting normal development of colon crypts, mucus production, and repair of injured mucosa. On the other hand, myeloid cell-derived CRAMP potently supported colon epithelial resistance to bacterial invasion during acute inflammation with exacerbated mucosal damage and higher rate of mouse mortality. Therefore, a well concerted cooperation of epithelial- and myeloid-derived CRAMP is essential for colon mucosal homeostasis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Células Epiteliais/metabolismo , Homeostase/fisiologia , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Animais , Colo/fisiologia , Camundongos , Camundongos Knockout , CatelicidinasRESUMO
Formyl peptide receptors (FPRs, mouse Fprs) belong to the G protein-coupled receptor superfamily and mediate phagocyte migration in response to bacteria- and host-derived chemoattractants; however, knowledge about their in vivo roles in bacterial pathogenesis is limited. In this study, we investigated the role of Fpr1 and Fpr2 in host defense against Escherichia coli infection. In vitro, we found that supernatants from E. coli cultures induced chemotaxis of wild-type (WT) mouse bone marrow-derived neutrophils and that the activity was significantly reduced in cells genetically deficient in either Fpr1 or Fpr2 and was almost absent in cells lacking both receptors. Consistent with this, E. coli supernatants induced chemotaxis and MAPK phosphorylation in HEK293 cells expressing either recombinant Fpr1 or Fpr2 but not untransfected parental cells. WT bone marrow -derived neutrophils could actively phagocytose and kill E. coli, whereas both activities were diminished in cells lacking Fpr1 or Fpr2; again, an additive effect was observed in cells lacking both receptors. In vivo, Fpr1 and Fpr2 deficiency resulted in reduced recruitment of neutrophils in the liver and peritoneal cavity of mice infected with inactivated E. coli Moreover, Fpr1-/- and Fpr2-/- mice had significantly increased mortality compared with WT mice after i.p. challenge with a virulent E. coli clinical isolate. These results indicate a critical role of Fprs in host defense against E. coli infection.
Assuntos
Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Escherichia coli/imunologia , Receptores de Formil Peptídeo/imunologia , Receptores de Formil Peptídeo/metabolismo , Animais , Medula Óssea/imunologia , Medula Óssea/metabolismo , Medula Óssea/microbiologia , Células Cultivadas , Quimiotaxia/imunologia , Células HEK293 , Humanos , Fígado/imunologia , Fígado/metabolismo , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Cavidade Peritoneal/microbiologia , Fagocitose/imunologia , Fosforilação/imunologiaRESUMO
OBJECTIVES: Trauma predisposes to systemic sterile inflammation (systemic inflammatory response syndrome) as well as infection, but the mechanisms linking injury to infection are poorly understood. Mitochondrial debris contains formyl peptides. These bind formyl peptide receptor-1, trafficking neutrophils to wounds, initiating systemic inflammatory response syndrome, and wound healing. Bacterial formyl peptides, however, also attract neutrophils via formyl peptide receptor-1. Thus, mitochondrial formyl peptides might suppress neutrophils antimicrobial function. Also, formyl peptide receptor-1 blockade used to mitigate systemic inflammatory response syndrome might predispose to sepsis. We examined how mitochondrial formyl peptides impact neutrophils functions contributing to antimicrobial responses and how formyl peptide receptor-1 antagonists affect those functions. DESIGN: Prospective study of human and murine neutrophils and clinical cohort analysis. SETTING: University research laboratory and level 1 trauma center. PATIENTS: Trauma patients, volunteer controls. ANIMAL SUBJECTS: C57Bl/6, formyl peptide receptor-1, and formyl peptide receptor-2 knockout mice. INTERVENTIONS: Human and murine neutrophils functions were activated with autologous mitochondrial debris, mitochondrial formyl peptides, or bacterial formyl peptides followed by chemokines or leukotrienes. The experiments were repeated using formyl peptide receptor-1 antagonist cyclosporin H, "designer" human formyl peptide receptor-1 antagonists (POL7178 and POL7200), or anti-formyl peptide receptor-1 antibodies. Mouse injury/lung infection model was used to evaluate effect of formyl peptide receptor-1 inhibition. MEASUREMENTS AND MAIN RESULTS: Human neutrophils cytosolic calcium, chemotaxis, reactive oxygen species production, and phagocytosis were studied before and after exposure to mitochondrial debris, mitochondrial formyl peptides, and bacterial formyl peptides. Mitochondrial formyl peptide and bacterial formyl peptides had similar effects on neutrophils. Responses to chemokines and leukotrienes were suppressed by prior exposure to formyl peptides. POL7200 and POL7178 were specific antagonists of human formyl peptide receptor-1 and more effective than cyclosporin H or anti-formyl peptide receptor-1 antibodies. Formyl peptides inhibited mouse neutrophils responses to chemokines only if formyl peptide receptor-1 was present. Formyl peptide receptor-1 blockade did not inhibit neutrophils bacterial phagocytosis or reactive oxygen species production. Cyclosporin H increased bacterial clearance in lungs after injury. CONCLUSIONS: Formyl peptides both activate and desensitize neutrophils. Formyl peptide receptor-1 blockade prevents desensitization, potentially both diminishing systemic inflammatory response syndrome and protecting the host against secondary infection after tissue trauma or primary infection.
Assuntos
Proteínas Mitocondriais/imunologia , Ativação de Neutrófilo/imunologia , Receptores de Formil Peptídeo/antagonistas & inibidores , Animais , Ciclosporina/farmacologia , Humanos , Lesão Pulmonar/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Infecções Respiratórias/fisiopatologiaRESUMO
Mouse cathelin-related antimicrobial peptide (CRAMP) and its homologue human cathelicidin (LL-37) play active roles in innate immune responses, angiogenesis, and wound healing. In addition, LL-37/CRAMP fends off microbes and protects against infections in the colon, where the epithelium is exposed to myriad of enteric pathogens. It is increasingly recognized that LL-37/CRAMP maintains colon mucosal barrier integrity, shapes the composition of microbiota, and protects the host from tumorigenesis. In this review, we discuss the importance of LL-37/CRAMP in the homeostasis of the host, with novel findings derived from mice deficient in CRAMP that support the proposition for this natural antimicrobial peptide and an immune modulator as a drug lead for therapeutic development.
Assuntos
Anti-Inflamatórios/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Colo/imunologia , Neoplasias do Colo/microbiologia , Microbioma Gastrointestinal/imunologia , Mucosa Intestinal/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Carcinogênese , Colo/microbiologia , Neoplasias do Colo/imunologia , Homeostase , Humanos , Imunidade nas Mucosas , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Knockout , CatelicidinasRESUMO
Commensal bacteria are critical for physiological functions in the gut, and dysbiosis in the gut may cause diseases. In this article, we report that mice deficient in cathelin-related antimicrobial peptide (CRAMP) were defective in the development of colon mucosa and highly sensitive to dextran sulfate sodium (DSS)-elicited colitis, as well as azoxymethane-mediated carcinogenesis. Pretreatment of CRAMP-/- mice with antibiotics markedly reduced the severity of DSS-induced colitis, suggesting CRAMP as a limiting factor on dysbiosis in the colon. This was supported by observations that wild-type (WT) mice cohoused with CRAMP-/- mice became highly sensitive to DSS-induced colitis, and the composition of fecal microbiota was skewed by CRAMP deficiency. In particular, several bacterial species that are typically found in oral microbiota, such as Mogibacterium neglectum, Desulfovibrio piger, and Desulfomicrobium orale, were increased in feces of CRAMP-/- mice and were transferred to WT mice during cohousing. When littermates of CRAMP+/- parents were examined, the composition of the fecal microbiota of WT pups and heterozygous parents was similar. In contrast, although the difference in fecal microbiota between CRAMP-/- and WT pups was small early on after weaning and single mouse housing, there was an increasing divergence with prolonged single housing. These results indicate that CRAMP is critical in maintaining colon microbiota balance and supports mucosal homeostasis, anti-inflammatory responses, and protection from carcinogenesis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Colo/metabolismo , Colo/microbiologia , Microbioma Gastrointestinal/fisiologia , Homeostase/fisiologia , Microbiota/fisiologia , Animais , Colite/metabolismo , Colite/microbiologia , Modelos Animais de Doenças , Fezes/microbiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas/metabolismo , CatelicidinasRESUMO
The Lin-c-Kit+ Sca-1+ cell population in the bone marrow (BM) serves as the direct precursor for differentiation of myeloid cells. In this study, we report that deficiency in Fpr2, a G protein-coupled chemoattractant receptor in mice, is associated with reduced BM nucleated cells, including CD31+Ly6C+ (granulocytes and monocytes), CD31-/Ly6Cint (granuloid cells), and CD31-/Ly6Chigh (predominantly monocytes) cells. In particular, the number of Lin-c-Kit+Sca-1+ (LKS) cells was reduced in Fpr2-/- mouse BM. This was supported by observations of the reduced incorporation of intraperitoneally injected bromodeoxyuridine by cells in the c-Kit+ population from Fpr2-/- mouse BM. Purified c-Kit+ cells from Fpr2-/- mice showed reduced expansion when cultured in vitro with stem cell factor (SCF). SCF/c-Kit-mediated phosphorylation of P38, STAT1, Akt (Thr-308), and Akt (Ser-473) was also significantly reduced in c-Kit+ cells from Fpr2-/- mice. Furthermore, Fpr2 agonists enhanced SCF-induced proliferation of c-Kit+ cells. Colony-forming unit assays revealed that CFU-granulocyte-macrophage formation of BM cells from Fpr2-/- mice was significantly reduced. After heat-inactivated bacterial stimulation in the airway, the expansion of c-kit+ Sca-1+ cells in BM and recruitment of Ly6G+ cells to the lungs and CD11b+Ly6C+TNFα+ cells to the spleen of Fpr2-/- mice was significantly reduced. These results demonstrate an important role for Fpr2 in the development of myeloid lineage precursors in mouse BM.
Assuntos
Antígenos Ly/análise , Deleção de Genes , Proteínas de Membrana/análise , Células Progenitoras Mieloides/citologia , Proteínas Proto-Oncogênicas c-kit/análise , Receptores de Formil Peptídeo/genética , Animais , Contagem de Células , Linhagem da Célula , Proliferação de Células , Feminino , Masculino , Camundongos , Células Progenitoras Mieloides/metabolismo , Receptores de Formil Peptídeo/análiseRESUMO
Phagocytic cells in fish secrete antimicrobial peptides (AMPs) such as piscidins, glycosaminoglycans such as heparin, and copper ions as first-line immune defenses. Recently, we established that Cu2+ coordination by piscidins 1 (P1) and 3 (P3) enhances their antibacterial activity against membranes and DNA. Interestingly, we noted that physicochemical similarities exist between both piscidins and other AMPs that interact with heparin and induce immune-cell chemotaxis through formyl peptide receptors (FPRs) involved in innate immunity. Thus, we postulated that P1 and P3 interact with heparin and FPRs but that these interactions distinctively depend on Cu2+ Here, we investigate the interactome potentiated by piscidins, heparin, FPR, and Cu2+ Utilizing FPR-transfected cells and neutrophils, we demonstrate that both piscidins exclusively use FPR1 and FPR2 to induce chemotaxis and that Cu2+ reduces their chemotaxis induction. P1 is more effective at activating FPR1 than P3 and other known AMP ligands. Furthermore, the expression of Fpr2 on the surface of neutrophils is down-regulated by both peptides. Copper conjugation of the peptides does not further increase down-regulation, suggesting that the conformational changes induced by the metal translate into reduced FPR efficacy without altering the binding affinity. Using surface plasmon resonance, we show that piscidin-heparin interactions are Cu2+-dependent and reduced at the acidic pH of phagosomes. Although heparin decreases the antimicrobial activity of P3-Cu2+, it does not affect bacterial killing by P1-Cu2+ Copper's effects on modulating the micromolar-range interactions of both piscidins with FPR and heparin suggest that the interactome of these distinct immune agents plays an important role in innate immunity. The interactions between diverse host-defense molecules uncovered here may help inform the design of novel therapeutics to treat immune-related diseases.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cobre/farmacologia , Proteínas de Peixes/farmacologia , Heparina/imunologia , Mastócitos/efeitos dos fármacos , Receptores de Formil Peptídeo/imunologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bass , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Quimiotaxia/efeitos dos fármacos , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Cobre/química , Cobre/metabolismo , Proteínas de Peixes/síntese química , Proteínas de Peixes/metabolismo , Células HEK293 , Heparina/química , Heparina/metabolismo , Humanos , Imunidade Inata , Mastócitos/citologia , Mastócitos/imunologia , Camundongos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Cultura Primária de Células , Isoformas de Proteínas/síntese química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Receptores de Formil Peptídeo/genética , Técnicas de Síntese em Fase SólidaRESUMO
Inflammation is associated with a variety of diseases. The hallmark of inflammation is leukocyte infiltration at disease sites in response to pathogen- or damage-associated chemotactic molecular patterns (PAMPs and MAMPs), which are recognized by a superfamily of seven transmembrane, Gi-protein-coupled receptors (GPCRs) on cell surface. Chemotactic GPCRs are composed of two major subfamilies: the classical GPCRs and chemokine GPCRs. Formyl-peptide receptors (FPRs) belong to the classical chemotactic GPCR subfamily with unique properties that are increasingly appreciated for their expression on diverse host cell types and the capacity to interact with a plethora of chemotactic PAMPs and MAMPs. Three FPRs have been identified in human: FPR1-FPR3, with putative corresponding mouse counterparts. FPR expression was initially described in myeloid cells but subsequently in many non-hematopoietic cells including cancer cells. Accumulating evidence demonstrates that FPRs possess multiple functions in addition to controlling inflammation, and participate in the processes of many pathophysiologic conditions. They are not only critical mediators of myeloid cell trafficking, but are also implicated in tissue repair, angiogenesis and protection against inflammation-associated tumorigenesis. A series recent discoveries have greatly expanded the scope of FPRs in host defense which uncovered the essential participation of FPRs in step-wise trafficking of myeloid cells including neutrophils and dendritic cells (DCs) in host responses to bacterial infection, tissue injury and wound healing. Also of great interest is the FPRs are exploited by malignant cancer cells for their growth, invasion and metastasis. In this article, we review the current understanding of FPRs concerning their expression in a vast array of cell types, their involvement in guiding leukocyte trafficking in pathophysiological conditions, and their capacity to promote the differentiation of immune cells, their participation in tumor-associated inflammation and cancer progression. The close association of FPRs with human diseases and cancer indicates their potential as targets for the development of therapeutics.
Assuntos
Inflamação/metabolismo , Inflamação/patologia , Receptores de Formil Peptídeo/metabolismo , Animais , Diferenciação Celular/fisiologia , Humanos , Leucócitos/metabolismo , Leucócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais/fisiologiaRESUMO
Mouse formylpeptide receptor 2 (Fpr2) is a homologue of the human G-protein coupled chemoattractant receptor FPR2, which interacts with pathogen and host-derived chemotactic agonists. Our previous studies revealed reduced allergic airway inflammation and immune responses in Fpr2-deficient (Fpr2(-/-)) mice in association with diminished dendritic cell (DC) recruitment into the airway and draining lymph nodes. These defects prompted us to investigate the potential changes in the differentiation and maturation of DCs caused by Fpr2 deficiency. Bone marrow monocytes from Fpr2(-/-) mouse mice incubated with GM-CSF and IL-4 in vitro showed normal expression of markers of immature DCs. However, upon stimulation with the TLR4 agonist LPS, Fpr2(-/-) mouse DCs failed to express normal levels of maturation markers with reduced production of IL-12 and diminished chemotaxis in response to the DC homing chemokine CCL21. Fpr2(-/-) DCs also failed to induce allogeneic T-cell proliferation in vitro, and their recruitment into the T-cell zones of the spleen was reduced after antigen immunization. The capacity of Fpr2 to sustain normal DC maturation was dependent on its interaction with an endogenous ligand CRAMP expressed by DCs, because neutralization of either Fpr2 or CRAMP inhibited DC maturation in response to LPS. We additionally observed that the presence of exogenous CRAMP in culture increased the sensitivity of WT mouse DCs to LPS stimulation. The importance of CRAMP for DC maturation was further demonstrated by the observations that DCs from CRAMP(-/-) mice expressed lower levels of costimulatory molecules and MHC II and exhibited poor chemotaxis in response to CCL21 after LPS stimulation. Our observations indicate a nonredundant role for Fpr2 and its agonist CRAMP in DC maturation in immune responses.
Assuntos
Catelicidinas/metabolismo , Células Dendríticas/metabolismo , Monócitos/metabolismo , Receptores de Formil Peptídeo/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos , Catelicidinas/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Quimiocina CCL21/genética , Quimiocina CCL21/metabolismo , Células Dendríticas/citologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interleucina-12/genética , Interleucina-12/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Knockout , Monócitos/citologia , Receptores de Formil Peptídeo/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Chemoattractant receptors regulate leukocyte accumulation at sites of inflammation. In allergic airway inflammation, although a chemokine receptor CCR2 was implicated in mediating monocyte-derived dendritic cell (DC) recruitment into the lung, we previously also discovered reduced accumulation of DCs in the inflamed lung in mice deficient in formylpeptide receptor Fpr2 (Fpr2(-/-)). We therefore investigated the role of Fpr2 in the trafficking of monocyte-derived DCs in allergic airway inflammation in cooperation with CCR2. We report that in allergic airway inflammation, CCR2 mediated the recruitment of monocyte-derived DCs to the perivascular region, and Fpr2 was required for further migration of the cells into the bronchiolar area. We additionally found that the bronchoalveolar lavage liquid from mice with airway inflammation contained both the CCR2 ligand CCL2 and an Fpr2 agonist CRAMP. Furthermore, similar to Fpr2(-/-) mice, in the inflamed airway of CRAMP(-/-) mice, DC trafficking into the peribronchiolar areas was diminished. Our study demonstrates that the interaction of CCR2 and Fpr2 with their endogenous ligands sequentially mediates the trafficking of DCs within the inflamed lung.
Assuntos
Asma/metabolismo , Células Dendríticas/metabolismo , Monócitos/metabolismo , Receptores CCR2/metabolismo , Receptores de Formil Peptídeo/metabolismo , Transdução de Sinais , Animais , Peptídeos Catiônicos Antimicrobianos , Asma/genética , Asma/patologia , Catelicidinas/genética , Catelicidinas/metabolismo , Movimento Celular/genética , Células Dendríticas/patologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Knockout , Monócitos/patologia , Receptores CCR2/genética , Receptores de Formil Peptídeo/genéticaRESUMO
Antigen-presenting cells (APCs) act as vehicles that transfer HIV to their target CD4(+) cells through an intercellular junction, termed the virologic synapse. The molecules that are involved in this process remain largely unidentified. In this study, we used photoaffinity labeling and a proteomic approach to identify new proteins that facilitate HIV-1 transfer. We identified ectopic mitochondrial ATP synthase as a factor that mediates HIV-1 transfer between APCs and CD4(+) target cells. Monoclonal antibodies against the ß-subunit of ATP synthase inhibited APC-mediated transfer of multiple strains HIV-1 to CD4(+) target cells. Likewise, the specific inhibitors of ATPase, citreoviridin and IF1, completely blocked APC-mediated transfer of HIV-1 at the APC-target cell interaction step. Confocal fluorescent microscopy showed localization of extracellular ATP synthase at junctions between APC and CD4(+) target cells. We conclude that ectopic ATP synthase could be an accessible molecular target for inhibiting HIV-1 proliferation in vivo.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , HIV-1/metabolismo , ATPases Mitocondriais Próton-Translocadoras/fisiologia , Anticorpos/farmacologia , Apresentação de Antígeno/fisiologia , Células Apresentadoras de Antígenos/imunologia , Transporte Biológico/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Células HEK293 , HIV-1/imunologia , Células HeLa , Humanos , Junções Intercelulares/imunologia , Junções Intercelulares/metabolismo , ATPases Mitocondriais Próton-Translocadoras/imunologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Marcadores de Fotoafinidade/farmacologia , Transporte Proteico/imunologia , Coloração e Rotulagem/métodos , Distribuição TecidualRESUMO
The plasma lipoprotein-associated apolipoproteins (apo) A-I and apoE have well described anti-inflammatory actions in the cardiovascular system, and mimetic peptides that retain these properties have been designed as therapeutics. The anti-inflammatory mechanisms of apolipoprotein mimetics, however, are incompletely defined. Whether circulating apolipoproteins and their mimetics regulate innate immune responses at mucosal surfaces, sites where transvascular emigration of leukocytes is required during inflammation, remains unclear. Herein, we report that Apoai(-/-) and Apoe(-/-) mice display enhanced recruitment of neutrophils to the airspace in response to both inhaled lipopolysaccharide and direct airway inoculation with CXCL1. Conversely, treatment with apoA-I (L-4F) or apoE (COG1410) mimetic peptides reduces airway neutrophilia. We identify suppression of CXCR2-directed chemotaxis as a mechanism underlying the apolipoprotein effect. Pursuing the possibility that L-4F might suppress chemotaxis through heterologous desensitization, we confirmed that L-4F itself induces chemotaxis of human PMNs and monocytes. L-4F, however, fails to induce a calcium flux. Further exploring structure-function relationships, we studied the alternate apoA-I mimetic L-37pA, a bihelical analog of L-4F with two Leu-Phe substitutions. We find that L-37pA induces calcium and chemotaxis through formyl peptide receptor (FPR)2/ALX, whereas its D-stereoisomer (i.e. D-37pA) blocks L-37pA signaling and induces chemotaxis but not calcium flux through an unidentified receptor. Taken together, apolipoprotein mimetic peptides are novel chemotactic agents that possess complex structure-activity relationships to multiple receptors, displaying anti-inflammatory efficacy against innate immune responses in the airway.
Assuntos
Apolipoproteína A-I/farmacologia , Apolipoproteínas E/farmacologia , Materiais Biomiméticos/farmacologia , Quimiotaxia/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Peptídeos/farmacologia , Animais , Apolipoproteína A-I/genética , Sinalização do Cálcio/efeitos dos fármacos , Quimiocina CXCL1/farmacologia , Quimiotaxia/fisiologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/fisiologia , Neutrófilos/citologia , Receptores de Formil Peptídeo/genética , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/genética , Receptores de Lipoxinas/metabolismo , Relação Estrutura-AtividadeRESUMO
The hepatitis C virus (HCV) nonstructural 5A, a phosphorylated zinc metalloprotein, is an essential component of the HCV replication complex. An amphipathic α-helical peptide (HCV peptide [C5A]) derived from nonstructural 5A membrane anchor domain possesses potent anti-HCV and anti-HIV activity in vitro. In this study, we aimed to investigate the potential of HCV peptide (C5A) to regulate host immune responses. The capacity of HCV peptide (C5A) in vitro to induce migration and calcium mobilization of human phagocytes and chemoattractant receptor-transfected cells was investigated. The recruitment of phagocytes in vivo induced by HCV peptide (C5A) and its adjuvant activity were examined. The results revealed that HCV peptide (C5A) was a chemoattractant and activator of human phagocytic leukocytes by using a G-protein coupled receptor, namely formyl peptide receptor. In mice, HCV peptide (C5A) induced massive phagocyte infiltration after injection in the air pouch or the s.c. region. HCV peptide (C5A) also acted as an immune adjuvant by enhancing specific T cell responses to Ag challenge in mice. Our results suggest that HCV peptide (C5A) derived from HCV regulates innate and adaptive immunity in the host by activating the formyl peptide receptor.
Assuntos
Fragmentos de Peptídeos/fisiologia , Fagócitos/imunologia , Fagócitos/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Proteínas não Estruturais Virais/fisiologia , Imunidade Adaptativa , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Imunidade Inata , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/virologia , Fragmentos de Peptídeos/química , Fagócitos/virologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Receptores de Formil Peptídeo/fisiologia , Receptores de Lipoxinas/fisiologia , Proteínas não Estruturais Virais/químicaRESUMO
Formyl peptide receptor 2 (FPR2) and its mouse counterpart Fpr2 are the members of the G protein-coupled receptor (GPCR) family. FPR2 is the only member of the FPRs that interacts with ligands from different sources. FPR2 is expressed in myeloid cells as well as epithelial cells, endothelial cells, neurons, and hepatocytes. During the past years, some unusual properties of FPR2 have attracted intense attention because FPR2 appears to possess dual functions by activating or inhibiting intracellular signal pathways based on the nature, concentration of the ligands, and the temporal and spatial settings of the microenvironment in vivo, the cell types it interacts with. Therefore, FPR2 controls an abundant array of developmental and homeostatic signaling cascades, in addition to its "classical" capacity to mediate the migration of hematopoietic and non-hematopoietic cells including malignant cells. In this review, we summarize recent development in FPR2 research, particularly in its role in diseases, therefore helping to establish FPR2 as a potential target for therapeutic intervention.
Assuntos
Células Endoteliais , Receptores de Formil Peptídeo , Camundongos , Animais , Receptores de Formil Peptídeo/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais , Receptores de Lipoxinas/metabolismoRESUMO
BACKGROUND: Formyl peptide receptor 2 (Fpr2) plays a crucial role in colon homeostasis and microbiota balance. Commensal E. coli is known to promote the regeneration of damaged colon epithelial cells. The aim of the study was to investigate the connection between E. coli and Fpr2 in the recovery of colon epithelial cells. RESULTS: The deficiency of Fpr2 was associated with impaired integrity of the colon mucosa and an imbalance of microbiota, characterized by the enrichment of Proteobacteria in the colon. Two serotypes of E. coli, O22:H8 and O91:H21, were identified in the mouse colon through complete genome sequencing. E. coli O22:H8 was found to be prevalent in the gut of mice and exhibited lower virulence compared to O91:H21. Germ-free (GF) mice that were pre-orally inoculated with E. coli O22:H8 showed reduced susceptibility to chemically induced colitis, increased proliferation of epithelial cells, and improved mouse survival. Following infection with E. coli O22:H8, the expression of Fpr2 in colon epithelial cells was upregulated, and the products derived from E. coli O22:H8 induced migration and proliferation of colon epithelial cells through Fpr2. Fpr2 deficiency increased susceptibility to chemically induced colitis, delayed the repair of damaged colon epithelial cells, and heightened inflammatory responses. Additionally, the population of E. coli was observed to increase in the colons of Fpr2-/- mice with colitis. CONCLUSION: Commensal E. coli O22:H8 stimulated the upregulation of Fpr2 expression in colon epithelial cells, and the products from E. coli induced migration and proliferation of colon epithelial cells through Fpr2. Fpr2 deficiency led to an increased E. coli population in the colon and delayed recovery of damaged colon epithelial cells in mice with colitis. Therefore, Fpr2 is essential for the effects of commensal E. coli on colon epithelial cell recovery.
RESUMO
Highly malignant human gliomas overexpress the G-protein-coupled chemoattractant receptor formyl peptide receptor (FPR1), which promotes tumor progression when activated. Our previous studies demonstrated that necrotic glioblastoma cells release chemotactic agonist(s) that activate FPR1 on viable tumor cells. In the present study, we identified an FPR1 agonist released by necrotic human glioblastoma cells. Necrotic tumor cell supernatant (NecSup) contained Annexin 1 (Anx A1), a chemotatic polypeptide agonist for FPR1. Immunoabsorption of Anx A1 with a specific antibody markedly reduced the chemotactic activity of NecSup for tumor cells and diminished its capacity to promote tumor cell growth, invasion, and colony formation on soft agar. In addition, Anx A1 was present in tumor xenografts formed by human glioblastoma cells in nude mice. Anx A1 knockdown significantly reduced the tumorigenicity of glioblastoma cells in nude mice, but FPR1/Anx A1 double knockdown diminished tumor growth even further. The clinical relevance of Anx A1 in gliomas was supported by the observation that Anx A1 was more highly expressed in poorly differentiated human primary gliomas compared with lower grade tumors. Our study implicates Anx A1 as a major component in necrotic tumor cell-derived stimulants of the growth of glioblastoma via the activation of FPR1.
Assuntos
Anexina A1/metabolismo , Glioblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Formil Peptídeo/agonistas , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica , Quimiotaxia/fisiologia , Técnicas de Silenciamento de Genes , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Necrose , Transplante de Neoplasias , Transplante HeterólogoRESUMO
The formylpeptide receptor-like 1, now officially termed FPR2, in human and its mouse homolog mFPR2 mediate leukocyte migration in response to agonists associated with inflammation and immune responses. To clarify the in vivo role of the receptor, we generated mice deficient in mFPR2. mFPR2(-/-) mice showed markedly reduced severity in OVA/alum-induced allergic airway inflammation. This was associated with diminished recruitment of CD11c(+) dendritic cells into the airway mucosa and secondary lymphoid organs, as well as reduced production of Type 2 cytokines and Igs. We also found that the bronchoalveolar lavage fluid from wild type mice with airway inflammation contained mFPR2 agonist activity. This study reveals a critical role for mFPR2 in the progression of allergic airway inflammation and immune responses.
Assuntos
Hipersensibilidade/imunologia , Inflamação/imunologia , Pulmão/imunologia , Receptores de Formil Peptídeo/imunologia , Hipersensibilidade Respiratória/imunologia , Alérgenos/imunologia , Alérgenos/farmacologia , Compostos de Alúmen/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Separação Celular , Quimiotaxia de Leucócito/imunologia , Citocinas/análise , Citocinas/biossíntese , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Imuno-Histoquímica , Inflamação/patologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Ovalbumina/farmacologia , Hipersensibilidade Respiratória/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Background: Hepatocellular carcinoma (HCC) is one of the most lethal human tumors with extensive intratumor heterogeneity (ITH). Serine protease 3 (PRSS3) is an indispensable member of the trypsin family and has been implicated in the pathogenesis of several malignancies, including HCC. However, the paradoxical effects of PRSS3 on carcinogenesis due to an unclear molecular basis impede the utilization of its biomarker potential. We hereby explored the contribution of PRSS3 transcripts to tumor functional heterogeneity by systematically dissecting the expression of four known splice variants of PRSS3 (PRSS3-SVs, V1~V4) and their functional relevance to HCC. Methods: The expression and DNA methylation of PRSS3 transcripts and their associated clinical relevance in HCC were analyzed using several publicly available datasets and validated using qPCR-based assays. Functional experiments were performed in gain- and loss-of-function cell models, in which PRSS3 transcript constructs were separately transfected after deleting PRSS3 expression by CRISPR/Cas9 editing. Results: PRSS3 was aberrantly differentially expressed toward bipolarity from very low (PRSS3Low ) to very high (PRSS3High ) expression across HCC cell lines and tissues. This was attributable to the disruption of PRSS3-SVs, in which PRSS3-V2 and/or PRSS3-V1 were dominant transcripts leading to PRSS3 expression, whereas PRSS3-V3 and -V4 were rarely or minimally expressed. The expression of PRSS3-V2 or -V1 was inversely associated with site-specific CpG methylation at the PRSS3 promoter region that distinguished HCC cells and tissues phenotypically between hypermethylated low-expression (mPRSS3-SVLow ) and hypomethylated high-expression (umPRSS3-SVHigh ) groups. PRSS3-SVs displayed distinct functions from oncogenic PRSS3-V2 to tumor-suppressive PRSS3-V1, -V3 or PRSS3-V4 in HCC cells. Clinically, aberrant expression of PRSS3-SVs was translated into divergent relevance in patients with HCC, in which significant epigenetic downregulation of PRSS3-V2 was seen in early HCC and was associated with favorable patient outcome. Conclusions: These results provide the first evidence for the transcriptional and functional characterization of PRSS3 transcripts in HCC. Aberrant expression of divergent PRSS3-SVs disrupted by site-specific CpG methylation may integrate the effects of oncogenic PRSS3-V2 and tumor-suppressive PRSS3-V1, resulting in the molecular diversity and functional plasticity of PRSS3 in HCC. Dysregulated expression of PRSS3-V2 by site-specific CpG methylation may have potential diagnostic value for patients with early HCC.