Signalling by senescent melanocytes hyperactivates hair growth.

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

Excessive nucleic acid R-loops induce mitochondria-dependent epithelial cell necroptosis and drive spontaneous intestinal inflammation.

Oxidative stress, which can be activated by a variety of environmental risk factors, has been implicated as an important pathogenic factor for inflammatory bowel disease (IBD). However, how oxidative stress drives IBD onset remains elusive. Here, we found that oxidative stress was strongly activated in inflamed tissues from both ulcerative colitis patients and Crohn's disease patients, and it caused nuclear-to-cytosolic TDP-43 transport and a reduction in the TDP-43 protein level. To investigate the function of TDP-43 in IBD, we inducibly deleted exons 2 to 3 of Tardbp (encoding Tdp-43) in mouse intestinal epithelium, which disrupted its nuclear localization and RNA-processing function. The deletion gave rise to spontaneous intestinal inflammation by inducing epithelial cell necroptosis. Suppression of the necroptotic pathway with deletion of Mlkl or the RIP1 inhibitor Nec-1 rescued colitis phenotypes. Mechanistically, disruption of nuclear TDP-43 caused excessive R-loop accumulation, which triggered DNA damage and genome instability and thereby induced PARP1 hyperactivation, leading to subsequent NAD+ depletion and ATP loss, consequently activating mitochondrion-dependent necroptosis in intestinal epithelial cells. Importantly, restoration of cellular NAD+ levels with NAD+ or NMN supplementation, as well as suppression of ALKBH7, an α-ketoglutarate dioxygenase in mitochondria, rescued TDP-43 deficiency-induced cell death and intestinal inflammation. Furthermore, TDP-43 protein levels were significantly inversely correlated with γ-H2A.X and p-MLKL levels in clinical IBD samples, suggesting the clinical relevance of TDP-43 deficiency-induced mitochondrion-dependent necroptosis. Taken together, these findings identify a unique pathogenic mechanism that links oxidative stress to intestinal inflammation and provide a potent and valid strategy for IBD intervention.

mTORC2-AKT signaling to PFKFB2 activates glycolysis that enhances stemness and tumorigenicity of intestinal epithelial cells.

Although elevated glycolysis has been widely recognized as a hallmark for highly proliferating cells like stem cells and cancer, its regulatory mechanisms are still being updated. Here, we found a previously unappreciated mechanism of mammalian target of rapamycin complex 2 (mTORC2) in regulating glycolysis in intestinal stem cell maintenance and cancer progression. mTORC2 key subunits expression levels and its kinase activity were specifically upregulated in intestinal stem cells, mouse intestinal tumors, and human colorectal cancer (CRC) tissues. Genetic ablation of its key scaffolding protein Rictor in both mouse models and cell lines revealed that mTORC2 played an important role in promoting intestinal stem cell proliferation and self-renewal. Moreover, utilizing mouse models and organoid culture, mTORC2 loss of function was shown to impair growth of gut adenoma and tumor organoids. Based on these findings, we performed RNA-seq and noticed significant metabolic reprogramming in Rictor conditional knockout mice. Among all the pathways, carbohydrate metabolism was most profoundly altered, and further studies demonstrated that mTORC2 promoted glycolysis in intestinal epithelial cells. Most importantly, we showed that a rate-limiting enzyme in regulating glycolysis, 6-phosphofructo-2-kinase (PFKFB2), was a direct target for the mTORC2-AKT signaling. PFKFB2 was phosphorylated upon mTORC2 activation, but not mTORC1, and this process was AKT-dependent. Together, this study has identified a novel mechanism underlying mTORC2 activated glycolysis, offering potential therapeutic targets for treating CRC.

Cathepsin B as a key regulator of ferroptosis in microglia following intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a subtype of stroke marked by elevated mortality and disability rates. Recently, mounting evidence suggests a significant role of ferroptosis in the pathogenesis of ICH. Through a combination of bioinformatics analysis and basic experiments, our goal is to identify the primary cell types and key molecules implicated in ferroptosis post-ICH. This aims to propel the advancement of ferroptosis research, offering potential therapeutic targets for ICH treatment. Our study reveals pronounced ferroptosis in microglia and identifies the target gene, cathepsin B (Ctsb), by analyzing differentially expressed genes following ICH. Ctsb, a cysteine protease primarily located in lysosomes, becomes a focal point in our investigation. Utilizing in vitro and in vivo models, we explore the correlation between Ctsb and ferroptosis in microglia post-ICH. Results demonstrate that ICH and hemin-induced ferroptosis in microglia coincide with elevated levels and activity of Ctsb protein. Effective alleviation of ferroptosis in microglia after ICH is achieved through the inhibition of Ctsb protease activity and protein levels using inhibitors and shRNA. Additionally, a notable increase in m6A methylation levels of Ctsb mRNA post-ICH is observed, suggesting a pivotal role of m6A methylation in regulating Ctsb translation. These research insights deepen our comprehension of the molecular pathways involved in ferroptosis after ICH, underscoring the potential of Ctsb as a promising target for mitigating brain damage resulting from ICH.

Dystrophin 71 deficiency causes impaired aquaporin-4 polarization contributing to glymphatic dysfunction and brain edema in cerebral ischemia.

OBJECTIVE: The glymphatic system serves as a perivascular pathway that aids in clearing liquid and solute waste from the brain, thereby enhancing neurological function. Disorders in glymphatic drainage contribute to the development of vasogenic edema following cerebral ischemia, although the molecular mechanisms involved remain poorly understood. This study aims to determine whether a deficiency in dystrophin 71 (DP71) leads to aquaporin-4 (AQP4) depolarization, contributing to glymphatic dysfunction in cerebral ischemia and resulting in brain edema. METHODS: A mice model of middle cerebral artery occlusion and reperfusion was used. A fluorescence tracer was injected into the cortex and evaluated glymphatic clearance. To investigate the role of DP71 in maintaining AQP4 polarization, an adeno-associated virus with the astrocyte promoter was used to overexpress Dp71. The expression and distribution of DP71 and AQP4 were analyzed using immunoblotting, immunofluorescence, and co-immunoprecipitation techniques. The behavior ability of mice was evaluated by open field test. Open-access transcriptome sequencing data were used to analyze the functional changes of astrocytes after cerebral ischemia. MG132 was used to inhibit the ubiquitin-proteasome system. The ubiquitination of DP71 was detected by immunoblotting and co-immunoprecipitation. RESULTS: During the vasogenic edema stage following cerebral ischemia, a decline in the efflux of interstitial fluid tracer was observed. DP71 and AQP4 were co-localized and interacted with each other in the perivascular astrocyte endfeet. After cerebral ischemia, there was a notable reduction in DP71 protein expression, accompanied by AQP4 depolarization and proliferation of reactive astrocytes. Increased DP71 expression restored glymphatic drainage and reduced brain edema. AQP4 depolarization, reactive astrocyte proliferation, and the behavior of mice were improved. After cerebral ischemia, DP71 was degraded by ubiquitination, and MG132 inhibited the decrease of DP71 protein level. CONCLUSION: AQP4 depolarization after cerebral ischemia leads to glymphatic clearance disorder and aggravates cerebral edema. DP71 plays a pivotal role in regulating AQP4 polarization and consequently influences glymphatic function. Changes in DP71 expression are associated with the ubiquitin-proteasome system. This study offers a novel perspective on the pathogenesis of brain edema following cerebral ischemia.

Secreted stromal protein ISLR promotes intestinal regeneration by suppressing epithelial Hippo signaling.

The Hippo-YAP signaling pathway plays an essential role in epithelial cells during intestinal regeneration and tumorigenesis. However, the molecular mechanism linking stromal signals to YAP-mediated intestinal regeneration and tumorigenesis is poorly defined. Here, we report a stroma-epithelium ISLR-YAP signaling axis essential for stromal cells to modulate epithelial cell growth during intestinal regeneration and tumorigenesis. Specifically, upon inflammation and in cancer, an oncogenic transcription factor ETS1 in stromal cells induces expression of a secreted protein ISLR that can inhibit Hippo signaling and activate YAP in epithelial cells. Deletion of Islr in stromal cells in mice markedly impaired intestinal regeneration and suppressed tumorigenesis in the colon. Moreover, the expression of stromal cell-specific ISLR and ETS1 significantly increased in inflamed mucosa of human IBD patients and in human colorectal adenocarcinoma, accounting for the epithelial YAP hyperactivation. Collectively, our findings provide new insights into the signaling crosstalk between stroma and epithelium during tissue regeneration and tumorigenesis.

Bclaf1 regulates c-FLIP expression and protects cells from TNF-induced apoptosis and tissue injury.

TNF stimulation generates pro-survival signals through activation of NF-κB that restrict the build-in death signaling triggered by TNF. The competition between TNF-induced survival and death signals ultimately determines the fate of a cell. Here, we report the identification of Bclaf1 as a novel component of the anti-apoptotic program of TNF. Bclaf1 depletion in multiple cells sensitizes cells to TNF-induced apoptosis but not to necroptosis. Bclaf1 exerts its anti-apoptotic function by promoting the transcription of CFLAR, a caspase 8 antagonist, downstream of NF-κB activation. Bclaf1 binds to the p50 subunit of NF-κB, which is required for Bclaf1 to stimulate CFLAR transcription. Finally, in Bclaf1 siRNA administered mice, TNF-induced small intestine injury is much more severe than in control mice with aggravated signs of apoptosis and pyroptosis. These results suggest Bclaf1 is a key regulator in TNF-induced apoptosis, both in vitro and in vivo.

APC and P53 mutations synergise to create a therapeutic vulnerability to NOTUM inhibition in advanced colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with the majority of cases initiated by inactivation of the APC tumour suppressor. This results in the constitutive activation of canonical WNT pathway transcriptional effector ß-catenin, along with induction of WNT feedback inhibitors, including the extracellular palmitoleoyl-protein carboxylesterase NOTUM which antagonises WNT-FZD receptor-ligand interactions. Here, we sought to evaluate the effects of NOTUM activity on CRC as a function of driver mutation landscape. DESIGN: Mouse and human colon organoids engineered with combinations of CRC driver mutations were used for Notum genetic gain-of-function and loss-of-function studies. In vitro assays, in vivo endoscope-guided orthotopic organoid implantation assays and transcriptomic profiling were employed to characterise the effects of Notum activity. Small molecule inhibitors of Notum activity were used in preclinical therapeutic proof-of-principle studies targeting oncogenic Notum activity. RESULTS: NOTUM retains tumour suppressive activity in APC-null adenomas despite constitutive ß-catenin activity. Strikingly, on progression to adenocarcinoma with P53 loss, NOTUM becomes an obligate oncogene. These phenotypes are Wnt-independent, resulting from differential activity of NOTUM on glypican 1 and 4 in early-stage versus late-stage disease, respectively. Ultimately, preclinical mouse models and human organoid cultures demonstrate that pharmacological inhibition of NOTUM is highly effective in arresting primary adenocarcinoma growth and inhibiting metastatic colonisation of distal organs. CONCLUSIONS: Our findings that a single agent targeting the extracellular enzyme NOTUM is effective in treating highly aggressive, metastatic adenocarcinomas in preclinical mouse models and human organoids make NOTUM and its glypican targets therapeutic vulnerabilities in advanced CRC.

Unbalanced regulation of Sec22b and Ykt6 blocks autophagosome axonal retrograde flux in neuronal ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury in ischemic penumbra is accountable for poor outcome of ischemic stroke patients receiving recanalization therapy. Compelling evidence previously demonstrated a dual role of autophagy in stroke. This study aimed to understand the traits of autophagy in the ischemic penumbra and the potential mechanism that switches the dual role of autophagy. We found that autophagy induction by rapamycin and lithium carbonate performed before ischemia reduced neurological deficits and infarction, while autophagy induction after reperfusion had the opposite effect in the male murine middle cerebral artery occlusion/reperfusion model, both of which were eliminated in mice lacking autophagy (Atg7flox/flox; Nestin-Cre). Autophagic flux determination showed that reperfusion led to a blockage of axonal autophagosome retrograde transport in neurons, which then led to autophagic flux damage. Then, we found that ischemia-reperfusion induced changes in the protein levels of Sec22b and Ykt6 in neurons, two autophagosome transport-related factors, in which Sec22b significantly increased and Ykt6 significantly decreased. In the absence of exogenous autophagy induction, Sec22b knockdown and Ykt6 overexpression significantly alleviated autophagic flux damage, infarction, and neurological deficits in neurons or murine exposed to cerebral ischemia-reperfusion in an autophagy-dependent manner. Furthermore, Sec22b knockdown and Ykt6 overexpression switched the outcome of rapamycin post-treatment from deterioration to neuroprotection. Thus, Sec22b and Ykt6 play key roles in neuronal autophagic flux, and modest regulation of Sec22b and Ykt6 may help to reverse the failure of targeting autophagy induction to improve the prognosis of ischemic stroke.Significance Statement:The highly polarized architecture of neurons with neurites presents challenges for material transport, such as autophagosomes, which form at the neurite tip and need to be transported to the cell soma for degradation. Here, we demonstrate that Sec22b and Ykt6 act as autophagosome porters and play an important role in maintaining the integrity of neuronal autophagic flux. Ischemia-reperfusion-induced excess Sec22b and loss of Ykt6 in neurons lead to axonal autophagosome retrograde trafficking failure, autophagic flux damage, and finally neuronal injury. Facilitated axonal autophagosome retrograde transport by Sec22b knockdown and Ykt6 overexpression may reduce ischemia-reperfusion-induced neuron injury and extend the therapeutic window of pharmacological autophagy induction for neuroprotection.

WDR34 affects PI3K/Akt and Wnt/ß-catenin pathways to regulates malignant biological behaviors of glioma cells.

PURPOSE: Glioma is the most prevalent primary intracranial tumor globally. WDR34, a member of the WDR superfamily with five WD40 repeats, is involved in the pathogenesis of several tumors. However, the role of WDR34 in glioma progression is unknown. METHODS: The expression and prognostic significance of WDR34 in glioma patients were analyzed using GEPIA. WDR34 expression was detected by qRT-PCR. Western blot was employed to determine the expression of Ki67, proliferating cell nuclear antigen (PCNA), matrix metallopeptidase (MMP)2, MMP9, phosphatase and tensin homolog, protein kinase B (Akt), phosphorylated Akt, ß-catenin, and c-Myc. CCK-8, BrdU incorporation assay, Transwell invasion assay, flow cytometry analysis, and measurement of caspase-3 and caspase-9 activities were conducted to examine the effects of WDR34 knockdown on glioma cells. RESULTS: WDR34 was upregulated in glioma, which predicted a poor prognosis in glioma patients. WDR34 knockdown inhibited cell proliferation and reduced the expression of Ki67 and PCNA in glioma cells. WDR34 knockdown repressed the invasive ability of glioma cells by decreasing MMP-2 and MMP-9 expression. WDR34 knockdown increased the apoptotic rate and caspase-3 and caspase-9 activities in glioma cells. The PI3K/Akt and Wnt/ß-catenin pathways were inhibited after WDR34 knockdown in glioma cells. Moreover, overexpression of Akt or ß-catenin reversed the function of WDR34 knockdown on proliferation, invasion, and apoptosis. WDR34 knockdown reduced tumor growth in vivo. CONCLUSIONS: WDR34 knockdown inhibited malignant biological behaviors of glioma cells by inactivating the PI3K/Akt and Wnt/ß-catenin signaling cascades.

CITEMOXMBD: A flexible single-cell multimodal omics analysis framework to reveal the heterogeneity of immune cells.

Simultaneous measurement of multiple modalities in single-cell analysis, represented by CITE-seq, is a promising approach to link transcriptional changes to cellular phenotype and function, requiring new computational methods to define cellular subtypes and states based on multiple data types. Here, we design a flexible single-cell multimodal analysis framework, called CITEMO, to integrate the transcriptome and antibody-derived tags (ADT) data to capture cell heterogeneity from the multi omics perspective. CITEMO uses Principal Component Analysis (PCA) to obtain a low-dimensional representation of the transcriptome and ADT, respectively, and then employs PCA again to integrate these low-dimensional multimodal data for downstream analysis. To investigate the effectiveness of the CITEMO framework, we apply CITEMO to analyse the cell subtypes of Cord Blood Mononuclear Cells (CBMC) samples. Results show that the CITEMO framework can comprehensively analyse single-cell multimodal samples and accurately identify cell subtypes. Besides, we find some specific immune cells that co-express multiple ADT markers. To better describe the co-expression phenomenon, we introduce the co-expression entropy to measure the heterogeneous distribution of the ADT combinations. To further validate the robustness of the CITEMO framework, we analyse Human Bone Marrow Cell (HBMC) samples and identify different states of the same cell type. CITEMO has an excellent performance in identifying cell subtypes and states for multimodal omics data. We suggest that the flexible design idea of CITEMO can be an inspiration for other single-cell multimodal tasks. The complete source code and dataset of the CITEMO framework can be obtained from https://github.com/studentiz/CITEMO.

Nfatc1+ colonic stem cells contribute to regeneration upon colitis.

BACKGROUND AND AIM: Colonic stem cells play important roles in both normal epithelial turnover and injury repair. Lgr5+ colonic stem cells are highly susceptible to DSS-induced damage. However, it is still unclear how colonic stem cells regenerate injured epithelium during colitis. Here, we explored the functions of a new population of NFATc1+ colonic stem cells in experimental colitis. METHODS: Nfatc1+ colonic stem cells were labeled using Nfatc1CreERT2 ;R26mTmG reporter mice. Immunostaining assays were used to detect Goblet cells, enteroendocrine cells, and intestinal stem/progenitor cells. We performed lineage tracing assay to investigate whether Nfatc1+ cells are real colonic stem cells using Nfatc1CreERT2 ;R26mTmG mice. The contribution of Nfatc1+ stem cells on epithelial regeneration was detected in experimental colitis induced by DSS. RESULTS: Nfatc1-reporter marked cells are enriched for +3 to +5 position in colonic crypts, and they are overlapped with Sox9+ cells and Hopx+ cells that have been identified as stem cells in small intestine. However, Nfatc1-reporter marked cells are not overlapped with Lgr5+ colonic stem cells, as well as differentiated goblet cells and enteroendocrine cells. Furthermore, Nfatc1-reporter marked cells are able to give rise to all lineages of the colonic epithelium, and they preferentially contribute to the regeneration of colonic epithelium in DSS-induced experimental colitis. CONCLUSION: Nfatc1+ cells were identified as a novel population of colonic stem cells that are primarily located at +3 to +5 position and contribute to epithelial regeneration during colitis.

Segmented carotid endarterectomy for treatment of Riles type 1A common carotid artery occlusion.

OBJECTIVE: Common carotid artery occlusion (CCAO) is a rare cause of cerebrovascular events. Symptomatic lesions are resistant to medical treatment and revascularization is often required, but there is no consensus on the treatment of CCAO at present. Riles type 1A CCAO is most likely to benefit from revascularization because it has patent outflow tract (internal carotid artery) which was supplied by patent external carotid artery (ECA) from collateral circulation. We described a novel surgical technique improved on the basis of the carotid endarterectomy (CEA) for treatment of Riles type 1A CCAO. METHODS: We rigorously screened ten patients with symptomatic Riles type1A CCAO for surgery from January 2017 to May 2019 and performed a full preoperative assessment of the inadequate collateral circulation compensation. Moreover, we retrospectively reviewed our experience of the segmented CEA in the treatment of them in our single center. RESULTS: Segmented CEA was performed on the left side in four cases and on the right side in six cases. The technical success rate of the procedure was 100%. Primary suture was used in nine cases. Only one patient (right CCAO) who had a history of neck radiotherapy was treated by the patch CEA. The mean temporary blocking time during surgery was 52.8 ± 9.15 min. The mean temporary blocking time for treating the upper segment of the common carotid artery (CCA) was 11.1 ± 2.64 min. In the postoperative period, cerebral perfusion on the ipsilateral site improved in all patients, myocardial infarction occurred in one patient, and recurrent laryngeal nerve damage occurred in another. No ischemic events or re-occlusion or restenosis (> 50%) of the treated CCA occurred during the mean follow-up of 32.6 ± 9.3 months. The preoperative mean modified Rankin Scale (mRS) score was 1.9 (range, 1-3; median, 2). At last follow-up for all patients, the mRS score was 1 (range, 0-3; median, 1). CONCLUSION: Segmented CEA, which utilizes the compensatory effect of collateral circulation, is an effective and safe technique to treat patients suffering from Riles type 1A CCAO with hemodynamic cerebrovascular compromise.

Nfatc1's Role in Mammary Epithelial Morphogenesis and Basal Stem/progenitor Cell Self-renewal.

Mammary gland is an outstanding system to study the regulatory mechanisms governing adult epithelial stem cell activity. Stem cells in the basal layer of the mammary gland fuel the morphogenesis and regeneration of a complex epithelial network during development and upon transplantation. The self-renewal of basal stem/progenitor cells is subjected to regulation by both cell-intrinsic and extrinsic mechanisms. Nfatc1 is a transcription factor that regulates breast tumorigenesis and metastasis, but its role in mammary epithelial development and stem cell function has not been investigated. Here we show that Nfatc1 is expressed in a small subset of mammary basal epithelial cells and its epithelial-specific deletion results in mild defects in side branching and basal-luminal cell balance. Moreover, Nfatc1-deficient basal cells exhibit reduced colony forming ability in vitro and somewhat compromised regenerative potential upon transplantation. Thus, our study provides evidence for a detectable yet non-essential role of Nfatc1 in mammary epithelial morphogenesis and basal stem/progenitor cell self-renewal.

Enhanced Transport of Shape and Rigidity-Tuned α-Lactalbumin Nanotubes across Intestinal Mucus and Cellular Barriers.

Mucus is a viscoelastic biological hydrogel that protects the epithelial surface from penetration by most nanoparticles, which limits the efficiency of oral drug delivery. Pursuing highly efficient, biocompatible, and biodegradable oral drug vehicles is of central importance to the development of promising nanomedicine. Here, we prepared five peptosomes (PSs) with various sizes, shapes, and rigidities based on self-assembly of amphiphilic α-lactalbumin (α-lac) peptides from partial enzymolysis and cross-linking. The mucus permeation of α-lac PSs and release of curcumin (Cur) encapsulated in these PSs were evaluated. Compared with a long nanotube, big nanosphere, small nanosphere, and cross-linked short nanotube, we demonstrated that a short nanotube (SNT) exhibits excellent permeability in mucus, which enables it to arrive at epithelial cells quickly. Besides, SNT exhibits the highest cellular uptake and transmembrane permeability on Caco-2/HT29-MTX (E12) 3D coculture model. In vivo pharmacokinetic evaluation revealed that SNT formulation shows the highest curcumin bioavailability, which is 6.85-folds higher than free Cur. Most importantly, Cur loaded in SNT exhibits the optimum therapeutic efficacy for in vivo treatment of dextran sulfate sodium (DSS)-induced ulcerative colitis. In the end, the mechanism of the high permeability of SNTs through mucus was explained by coarse-grained molecular dynamics simulations, which indicated that short time scale jiggling and flying across pores of mucus network played key roles. These findings revealed the tubular α-lac PSs could be a promising oral drug delivery system targeted to mucosal for improving absorption and bioavailability of hydrophobic bioactive ingredients.

Aberrant gut microbiota alters host metabolome and impacts renal failure in humans and rodents.

OBJECTIVE: Patients with renal failure suffer from symptoms caused by uraemic toxins, possibly of gut microbial origin, as deduced from studies in animals. The aim of the study is to characterise relationships between the intestinal microbiome composition, uraemic toxins and renal failure symptoms in human end-stage renal disease (ESRD). DESIGN: Characterisation of gut microbiome, serum and faecal metabolome and human phenotypes in a cohort of 223 patients with ESRD and 69 healthy controls. Multidimensional data integration to reveal links between these datasets and the use of chronic kidney disease (CKD) rodent models to test the effects of intestinal microbiome on toxin accumulation and disease severity. RESULTS: A group of microbial species enriched in ESRD correlates tightly to patient clinical variables and encode functions involved in toxin and secondary bile acids synthesis; the relative abundance of the microbial functions correlates with the serum or faecal concentrations of these metabolites. Microbiota from patients transplanted to renal injured germ-free mice or antibiotic-treated rats induce higher production of serum uraemic toxins and aggravated renal fibrosis and oxidative stress more than microbiota from controls. Two of the species, Eggerthella lenta and Fusobacterium nucleatum, increase uraemic toxins production and promote renal disease development in a CKD rat model. A probiotic Bifidobacterium animalis decreases abundance of these species, reduces levels of toxins and the severity of the disease in rats. CONCLUSION: Aberrant gut microbiota in patients with ESRD sculpts a detrimental metabolome aggravating clinical outcomes, suggesting that the gut microbiota will be a promising target for diminishing uraemic toxicity in those patients. TRIAL REGISTRATION NUMBER: This study was registered at ClinicalTrials.gov (NCT03010696).

MicroRNA-31 Reduces Inflammatory Signaling and Promotes Regeneration in Colon Epithelium, and Delivery of Mimics in Microspheres Reduces Colitis in Mice.

BACKGROUND & AIMS: Levels of microRNA 31 (MIR31) are increased in intestinal tissues from patients with inflammatory bowel diseases and colitis-associated neoplasias. We investigated the effects of this microRNA on intestinal inflammation by studying mice with colitis. METHODS: We obtained colon biopsy samples from 82 patients with ulcerative colitis (UC), 79 patients with Crohn's disease (CD), and 34 healthy individuals (controls) at Shanghai Tenth People's Hospital. MIR31- knockout mice and mice with conditional disruption of Mir31 specifically in the intestinal epithelium (MIR31 conditional knockouts) were given dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS) to induce colitis. We performed chromatin immunoprecipitation and luciferase assays to study proteins that regulate expression of MIR31, including STAT3 and p65, in LOVO colorectal cancer cells and organoids derived from mouse colon cells. Partially hydrolyzed alpha-lactalbumin was used to generate peptosome nanoparticles, and MIR31 mimics were loaded onto their surface using electrostatic adsorption. Peptosome-MIR31 mimic particles were encapsulated into oxidized konjac glucomannan (OKGM) microspheres, which were administered by enema into the large intestines of mice with DSS-induced colitis. Intestinal tissues were collected and analyzed by histology and immunohistochemistry. RESULTS: Levels of MIR31 were increased in inflamed mucosa from patients with CD or UC, and from mice with colitis, compared with controls. STAT3 and nuclear factor-κB activated transcription of MIR31 in colorectal cancer cells and organoids in response to tumor necrosis factor and interleukin (IL)6. MIR31-knockout and conditional-knockout mice developed more severe colitis in response to DSS and TNBS, with increased immune responses, compared with control mice. MIR31 bound to 3' untranslated regions of Il17ra and Il7r messenger RNAs (RNAs) (which encode receptors for the inflammatory cytokines IL17 and IL7) and Il6st mRNA (which encodes GP130, a cytokine signaling protein). These mRNAs and proteins were greater in MIR31-knockout mice with colitis, compared with control mice; MIR31 and MIR31 mimics inhibited their expression. MIR31 also promoted epithelial regeneration by regulating the WNT and Hippo signaling pathways. OKGM peptosome-MIR31 mimic microspheres localized to colonic epithelial cells in mice with colitis; they reduced the inflammatory response, increased body weight and colon length, and promoted epithelial cell proliferation. CONCLUSIONS: MIR31, increased in colon tissues from patients with CD or UC, reduces the inflammatory response in colon epithelium of mice by preventing expression of inflammatory cytokine receptors (Il7R and Il17RA) and signaling proteins (GP130). MIR31 also regulates the WNT and Hippo signaling pathways to promote epithelial regeneration following injury. OKGM peptosome-MIR31 microspheres localize to the colon epithelium of mice to reduce features of colitis. Transcript Profiling: GSE123556.

CX3CL1/CX3CR1 axis attenuates early brain injury via promoting the delivery of exosomal microRNA-124 from neuron to microglia after subarachnoid hemorrhage.

BACKGROUND: Microglial activation-mediated neuroinflammation is a major contributor to early brain injury (EBI) after subarachnoid hemorrhage (SAH). MicroRNA-124 (miR-124) is the most abundant miRNAs in the central nervous system (CNS) and plays a vital role in microglial activation by targeting protein CCAAT-enhancer-binding protein α (C/EBPα). It has been reported that the CX3CL1/CX3CR1 axis is involved in the delivery of miR-124 from neurons to microglia. METHODS: An experimental rat SAH model was established by injecting autologous arterial blood into the prechiasmatic cistern, and cultured primary neurons and microglia were exposed to oxyhemoglobin to mimic SAH in vitro. We additionally exploited specific expression plasmids encoding CX3CL1 and CX3CR1. RESULTS: We observed significant decreases in CX3CL1 and CX3CR1 in the brain tissues of SAH patients. We also observed decreases in the levels of CX3CL1 in neurons and CX3CR1 in microglia after SAH in rats. Moreover, microglia exhibited an activated phenotype with macrophage-like morphology and high levels of CD45 and major histocompatibility complex (MHC) class II after SAH. After overexpression of CX3CL1/CX3CR1, the level of CD45 and MHC class II and the release of inflammatory factors tumor necrosis factor α, interleukin 1α and complement 1q were significantly decreased. There was also increased neuronal degeneration and behavior dysfunction after SAH, both of which were inhibited by CX3CL1/CX3CR1 overexpression. Additionally, we found that the delivery of exosomal miR-124 from neurons to microglia was significantly reduced after SAH, accompanied by an increase in C/EBPα expression, and was inhibited by CX3CL1/CX3CR1 overexpression. In conclusion, the CX3CL1/CX3CR1 axis may play protective roles after SAH by promoting the delivery of exosomal miR-124 to microglia and attenuate microglial activation and neuroinflammation. CONCLUSIONS: CX3CL1/CX3CR1 axis may be a potential intervention target for the inhibition of SAH-induced EBI by promoting exosome transport of miR-124 to microglia.

RIP3 participates in early brain injury after experimental subarachnoid hemorrhage in rats by inducing necroptosis.

Necroptosis is a regulated form of necrosis that is mediated by a variety of proteins including tumor necrosis factor-α (TNF-α) and receptor-interacting proteins (RIPs). TNF-α, a critical inflammatory molecule, is one of the initiating signals in the necroptosis pathway, and RIP3 acts as a switch that commits the cell to necroptosis. Subarachnoid hemorrhage (SAH) is a common type of hemorrhagic stroke with high mortality and disability rates. RIP3 has been studied in many central nervous system (CNS) diseases, but its role in SAH has not been investigated in depth. Here, we used an autologous-blood injection model to study the role of RIP3 in brain injury induced by SAH in rats. Several indexes such as brain edema, loss of blood-brain barrier (BBB) integrity, and behavioral tests of neurological function were used to evaluate brain damage in SAH-injured rats. We found that the expression of RIP3 was increased in the rat brain after SAH, reaching the highest point 24 h post-injury. We also showed that genetic or pharmacological inhibition of RIP3 or TNF-α reduced the brain damage induced by SAH, whereas overexpression of RIP3 aggravated brain injury and neurological damage. Additionally, we verified the presence of RIP3-mediated necroptosis in an in vitro SAH model of primary cultured neurons treated with conditioned medium from primary microglia activated by oxygen hemoglobin (OxyHb). Collectively, our findings indicated that RIP3 contributed to brain damage after SAH by inducing necroptosis.