Inhibition of HDAC2 sensitises antitumour therapy by promoting NLRP3/GSDMD-mediated pyroptosis in colorectal cancer.

BACKGROUND: Although numerous studies have indicated that activated pyroptosis can enhance the efficacy of antitumour therapy in several tumours, the precise mechanism of pyroptosis in colorectal cancer (CRC) remains unclear. METHODS: Pyroptosis in CRC cells treated with antitumour agents was assessed using various techniques, including Western blotting, lactate dehydrogenase release assay and microscopy analysis. To uncover the epigenetic mechanisms that regulate NLRP3, chromatin changes and NLRP3 promoter histone modifications were assessed using Assay for Transposase-Accessible Chromatin using sequencing and RNA sequencing. Chromatin immunoprecipitation‒quantitative polymerase chain reaction was used to investigate the NLRP3 transcriptional regulatory mechanism. Additionally, xenograft and patient-derived xenograft models were constructed to validate the effects of the drug combinations. RESULTS: As the core molecule of the inflammasome, NLRP3 expression was silenced in CRC, thereby limiting gasdermin D (GSDMD)-mediated pyroptosis. Supplementation with NLRP3 can rescue pyroptosis induced by antitumour therapy. Overexpression of HDAC2 in CRC silences NLRP3 via epigenetic regulation. Mechanistically, HDAC2 suppressed chromatin accessibility by eliminating H3K27 acetylation. HDAC2 knockout promotes H3K27ac-mediated recruitment of the BRD4-p-P65 complex to enhance NLRP3 transcription. Inhibiting HDAC2 by Santacruzamate A in combination with classic antitumour agents (5-fluorouracil or regorafenib) in CRC xenograft-bearing animals markedly activated pyroptosis and achieved a significant therapeutic effect. Clinically, HDAC2 is inversely correlated with H3K27ac/p-P65/NLRP3 and is a prognostic factor for CRC patients. CONCLUSION: Collectively, our data revealed a crucial role for HDAC2 in inhibiting NLRP3/GSDMD-mediated pyroptosis in CRC cells and highlighted HDAC2 as a potential therapeutic target for antitumour therapy. HIGHLIGHTS: Silencing of NLRP3 limits the GSDMD-dependent pyroptosis in colorectal cancer. HDAC2-mediated histone deacetylation leads to epigenetic silencing of NLRP3. HDAC2 suppresses the NLRP3 transcription by inhibiting the formation of H3K27ac/BRD4/p-P65 complex. Targeting HDAC2 activates pyroptosis and enhances therapeutic effect.

Heterogeneous expression of ARID1A in colorectal cancer indicates distinguish immune landscape and efficacy of immunotherapy.

OBJECTIVE: AT-rich interaction domain 1A (ARID1A) mutant tumors show active anti-tumor immune response, which is the potential indication of immunotherapy. However, the relationship between the heterogeneous ARID1A expression and the immune response and immunotherapy efficacy in colorectal cancer (CRC) is still unclear. METHODS: We collected 1113 cases of patients with stage I-IV CRC who underwent primary resection at Harbin Medical University Cancer Hospital. ARID1A expression in CRC tissues was assessed via immunohistochemistry (IHC). CD8, CD163 and FOXP3 were stained by IHC to identify the immune landscape. Clinicopathological features of patients were compared using statistical tests like the Wilcoxon-Mann-Whitney test or χ2 tests. Kaplan-Meier survival analysis with log-rank tests were employed. RESULTS: Heterogeneous ARID1A expression was categorized into integrity expression, complete expression deficiency (cd-ARID1A), partial expression deficiency (pd-ARID1A), and clonal expression deficiency (cld-ARID1A). ARID1A-deficient expression was significant association with dMMR (P value < 0.001). Patients with ARID1A deficiency, compared to ARID1A-proficient patients, exhibited increased infiltration levels of CD8 + P value < 0.0001), CD163 + P value < 0.001), and FOXP3 + P value < 0.001).cells within the tumor tissue. However, in different subgroups, only samples with complete or partial deficiency of ARID1A showed a higher abundance of lymphocyte infiltration. In patients with ARID1A-clonal expression deficiency tumor, the infiltration patterns of three immune cell types were comparable to those in ARID1A-proficient patients. Heterogeneous ARID1A expression is related to the different prognosis and immunotherapythe efficacy in CRC patients. CONCLUSION: Heterogeneous ARID1A expression is accompanied by a different immune landscape. CRC patients with ARID1A-clonal expression deficiency do not benefit from the treatment of immune checkpoint inhibitors (ICIs).

The effect of selenium on the proliferation of bovine endometrial epithelial cells in a lipopolysaccharide-induced damage model.

BACKGROUND: Endometritis is a common bovine postpartum disease. Rapid endometrial repair is beneficial for forming natural defense barriers and lets cows enter the next breeding cycle as soon as possible. Selenium (Se) is an essential trace element closely related to growth and development in animals. This study aims to observe the effect of Se on the proliferation of bovine endometrial epithelial cells (BEECs) induced by lipopolysaccharide (LPS) and to elucidate the possible underlying mechanism. RESULTS: In this study, we developed a BEECs damage model using LPS. Flow cytometry, cell scratch test and EdU proliferation assay were used to evaluate the cell cycle, migration and proliferation. The mRNA transcriptions of growth factors were detected by quantitative reverse transcription-polymerase chain reaction. The activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/ß-catenin pathways were detected by Western blotting and immunofluorescence. The results showed that the cell viability and BCL-2/BAX protein ratio were significantly decreased, and the cell apoptosis rate was significantly increased in the LPS group. Compared with the LPS group, Se promoted cell cycle progression, increased cell migration and proliferation, and significantly increased the gene expressions of TGFB1, TGFB3 and VEGFA. Se decreased the BCL-2/BAX protein ratio, promoted ß-catenin translocation from the cytoplasm to the nucleus and activated the Wnt/ß-catenin and PI3K/AKT signaling pathways inhibited by LPS. CONCLUSIONS: In conclusion, Se can attenuate LPS-induced damage to BEECs and promote cell proliferation and migration in vitro by enhancing growth factors gene expression and activating the PI3K/AKT and Wnt/ß-catenin signaling pathways.

Staphylococcus pseudintermedius induces pyroptosis of canine corneal epithelial cells by activating the ROS-NLRP3 signalling pathway.

Staphylococcus pseudintermedius (S. pseudintermedius) is a common pathogen that causes canine corneal ulcers. However, the pathogenesis remained unclear. In this study, it has been demonstrated that S. pseudintermedius invaded canine corneal epithelial cells (CCECs) intracellularly, mediating oxidative damage and pyroptosis by promoting the accumulation of intracellular reactive oxygen species (ROS) and activating the NLRP3 inflammasome. The canine corneal stroma was infected with S. pseudintermedius to establish the canine corneal ulcer model in vivo. The intracellular infectious model in CCECs was established in vitro to explore the mechanism of the ROS - NLRP3 signalling pathway during the S. pseudintermedius infection by adding NAC or MCC950. Results showed that the expression of NLRP3 and gasdermin D (GSDMD) proteins increased significantly in the infected corneas (p < 0.01). The intracellular infection of S. pseudintermedius was confirmed by transmission electron microscopy and immunofluorescent 3D imaging. Flow cytometry analysis revealed that ROS and pyroptosis rates increased in the experimental group in contrast to the control group (p < 0.01). Furthermore, NAC or MCC950 inhibited activation of the ROS - NLRP3 signalling pathway and pyroptosis rate significantly, by suppressing pro-IL-1ß, cleaved-IL-1ß, pro-caspase-1, cleaved-caspase-1, NLRP3, GSDMD, GSDMD-N, and HMGB1 proteins. Thus, the research confirmed that oxidative damage and pyroptosis were involved in the process of CCECs infected with S. pseudintermedius intracellularly by the ROS - NLRP3 signalling pathway. The results enrich the understanding of the mechanisms of canine corneal ulcers and facilitate the development of new medicines and prevention measures.

Autophagy activation alleviates the LPS-induced inflammatory response in endometrial epithelial cells in dairy cows.

PROBLEM: Endometritis is a common disease that affects dairy cow reproduction. Autophagy plays a vital role in cellular homeostasis and modulates inflammation by regulating interactions with innate immune signaling pathways. However, little is known about the regulatory relationship between autophagy and inflammation in bovine endometrial epithelial cells (BEECs). Thus, we aimed to determine the role of autophagy in the inflammatory response in BEECs. METHODS OF STUDY: In the present study, the expression levels of proinflammatory cytokines were measured by quantitative real-time polymerase chain reaction. Changes in the nuclear factor-κB (NF-κB) pathway and autophagy were determined using immunoblotting and immunocytochemistry. The induction of autophagosome formation was visualized by transmission electron microscopy. RESULTS: Our results demonstrated that autophagy activation was inhibited in LPS-treated BEECs, while activation of the NF-κB pathway and the mRNA expression of IL-6, IL-8, and TNF-α were increased. Furthermore, blocking autophagy with the inhibitor chloroquine increased NF-κB signaling pathway activation and proinflammatory factor expression in LPS-treated BEECs. Conversely, activation of autophagy with the agonist rapamycin inhibited the NF-κB signaling pathway and downregulated proinflammatory factors. CONCLUSIONS: These data indicated that LPS-induced inflammation was related to the inhibition of autophagy in BEECs. Thus, the activation of autophagy may represent a novel therapeutic strategy for eliminating inflammation in BEECs.

The effect and mechanism of selenium supplementation on the proliferation capacity of bovine endometrial epithelial cells exposed to lipopolysaccharide in vitro under high cortisol background.

Bovine endometritis severely inhibits uterine repair and causes considerable economic loss. Besides, parturition-induced high cortisol levels inhibit immune function, reduce cell proliferation, and further inhibit tissue repair. Selenium (Se) is an essential trace element for animals to maintain normal physiological function and has powerful antioxidant functions. This study investigated whether Se supplementation reduces endometrial damage and promotes tissue repair in cows with endometritis under stress and explored the underlying mechanism. Primary bovine endometrial epithelial cells were isolated and purified from healthy cows. The cells were treated with different combinations of lipopolysaccharide (LPS), cortisol, and various concentrations of Se. Data showed that LPS stimulation inhibited cell proliferation and increased cell apoptosis. High levels of cortisol further exacerbated these effects. Flow cytometry, scratch wound healing tests, and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays showed that Se supplementation promoted cell cycle progression, cell migration, and cell proliferation in the presence of LPS and cortisol. The quantitative PCR results showed that the expression of related growth factors was increased after Se supplementation. After administering various inhibitors, we further demonstrated that Se supplementation decreased the activity of glycogen synthetase kinase 3ß (GSK-3ß) through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway to reduce the degradation of ß-catenin except the Wnt signal to promote cell proliferation. In conclusion, Se supplementation attenuated the cell damage induced by LPS at high cortisol levels and increased cell proliferation to promote uterine repair by elevating the mRNA expression of TGFB3 and VEGFA and activating the PI3K/AKT/GSK-3ß/ß-catenin signaling pathway.

After parturition, endometritis is a common bovine disease, which hinders endometrial repair and reduces bovine economic value. Besides, parturition-induced high cortisol levels cause immunosuppression, aggravate infection, and further inhibit cell proliferation and tissue repair. As an essential trace element, adding selenium to feed helps to maintain the normal physiological function of animals. This study developed a cellular model using lipopolysaccharide (LPS) and cortisol to simulate cows with endometritis in stress conditions. The results showed that Se supplementation attenuated bovine endometrial epithelial cell damage and promoted their proliferation in the presence of LPS and high cortisol levels, which are positively correlated with the concentration of Se. Besides, this study proved another molecular mechanism for Se to regulate ß-catenin except for the Wnt signal by affecting the ß-catenin degradation pathway.

Selenomethionine Inhibits NF-κB-mediated Inflammatory Responses of Bovine Mammary Epithelial Cells Caused by Klebsiella pneumoniae by Increasing Autophagic Flux.

Klebsiella pneumoniae (K. pneumoniae) is one of the major pathogens causing bovine clinical mastitis. Autophagy maintains cellular homeostasis and resists excessive inflammation in eukaryotic organisms. Selenomethionine (Se-Met) is commonly used as a source of selenium supplementation for dairy cows. This study aimed to investigate the effects of Se-Met on inflammatory responses mediated by nuclear factor-kappa B (NF-κB) through autophagy. We infected bovine mammary epithelial cell line (MAC-T) with K. pneumoniae and examined the expression of autophagy-related proteins and changes in autophagic vesicles, LC3 puncta, and autophagic flux at various intervals. The results showed that K. pneumoniae activated the early-stage autophagy of MAC-T cells. The levels of LC3-II, Beclin1, and ATG5, as well as the number of LC3 puncta and autophagic vesicles, increased after 2 h post-treatment. However, the late-stage autophagic flux was blocked. Furthermore, the effect of autophagy on NF-κB-mediated inflammation was investigated with different autophagy levels. The findings showed that enhanced autophagy inhibited the K. pneumoniae-induced inflammatory responses of MAC-T cells. The opposite results were found with the inhibition of autophagy. Finally, we examined the effect of Se-Met on NF-κB-mediated inflammation based on autophagy. The results indicated that Se-Met alleviated K. pneumoniae-induced autophagic flux blockage, inhibited NF-κB-mediated inflammation, and decreased the adhesion of K. pneumoniae to MAC-T cells. The inhibitory effect of Se-Met on NF-κB-mediated inflammation could be partially blocked by the autophagy inhibitor chloroquine (CQ). Overall, Se-Met attenuated K. pneumoniae-induced NF-κB-mediated inflammatory responses by enhancing autophagic flux.

EDARADD promotes colon cancer progression by suppressing E3 ligase Trim21-mediated ubiquitination and degradation of Snail.

Tumor cell migration, specifically epithelial-mesenchymal transition (EMT), serves as a key contributor to treatment failure in colon cancer patients. However, the limited comprehension of its genetic and biological aspects presents challenges for its investigation. EDAR-associated death domain (EDARADD), an important TNFR superfamily member, is elevated in colon cancer. However, it remains unclear about the exact role of EDARADD in the progression of colon cancer metastasis. In this study, we initially demonstrated that both protein and mRNA levels of EDDARADD are elevated in colon cancer tissues and cells, associated with reduced overall survival. Furthermore, functional experiments demonstrated that EDARADD promotes colon cancer cell proliferation and participates in EMT both in vitro and vivo. Mechanistically, Co-IP verified EDARADD could stabilize Snail1 by interacting with E3 ubiquitin ligase Trim21 to inhibit ubiquitination of Snail1. Interestingly, RNA-seq and ubiquitination assay revealed EDARADD's dual downregulation of Trim21 expression at the translational level via Cul1-mediated ubiquitin degradation, and at the transcriptional level through PPARa regulation. Moreover, EDARADD activates NF-κB signaling and experiences feedback transcriptional regulation by p65. In conclusion, this study highlights the signal pathway of EDARADD-PPARa-Trim21-Snail1-EMT and a feedback regulation of NF-κB signaling on EDARADD, which indicated EDARADD as an emerging therapeutic target for colon cancer.

The Effect of Selenium on Endometrial Repair in Goats with Endometritis at High Cortisol Levels.

Endometritis is a common postpartum disease of female animals that causes significant losses to the goat industry. High levels of cortisol induced by various stresses after delivery severely inhibit innate immunity and tissue repair. The repair ability of the endometrium is closely related to the reproductive performance of goats. Selenium (Se) is an essential trace element in animals that has powerful antioxidant and immunity-enhancing functions. In this study, we established a goat model of endometritis at high cortisol (Hydrocortisone) levels to investigate the effect of Se (supplement additive) on endometrial repair. The results showed that the clinical symptoms, %PMN in uterine secretions, morphological endometrial damage, and the gene expression of BAX were reduced in the goats with Se supplementation compared with those in the model group. Se increased the gene expression of BCL2, VEGFA, TGFB1, and PCNA and activated the PI3K/AKT and Wnt/ß-catenin signaling pathways in goats with Se supplementation. In conclusion, Se reduced the inflammatory response, increased the proliferation, and decreased the apoptosis of endometrial cells to promote endometrial tissue repair in goats with endometritis at high cortisol levels. It probably achieved this effect of promoting repair by activating the Wnt/ß-catenin and PI3K/AKT pathways and affecting the gene expression of VEGFA, TGFB1, PCNA, BCL2, and BAX.

ACVRL1 drives resistance to multitarget tyrosine kinase inhibitors in colorectal cancer by promoting USP15-mediated GPX2 stabilization.

BACKGROUND: Multitarget tyrosine kinase inhibitors (mTKIs) such as Regorafenib and Sorafenib have already been approved for the treatment of many solid tumours. However, the efficacy of mTKIs in colorectal cancer (CRC) is limited; the underlined mechanism remains largely elusive. Our study was aimed to find out the resistance mechanism of mTKIs in CRC. METHODS: RNA sequencing was used to identify the expression of Activin A receptor-like type 1 (ACVRL1) under the treatment of mTKIs. Gain/loss-of-function experiments were performed to assess the biological function of ACVRL1 in resistance to mTKIs. The underlying mechanisms of ACVRL1-mediated mTKI resistance were investigated by using liquid chromatography-mass spectrometry assays (LC-MS), co-immunoprecipitation assays (Co-IP), chromatin immunoprecipitation assays, ubiquitination assays, dual luciferase reporter assays, etc. RESULTS: RNA sequencing identified the activation of ACVRL1 under the treatment of mTKIs in CRC cells. ACVRL1 knockdown and overexpression significantly affects the sensitivity of CRC cells to mTKIs both in vitro and vivo. Mechanistically, we found the ß-catenin/TCF-1-KCNQ1OT1/miR-7-5p axis mediated the activation of ACVRL1. Furthermore, LC-MS assays indicated the interaction between ACVRL1 and glutathione peroxidase 2(GPX2) protein. IP assay defined ACVRL1 truncation (282-503aa) could be responsible for interacting with GPX2, and rescue experiments with ACVRL1 truncations confirmed the importance of this interaction in driving mTKI resistance. Co-IP assays confirmed that ACVRL1 associates with ubiquitin-specific peptidase 15(USP15) which directly deubiquinates GPX2 at the K187(K, lysine) site, leading to the accumulation of GPX2 protein. Rescue experiments performed with the lysine mutants in GPX2 CRISPR knockout cell model confirmed the importance of GPX2 K187 mutant. As a result, the increased ROS clearance and decreased cell apoptosis eventually lead to mTKI resistance in CRC. CONCLUSIONS: Our results demonstrate that the Wnt/ß-catenin/KCNQ1OT1/miR-7-5p/ACVRL1/GPX2 biological axis plays a vital role in CRC, targeting which may be an effective approach for overcoming mTKI resistance.

Selenium suppressed the LPS-induced inflammation of bovine endometrial epithelial cells through NF-κB and MAPK pathways under high cortisol background.

The bovine uterus is susceptible to infection, and the elevated cortisol level due to stress are common in cows after delivery. The essential trace element selenium plays a pivotal role in the antioxidant and anti-inflammatory defence system of body. This study investigated whether selenium supplementation protected endometrial cells from inflammation in the presence of high-level cortisol. The primary bovine endometrial epithelial cells were subjected to Escherichia coli lipopolysaccharide to establish cellular inflammation model. The gene expression of inflammatory mediators and proinflammatory cytokines was measured by quantitative PCR. The key proteins of NF-κB and MAPK signalling pathways were detected by Western blot and immunofluorescence. The result showed that pre-treatment of Na2 SeO3 (1, 2 and 4 µΜ) decreased the mRNA expression of proinflammatory genes, inhibited the activation of NF-κB and suppressed the phosphorylation of extracellular signal-regulated kinase, P38MAPK and c-Jun N-terminal kinase. This inhibition of inflammation was more apparent in the presence of high-level cortisol (30 ng/mL). These results indicated that selenium has an anti-inflammatory effect, which is mediated via NF-κB and MAPK signalling pathways and is augmented by cortisol in bovine endometrial epithelial cells.

China special issue on gastrointestinal tumors-Regulatory-immunoscore-A novel indicator to guide precision adjuvant chemotherapy in colorectal cancer.

Novel biomarkers are essential to improve the treatment efficacy and overall survival of stage II and III colorectal cancer (CRC), allowing for personalized treatment decisions. Here, the densities of CD8+ and FOXP3+ T cells in the tumor and invasive margin were processed by immunohistochemistry and digital pathology to form a scoring system named regulatory-Immunoscore (RIS). Cox proportional hazards regression models were used to determine the risk factors associated with time to recurrence. Harrell's concordance index and the time-dependent area under the curve were used to assess model performance. A total of 1213 stage I-III DNA mismatch repair-proficient colorectal cancer (pMMR CRC) patients were randomly assigned to a training set (n = 642) and a validation set (n = 571). From the Cox multivariable analysis, the association of RIS with survival was independent of patient age, sex and anatomy-based tumor risk parameters (P < .0001). For stage II patients, chemotherapy was significantly associated with better recurrence time in patients with low (95% confidence interval [CI]: 0.11-0.54, P = .001) and intermediate (95% CI = 0.25-0.57, P < .001) RIS values. In stage III patients treated with adjuvant chemotherapy, a treatment duration of 6 or more months was significantly associated with better recurrence time in patients with intermediate RIS values (95% CI = 0.38-0.90, P = .016) when compared with duration under 6 months. Therefore, these findings suggest that RIS is reliable for predicting recurrence risk and treatment responsiveness for patients with stage I-III pMMR CRC.

Meloxicam inhibited oxidative stress and inflammatory response of LPS-stimulated bovine endometrial epithelial cells through Nrf2 and NF-κB pathways.

Meloxicam is a selective cyclooxygenase-2 inhibitor and has been widely used in combination with antibiotics to alleviate uterine inflammation and provide analgesia in postpartum cows. Studies have shown that meloxicam has antioxidant and anti-inflammatory effects. However, the link between meloxicam and uterine inflammation and oxidative stress in dairy cows has not been studied. The purpose of this study was to research the effects of meloxicam (0.5 or 5 µM) on oxidative stress and inflammatory response of primary bovine endometrial epithelial cells (BEEC) stimulated by Escherichia coli lipopolysaccharide (1 µg/mL LPS). As a result, LPS stimulated the production of oxidative stress markers and the expression of inflammatory factors, accompanied by a decrease in the activity and the gene transcription of antioxidant enzymes. Co-treatment of meloxicam and LPS reduced the content of oxidative stress markers and the mRNA levels of the pro-inflammatory genes, and improved antioxidant enzyme activities and the corresponding gene expression as compared with the cells treated with LPS alone. Meloxicam attenuated the inhibitory effect of the Nrf2 pathway and the phosphorylation levels of p65 and IκBα caused by LPS. In conclusion, meloxicam alone had no effect on BEEC, but prevented oxidative stress and inflammatory response in LPS-stimulated BEEC.

PINK1/Parkin-mediated mitophagy enhances the survival of Staphylococcus aureus in bovine macrophages.

Mitochondria are cellular organelles that are involved in various metabolic processes, and damage to mitochondria can affect cell health and even lead to disease. Mitophagy is a mechanism by which cells selectively wrap and degrade damaged mitochondria to maintain cell homeostasis. However, studies have not focused on whether mitophagy is involved in the occurrence of Staphylococcus aureus (S. aureus)-induced mastitis in dairy cows. Here, we found that S. aureus infection of bovine macrophages leads to oxidative damage and mitochondria damage. The expression of LC3, PINK1 and Parkin was significantly increased after intracellular infection. We observed changes in the morphology of mitochondria and the emergence of mitochondrial autolysosomes in bovine macrophages by transmission electron microscopy and found that enhanced mitophagy promoted bacterial proliferation in the cell. In conclusion, this study demonstrates that S. aureus infection of bovine macrophages induces mitophagy through the PINK1/Parkin pathway, and this mechanism is used by the bacteria to avoid macrophage-induced death. These findings provide new ideas and references for the prevention and treatment of S. aureus infection.

Epigenetic-related gene mutations serve as potential biomarkers for immune checkpoint inhibitors in microsatellite-stable colorectal cancer.

Background: Combination therapy with immune checkpoint inhibitors (ICIs) may benefit approximately 10-20% of microsatellite-stable colorectal cancer (MSS-CRC) patients. However, there is a lack of optimal biomarkers. This study aims to understand the predictive value of epigenetic-related gene mutations in ICIs therapy in MSS-CRC patients. Methods: We analyzed DNA sequences and gene expression profiles from The Cancer Genome Atlas (TCGA) to examine their immunological features. The Harbin Medical University Cancer Hospital (HMUCH) clinical cohort of MSS-CRC patients was used to validate the efficacy of ICIs in patients with epigenetic-related gene mutations (Epigenetic_Mut). Results: In TCGA, 18.35% of MSS-CRC patients (78/425) had epigenetic-related gene mutations. The Epigenetic_Mut group had a higher tumor mutation burden (TMB) and frameshift mutation (FS_mut) rates. In all MSS-CRC samples, Epigenetic_Mut was elevated in the immune subtype (CMS1) and had a strong correlation with immunological features. Epigenetic_Mut was also associated with favorable clinical outcomes in MSS-CRC patients receiving anti-PD-1-based therapy from the HMUCH cohort. Using immunohistochemistry and flow cytometry, we demonstrated that Epigenetic_Mut samples were associated with increased anti-tumor immune cells both in tumor tissues and peripheral blood. Conclusion: MSS-CRC patients with epigenetic regulation impairment exhibit an immunologically active environment and may be more susceptible to treatment strategies based on ICIs.

MCC950 inhibits the inflammatory response and excessive proliferation of canine corneal stromal cells induced by Staphylococcus pseudintermedius.

BACKGROUND: The stromal layer is the thickest layer of the cornea, and corneal stromal cells play an important role in the inflammatory response and wound repair. This study investigated the effect of MCC950, an inhibitor of NLRP3 inflammasome, on the inflammatory response and proliferation of canine corneal stromal cells (CCSCs) induced by Staphylococcus pseudintermedius (S. pseudintermedius). METHODS: CCSCs were pretreated with MCC950 and infected with S. pseudintermedius. The phosphorylation of p65, IκBα, PI3K, and AKT and the expression of NLRP3, caspase-1 p20, cleaved IL-1ß, ASC, ß-catenin, c-Myc, and CyclinD1 were detected by western blotting. The expression of inflammatory factors (IL-1ß, IL-6, IL-8, IL-18, and TNF-α) and growth factors (EGF, FGF, TGF-ß1, VEGF, and CTGF) were measured by RT-PCR. The levels of MDA content and LDH activity were detected by an assay kit. The cell cycle was detected by flow cytometry. RESULTS: MCC950 down-regulated the phosphorylation of p65, IκBα, PI3K, and AKT and decreased the expression of NLRP3, caspase-1 p20, cleaved IL-1ß, ASC, ß-catenin, c-Myc, and CyclinD1 compared to those in the S. pseudintermedius infection group (p < 0.05). MCC950 significantly inhibited the expression of inflammatory factors (IL-1ß, IL-6, IL-8, IL-18, and TNF-α) and growth factors (EGF, FGF, TGF-ß1, VEGF, and CTGF) induced by S. pseudintermedius (p < 0.01). Compared to the S. pseudintermedius infection group, the MDA content and LDH activity of CCSCs were significantly decreased after treatment with MCC950 (p < 0.01). CONCLUSION: MCC950 attenuates S. pseudintermedius-induced inflammatory responses in CCSCs. At the same time, MCC950 can inhibit excessive proliferation of cells, which is beneficial for alleviating corneal fibrosis healing.

PINK1/parkin-mediated mitophagy alleviates Staphylococcus aureus-induced NLRP3 inflammasome and NF-κB pathway activation in bovine mammary epithelial cells.

Staphylococcus aureus (S. aureus) is known to induce chronic and persistent bovine mammary infection, which affects milk quality and leads to premature culling. The ability of S. aureus to invade mammalian cells protects it from clearance by the immune system. Mitophagy is important in cell homeostasis, and can be utilized by pathogens for immune escape. However, mitophagy's role in S. aureus-associated bovine mastitis remains unclear. Here, S. aureus infection induced mitophagy and enhanced mitochondrial translocation of parkin in MAC-T cells. After mitophagy inhibition by Mdivi-1 treatment or PTEN-induced putative kinase 1 (PINK1) silencing in MAC-T cells infected with S. aureus, NOD-like receptor protein 3 (NLRP3) inflammasome activation and p65 and IκBα phosphorylation were increased. Meanwhile, PINK1 overexpression had the opposite effects. In addition, NLRP3 inflammasome overactivation and enhanced p65 and IκBα phosphorylation caused by PINK1 silencing were reversed by MitoTEMPO. Furthermore, PINK1/parkin-mediated mitophagy promoted S. aureus survival and contributed to persistent S. aureus infection. These findings provide new insights into S. aureus invasion in bovine mastitis.

Antibiotic resistance, biofilm formation, and virulence factors of isolates of staphylococcus pseudintermedius from healthy dogs and dogs with keratitis.

Canine bacterial keratitis is a common infection that can potentially threaten vision. Staphylococcus pseudintermedius (S. pseudintermedius) is an opportunistic pathogen that has been isolated from the canine conjunctival sac but there are only a few reports on the role of this bacterium in canine keratitis. This study focused on the distribution rate of S. pseudintermedius in the canine conjunctival sac, and the antibiotic resistance, biofilm-producing ability, and dissemination of virulence factors in strains of S. pseudintermedius isolated from healthy dogs and dogs with keratitis. The study included 35 healthy dogs and 40 dogs with keratitis. Bacterial species were confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Strains of S. pseudintermedius were screened for resistance against nine different antibiotics by the Kirby-Bauer assay. The ability to produce biofilm was investigated by microtiter plate assay (MtP) and amplification of icaA and icaD genes. Virulence factors in the strains were also evaluated. A total of 132 aerobic bacteria were isolated from the 119 samples in the study. Among them, 67 bacterial strains were isolated from 70 eyes of healthy dogs, and 65 bacterial strains were isolated from 49 eyes of dogs with keratitis. The prevalence of S. pseudintermedius, which was the most frequent bacterial isolate in both the groups, was 20.9% in the healthy group and 23.08% in the keratitis group. Most of the isolates of S. pseudintermedius were sensitive to rifampin (96.6%), oxacillin (100%), and neomycin (96.6%), and resistant to tetracycline (96.6%). Virulence factors such as lip (96.6%), hlgB (96.6%), and hlgA (96.6%) were found in most of the isolates, and 89.66% of isolates were classed as biofilm producers. In conclusion, S. pseudintermedius was the common bacterium in the conjunctivital sac of the healthy dogs and dogs with keratitis in Yangzhou, China, and the presence of virulence factors and biofilm-formation ability were high in the strains isolated from the dogs with keratitis.

Selenomethionine alleviates NF-κB-mediated inflammation in bovine mammary epithelial cells induced by Escherichia coli by enhancing autophagy.

Autophagy is crucial for the maintenance of homeostasis under stimuli related to infection. Selenium (Se) plays variable roles in defence against infection and Selenomethionine (Se-Met) is a common Se supplementation. This study aimed to understand whether Se-Met could regulate the nuclear factor-kappa B (NF-κB) signaling pathway through autophagy. Mammary alveolar cell-T (MAC-T) was challenged with Escherichia coli (E. coli). Western blotting and real-time quantitative PCR (RT-qPCR) were used to detect the protein expression and mRNA expression of cytokines. Immunofluorescence assays were performed to observe the expression of intracellular LC3. The results showed that E. coli inhibited autophagy by decreasing the LC3-Ⅱ protein levels, and the Atg5 and Beclin1 protein levels were increased after 4 h. Infection also decreased the number of LC3 puncta. E. coli increased the phosphorylation of p65 and IκBα protein. Concomitantly, the levels of interleukin (IL)-1ß, IL-6, IL-8 and tumour necrosis factor (TNF)-α mRNA increased at 3 and 4 h post-infection. We further explored the regulatory role of autophagy on NF-κB-mediated inflammation with autophagy modulators and shAtg5. The results indicated that the autophagy activator reduced the phosphorylation of p65 and IκBα and the mRNA expression of IL-1ß, IL-6, IL-8 and TNF-α. Additionally, activating autophagy weakened the adhesion to MAC-T of E. coli. Autophagy inhibitors exacerbated NF-κB-mediated inflammation and strengthened the adhesion of E. coli to cells. We then examined the effects of Se-Met on NF-κB-mediated inflammation through autophagy. The data suggested that Se-Met enhanced LC3-II expression, inhibited the E. coli-induced phosphorylation of p65 and IκBα, and suppressed the adhesion ability of E. coli to MAC-T and that the effects of Se-Met in attenuating NF-κB-mediated inflammation were partially blocked by an autophagy inhibitor. In summary, Se-Met alleviated NF-κB-mediated inflammation induced by E. coli by enhancing autophagy in bovine mammary epithelial cells.

Selenomethionine protected BMECs from inflammatory injury and oxidative damage induced by Klebsiella pneumoniae by inhibiting the NF-κB and activating the Nrf2 signaling pathway.

Klebsiella pneumoniae (K. pneumoniae) is one of the main environmental pathogens causing bovine mastitis. The incidence of bovine mastitis caused by K. pneumoniae is increasing worldwide. Selenium is an essential trace element that has multiple physiological functions, such as antioxidant and anti-inflammatory activities. Therefore, this study aimed to verify whether selenomethionine (SeMet) could contribute to alleviating the inflammatory injury and oxidative damage induced by K. pneumoniae. Bovine mammary epithelial cells were cultured in vitro and pretreated with 4 µM SeMet before being infected with K. pneumoniae. Western blot analysis was used to detect the expression of the related proteins in the NF-κB and Nrf2 signaling pathways. The gene expression levels of IL-1ß, IL-6, IL-8, TNF-α, Nrf2, Keap1, NQO-1 and HO-1 were detected using RT-qPCR. The levels of MDA, GSH-PX, SOD, CAT and T-AOC were detected by commercial assay kits. Flow cytometry was used to determine the level of intracellular ROS, and immunofluorescence was used to detect the nuclear localization of Nrf2 protein. Briefly, SeMet downregulated the phosphorylation levels of IκBα and p65 proteins and the gene expression levels of IL-1ß, IL-6, IL-8 and TNF-α were also decreased. Moreover, the protein and gene expression levels of Nrf2, NQO-1 and HO-1 were upregulated, and the nuclear expression of Nrf2 protein was also promoted, which enhanced the activity of antioxidant enzymes. In conclusion, SeMet protected BMECs from inflammatory injury and oxidative stress induced by K. pneumoniae by inhibiting the NF-κB and activating the Nrf2 signaling pathway.