Caspase-3/Gasdermin E-mediated pyroptosis contributes to Ricin toxin-induced inflammation.

Ricin toxin (RT) is highly cytotoxic and can release a considerable amount of pro-inflammatory factors due to depurination, causing excessive inflammation that may aggravate the harm to the body. Pyroptosis, a type of gasdermin-mediated cell death, is a contributor to the exacerbation of inflammation. Accumulating evidence indicate that pyroptosis plays a significant role in the pathogen infection and tissue injury, suggesting a potential correlation between pyroptosis and RT-induced inflammation. Here, we aim to demonstrate this correlation and explore its molecular mechanisms. Results showed that RT triggers mouse alveolar macrophage MH-S cells pyroptosis by activating caspase-3 and cleaving Gasgermin E (GSDME). In contrast, inhibition of caspase-3 with Z-DEVD-FMK (inhibitor of caspase-3) or knockdown of GSDME attenuates this process, suggesting the essential role of caspase-3/GSDME-mediated pyroptosis in contributing to RT-induced inflammation. Collectively, our study enhances our understanding of a novel mechanism of ricin cytotoxicity, which may emerge as a potential target in immunotherapy to control the RT-induced inflammation.

Crosstalk between autophagy and inflammasomes in ricin-induced inflammatory injury.

Ricin (ricin toxin, RT) has the potential to cause damage to multiple organs and systems. Currently, there are no existing antidotes, vaccinations, or effective therapies to prevent or treat RT intoxication. Apart from halting protein synthesis, RT also induces oxidative stress, inflammation and autophagy. To explore the mechanisms of RT-induced inflammatory injury and specific targets of prevention and treatment for RT poisoning, we characterized the role of cross-talk between autophagy and NLRP3 inflammasome in RT-induced damage and elucidated the underlying mechanisms. We showed that RT-induced inflammation was attributed to activation of the TLR4/MyD88/NLRP3 signaling and ROS production, evidenced by increased ASC speck formation and attenuated TXNIP/TRX-1 interaction, as well as pre-treatment with MCC950, MyD88 knockdown and NAC significantly reduced IL-1ß, IL-6 and TNF-α mRNA expression. In addition, autophagy is also enhanced in RT-triggered MLE-12 cells. RT elevated the levels of ATG5, p62 and Beclin1 protein, provoked the accumulation of LC3 puncta detected by immunofluorescence staining. Treatment with rapamycin (Rapa) reversed the RT-caused TLR4/MyD88/NLRP3 signaling activation, ASC specks formation as well as the levels of IL-1ß, IL-6 and TNF-α mRNA. In conclusion, RT promoted NLRP3 inflammasome activation and autophgay. Inflammation induced by RT was attenuated by autophagy activation, which suppressed the NLRP3 inflammasome. These findings suggest Rapa as a potential therapeutic drug for the treatment of RT-induced inflammation-related diseases.

The current landscape of the antimicrobial peptide melittin and its therapeutic potential.

Melittin, a main component of bee venom, is a cationic amphiphilic peptide with a linear α-helix structure. It has been reported that melittin can exert pharmacological effects, such as antitumor, antiviral and anti-inflammatory effects in vitro and in vivo. In particular, melittin may be beneficial for the treatment of diseases for which no specific clinical therapeutic agents exist. Melittin can effectively enhance the therapeutic properties of some first-line drugs. Elucidating the mechanism underlying melittin-mediated biological function can provide valuable insights for the application of melittin in disease intervention. However, in melittin, the positively charged amino acids enables it to directly punching holes in cell membranes. The hemolysis in red cells and the cytotoxicity triggered by melittin limit its applications. Melittin-based nanomodification, immuno-conjugation, structural regulation and gene technology strategies have been demonstrated to enhance the specificity, reduce the cytotoxicity and limit the off-target cytolysis of melittin, which suggests the potential of melittin to be used clinically. This article summarizes research progress on antiviral, antitumor and anti-inflammatory properties of melittin, and discusses the strategies of melittin-modification for its future potential clinical applications in preventing drug resistance, enhancing the selectivity to target cells and alleviating cytotoxic effects to normal cells.

Corrigendum: A neutralizing bispecific single-chain antibody against SARS-CoV-2 Omicron variant produced based on CR3022.

[This corrects the article DOI: 10.3389/fcimb.2023.1155293.].

γ-Bungarotoxin impairs the vascular endothelial barrier function by inhibiting integrin α5.

γ-bungarotoxin (γ-BGT) is an RGD motif-containing protein, derived from the venom of Bungarus multicinctus, leading to acute death in mice. These RGD motif-containing proteins from snake venom belonging to the disintegrin family can interfere with vascular endothelial homeostasis by directly binding cell surface integrins. Targeting integrins that generate vascular endothelial dysfunction may contribute to γ-BGT poisoning, however, the underlying mechanisms have not been investigated in detail. In this study, the results showed that γ-BGT played a role in -promoting the permeability of the vascular endothelial barrier. Depending on its selective binding to integrin α5 in vascular endothelium (VE), γ-BGT initiated downstream events, including focal adhesion kinase dephosphorylation and cytoskeleton remodeling, resulting in the intercellular junction interruption. Those alternations facilitated paracellular permeability of VE and barrier dysfunction. Proteomics profiling identified that as a downstream effector of the integrin α5 / FAK signaling pathway cyclin D1 partially mediated the cellular structural changes and barrier dysfunction. Furthermore, VE-released plasminogen activator urokinase and platelet-derived growth factor D could serve as potential diagnostic biomarkers for γ-BGT-induced vascular endothelial dysfunction. Our results indicate the mechanisms through which γ-BGT as a novel disintegrin directly interacts with the VE, with consequences for barrier dysfunction.

Anti-ricin toxin human neutralizing antibodies and DMAbs protection against ricin toxin poisoning.

DNA-encoded monoclonal antibodies (DMAbs) and in vivo expression of antibody therapeutics presents an innovative alternative to conventional delivery methods. Therefore, in order to prevent the lethal dose of ricin toxin (RT) and to avoid human anti-mouse antibody (HAMA) reaction, we developed the human neutralizing antibody 4-4E against RT and constructed DMAb-4-4E. The human neutralizing antibody 4-4E could neutralize RT in vitro and in vivo, while the mice in RT group all died. Using intramuscular electroporation (IM EP), antibodies were rapidly expressed in vivo within 7 days and were enriched in intestine and gastrocnemius muscle mostly. Besides, we found that DMAbs have shown a broad protective efficacy of RT poisoning prophylaxis. Driven by plasmids for IgG expression, mice were survived and the blood glucose level of mice in DMAb-IgG group returned to normal at 72 h post RT challenge, and the RT group died within 48 h. Furthermore, hindrance of protein disulfide isomerase (PDI) and accumulation of RT in endosomes were found in IgG-protected cells, revealing the possible mechanism of neutralization details. These data support the further study of RT-neutralizing monoclonal antibodies (mAbs) in the development.

A neutralizing bispecific single-chain antibody against SARS-CoV-2 Omicron variant produced based on CR3022.

Introduction: The constantly mutating SARS-CoV-2 has been infected an increasing number of people, hence the safe and efficacious treatment are urgently needed to combat the COVID-19 pandemic. Currently, neutralizing antibodies (Nabs), targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are potentially effective therapeutics against COVID-19. As a new form of antibody, bispecific single chain antibodies (BscAbs) can be easily expressed in E. coli and exhibits broad-spectrum antiviral activity. Methods: In this study, we constructed two BscAbs 16-29, 16-3022 and three single chain variable fragments (scFv) S1-16, S2-29 and S3022 as a comparison to explore their antiviral activity against SARS-CoV-2. The affinity of the five antibodies was characterized by ELISA and SPR and the neutralizing activity of them was analyzed using pseudovirus or authentic virus neutralization assay. Bioinformatics and competitive ELISA methods were used to identify different epitopes on RBD. Results: Our results revealed the potent neutralizing activity of two BscAbs 16-29 and 16-3022 against SARS-CoV-2 original strain and Omicron variant infection. In addition, we also found that SARS-CoV RBD-targeted scFv S3022 could play a synergistic role with other SARS-CoV-2 RBD-targeted antibodies to enhance neutralizing activity in the form of a BscAb or in cocktail therapies. Discussion: This innovative approach offers a promising avenue for the development of subsequent antibody therapies against SARSCoV-2. Combining the advantages of cocktails and single-molecule strategies, BscAb therapy has the potential to be developed as an effective immunotherapeutic for clinical use to mitigate the ongoing pandemic.

A novel circular RNA circNLRP3 alleviated ricin toxin-induced TNF-α production through sponging miR-221-5p.

Acting as microRNA (miRNA) sponges, circular RNAs (circRNAs) have been discovered to be critical modulators of inflammatory processes. Ricin Toxin (RT) is highly toxic to mammalian cells and low doses of RT can induce acute inflammation. However, current researches on the underlying mechanism and function of circRNA/miRNA network in RT-induced inflammation are limited. Previously, we found miR-221-5p was aberrant and associated with the inflammation of RT induction. In this study, based on the circRNA high-throughput sequencing (circRNA-seq), we obtained a novel circRNA termed circNLRP3 and revealed that circNLRP3 can sponge miR-221-5p, release its target mRNA A20, and further suppress NF-κB signaling pathway to alleviated RT-induced TNF-α production. Our findings elucidated a possible mechanistic link between the circNLRP3/miR-221-5p/A20 axis and RT-induced inflammatory response, which may broaden our understanding of RT poisoning.

MicroRNA-221-5p Promotes Ricin Toxin-induced Inflammation via PI3K/Akt signaling pathway by targeting COL4a5.

Ricin toxin (RT) is one of the most lethal type II ribosome-inactivating proteins (RIP), and is classified as a potential bioterror agent due to its severe cytotoxicity and high availability. The toxicity of RT is dependent on both dose and route of exposure. Increasing evidence demonstrates that sub-lethal RT induces acute inflammation and increases the release of pro-inflammatory cytokines. However, current studies on mechanism of RT-induced inflammation are limited. In this study, to evaluate the relationship between miRNAs and RT-induced inflammation, RNA sequencing (RNA-Seq) was used to analyze the expression of miRNAs and mRNAs in RT-treated RAW264.7 macrophage cells. A total of 14 significantly differently expressed (DE) miRNAs and 323 miRNA-mRNA interaction pairs were predicted by bioinformatics analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that majority of those interaction pairs were involved in PI3K/Akt pathway. In addition, overexpression of miR-221-5p promoted the inflammatory response by inhibiting the mRNA expression of COL4a5. This work contributes to our understanding of RT-induced inflammation and demonstrates the potential role of miRNAs in innate immunity, which may be regarded as potential targets in developing therapies for RT poisoning.

Bioinspired fabrication of bifunctional antibody-enzyme co-assembled nanocomposites for chemiluminescence immunoassays of E. coli O157:H7.

Immunoassays based on enzyme-labeled antibodies have been widely used in the food safety field. However, the production process of enzyme-labeled antibodies is complicated and the low storage stability limits their application. Herein, antibody-horseradish peroxidase (HRP) co-assembled nanocomposites (AHC NCs) with outstanding advantages such as enhanced stability, lower cost, and substrate affinity were successfully prepared via a one-pot green method. Then the AHC NCs were employed as an alternative to traditional enzyme-labeled antibodies to develop a chemiluminescence enzyme immunoassay (CLEIA) toward Escherichia coli (E. coli) O157:H7. Under optimal conditions, E. coli O157:H7 can be detected in a linear range from 1 × 103 CFU mL-1 to 5 × 106 CFU mL-1, while the limit of detection (LOD) is as low as 2.2 × 102 CFU mL-1 (3σ). A series of repeatability studies showed reproducible results with a coefficient of variation of less than 7%. In addition, the proposed CLEIA was successfully applied to the analysis of spiked samples (tap water) and gave quantitative recoveries from 93.72% to 100.72%. This work demonstrates that the developed CLEIA can be applied as a universal platform for specific detection of diversified analytes.

CircEpc1 Promotes Ricin Toxin-Induced Inflammation via Activation of NF-κB and MAPK Signaling Pathways by Sponging miR-5114.

Increasing studies have concentrated on investigating circular RNAs (circRNAs) as pivotal regulators in the progression of numerous diseases and biological processes and abundant evidence shows that circRNAs are participated in the regulation of innate immune responses. Several studies showed that Ricin Toxin (RT) could induce inflammatory injury. There was no research on the particular functions and underlying mechanisms of circRNAs in RT-induced inflammation. In this study, RNA sequencing performed on RT-treated and normal RAW264.7 macrophage cells was used to investigated the differentially expressed circRNAs. Based on the dataset, the expression of circEpc1 (mmu_circ_0,000,842) was identified higher in RT-treated cells. Moreover, gain-and-loss function assays showed that circEpc1 function as a promoter in RT-induced inflammation in vivo and in vitro. Mechanistically, circEpc1 acted as a miR-5114 sponge to relieve the suppressive effect of miR-5114 on its target NOD2 and thereby activating NF-κB and MAPK signaling pathways. Our results illuminated a link between RT-induced inflammation and the circEpc1 regulatory loop and provided novel insight into the functions of circRNA in innate immune, which may emerge as a potential target in immunotherapy to control the RT-induced inflammatory injury.

Identification of a lncRNA/circRNA-miRNA-mRNA network to explore the effects of ricin toxin-induced inflammation in RAW264.7 cells.

Ricin toxin (RT) is a ribosome-inactivating protein derived from the beans of the castor oil plant. Our previous studies have reported that RT can induce the production of inflammatory cytokines and cause inflammatory injury in RAW264.7 cells. In order to explore the various biological processes that long noncoding RNA (lncRNA), circular RNA (circRNA) and micro RNA (miRNA) as endogenous non-coding RNAs (ceRNAs) may participate in the pro-inflammatory mechanism, RT (20 ng/mL) treated and normal RAW264.7 cells were firstly sequenced by RNA-seq. By comparing the differentially expressed genes, we obtained 10 hub genes and enriched the inflammatory-related signaling pathways. Based on our results, we concluded a lncRNA/circRNA-miRNA-mRNA network. Finally, we verified the key genes and pathways by qRT-PCR, WB and ELISA. From the experiment results, an opening MAPK signaling pathway in TNF signaling pathway via TNFR2 was found involved in RT-induced inflammation. This work provides a reference for searching for ceRNA targets or therapeutic drugs in RT-induced inflammatory injury in the future.

Recombinant Ricin Toxin Binding Subunit B (RTB) Stimulates Production of TNF-α by Mouse Macrophages Through Activation of TLR4 Signaling Pathway.

Ricin toxin binding subunit B (RTB) is a galactose-binding lectin protein derived from the beans of the castor oil plant (Ricinus communis). Our previous studies have reported a direct immunomodulatory effect of recombinant RTB, which stimulates RAW264.7 cells to produce cytokines including TNF-α. However, the role of RTB in innate immune response and its specific mechanism have not been reported in detail. In this work, the results showed that RTB treatment of macrophages significantly increased TLR4 protein levels. RTB also activated TLR4 downstream events, including MyD88, IRAK, and TRAF6, resulting in macrophage activation and TNF-α production. This process is reflected in the increase of IκB phosphorylation. TLR4 knockdown macrophages treated with RTB exhibited greatly reduced IκB phosphorylation and TNF-α secretion. Moreover, treatment with MyD88 inhibitor also suppressed TNF-α production. The docking of RT and TLR4 was simulated by computer, and the contact residues were concentrated on RTB. Our results suggest that recombinant RTB can activate mouse macrophages to secrete TNF-α through activation of NF-κB via the TLR4 signaling pathways.

Enhancing the antivirus activity of chimeric canine interferon with ricin subunit B by using nanoparticle formulations.

Despite interferon alpha having a broad spectrum of antiviral activity and strong antiproliferative activity, its applications are severely limited due to the intrinsic properties of proteins, such as poor stability and short serum half-life. In our study, canine interferon alpha (CaIFNα) gene was fused with the ricin toxin B chain (RTB) to form rCaIFNα/RTB, which encodes a 463-amino acid protein containing a 15-amino acid encoded (G4S)3 flexible linker. After expression in prokaryote, purification and renaturation, the cytotoxicity and antiviral activity of rCaIFNα/RTB were investigated in Madin-Darby canine kidney (MDCK) cells. rCaIFNα/RTB exerted a superior anti-vesicular stomatitis virus (VSV) activity on MDCK cells. Furthermore, we have developed a nanoparticle formulation of rCaIFNα/RTB by using polyethylenimine (PEI) through electrostatic interaction. rCaIFNα/RTB@PEI10000 is more stable than rCaIFNα/RTB at various pH and temperature levels, and it possesses enhanced antiviral activity. Our findings facilitate further research on the role of type I IFN in antiviral defense responses in Canis lupus familiaris.

Critical role of toll-like receptor 4 (TLR4) in ricin toxin-induced inflammatory responses in macrophages.

Ricin toxin (RT) is a natural plant-derived protein toxin from the seed of castor beans that belongs to a family of type II ribosome-inactivating proteins (RIPs). In addition to its main toxic mechanism of inhibiting the synthesis of cellular proteins, RT can induce the production of inflammatory cytokines and cause inflammatory injury. Macrophages play a crucial role in innate immunity and the adaptive immune response as the first line of host defense against bacterial infections and various types of invading pathogens. Upon activation, macrophages release types of cytokines to remove pathogens. However, the effect of RT on the immune response and its mechanism are not well characterized. In the current study, we investigated the activation of the TLR4-mediated signaling pathway by low-dose RT treatment and its interaction with signaling molecules in the transduction pathway. We found that low-dose RT can activate MyD88- and TRIF-dependent signaling pathways, revealing a possible mechanism by which low-dose RT-activates TLR4-mediated signaling pathways. We also confirmed that the TLR4-induced activation of the inflammatory signaling pathways was produced via its binding to RT. This study may help to identify the most important target molecules and clarify the mechanism of inflammatory injury of ricin.

GLP-1 Gene-Modified Human Umbilical Cord Mesenchymal Stem Cell Line Improves Blood Glucose Level in Type 2 Diabetic Mice.

Type 2 diabetes constitutes a serious threat to the health of patients, but there is currently no ideal treatment in the clinic. Glucagon-like peptide-1 and human umbilical cord mesenchymal stem cells have been confirmed to have antidiabetic effects, but both of them have certain defects in the process of antidiabetes, which cannot meet the need of clinical treatment. We hypothesized that human umbilical cord mesenchymal stem cells can be used as a vector to construct a novel cell line that expresses GLP-1 in vivo for a long time. And this cell strain results in lowering blood glucose in type 2 diabetic mice. The results showed that after 3 weeks of intramuscular injection of the new cell line, the fasting blood glucose of type 2 diabetic mice returned to the normal range, and the hypoglycemic effect was maintained within 3 weeks after putting an end to the drug. At the same time, during the administration, the mice lost weight, the food intake decreased, the half-life of GLP-1 in the body prolonged, the IR reduced, and the pancreatic function recovered. The results of this study indicate that the novel cell line can prolong the half-life of GLP-1 in vivo and effectively lower blood sugar, which is a feasible method to improve type 2 diabetes.

Hybrids of carbon dots with subunit B of ricin toxin for enhanced immunomodulatory activity.

Although Ricin toxin binding subunit B (RTB) can promote the activation of macrophages and modulate the cell-mediated immunity, its applications are severely limited due to the intrinsic properties of proteins, like poor stability and low efficacy of cellular uptake. In this work, the stable nanoparticles were prepared by supramolecular assembling of carbon dots (CDs) and RTB. The formed CDs-RTB possesses robust stability and can protect RTB against enzymatic hydrolysis. More importantly, CDs-RTB can promote macrophages proliferation, improve the generation of NO, IL-6 and TNF-α in RAW264.7 cells and increase the expression of mRNA, indicating the enhanced immunomodulatory activity of CDs-RTB. This work highlights the potential of using CDs as a simple and stable platform to assemble RTB and effectively promotes the application of RTB as the immunostimulant.