Epithelial Cell Death Research Articles

S-RBD-modified and miR-486-5p-engineered exosomes derived from mesenchymal stem cells suppress ferroptosis and alleviate radiation-induced lung injury and long-term pulmonary fibrosis

BackgroundRadiation-induced lung injury (RILI) is associated with alveolar epithelial cell death and secondary fibrosis in injured lung. Mesenchymal stem cell (MSC)-derived exosomes have regenerative effect against lung injury and the potential to intervene of RILI. However, their intervention efficacy is limited because they lack lung targeting characters and do not carry sufficient specific effectors. SARS-CoV-2 spike glycoprotein (SARS-CoV-2-S-RBD) binds angiotensin-converting enzyme 2 (ACE2) receptor and mediates interaction with host cells. MiR-486-5p is a multifunctional miRNA with angiogenic and antifibrotic potential and acts as an effector in MSC-derived exosomes. Ferroptosis is a form of cell death associated with radiation injury, its roles and mechanisms in RILI remain unclear. In this study, we developed an engineered MSC-derived exosomes with SARS-CoV-2-S-RBD- and miR-486-5p- modification and investigated their intervention effects on RIPF and action mechanisms via suppression of epithelial cell ferroptosis.ResultsAdenovirus-mediated gene modification led to miR-486-5p overexpression in human umbilical cord MSC exosomes (p < 0.05), thereby constructing miR-486-5p engineered MSC exosomes (miR-486-MSC-Exo). MiR-486-MSC-Exo promoted the proliferation and migration of irradiated mouse lung epithelial (MLE-12) cells in vitro and inhibited RILI in vivo (all p < 0.05). MiR-486-MSC-Exo suppressed ferroptosis in MLE-12 cells, and an in vitro assay revealed that the expression of fibrosis-related genes is up-regulated following ferroptosis (both p < 0.05). MiR-486-MSC-Exo reversed the up-regulated expression of fibrosis-related genes induced by TGF-β1 in vitro and improved pathological fibrosis in RIPF mice in vivo (all p < 0.05). SARS-CoV-2-S-RBD-modified and miR-486-5p-engineered MSC exosomes (miR-486-RBD-MSC-Exo) were also constructed, and the distribution of DiR dye-labeled miR-486-RBD-MSC-Exo in hACE2CKI/CKI Sftpc-Cre+ mice demonstrated long-term retention in the lung (p < 0.05). MiR-486-RBD-MSC-Exo significantly improved the survival rate and pathological changes in hACE2CKI/CKI Sftpc-Cre+ RIPF mice (all p < 0.05). Furthermore, miR-486-MSC-Exo exerted anti-fibrotic effects via targeted SMAD2 inhibition and Akt phosphorylation activation (p < 0.05).ConclusionsEngineered MSC exosomes with SARS-CoV-2-S-RBD- and miR-486-5p-modification were developed. MiR-486-RBD-MSC-Exo suppressed ferroptosis and fibrosis of MLE-12 cells in vitro, and alleviated RILI and long-term RIPF in ACE2 humanized mice in vivo. MiR-486-MSC-Exo exerted anti-fibrotic effects via SMAD2 inhibition and Akt activation. This study provides a potential approach for RIPF intervention.Graphical

A sphingolipid rheostat controls apoptosis versus apical cell extrusion as alternative tumour-suppressive mechanisms

Evasion of cell death is a hallmark of cancer, and consequently the induction of cell death is a common strategy in cancer treatment. However, the molecular mechanisms regulating different types of cell death are poorly understood. We have formerly shown that in the epidermis of hypomorphic zebrafish hai1a mutant embryos, pre-neoplastic transformations of keratinocytes caused by unrestrained activity of the type II transmembrane serine protease Matriptase-1 heal spontaneously. This healing is driven by Matriptase-dependent increased sphingosine kinase (SphK) activity and sphingosine-1-phosphate (S1P)-mediated keratinocyte loss via apical cell extrusion. In contrast, amorphic hai1afr26 mutants with even higher Matriptase-1 and SphK activity die within a few days. Here we show that this lethality is not due to epidermal carcinogenesis, but to aberrant tp53-independent apoptosis of keratinocytes caused by increased levels of pro-apoptotic C16 ceramides, sphingolipid counterparts to S1P within the sphingolipid rheostat, which severely compromises the epidermal barrier. Mathematical modelling of sphingolipid rheostat homeostasis, combined with in vivo manipulations of components of the rheostat or the ceramide de novo synthesis pathway, indicate that this unexpected overproduction of ceramides is caused by a negative feedback loop sensing ceramide levels and controlling ceramide replenishment via de novo synthesis. Therefore, despite their initial decrease due to increased conversion to S1P, ceramides eventually reach cell death-inducing levels, making transformed pre-neoplastic keratinocytes die even before they are extruded, thereby abrogating the normally barrier-preserving mode of apical live cell extrusion. Our results offer an in vivo perspective of the dynamics of sphingolipid homeostasis and its relevance for epithelial cell survival versus cell death, linking apical cell extrusion and apoptosis. Implications for human carcinomas and their treatments are discussed.

Dietary titanium dioxide nanoparticles impair intestinal epithelial regeneration by perturbating the function of intestinal stem cells

Intestinal health is closely linked to intestinal stem cells (ISCs), which are highly sensitive to the harmful substances in the lumen. However, there is limited knowledge regarding the effects of food additives on ISCs. This study aims to investigate the impact of dietary titanium dioxide nanoparticles (TiO2 NPs) compared with titanium dioxide microparticles (TiO2 MPs) on intestinal health associated with ISCs in response to dextran sodium sulfate (DSS)-induced enteritis in mice, as well as the related mechanism. We found that exposure to 1% (w/w) TiO2 NPs aggravated DSS-induced enteritis in mice, while this effect could not be observed under exposure to TiO2 MPs. Additionally, 1% (w/w) TiO2 NPs exposure under DSS-induced enteritis worsened the ISC-mediated regeneration of intestinal epithelium by decreasing the epithelial cell proliferation and epithelial turnover rate while increasing epithelial cell death. Meanwhile, using a 3D intestinal organoid model, we discovered that 20 μg/mL TiO2 NPs impaired ISC function and disrupted ISC fate specification both ex vivo and in vitro. Furthermore, TiO2 NPs hindered the nuclear translocation of β-catenin, reducing the overall output of Wnt signaling. Together, TiO2 NPs deteriorated the intestinal epithelial regeneration of mice with DSS-induced enteritis by perturbating ISC function and fate specification through a mechanism involving Wnt signaling. These findings highlight the adverse effect of dietary TiO2 NPs on ISCs and shed light on the particle size optimization of TiO2 food additive.

NINJ1-mediated Macrophage Plasma Membrane Rupture and Neutrophil Extracellular Trap Formation Contribute to Oxalate Nephropathy.

Oxalate nephropathy is characterized by calcium oxalate crystals deposition, which triggers necrosis of renal tubular epithelial cells, initiates an inflammatory cascade characterized by neutrophil and macrophage activation within the renal microenvironment. Despite the close association of immune cells with acute oxalate nephropathy, the underlying mechanisms still remain unclear. Nerve injury-induced protein 1 (NINJ1) plays an essential role in the induction of plasma membrane rupture (PMR), leading to damage-associated molecular patterns (DAMPs) release and triggering inflammation. We hypothesize that NINJ1-mediated high mobility group box 1 (HMGB1) release from macrophage PMR and neutrophil extracellular traps (NETs) formation synergistically contribute to the progression of acute oxalate nephropathy. Using a murine model of acute oxalate nephropathy, myeloid cell-specific deletion of Ninj1 mice (Ninj1fl/flvavcre) and their wild-type littermate control mice (Ninj1wt/wtvavcre) were administered intraperitoneal injection of 100 mg/kg sodium oxalate followed by drinking water with 3% sodium oxalate. Evaluation was conducted on tubular injury and inflammatory cell infiltration. In vitro studies involved isolation and culture of renal proximal tubular epithelial cells (RTECs), bone marrow-derived macrophages, and neutrophils to investigate NETs formation and HMGB1 release. Targeted deletion of Ninj1 in myeloid cells significantly mitigated oxalate-induced acute kidney injury by suppressing both HMGB1 release and NETs formation in vivo. In vitro investigations demonstrated that HMGB1 release from macrophage PMR and NETs formation in neutrophils mediated by NINJ1 oligomerization, which consequently coordinated to enhance renal tubular epithelial cell death. Our findings elucidate the pivotal role of NINJ1-dependent macrophage PMR and NETs formation in the progression of acute oxalate nephropathy, providing novel insights for its prevention and therapeutic targets.

A new ex vivo model system to analyze factors affecting the integrity of fetal membranes in fetoscopic surgery

We developed an ex vivo model system to analyze the influence of relevant environmental and mechanical factors potentially affecting the integrity of fetal membranes during fetoscopic surgery. The set-up exposes amniochorion membranes to insufflation at predefined levels of gas pressure, flow, humidity, and temperature. Change in fetal membranes stiffness is quantified during the phase mimicking surgery through measurement of membranes’ strain in response to cyclic overpressure. The trocar induced perforation creates a mechanical weakness whose stability is assessed by increasing the insufflation pressure until membrane rupture. Damage of the epithelial cells lining the amnion is assessed through live-dead staining. Initial experiments demonstrated the functionality of the new apparatus and the feasibility of the proposed protocols. Fetal membranes exposed to air with low humidity for approximately 1 h demonstrated significant embrittlement, while their mechanical integrity was maintained in case of gas insufflation at high humidity (air as well as CO2). Under dry circumstances, there was a significant rate of epithelial cell death. Separation of amnion and chorion in the region of the trocar site was visible in all experiments. This new model is a versatile platform for analyzing the mechanical, histological, and biological implications of fetoscopic surgery on fetal membranes.

The Effect of In Vitro Digestion on the Anti-allergic, Anti-Inflammatory and Antioxidant Properties of Purple Rice and Purple Barley Phenolic Extracts in Caco-2 and RBL-2H3 Cells

Gastrointestinal diseases are associated with increased oxidative stress and inflammation. Pigmented cereal polyphenols are believed to have therapeutic potential. However, the impact of digestion on their bioactivity has not been fully elucidated. This study investigated the impact of digestion on the ability of purple rice extract (PRx) and purple barley (PBx) to alleviate oxidative stress-induced intestinal epithelial cell death and allergic inflammation. The study used a human colon adenocarcinoma (Caco-2) cell line to simulate intestinal oxidative stress for 4 h using 1 mM hydrogen peroxide. To evaluate the protective effects of digested and undigested cereal extracts, cells were first pre-treated with varying concentrations (50, 100, 200, and 500 μg/ml) of both purple rice (PRx) and purple barley (PBx) extracts. A rat basophilic leukemia (RBL-2H3) cell line was used to investigate the ability of the extracts to prevent calcium ionophore-induced histamine and cytokine release. The results demonstrated that both digested and undigested phenolic extracts prevented oxidative stress-induced cell death in Caco-2 cells and reduced Interleukin (IL)-8 secretion. PRx showed greater cytoprotective effects than PBx. Both digested and undigested extracts showed a time-dependent antihistamine effect, with the greatest inhibitory effects observed after 2 h and 4 h of treatment with PRx. Additionally, the extracts significantly attenuated the release of several cytokines including IL-2, IL-4, IL-13, monocyte chemoattractant protein-1, interferon-gamma, and tumour necrosis factor-alpha. Overall, the study demonstrates that both digested and undigested pigmented rice and barley extracts are potential sources of polyphenols which may promote intestinal health and alleviate allergic inflammation.

STING Promotes the Progression of ADPKD by Regulating Mitochondrial Function, Inflammation, Fibrosis, and Apoptosis.

Autosomal dominant polycystic kidney disease (ADPKD) is a predominant genetic disease, which is caused by mutations in PKD genes and is associated with DNA damage in cystic cells. The intrinsic stimulator of interferon genes (STING) pathway is crucial for recognizing damaged DNA in the cytosol, triggering the expression of inflammatory cytokines to activate defense mechanisms. However, the precise roles and mechanisms of STING in ADPKD remain elusive. In this study, we show that Pkd1 mutant mouse kidneys show upregulation of STING, which is stimulated by the DNAs of nuclear and mitochondrial origin. The activation of STING promotes cyst growth through increasing (1) the activation of NF-κB in Pkd1 mutant cells and (2) the recruitment of macrophages in the interstitial and peri-cystic regions in Pkd1 mutant mouse kidneys via NF-κB mediating the upregulation of TNF-α and MCP-1. Targeting STING with its specific inhibitor C-176 delays cyst growth in an early-stage aggressive Pkd1 conditional knockout mouse model and a milder long-lasting Pkd1 mutant mouse model. Targeting STING normalizes mitochondrial structure and function, decreases the formation of micronuclei, induces Pkd1 mutant renal epithelial cell death via p53 signaling, and decreases renal fibrosis in Pkd1 mutant mouse kidneys. These results support that STING is a novel therapeutic target for ADPKD treatment.

The IDH1-R132H mutation aggravates cisplatin-induced acute kidney injury by promoting ferroptosis through disrupting NDUFA1 and FSP1 interaction

AbstractThe IDH1-R132H mutation is implicated in the development of various tumors. Whether cisplatin, a common chemotherapeutic agent, induces more significant renal toxicity in individuals with the IDH1-R132H mutation remains unclear. In this study, we observed that the IDH1-R132H mutation exacerbates mitochondrial lipid peroxidation and dysfunction in renal tubules, rendering the kidneys more susceptible to cisplatin-induced ferroptosis. The IDH1-R132H mutation increases methylation of the Ndufa1 promoter, thereby suppressing NDUFA1 transcription and translation. This suppression disrupts NDUFA1’s interaction with FSP1, reducing its resistance to cisplatin-induced tubular epithelial cell death. As a consequence, ROS accumulates, lipid peroxidation occurs, and ferroptosis is triggered, thereby promoting acute kidney injury. In summary, this study elucidates a novel mechanism underlying cisplatin-induced nephrotoxicity and provides valuable insights for the development of personalized treatment strategies for tumor patients carrying the IDH1-R132H mutation.

Transcriptomic analysis and validation study of key genes and the HIF‑1α/HO‑1 pathway associated with ferroptosis in neutrophilic asthma.

Ferroptosis, as a unique form of cell death caused by iron overload and lipid peroxidation, is involved in the pathogenesis of various inflammatory diseases of the airways. Inhibition of ferroptosis has become a novel strategy for reducing airway epithelial cell death and improving airway inflammation. The aim of the present study was to analyze and validate the key genes and signaling pathways associated with ferroptosis by bioinformatic methods combined with experimental analyzes in vitro and in vivo to aid the diagnosis and treatment of neutrophilic asthma. A total of 1,639 differentially expressed genes (DEGs) were identified in the transcriptome dataset. After overlapping with ferroptosis-related genes, 11 differentially expressed ferroptosis-related genes (DE-FRGs) were obtained. A new diagnostic model was constructed by these DE-FRGs from the transcriptome dataset with those from the GSE108417 dataset. The receiver operating characteristic curve analysis indicated that the area under the curve had good diagnostic performance (>0.8). As a result, four key DE-FRGs (CXCL2, HMOX1, IL-6 and SLC7A5) and biological pathway [hypoxia-inducible factor 1 (HIF-1) signaling pathway] associated with ferroptosis in neutrophilic asthma were identified by the bioinformatics analysis combined with experimental validation. The upstream regulatory network of key DE-FRGs and target drugs were predicted and the molecular docking results from screened 37 potential therapeutic drugs revealed that the 13 small-molecule drugs exhibited a higher stable binding to the primary proteins of key DE-FRGs. The results suggested that four key DE-FRGs and the HIF-1α/heme oxygenase 1 pathway associated with ferroptosis have potential as novel markers or targets for the diagnosis or treatment of neutrophilic asthma.

Chitinase 3-like-1 Inhibits Innate Antitumor and Tissue Remodeling Immune Responses by Regulating CD47-SIRPα- and CD24-Siglec10-Mediated Phagocytosis.

Innate immune responses such as phagocytosis are critically linked to the generation of adaptive immune responses against the neoantigens in cancer and the efferocytosis that is essential for homeostasis in diseases characterized by lung injury, inflammation, and remodeling as in chronic obstructive pulmonary disease (COPD). Chitinase 3-like-1 (CHI3L1) is induced in many cancers where it inhibits adaptive immune responses by stimulating immune checkpoint molecules (ICPs) and portends a poor prognosis. CHI3L1 is also induced in COPD where it regulates epithelial cell death. In this study, we demonstrate that pulmonary melanoma metastasis inhibits macrophage phagocytosis by stimulating the CD47-SIRPα and CD24-Siglec10 phagocytosis checkpoint pathways while inhibiting macrophage "eat me" signals from calreticulin and HMGB1. We also demonstrate that these effects on macrophage phagocytosis are associated with CHI3L1 stimulation of the SHP-1 and SHP-2 phosphatases and inhibition of the accumulation and phosphorylation of cytoskeleton-regulating nonmuscle myosin IIa. This inhibition of innate immune responses such as phagocytosis provides a mechanistic explanation for the ability of CHI3L1 to stimulate ICPs and inhibit adaptive immune responses in cancer and diseases such as COPD. The ability of CHI3L1 to simultaneously inhibit innate immune responses, stimulate ICPs, inhibit T cell costimulation, and regulate a number of other oncogenic and inflammation pathways suggests that CHI3L1-targeted therapeutics are promising interventions in cancer, COPD, and other disorders.

Capsaicin mitigates ventilator-induced lung injury by suppressing ferroptosis and maintaining mitochondrial redox homeostasis through SIRT3-dependent mechanisms

BackgroundVentilator-induced lung injury (VILI) is one of the severe complications in the clinic concerning mechanical ventilation (MV). Capsaicin (CAP) has anti-inflammatory and inhibitory effects on oxidative stress, which is a significant element causing cellular ferroptosis. Nevertheless, the specific role and potential mechanistic pathways through which CAP modulates ferroptosis in VILI remain elusive.MethodsVILI was established in vivo, and the pulmonary epithelial cell injury model induced by circulation stretching (CS) was established in vitro. Both mice and cells were pretreated with CAP. Transmission electron microscopy, ELISA, Western blot, immunofluorescence, RT-PCR, fluorescent probes, and other experimental methods were used to clarify the relationship between iron death and VILI in alveolar epithelial cells, and whether capsaicin alleviates VILI by inhibiting iron death and its specific mechanism.ResultsFerroptosis was involved in VILI by utilizing in vivo models. CAP inhibited ferroptosis and alleviated VILI's lung damage and inflammation, and this protective effect of CAP was dependent on maintaining mitochondrial redox system through SITR3 signaling. In the CS-caused lung epithelial cell injury models, CAP reduced pathological CS-caused ferroptosis and cell injury. Knockdown SIRT3 reversed the role of CAP on the maintaining mitochondria dysfunction under pathological CS and eliminated its subsequent advantageous impacts for ferroptosis against overstretching cells.ConclusionThe outcomes showed that CAP alleviated ferroptosis in VILI via improving the activity of SITR3 to suppressing mitochondrial oxidative damage and maintaining mitochondrial redox homeostasis, illustrating its possibility as a novel therapeutic goal for VILI.Graphical

Subpopulation dynamics of T and B lymphocytes in Sjögren's syndrome: implications for disease activity and treatment.

Sjögren's syndrome (SS) is an autoimmune disorder primarily affecting the body's exocrine glands, particularly the salivary and lacrimal glands, which lead to severe symptoms of dry eyes and mouth. The pathogenesis of SS involves the production of autoantibodies by activated immune cells, and secretion of multiple cytokines, which collectively lead to tissue damage and functional impairment. In SS, the Immune interaction among T and B cells is particularly significant. Lymphocytic infiltration in the salivary glands is predominantly composed of CD4+ T cells, whose activation cause the death of glandular epithelial cells and subsequent tissue destruction. The excessive activity of T cells contributes significantly to the disease mechanism, with helper T cells (CD4+) differentiating into various subgroups including Th1/Th2, Th17, as well as Treg, each contributing to the pathological process through distinct cytokine secretion. In patients with SS, B cells are excessively activated, leading to substantial production of autoantibodies. These antibodies can attack self-tissues, especially the lacrimal and salivary glands, causing inflammation and tissue damage. Changes in B cell subpopulations in SS patients, such as increases in plasmablasts and plasma cells, correlate positively with serum autoantibody levels and disease progression. Therapies targeting T cells and B cells are extensively researched with the aim of alleviating symptoms and improving the quality of life for patients. Understanding how these cells promote disease development through various mechanisms, and further identifying novel T and B cell subgroups with functional characterization, will facilitate the development of more effective strategies to treat SS.

Metabolite-based inter-kingdom communication controls intestinal tissue recovery following chemotherapeutic injury

Cytotoxic chemotherapies have devastating side effects, particularly within the gastrointestinal tract. Gastrointestinal toxicity includes the death and damage of the epithelium and an imbalance in the intestinal microbiota, otherwise known as dysbiosis. Whether dysbiosis is a direct contributor to tissue toxicity is a key area of focus. Here, from both mammalian and bacterial perspectives, we uncover an intestinal epithelial cell death-Enterobacteriaceae signaling axis that fuels dysbiosis. Specifically, our data demonstrate that chemotherapy-induced epithelial cell apoptosis and the purine-containing metabolites released from dying cells drive the inter-kingdom transcriptional re-wiring of the Enterobacteriaceae, including fundamental shifts in bacterial respiration and promotion of purine utilization-dependent expansion, which in turn delays the recovery of the intestinal tract. Inhibition of epithelial cell death or restriction of the Enterobacteriaceae to homeostatic levels reverses dysbiosis and improves intestinal recovery. These findings suggest that supportive therapies that maintain homeostatic levels of Enterobacteriaceae may be useful in resolving intestinal disease.

Mesoporous zinc-polyphenol nanozyme for attenuating renal ischemia-reperfusion injury.

Aim: To target the reactive oxygen species (ROS) accumulation and renal tubular epithelial cell (rTEC) death in renal ischemia-reperfusion injury (IRI), we constructed a nanoparticle that offers ROS scavenging and rTEC-death inhibition: mesoporous zinc-tannic acid nanozyme (ZnTA).Materials & methods: After successfully constructing ZnTA, we proceeded to examine its effect on ROS accumulation, cellular ferroptosisand apoptosis, as well as injury severity.Results: Malondialdehyde, Fe2+ amounts and 4-HNE staining demonstrated that ZnTA effectively attenuated rTEC ferroptosis. TUNEL staining confirmed that Zn2+ carried by ZnTA could effectively inhibit caspase 3 and caspase 9, mitigating apoptosis. Finally, it reduced renal IRI through the synergistic effect of ROSscavenging and cell-death inhibition.Conclusion: This study is expected to provide a paradigm for a combined therapeutic strategy for renal IRI.

Astragaloside IV attenuates ferroptosis and protects against iron overload-induced retinal injury

Retinal injury may be exacerbated by iron overload. Astragaloside IV (AS-IV) has potential applications in the food and healthcare industry to promote eye health. We sought to determine the mechanisms responsible for the protective effects of AS-IV on photoreceptor and retinal pigment epithelium cell death induced by iron overload. We conducted in vitro and in vivo experiments involving AS-IV pretreatment. We tested AS-IV for its ability to protect iron-overload mice from retinal injury. In particular, we analyzed the effects of AS-IV on iron overload-induced ferroptosis in 661W and ARPE-19 cells. AS-IV not only attenuated iron deposition and retinal injury in iron-overload mice but also effectively reduced iron overload-induced ferroptotic cell death in 661W and ARPE-19 cells. AS-IV effectively prevented ferroptosis by inhibiting iron accumulation and lipid peroxidation. In addition, inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2) eliminated the protective effect of AS-IV against ferroptosis. The results suggest that ferroptosis might be a significant cause of retinal cell death associated with iron overload. AS-IV provides protection from iron overload-induced ferroptosis, partly by activating the Nrf2 signaling pathway.

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy.

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-α and IFN-γ, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.

METTL3 restricts RIPK1-dependent cell death via the ATF3-cFLIP axis in the intestinal epithelium

Intestinal epithelial cells (IECs) are pivotal for maintaining intestinal homeostasis through self-renewal, proliferation, differentiation, and regulated cell death. While apoptosis and necroptosis are recognized as distinct pathways, their intricate interplay remains elusive. In this study, we report that Mettl3-mediated m6A modification maintains intestinal homeostasis by impeding epithelial cell death. Mettl3 knockout induces both apoptosis and necroptosis in IECs. Targeting different modes of cell death with specific inhibitors unveils that RIPK1 kinase activity is critical for the cell death triggered by Mettl3 knockout. Mechanistically, this occurs via the m6A-mediated transcriptional regulation of Atf3, a transcription factor that directly binds to Cflar, the gene encoding the anti-cell death protein cFLIP. cFLIP inhibits RIPK1 activity, thereby suppressing downstream apoptotic and necroptotic signaling. Together, these findings delineate the essential role of the METTL3-ATF3-cFLIP axis in homeostatic regulation of the intestinal epithelium by blocking RIPK1 activity.

Inducible RPE-specific GPX4 knockout causes oxidative stress and retinal degeneration with features of age-related macular degeneration

Age-related macular degeneration (AMD) is one of the leading causes of vision loss in the elderly. This disease involves oxidative stress burden in the retina leading to death of retinal pigment epithelial (RPE) cells and photoreceptors. The retina is susceptible to oxidative stress, in part due to high metabolic activity and high concentration of polyunsaturated fatty acids that undergo lipid peroxidation chain reactions. Antioxidant enzymes exist in the retina to combat this stress, including glutathione peroxidase 4 (GPX4). GPX4 specifically reduces oxidized lipids, protecting against lipid peroxidation-induced oxidative stress, which is noted in dry AMD. We hypothesize that Gpx4 knockout within the RPE will result in an environment of chronic oxidative stress yielding degeneration akin to AMD. C57BL/6J mice with a floxed Gpx4 gene were mated with Rpe65Cre/ER mice. Offspring containing Rpe65Cre ± alleles and either Gpx4 WT or Gpx4 fl/fl alleles were administered tamoxifen to induce Gpx4 knockout in Gpx4 fl/fl mice. At sequential timepoints, retinal phenotypes were assessed via in vivo imaging utilizing confocal scanning laser ophthalmoscopy and optical coherence tomography (OCT), and visual function was probed by electroretinography. Retinas were studied post-mortem by immunohistochemical analyses, electron microscopy, plastic sectioning, and quantitative polymerase chain reaction and Western analyses. The RPE-specific Gpx4 knockout model was validated via Western analysis indicating diminished GPX4 protein only within the RPE and not the neural retina. Following Gpx4 knockout, RPE cells became dysfunctional and died, with significant cell loss occurring 2 weeks post-knockout. Progressive thinning of the photoreceptor layer followed RPE degeneration and was accompanied by loss of visual function. OCT and light microscopy showed hyperreflective foci and enlarged, pigmented cells in and above the RPE layer. Electron microscopy revealed decreased mitochondrial cristae and loss of basal and apical RPE ultrastructure. Finally, there was increased carboxyethylpyrrole staining, indicating oxidation of docosahexaenoic acid, and increased levels of mRNAs encoding oxidative stress-associated genes in the RPE and photoreceptors. Overall, we show that RPE-localized GPX4 is necessary for the health of the RPE and outer retina, and that knockout recapitulates phenotypes of dry AMD.

Distinct cell death pathways induced by granzymes collectively protect against intestinal Salmonella infection

Intestinal intraepithelial T lymphocytes (IEL) constitutively express high amounts of the cytotoxic proteases Granzymes (Gzm) A and B and are therefore thought to protect the intestinal epithelium against infection by killing infected epithelial cells. However, the role of IEL granzymes in a protective immune response has yet to be demonstrated. We show that GzmA and GzmB are required to protect mice against oral, but not intravenous, infection with Salmonella enterica serovar Typhimurium, consistent with an intestine-specific role. IEL-intrinsic granzymes mediate the protective effects by controlling intracellular bacterial growth and aiding in cell-intrinsic pyroptotic cell death of epithelial cells. Surprisingly, we found that both granzymes play non-redundant roles. GzmB-/- mice carried significantly lower burdens of Salmonella, as predominant GzmA-mediated cell death effectively reduced bacterial translocation across the intestinal barrier. Conversely, in GzmA-/- mice, GzmB-driven apoptosis favored luminal Salmonella growth by providing nutrients, while still reducing translocation across the epithelial barrier. Together, the concerted actions of both GzmA and GzmB balance cell death mechanisms at the intestinal epithelium to provide optimal control that Salmonella cannot subvert.

Modified Jiaoqi Powder enhances epithelial autophagy against TNF-triggered apoptosis in chronic ulcerative colitis

BackgroundA vicious cycle of dysregulated intestinal epithelial cell death, intestinal barrier defect, and subsequent inflammation response is core to chronic ulcerative colitis (UC). Modified Jiaoqi Powder (MJQP), a traditional Chinese medicine formula, has been clinically applied to treat chronic relapsing type and chronic persistent type of UC . Nevertheless, the underlying mechanism of MJPQ in chronic UC remains unknown. PurposeThe present study aimed to demonstrate the favorable effect and potential molecular mechanism of MJQP in chronic UC. MethodsThe chemical components of MJQP and MJQP drug serum were identified by LC-MS/MS. The curative effect of MJQP was evaluated in a well-established DSS-induced chronic UC mice model by measuring body weight, colon length, disease activity index (DAI) and histological scores . Serum cytokines, including interleukin (IL)-1β, IL-12, IL-13, IL-4, tumor necrosis factor-alpha (TNF-α) and INF-γ were measured using enzyme-linked immunosorbent assay. Western blotting, immunofluorescence, and MTT assay were used to analyze the effects of MJQP on colonic barrier function in chronic UC mice and human epithelial cell lines. TUNEL assay, western blotting, and flow cytometry were used to examine the related apoptosis indicators. An electron microscope was used to observe autophagosomes and autolysosomes, while western blotting and immunofluorescence were used to detect autophagy-associated proteins. Network pharmacology was used to predict potential targets and pathways of MJQP in UC. Finally, the TNF pathway-related proteins were detected by immunohistochemistry and western blotting. ResultsMJQP administration prevented the UC progression with faster weight gain, longer colon length, lower histological scores and DAI, and up/down- regulation of inflammatory factors. The expressions of tight junction proteins ki67 and E-cadherin increased dose-dependently after MJQP intervention. Moreover, MJQP treatment promoted the viability of NCM460 and Caco2 cells in a concentration-dependent manner. MJQP dose-dependently decreased the proportion of TUNEL-positive cells and attenuated the pro-apoptotic proteins cleaved-caspase 8 and cleaved-caspase 3 in colonic tissues. Flow cytometry also showed that MJQP dose-dependently decreased the apoptotic cell population of LPS-induced NCM460 and Caco2 cells. Electron microscopy revealed that autophagosomes and autolysosomes were significantly improved in the MJQP-treated groups. Additionally, autophagy-related proteins were significantly expressed after MJQP treatment. Network pharmacological analysis predicted that MJQP may alleviate chronic UC by affecting TNF-related signaling pathways and promoting intestinal epithelial cell proliferation. As anticipated, the TNF pathway-associated proteins were attenuated dose-dependently in colonic tissues after MJQP treatment. ConclusionThese results provide novel therapeutic strategies, indicating that MJQP may be a promising candidate treatment for chronic UC by promoting epithelial barrier restitution by enhancing epithelial autophagy against TNF–mediated apoptosis.