RESUMO
Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, has developed resistance to almost every conventional antibiotic. There is an urgent need to develop novel therapies against gonorrhea. Many pathogens, including N. gonorrhoeae, bind the complement inhibitor factor H (FH) to evade complement-dependent killing. Sialylation of gonococcal lipooligosaccharide, as occurs in vivo, augments binding of human FH through its domains 18-20 (FH18-20). We explored the use of fusing FH18-20 with IgG Fc (FH18-20/Fc) to create a novel anti-infective immunotherapeutic. FH18-20 also binds to select host glycosaminoglycans to limit unwanted complement activation on host cells. To identify mutation(s) in FH18-20 that eliminated complement activation on host cells, yet maintained binding to N. gonorrhoeae, we created four mutations in domains 19 or 20 described in atypical hemolytic uremic syndrome that prevented binding of mutated fH to human erythrocytes. One of the mutant proteins (D to G at position 1119 in domain 19; FHD1119G/Fc) facilitated complement-dependent killing of gonococci similar to unmodified FH18-20/Fc but, unlike FH18-20/Fc, did not lyse human erythrocytes. FHD1119G/Fc bound to all (100%) of 15 sialylated clinical N. gonorrhoeae isolates tested (including three contemporary ceftriaxone-resistant strains), mediated complement-dependent killing of 10 of 15 (67%) strains, and enhanced C3 deposition (≥10-fold above baseline levels) on each of the five isolates not directly killed by complement. FHD1119G/Fc facilitated opsonophagocytic killing of a serum-resistant strain by human polymorphonuclear neutrophils. FHD1119G/Fc administered intravaginally significantly reduced the duration and burden of gonococcal infection in the mouse vaginal colonization model. FHD1119G/Fc represents a novel immunotherapeutic against multidrug-resistant N. gonorrhoeae.
Assuntos
Fator H do Complemento/imunologia , Gonorreia/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoterapia/métodos , Proteínas Recombinantes de Fusão/imunologia , Animais , Fator H do Complemento/farmacologia , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Humanos , Fragmentos Fc das Imunoglobulinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Neisseria gonorrhoeae/imunologia , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Aeroallergy results from maladaptive immune responses to ubiquitous, otherwise innocuous environmental proteins. Although the proteins targeted by aeroallergic responses represent a tiny fraction of the airborne proteins humans are exposed to, allergenicity is a quite public phenomenon-the same proteins typically behave as aeroallergens across the human population. Why particular proteins tend to act as allergens in susceptible hosts is a fundamental mechanistic question that remains largely unanswered. The main house-dust-mite allergen, Der p 2, has structural homology with MD-2 (also known as LY96), the lipopolysaccharide (LPS)-binding component of the Toll-like receptor (TLR) 4 signalling complex. Here we show that Der p 2 also has functional homology, facilitating signalling through direct interactions with the TLR4 complex, and reconstituting LPS-driven TLR4 signalling in the absence of MD-2. Mirroring this, airway sensitization and challenge with Der p 2 led to experimental allergic asthma in wild type and MD-2-deficient, but not TLR4-deficient, mice. Our results indicate that Der p 2 tends to be targeted by adaptive immune responses because of its auto-adjuvant properties. The fact that other members of the MD-2-like lipid-binding family are allergens, and that most defined major allergens are thought to be lipid-binding proteins, suggests that intrinsic adjuvant activity by such proteins and their accompanying lipid cargo may have some generality as a mechanism underlying the phenomenon of allergenicity.
Assuntos
Alérgenos/imunologia , Alérgenos/metabolismo , Antígenos de Dermatophagoides/imunologia , Antígenos de Dermatophagoides/metabolismo , Mimetismo Molecular/imunologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Ar , Alérgenos/química , Alérgenos/genética , Animais , Antígenos de Dermatophagoides/química , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes , Asma/genética , Asma/imunologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Lipopolissacarídeos/imunologia , Antígeno 96 de Linfócito/química , Antígeno 96 de Linfócito/deficiência , Antígeno 96 de Linfócito/genética , Antígeno 96 de Linfócito/imunologia , Antígeno 96 de Linfócito/metabolismo , Camundongos , Ligação Proteica , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genéticaRESUMO
Combinatorial strategies, such as targeting different immune checkpoint receptors, hold promise to increase the breadth and duration of the response to cancer therapy. Here we describe the preclinical evaluation of CTX-8371, a protein construct which combines PD-1 and PD-L1 targeting in one bispecific, tetravalent antibody. CTX-8371 matched or surpassed the activity of anti-PD-1 and PD-L1 benchmark antibodies in several in vitro T cell activation assays and outperformed clinically approved benchmarks in the subcutaneous MC38 colon and the B16F10 lung metastasis mouse tumor models. Investigation into the mechanism of action revealed that CTX-8371 co-engagement of PD-1 and PD-L1 induced the proteolytic cleavage and loss of cell surface PD-1, which is a novel and non-redundant mechanism that adds to the PD-1/PD-L1 signaling axis blockade. The combination of CTX-8371 and an agonistic anti-CD137 antibody further increased the anti-tumor efficacy with long-lasting curative therapeutic effect. In summary, CTX-8371 is a novel checkpoint inhibitor that might provide greater clinical benefit compared to current anti-PD-1 and PD-L1 antibodies, especially when combined with agents with orthogonal mechanisms of action, such as agonistic anti-CD137 antibodies.
Assuntos
Anticorpos Biespecíficos , Neoplasias Pulmonares , Camundongos , Animais , Antígeno B7-H1 , Receptor de Morte Celular Programada 1 , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Ativação LinfocitáriaRESUMO
TLR7 is the mammalian receptor for ssRNA and some nucleotide-like small molecules. We have generated a mouse by N-nitrose-N'-ethyl urea mutagenesis in which threonine 68 of TLR7 was substituted with isoleucine. Cells bearing this mutant TLR7 lost the sensitivity to the small-molecule TLR7 agonist resiquimod, hence the name TLR7(rsq1). In this work, we report the characterization of this mutant protein. Similar to the wild-type counterpart, TLR7(rsq1) localizes to the endoplasmic reticulum and is expressed at normal levels in both primary cells and reconstituted 293T cells. In addition to small-molecule TLR7 agonists, TLR7(rsq1) fails to be activated by ssRNA. Whole-transcriptome analysis demonstrates that TLR7 is the exclusive and indispensable receptor for both classes of ligands, consistent with the fact that both ligands induce highly similar transcriptional signatures in TLR7(wt/wt) splenocytes. Thus, TLR7(rsq1) is a bona fide phenocopy of the TLR7 null mouse. Because TLR7(rsq1) binds to ssRNA, our studies imply that the N-terminal portion of TLR7 triggers a yet to be identified event on TLR7. TLR7(rsq1) mice might represent a valuable tool to help elucidate novel aspects of TLR7 biology.
Assuntos
Mutação Puntual/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Células HEK293 , Humanos , Imidazóis/farmacologia , Ligantes , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ligação Proteica/imunologia , Transdução de Sinais/efeitos dos fármacos , Receptor 7 Toll-Like/deficiênciaRESUMO
Human factor H (HufH), a key inhibitor of the alternative pathway of complement, binds to Neisseria gonorrhoeae and constitutes an important mechanism of human-specific complement evasion. The C-terminal domain 20 of HufH contains the binding site for sialylated gonococci. We exploited differences in amino acid sequences between human and non-binding chimpanzee fH domain 20 to create cross-species mutations to define amino acids important for binding to sialylated gonococci. We used fH/Fc fusion constructs that contained contiguous fH domains 18-20 fused to Fc fragments of murine IgG2a. The Fc region was used both as a tag for detection of each fusion molecule on the bacterial surface and as an indicator for complement-dependent killing. Arg-1203 was critical for binding to both porin (Por) B.1A and PorB.1B strains. Modeling of the R1203N human-to-chimpanzee mutation using the crystal structure of HufH19-20 as a template showed a loss of positive charge that protrudes at the C terminus of domain 20. We tested the functional importance of Arg-1203 by incubating sialylated gonococci with normal human serum, in the presence of wild-type HufH18-20/Fc or its R1203A mutant. Gonococci bound and were killed by wild-type HufH18-20/Fc but not by the R1203A mutant. A recombinant fH/Fc molecule that contained chimpanzee domain 20, humanized only at amino acid 1203 (N1203R) also bound to sialylated gonococci and restored killing. These findings provide further insights into the species specificity of gonococcal infections and proof-of-concept of a novel therapeutic approach against gonorrhea, a disease rapidly becoming resistant to conventional antibiotics.
Assuntos
Fator H do Complemento/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neisseria gonorrhoeae/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Arginina , Sítios de Ligação , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/farmacologia , Heparina/farmacologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Neisseria gonorrhoeae/efeitos dos fármacos , Pan troglodytes , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Especificidade da EspécieRESUMO
Soluble proteins that bind LPS, like myeloid differentiation-2 (MD-2) and CD14, have essential roles in regulating LPS signaling through TLR4. During a gram-negative bacterial infection, the host may control the response by adjusting the levels of soluble MD-2 and CD14. To address the surface expression of MD-2 on human leukocytes, we developed a mAb, IIC1, that recognized MD-2 both free and when bound to TLR4. MD-2 was found on the surface of freshly isolated monocytes, on a subpopulation of CD19(+) B-cells and on CD15(+) neutrophils. LPS transiently reduced the MD-2 levels on monocytes, which is most likely due to endocytosis of the LPS receptor complex since MD-2 colocalized with TLR4 in early endosomes after LPS stimulation. In the absence of LPS, MD-2 partly colocalized with TLR4 in Golgi trans and medial compartments. Cultivating monocytes for 18-20 h resulted in loss of MD-2 expression on the surface, which was reversed either by LPS or IL-10. Furthermore, addition of IL-10, but not LPS, resulted in a considerable increase in mRNA for both MD-2 and CD14. Using ELISA, we demonstrated that IL-10 had a profound dose- and time-related effect on the release of soluble MD-2 and soluble CD14 from monocytes. In HIV-infected patients, the amounts of MD-2, CD14, and IL-10 increased significantly in the patient group with AIDS. Of interest, we found that IL-10, CD14, and MD-2 levels were positively correlated, suggesting that IL-10 may be a driving force for increased release of MD-2 and CD14 during systemic inflammation.
Assuntos
Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Interleucina-10/fisiologia , Receptores de Lipopolissacarídeos/biossíntese , Antígeno 96 de Linfócito/biossíntese , Monócitos/imunologia , Monócitos/metabolismo , Regulação para Cima/imunologia , Adulto , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Feminino , Humanos , Mediadores da Inflamação/fisiologia , Interleucina-10/sangue , Receptores de Lipopolissacarídeos/sangue , Receptores de Lipopolissacarídeos/genética , Antígeno 96 de Linfócito/sangue , Antígeno 96 de Linfócito/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/biossínteseRESUMO
Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM) is the fourth TIR domain-containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-kappaB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-alpha/beta, regulated on activation, normal T cell expressed and secreted (RANTES), and gamma interferon-inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/fisiologia , NF-kappa B/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Quimiocina CCL5/metabolismo , Humanos , Fator Regulador 3 de Interferon , Fator Regulador 7 de Interferon , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA de Cadeia Dupla/farmacologia , RNA Interferente Pequeno/fisiologia , Receptor 3 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
Both Toll-like receptor 4 (TLR4)- and MD-2-deficient mice succumb to otherwise nonfatal gram-negative bacteria inocula, demonstrating the pivotal role played by these proteins in antibacterial defense in mammals. MD-2 is a soluble endogenous ligand for TLR4 and a receptor for lipopolysaccharide (LPS). LPS-bound MD-2 transmits an activating signal onto TLR4. In this report, we show that both recombinant and endogenous soluble MD-2 bind tightly to the surface of live gram-negative bacteria. As a consequence, MD-2 enhances cellular activation, bacterial internalization, and intracellular killing, all in a TLR4-dependent manner. The enhanced internalization of MD-2-coated bacteria was not observed in macrophages expressing Lps(d), a signaling-incompetent mutant form of TLR4, suggesting that the enhanced phagocytosis observed is dependent on signal transduction. The data confirm the notion that soluble MD-2 is a genuine opsonin that enhances proinflammatory opsonophagocytosis by bridging live gram-negative bacteria to the LPS transducing complex. The presented results extend our understanding of the role of the TLR4/MD-2 signaling axis in bacterial recognition by phagocytes.
Assuntos
Bactérias Gram-Negativas/imunologia , Antígeno 96 de Linfócito/imunologia , Fagocitose , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/fisiologia , Animais , Camundongos , Proteínas Opsonizantes/metabolismoRESUMO
The cell surface receptor complex formed by TLR4 and myeloid differentiation 2 (MD-2) is engaged when cells are exposed to LPS. Recent studies suggested that surface localization of functional mouse TLR4 (mTLR4) depends on the simultaneous expression of MD-2. As we did not observe a similar requirement, we conducted a comparative study of human TLR4 and mTLR4 surface expression in immune cells derived from the MD-2 knockout mouse and LPS-responsive cell lines and in cells that ectopically express TLR4. Our results indicate that in the human and mouse models, neither TLR4 function nor TLR4 surface targeting requires MD-2 coexpression. Accordingly, we report on one human cell line, which constitutively expresses functional TLR4 on the cell surface in the absence of MD-2 expression.
Assuntos
Regulação da Expressão Gênica , Antígeno 96 de Linfócito/biossíntese , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/deficiência , Antígeno 96 de Linfócito/imunologia , Camundongos , Camundongos Knockout , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/imunologiaRESUMO
Toll-like receptors (TLRs) are a small family of type-I glycoproteins that bind to and are activated by conserved non-self molecular signatures carried by microorganisms. Toll-like receptor 4 is triggered by most lipopolysaccharides (LPS). LPS is a complex amphipathic saccharolipidic glycan derived from Gram-negative bacteria. Unique among TLRs, TLR4 activity and interaction with its natural ligand(s) strictly depends on the presence of the extracellular adaptor MD-2. MD-2 is a small secreted glycoprotein that binds with cytokine-like affinities to both the hydrophobic portion of LPS and to the extracellular domain of TLR4. The interaction between MD-2 and LPS induces a triggering event on TLR4, which involves the molecular rearrangement of the receptor complex and its homotypic aggregation. In silico analysis suggests that MD-2 and MD-1 are paralogs derived from a common predecessor at the level of early vertebrates. In this review, we summarize the current state of knowledge concerning MD-2.
Assuntos
Antígeno 96 de Linfócito/fisiologia , Sequência de Aminoácidos , Animais , Bactérias/imunologia , Humanos , Imunidade Inata/fisiologia , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Antígeno 96 de Linfócito/genética , Dados de Sequência Molecular , Receptor 4 Toll-Like/fisiologiaRESUMO
Intracellular signals, delivered in professional antigen-presenting cells following the engagement of major histocompatibility complex (MHC) class II molecules, activate a variety of cellular functions that also contribute to efficient antigen presentation. As far as human malignancies, the signaling ability of human leukocyte antigens (HLA) class II molecules is a rather well-characterized event in hematologic tumors; in contrast, very limited evidences are available in solid neoplasias of different histotypes that may constitutively express HLA class II antigens. Among solid malignancies, a significant proportion of human cutaneous melanomas have been shown to express HLA class II molecules, and cutaneous melanoma undoubtedly represents a 'model disease' to investigate tumor immunobiology, to unveil the molecular basis underlying the interactions between neoplastic cells and host's immune system, and ultimately to set up new bio-immunotherapeutic approaches. Upcoming preclinical evidences unveil a signaling potential of HLA-DR antigens expressed on melanoma cells, and suggest for the clinical implication of HLA class II molecules as novel therapeutic targets. Therefore, in this review, we will focus on the emerging role of HLA class II antigens as intracellular signal transducing elements in neoplastic cells of the melanocytic lineage, emphasizing their foreseeable role in targeted therapy of human melanoma and potentially of HLA class II antigens-positive tumors of different histology.
Assuntos
Antígenos HLA-D/imunologia , Neoplasias/imunologia , Células Apresentadoras de Antígenos/imunologia , Divisão Celular/imunologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Antígenos HLA-D/genética , Antígenos HLA-DR/imunologia , Humanos , Neoplasias/genética , Transdução de Sinais/imunologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologiaRESUMO
Activation of Toll-like receptor (TLR) signaling by microbial and host molecular signatures is critical to the induction of immune responses. Such signaling is, perforce, kept under tight control. We recently discovered a novel endogenous inhibitor of TLR-4 - RP105. Initially identified as a B-cell-specific molecule with a role in B-cell proliferation in response to RP105 mAb and LPS, RP105 is a TLR-4 homologue. Further, like TLR-4 whose surface expression and signaling depends upon co-expression of the secreted protein MD-2, surface expression of RP105 is dependent upon co-expression of the MD2 homologue, MD-1. Unlike the TLRs, however, RP105 lacks a signaling domain, having the apparent structure of a TLR inhibitor. Further, RP105 is not B-cell-specific; its expression directly mirrors that of TLR-4 on dendritic cells and macrophages. These considerations suggested a role for RP105 as a physiological inhibitor of TLR-4 signaling. Indeed, we have recently found that: (i) RP105 is a specific inhibitor of TLR-4 signaling in HEK293 cells; (ii) RP105/MD-1 interacts directly with TLR-4/MD-2, inhibiting the ability of this signaling complex to bind LPS; (iii) RP105 regulates TLR-4 signaling in dendritic cells and macrophages; and (iv) RP105 regulates in vivo responses to LPS.
Assuntos
Antígenos de Superfície/metabolismo , Linfócitos B/imunologia , Antígeno 96 de Linfócito/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Superfície/genética , Linhagem Celular , Células Dendríticas/imunologia , Humanos , Antígeno 96 de Linfócito/genética , Macrófagos Peritoneais/imunologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
Antibody-drug conjugates (ADC) represent a promising therapeutic modality for managing cancer. Here, we report a novel humanized ADC that targets the tetraspanin-like protein TM4SF1. TM4SF1 is highly expressed on the plasma membranes of many human cancer cells and also on the endothelial cells lining tumor blood vessels. TM4SF1 is internalized upon interaction with antibodies. We hypothesized that an ADC against TM4SF1 would inhibit cancer growth directly by killing cancer cells and indirectly by attacking the tumor vasculature. We generated a humanized anti-human TM4SF1 monoclonal antibody, v1.10, and armed it with an auristatin cytotoxic agent LP2 (chemical name mc-3377). v1.10-LP2 selectively killed cultured human tumor cell lines and human endothelial cells that express TM4SF1. Acting as a single agent, v1.10-LP2 induced complete regression of several TM4SF1-expressing tumor xenografts in nude mice, including non-small cell lung cancer and pancreas, prostate, and colon cancers. As v1.10 did not react with mouse TM4SF1, it could not target the mouse tumor vasculature. Therefore, we generated a surrogate anti-mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2. At 3 mpk, 2A7A-LP2 regressed several tumor xenografts without noticeable toxicity. Combination therapy with v1.10-LP2 and 2A7A-LP2 together was more effective than either ADC alone. These data provide proof-of-concept that TM4SF1-targeting ADCs have potential as anticancer agents with dual action against tumor cells and the tumor vasculature. Such agents could offer exceptional therapeutic value and warrant further investigation. Mol Cancer Ther; 14(8); 1868-76. ©2015 AACR.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores da Angiogênese/toxicidade , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Expressão Gênica , Humanos , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica , Coelhos , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Non-methylated CpG-motifs in bacterial or viral DNA are recognized by TLR9 as foreign. The activation of TLR9 by microbial DNA or synthetic oligonucleotides based on these motifs leads to the induction of innate immune responses. We have compared the subcellular localization of fluorescent versions of TLR9 and TLR4 and found that TLR9 is expressed in the endoplasmic reticulum while TLR4 is expressed on the plasma membrane. Fluorescently tagged bacterial DNA or CpG-DNA was observed to traffic to a tubular lysosomal compartment in human pDCs. In stimulated cells, TLR9 translocated to CpG-DNA or microbial DNA containing structures in the endosome, where TLR9 binds to DNA and initiates signaling.
Assuntos
Ilhas de CpG/imunologia , DNA Bacteriano/imunologia , Leucócitos Mononucleares/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Transdução de Sinais/imunologia , Membrana Celular/imunologia , Células Cultivadas , Ilhas de CpG/genética , DNA Bacteriano/genética , Retículo Endoplasmático/imunologia , Escherichia coli/genética , Escherichia coli/imunologia , Humanos , Lisossomos/genética , Lisossomos/imunologia , Lisossomos/ultraestrutura , Glicoproteínas de Membrana/genética , Microscopia Confocal , Receptores de Superfície Celular/genética , Transdução de Sinais/genética , Receptor 4 Toll-Like , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMO
Bacterial lipopolysaccharides (LPSs) are recognized in mammals by a receptor complex composed of CD14, Toll-like receptor 4 (TLR4), and MD-2. The mechanism of TLR4 function remains to be elucidated. We constructed chimeric TLR molecules C-terminally fused to fluorescent proteins and stably expressed these chimeric constructs in cells. Confocal microscopy revealed TLR4 to be expressed on the plasma membrane and the Golgi apparatus. Time-lapse confocal imaging showed rapid recycling of TLR4/CD14/MD-2 complexes between the Golgi and the plasma membrane. Membrane TLR4 engagement by antibody was sufficient to induce signaling and pharmacological disruption of the Golgi did not affect cellular responses to LPS. Thus, LPS signaling commences after LPS recognition by surface-expressed TLR4 independent of LPS trafficking to the Golgi.
Assuntos
Membrana Celular/metabolismo , Inflamação , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Transdução de Sinais , Linhagem Celular , Meios de Cultura , Ensaio de Imunoadsorção Enzimática , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde , Humanos , Interleucina-8/metabolismo , Rim/citologia , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
To gain insight into the functional antibody repertoire of rabbits, the VH and VL repertoires of bone marrow (BM) and spleen (SP) of a naïve New Zealand White rabbit (NZW; Oryctolagus cuniculus) and that of lymphocytes collected from a NZW rabbit immunized (IM) with a 16-mer peptide were deep-sequenced. Two closely related genes, IGHV1S40 (VH1a3) and IGHV1S45 (VH4), were found to dominate (~90%) the VH repertoire of BM and SP, whereas, IGHV1S69 (VH1a1) contributed significantly (~40%) to IM. BM and SP antibodies recombined predominantly with IGHJ4. A significant proportion (~30%) of IM sequences recombined with IGHJ2. The VK repertoire was encoded by nine IGKV genes recombined with one IGKJ gene, IGKJ1. No significant bias in the VK repertoire of the BM, SP and IM samples was observed. The complementarity-determining region (CDR)-H3 and -L3 length distributions were similar in the three samples following a Gaussian curve with average length of 12.2 ± 2.4 and 11.1 ± 1.1 amino acids, respectively. The amino acid composition of the predominant CDR-H3 and -L3 loop lengths was similar to that of humans and mice, rich in Tyr, Gly, Ser and, in some specific positions, Asp. The average number of mutations along the IGHV/KV genes was similar in BM, SP and IM; close to 12 and 15 mutations for VH and VL, respectively. A monoclonal antibody specific for the peptide used as immunogen was obtained from the IM rabbit. The CDR-H3 sequence was found in 1,559 of 61,728 (2.5%) sequences, at position 10, in the rank order of the CDR-H3 frequencies. The CDR-L3 was found in 24 of 11,215 (0.2%) sequences, ranking 102. No match was found in the BM and SP samples, indicating positive selection for the hybridoma sequence. Altogether, these findings lay foundations for engineering of rabbit V regions to enhance their potential as therapeutics, i.e., design of strategies for selection of specific rabbit V regions from NGS data mining, humanization and design of libraries for affinity maturation campaigns.
Assuntos
Anticorpos/genética , Anticorpos/imunologia , Coelhos/genética , Coelhos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Medula Óssea/imunologia , Regiões Determinantes de Complementaridade/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridomas/imunologia , Imunização , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Mutação , Peptídeos/imunologia , Engenharia de Proteínas , Homologia de Sequência de Aminoácidos , Baço/imunologiaRESUMO
Complement forms a key arm of innate immune defenses against gonococcal infection. Sialylation of gonococcal lipo-oligosaccharide, or expression of porin 1A (Por1A) protein, enables Neisseria gonorrhoeae to bind the alternative pathway complement inhibitor, factor H (fH), and evade killing by human complement. Using recombinant fH fragment-murine Fc fusion proteins, we localized two N. gonorrhoeae Por1A-binding regions in fH: one in complement control protein domain 6, the other in complement control proteins 18-20. The latter is similar to that reported previously for sialylated Por1B gonococci. Upon incubation with human serum, Por1A and sialylated Por1B strains bound full-length human fH (HufH) and fH-related protein 1. In addition, Por1A strains bound fH-like protein 1 weakly. Only HufH, but not fH from other primates, bound directly to gonococci. Consistent with direct HufH binding, unsialylated Por1A gonococci resisted killing only by human complement, but not complement from other primates, rodents or lagomorphs; adding HufH to these heterologous sera restored serum resistance. Lipo-oligosaccharide sialylation of N. gonorrhoeae resulted in classical pathway regulation as evidenced by decreased C4 binding in human, chimpanzee, and rhesus serum but was accompanied by serum resistance only in human and chimpanzee serum. Direct-binding specificity of HufH only to gonococci that prevents serum killing is restricted to humans and may in part explain species-specific restriction of natural gonococcal infection. Our findings may help to improve animal models for gonorrhea while also having implications in the choice of complement sources to evaluate neisserial vaccine candidates.
Assuntos
Atividade Bactericida do Sangue/imunologia , Fator H do Complemento/metabolismo , Proteínas Inativadoras do Complemento/metabolismo , Via Clássica do Complemento/imunologia , Neisseria gonorrhoeae/imunologia , Motivos de Aminoácidos/imunologia , Animais , Fator H do Complemento/fisiologia , Proteínas Inativadoras do Complemento/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Lipopolissacarídeos/sangue , Macaca mulatta , Camundongos , Neisseria gonorrhoeae/metabolismo , Oligossacarídeos/sangue , Pan troglodytes , Papio , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Porinas/metabolismo , Ligação Proteica/imunologia , Especificidade da EspécieRESUMO
The major and minor fimbriae proteins produced by the human pathogen Porphyromonas gingivalis are required for invasion of human aortic endothelial cells and for the stimulation of potent inflammatory responses. In this study, we report that native forms of both the major and minor fimbriae proteins bind to and signal through TLR2 for this response. Major and minor fimbriae bound to a human TLR2:Fc chimeric protein with an observed K(d) of 28.9 nM and 61.7 nM, respectively. Direct binding of the major and minor fimbriae to a human chimeric CD14-Fc protein also established specific binding of the major and minor fimbriae to CD14 with classic saturation kinetics. Using a P. gingivalis major and minor fimbriae mutant, we confirmed that TLR2 binding in whole cells is dependent on the expression of the major and minor fimbriae. Although we did not observe binding with the major or minor fimbriae to the TLR4-Fc chimeric protein, signaling through TLR4 for both proteins was demonstrated in human embryonic kidney 293 cells transfected with TLR4 and only in the presence MD-2. Transient transfection of dominant-negative forms of TLR2 or TLR4 reduced IL-8 production by human aortic endothelial cells following stimulation with major or minor fimbriae. The ability of two well-defined microbe-associated molecular patterns to select for innate immune recognition receptors based on accessory proteins may provide a novel way for a pathogen to sense and signal in appropriate host environments.
Assuntos
Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Fímbrias Bacterianas/imunologia , Mediadores da Inflamação/metabolismo , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Anticorpos Bloqueadores/fisiologia , Linhagem Celular , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Proteínas de Fímbrias/metabolismo , Proteínas de Fímbrias/fisiologia , Fímbrias Bacterianas/metabolismo , Humanos , Interleucina-8/antagonistas & inibidores , Interleucina-8/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Porphyromonas gingivalis/química , Porphyromonas gingivalis/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , TransfecçãoRESUMO
Hemozoin (HZ) is an insoluble crystal formed in the food vacuole of malaria parasites. HZ has been reported to induce inflammation by directly engaging Toll-like receptor (TLR) 9, an endosomal receptor. "Synthetic" HZ (beta-hematin), typically generated from partially purified extracts of bovine hemin, is structurally identical to natural HZ. When HPLC-purified hemin was used to synthesize the crystal, beta-hematin had no inflammatory activity. In contrast, natural HZ from Plasmodium falciparum cultures was a potent TLR9 inducer. Natural HZ bound recombinant TLR9 ectodomain, but not TLR2. Both TLR9 stimulation and TLR9 binding of HZ were abolished by nuclease treatment. PCR analysis demonstrated that natural HZ is coated with malarial but not human DNA. Purified malarial DNA activated TLR9 but only when DNA was targeted directly to the endosome with a transfection reagent. Stimulatory quantities of natural HZ contain <1 microg of malarial DNA; its potency in activating immune responses was even greater than transfecting malarial DNA. Thus, although the malarial genome is extremely AT-rich, its DNA is highly proinflammatory, with the potential to induce cytokinemia and fever during disease. However, its activity depends on being bound to HZ, which we propose amplifies the biological responses to malaria DNA by targeting it to a TLR9(+) intracellular compartment.
Assuntos
Apresentação de Antígeno , DNA de Protozoário/metabolismo , Hemeproteínas/fisiologia , Imunidade Inata , Plasmodium falciparum/genética , Receptor Toll-Like 9/metabolismo , Animais , DNA de Protozoário/imunologia , Humanos , Ativação Linfocitária/imunologia , Melanoma Experimental , Camundongos , Plasmodium falciparum/imunologia , Receptor Toll-Like 9/imunologiaRESUMO
Microbial and synthetic DNA rich in CpG dinucleotides stimulates Toll-like receptor 9 (TLR9), whereas DNA lacking CpG either is inert or can inhibit TLR9 activation. The molecular mechanisms by which TLR9 becomes activated or is inhibited are not well understood. Here we show that TLR9 bound to stimulatory and inhibitory DNA; however, only stimulatory DNA led to substantial conformational changes in the TLR9 ectodomain. In the steady state, 'inactive' TLR9 homodimers formed in an inactivated conformation. Binding of DNA containing CpG, but not of DNA lacking CpG, to TLR9 dimers resulted in allosteric changes in the TLR9 cytoplasmic signaling domains. In endosomes, conformational changes induced by DNA containing CpG resulted in close apposition of the cytoplasmic signaling domains, a change that is probably required for the recruitment of signaling adaptor molecules. Our results indicate that the formation of TLR9 dimers is not sufficient for its activation but instead that TLR9 activation is regulated by conformational changes induced by DNA containing CpG.