RESUMEN
Single-cell RNA-seq enabled in-depth study on tissue micro-environment and immune-profiling, where a crucial step is to annotate cell identity. Immune cells play key roles in many diseases, whereas their activities are hard to track due to their diverse and highly variable nature. Existing cell-type identifiers had limited performance for this purpose. We present HiCAT, a hierarchical, marker-based cell-type identifier utilising gene set analysis for statistical scoring for given markers. It features successive identification of major-type, minor-type and subsets utilising subset markers structured in a three-level taxonomy tree. Comparison with manual annotation and pairwise match test showed HiCAT outperforms others in major- and minor-type identification. For subsets, we qualitatively evaluated the marker expression profile demonstrating that HiCAT provide the clearest immune-cell landscape. HiCAT was also used for immune-cell profiling in ulcerative colitis and discovered distinct features of the disease in macrophage and T-cell subsets that could not be identified previously.
Asunto(s)
Perfilación de la Expresión Génica , Macrófagos , ARNRESUMEN
The orphan nuclear receptor estrogen-related receptor α (ERRα; NR3B1) is a key metabolic regulator, but its function in regulating inflammation remains largely unknown. Here, we demonstrate that ERRα negatively regulates Toll-like receptor (TLR)-induced inflammation by promoting Tnfaip3 transcription and fine-tuning of metabolic reprogramming in macrophages. ERRα-deficient (Esrra(-/-)) mice showed increased susceptibility to endotoxin-induced septic shock, leading to more severe pro-inflammatory responses than control mice. ERRα regulated macrophage inflammatory responses by directly binding the promoter region of Tnfaip3, a deubiquitinating enzyme in TLR signaling. In addition, Esrra(-/-) macrophages showed an increased glycolysis, but impaired mitochondrial respiratory function and biogenesis. Further, ERRα was required for the regulation of NF-κB signaling by controlling p65 acetylation via maintenance of NAD(+) levels and sirtuin 1 activation. These findings unravel a previously unappreciated role for ERRα as a negative regulator of TLR-induced inflammatory responses through inducing Tnfaip3 transcription and controlling the metabolic reprogramming.
Asunto(s)
Cisteína Endopeptidasas/biosíntesis , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Macrófagos/metabolismo , Receptores de Estrógenos/genética , Receptor Toll-Like 4/inmunología , Acetilación , Animales , Calcio/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Células Cultivadas , Cisteína Endopeptidasas/genética , Activación Enzimática/genética , Glucólisis/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Lipopolisacáridos , Macrófagos/inmunología , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/metabolismo , NAD/metabolismo , Fosforilación Oxidativa , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/inmunología , Choque Séptico/inmunología , Transducción de Señal , Sirtuina 1/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción ReIA/metabolismo , Transcripción Genética/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Ubiquitinación , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
The link between chronic inflammation and cancer development is well acknowledged. Inflammatory bowel disease including ulcerative colitis and Crohn's disease frequently promotes colon cancer development. Thus, control of intestinal inflammation is a therapeutic strategy to prevent and manage colitis-associated colorectal cancer (CRC). Recently, gut mucosal damage-associated molecular patterns S100A8 and S100A9, acting via interactions with their pattern recognition receptors (PRRs), especially TLR4 and RAGE, have emerged as key players in the pathogenesis of colonic inflammation. We found elevated serum levels of S100A8 and S100A9 in both colitis and colitis-associated CRC mouse models along with significant increases in their binding with PRR, TLR4, and RAGE. In this study we developed a dual PRR-inhibiting peptide system (rCT-S100A8/A9) that consisted of TLR4- and RAGE-inhibiting motifs derived from S100A8 and S100A9, and conjugated with a CT peptide (TWYKIAFQRNRK) for colon-specific delivery. In human monocyte THP-1 and mouse BMDMs, S100A8/A9-derived peptide comprising TLR4- and RAGE-interacting motif (0.01, 0.1, 1 µM) dose-dependently inhibited the binding of S100 to TLR4 or RAGE, and effectively inhibited NLRP3 inflammasome activation. We demonstrated that rCT-S100A8/A9 had appropriate drug-like properties including in vitro stabilities and PK properties as well as pharmacological activities. In mouse models of DSS-induced acute and chronic colitis, injection of rCT-S100A8/A9 (50 µg·kg-1·d-1, i.p. for certain consecutive days) significantly increased the survival rates and alleviated the pathological injuries of the colon. In AOM/DSS-induced colitis-associated colorectal cancer (CAC) mouse model, injection of rCT-S100A8/A9 (50 µg·kg-1·d-1, i.p.) increased the body weight, decreased tumor burden in the distal colon, and significantly alleviated histological colonic damage. In mice bearing oxaliplatin-resistant CRC xenografts, injection of rCT-S100A8/A9 (20 µg/kg, i.p., every 3 days for 24-30 days) significantly inhibited the tumor growth with reduced EMT-associated markers in tumor tissues. Our results demonstrate that targeting the S100-PRR axis improves colonic inflammation and thus highlight this axis as a potential therapeutic target for colitis and CRC.
Asunto(s)
Neoplasias Asociadas a Colitis , Colitis , Humanos , Ratones , Animales , Receptor Toll-Like 4/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Inflamación/metabolismo , Péptidos/farmacología , Péptidos/uso terapéutico , Péptidos/metabolismoRESUMEN
The orphan nuclear receptor SHP (small heterodimer partner) is a transcriptional corepressor that regulates hepatic metabolic pathways. Here we identified a role for SHP as an intrinsic negative regulator of Toll-like receptor (TLR)-triggered inflammatory responses. SHP-deficient mice were more susceptible to endotoxin-induced sepsis. SHP had dual regulatory functions in a canonical transcription factor NF-κB signaling pathway, acting as both a repressor of transactivation of the NF-κB subunit p65 and an inhibitor of polyubiquitination of the adaptor TRAF6. SHP-mediated inhibition of signaling via the TLR was mimicked by macrophage-stimulating protein (MSP), a strong inducer of SHP expression, via an AMP-activated protein kinase-dependent signaling pathway. Our data identify a previously unrecognized role for SHP in the regulation of TLR signaling.
Asunto(s)
FN-kappa B/inmunología , Receptores Citoplasmáticos y Nucleares/inmunología , Sepsis/inmunología , Receptores Toll-Like/inmunología , Proteínas Quinasas Activadas por AMP/inmunología , Animales , Inmunoprecipitación de Cromatina , Femenino , Immunoblotting , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/inmunología , Ubiquitinación/inmunologíaRESUMEN
Antibodies are recognition molecules that can bind to diverse targets ranging from pathogens to small analytes with high binding affinity and specificity, making them widely employed for sensing and therapy. However, antibodies have limitations of low stability, long production time, short shelf life, and high cost. Here, we report a facile approach for the design of luminescent artificial antibodies with nonbiological polymeric recognition phases for the sensitive detection, rapid identification, and effective inactivation of pathogenic bacteria. Transition-metal dichalcogenide (TMD) nanosheets with a neutral dextran phase at the interfaces selectively recognized S. aureus, whereas the nanosheets bearing a carboxymethylated dextran phase selectively recognized E. coli O157:H7 with high binding affinity. The bacterial binding sites recognized by the artificial antibodies were thoroughly identified by experiments and molecular dynamics simulations, revealing the significance of their multivalent interactions with the bacterial membrane components for selective recognition. The luminescent WS2 artificial antibodies could rapidly detect the bacteria at a single copy from human serum without any purification and amplification. Moreover, the MoSe2 artificial antibodies selectively killed the pathogenic bacteria in the wounds of infected mice under light irradiation, leading to effective wound healing. This work demonstrates the potential of TMD artificial antibodies as an alternative to antibodies for sensing and therapy.
Asunto(s)
Antibacterianos/uso terapéutico , Infecciones por Escherichia coli/tratamiento farmacológico , Sustancias Luminiscentes/uso terapéutico , Nanoestructuras/uso terapéutico , Animales , Antibacterianos/química , Antibacterianos/efectos de la radiación , Dextranos/química , Escherichia coli O157/efectos de los fármacos , Escherichia coli O157/aislamiento & purificación , Luz , Sustancias Luminiscentes/química , Sustancias Luminiscentes/efectos de la radiación , Ratones , Simulación de Dinámica Molecular , Molibdeno/química , Molibdeno/efectos de la radiación , Molibdeno/uso terapéutico , Nanoestructuras/química , Nanoestructuras/efectos de la radiación , Terapia Fototérmica , Compuestos de Selenio/química , Compuestos de Selenio/efectos de la radiación , Compuestos de Selenio/uso terapéutico , Piel/microbiología , Espectrometría Raman , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Sulfuros/química , Sulfuros/efectos de la radiación , Sulfuros/uso terapéutico , Compuestos de Tungsteno/química , Compuestos de Tungsteno/efectos de la radiación , Compuestos de Tungsteno/uso terapéutico , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Dense granule proteins (GRAs) are essential components in Toxoplasma gondii, which are suggested to be promising serodiagnostic markers in toxoplasmosis. In this study, we investigated the function of GRA9 in host response and the associated regulatory mechanism, which were unknown. We found that GRA9 interacts with NLR family pyrin domain containing 3 (NLRP3) involved in inflammation by forming the NLRP3 inflammasome. The C-terminal of GRA9 (GRA9C) is essential for GRA9-NLRP3 interaction by disrupting the NLRP3 inflammasome through blocking the binding of apoptotic speck-containing (ASC)-NLRP3. Notably, Q200 of GRA9C is essential for the interaction of NLRP3 and blocking the conjugation of ASC. Recombinant GRA9C (rGRA9C) showed an anti-inflammatory effect and the elimination of bacteria by converting M1 to M2 macrophages. In vivo, rGRA9C increased the anti-inflammatory and bactericidal effects and subsequent anti-septic activity in CLP- and E. coli- or P. aeruginosa-induced sepsis model mice by increasing M2 polarization. Taken together, our findings defined a role of T. gondii GRA9 associated with NLRP3 in host macrophages, suggesting its potential as a new candidate therapeutic agent for sepsis.
Asunto(s)
Inflamasomas/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Proteínas Protozoarias/inmunología , Sepsis/terapia , Toxoplasma/inmunología , Animales , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Proteínas Adaptadoras de Señalización CARD/inmunología , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Parásitos/inmunología , Macrófagos/clasificación , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/inmunología , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Sepsis/inmunología , Sepsis/prevención & control , Toxoplasma/genética , Toxoplasma/patogenicidadRESUMEN
The increase in drug-resistant Mycobacterium abscessus, which has become resistant to existing standard-of-care agents, is a major concern, and new antibacterial agents are strongly needed. In this study, we introduced etamycin that showed an excellent activity against M. abscessus. We found that etamycin significantly inhibited the growth of M. abscessus wild-type strain, three subspecies, and clinical isolates in vitro and inhibited the growth of M. abscessus that resides in macrophages without cytotoxicity. Furthermore, the in vivo efficacy of etamycin in the zebrafish (Danio rerio) infection model was greater than that of clarithromycin, which is recommended as the core agent for treating M. abscessus infections. Thus, we concluded that etamycin is a potential anti-M. abscessus candidate for further development as a clinical drug candidate.
Asunto(s)
Enfermedades de los Peces , Macrólidos/farmacología , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Mycobacterium abscessus/crecimiento & desarrollo , Pez Cebra/microbiología , Animales , Femenino , Enfermedades de los Peces/tratamiento farmacológico , Enfermedades de los Peces/microbiología , RatonesRESUMEN
Mycobacterium abscessus is the most difficult-to-treat nontuberculous mycobacteria because of its resistance to many antibiotics. In this study, we screened the Korea Chemical Bank library for a bioluminescent reporter assay to identify molecules capable of acting against M. abscessus. On application of the assay, rifamycin O showed excellent in vitro activity with a narrow range of the minimum inhibitory concentration required to inhibit the growth of 90% of the bacterium (MIC90 = 4.0-6.2 µM); its in vivo efficacy in the zebrafish (Danio rerio) infection model was comparable to that of rifabutin at 25 µM. Furthermore, rifamycin O did not show significant toxicity in cells and the zebrafish model. These results are the first in vivo indication that rifamycin O may be a drug candidate for treating M. abscessus infections.
Asunto(s)
Antibacterianos/farmacología , Mycobacterium abscessus/efectos de los fármacos , Rifamicinas/farmacología , Animales , Antibacterianos/química , Humanos , Mediciones Luminiscentes , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Rifamicinas/química , Pez CebraRESUMEN
Tuberculosis is a global health problem and at least one-third of the world's population is infected with Mycobacterium tuberculosis (MTB). MTB is a successful pathogen that enhances its own intracellular survival by inhibiting inflammation and arresting phago-lysosomal fusion. We previously demonstrated that Toxoplasma gondii (T. gondii) dense granule antigen (GRA) 7 interacts with TNF receptor-associated factor 6 via Myeloid differentiation primary response gene 88, enabling innate immune responses in macrophages. To extend these studies, we found that GRA7 interacts with host proteins involved in antimicrobial host defense mechanisms as a therapeutic strategy for tuberculosis. Here, we show that protein kinase C (PKC)α-mediated phosphorylation of T. gondii GRA7-I (Ser52) regulates the interaction of GRA7 with PYD domain of apoptosis-associated speck-like protein containing a carboxy-terminal CARD, which is capable of oligomerization and inflammasome activation can lead to antimicrobial defense against MTB. Furthermore, GRA7-III interacted with the PX domain of phospholipase D1, facilitating its enzyme activity, phago-lysosomal maturation, and subsequent antimicrobial activity in a GRA7-III (Ser135) phosphorylation-dependent manner via PKCα. Taken together, these results underscore a previously unrecognized role of GRA7 in modulating antimicrobial host defense mechanism during mycobacterial infection.
Asunto(s)
Antígenos de Protozoos/metabolismo , Mycobacterium/inmunología , Proteína Quinasa C-alfa/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/fisiología , Tuberculosis/inmunología , Animales , Antígenos de Protozoos/genética , Dominio de Reclutamiento y Activación de Caspasas , Diferenciación Celular , Humanos , Inmunidad Innata , Inflamasomas/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Fosforilación , Proteína Quinasa C-alfa/genética , Proteínas Protozoarias/genética , Dominio Pirina , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Toxoplasma/genética , Toxoplasma/inmunología , Tuberculosis/microbiologíaRESUMEN
The role of peroxisome proliferator-activated receptor α (PPAR-α) in innate host defense is largely unknown. In this study, we show that PPAR-α is essential for antimycobacterial responses via activation of transcription factor EB (TFEB) transcription and inhibition of lipid body formation. PPAR-α deficiency resulted in an increased bacterial load and exaggerated inflammatory responses during mycobacterial infection. PPAR-α agonists promoted autophagy, lysosomal biogenesis, phagosomal maturation, and antimicrobial defense against Mycobacterium tuberculosis or M. bovis bacillus Calmette-Guérin. PPAR-α agonists regulated multiple genes involved in autophagy and lysosomal biogenesis, including Lamp2, Rab7, and Tfeb in bone marrow-derived macrophages. Silencing of TFEB reduced phagosomal maturation and antimicrobial responses, but increased macrophage inflammatory responses during mycobacterial infection. Moreover, PPAR-α activation promoted lipid catabolism and fatty acid ß-oxidation in macrophages during mycobacterial infection. Taken together, our data indicate that PPAR-α mediates antimicrobial responses to mycobacterial infection by inducing TFEB and lipid catabolism.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/inmunología , Inmunidad Innata/inmunología , Metabolismo de los Lípidos/inmunología , Infecciones por Mycobacterium/inmunología , PPAR alfa/inmunología , Animales , Autofagia/fisiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunohistoquímica , Gotas Lipídicas/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium , PPAR alfa/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
Mycobacterium abscessus is a highly pathogenic drug-resistant rapidly growing mycobacterium. In this study, we evaluated the in vitro, intracellular, and in vivo activities of LCB01-0371, a novel and safe oxazolidinone derivative, for the treatment of M. abscessus infection and compared its resistance to that of other oxazolidinone drugs. LCB01-0371 was effective against several M. abscessus strains in vitro and in a macrophage model of infection. In the murine model, a similar efficacy to linezolid was achieved, especially in the lungs. We induced laboratory-generated resistance to LCB01-0371; sequencing analysis revealed mutations in rplC of T424C and G419A and a nucleotide insertion at the 503 position. Furthermore, LCB01-0371 inhibited the growth of amikacin-, cefoxitin-, and clarithromycin-resistant strains. Collectively, our data indicate that LCB01-0371 might represent a promising new class of oxazolidinones with improved safety, which may replace linezolid for the treatment of M. abscessus.
Asunto(s)
Antibacterianos/uso terapéutico , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Mycobacterium abscessus/efectos de los fármacos , Oxazolidinonas/uso terapéutico , Animales , Farmacorresistencia Bacteriana/genética , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Linezolid/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Mycobacterium abscessus/aislamiento & purificaciónRESUMEN
MicroRNAs (miRNAs) are small noncoding nucleotides that play critical roles in the regulation of diverse biological functions, including the response of host immune cells. Autophagy plays a key role in activating the antimicrobial host defense against Mycobacterium tuberculosis. Although the pathways associated with autophagy must be tightly regulated at a posttranscriptional level, the contribution of miRNAs and whether they specifically influence the activation of macrophage autophagy during M. tuberculosis infection are largely unknown. In this study, we demonstrate that M. tuberculosis infection of macrophages leads to increased expression of miRNA-125a-3p (miR-125a), which targets UV radiation resistance-associated gene (UVRAG), to inhibit autophagy activation and antimicrobial responses to M. tuberculosis. Forced expression of miR-125a significantly blocked M. tuberculosis-induced activation of autophagy and phagosomal maturation in macrophages, and inhibitors of miR-125a counteracted these effects. Both TLR2 and MyD88 were required for biogenesis of miR-125a during M. tuberculosis infection. Notably, activation of the AMP-activated protein kinase significantly inhibited the expression of miR-125a in M. tuberculosis-infected macrophages. Moreover, either overexpression of miR-125a or silencing of UVRAG significantly attenuated the antimicrobial effects of macrophages against M. tuberculosis. Taken together, these data indicate that miR-125a regulates the innate host defense by inhibiting the activation of autophagy and antimicrobial effects against M. tuberculosis through targeting UVRAG.
Asunto(s)
Autofagia/genética , Macrófagos/inmunología , MicroARNs/fisiología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Proteínas Supresoras de Tumor/genética , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/inmunología , Animales , Línea Celular , Regulación de la Expresión Génica , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Factor 88 de Diferenciación Mieloide , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 2 , Tuberculosis Pulmonar/genéticaRESUMEN
The intracellular parasite Toxoplasma gondii has unique dense granule antigens (GRAs) that are crucial for host infection. Emerging evidence suggests that GRA7 of T. gondii is a promising serodiagnostic marker and an effective toxoplasmosis vaccine candidate; however, little is known about the intracellular regulatory mechanisms involved in the GRA7-induced host responses. Here we show that GRA7-induced MyD88 signaling through the activation of TRAF6 and production of reactive oxygen species (ROS) is required for the induction of NF-κB-mediated proinflammatory responses by macrophages. GRA7 stimulation resulted in the rapid activation of mitogen-activated protein kinases and an early burst of ROS in macrophages in a MyD88-dependent manner. GRA7 induced a physical association between GRA7 and TRAF6 via MyD88. Remarkably, the C terminus of GRA7 (GRA7-V) was sufficient for interaction with and ubiquitination of the RING domain of TRAF6, which is capable of inflammatory cytokine production. Interestingly, the generation of ROS and TRAF6 activation are mutually dependent on GRA7/MyD88-mediated signaling in macrophages. Furthermore, mice immunized with GRA7-V showed markedly increased Th1 immune responses and protective efficacy against T. gondii infection. Collectively, these results provide novel insight into the crucial role of GRA7-TRAF6 signaling in innate immune responses.
Asunto(s)
Antígenos de Protozoos/metabolismo , Macrófagos/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Proteínas Protozoarias/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Toxoplasma/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Línea Celular , Citocinas/biosíntesis , Activación Enzimática , Células HEK293 , Humanos , Inmunidad Innata/inmunología , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , NADPH Oxidasa 2 , NADPH Oxidasas/genética , FN-kappa B/inmunología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/inmunología , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/parasitología , UbiquitinaciónRESUMEN
The strongest anaphylatoxin, C5a, plays a critical role in the proinflammatory responses, causing the pathogenesis of a number of inflammatory diseases including sepsis, asthma, and rheumatoid arthritis. Inhibitors of C5a thus have great potential as therapeutics for various inflammatory disorders. Herein, we present the development of a high-affinity repebody against human C5a (hC5a), which effectively suppresses the proinflammatory response. A repebody scaffold composed of leucine-rich repeat (LRR) modules was previously developed as an alternative protein scaffold. A repebody specifically binding to hC5a was selected through a phage display, and its affinity was increased up to 5 nM using modular engineering. The repebody was shown to effectively inhibit the production of C5a-induced proinflammatory cytokines by human monocytes. To obtain insight into a mode of action by the repebody, we determined its crystal structure in complex with hC5a. A structural analysis revealed that the repebody binds to the D1 and D3 regions of hC5a, overlapping several epitope residues with the hC5a receptor (hC5aR). It is thus likely that the repebody suppresses the hC5a-mediated immune response in monocytes by blocking the binding of hC5a to its receptor. The anti-hC5a repebody can be developed as a potential therapeutic for C5a-involved inflammatory diseases.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Complemento C5a/química , Complemento C5a/inmunología , Mediadores de Inflamación/inmunología , Receptor de Anafilatoxina C5a/química , Receptor de Anafilatoxina C5a/inmunología , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Células Cultivadas , Humanos , Factores Inmunológicos/química , Factores Inmunológicos/inmunología , Mediadores de Inflamación/química , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Unión Proteica , Conformación ProteicaRESUMEN
Intracellular reactive oxygen species (ROS) are essential secondary messengers in many signaling cascades governing innate immunity and cellular functions. TLR3 signaling is crucially involved in antiviral innate and inflammatory responses; however, the roles of ROS in TLR3 signaling remain largely unknown. In this study, we show that TLR3-induced ROS generation is required for the activation of NF-κB, IFN-regulatory factor 3, and STAT1-mediated innate immune responses in macrophages. TLR3 induction led to a rapid increase in ROS generation and a physical association between components of the NADPH oxidase (NOX) enzyme complex (NOX2 and p47(phox)) and TLR3 via a Ca(2+)-c-Src tyrosine kinase-dependent pathway. TLR3-induced ROS generation, NOX2, and p47(phox) were required for the phosphorylation and nuclear translocation of STAT1 and STAT2. TLR3-induced activation of STAT1 contributed to the generation of inflammatory mediators, which was significantly attenuated in NOX2- and p47(phox)-deficient macrophages, suggesting a role for ROS-STAT1 in TLR3-mediated innate immune responses. Collectively, these results provide a novel insight into the crucial role that TLR3-ROS signaling plays in innate immune responses by activating STAT1.
Asunto(s)
Inmunidad Innata/inmunología , Especies Reactivas de Oxígeno/inmunología , Factor de Transcripción STAT1/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 3/inmunología , Animales , Western Blotting , Células Cultivadas , Femenino , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Inflamación/inmunología , Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/metabolismo , Receptor Toll-Like 3/metabolismoRESUMEN
Interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune responses for host defense and tumorigenic process. Upregulation of IL-6 is known to constitutively phosphorylate signal transducer and activator of transcription 3 (STAT3), leading to activation of multiple oncogene pathways and inflammatory cascade. Here, we present the development of a high-affinity protein binder, termed repebody, which effectively suppresses non-small cell lung cancer in vivo by blocking the IL-6/STAT3 signaling. We selected a repebody that prevents human IL-6 (hIL-6) from binding to its receptor by a competitive immunoassay, and modulated its binding affinity for hIL-6 up to a picomolar range by a modular approach that mimics the combinatorial assembly of diverse modules to form antigen-specific receptors in nature. The resulting repebody was highly specific for hIL-6, effectively inhibiting the STAT3 phosphorylation in a dose- and binding affinity-response manner in vitro. The repebody was shown to have a remarkable suppression effect on the growth of tumors and STAT3 phosphorylation in xenograft mice with non-small cell lung cancer by blocking the hIL-6/STAT3 signaling. Structural analysis of the repebody and IL-6 complex revealed that the repebody binds the site 2a of hIL-6, overlapping a number of epitope residues at site 2a with gp130, and consequently causes a steric hindrance to the formation of IL-6/IL-6Rα complex. Our results suggest that high-affinity repebody targeting the IL-6/STAT3 pathway can be developed as therapeutics for non-small cell lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Femenino , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Upon phagocytosis, Legionella pneumophila translocates numerous effector proteins into host cells to perturb cellular metabolism and immunity, ultimately establishing intracellular survival and growth. VipD of L. pneumophila belongs to a family of bacterial effectors that contain the N-terminal lipase domain and the C-terminal domain with an unknown function. We report the crystal structure of VipD and show that its C-terminal domain robustly interferes with endosomal trafficking through tight and selective interactions with Rab5 and Rab22. This domain, which is not significantly similar to any known protein structure, potently interacts with the GTP-bound active form of the two Rabs by recognizing a hydrophobic triad conserved in Rabs. These interactions prevent Rab5 and Rab22 from binding to downstream effectors Rabaptin-5, Rabenosyn-5 and EEA1, consequently blocking endosomal trafficking and subsequent lysosomal degradation of endocytic materials in macrophage cells. Together, this work reveals endosomal trafficking as a target of L. pneumophila and delineates the underlying molecular mechanism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Legionella pneumophila/metabolismo , Legionelosis/metabolismo , Macrófagos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transporte Biológico/genética , Proteínas Portadoras/genética , Proteínas de Unión al ADN , Endosomas/genética , Endosomas/microbiología , Endosomas/patología , Células HeLa , Humanos , Legionella pneumophila/química , Legionella pneumophila/genética , Legionelosis/genética , Legionelosis/patología , Lisosomas/genética , Lisosomas/metabolismo , Lisosomas/microbiología , Lisosomas/patología , Macrófagos/microbiología , Macrófagos/patología , Ratones , Proteínas Nucleares/genética , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab5/genéticaRESUMEN
Immune-mediated inflammatory diseases are various groups of conditions that result in immune system disorders and increased cancer risk. Despite the identification of causative cytokines and pathways, current clinical treatment for immune-mediated inflammatory diseases is limited. In addition, immune-mediated inflammatory disease treatment can increase the risk of cancer. Several previous studies have demonstrated that Toxoplasma gondii manipulates the immune response by inhibiting or stimulating cytokines, suggesting the potential for controlling and maintaining a balanced immune system. Additionally, T. gondii also has the unique characteristic of being a so-called "Trojan horse" bacterium that can be used as a drug delivery system to treat regions that have been resistant to previous drug delivery therapies. In this study, we reviewed the potential of T. gondii in drug development and as a delivery system through current research on inflammation-regulating mechanisms in immune-mediated inflammatory diseases.
Asunto(s)
Neoplasias , Toxoplasma , Humanos , Toxoplasma/metabolismo , Citocinas/metabolismo , Inflamación/tratamiento farmacológico , Desarrollo de MedicamentosRESUMEN
Conditions such as acute pancreatitis, ulcerative colitis, delayed graft function and infections caused by a variety of microorganisms, including gram-positive and gram-negative organisms, increase the risk of sepsis and therefore mortality. Immune dysfunction is a characterization of sepsis, so timely and effective treatment strategies are needed. The conventional approaches, such as antibiotic-based treatments, face challenges such as antibiotic resistance, and cytokine-based treatments have shown limited efficacy. To address these limitations, a novel approach focusing on membrane receptors, the initiators of the inflammatory cascade, is proposed. Membrane receptors such as Toll-like receptors, interleukin-1 receptor, endothelial protein C receptor, µ-opioid receptor, triggering receptor expressed on myeloid cells 1, and G-protein coupled receptors play pivotal roles in the inflammatory response, offering opportunities for rapid regulation. Various membrane receptor blockade strategies have demonstrated efficacy in both preclinical and clinical studies. These membrane receptor blockades act as early stage inflammation modulators, providing faster responses compared to conventional therapies. Importantly, these blockers exhibit immunomodulatory capabilities without inducing complete immunosuppression. Finally, this review underscores the critical need for early intervention in acute inflammatory and infectious diseases, particularly those posing a risk of progressing to sepsis. And, exploring membrane receptor blockade as an adjunctive treatment for acute inflammatory and infectious diseases presents a promising avenue. These novel approaches, when combined with antibiotics, have the potential to enhance patient outcomes, particularly in conditions prone to sepsis, while minimizing risks associated with antibiotic resistance and immune suppression.
Asunto(s)
Inflamación , Sepsis , Humanos , Sepsis/tratamiento farmacológico , Sepsis/inmunología , Animales , Inflamación/inmunología , Inflamación/tratamiento farmacológico , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismoRESUMEN
Ulcerative colitis (UC) is a significant inflammatory bowel disease caused by an abnormal immune response to gut microbes. However, there are still gaps in our understanding of how immune and metabolic changes specifically contribute to this disease. Our research aims to address this gap by examining mouse colons after inducing ulcerative colitis-like symptoms. Employing single-cell RNA-seq and 16 s rRNA amplicon sequencing to analyze distinct cell clusters and microbiomes in the mouse colon at different time points after induction with dextran sodium sulfate. We observe a significant reduction in epithelial populations during acute colitis, indicating tissue damage, with a partial recovery observed in chronic inflammation. Analyses of cell-cell interactions demonstrate shifts in networking patterns among different cell types during disease progression. Notably, macrophage phenotypes exhibit diversity, with a pronounced polarization towards the pro-inflammatory M1 phenotype in chronic conditions, suggesting the role of macrophage heterogeneity in disease severity. Increased expression of Nampt and NOX2 complex subunits in chronic UC macrophages contributes to the inflammatory processes. The chronic UC microbiome exhibits reduced taxonomic diversity compared to healthy conditions and acute UC. The study also highlights the role of T cell differentiation in the context of dysbiosis and its implications in colitis progression, emphasizing the need for targeted interventions to modulate the inflammatory response and immune balance in colitis.