ABSTRACT
The cytokine interleukin-6 (IL-6) plays a crucial role in autoimmune and inflammatory diseases. Understanding the precise mechanism of IL-6 interaction at the amino acid level is essential to develop IL-6-inhibiting compounds. In this study, we employed computer-guided drug design tools to predict the key residues that are involved in the interaction between IL-6 and its receptor IL-6R. Subsequently, we generated IL-6 mutants and evaluated their binding affinity to IL-6R and the IL-6R - gp130 complex, as well as monitoring their biological activities. Our findings revealed that the R167A mutant exhibited increased affinity for IL-6R, leading to enhanced binding to IL-6R - gp130 complex and subsequently elevated intracellular phosphorylation of STAT3 in effector cells. On the other hand, although E171A reduced its affinity for IL-6R, it displayed stronger binding to the IL-6R - gp130 complex, thereby enhancing its biological activity. Furthermore, we identified the importance of R178 and R181 for the precise recognition of IL-6 by IL-6R. Mutants R181A/V failed to bind to IL-6R, while maintaining an affinity for the IL-6 - gp130 complex. Additionally, deletion of the D helix resulted in complete loss of IL-6 binding affinity for IL-6R. Overall, this study provides valuable insights into the binding mechanism of IL-6 and establishes a solid foundation for future design of novel IL-6 inhibitors.
Subject(s)
Interleukin-6 , Molecular Docking Simulation , Protein Binding , Receptors, Interleukin-6 , Interleukin-6/metabolism , Interleukin-6/genetics , Humans , Receptors, Interleukin-6/metabolism , Receptors, Interleukin-6/genetics , Receptors, Interleukin-6/chemistry , Cytokine Receptor gp130/metabolism , Cytokine Receptor gp130/genetics , Cytokine Receptor gp130/chemistry , Mutagenesis, Site-Directed , Binding Sites , STAT3 Transcription Factor/metabolism , Phosphorylation , MutationABSTRACT
Background: CD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v. Methods: In this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping. Results: An optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation. Conclusions: This study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.
Subject(s)
African Swine Fever Virus , African Swine Fever , Antibodies, Monoclonal , Epitope Mapping , NF-kappa B , Animals , African Swine Fever Virus/immunology , NF-kappa B/metabolism , NF-kappa B/immunology , Swine , Mice , African Swine Fever/immunology , African Swine Fever/virology , Antibodies, Monoclonal/immunology , Viral Envelope Proteins/immunology , Epitopes/immunology , Antibodies, Viral/immunology , Mice, Inbred BALB CABSTRACT
Influenza A viruses (IAVs) continue to pose a huge threat to public health, and their prevention and treatment remain major international issues. Neuraminidase (NA) is the second most abundant surface glycoprotein on influenza viruses, and antibodies to NA have been shown to be effective against influenza infection. In this study, we generated a monoclonal antibody (mAb), named FNA1, directed toward N1 NAs. FNA1 reacted with H1N1 and H5N1 NA, but failed to react with the NA proteins of H3N2 and H7N9. In vitro, FNA1 displayed potent antiviral activity that mediated both NA inhibition (NI) and blocking of pseudovirus release. Moreover, residues 219, 254, 358, and 388 in the NA protein were critical for FNA1 binding to H1N1 NA. However, further validation is necessary to confirm whether FNA1 mAb is indeed a good inhibitor against NA for application against H1N1 and H5N1 viruses.
Subject(s)
Antibodies, Monoclonal , Influenza A Virus, H1N1 Subtype , Neuraminidase , Neuraminidase/immunology , Neuraminidase/metabolism , Neuraminidase/antagonists & inhibitors , Antibodies, Monoclonal/immunology , Influenza A Virus, H1N1 Subtype/immunology , Humans , Animals , Antibodies, Viral/immunology , Mice , Influenza A Virus, H5N1 Subtype/immunology , Mice, Inbred BALB C , Antiviral Agents/pharmacology , Viral Proteins/immunology , Viral Proteins/metabolism , Influenza A Virus, H3N2 Subtype/immunology , Influenza A Virus, H7N9 Subtype/immunologyABSTRACT
INTRODUCTION: Excessive immune activation induces tissue damage during infection. Compared to external strategies to reconstruct immune homeostasis, host balancing ways remain largely unclear. OBJECTIVES: Here we found a neuroimmune way that prevents infection-induced tissue damage. METHODS: By FACS and histopathology analysis of brain Streptococcus pneumonia meningitis infection model and behavioral testing. Western blot, co-immunoprecipitation, and ubiquitination analyze the Fluoxetine initiate 5-HT7R-STUB1-CCR5 K48-linked ubiquitination degradation. RESULTS: Fluoxetine, a selective serotonin reuptake inhibitor, or the agonist of serotonin receptor 5-HT7R, protects mice from meningitis by inhibiting CCR5-mediated excessive immune response and tissue damage. Mechanistically, the Fluoxetine-5-HT7R axis induces proteasome-dependent degradation of CCR5 via mTOR signaling, and then recruits STUB1, an E3 ubiquitin ligase, to initiate K48-linked polyubiquitination of CCR5 at K138 and K322, promotes its proteasomal degradation. STUB1 deficiency blocks 5-HT7R-mediated CCR5 degradation. CONCLUSION: Our results reveal a neuroimmune pathway that balances anti-infection immunity via happiness neurotransmitter receptor and suggest the 5-HT7R-CCR5 axis as a potential target to promote neuroimmune resilience.
ABSTRACT
Sleep deprivation (SD) weakens the immune system and leads to increased susceptibility to infectious or inflammatory diseases. However, it is still unclear how SD affects humoral immunity. In the present study, sleep disturbance was conducted using an sleep deprivation instrument, and the bacterial endotoxin lipopolysaccharide (LPS) was used to activate the immune response. It was found that SD-pretreatment reduced LPS-induced IgG2b+ B cells and IgG2b isotype antibody production in lymphocytes of spleen. And, SD-pretreatment decreased the proportion of CD4+T cells, production of CD4+T cells derived TGF-ß1 and its contribution in helping IgG2b production. Additionally, BMAL1 and CLOCK were selectively up-regulated in lymphocytes after SD. Importantly, BMAL1 and CLOCK deficiency contributed to TGF-ß1 expression and production of IgG2b+ B cells. Thus, our results provide a novel insight to explain the involvement of BMAL1 and CLOCK under SD stress condition, and their roles in inhibiting TGF-ß1 expression and contributing to reduction of LPS induced IgG2b production.
Subject(s)
ARNTL Transcription Factors , Antibody Formation , CLOCK Proteins , Immunoglobulin G , Sleep Deprivation , Sleep Deprivation/genetics , Sleep Deprivation/immunology , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Rats, Sprague-Dawley , Mice, Inbred C57BL , ARNTL Transcription Factors/genetics , ARNTL Transcription Factors/immunology , CLOCK Proteins/genetics , CLOCK Proteins/immunology , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , Antibody Formation/drug effects , Antibody Formation/genetics , Stress, Physiological/immunology , Animals , Mice , Rats , Cells, CulturedABSTRACT
IMPORTANCE: Staphylococcus aureus is a Gram-positive opportunistic bacterium that is responsible for the majority of skin infections in humans. Our study provides important molecular insights into the pathogenesis of S. aureus skin infections and identifies a potential therapeutic target for the treatment of these infections. Our findings also indicate that ß-hemolysin (Hlb) secreted by colonized S. aureus is a risk factor for epidermal growth factor receptor (EGFR)-related diseases by acting as an agonist of EGFR. The neutralized monoclonal antibody we have developed for the first time will provide a functional inhibitor of Hlb. This study provides important insights to better understand the relationship between the skin colonization of S. aureus and inflammatory skin diseases.
Subject(s)
Staphylococcal Infections , Staphylococcus aureus , Humans , Staphylococcus aureus/metabolism , Hemolysin Proteins/metabolism , Skin/microbiology , ErbB Receptors/metabolism , Staphylococcal Infections/microbiology , Inflammation/pathologyABSTRACT
Smallpox is an infectious disease caused by the variola virus, and it has a high mortality rate. Historically it has broken out in many countries and it was a great threat to human health. Smallpox was declared eradicated in 1980, and Many countries stopped nation-wide smallpox vaccinations at that time. In recent years the potential threat of bioterrorism using smallpox has led to resumed research on the treatment and prevention of smallpox. Effective ways of preventing and treating smallpox infection have been reported, including vaccination, chemical drugs, neutralizing antibodies, and clinical symptomatic therapies. Antibody treatments include anti-sera, murine monoclonal antibodies, and engineered humanized or human antibodies. Engineered antibodies are homologous, safe, and effective. The development of humanized and genetically engineered antibodies against variola virus via molecular biology and bioinformatics is therefore a potentially fruitful prospect with respect to field application. Natural smallpox virus is inaccessible, therefore most research about prevention and/or treatment of smallpox were done using vaccinia virus, which is much safer and highly homologous to smallpox. Herein we summarize vaccinia virus epitope information reported to date, and discuss neutralizing antibodies with potential value for field application.
ABSTRACT
PURPOSE: Mitogen-activated protein kinases (MAPK), specifically the c-Jun N-terminal kinase (JNK)-MAPK subfamily, play a crucial role in the development of various cancers, including hepatocellular carcinoma (HCC). However, the specific roles of JNK1/2 and their upstream regulators, MKK4/7, in HCC carcinogenesis remain unclear. METHODS: In this study, we performed differential expression analysis of JNK-MAPK components at both the transcriptome and protein levels using TCGA and HPA databases. We utilized Kaplan-Meier survival plots and receiver operating characteristic (ROC) curve analysis to evaluate the prognostic performance of a risk scoring model based on these components in the TCGA-HCC cohort. Additionally, we conducted immunoblotting, apoptosis analysis with FACS and soft agar assays to investigate the response of JNK-MAPK pathway components to various death stimuli (TRAIL, TNF-α, anisomycin, and etoposide) in HCC cell lines. RESULTS: JNK1/2 and MKK7 levels were significantly upregulated in HCC samples compared to paracarcinoma tissues, whereas MKK4 was downregulated. ROC analyses suggested that JNK2 and MKK7 may serve as suitable diagnostic genes for HCC, and high JNK2 expression correlated with significantly poorer overall survival. Knockdown of JNK1 enhanced TRAIL-induced apoptosis in hepatoma cells, while JNK2 knockdown reduced TNF-α/cycloheximide (CHX)-and anisomycin-induced apoptosis. Neither JNK1 nor JNK2 knockdown affected etoposide-induced apoptosis. Furthermore, MKK7 knockdown augmented TNF-α/CHX- and TRAIL-induced apoptosis and inhibited colony formation in hepatoma cells. CONCLUSION: Targeting MKK7, rather than JNK1/2 or MKK4, may be a promising therapeutic strategy to inhibit the JNK-MAPK pathway in HCC therapy.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Carcinoma, Hepatocellular/genetics , Tumor Necrosis Factor-alpha , Etoposide , Anisomycin , MAP Kinase Kinase 7/genetics , MAP Kinase Kinase 7/metabolism , Liver Neoplasms/genetics , ApoptosisABSTRACT
The mechanisms by which retinoic acid-inducible gene I (RIG-I), a critical RNA virus sensor, is regulated in many biological and pathological processes remain to be determined. Here, we demonstrate that T cell immunoglobulin and mucin protein-3 (Tim-3), an immune checkpoint inhibitor, mediates infection tolerance by suppressing RIG-I-type I interferon pathway. Overexpression or blockade of Tim-3 affects type I interferon expression, virus replication, and tissue damage in mice following H1N1 infection. Tim-3 signaling decreases RIG-I transcription via STAT1 in macrophages and promotes the proteasomal dependent degradation of RIG-I by enhancing K-48-linked ubiquitination via the E3 ligase RNF-122. Silencing RIG-I reversed Tim-3 blockage-mediated upregulation of type I interferon in macrophages. We thus identified a new mechanism through which Tim-3 mediates the immune evasion of H1N1, which may have clinical implications for the treatment of viral diseases.
Subject(s)
Influenza A Virus, H1N1 Subtype , Interferon Type I , Mice , Animals , Hepatitis A Virus Cellular Receptor 2/genetics , Hepatitis A Virus Cellular Receptor 2/metabolism , Macrophages , Interferon Type I/genetics , Ubiquitin-Protein Ligases/geneticsABSTRACT
AIM: To investigate the impact of deficiency of LIG4 gene on site-specific integration in CHO cells. RESULTS: CHO cells are considered the most valuable mammalian cells in the manufacture of biological medicines, and genetic engineering of CHO cells can improve product yield and stability. The traditional method of inserting foreign genes by random integration (RI) requires multiple rounds of screening and selection, which may lead to location effects and gene silencing, making it difficult to obtain stable, high-yielding cell lines. Although site-specific integration (SSI) techniques may overcome the challenges with RI, its feasibility is limited by the very low efficiency of the technique. Recently, SSI efficiency has been enhanced in other mammalian cell types by inhibiting DNA ligase IV (Lig4) activity, which is indispensable in DNA double-strand break repair by NHEJ. However, this approach has not been evaluated in CHO cells. In this study, the LIG4 gene was knocked out of CHO cells using CRISPR/Cas9-mediated genome editing. Efficiency of gene targeting in LIG4-/--CHO cell lines was estimated by a green fluorescence protein promoterless reporter system. Notably, the RI efficiency, most likely mediated by NHEJ in CHO, was inhibited by LIG4 knockout, whereas SSI efficiency strongly increased 9.2-fold under the precise control of the promoter in the ROSA26 site in LIG4-/--CHO cells. Moreover, deletion of LIG4 had no obvious side effects on CHO cell proliferation. CONCLUSIONS: Deficiency of LIG4 represents a feasible strategy to improve SSI efficiency and suggests it can be applied to develop and engineer CHO cell lines in the future.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Animals , CHO Cells , CRISPR-Cas Systems/genetics , Cricetinae , Cricetulus , DNA End-Joining Repair/genetics , DNA Ligase ATP/geneticsABSTRACT
Cancer vaccines have gradually attracted attention for their tremendous preclinical and clinical performance. With the development of next-generation sequencing technologies and related algorithms, pipelines based on sequencing and machine learning methods have become mainstream in cancer antigen prediction; of particular focus are neoantigens, mutation peptides that only exist in tumor cells that lack central tolerance and have fewer side effects. The rapid prediction and filtering of neoantigen peptides are crucial to the development of neoantigen-based cancer vaccines. However, due to the lack of verified neoantigen datasets and insufficient research on the properties of neoantigens, neoantigen prediction algorithms still need to be improved. Here, we recruited verified cancer antigen peptides and collected as much relevant peptide information as possible. Then, we discussed the role of each dataset for algorithm improvement in cancer antigen research, especially neoantigen prediction. A platform, Cancer Antigens Database (CAD, http://cad.bio-it.cn/), was designed to facilitate users to perform a complete exploration of cancer antigens online.
ABSTRACT
Sialic acid-binding Ig-like lectin-15 is an important immunosuppressive molecule considered to be a key target in next-generation tumor immunotherapy. In this study, we screened 22 high-affinity antibodies that specifically recognize human Siglec-15 by using a large human phage antibody library, and five representative sequences were selected for further study. The results showed the binding activity of five antibodies to Siglec-15 (EC50 ranged from 0.02368 µg/mL to 0.07949 µg/mL), and in two Siglec-15-overexpressed cell lines, three antibodies had the strongest binding activity, so the two clones were discarded for further study. Subsequently, the affinity of three antibodies were measured by bio-layer interferometry technology (5-9 × 10E-09M). As the reported ligands of Siglec-15, the binding activity of Siglec-15 and sialyl-Tn, cluster of differentiation 44, myelin-associated glycoprotein, and leucine-rich repeat-containing protein 4C can be blocked by three of the antibodies. Among these, 3F1 had a competitive advantage. Then, the antibody 3F1 showed an obvious antibody-dependent cell-mediated cytotoxicity effect (EC50 was 0.85 µg/mL). Further, antibody 3F1 can reverse the inhibitory effect of Siglec-15 on lymphocyte proliferation (especially CD4+T and CD8+T) and cytokine release Interferon-γ. Given the above results, 3F1 was selected as a candidate for the in vivo pharmacodynamics study. In the tumor model of Balb/c Nude mice, 3F1 (10 mg/kg) showed certain antitumor effects [tumor growth inhibition (TGI) was 31.5%], while the combination of 3F1 (5 mg/kg) and Erbitux (5 mg/kg) showed significant antitumor effects (TGI was 48.7%) compared with the PBS group. In conclusion, novel human antibody 3F1 has antitumor activity and is expected to be an innovative candidate drug targeting Siglec-15 for tumor immunotherapy. SIGNIFICANCE STATEMENT: Siglec-15 is considered as an important target in the next generation of tumor immunotherapy. 3F1 is expected to be the most promising potential candidate for targeting Siglec-15 for cancer treatment and could provide a reference for the development of antitumor drugs.
Subject(s)
Antigens, CD , Neoplasms , Animals , Antigens, CD/metabolism , Humans , Immunoglobulins , Lectins/chemistry , Lectins/metabolism , Ligands , Membrane Proteins , Mice , Mice, Nude , Neoplasms/drug therapyABSTRACT
The continuous mutation of SARS-CoV-2 has presented enormous challenges to global pandemic prevention and control. Recent studies have shown evidence that the genome sequence of SARS-CoV-2 nucleocapsid proteins is relatively conserved, and their biological functions are being confirmed. There is increasing evidence that the N protein will not only provide a specific diagnostic marker but also become an effective treatment target. In this study, 2G4, which specifically recognizes the N protein, was identified by screening a human phage display library. Based on the computer-guided homology modelling and molecular docking method used, the 3-D structures for the 2G4 scFv fragment (VH-linker-VL structure, with (G4S)3 as the linker peptide in the model), SARS-CoV-2 N protein and its complex were modelled and optimized with a suitable force field. The binding mode and key residues of the 2G4 and N protein interaction were predicted, and three mutant antibodies (named 2G4-M1, 2G4-M2 and 2G4-M3) with higher affinity were designed theoretically. Using directed point mutant technology, the three mutant antibodies were prepared, and their affinity was tested. Their affinity constants of approximately 0.19 nM (2G4-M1), 0.019 nM (2G4-M2) and 0.075 nM (2G4-M3) were at least one order of magnitude lower than that of the parent antibody (3 nM; 2G4, parent antibody), as determined using a biolayer interferometry (BLI) assay. It is expected that high-affinity candidates will be used for diagnosis and even as potential therapeutic drugs for the SARS-CoV-2 pandemic.
Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Monoclonal , Antibody Affinity , Cell Surface Display Techniques , Humans , Molecular Docking Simulation , SARS-CoV-2/geneticsABSTRACT
Staphylococcus aureus is a major pathogenic bacterium that causes a variety of clinical infections. The emergence of multi-drug resistant mechanisms requires novel strategies to mitigate S. aureus infection. Alpha-hemolysin (Hla) is a key virulence factor that is believed to play a significant role in the pathogenesis of S. aureus infections. In this study, we screened a naïve human Fab library for identification of monoclonal antibodies targeting Hla by phage display technology. We found that the monoclonal antibody YG1 blocked the Hla-mediated lysis of rabbit red blood cells and inhibited Hla binding to A549 cells in a concentration-dependent manner. YG1 also provided protection against acute peritoneal infection, bacteremia, and pneumonia in murine models. We further characterized its epitope using different Hla variants and found that the amino acids N209 and F210 of Hla were functionally and structurally important for YG1 binding. Overall, these results indicated that targeting Hla with YG1 could serve as a promising protective strategy against S. aureus infection.
ABSTRACT
Phagocytic resistance plays a key role in tumor-mediated immune escape, so phagocytosis immune checkpoints are a potential target for cancer immunotherapy. CD47 is one of the important phagocytosis immune checkpoints; thus, blocking the interaction between CD47 and signal regulatory protein α (SIRPα) may provide new options for cancer treatment. Using computer-aided targeted epitope mammalian cell-displayed antibody library, we screened and obtained an engineered SIRPα variant fragment crystallizable fusion protein, FD164, with higher CD47-binding activity than wild-type SIRPα Compared with wild-type SIRPα, FD164 has approximately 3-fold higher affinity for binding to CD47, which further enhanced its phagocytic effect in vitro and tumor suppressor activity in vivo. FD164 maintains the similar antitumor activity of the clinical research drug Hu5F9 in the mouse xenograft model. Furthermore, FD164 combined with rituximab can significantly improve the effect of single-agent therapy. On the other hand, compared with Hu5F9, FD164 does not cause hemagglutination, and its ability to bind to red blood cells or white blood cells is weaker at the same concentration. Finally, it was confirmed by computer structure prediction and alanine scanning experiments that the N45, E47, 52TEVYVK58, K60, 115EVTELTRE122, and E124 residues of CD47 are important for SIRPα or FD164 recognition. Briefly, we obtained a high-affinity SIRPα variant FD164 with balanced safety and effectiveness. SIGNIFICANCE STATEMENT: Up to now, few clinically marketed drugs targeting CD47 have been determined to be effective and safe. FD164, a potential signal regulatory protein α variant fragment crystallizable protein with balanced safety and effectiveness, could provide a reference for the development of antitumor drugs.
Subject(s)
Antigens, Differentiation/genetics , Antigens, Differentiation/immunology , CD47 Antigen/immunology , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , Animals , Antibodies, Monoclonal, Humanized/adverse effects , Antibodies, Monoclonal, Humanized/therapeutic use , Antigens, Differentiation/adverse effects , Antigens, Differentiation/chemistry , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Burkitt Lymphoma/drug therapy , Burkitt Lymphoma/immunology , Burkitt Lymphoma/pathology , CD47 Antigen/chemistry , CHO Cells , Cell Line , Cricetulus , Drug Design , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Hemagglutination/drug effects , Immunotherapy , Mice, SCID , Models, Molecular , Phagocytosis/drug effects , Phagocytosis/immunology , Receptors, Immunologic/chemistry , Recombinant Fusion Proteins/adverse effects , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Rituximab/therapeutic use , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Nuclear factor 90 (NF90) is a novel virus sensor that serves to initiate antiviral innate immunity by triggering stress granule (SG) formation. However, the regulation of the NF90-SG pathway remains largely unclear. We found that Tim-3, an immune checkpoint inhibitor, promotes the ubiquitination and degradation of NF90 and inhibits NF90-SG-mediated antiviral immunity. Vesicular stomatitis virus (VSV) infection induces the up-regulation and activation of Tim-3 in macrophages, which in turn recruit the E3 ubiquitin ligase TRIM47 to the zinc finger domain of NF90 and initiate a proteasome-dependent degradation via K48-linked ubiquitination at Lys297. Targeted inactivation of Tim-3 enhances the NF90 downstream SG formation by selectively increasing the phosphorylation of protein kinase R and eukaryotic translation initiation factor 2α, the expression of SG markers G3BP1 and TIA-1, and protecting mice from VSV challenge. These findings provide insights into the crosstalk between Tim-3 and other receptors in antiviral innate immunity and its related clinical significance.
Subject(s)
Hepatitis A Virus Cellular Receptor 2 , Immunity, Innate/immunology , Nuclear Factor 90 Proteins , Ubiquitination/immunology , Virus Diseases/immunology , Animals , Cytoplasmic Granules/immunology , Cytoplasmic Granules/metabolism , Hepatitis A Virus Cellular Receptor 2/immunology , Hepatitis A Virus Cellular Receptor 2/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice , Nuclear Factor 90 Proteins/immunology , Nuclear Factor 90 Proteins/metabolism , Rhabdoviridae Infections/immunology , VesiculovirusABSTRACT
Viral encephalitis is the most common cause of encephalitis. It is responsible for high morbidity rates, permanent neurological sequelae, and even high mortality rates. The host immune response plays a critical role in preventing or clearing invading pathogens, especially when effective antiviral treatment is lacking. However, due to blockade of the blood-brain barrier, it remains unclear how peripheral immune cells contribute to the fight against intracerebral viruses. Here, we report that peripheral injection of an antibody against human Tim-3, an immune checkpoint inhibitor widely expressed on immune cells, markedly attenuated vesicular stomatitis virus (VSV) encephalitis, marked by decreased mortality and improved neuroethology in mice. Peripheral injection of Tim-3 antibody enhanced the recruitment of immune cells to the brain, increased the expression of major histocompatibility complex-I (MHC-I) on macrophages, and as a result, promoted the activation of VSV-specific CD8+ T cells. Depletion of macrophages abolished the peripheral injection-mediated protection against VSV encephalitis. Notably, for the first time, we found a novel post-translational modification of MHC-I by Tim-3, wherein, by enhancing the expression of MARCH9, Tim-3 promoted the proteasome-dependent degradation of MHC-I via K48-linked ubiquitination in macrophages. These results provide insights into the immune response against intracranial infections; thus, manipulating the peripheral immune cells with Tim-3 antibody to fight viruses in the brain may have potential applications for combating viral encephalitis.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Antibodies, Neutralizing/administration & dosage , Antigen-Presenting Cells/drug effects , Brain/drug effects , Encephalitis, Viral/prevention & control , Hepatitis A Virus Cellular Receptor 2/antagonists & inhibitors , Macrophages/drug effects , Rhabdoviridae Infections/prevention & control , Vesiculovirus/immunology , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Antigen-Presenting Cells/virology , Brain/immunology , Brain/metabolism , Brain/virology , Chlorocebus aethiops , Disease Models, Animal , Encephalitis, Viral/immunology , Encephalitis, Viral/metabolism , Encephalitis, Viral/virology , HEK293 Cells , Hepatitis A Virus Cellular Receptor 2/immunology , Histocompatibility Antigens Class I/metabolism , Host-Pathogen Interactions , Humans , Injections, Intraperitoneal , Macrophages/immunology , Macrophages/metabolism , Macrophages/virology , Male , Mice , Mice, Inbred C57BL , Proteasome Endopeptidase Complex/metabolism , Proteolysis , RAW 264.7 Cells , Rhabdoviridae Infections/immunology , Rhabdoviridae Infections/metabolism , Rhabdoviridae Infections/virology , Ubiquitination , Vero Cells , Vesiculovirus/pathogenicity , Viral LoadABSTRACT
Ornithine transcarbamylases (OTC), a key enzyme in urea cycle, is an important marker for some liver injury or diseases. However, whether OTC could be a sensitive indicator for liver dysfunction under sleep disturbance condition remains unknown. The present study aimed to explore the circadian oscillation expression of OTC and its significance in disturbed sleep condition. Sleep disturbance was conducted by a sleep deprivation (SD) instrument. Our results found that SD for 72h induced abnormal increasing of OTC levels in serum and liver of rats. And, serum OTC concentration and liver OTC expression could return to normal levels after recovery sleep following SD. Moreover, hepatic OTC expression showed circadian oscillation in day and night, characterized with occurrence of a peak between ZT 22 and ZT 2, and a nadir between ZT 14 and ZT 18. Further analysis suggested the existence of ROR response element (RORE) for potential RORÉ binding sites in OTC promoter region, and elevated RORÉ expression in rat livers under sleep disturbance condition. Additionally, oscillation expression of OTC induced by serum shock in HepG2 cells was characterized with a peak occurred between ZT 12 and ZT 16, and RORÉ knockdown at ZT 16 significantly lowered OTC expression. The results together indicate that OTC is closely correlated with circadian clock, and could be a sensitive indicator for sleep disturbance stress.
Subject(s)
Circadian Rhythm , Ornithine Carbamoyltransferase/metabolism , Sleep Wake Disorders/enzymology , Sleep Wake Disorders/physiopathology , Animals , Base Sequence , Gene Expression Regulation, Enzymologic , Hep G2 Cells , Homeostasis , Humans , Liver/enzymology , Male , Nuclear Receptor Subfamily 1, Group F, Member 1/metabolism , Ornithine Carbamoyltransferase/genetics , Rats, Sprague-Dawley , Sleep/genetics , Sleep Wake Disorders/geneticsABSTRACT
Extracellular traps released by neutrophils (NETs) are essential for the clearance of Pseudomonas aeruginosa. Alkaline protease (AprA) secreted by P. aeruginosa negatively correlates with clinical improvement. Moreover, anti-AprA in patients with cystic fibrosis (CF) can help identify patients with aggressive forms of chronic infection. However, the mechanism underlying the clinical outcomes remains unclear. We demonstrated that aprA deficiency in P. aeruginosa decreased the bacterial burden and reduced lung infection. AprA degraded NET components in vitro and in vivo but did not affect NET formation. Importantly, antibodies induced by AprA acted as an agonist and directly enhanced the degrading activities of AprA. Moreover, antisera from patients with P. aeruginosa infection exhibited antibody-dependent enhancement (ADE) similar to that of the antibodies we prepared. Our further investigations showed that the interaction between AprA and the specific antibodies might make the enzyme active sites better exposed, and subsequently enhance the recognition of substrates and accelerate the degradation. Our findings revealed that AprA secreted by P. aeruginosa may aggravate infection by destroying formed NETs, an effect that was further enhanced by its antibodies.
Subject(s)
Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Endopeptidases/immunology , Extracellular Traps/immunology , Pseudomonas Infections/immunology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/immunology , Animals , Antibody-Dependent Cell Cytotoxicity , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Disease Models, Animal , Endopeptidases/genetics , Endopeptidases/metabolism , Extracellular Traps/enzymology , Female , Host-Pathogen Interactions/immunology , Humans , Mice , Neutrophils/immunology , Neutrophils/metabolism , Pneumonia, Bacterial/immunology , Pneumonia, Bacterial/metabolism , Pneumonia, Bacterial/microbiology , Pneumonia, Bacterial/pathology , Pseudomonas Infections/metabolism , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/pathogenicityABSTRACT
Staphylococcal enterotoxin B (SEB), one of the exotoxins produced by Staphylococcus aureus, is the key toxin that causes poisoning reactions and toxic shock syndrome. In the current research work, a novel human antibody named LXY8 was screened from a human phage display antibody library, and LXY8 blocked the interaction between SEB and the T cell receptor (TCR). The binding activity between LXY8 and SEB was 0.525 nM. Furthermore, LXY8 could effectively inhibit the SEB-induced activation of peripheral blood mononuclear cells and release of cytokines. In the BALB/c mouse model, LXY8 effectively neutralized SEB toxicity in vivo. Finally, based on computer-guided molecular modeling, we designed a series of SEB mutation sites; these sites facilitated the determination of the key residues (i.e.176EFNN179) of SEB recognized by LXY8. The research revealed that the 176EFNN179 residues of SEB are important for specific antibody-antigen recognition. The results may be helpful for the development of antibody-based therapy for SEB-induced toxic shock syndrome.