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BACKGROUND: Human carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is an inhibitory cell surface protein that functions through homophilic and heterophilic ligand binding. Its expression on immune cells in human tumors is poorly understood. METHODS: An antibody that distinguishes human CEACAM1 from other highly related CEACAM family members was labeled with 159Tb and inserted into a panel of antibodies that included specificity for programmed cell death protein 1 (PD1) and PD-L1, which are targets of immunotherapy, to gain a data-driven immune cell atlas using cytometry by time-of-flight (CyTOF). A detailed inventory of CEACAM1, PD1, and PD-L1 expression on immune cells in metastatic lesions to lymph node or soft tissues and peripheral blood samples from patients with treatment-naive and -resistant melanoma as well as peripheral blood samples from healthy controls was performed. RESULTS: CEACAM1 is absent or at low levels on healthy circulating immune cells but is increased on immune cells in peripheral blood and tumors of melanoma patients. The majority of circulating PD1-positive NK cells, innate T cells, B cells, monocytic cells, dendritic cells, and CD4+ T cells in the peripheral circulation of treatment-resistant disease co-express CEACAM1 and are demonstrable as discrete populations. CEACAM1 is present on distinct types of cells that are unique to the tumor microenvironment and exhibit expression levels that are highest in treatment resistance; this includes tumor-infiltrating CD8+ T cells. CONCLUSIONS: To the best of our knowledge, this work represents the first comprehensive atlas of CEACAM1 expression on immune cells in a human tumor and reveals an important correlation with treatment-resistant disease. These studies suggest that agents targeting CEACAM1 may represent appropriate partners for PD1-related pathway therapies.
Some proteins, such as programmed cell death protein 1 (PD1), can stop the immune system from attacking cancer cells, allowing cancers to grow. Therapies targeting these proteins can be highly effective, but tumors can become resistant. It is important to identify factors involved in this resistance to develop improved cancer therapies. Human carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a protein that inhibits an immune response and its levels have been associated with poor patient outcomes. We applied a method that allows for the detection of proteins on a single cell to uncover CEACAM1 patterns in melanoma. We found that increased CEACAM1 expression levels on multiple different immune cell types was associated with tumors that were resistant to therapy. These findings may help us to understand the role of CEACAM1 in cancer and to develop better cancer therapies.
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Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. Developing methods that can provide insight into correlations between membrane protein colocalization and interactions is challenging. We report here on a correlated stochastic optical reconstruction microscopy/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Using live cell homoFRET imaging of carcinoembryonic antigen-related cellular adhesion molecule 1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, we revealed highly heterogeneous diffraction-limited structures on the surface of HeLa cells. Furthermore, correlated super-resolved stochastic optical reconstruction microscopy imaging showed that these structures comprised a complex mixture and spatial distribution of self-associated carcinoembryonic antigen-related cellular adhesion molecule 1 molecules. In conclusion, this correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function.
Assuntos
Microscopia , Humanos , Células HeLa , Proteínas de Membrana , Microscopia/métodos , Imagem Óptica , Técnicas CitológicasRESUMO
The commensal bacteria that make up the gut microbiota impact the health of their host on multiple levels. In particular, the interactions taking place between the microbe-associated molecule patterns (MAMPs) and pattern recognition receptors (PRRs), expressed by intestinal epithelial cells (IECs), are crucial for maintaining intestinal homeostasis. While numerous studies showed that TLRs and NLRs are involved in the control of gut homeostasis by commensal bacteria, the role of additional innate immune receptors remains unclear. Here, we seek for novel MAMP-PRR interactions involved in the beneficial effect of the commensal bacterium Akkermansia muciniphila on intestinal homeostasis. We show that A. muciniphila strongly activates NF-κB in IECs by releasing one or more potent activating metabolites into the microenvironment. By using drugs, chemical and gene-editing tools, we found that the released metabolite(s) enter(s) epithelial cells and activate(s) NF-κB via an ALPK1, TIFA and TRAF6-dependent pathway. Furthermore, we show that the released molecule has the biological characteristics of the ALPK1 ligand ADP-heptose. Finally, we show that A. muciniphila induces the expression of the MUC2, BIRC3 and TNFAIP3 genes involved in the maintenance of the intestinal barrier function and that this process is dependent on TIFA. Altogether, our data strongly suggest that the commensal A. muciniphila promotes intestinal homeostasis by activating the ALPK1/TIFA/TRAF6 axis, an innate immune pathway exclusively described so far in the context of Gram-negative bacterial infections.
Assuntos
Microbioma Gastrointestinal , NF-kappa B , Difosfato de Adenosina , Akkermansia , Heptoses , Imunidade Inata , Fator 6 Associado a Receptor de TNF , VerrucomicrobiaRESUMO
BACKGROUND: The ongoing COVID-19 pandemic has resulted in 185 million recorded cases and over 4 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. RESULTS: Here, we describe a novel, intranasally delivered COVID-19 vaccine based on a helper-dependent adenoviral (HD-Ad) vector. The vaccine (HD-Ad_RBD) produces a soluble secreted form of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and we show it induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. CONCLUSION: Our approaches provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.
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While infection by Neisseria gonorrhoeae is often asymptomatic in women, undetected infections can ascend into the upper genital tract to elicit an inflammatory response that manifests as pelvic inflammatory disease, with the outcomes depending on the intensity and duration of inflammation and whether it is localized to the endometrial, fallopian tube, ovarian, and/or other tissues. This review examines the contribution of N. gonorrhoeae versus other potential causes of pelvic inflammatory disease by considering new insights gained through molecular, immunological, and microbiome-based analyses, and the current epidemiological burden of infection, with an aim to highlighting key areas for future study.
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Infecções por Chlamydia/epidemiologia , Gonorreia/epidemiologia , Neisseria gonorrhoeae/isolamento & purificação , Doença Inflamatória Pélvica/epidemiologia , Infecções por Chlamydia/complicações , Endometrite/microbiologia , Endométrio/microbiologia , Endométrio/patologia , Tubas Uterinas/microbiologia , Feminino , Gonorreia/diagnóstico , Humanos , Doença Inflamatória Pélvica/diagnóstico , Doença Inflamatória Pélvica/microbiologiaRESUMO
Sexually transmitted infections are a critical public health issue. However, the mechanisms underlying sexually transmitted infections in women and the link between the infection mechanism and the wide range of clinical outcomes remain elusive due to a lack of research models mimicking human infection in vivo. We established a human cervical tissue explant model to mimic local Neisseria gonorrhoeae (GC) infections. We found that GC preferentially colonize the ectocervix by activating integrin-ß1, which inhibits epithelial shedding. GC selectively penetrate into the squamocolumnar junction (TZ) and endocervical epithelia by inducing ß-catenin phosphorylation, which leads to E-cadherin junction disassembly. Epithelial cells in various cervical regions differentially express carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), the host receptor for GC opacity-associated proteins (OpaCEA). Relatively high levels were detected on the luminal membrane of ecto/endocervical epithelial cells but very low levels intracellularly in TZ epithelial cells. CEACAM-OpaCEA interaction increased ecto/endocervical colonization and reduced endocervical penetration by increasing integrin-ß1 activation and inhibiting ß-catenin phosphorylation respectively, through CEACAM downstream signaling. Thus, the intrinsic properties of cervical epithelial cells and phase-variation of bacterial surface molecules both play a role in controlling GC infection mechanisms and infectivity, preferential colonization or penetration, potentially leading to asymptomatic or symptomatic infection.
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Aderência Bacteriana/fisiologia , Colo do Útero/microbiologia , Gonorreia/microbiologia , Mucosa/microbiologia , Neisseria gonorrhoeae/patogenicidade , Colo do Útero/metabolismo , Feminino , Humanos , Mucosa/metabolismo , Técnicas de Cultura de ÓrgãosRESUMO
The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection.IMPORTANCECandida albicans is a natural member of the human mucosal microbiota that can also cause superficial infections and life-threatening systemic infections, both of which are characterized by inflammation. Host defense relies mainly on the ingestion and destruction of C. albicans by innate immune cells, such as macrophages and neutrophils. Although some C. albicans cells are killed by macrophages, most undergo a morphological change and escape by inducing macrophage pyroptosis. Here, we investigated the C. albicans genes and host factors that promote macrophage pyroptosis in response to intracellular fungi. This work provides a foundation for understanding how host immune cells interact with C. albicans and may lead to effective strategies to modulate inflammation induced by fungal infections.
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Candida albicans/genética , Genes Fúngicos , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Piroptose , Animais , Candida albicans/patogenicidade , Feminino , Ensaios de Triagem em Larga Escala , Evasão da Resposta Imune , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , FagocitoseRESUMO
The gonococcal Opa proteins are an antigenically variable family of surface adhesins that bind human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CEACAM3, CEACAM5, and/or CEACAM6, cell surface glycoproteins that are differentially expressed on a broad spectrum of human cells and tissues. While they are presumed to be important for infection, the significance of various Opa-CEACAM-mediated cellular interactions in the context of the genital tract has remained unclear. Here, we observed that CEACAM1 and CEACAM5 are differentially expressed on epithelia lining the upper and lower portions of the human female genital tract, respectively. Using transgenic mouse lines expressing human CEACAMs in a manner that reflects this differential pattern, we considered the impact of Opa-CEACAM interactions during uncomplicated lower genital tract infections versus during pelvic inflammatory disease. Our results demonstrate that Opa-CEACAM5 binding on vaginal epithelia facilitates the long-term colonization of the lower genital tract, while Opa protein binding to CEACAM1 on uterine epithelia enhances gonococcal association and penetration into these tissues. While these Opa-dependent interactions with CEACAM-expressing epithelial surfaces promote infection, Opa binding by neutrophil-expressed CEACAMs counterbalances this by facilitating more effective gonococcal clearance. Furthermore, during uterine infections, CEACAM-dependent tissue invasion aggravates disease pathology by increasing the acute inflammatory response. Together, these findings demonstrate that the outcome of infection is determined by both the cell type-specific expression of human CEACAMs and the CEACAM specificity of the Opa variants expressed, which combine to determine the level of gonococcal association with the genital mucosa versus the extent of CEACAM-dependent inflammation and gonococcal clearance by neutrophils.
Assuntos
Antígenos CD/metabolismo , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Antígeno Carcinoembrionário/metabolismo , Moléculas de Adesão Celular/metabolismo , Genitália Feminina/patologia , Gonorreia/fisiopatologia , Infecções do Sistema Genital/fisiopatologia , Animais , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Feminino , Proteínas Ligadas por GPI/metabolismo , Perfilação da Expressão Gênica , Genitália Feminina/microbiologia , Gonorreia/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neisseria gonorrhoeae/fisiologia , Infecções do Sistema Genital/microbiologia , Resultado do Tratamento , Útero/microbiologia , Útero/patologia , Vagina/microbiologia , Vagina/patologiaRESUMO
HIV synergy with sexually transmitted co-infections is well-documented in the clinic. Co-infection with Neisseria gonorrhoeae in particular, increases genital HIV shedding and mucosal transmission. However, no animal model of co-infection currently exists to directly explore this relationship or to bridge the gap in understanding between clinical and in vitro studies of this interaction. This study aims to test the feasibility of using a humanized mouse model to overcome this barrier. Combining recent in vivo modelling advancements in both HIV and gonococcal research, we developed a co-infection model by engrafting immunodeficient NSG mice with human CD34+ hematopoietic stem cells to generate humanized mice that permit both systemic HIV infection and genital N. gonorrhoeae infection. Systemic plasma and vaginal lavage titres of HIV were measured in order to assess the impact of gonococcal challenge on viral plasma titres and genital shedding. Engrafted mice showed human CD45+ leukocyte repopulation in blood and mucosal tissues. Systemic HIV challenge resulted in 104-105 copies/mL of viral RNA in blood by week 4 post-infection, as well as vaginal shedding of virus. Subsequent gonococcal challenge resulted in unchanged plasma HIV levels but higher viral shedding in the genital tract, which reflects published clinical observations. Thus, human CD34+ stem cell-transplanted NSG mice represent an experimentally tractable animal model in which to study HIV shedding during gonococcal co-infection, allowing dissection of molecular and immunological interactions between these pathogens, and providing a platform to assess future therapeutics aimed at reducing HIV transmission.
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Antígenos CD34/imunologia , Gonorreia/complicações , Infecções por HIV/complicações , HIV/fisiologia , Neisseria gonorrhoeae/isolamento & purificação , Vagina/virologia , Carga Viral , Eliminação de Partículas Virais , Animais , Feminino , Infecções por HIV/virologia , Humanos , CamundongosRESUMO
The neutrophil-specific innate immune receptor CEACAM3 functions as a decoy to capture Gram-negative pathogens, such as Neisseria gonorrhoeae, that exploit CEACAM family members to adhere to the epithelium. Bacterial binding to CEACAM3 results in their efficient engulfment and triggers activation of an nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-dependent inflammatory response by human neutrophils. Herein, we report that CEACAM3 cross-linking is not sufficient for induction of cytokine production and show that the inflammatory response induced by Neisseria gonorrhoeae infection is elicited by an integration of signals from CEACAM3 and toll-like receptors. Using neutrophils from a human CEACAM-expressing mouse line (CEABAC), we use a genetic approach to reveal a molecular bifurcation of the CEACAM3-mediated antimicrobial and inflammatory responses. Ex vivo experiments with CEABAC-Rac2-/- , CEABAC-Bcl10-/- , and CEABAC-Malt1-/- neutrophils indicate that these effectors are not necessary for gonococcal engulfment, yet all 3 effectors contribute to CEACAM3-mediated cytokine production. Interestingly, although Bcl10 and Malt1 are often inextricably linked, Bcl10 enabled synergy between toll-like receptor 4 and CEACAM3, whereas Malt1 did not. Together, these findings reveal an integration of the specific innate immune receptor CEACAM3 into the network of more conventional pattern recognition receptors, providing a mechanism by which the innate immune system can unleash its response to a relentless pathogen.
Assuntos
Proteína 10 de Linfoma CCL de Células B/genética , Antígeno Carcinoembrionário/imunologia , Gonorreia/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Neisseria gonorrhoeae/imunologia , Neutrófilos/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Antígenos CD/imunologia , Proteína 10 de Linfoma CCL de Células B/imunologia , Moléculas de Adesão Celular/imunologia , Células Cultivadas , Proteínas Ligadas por GPI/imunologia , Gonorreia/microbiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/imunologia , Proteína RAC2 de Ligação ao GTPRESUMO
d-glycero-ß-d-manno-heptose 1,7-biphosphate (HBP) is an enzymatic intermediate in the biosynthesis of the heptose component of lipopolysaccharide (LPS), and was recently revealed to be a pathogen-associated molecular pattern (PAMP) that allows detection of Gram-negative bacteria by the mammalian immune system. Cellular detection of HBP depends upon its stimulation of a cascade that leads to the phosphorylation and assembly of the TRAF-interacting with forkhead-associated domain protein A (TIFA), which activates the transcription factor NF-κB. In this note, an alternate chemical synthesis of HBP is described and its biological activity is established, providing pure material for further assessing and exploiting the biological activity of this compound.
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Heptoses/química , Heptoses/síntese química , Fosfatos/química , Fosfatos/síntese química , Linhagem Celular Tumoral , Técnicas de Química Sintética , Heptoses/farmacologia , Humanos , NF-kappa B/metabolismo , Fosfatos/farmacologiaRESUMO
Helicobacter pylori is a bacterial pathogen that colonizes the human stomach, causing inflammation which, in some cases, leads to gastric ulcers and cancer. The clinical outcome of infection depends on a complex interplay of bacterial, host genetic, and environmental factors. Although H. pylori is recognized by both the innate and adaptive immune systems, this rarely results in bacterial clearance. Gastric epithelial cells are the first line of defense against H. pylori and alert the immune system to bacterial presence. Cytosolic delivery of proinflammatory bacterial factors through the cag type 4 secretion system (cag-T4SS) has long been appreciated as the major mechanism by which gastric epithelial cells detect H. pylori Classically attributed to the peptidoglycan sensor NOD1, recent work has highlighted the role of NOD1-independent pathways in detecting H. pylori; however, the bacterial and host factors involved have remained unknown. Here, we show that bacterially derived heptose-1,7-bisphosphate (HBP), a metabolic precursor in lipopolysaccharide (LPS) biosynthesis, is delivered to the host cytosol through the cag-T4SS, where it activates the host tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA)-dependent cytosolic surveillance pathway. This response, which is independent of NOD1, drives robust NF-κB-dependent inflammation within hours of infection and precedes NOD1 activation. We also found that the CagA toxin contributes to the NF-κB-driven response subsequent to TIFA and NOD1 activation. Taken together, our results indicate that the sequential activation of TIFA, NOD1, and CagA delivery drives the initial inflammatory response in gastric epithelial cells, orchestrating the subsequent recruitment of immune cells and leading to chronic gastritis.IMPORTANCEH. pylori is a globally prevalent cause of gastric and duodenal ulcers and cancer. H. pylori antibiotic resistance is rapidly increasing, and a vaccine remains elusive. The earliest immune response to H. pylori is initiated by gastric epithelial cells and sets the stage for the subsequent immunopathogenesis. This study revealed that host TIFA and H. pylori-derived HBP are critical effectors of innate immune signaling that account for much of the inflammatory response to H. pylori in gastric epithelial cells. HBP is delivered to the host cell via the cag-T4SS at a time point that precedes activation of the previously described NOD1 and CagA inflammatory pathways. Manipulation of the TIFA-driven immune response in the host and/or targeting of ADP-heptose biosynthesis enzymes in H. pylori may therefore provide novel strategies that may be therapeutically harnessed to achieve bacterial clearance.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Epiteliais/microbiologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Helicobacter pylori/imunologia , Transdução de Sinais , Sistemas de Secreção Tipo IV/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Citosol/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Mucosa Gástrica/imunologia , Gastrite/imunologia , Gastrite/microbiologia , Ilhas Genômicas , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Lipopolissacarídeos/biossíntese , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
CEACAM1 is an extensively studied cell surface molecule with established functions in multiple cancer types, as well as in various compartments of the immune system. Due to its multi-faceted role as a recently appreciated immune checkpoint inhibitor and tumor marker, CEACAM1 is an attractive target for cancer immunotherapy. Herein, we highlight CEACAM1's function in various immune compartments and cancer types, including in the context of metastatic disease. This review outlines CEACAM1's role as a therapeutic target for cancer treatment in light of these properties.
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The engraftment of circulating cancer cells at distal sites represents a key step in the metastatic cascade, yet remains an unexplored target for therapeutic intervention. In this study, we establish that a vaccination strategy yielding an antigen-specific TH 9 response induces long term host surveillance and prevents the engraftment of circulating cancer cells. Specifically, we show that vaccination with a recombinant CEA IgV-like N domain, formulated with the TLR3 ligand poly I:C, elicits a CEA-specific TH 9 response, wherein IL-9 secreting TH cells act in concert with CEA N domain-specific antibodies as well as activated mast cells in preventing tumor cell engraftment. The development of this immune response was dependent on TLR3, since interference with the TLR3-dsRNA complex formation led to a reduction in vaccine-imparted protection and a shift in the resulting immune response toward a TH 2 response. These findings point to the existence of an alternate tumor targeting immune mechanism that can be exploited for the purpose of developing vaccine therapies targeting tumor dissemination and engraftment.
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Antígenos de Neoplasias/imunologia , Mastócitos/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Neoplasias/genética , Vacinas Anticâncer/imunologia , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Xenoenxertos , Humanos , Interleucina-9/biossíntese , Mastócitos/metabolismo , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Neoplasias/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/metabolismo , Receptor 3 Toll-Like/metabolismoRESUMO
Infections by Neisseria gonorrhoeae are increasingly common, are often caused by antibiotic-resistant strains, and can result in serious and lasting sequelae, prompting the reemergence of gonococcal disease as a leading global health concern. N. gonorrhoeae is a human-restricted pathogen that primarily colonizes urogenital mucosal surfaces. Disease progression varies greatly between the sexes: men usually present with symptomatic infection characterized by a painful purulent urethral discharge, while in women, the infection is often asymptomatic, with the most severe pathology occurring when the bacteria ascend from the lower genital tract into the uterus and fallopian tubes. Classical clinical studies demonstrated that clinically infectious strains uniformly express Opa adhesins; however, their specificities were unknown at the time. While in vitro studies have since identified CEACAM proteins as the primary target of Opa proteins, the gonococcal specificity for this human family of receptors has not been addressed in the context of natural infection. In this study, we characterize a collection of low-passage-number clinical-specimen-derived N. gonorrhoeae isolates for Opa expression and assess their CEACAM-binding profiles. We report marked in vivo selection for expression of phase-variable Opa proteins that bind CEACAM1 and CEACAM5 but selection against expression of Opa variants that bind to the neutrophil-restricted decoy receptor CEACAM3. This is the first study showing phenotypic selection for distinct CEACAM-binding phenotypes in vivo, and it supports the opposing functions of CEACAMs that facilitate infection versus driving inflammation within the genital tract.
Assuntos
Antígenos CD/metabolismo , Aderência Bacteriana/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Antígeno Carcinoembrionário/metabolismo , Moléculas de Adesão Celular/metabolismo , Gonorreia/imunologia , Adesinas Bacterianas/metabolismo , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Degranulação Celular/imunologia , Linhagem Celular , Colo do Útero/microbiologia , Feminino , Proteínas Ligadas por GPI/metabolismo , Gonorreia/microbiologia , Humanos , Inflamação/imunologia , Masculino , Neisseria gonorrhoeae/imunologia , Neisseria gonorrhoeae/isolamento & purificação , Neutrófilos/imunologia , Ligação Proteica , Isoformas de Proteínas/metabolismo , Uretra/microbiologiaRESUMO
An overwhelming neutrophil-driven response causes both acute symptoms and the lasting sequelae that result from infection with Neisseria gonorrhoeae. Neutrophils undergo an aggressive opsonin-independent response to N. gonorrhoeae, driven by the innate decoy receptor CEACAM3. CEACAM3 is exclusively expressed by human neutrophils, and drives a potent binding, phagocytic engulfment and oxidative killing of Opa-expressing bacteria. In this study, we sought to explore the contribution of neutrophils to the pathogenic inflammatory process that typifies gonorrhea. Genome-wide microarray and biochemical profiling of gonococcal-infected neutrophils revealed that CEACAM3 engagement triggers a Syk-, PKCδ- and Tak1-dependent signaling cascade that results in the activation of an NF-κB-dependent transcriptional response, with consequent production of pro-inflammatory cytokines. Using an in vivo model of N. gonorrhoeae infection, we show that human CEACAM-expressing neutrophils have heightened migration toward the site of the infection where they may be further activated upon Opa-dependent binding. Together, this study establishes that the role of CEACAM3 is not restricted to the direct opsonin-independent killing by neutrophils, since it also drives the vigorous inflammatory response that typifies gonorrhea. By carrying the potential to mobilize increasing numbers of neutrophils, CEACAM3 thereby represents the tipping point between protective and pathogenic outcomes of N. gonorrhoeae infection.
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Biomarcadores/metabolismo , Gonorreia/imunologia , Mediadores da Inflamação/metabolismo , Inflamação/etiologia , Neisseria gonorrhoeae/patogenicidade , Neutrófilos/imunologia , Animais , Aderência Bacteriana , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Gonorreia/metabolismo , Gonorreia/microbiologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Neisseria gonorrhoeae/imunologia , Neutrófilos/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Fagocitose/fisiologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Quinase SykRESUMO
Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) can engage in both cis-homophilic (parallel) oligomerization and trans-homophilic (anti-parallel) binding. In this study, we establish that the CEACAM1 transmembrane domain has a propensity to form cis-dimers via the transmembrane-embedded (432)GXXXG(436) motif and that this basal state is overcome when activated calmodulin binds to the CEACAM1 cytoplasmic domain. Although mutation of the (432)GXXXG(436) motif reduced CEACAM1 oligomerization, it did not affect surface localization of the receptor or influence CEACAM1-dependent cellular invasion by the pathogenic Neisseria. The mutation did, however, have a striking effect on CEACAM1-dependent cellular aggregation, increasing both the kinetics of cell-cell association and the size of cellular aggregates formed. CEACAM1 association with tyrosine kinase c-Src and tyrosine phosphatases SHP-1 and SHP-2 was not affected by the (432)GXXXG(436) mutation, consistent with their association with the monomeric form of wild type CEACAM1. Collectively, our results establish that a dynamic oligomer-to-monomer shift in surface-expressed CEACAM1 facilitates trans-homophilic binding and downstream effector signaling.
Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Multimerização Proteica , Transdução de Sinais , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Antígenos CD/genética , Cálcio/metabolismo , Calmodulina/metabolismo , Adesão Celular , Moléculas de Adesão Celular/genética , Membrana Celular/metabolismo , Células HeLa , Humanos , Immunoblotting , Microscopia Confocal , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Quinases da Família src/metabolismoRESUMO
Neisseria meningitidis (Nme) asymptomatically colonizes the human nasopharynx, yet can initiate rapidly-progressing sepsis and meningitis in rare instances. Understanding the meningococcal lifestyle within the nasopharyngeal mucosa, a phase of infection that is prerequisite for disease, has been hampered by the lack of animal models. Herein, we compare mice expressing the four different human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) that can bind the neisserial Opa protein adhesins, and find that expression of human CEACAM1 is necessary and sufficient to establish intranasal colonization. During infection, in vivo selection for phase variants expressing CEACAM1-specific Opa proteins occurs, allowing mucosal attachment and entry into the subepithelial space. Consistent with an essential role for Opa proteins in this process, Opa-deficient meningococci were unable to colonize the CEACAM1-humanized mice. While simple Opa-mediated attachment triggered an innate response regardless of meningococcal viability within the inoculum, persistence of viable Opa-expressing bacteria within the CEACAM1-humanized mice was required for a protective memory response to be achieved. Parenteral immunization with a capsule-based conjugate vaccine led to the accumulation of protective levels of Nme-specific IgG within the nasal mucus, yet the sterilizing immunity afforded by natural colonization was instead conferred by Nme-specific IgA without detectable IgG. Considered together, this study establishes that the availability of CEACAM1 helps define the exquisite host specificity of this human-restricted pathogen, displays a striking example of in vivo selection for the expression of desirable Opa variants, and provides a novel model in which to consider meningococcal infection and immunity within the nasopharyngeal mucosa.
Assuntos
Adaptação Fisiológica , Infecções Meningocócicas/microbiologia , Nasofaringe/microbiologia , Neisseria meningitidis/crescimento & desenvolvimento , Mucosa Respiratória/microbiologia , Infecções Respiratórias/microbiologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Aderência Bacteriana , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Escherichia coli/metabolismo , Células HeLa , Humanos , Imunidade nas Mucosas , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/metabolismo , Camundongos , Camundongos Transgênicos , Viabilidade Microbiana , Mutação , Nasofaringe/imunologia , Nasofaringe/metabolismo , Nasofaringe/patologia , Neisseria meningitidis/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Neutrófilos/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/metabolismo , Infecções Respiratórias/patologiaRESUMO
Infection with Neisseria gonorrhoeae (N. gonorrhoeae) can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa) proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on CD4⺠T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1)-specific cytotoxic T-lymphocyte (CTL) responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific Opa(CEA), but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, Opa(CEA) binding to CEACAM1 reduced the DCs' capacity to stimulate an allogeneic T cell proliferative response. Moreover, Opa(CEA)-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with Opa(CEA)-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain why infection of N. gonorrhoeae fails to trigger an effective specific immune response or develop immune memory, and may affect the potent synergy between gonorrhea and HIV-1 infection.
Assuntos
Antígenos CD/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , HIV-1/imunologia , Neisseria gonorrhoeae/metabolismo , Linfócitos T Citotóxicos/imunologia , Imunidade Adaptativa , Proliferação de Células , Células Dendríticas/microbiologia , Células Dendríticas/virologia , Regulação para Baixo , Fímbrias Bacterianas/fisiologia , Humanos , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Monócitos/citologia , Neisseria gonorrhoeae/fisiologia , Especificidade da Espécie , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/virologia , Antígeno CD83RESUMO
Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with Crohn disease. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. The aim of this study was to determine whether activation of STAT1 by IFNγ was prevented by AIEC infection, and to define the mechanisms used. Human epithelial cells were infected with three different AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ, and STAT1 activation was monitored by immunoblotting. Our data show that live AIEC with active protein synthesis machinery is able to prevent IFNγ-mediated STAT1 phosphorylation, and that a secreted factor may be involved. We conclude that the suppression of epithelial cell STAT1 signal transduction by AIEC strains isolated from patients with Crohn disease represents a novel mechanism by which the pathogen evades host immune responses to the infection.