RESUMEN
BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins. RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-ß activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV. CONCLUSION: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.
Asunto(s)
Anticuerpos Neutralizantes , Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Péptidos , Virus del Síndrome Respiratorio y Reproductivo Porcino , Receptores de Superficie Celular , Anticuerpos de Dominio Único , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Animales , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/química , Porcinos , Antígenos de Diferenciación Mielomonocítica/inmunología , Antígenos de Diferenciación Mielomonocítica/metabolismo , Receptores de Superficie Celular/inmunología , Antígenos CD/inmunología , Antígenos CD/metabolismo , Anticuerpos Neutralizantes/inmunología , Péptidos/química , Péptidos/farmacología , Péptidos/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Ratones , Replicación Viral/efectos de los fármacos , Línea CelularRESUMEN
Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing acute intestinal infection in pigs, with high mortality often seen in neonatal pigs. The newborns rely on innate immune responses against invading pathogens because of lacking adaptive immunity. However, how PEDV disables the innate immunity of newborns toward severe infection remains unknown. We found that PEDV infection led to reduced expression of histone deacetylases (HDACs), especially HDAC1, in porcine IPEC-J2 cells. HDACs are considered important regulators of innate immunity. We hypothesized that PEDV interacts with certain host factors to regulate HDAC1 expression in favor of its replication. We show that HDAC1 acted as a negative regulator of PEDV replication in IPEC-J2 cells, as shown by chemical inhibition, gene knockout, and overexpression. A GC-box (GCCCCACCCCC) within the HDAC1 promoter region was identified for Sp1 binding in IPEC-J2 cells. Treatment of the cells with Sp1 inhibitor mithramycin A inhibited HDAC1 expression, indicating direct regulation of HDAC1 expression by Sp1. Of the viral proteins that were overexpressed in IPEC-J2 cells, the N protein was found to be present in the nuclei and more inhibitory to HDAC1 transcription. The putative nuclear localization sequence 261PKKNKSR267 contributed to its nuclear localization. The N protein interacted with Sp1 and interfered with its binding to the promoter region, thereby inhibiting its transcriptional activity for HDAC1 expression. Our findings reveal a novel mechanism of PEDV evasion of the host responses, offering implications for studying the infection processes of other coronaviruses. IMPORTANCE The enteric coronavirus porcine epidemic diarrhea virus (PEDV) causes fatal acute intestinal infection in neonatal pigs that rely on innate immune responses. Histone deacetylases (HDACs) play important roles in innate immune regulation. Our study found PEDV suppresses HDAC1 expression via the interaction of its N protein and porcine Sp1, which identified a novel mechanism of PEDV evasion of the host responses to benefit its replication. This study suggests that other coronaviruses, including SARS-CoV and SARS-CoV-2, also make use of their N proteins to intercept the host immune responses in favor of their infection.
Asunto(s)
Infecciones por Coronavirus/veterinaria , Células Epiteliales/virología , Histona Desacetilasa 1/antagonistas & inhibidores , Mucosa Intestinal/virología , Factor de Transcripción Sp1/metabolismo , Enfermedades de los Porcinos/virología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Células Cultivadas , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Virus de la Diarrea Epidémica Porcina/patogenicidad , Factor de Transcripción Sp1/genética , Porcinos , Enfermedades de los Porcinos/metabolismo , Enfermedades de los Porcinos/patología , Proteínas no Estructurales Virales/genéticaRESUMEN
Porcine epidemic diarrhea virus (PEDV) can infect pigs of all ages, especially piglets. PEDV has spread across Asia since the 1980s. The highly virulent variant PEDV broke out on a large scale and caused huge economic losses to the pig industry in late 2010 in China. Rapid detection methods with high specificity and sensitivity are urgently needed for the diagnosis and control of the disease. In this study, we divided the PEDV S1 gene into three segments and constructed the recombinant plasmids pFastBac1-S1T1 (aa 21-279), pFastBac1-S1T2 (aa 280-539) and pFastBac1-S1T3 (aa 540-788), which carry the different antigenic regions of the S1 gene. Truncated S1 proteins PEDV-S1T1/S1T2/S1T3 were obtained by a Bac-to-Bac expression system, with protein sizes of 36 kDa, 38 kDa and 38 kDa, respectively. Recombinant proteins presented high reactivity with the monoclonal antibody against PEDV and positive pig serum. Based on full-length S1 protein and these truncated proteins, we established indirect ELISA methods for the detection of PEDV IgA antibody. A total of 213 clinical serum samples were tested by the above indirect ELISA methods, and IFA was used as the gold standard. ROC curves revealed a significant correlation between S1-ELISA and S1T2-ELISA with a 0.9134 correlation coefficient and favourable sensitivity and specificity of S1-ELISA (93.24%, 95.68%) and S1T2-ELISA (89.33%, 94.16%). Our results also indicated that serum with higher neutralizing activity (SNT ≥ 40) had a higher IgA antibody level based on S1-ELISA, S1T1-ELISA and S1T2-ELISA. In conclusion, both S1-ELISA and S1T2-ELISA can be used as candidate systems for detecting anti-PEDV IgA antibody titers in serum, which can reflect the level of neutralizing activity in pigs after natural infection or vaccination. The above research results provide a basis for the prevention and control of PEDV and can be used in the detection of host anti-infective immunity and evaluation of vaccine immune effects.
Asunto(s)
Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Animales , Anticuerpos Antivirales , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Inmunoglobulina A , Virus de la Diarrea Epidémica Porcina/genética , PorcinosRESUMEN
Ginsenoside Rg3 is one of the main constituents of Panax ginseng. Compelling evidence has demonstrated that ginsenoside Rg3 is capable of inhibiting inflammation. However, the mechanism mediating its anti-inflammatory effects remain unclear. Here we show that ginsenoside Rg3 blocks IL-1ß secretion and caspase-1 activation through inhibiting LPS priming and the NLRP3 inflammasome activation in human and mouse macrophages. Rg3 specifically inhibits activation of NLRP3 but not the NLRC4 or AIM2 inflammasomes. In addition, Rg3 has no effect on upstream regulation of NLRP3 inflammasome, such as K+ efflux, ROS production, or mitochondrial membrane potential. Mechanistically, Rg3 abrogates NEK7-NLRP3 interaction, and subsequently inhibits NLRP3-ASC interaction, ASC oligomerization, and speckle formation. More importantly, Rg3 can reduce IL-1ß secretion induced by LPS in mice and protect mice from lethal endotoxic shock. Thus, our findings reveal an anti-inflammatory mechanism for Rg3 and suggest its potential use in NLRP3-driven diseases.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Ginsenósidos/farmacología , Inflamasomas/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Humanos , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
Pathogenic mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium marinum, can trigger NLRP3 inflammasome activation leading to maturation and secretion of interleukin 1ß (IL-1ß). However, the mycobacterial factors involved in the activation of NLRP3 inflammasome are not fully understood. Here, we identified that the PPE family protein PPE13 was responsible for the induction of IL-1ß secretion in a NLRP3 inflammasome-dependent manner. We found that the recombinant Mycobacterium smegmatis expressing PPE13 activates NLRP3 inflammasome, thereby inducing caspase-1 cleavage and IL-1ß secretion in J774A.1, BMDMs, and THP-1 macrophages. To examine whether this inflammasome activation was triggered by PPE13 rather than components of M. smegmatis, PPE13 was introduced into the aforementioned macrophages by lentivirus as a delivery vector. Similarly, this led to the activation of NLRP3 inflammasome, indicating that PPE13 is a direct activator of NLRP3 cascade. We further demonstrated that the NLRP3 complex activated the inflammasome cascade, and the assembly of this complex was facilitated by PPE13 through interacting with the LRR and NATCH domains of NLRP3. Finally, we found that all PPE13 proteins isolated from M. tuberculosis, M. bovis, and M. marinum can activate NLRP3 inflammasome through binding to NLRP3, which requires C-terminal repetitive MPTR domain of PPE13. Thus, we, for the first time, revealed that PPE13 triggers the inflammasome-response by interacting with the MPTR domain of PPE13 and the LRR and NATCH domains of NLRP3. These findings provide a novel perspective on the function of PPE proteins in the immune system during mycobacteria invasion.
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Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Mycobacterium/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Animales , Línea Celular , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Dominios Proteicos , Células THP-1RESUMEN
In the present study, the adjuvant effect of soybean oil containing ginseng root saponins (SO-GS-R) on the immune response to foot-and-mouth disease vaccine (FMDV) in mice was investigated. When immunized with FMDV antigen emulsified in an SO-GS-R formulation, mice generated remarkably higher serum antibody and cytokine responses than mice immunized with FMDV antigen alone. To elucidate the mechanisms underlying the adjuvant effect of SO-GS-R, we measured cytokines in serum and muscle tissue after intramuscular injection of SO-GS-R. The results showed that injection of SO-GS-R significantly increased the levels of IL-1ß, IL-5, IL-6, G-CSF, KC, MCP-1, MIP-1α, and MIP-1ß in both serum and muscle. These results suggested that SO-GS-R recruits neutrophils, eosinophils, T cells and macrophages, causing immune cell recruitment at the injection site, driving antigen-presenting cells to actively participate in the onset of immunity, and amplifying the immune responses. Considering its adjuvant activity and plant-derived properties, SO-GS-R should be further studied for its adjuvant effect on vaccines used in food animals.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Citocinas/biosíntesis , Fiebre Aftosa/prevención & control , Inmunización , Panax/inmunología , Saponinas/inmunología , Aceite de Soja/inmunología , Vacunas Virales/inmunología , Alimentación Animal , Animales , Anticuerpos Antivirales/sangre , Quimiocina CCL2/sangre , Quimiocina CCL3/sangre , Quimiocina CCL4/sangre , Quimiocina CXCL1/sangre , Citocinas/sangre , Femenino , Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/inmunología , Factor Estimulante de Colonias de Granulocitos/sangre , Inmunoglobulina G/sangre , Inyecciones Intramusculares , Interleucina-1beta/sangre , Interleucina-5/sangre , Interleucina-6/sangre , Ratones , Ratones Endogámicos BALB C , Músculos/inmunología , Aceites de Plantas/farmacología , Saponinas/farmacología , Aceite de Soja/química , Factores de Tiempo , VacunaciónRESUMEN
BACKGROUND: Mycobacterium bovis, the causative agent of bovine tuberculosis, infects host macrophages and triggers production of the proinflammatory cytokine interleukin 1ß (IL-1ß). The mechanism by which macrophages become activated and secrete IL-1ß in tuberculosis has not yet been elucidated. METHODS: In this study, we investigated the role of the absence in melanoma 2 (AIM2) inflammasome in IL-1ß release from macrophages infected with pathogenic M. bovis strain. RESULTS: We found that the AIM2 inflammasome activation is involved in the production of IL-1ß in primary and immortalized mouse macrophage upon M. bovis infection; that the activation process requires cytoplasmic potassium efflux, mycobacterial internalization, but not reactive oxygen species (ROS) or IFN-ß release; that the AIM2 inflammasome contributes to the synthesis of proinflammatory and chemotatic factors in M. bovis-infected macrophages; and that the activation of the AIM2 inflammasome is due, at least in part, to mycobacterial translocation into the cytosol. CONCLUSIONS: We conclude that the AIM2 inflammasome is involved in macrophage activation during infection with virulent M. bovis strain. To our knowledge, this is the first evidences for the involvement of the AIM2 inflammasome in M. bovis infection.
Asunto(s)
Inflamasomas/metabolismo , Activación de Macrófagos , Mycobacterium bovis/inmunología , Proteínas Nucleares/metabolismo , Tuberculosis Bovina/inmunología , Animales , Caspasa 1/genética , Caspasa 1/metabolismo , Bovinos , Línea Celular , Citocinas/metabolismo , Citosol/microbiología , Proteínas de Unión al ADN , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Mycobacterium bovis/patogenicidad , Proteínas Nucleares/genética , Fagosomas/microbiología , Potasio/metabolismo , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/inmunología , Tuberculosis Bovina/microbiología , Regulación hacia Arriba , VirulenciaRESUMEN
Gasdermin D (GSDMD) functions as a pivotal executor of pyroptosis, eliciting cytokine secretion following cleavage by inflammatory caspases. However, the role of posttranslational modifications (PTMs) in GSDMD-mediated pyroptosis remains largely unexplored. In this study, we demonstrate that GSDMD can undergo acetylation at the Lysine 248 residue, and this acetylation enhances pyroptosis. We identify histone deacetylase 4 (HDAC4) as the specific deacetylase responsible for mediating GSDMD deacetylation, leading to the inhibition of pyroptosis both in vitro and in vivo. Deacetylation of GSDMD impairs its ubiquitination, resulting in the inhibition of pyroptosis. Intriguingly, phosphorylation of HDAC4 emerges as a critical regulatory mechanism promoting its ability to deacetylate GSDMD and suppress GSDMD-mediated pyroptosis. Additionally, we implicate Protein phosphatase 1 (PP1) catalytic subunits (PP1α and PP1γ) in the dephosphorylation of HDAC4, thereby nullifying its deacetylase activity on GSDMD. This study reveals a complex regulatory network involving HDAC4, PP1, and GSDMD. These findings provide valuable insights into the interplay among acetylation, ubiquitination, and phosphorylation in the regulation of pyroptosis, offering potential targets for further investigation in the field of inflammatory cell death.
Asunto(s)
Gasderminas , Histona Desacetilasas , Proteína Fosfatasa 1 , Piroptosis , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Procesamiento Proteico-Postraduccional , Humanos , Animales , Ratones , Gasderminas/metabolismoRESUMEN
The cellular prion protein (PrP(C) ) is a glycoprotein anchored by glycosylphosphatidylinositol (GPI) to the cell surface and is abundantly expressed in the central nervous system. Numerous studies have suggested a protective function for PrP(C) , including protection from ischemic and excitotoxic lesions and several apoptotic insults, and recent reports have shown that PrP(C) has a context-dependent neuroprotective function. In this study, we investigated the effect of PPNP down-regulation on various forms of microglial activation. We first examined the mRNA expression of PRNP upon exposure to IFN-γ, IL-4, or IL-10 in BV2 microglia. We then analyzed the effect of si-RNA-mediated disruption of PRNP on different parameters of microglial activation in IFN-γ-, IL-4-, or IL-10-stimulated microglia. The results showed that PRNP mRNA expression was invariably down-regulated in microglia upon exposure to IFN-γ, IL-4, or IL-10. PRNP silencing prior to cytokines treatment reduced the responsiveness of microglia to INF-γ treatment, significantly altered IL-4-induced microglial activation phenotype, and had no effect on IL-10-induced microglial activation. Together, these results support a role of PrP(C) in the modulation of the shift of microglia from a quiescent state to an activated phenotype and in the regulation of the microglial response during classical and alternative activation.
Asunto(s)
Microglía/citología , Microglía/fisiología , Priones/fisiología , Animales , Línea Celular , Regulación hacia Abajo/genética , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos C57BL , Inhibición Neural/genética , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/metabolismo , Proteínas Priónicas , Priones/antagonistas & inhibidores , Priones/genética , ARN Interferente Pequeño/genéticaRESUMEN
The inflammatory responses in Alzheimer's disease and prion diseases are dominated by microglia activation. Three different phenotypes of microglial activation, namely classical activation, alternative activation, and acquired deactivation, have been described. In this study, we investigated the effect of amyloidogenic fragments of amyloid ß and prion proteins (Aß1-42 and PrP106-126) on various forms of microglial activation. We first examined the effect of Aß1-42 and PrP106-126 stimulation on the mRNA expression levels of several markers of microglial activation, as well as the effect of cytochalasin D, a phagocytosis inhibitor, on microglial activation in Aß1-42- and PrP106-126-stimulated BV2 microglia. results showed that Aß1-42 and PrP106-126 induced the classical activation of BV2 microglia, decreased the expression level of alternative expression markers, and had no effect on the expression of acquired deactivation markers. Cytochalasin D treatment significantly reduced Aß1-42- and PrP106-126-induced up-regulation of proinflammatory factors, but did not change the expression profile of the markers of alternative activation or acquired deactivation in BV2 cells which were exposed to Aß1-42 and PrP106-126. Our results suggested that microglia interact with amyloidogenic peptides in the extracellular milieu-stimulated microglial classical activation and reduce its alternative activation, and that the uptake of amyloidogenic peptides from the extracellular milieu amplifies the classical microglial activation.
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Péptidos beta-Amiloides/metabolismo , Microglía/patología , Fagocitosis , Priones/metabolismo , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
Porcine epidemic diarrhea virus (PEDV) is the main etiologic agent causing acute swine epidemic diarrhea, leading to severe economic losses to the pig industry. PEDV has evolved to deploy complicated antagonistic strategies to escape from host antiviral innate immunity. Our previous study demonstrated that PEDV downregulates histone deacetylase 1 (HDAC1) expression by binding viral nucleocapsid (N) protein to the transcription factor Sp1, inducing enhanced protein acetylation. We hypothesized that PEDV inhibition of HDAC1 expression would enhance acetylation of the molecules critical in innate immune signaling. Signal transducer and activator of transcription 1 (STAT1) is a crucial transcription factor regulating expression of interferon (IFN)-stimulated genes (ISGs) and anti-PEDV immune responses, as shown by overexpression, chemical inhibition, and gene knockdown in IPEC-J2 cells. We further show that PEDV infection and its N protein overexpression, although they upregulated STAT1 transcription level, could significantly block poly(I·C) and IFN-λ3-induced STAT1 phosphorylation and nuclear localization. Western blotting revealed that PEDV and its N protein promote STAT1 acetylation via downregulation of HDAC1. Enhanced STAT1 acetylation due to HDAC1 inhibition by PEDV or MS-275 (an HDAC1 inhibitor) impaired STAT1 phosphorylation, indicating that STAT1 acetylation negatively regulated its activation. These results, together with our recent report on PEDV N-mediated inhibition of Sp1, clearly indicate that PEDV manipulates the Sp1-HDAC1-STAT1 signaling axis to inhibit transcription of OAS1 and ISG15 in favor of its replication. This novel immune evasion mechanism is realized by suppression of STAT1 activation through preferential modulation of STAT1 acetylation over phosphorylation as a result of HDAC1 expression inhibition. IMPORTANCE PEDV has developed sophisticated evasion mechanisms to escape host IFN signaling via its structural and nonstructural proteins. STAT1 is one of the key transcription factors in regulating expression of ISGs. We found that PEDV and its N protein inhibit STAT1 phosphorylation and nuclear localization via inducing STAT1 acetylation as a result of HDAC1 downregulation, which, in turn, dampens the host IFN signaling activation. Our study demonstrates a novel mechanism that PEDV evades host antiviral innate immunity through manipulating the reciprocal relationship of STAT1 acetylation and phosphorylation. This provides new insights into the pathogenetic mechanisms of PEDV and even other coronaviruses.
Asunto(s)
Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Animales , Porcinos , Interferón lambda , Fosforilación , Línea Celular , Acetilación , Antivirales , Factores de Transcripción , Factor de Transcripción STAT1RESUMEN
PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) is a major swine pathogen causing economic losses. To the date, effective broad PRRSV inhibitory strategies have not been available in practice yet. Targeting the key viral receptor CD163 to block PRRSV entry has emerged as an alternative approach beside vaccines for PRRSV inhibition. As an effective therapeutic tool, nanoantibodies (Nbs) have been widely used in antiviral research. In this study, a phage display VHH library was constructed for the selection of Nbs against porcine CD163 scavenger receptor cysteine-rich 5-9 domain (SRCR5-9). After five rounds of bio-panning and indirect ELISA, seven CD163-specific Nbs (Nb1-Nb7) were identified. All obtained Nbs displayed strong affinity to CD163 receptor and excellent antiviral activity. In particular, Nb2 exhibited significant broad inhibitory effects on variable PRRSV lineages and downregulated virus-related NF-κB signaling. Further studies suggested that Nbs mainly exerted antiviral functions by interfering with virus attachment stage, and also decreased the transcription of CD163. The conformational epitopes recognized by Nbs were localized in the SRCR5 domain of CD163, a crucial region in PRRSV infection. Overall, our findings provide a novel insight into the biofunction of CD163 in antiviral infection and the development of broad-spectrum strategies against PRRSV.
Asunto(s)
Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Anticuerpos de Dominio Único , Porcinos , Animales , Anticuerpos de Dominio Único/farmacología , Antivirales/farmacologíaRESUMEN
The microbiota-associated factors that influence host susceptibility and immunity to enteric viral infections remain poorly defined. We identified that the herbal monomer ginsenoside Rg3 (Rg3) can shape the gut microbiota composition, enriching robust short-chain fatty acid (SCFA)-producing Blautia spp. Colonization by representative Blautia coccoides and Blautia obeum could protect germ-free or vancomycin (Van)-treated mice from enteric virus infection, inducing type I interferon (IFN-I) responses in macrophages via the MAVS-IRF3-IFNAR signaling pathway. Application of exogenous SCFAs (acetate/propionate) reproduced the protective effect of Rg3 and Blautia spp. in Van-treated mice, enhancing intracellular Ca2+- and MAVS-dependent mtDNA release and activating the cGAS-STING-IFN-I axis by stimulating GPR43 signaling in macrophages. Our findings demonstrate that macrophage sensing of metabolites from specific commensal bacteria can prime the IFN-I signaling that is required for antiviral functions.
Asunto(s)
Interferón Tipo I , Virosis , Ratones , Animales , Inmunidad Innata/genética , Proteínas de la Membrana/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Ácidos Grasos VolátilesRESUMEN
Currently, antibiotics are the most common treatment for bacterial infections in clinical practice. However, with the abuse of antibiotics and the emergence of drug-resistant bacteria, the use of antibiotics has faced an unprecedented challenge. It is imminent to develop nonantibiotic antimicrobial agents. Based on the cation-π structure of barnacle cement protein, a polyphosphazene-based polymer poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl p-ammonium bromide (ammonium bromide)-g-(N,N,N,N-dimethylaminoethyl acetate ethylammonium bromide)] (PZBA) with potential adhesion and inherent antibacterial properties was synthesized, and a series of injectable antibacterial adhesive hydrogels (PZBA-PVA) were prepared by cross-linking with poly(vinyl alcohol) (PVA). PZBA-PVA hydrogels showed good biocompatibility, and the antibacterial rate of the best-performed hydrogel reached 99.81 ± 0.04% and 98.80 ± 2.16% against Staphylococcus aureus and Escherichia coli within 0.5 h in vitro, respectively. In the infected wound model, the healing rate of the PZBA-PVA-treated group was significantly higher than that of the Tegaderm film group due to the fact that the hydrogel suppressed inflammatory responses and modulated the infiltration of immune cells. Moreover, the wound healing mechanism of the PZBA-PVA hydrogel was further evaluated by real-time polymerase chain reaction and total RNA sequencing. The results indicated that the process of hemostasis and tissue development was prompted and the inflammatory and immune responses were suppressed to accelerate wound healing. Overall, the PZBA-PVA hydrogel is shown to have the potential for infected wound healing application.
Asunto(s)
Infecciones Estafilocócicas , Adhesivos Tisulares , Humanos , Hidrogeles/farmacología , Hidrogeles/química , Antibacterianos/farmacología , Antibacterianos/químicaRESUMEN
BACKGROUND: Prion diseases are neurodegenerative disorders characterized by the accumulation of an abnormal disease-associated prion protein, PrPSc. In prion-infected brains, activated microglia are often present in the vicinity of PrPSc aggregates, and microglial activation is thought to play a key role in the pathogenesis of prion diseases. Although interleukin (IL)-1ß release by prion-induced microglia has been widely reported, the mechanism by which primed microglia become activated and secrete IL-1ß in prion diseases has not yet been elucidated. In this study, we investigated the role of the NACHT, LRR and PYD domains-containing protein (NALP)3 inflammasome in IL-1ß release from lipopolysaccharide (LPS)-primed microglia after exposure to a synthetic neurotoxic prion fragment (PrP106-126). METHODS: The inflammasome components NALP3 and apoptosis-associated speck-like protein (ASC) were knocked down by gene silencing. IL-1ß production was assessed using ELISA. The mRNA expression of NALP3, ASC, and pro-inflammatory factors was measured by quantitative PCR. Western blot analysis was used to detect the protein level of NALP3, ASC, caspase-1 and nuclear factor-κB. RESULTS: We found that that PrP106-126-induced IL-1ß release depends on NALP3 inflammasome activation, that inflammasome activation is required for the synthesis of pro-inflammatory and chemotactic factors by PrP106-126-activated microglia, that inhibition of NF-κB activation abrogated PrP106-126-induced NALP3 upregulation, and that potassium efflux and production of reactive oxygen species were implicated in PrP106-126-induced NALP3 inflammasome activation in microglia. CONCLUSIONS: We conclude that the NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation. To our knowledge, this is the first time that strong evidence for the involvement of NALP3 inflammasome in prion-associated inflammation has been found.
Asunto(s)
Proteínas Portadoras/fisiología , Inflamasomas/fisiología , Microglía/efectos de los fármacos , Microglía/patología , Fragmentos de Péptidos/toxicidad , Priones/toxicidad , Animales , Animales Recién Nacidos , Células Cultivadas , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLRRESUMEN
Porcine epidemic diarrhea virus (PEDV) infection causes huge economic losses to the pig industry worldwide. DNAJA3, a member of the Hsp40 family proteins, is known to play an important role in the replication of several viruses. However, it remains unknown if it interacts with PEDV. We found that DNAJA3 interacted with PEDV S1, initially with yeast two-hybrid screening and later with Co-IP, GST pull-down, and confocal imaging. Further experiments showed the functional relationship between DNAJA3 and PEDV in the infected IPEC-J2 cells. DNAJA3 overexpression significantly inhibited PEDV replication while its knockdown had the opposite effect, suggesting that it is a negative regulator of PEDV replication. In addition, DNAJA3 expression could be downregulated by PEDV infection possibly as the viral strategy to evade the suppressive role of DNAJA3. By gene silencing and overexpression, we were able to show that DNAJA3 inhibited PEDV adsorption to IPEC-J2 cells but did not affect virus invasion. In conclusion, our study provides clear evidence that DNAJA3 mediates PEDV adsorption to host cells and plays an antiviral role in IPEC-J2 cells.
Asunto(s)
Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Porcinos , Animales , Chlorocebus aethiops , Virus de la Diarrea Epidémica Porcina/genética , Adsorción , Replicación Viral , Células Vero , Proteínas/farmacologíaRESUMEN
The current study was conducted to analyze the functions of blood neutrophils in transition cows and their association with postpartum mastitis risk as indicated by somatic cell counts (SCCs) in milk. Seventy-six healthy Holstein dairy cows were monitored from Week 4 prepartum to Week 4 postpartum. Five dairy cows with low SCCs (38 ± 6.0 × 103/mL) and five with high SCCs (3,753 ± 570.0 × 103/mL) were selected based on milk SCCs during the first three weeks of lactation. At Week 1 pre- and postpartum, serum samples were obtained from each cow to measure neutrophil extracellular trap (NET)-related variables, and blood neutrophils were collected for transcriptome analysis by RNA sequencing. The serum concentration of NETs was significantly higher (P < 0.05) in cows with high SCCs than in cows with low SCCs (36.5 ± 2.92 vs. 18.4 ± 1.73 ng/mL). The transcriptomic analysis revealed that the transcriptome differences in neutrophils between high- and low-SCC cows were mainly in cell cycle-related pathways (42.6%), including the cell cycle, DNA damage, and chromosomal conformation, at Week 1 prepartum. The hub genes of these pathways were mainly involved in both the cell cycle and NETosis. These results indicated that the formation of NETs in the blood of transition dairy cows was different between cows with low and high SCCs, which may be used as a potential indicator for the prognosis of postpartum mastitis risk and management strategies of perinatal dairy cows.
Asunto(s)
Trampas Extracelulares , Mastitis Bovina , Animales , Bovinos , Femenino , Humanos , Lactancia , Leche , EmbarazoRESUMEN
Gasdermin D (GSDMD) participates in the activation of inflammasomes and pyroptosis. Meanwhile, ubiquitination strictly regulates inflammatory responses. However, how ubiquitination regulates Gasdermin D activity is not well understood. In this study, we show that pyroptosis triggered by Gasdermin D is regulated through ubiquitination. Specifically, SYVN1, an E3 ubiquitin ligase of gasdermin D, promotes GSDMD-mediated pyroptosis. SYVN1 deficiency inhibits pyroptosis and subsequent LDH release and PI uptake. SYVN1 directly interacts with GSDMD, and mediates K27-linked polyubiquitination of GSDMD on K203 and K204 residues, promoting GSDMD-induced pyroptotic cell death. Thus, our findings revealed the essential role of SYVN1 in GSDMD-mediated pyroptosis. Overall, GSDMD ubiquitination is a potential therapeutic module for inflammatory diseases.
Asunto(s)
Piroptosis , Ubiquitina-Proteína Ligasas , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Piroptosis/fisiología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Coronaviruses (CoVs) are a family of RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans. Here, we use porcine epidemic diarrhea virus (PEDV) as a model of CoVs to illustrate the reciprocal regulation between CoV infection and pyroptosis. For the first time, we elucidate the molecular mechanism of porcine gasdermin D (pGSDMD)-mediated pyroptosis and demonstrate that amino acids R238, T239, and F240 within pGSDMD-p30 are critical for pyroptosis. Furthermore, 3C-like protease Nsp5 from SARS-CoV-2, MERS-CoV, PDCoV, and PEDV can cleave pGSDMD at the Q193-G194 junction to produce two fragments unable to trigger pyroptosis. The two cleaved fragments could not inhibit PEDV replication. In addition, Nsp5 from SARS-CoV-2 and MERS-CoV also cleave human GSDMD (hGSDMD). Therefore, we provide clear evidence that PEDV may utilize the Nsp5-GSDMD pathway to inhibit pyroptosis and, thus, facilitate viral replication during the initial period, suggesting an important strategy for the coronaviruses to sustain their infection. IMPORTANCE Recently, GSDMD has been reported as a key executioner for pyroptosis. This study first demonstrates the molecular mechanism of pGSDMD-mediated pyroptosis and that the pGSDMD-mediated pyroptosis protects host cells against PEDV infection. Notably, PEDV employs its Nsp5 to directly cleave pGSDMD in favor of its replication. We found that Nsp5 proteins from other coronaviruses, such as porcine deltacoronavirus, severe acute respiratory syndrome coronavirus 2, and Middle East respiratory syndrome coronavirus, also had the protease activity to cleave human and porcine GSDMD. Thus, we provide clear evidence that the coronaviruses might utilize Nsp5 to inhibit the host pyroptotic cell death and facilitate their replication during the initial period, an important strategy for their sustaining infection. We suppose that GSDMD is an appealing target for the design of anticoronavirus therapies.
Asunto(s)
COVID-19 , Virus de la Diarrea Epidémica Porcina , Animales , Humanos , Gasderminas , Péptido Hidrolasas , Piroptosis , SARS-CoV-2 , PorcinosRESUMEN
Porcine epidemic diarrhea virus (PEDV) is a swine enterovirus that causes huge economic losses to the swine industry. It is of great interest to understand the gene expression patterns of host responses to PEDV infection and the mechanistic insights. Here, we report the differences of gene expression profiles by RNA-seq in the porcine small intestinal 2-D enteroids cells infected with low-passage (16 passages, P16) and high-passage (120 passages, P120) PEDV strains for 12, 24 and 36 h. Of the 57 genes differentially expressed in P16 PEDV infected enteroids, 49 were upregulated and 7 downregulated at all time points. There were 247 genes with different patterns of expression in the enteroids infected with P120 PEDV: upregulation seen with 105 genes and downregulation with the remaining majority at all time points. Infection of both P16 and P120 PEDV strains led to significant upregulation of ISGs, such as ISG15, MX1 and RSAD2. In particular, P120 PEDV infection inhibited transcription of genes related to lipid metabolism, including those involved in lipid decomposition, absorption, bile secretion and cholesterol metabolism. Treatment of the infected enteroids with palmitic acid resulted in marked reduction of replication of both P16 and P120 PEDV strains. These results indicate that PEDV might manipulate lipid metabolism of the host to benefit its replication. Further research is warranted to study the mechanisms how palmitic acid inhibits PEDV replication.