SETDB1-Mediated Chromatin Regulation in Intestinal Epithelial Cells During Intestinal Ischemia-Reperfusion Injury

Ikenoue M., Choijookhuu N., Yano K., Fidya, Takahashi N., Ishizuka T., Shirouzu S., Yamaguma Y., Kai K., Higuchi K., Sawaguchi A., Nanashima A., Hishikawa Y.

Su (var) 3–9, enhancer of seste, trithorax (SET)-domain bifurcated histone lysine methyltransferase (SETDB1) plays a crucial role in maintaining intestinal stem cell homeostasis; however, its physiological function in epithelial injury is largely unknown. In this study, we investigated the role of SETDB1 in epithelial regeneration using an intestinal ischemia/reperfusion injury (IRI) mouse model. Jejunum tissues were sampled after 75 min of ischemia followed by 3, 24, and 48 h of reperfusion. Morphological evaluations were performed using light microscopy and electron microscopy, and the involvement of SETDB1 in epithelial remodeling was investigated by immunohistochemistry. Expression of SETDB1 was increased following 24 h of reperfusion and localized in not only the crypt bottom but also in the transit amplifying zone and part of the villi. Changes in cell lineage, repression of cell adhesion molecule expression, and decreased histone H3 methylation status were detected in the crypts at the same time. Electron microscopy also revealed aberrant alignment of crypt nuclei and fusion of adjacent villi. Furthermore, increased SETDB1 expression and epithelial remodeling were confirmed with loss of stem cells, suggesting SETDB1 affects epithelial cell plasticity. In addition, crypt elongation and increased numbers of Ki-67 positive cells indicated active cell proliferation after IRI; however, the expression of PCNA was decreased compared to sham mouse jejunum. These morphological changes and the aberrant expression of proliferation markers were prevented by sinefungin, a histone methyltransferase inhibitor. In summary, SETDB1 plays a crucial role in changes in the epithelial structure after IRI-induced stem cell loss.

Xia K., Wang T., Chen Z., Guo J., Yu B., Chen Q., Qiu T., Zhou J., Zheng S.

Kitazawa R., Haraguchi R., Kitazawa S.

Chen L., Dai M., Zuo W., Dai Y., Yang Q., Yu S., Huang M., Liu H.

Macrophages exhibit distinct phenotypes that are pro-inflammatory (M1) or anti-inflammatory (M2) in response to inflammation. In this study, we tried to identify the roles and mechanisms of interferon regulatory factor 7 (IRF7) in modulating the phenotypes of macrophages in lipopolysaccharide (LPS)-induced intestinal inflammation. The mouse model of intestinal inflammation was induced by lipopolysaccharide (LPS), and mouse bone marrow-derived macrophages (BMDMs) and mouse intestinal epithelial cells were selected for experimental verification in vitro. Results demonstrated that IRF7 was highly expressed in the mouse model of intestinal inflammation, while IRF7 deficiency repressed macrophage M1 polarization and attenuated intestinal inflammation in mice. p65 and SET domain bifurcated 1 (SETDB1) synergistically promoted histone 3 lysine 4 trimethylation (H3K4me3) methylation to elevate IRF7 expression, which activated the Nod-like receptor (NLR) pathway to induce macrophage M1 polarization. Through this mechanism, IRF7 in BMDMs functioned to accelerate intestinal epithelial cell apoptosis and their release of pro-inflammatory proteins. Furthermore, the promoting effect of p65 and SETDB1 on LPS-induced intestinal inflammation was validated in vivo. To sum up, NF-κB p65 and SETDB1 facilitated IRF7-mediated macrophage M1 polarization, thereby aggravating the LPS-induced intestinal inflammation. Hence, this study highlights the appealing value of these factors as anti-inflammatory targets.

Rungratanawanich W., Lin Y., Wang X., Kawamoto T., Chidambaram S.B., Song B.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is the major enzyme responsible for metabolizing toxic acetaldehyde to acetate and acts as a protective or defensive protein against various disease states associated with alcohol use disorder (AUD), including alcohol-related liver disease (ARLD). We hypothesized that Aldh2-knockout (KO) mice are more susceptible to binge alcohol-mediated liver injury than wild-type (WT) mice through increased oxidative stress, gut leakiness and endotoxemia. Therefore, this study aimed to investigate the protective role of ALDH2 in binge alcohol-induced gut permeability, endotoxemia, and acute inflammatory liver injury by exposing Aldh2-KO or WT mice to a single oral dose of binge alcohol 3.5, 4.0, or 5.0 g/kg. Our findings showed for the first time that ALDH2 deficiency in Aldh2-KO mice increases their sensitivity to binge alcohol-induced oxidative and nitrative stress, enterocyte apoptosis, and nitration of gut tight junction (TJ) and adherent junction (AJ) proteins, leading to their degradation. These resulted in gut leakiness and endotoxemia in Aldh2-KO mice after exposure to a single dose of ethanol even at 3.5 g/kg, while no changes were observed in the corresponding WT mice. The elevated serum endotoxin (lipopolysaccharide, LPS) and bacterial translocation contributed to systemic inflammation, hepatocyte apoptosis, and subsequently acute liver injury through the gut-liver axis. Treatment with Daidzin, an ALDH2 inhibitor, exacerbated ethanol-induced cell permeability and reduced TJ/AJ proteins in T84 human colon cells. These changes were reversed by Alda-1, an ALDH2 activator. Furthermore, CRISPR/Cas9-mediated knockout of ALDH2 in T84 cells increased alcohol-mediated cell damage and paracellular permeability. All these findings demonstrate the critical role of ALDH2 in alcohol-induced epithelial barrier dysfunction and suggest that ALDH2 deficiency or gene mutation in humans is a risk factor for alcohol-mediated gut and liver injury, and that ALDH2 could be an important therapeutic target against alcohol-associated tissue or organ damage.

Fan X., Shi G., Feng J., Jian L.

Zhao Z., Feng L., Peng X., Ma T., Tong R., Zhong L.

Epigenetic alterations are implicated in tumour immune evasion and immune checkpoint blockade (ICB) resistance. SET domain bifurcated histone methyltransferase 1 (SETDB1) is a histone lysine methyltransferase that catalyses histone H3K9 di- and tri-methylation on euchromatin, and growing evidence indicates that SETDB1 amplification and abnormal activation are significantly correlated with the unfavourable prognosis of multiple malignant tumours and contribute to tumourigenesis and progression, immune evasion and ICB resistance. The main underlying mechanism is H3K9me3 deposition by SETDB1 on tumour-suppressive genes, retrotransposons, and immune genes. SETDB1 targeting is a promising approach to cancer therapy, particularly immunotherapy, because of its regulatory effects on endogenous retroviruses. However, SETDB1-targeted therapy remains challenging due to potential side effects and the lack of antagonists with high selectivity and potency. Here, we review the role of SETDB1 in tumourigenesis and immune regulation and present the current challenges and future perspectives of SETDB1 targeted therapy.

Przepiórska K., Wnuk A., Beyer C., Kajta M.

Amorfrutin B is a selective modulator of the PPARγ receptor, which has recently been identified as an effective neuroprotective compound that protects brain neurons from hypoxic and ischemic damage. Our study demonstrated for the first time that a 6-h delayed post-treatment with amorfrutin B prevented hypoxia/ischemia-induced neuronal apoptosis in terms of the loss of mitochondrial membrane potential, heterochromatin foci formation, and expression of specific genes and proteins. The expression of all studied apoptosis-related factors was decreased in response to amorfrutin B, both during hypoxia and ischemia, except for the expression of anti-apoptotic BCL2, which was increased. After post-treatment with amorfrutin B, the methylation rate of the pro-apoptotic Bax gene was inversely correlated with the protein level, which explained the decrease in the BAX/BCL2 ratio as a result of Bax hypermethylation. The mechanisms of the protective action of amorfrutin B also involved the inhibition of autophagy, as evidenced by diminished autophagolysosome formation and the loss of neuroprotective properties of amorfrutin B after the silencing of Becn1 and/or Atg7. Although post-treatment with amorfrutin B reduced the expression levels of Becn1, Nup62, and Ambra1 during hypoxia, it stimulated Atg5 and the protein levels of MAP1LC3B and AMBRA1 during ischemia, supporting the ambiguous role of autophagy in the development of brain pathologies. Furthermore, amorfrutin B affected the expression levels of apoptosis-focused and autophagy-related miRNAs, and many of these miRNAs were oppositely regulated by amorfrutin B and hypoxia/ischemia. The results strongly support the position of amorfrutin B among the most promising anti-stroke and wide-window therapeutics.

Kase K., Reintam Blaser A., Tamme K., Mändul M., Forbes A., Talving P., Murruste M.

There is a lack of population-based studies on acute mesenteric ischemia (AMI). We have therefore performed a nationwide epidemiological study in Estonia, addressing incidence, demographics, interventions and mortality of AMI. A retrospective population-based review was conducted of all adult cases of AMI accrued from the digital Estonian Health Insurance Fund and Causes of Death Registry for 2016–2020 based on international classification of diseases (ICD-10) diagnostic codes and procedure codes (NOMESCO). Overall, 577 cases of AMI were identified—an annual incidence of 8.7 per 100,000. The median age was 79 (range 32–104) and 57% were female. Predominating comorbidities included hypertensive disease (81%), atherosclerosis (67%), and atrial fibrillation (52%). The majority of cases (60%) were caused by superior mesenteric artery occlusion (thrombosis 54%, embolism 12%, and unclear 34%). Inferior mesenteric artery occlusion occurred in 7%, non-occlusive mesenteric ischemia in 7%, venous thrombosis in 4%, whereas the type remained unclear in 21% of cases. 40% of patients received intervention (revascularization and/or intestinal resection) and 13% active non-operative treatment. In 21% an exploratory laparotomy or laparoscopy revealed unsalvageable bowel prompting end-of-life care, which was the only management in a further 25% of cases. The population-based annual incidence of AMI in Estonia was 8.7 per 100,000 during the study period. The overall hospital mortality and 1 year mortality were 64% and 74%, respectively. In the 53% of patients who received active treatment hospital mortality was 32% and 1 year all-cause mortality was 51%. ClinicalTrials.gov Identifier NCT04867499.

Yano K., Choijookhuu N., Ikenoue M., Fidya, Fukaya T., Sato K., Lee D., Taniguchi N., Chosa E., Nanashima A., Hishikawa Y.

Liver regeneration is an extraordinarily complex process involving a variety of factors; however, the role of chromatin protein in hepatocyte proliferation is largely unknown. In this study, we investigated the functional role of high-mobility group box 2 (HMGB2), a chromatin protein in liver regeneration using wild-type and HMGB2-knockout (KO) mice. Liver tissues were sampled after 70% partial hepatectomy (PHx), and analyzed by immunohistochemistry, western blotting and flow cytometry using various markers of cell proliferation. In WT mice, hepatocyte proliferation was strongly correlated with the spatiotemporal expression of HMGB2; however, cell proliferation was significantly delayed in hepatocytes of HMGB2-KO mice. Quantitative PCR demonstrated that cyclin D1 and cyclin B1 mRNAs were significantly decreased in HMGB2-KO mice livers. Interestingly, hepatocyte size was significantly larger in HMGB2-KO mice at 36–72 h after PHx, and these results suggest that hepatocyte hypertrophy appeared in parallel with delayed cell proliferation. In vitro experiments demonstrated that cell proliferation was significantly decreased in HMGB2-KO cells. A significant delay in cell proliferation was also found in HMGB2-siRNA transfected cells. In summary, spatiotemporal expression of HMGB2 is important for regulation of hepatocyte proliferation and cell size during liver regeneration.

Miyazaki S., Funamoto T., Sekimoto T., Kurogi S., Ohta T., Nagai T., Tajima T., Imasaka M., Yoshinobu K., Araki K., Araki M., Choijookhuu N., Hishikawa Y., Chosa E.

Di Tomaso M.V., Vázquez Alberdi L., Olsson D., Cancela S., Fernández A., Rosillo J.C., Reyes Ábalos A.L., Álvarez Zabaleta M., Calero M., Kun A.

Myelination of the peripheral nervous system requires Schwann cells (SC) differentiation into the myelinating phenotype. The peripheral myelin protein-22 (PMP22) is an integral membrane glycoprotein, expressed in SC. It was initially described as a growth arrest-specific (gas3) gene product, up-regulated by serum starvation. PMP22 mutations were pathognomonic for human hereditary peripheral neuropathies, including the Charcot-Marie-Tooth disease (CMT). Trembler-J (TrJ) is a heterozygous mouse model carrying the same pmp22 point mutation as a CMT1E variant. Mutations in lamina genes have been related to a type of peripheral (CMT2B1) or central (autosomal dominant leukodystrophy) neuropathy. We explore the presence of PMP22 and Lamin B1 in Wt and TrJ SC nuclei of sciatic nerves and the colocalization of PMP22 concerning the silent heterochromatin (HC: DAPI-dark counterstaining), the transcriptionally active euchromatin (EC), and the nuclear lamina (H3K4m3 and Lamin B1 immunostaining, respectively). The results revealed that the number of TrJ SC nuclei in sciatic nerves was greater, and the SC volumes were smaller than those of Wt. The myelin protein PMP22 and Lamin B1 were detected in Wt and TrJ SC nuclei and predominantly in peripheral nuclear regions. The level of PMP22 was higher, and those of Lamin B1 lower in TrJ than in Wt mice. The level of PMP22 was higher, and those of Lamin B1 lower in TrJ than in Wt mice. PMP22 colocalized more with Lamin B1 and with the transcriptionally competent EC, than the silent HC with differences between Wt and TrJ genotypes. The results are discussed regarding the probable nuclear role of PMP22 and the relationship with TrJ neuropathy.

Funken D., Yu Y., Feng X., Imvised T., Gueler F., Prinz I., Madadi-Sanjani O., Ure B.M., Kuebler J.F., Klemann C.

T-cells have been demonstrated to modulate ischemia–reperfusion injury (IRI) in the kidney, lung, liver, and intestine. Whereas most T-cell subpopulations contribute primarily to the antigen-specific effector and memory phases of immunity, γδ-T-cells combine adaptive features with rapid, innate-like responses that can place them in the initiation phase of immune reactions. Therefore, we aimed to clarify the role of γδ-T-cells in intestinal IRI. Adult wild-type (WT) and γδ-T-cell-deficient mice were subjected to acute intestinal IRI. Gene expression of pro-inflammatory cytokines and influx of leukocyte subpopulations in the gut were assessed by qPCR and flow cytometry. Serum transaminases were measured as an indicator of distant organ IRI. Intestinal IRI led to increased influx of neutrophils, pro-inflammatory cytokine expression and LDH/ALT/AST elevation. Selective deficiency of γδ-T-cells significantly decreased pro-inflammatory cytokine levels and neutrophil infiltration in the gut following IRI compared to controls. Furthermore, γδ-T-cell deficiency resulted in decreased LDH and transaminases levels in sera, indicating amelioration of distant organ injury. Increasing evidence demonstrates a key role of T-cell subpopulations in IRI. We demonstrate that γδ-T-cell deficiency ameliorated pro-inflammatory cytokine production, neutrophil recruitment and distant organ injury. Thus, γδ-T-cells may be considered as mediators contributing to the inflammatory response in the acute phase of intestinal IRI.

Markouli M., Strepkos D., Piperi C.

The SET Domain Bifurcated Histone Lysine Methyltransferase 1 (SETDB1) is a prominent member of the Suppressor of Variegation 3–9 (SUV39)-related protein lysine methyltransferases (PKMTs), comprising three isoforms that differ in length and domain composition. SETDB1 is widely expressed in human tissues, methylating Histone 3 lysine 9 (H3K9) residues, promoting chromatin compaction and exerting negative regulation on gene expression. SETDB1 has a central role in normal physiology and nervous system development, having been implicated in the regulation of cell cycle progression, inactivation of the X chromosome, immune cells function, expression of retroelements and formation of promyelocytic leukemia (PML) nuclear bodies (NB). SETDB1 has been frequently deregulated in carcinogenesis, being implicated in the pathogenesis of gliomas, melanomas, as well as in lung, breast, gastrointestinal and ovarian tumors, where it mainly exerts an oncogenic role. Aberrant activity of SETDB1 has also been implicated in several neuropsychiatric, cardiovascular and gastrointestinal diseases, including schizophrenia, Huntington’s disease, congenital heart defects and inflammatory bowel disease. Herein, we provide an update on the unique structural and biochemical features of SETDB1 that contribute to its regulation, as well as its molecular and cellular impact in normal physiology and disease with potential therapeutic options.

Yang J., Wu Y., Xu Y., Jia J., Xi W., Deng H., Tu W.

AbstractBackground Intestinal ischemia/reperfusion (I/R) is a common clinical problem that occurs during various clinical pathological processes. Dexmedetomidine (DEX), a widely used anesthetic adjuvant agent, can induce protection against intestinal I/R in vivo; however, the underlying mechanism is not fully understood. In the present study, we aimed to investigate the protective effects of DEX and examine whether its mechanism was associated with the TLR4/MyD88/NF-κB signaling pathway. Methods Sprague–Dawley rats were pretreated with DEX and then subjected to I/R-induced intestinal injury. In vivo, intestinal histopathological examination and scoring were performed, the levels of serum intestinal fatty acid-binding protein (I-FABP), intestinal tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and expression levels of TLR4, MyD88, and NF-κB in the intestine were determined. In in vitro experiments, the human colon carcinoma cell line (Caco-2) was incubated with DEX before deprivation/reoxygenation (OGD/R) treatment. The cell viability of Caco-2 cells, the levels of lactate dehydrogenase (LDH), TNF-α, and IL-1β in the supernatant, as well as protein expression of TLR4, MyD88, and NF-κB in Caco-2 cells, were measured. Statistical analysis was performed using SPSS version 21.0. Results DEX preconditioning significantly reduced the intestinal pathological Chiu's score, serum I-FABP, intestinal TNF-α, IL-1β levels, and the protein expression of TLR4, MyD88, and NF-κB in the rats with intestinal I/R injury. Similarly, in vitro, DEX pretreatment protected against OGD/R-induced Caco-2 cell damage and inhibited TLR4/MyD88/NF-κB signaling, as evidenced by increased cell viability, decreased LDH activity, reduced TNF-α and IL-1β levels, as well as downregulated TLR4, MyD88, and NF-κB protein levels. Conclusions Our findings suggested that DEX could reduce intestinal I/R injury in rats and OGD/R damage in Caco-2 cells, and this protection might be attributed to antiinflammatory effects and inhibition of the TLR4/MyD88/NF-κB signaling pathway.