Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed ...mechanisms regulating circadian physiology of the intestine remain largely unknown. In this report, we show that both pluripotent stem cell‐derived human intestinal organoids engrafted into mice and patient‐derived human intestinal enteroids possess circadian rhythms and demonstrate circadian phase‐dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti‐phasic necrotic cell death responses to TcdB. RNA‐Seq analysis shows that ~3–10% of the detectable transcripts are rhythmically expressed in mouse and human enteroids. Remarkably, we observe anti‐phasic gene expression of Rac1, a small GTPase directly inactivated by TcdB, between mouse and human enteroids, and disruption of Rac1 abolishes clock‐dependent necrotic cell death responses. Our findings uncover robust functions of circadian rhythms regulating clock‐controlled genes in both mouse and human enteroids governing organism‐specific, circadian phase‐dependent necrotic cell death responses, and lay a foundation for human organ‐ and disease‐specific investigation of clock functions using human organoids for translational applications.
SYNOPSIS
How rhythmicity of gene expression in the gut epithelium develops and which molecular mechanisms contribute to its maintenance remains unclear. This analysis of mouse and human organoid models uncovers species‐specific oscillatory behavior of circadian genes in the intestine.
Maturation of pluripotent stem cell‐derived human intestinal organoids is required to develop robust circadian rhythms.
Approximately 3–10% of transcripts in mouse and human intestinal enteroids are clock‐controlled genes.
Phase set enrichment analysis uncovers eight conserved rhythmic signaling pathways.
Rac1 small GTPase oscillations are anti‐phasic between mouse and human enteroids, regulating distinct necrotic cell death responses to Clostridium difficile toxin B.
Mouse and human 3D in vitro models show tissue maturation to determine species‐specific circadian rhytms in the intestine.
The occurrence of gastritis, gastric ulcers, distal gastric cancer, and gastric mucosal lymphoma in humans is strongly associated with Helicobacter pylori (H. pylori). Vaccination is an effective ...preventive measure due to the increasing prevalence of antibiotic resistance. Fusion vaccination is a potentially practical approach. A fusion vaccine was created in this study by combining the cholera toxin B subunit (CTB) with the antigenic H. pylori urease I subunit (CTB-UreI). The CTB-UreI DNA vaccine was chemically cloned into pIRES2-EGFP, and the success of the cloning was validated using PCR and restriction enzyme digestion. An investigation was conducted on the induction of CTB-UreI in Escherichia coli BL21(DE3). The immunogenicity and immune-protective efficacy of the vaccination were assessed in BALB/c mice. The Western blot assay successfully identified the activation of CTB-UreI.
In comparison, BALB/c mice receiving pIRES2-EGFP/CTB-UreI vaccination exhibited higher IgG, IgA, IFN-γ, IL-4, and IL-17 levels in their blood samples. In addition, there was a decrease in stomach injuries and bacterial loads. Furthermore, BALB/c mice inoculated with pIRES2-EGFP/CTB-UreI showed a high level of immunity (100%) against the H. pylori challenge. The pIRES2-EGFP/CTB-UreI elicited a combination of Th1/Th2/Th17 immune responses, possibly contributing to an effective defence mechanism. Our data suggests that using this fusion vaccine to prevent H. pylori infection is a promising option.
Mesenchymal stem cell (MSC), a widely used adult stem cell candidate for regenerative medicine, has been shown to exert some of its therapeutic effects through the secretion of extracellular vesicles ...(EVs). These homogenously sized EVs of 100-150 ηm exhibited many exosome-like biophysical and biochemical properties and carry both proteins and RNAs. Recently, exosome-associated proteins in this MSC EV preparation were found to segregate primarily to those EVs that bind cholera toxin B chain (CTB), a GM1 ganglioside-specific ligand, and pulse-chase experiments demonstrated that these EVs have endosomal origin and carried many of the exosome-associated markers. Here, we report that only a fraction of the MSC EV proteome was found in CTB-bound EVs. Using Annexin V (AV) and Shiga toxin B subunit (ST) with affinities for phosphatidylserine and globotriaosylceramide, respectively, AV- and a ST-binding EV were identified. CTB-, AV- and ST-binding EVs all carried actin. However, the AV-binding EVs carried low or undetectable levels of the exosome-associated proteins. Only the ST-binding EVs carried RNA and EDA-containing fibronectin. Proteins in AV-binding EVs were also different from those released by apoptotic MSCs. CTB- and AV-binding activities were localized to the plasma membrane and cytoplasm of MSCs, while ST-binding activity was localized to the nucleus. Together, this study demonstrates that cells secrete many types of EVs. Specifically, MSCs secrete at least 3 types. They can be differentially isolated based on their affinities for membrane lipid-binding ligands. As the subcellular sites of the binding activities of these ligands and cargo load are different for each EV type, they are likely to have a different biogenesis pathway and possibly different functions.
Clostridioides difficile
infection (CDI) causes nosocomial/antibiotic-associated gastrointestinal diseases with dramatically increasing global incidence and mortality rates. The main
C. difficile
...virulence factors, toxins A and B (TcdA/TcdB), cause cytopathic/cytotoxic effects and inflammation. We demonstrated that TcdB induces caspase-dependent, mitochondria-independent enteric glial cell (EGC) apoptosis that is enhanced by the pro-inflammatory cytokines TNF-α and IFN-γ (CKs) by increasing caspase-3/7/9 and PARP activation. Because this cytotoxic synergism is important for CDI pathogenesis, we investigated the apoptotic pathways involved in TcdB- and TcdB + CK-induced apoptosis indepth. EGCs were pre-treated with the inhibitors BAF or Q-VD-OPh (pan-caspase), Z-DEVD-fmk (caspase-3/7), Z-IETD-fmk (caspase-8), PD150606 (calpains), and CA-074Me (cathepsin B) 1 h before TcdB exposure, while CKs were given 1.5 h after TcdB exposure, and assays were performed at 24 h. TcdB and TcdB + CKs induced apoptosis through three signalling pathways activated by calpains, caspases and cathepsins, which all are involved both in induction and execution apoptotic signalling under both conditions but to different degrees in TcdB and TcdB + CKs especially as regards to signal transduction mediated by these proteases towards downstream effects (apoptosis). Calpain activation by Ca
2+
influx is the first pro-apoptotic event in TcdB- and TcdB + CK-induced EGC apoptosis and causes caspase-3, caspase-7 and PARP activation. PARP is also directly activated by calpains which are responsible of about 75% of apoptosis in TcdB and 62% in TcdB + CK which is both effector caspase-dependent and -independent. Initiator caspase-8 activation mediated by TcdB contributes to caspase-3/caspase-7 and PARP activation and is responsible of about 28% of apoptosis in both conditions. Caspase-3/caspase-7 activation is weakly responsible of apoptosis, indeed we found that it mediates 27% of apoptosis only in TcdB. Cathepsin B contributes to triggering pro-apoptotic signal and is responsible in both conditions of about 35% of apoptosis by a caspase-independent manner, and seems to regulate the caspase-3 and caspase-7 cleaved fragment levels, highlighting the complex interaction between these cysteine protease families activated during TcdB-induced apoptosis. Further a relevant difference between TcdB- and TcdB + CK-induced apoptosis is that TcdB-induced apoptosis increased slowly reaching at 72 h the value of 18.7%, while TcdB + CK-induced apoptosis increased strongly reaching at 72 h the value of 60.6%. Apoptotic signalling activation by TcdB + CKs is enriched by TNF-α-induced NF-κB signalling, inhibition of JNK activation and activation of AKT. In conclusion, the ability of
C. difficile
to activate three apoptotic pathways represents an important strategy to overcome resistance against its cytotoxic activity.
The therapeutic efficacy of mesenchymal stromal cells (MSCs), multipotent progenitor cells, is attributed to small (50–200 nm) extracellular vesicles (EVs). The presence of a lipid membrane ...differentiates exosomes and EVs from other macromolecules. Analysis of this lipid membrane revealed three distinct small MSC EV subtypes, each with a differential affinity for cholera toxin B chain (CTB), annexin V (AV), and Shiga toxin B chain (ST) that bind GM1 ganglioside, phosphatidylserine, and globotriaosylceramide, respectively. Similar EV subtypes are also found in biologic fluids and are independent sources of disease biomarkers. Here, we compare and contrast these three EV subtypes. All subtypes carry β-actin, but only CTB-binding EVs (CTB-EVs) are true exosomes, enriched with exosome proteins and derived from endosomes. No unique protein has been identified yet in AV-binding EVs (AV-EVs); ST-binding EVs (ST-EVs) carry RNA and a high level of extra domain A-containing fibronectin. Based on the CTB, AV, and ST subcellular binding sites, the origins of CTB-, AV-, and ST-EV biogenesis are the plasma membrane, cytoplasm, and nucleus, respectively. The differentiation of EV subtypes through membrane lipids underlies the importance of membrane lipids in defining EVs and implies an influence on EV biology and functions.
Assessment of ASC Oligomerization by Flow Cytometry Hurtado-Navarro, Laura; Baroja-Mazo, Alberto; Martínez-Banaclocha, Helios ...
Methods in molecular biology (Clifton, N.J.),
2022, Volume:
2459
Journal Article
Inflammasomes are multiprotein complexes that critically control different aspects of innate and adaptive immunity. Upon activation, inflammasome proteins oligomerize forming scaffolds to nucleate ...the apoptosis-associated speck-like protein containing a CARD (ASC) in filaments that will finally result in large ASC oligomers that are commonly named as ASC specks. In this chapter, we present a method to monitor NLRP3 or pyrin inflammasome activation in human monocytes upon extracellular ATP or Clostridium difficile toxin B treatment, respectively, by detecting intracellular oligomers of ASC by flow cytometry. This method could be used to evaluate the degree of inflammasome activation in blood samples from patients suffering from different chronic inflammatory diseases.
•BTX-B suppresses the progression of psoriasis-like dermatitis in IMQ-treated mice.•BTX-B suppressed the type 17 inflammation in IMQ-treated skin lesions.•BTX-B regulates psoriasis via neuroimmune ...system.•Neuropeptide inhibitor, including BTX can be applied to the treatment of psoriasis.
Psoriasis is a multifactorial disease arises from a complex interaction of genetics, immune system, and environmental aspects. IL-23/Th17 immune axis has been considered as a primary modulator in psoriasis. In addition, several findings imply that nervous system may take a part in the pathogenesis of psoriasis, suggesting that nervous system, through its neuropeptide, may interact with immune system and lead to the formation of psoriasis.
We aimed to ascertain the role of neuropeptides secreted from neurons in the pathogenesis of psoriasis in vivo.
The release of neuropeptide was inhibited by injecting Botulinum toxin B (BTX-B) on Imiquimod (IMQ)-induced psoriasis-like dermatitis mice model. Quantification of skin dermatitis, infiltrating inflammatory cells, and the production of cytokines at the lesional skin area were performed by PSI score, immunostaining, and real-time PCR. We also tested the effect of selective CGRP antagonist (CGRP8−37) on psoriasis-like dermatitis in IMQ-treated mice.
BTX-B injection significantly suppressed PSI score and reduced the number of CD4+ T cells, CD11c+ dendritic cells, and the production of IL-17A/F in the lesional skin. The expressions of PGP9.5+ nerve fibers and neuropeptides (SP, CGRP) were also significantly reduced following BTX-B injection. Additionally, CGRP antagonist also suppressed the development of IMQ-induced psoriasis-like dermatitis in mice.
The suppression of neuropeptide secretion in the skin by BTX injection might inhibit nerve elongation, the infiltration of immune cells, as well as IL-17 production, resulting in the improvement of psoriasis. Neuropeptide inhibitor could also be applied to the treatment of psoriasis.
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