RESUMO
Body fluid detection is an important component in the toolbox of forensic scientists, with saliva playing a particularly critical role in forensic evidence. Given that each body fluid possesses a distinct microbiome, the identification of body fluid based on specific representatives of the microbiota presents an appealing approach for forensic applications. In this study, we have developed a real-time polymerase chain reaction (RT-PCR)-based method for the precise identification of saliva, focusing on three bacteria highly associated with saliva but not with other tested body fluids -Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus salivarius. The inclusion of these three bacterial species enhances the accuracy of detection and reinforces validation. Notably, specific identification of saliva was achievable even at low concentrations where Phadebas, a commonly used method for saliva detection, proved ineffective. Importantly, bacteria-based saliva detection utilizes DNA generated for small tandem repeats (STR) profiling, facilitating seamless integration into forensic laboratories and optimizing DNA sample utilization. This study collectively proposes an effective bacterial DNA-based approach for saliva identification, demonstrating promising potential for forensic applications.
Assuntos
DNA Bacteriano , Fusobacterium nucleatum , Porphyromonas gingivalis , Reação em Cadeia da Polimerase em Tempo Real , Saliva , Streptococcus salivarius , Saliva/microbiologia , Humanos , Fusobacterium nucleatum/isolamento & purificação , Fusobacterium nucleatum/genética , Porphyromonas gingivalis/isolamento & purificação , Porphyromonas gingivalis/genética , Streptococcus salivarius/isolamento & purificação , Streptococcus salivarius/genética , Repetições de Microssatélites , Impressões Digitais de DNA , RNA Ribossômico 16S/genéticaRESUMO
The postnatal interaction between microbiota and the immune system establishes lifelong homeostasis at mucosal epithelial barriers, however, the barrier-specific physiological activities that drive the equilibrium are hardly known. During weaning, the oral epithelium, which is monitored by Langerhans cells (LC), is challenged by the development of a microbial plaque and the initiation of masticatory forces capable of damaging the epithelium. Here we show that microbial colonization following birth facilitates the differentiation of oral LCs, setting the stage for the weaning period, in which adaptive immunity develops. Despite the presence of the challenging microbial plaque, LCs mainly respond to masticatory mechanical forces, inducing adaptive immunity, to maintain epithelial integrity that is also associated with naturally occurring alveolar bone loss. Mechanistically, masticatory forces induce the migration of LCs to the lymph nodes, and in return, LCs support the development of immunity to maintain epithelial integrity in a microbiota-independent manner. Unlike in adult life, this bone loss is IL-17-independent, suggesting that the establishment of oral mucosal homeostasis after birth and its maintenance in adult life involve distinct mechanisms.
Assuntos
Células de Langerhans , Microbiota , Adulto , Humanos , Interleucina-17 , Homeostase , Imunidade Adaptativa , Placa AmiloideRESUMO
While saliva regulates the interplay between the microbiota and the oral immune system, the mechanisms establishing postnatal salivary immunity are ill-defined. Here, we show that high levels of neutrophils and neonatal Fc receptor (FcRn)-transferred maternal IgG are temporarily present in the neonatal murine salivary glands in a microbiota-independent manner. During weaning, neutrophils, FcRn, and IgG decrease in the salivary glands, while the polymeric immunoglobulin receptor (pIgR) is upregulated in a growth arrest-specific 6 (GAS6)-dependent manner independent of the microbiota. Production of salivary IgA begins following weaning and relies on CD4-help, IL-17, and the microbiota. The weaning phase is characterized by a transient accumulation of dendritic cells capable of migrating from the oral mucosa to the salivary glands upon exposure to microbial challenges and activating T cells. This study reveals the postnatal mechanisms developed in the salivary glands to induce immunity and proposes the salivary glands as an immune inductive site.
Assuntos
Microbiota , Receptores de Imunoglobulina Polimérica , Camundongos , Animais , Saliva , Glândulas Salivares , Imunoglobulina GRESUMO
Unlike epidermal Langerhans cells (LCs) that originate from embryonic precursors and are self-renewed locally, mucosal LCs arise and are replaced by circulating bone marrow (BM) precursors throughout life. While the unique lifecycle of epidermal LCs is associated with an age-dependent decrease in their numbers, whether and how aging has an impact on mucosal LCs remains unclear. Focusing on gingival LCs we found that mucosal LCs are reduced with age but exhibit altered morphology with that observed in aged epidermal LCs. The reduction of gingival but not epidermal LCs in aged mice was microbiota-dependent; nevertheless, the impact of the microbiota on gingival LCs was indirect. We next compared the ability of young and aged BM precursors to differentiate to mucosal LCs. Mixed BM chimeras, as well as differentiation cultures, demonstrated that aged BM has intact if not superior capacity to differentiate into LCs than young BM. This was in line with the higher percentages of mucosal LC precursors, pre-DCs, and monocytes, detected in aged BM. These findings suggest that while aging is associated with reduced LC numbers, the niche rather than the origin controls this process in mucosal barriers.
Assuntos
Diferenciação Celular , Microambiente Celular/imunologia , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Mucosa/imunologia , Mucosa/metabolismo , Fatores Etários , Envelhecimento/fisiologia , Animais , Biomarcadores , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Microambiente Celular/genética , Senescência Celular/genética , Senescência Celular/imunologia , Células Epidérmicas/imunologia , Células Epidérmicas/metabolismo , Epiderme/imunologia , Epiderme/metabolismo , Epiderme/microbiologia , Expressão Gênica , Gengiva/imunologia , Gengiva/metabolismo , Gengiva/microbiologia , Imunofenotipagem , Células de Langerhans/citologia , Camundongos , Microbiota , Mucosa/microbiologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
NK cells interact with a wide variety of hazardous cells including pathogen-infected and tumor cells. NKp46 is a specific NK killer receptor that recognizes various influenza hemagglutinins and unknown tumor ligands. It was recently shown that NKp46 plays a significant role in the in vivo eradication of tumor cells; however, the role played by NKp46 in vivo with regard to tumor development is still unclear. In this study, we used the 3-methylcholanthrene (MCA)-induced fibrosarcoma model in NKp46-deficient mice to test the NKp46 recognition of carcinogen-induced tumors. We show that although the rate of MCA-induced tumor formation was similar in the presence and in the absence of NKp46, the expression of its unknown ligands was NKp46 dependent. The unknown NKp46 ligands were nearly absent in tumors that originated in wild-type mice, whereas they were detected in tumors that originated in the NKp46-deficient mice. We demonstrate that the interactions between NKp46 and its MCA tumor-derived ligands lead to the secretion of IFN-gamma but not to the elimination of the MCA-derived tumor cells. In addition, we show that the in vivo growth of MCA-derived tumor cells expressing high levels of the NKp46 ligands is NKp46 and IFN-gamma dependent. Thus, we present in this study a novel NKp46-mediated mechanism of tumor editing.
Assuntos
Antígenos Ly/fisiologia , Fibrossarcoma/imunologia , Fibrossarcoma/metabolismo , Vigilância Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/fisiologia , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Técnicas de Cocultura , Fibrossarcoma/induzido quimicamente , Fibrossarcoma/patologia , Interferon gama/metabolismo , Células Matadoras Naturais/patologia , Ligantes , Metilcolantreno/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptor 1 Desencadeador da Citotoxicidade Natural/deficiência , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Evasão Tumoral/imunologiaRESUMO
The killing activity of NK cells is carried out by several activating NK receptors, which includes NKp46, NKp44, NKp30, NKp80, NKG2D, and 2B4. The ligands of these receptors are either self-derived, pathogen-derived, stress-induced ligands or tumor ligands. Importantly, none of these killer ligands are expressed on NK cells and thus self-killing of NK cells is prevented. A notable exception with this regard, is the ligand of the 2B4 receptor. This unusual receptor can exert both activating and inhibiting signals; however, in human NK cells, it serves mainly as an activating receptor. The ligand of 2B4 is CD48 and in contrast to the ligands of all the other NK activating receptors, CD48 is also present on NK cells. Thus, NK cells might be at risk for self-killing that is mediated via the 2B4-CD48 interaction. In this study, we identify a novel mechanism that prevents this self-killing as we show that the association of the MHC class I proteins with the 2B4 receptor, both present on NK cells, results in the attenuation of the 2B4-mediated self-killing of NK cells.
Assuntos
Antígenos CD/metabolismo , Testes Imunológicos de Citotoxicidade , Regulação para Baixo/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/metabolismo , Animais , Antígenos CD/imunologia , Antígenos CD/fisiologia , Antígeno CD48 , Linhagem Celular Transformada , Linhagem Celular Tumoral , Inibidores do Crescimento/antagonistas & inibidores , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/fisiologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Humanos , Células Matadoras Naturais/citologia , Ligantes , Camundongos , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/fisiologia , Transdução de Sinais/imunologia , Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
The activity of NK cells is regulated by activating receptors that recognize mainly stress-induced ligands and by inhibitory receptors that recognize mostly MHC class I proteins on target cells. Comparing the cytoplasmic tail sequences of various MHC class I proteins revealed the presence of unique cysteine residues in some of the MHC class I molecules which are absent in others. To study the role of these unique cysteines, we performed site specific mutagenesis, generating MHC class I molecules lacking these cysteines, and demonstrated that their expression on the cell surface was impaired. Surprisingly, we demonstrated that these cysteines are crucial for the surface binding of the leukocyte Ig-like receptor 1 inhibitory receptor to the MHC class I proteins, but not for the binding of the KIR2DL1 inhibitory receptor. In addition, we demonstrated that the cysteine residues in the cytoplasmic tail of MHC class I proteins are crucial for their egress from the endoplasmic reticulum and for their palmitoylation, thus probably affecting their expression on the cell surface. Finally, we show that the cysteine residues are important for proper extracellular conformation. Thus, although the interaction between leukocyte Ig-like receptor 1 and MHC class I proteins is formed between two extracellular surfaces, the intracellular components of MHC class I proteins play a crucial role in this recognition.
Assuntos
Cisteína/fisiologia , Líquido Extracelular/imunologia , Antígeno HLA-B7/imunologia , Antígenos HLA-C/imunologia , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Linhagem Celular Transformada , Membrana Celular/genética , Membrana Celular/imunologia , Cisteína/genética , Citotoxicidade Imunológica/genética , Líquido Extracelular/metabolismo , Antígeno HLA-B7/biossíntese , Antígeno HLA-B7/genética , Antígeno HLA-B7/metabolismo , Antígenos HLA-C/biossíntese , Antígenos HLA-C/genética , Antígenos HLA-C/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/imunologia , Receptores KIR2DL1RESUMO
The inhibition of NK cell killing is mainly mediated via the interaction of NK inhibitory receptors with MHC class I proteins. In addition, we have previously demonstrated that NK cells are inhibited in a class I MHC-independent manner via homophilic carcinoembryonic Ag (CEA) cell adhesion molecules (CEACAM1)-CEACAM1 and heterophilic CEACAM1-CEA interactions. However, the cross-talk between immune effector cells and their target cells is not limited to cell interactions per se, but also involves a specific exchange of proteins. The reasons for these molecular exchanges and the functional outcome of this phenomenon are still mostly unknown. In this study, we show that NK cells rapidly and specifically acquire CEA molecules from target cells. We evaluated the role of cytotoxicity in the acquisition of CEA and demonstrated it to be mostly killing independent. We further demonstrate that CEA transfer requires a specific interaction with an unknown putative NK cell receptor and that carbohydrates are probably involved in CEA recognition and acquisition by NK cells. Functionally, the killing of bulk NK cultures was inhibited by CEA-expressing cells, suggesting that this putative receptor is an inhibitory receptor.
Assuntos
Antígeno Carcinoembrionário/metabolismo , Células Matadoras Naturais/metabolismo , Neoplasias/metabolismo , Comunicação Parácrina , Apoptose , Metabolismo dos Carboidratos , Carboidratos/química , Antígeno Carcinoembrionário/química , Membrana Celular/metabolismo , Células Cultivadas , Técnicas de Cocultura , Antígenos HLA-C/metabolismo , Humanos , Células Matadoras Naturais/citologiaRESUMO
As initially described by K. Karre and colleagues in the missing self hypothesis, cells expressing self-MHC class I proteins are protected from NK cells attack. In contrast, reduction in the expression of MHC class I molecules due to viral infection or tumor transformation result in the killing of these "abnormal" cells by NK cells via NK-activating receptors. Thus, NK killing of target cells is determined by both negative signals coming from MHC class I proteins and by positive signals derived from the activating ligands. The bound peptide in MHC class I play an important role in the balanced recognition of NK cells. The peptide stabilizes the MHC complex and interacts directly with the NK inhibitory receptors, thus participating in the determination of the fate of the target cells. In this study we demonstrate that posttranslational modifications such as phosphorylation of the presented peptide altered the ability of NK cells to recognize MHC class I molecules. By using a consensus peptide (QYDDAVYKL) that binds HLA-Cw4 in which different positions in the bound peptide were modified by serine phosphorylation, we observed a reduction in KIR2DL1 binding that led to decreased protection from NK killing. Therefore, it might be possible that alteration in the phosphorylation pattern during tumor transformation or viral infection may result in less inhibition and, consequently, improved NK cell killing.