Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy.

Intravesical treatment using either reovirus or natural killer (NK) cells serves as an efficient strategy for the treatment of bladder cancer cells (BCCs); however, corresponding monotherapies have often shown modest cytotoxicity. The potential of a locoregional combination using high-dose reovirus and NK cell therapy in an intravesical approach has not yet been studied. In this study, we evaluated the effectiveness of reoviruses and expanded NK cells (eNK) as potential strategies for the treatment of bladder cancer. The anti-tumor effects of mono-treatment with reovirus type 3 Dearing strain (RC402 and RP116) and in combination with interleukin (IL)-18/-21-pretreated eNK cells were investigated on BCC lines (5637, HT-1376, and 253J-BV) using intravesical therapy to simulate in vitro model. RP116 and IL-18/-21-pretreated eNK cells exhibited effective cytotoxicity against grade 1 carcinoma (5637 cells) when used alone, but not against HT-1376 (grade 2 carcinoma) and 253J-BV cells (derived from a metastatic site). Notably, combining RP116 with IL-18/-21-pretreated eNK cells displayed effective cytotoxicity against both HT-1376 and 253J-BV cells. Our findings underscore the potential of a combination therapy using reoviruses and NK cells as a promising strategy for treating bladder cancer.

Gegen Qinlian decoction ameliorates TNBS-induced ulcerative colitis by regulating Th2/Th1 and Tregs/Th17 cells balance, inhibiting NLRP3 inflammasome activation and reshaping gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese herbal medicine Gegen Qinlian Decoction (GQD) has been clinically shown to be an effective treatment of ulcerative colitis (UC) in China. However, the underlying mechanism of GQD's anti-ulcerative colitis properties and its effect on gut microbiota still deserve further exploration. AIM OF THE STUDY: This study observed the regulatory effects of GQD on Th2/Th1 and Tregs/Th17 cells balance, the NOD-like receptor family pyrin domain containing 3 (NLRP3) infammasome and gut microbiota in TNBS-induced UC in BALB/c mice. MATERIALS AND METHODS: 61 main chemical compounds in the GQD were determined by UPLC-Q-TOF/MS. The UC BALB/c model was established by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and GQD was orally administered at low and high dosages of 2.96 and 11.83 g/kg/day, respectively. The anti-inflammatory effects of GQD for ulcerative colitis were evaluated by survival rate, body weight, disease activity index (DAI) score, colonic weight and index, spleen index, hematoxylin-eosin (HE) staining and histopathological scores. Flow cytometry was used to detect the percentage of CD4, Th1, Th2, Th17 and Tregs cells. The levels of Th1-/Th2-/Th17-/Tregs-related inflammatory cytokines and additional proinflammatory cytokines (IL-1ß, IL-18) were detected by CBA, ELISA, and RT-PCR. The expressions of GATA3, T-bet, NLRP3, Caspase-1, IL-Iß, Occludin and Zonula occludens-1 (ZO-1) on colon tissues were detected by Western blot and RT-PCR. Transcriptome sequencing was performed using colon tissue and 16S rRNA gene sequencing was performed on intestinal contents. Fecal microbiota transplantation (FMT) was employed to assess the contribution of intestinal microbiota and its correlation with CD4 T cells and the NLRP3 inflammasome. RESULTS: GQD increased the survival rate of TNBS-induced UC in BALB/c mice, and significantly improved their body weight, DAI score, colonic weight and index, spleen index, and histological characteristics. The intestinal barrier dysfunction was repaired after GQD administration through promoting the expression of tight junction proteins (Occludin and ZO-1). GQD restored the balance of Th2/Th1 and Tregs/Th17 cells immune response of colitis mice, primarily inhibiting the increase in Th2/Th1 ratio and their transcription factor production (GATA3 and T-bet). Morever, GQD changed the secretion of Th1-/Th2-/Th17-/Tregs-related cytokines (IL-2, IL-12, IL-5, IL-13, IL-6, IL-10, and IL-17A) and reduced the expressions of IL-1ß, IL-18. Transcriptome results suggested that GQD could also remodel the immune inflammatory response of colitis by inhibiting NOD-like receptor signaling pathway, and Western blot, immunohistochemistry and RT-PCR further revealed that GQD exerted anti-inflammatory effects by inhibiting the NLRP3 inflammasome, such as down-regulating the expression of NLRP3, Caspase-1 and IL-1ß. More interestingly, GQD regulated gut microbiota dysbiosis, suppressed the overgrowth of conditional pathogenic gut bacteria like Helicobacter, Proteobacteria, and Mucispirillum, while the probiotic gut microbiota, such as Lactobacillus, Muribaculaceae, Ruminiclostridium_6, Akkermansia, and Ruminococcaceae_unclassified were increased. We further confirmed that GQD-treated gut microbiota was sufficient to relieve TNBS-induced colitis by FMT, involving the modulation of Th2/Th1 and Tregs/Th17 balance, inhibition of NLRP3 inflammasome activation, and enhancement of colonic barrier function. CONCLUSIONS: GQD might alleviate TNBS-induced UC via regulating Th2/Th1 and Tregs/Th17 cells Balance, inhibiting NLRP3 inflammasome and reshaping gut microbiota, which may provide a novel strategy for patients with colitis.

TRIM6 Promotes ROS-Mediated Inflammasome Activation and Pyroptosis in Renal Tubular Epithelial Cells via Ubiquitination and Degradation of GPX3 Protein.

BACKGROUND: Pyroptosis is a critical form of cell death during the development of chronic kidney disease (CKD). Tripartite motif 6 (TRIM6) is an E3-ubiquitin ligase that participates in the progression renal fibrosis (RF). The aim of this study was to investigate the roles of TRIM6 and Glutathione peroxidase 3 (GPX3) in oxidative stress-induced inflammasome activation and pyroptosis in Ang-II treated renal tubular epithelial cells. METHODS: To study its role in RF, TRIM6 expression was either reduced or increased in human kidney-2 (HK2) cells using lentivirus, and Ang-II, NAC and BMS-986299 were served as reactive oxygen species (ROS) inducer, ROS scavenger and NLRP3 agonist respectively. Pyroptosis and mitochondrial ROS were measured by flow cytometry. The levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were determined using commercial kits, while the levels of IL-1ß, IL-18, IL-6, and tumor necrosis factor-α (TNF-α) were determined by Enzyme-Linked Immunosorbent Assay (ELISA). Co-immunoprecipitation (Co-IP) assay was used to evaluate the interaction between TRIM6 and GPX3. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to measure mRNA and protein expression, respectively. RESULTS: Treatment with Angiotensin II (Ang II) increased the protein and mRNA levels of TRIM6 in HK2 cells. Ang II also increased mitochondrial ROS production and the malondialdehyde (MDA) level, but decreased the levels of GSH and SOD. In addition, Ang II enhanced HK2 cell pyroptosis, increased the levels of IL-1ß, IL-18, IL-6, and TNF-α, and promoted the expression of active IL-1ß, NLRP3, caspase-1, and GSDMD-N proteins. These effects were reversed by knockdown of TRIM6 and by treatment with N-acetyl-L-cysteine (NAC), a ROS scavenger. BMS-986299, an NLRP3 agonist treatment, did not affect ROS production in HK2 cells exposed to Ang II combined with NAC, but cell pyroptosis and inflammation were aggravated. Moreover, the overexpression of TRIM6 in HK2 cells resulted in similar effects to Ang II. NAC and GPX3 overexpression in HK2 cells could reverse ROS production, inflammation, and pyroptosis induced by TRIM6 overexpression. TRIM6 overexpression decreased the GPX3 protein level by promoting its ubiquitination, without affecting the GPX3 mRNA level. Thus, TRIM6 facilitates GPX3 ubiquitination, contributing to increased ROS levels and pyroptosis in HK2 cells. CONCLUSIONS: TRIM6 increases oxidative stress and promotes the pyroptosis of HK2 cells by regulating GPX3 ubiquitination. These findings could contribute to the development of novel drugs for the treatment of RF.

Elucidating the mechanisms underlying astrocyte-microglia crosstalk in hippocampal neuroinflammation induced by acute diquat exposure.

The transition from paraquat (PQ) to diquat (DQ), both organic dication herbicides, in China has led to significant increases in the number of acute DQ poisoning cases. Case studies have shown that acute DQ poisoning resulted in injury to the central nervous system (CNS), but the mechanism underlying the injury remains to be explored. The present study aimed to investigate how DQ influenced purinergic signaling between astrocytes and microglia and whether extracellular ATP (eATP) was involved in promoting neuroinflammation induced by acute DQ toxicity through the activation of the P2X4/NLRP3 signaling pathway. We constructed a rat model of acute DQ toxicity to observe the pathological changes in hippocampal tissues after DQ exposure and measure the expression levels of IL-1ß and TNF-α in the hippocampal tissue. We also established an in vitro co-culture model of C6 astrocytes and BV-2 microglia using transwell chambers, measured the amount of eATP secreted into C6 astrocytes after DQ treatment, and assessed the inflammatory response and changes in the P2X4/NLRP3 signaling pathway in BV-2 microglia. The results showed that the neurons in the hippocampal tissue of rats exhibited loose arrangement, nuclear consolidation, and necrosis after DQ exposure, and IL-1ß and TNF-α levels were signification higher in the hippocampal tissue after DQ exposure. DQ exposure to the co-cultured cells induced an increase in ATP secretion from C6 astrocytes as well as a significant increase of P2X4, NLRP3, IL-1ß, and IL-18 expression in BV-2 microglia. In contrast, pretreatment of C6 astrocytes with apyrase (an ATP hydrolase) resulted in a significant decrease of P2X4, NLRP3, IL-1ß, and IL-18 expression in BV-2 microglia. Furthermore, inhibition of P2X4 expression in BV-2 microglia by transfection with si-P2X4 effectively reversed the increase of NLRP3, IL-1ß, and IL-18 in BV-2 microglia induced by DQ when co-cultured with C6 astrocytes. These results indicate that astrocytes can activate the P2X4/NLRP3 signaling pathway in microglia through the DQ-induced extracellular release of ATP to promote neuroinflammation in rat hippocampal tissue.

Ochratoxin A stimulates release of IL-1ß, IL-18 and CXCL8 from cultured human microglia.

Exposure to mycotoxins has been associated with the development of neuropsychiatric symptoms and Ochratoxin A (OTA) has emerged as one of the main mycotoxins associated with neurotoxicity. However, the mechanism via OTA exerts its neurotoxic effects is not well understood, especially the importance of activated microglia and their contribution to neuroinflammation. Here we report the effect of OTA on cultured immortalized human microglia-SV40, as compared to the effect of neurotensin (NT) and lipopolysaccharide (LPS) used as "positive" triggers. OTA (1, 10 and 100 nM for 24 hrs) stimulated microglia to release in the supernatant fluids statistically significant amounts of IL-1ß, IL-18 and CXCL8 assayed with ELISA. Preventing or inhibiting OTA-stimulated activation of microglia by luteolin could be an important way to limit mycotoxin-induced neuroinflammation and improve associated neuropsychiatric diseases.

Interleukin-18 in chronic pain: Focus on pathogenic mechanisms and potential therapeutic targets.

Chronic pain has been proven to be an independent disease, other than an accompanying symptom of certain diseases. Interleukin-18 (IL-18), a pro-inflammatory cytokine with pleiotropic biological effects, participates in immune modulation, inflammatory response, tumor growth, as well as the process of chronic pain. Compelling evidence suggests that IL-18 is upregulated in the occurrence of chronic pain. Antagonism or inhibition of IL-18 expression can alleviate the occurrence and development of chronic pain. And IL-18 is located in microglia, while IL-18R is mostly located in astrocytes in the spinal cord. This indicates that the interaction between microglia and astrocytes mediated by the IL-18/IL-18R axis is involved in the occurrence of chronic pain. In this review, we described the role and mechanism of IL-18 in different types of chronic pain. This review provides strong evidence that IL-18 is a potential therapeutic target in pain management.

M1 polarization of macrophages promotes stress-induced hair loss via interleukin-18 and interleukin-1ß.

Stress-induced hair loss is a prevalent health concern, with mechanisms that remain unclear, and effective treatment options are not yet available. In this study, we investigated whether stress-induced hair loss was related to an imbalanced immune microenvironment. Screening the skin-infiltrated immune cells in a stressed mouse model, we discovered a significant increase in macrophages upon stress induction. Clearance of macrophages rescues mice from stress-induced hair shedding and depletion of hair follicle stem cells (HFSCs) in the skin, demonstrating the role of macrophages in triggering hair loss in response to stress. Further flow cytometry analysis revealed a significant increase in M1 phenotype macrophages in mice under stressed conditions. In searching for humoral factors mediating stress-induced macrophage polarization, we found that the hormone Norepinephrine (NE) was elevated in the blood of stressed mice. In addition, in-vivo and in-vitro studies confirm that NE can induce macrophage polarization toward M1 through the ß-adrenergic receptor, Adrb2. Transcriptome, enzyme-linked immunosorbent assay (ELISA), and western blot analyses reveal that the NLRP3/caspase-1 inflammasome signaling and its downstream effector interleukin 18 (IL-18) and interleukin 1 beta (IL-1ß) were significantly upregulated in the NE-treated macrophages. However, inhibition of the NE receptor Adrb2 with ICI118551 reversed the upregulation of NLRP3/caspase-1, IL-18, and IL-1ß. Indeed, IL-18 and IL-1ß treatments lead to apoptosis of HFSCs. More importantly, blocking IL-18 and IL-1ß signals reversed HFSCs depletion in skin organoid models and attenuated stress-induced hair shedding in mice. Taken together, this study demonstrates the role of the neural (stress)-endocrine (NE)-immune (M1 macrophages) axis in stress-induced hair shedding and suggestes that IL-18 or IL-1ß may be promising therapeutic targets.

Combination of Doxycycline with Metronidazole Protects against Pyroptosis in Rats with Endometritis through the Modulation of TLR4/NF-κB Pathway.

BACKGROUND: Endometritis is a condition usually resulted from the bacterial infection of uterus, causing pelvic disease, sepsis, shock, uterine necrosis and even death if it is inappropriately treated. The aim of this study is to explore the pathogenesis of endometritis, and investigate whether the combination of doxycycline and metronidazole offers stronger protection against lipopolysaccharide (LPS)-induced endometritis, and decipher more about the mechanisms underlying endometritis-related pyroptosis. METHODS: Sprague-Dawley (SD) rats were divided into five groups (n = 8 per group): control, model, metronidazole, doxycycline, and combination groups. In control group, the rats were injected with saline, while in other groups, lipopolysaccharide was injected into uterus of the rats to establish endometritis. Hematoxylin-eosin (H&E) staining was performed as part of the histopathological examination of endometrium. The integrity of chromatin and pyroptosis were evaluated by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. Western blot and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) were performed to ascertain the activation of toll-like receptors (TLR4)/nuclear factor-kappa B (NF-κB) pathway by detecting protein levels of phosphorylated p50 (p-p50)/p50, phosphorylated nuclear factor-kappa B (p-NF-κB)/NF-κB, phosphorylated IkappaB (p-IκB), and TLR4 protein and mRNA. Development of pyroptosis was also detected by determining the levels of caspase-1 and caspase-5 through Western blot and qRT-PCR. Enzyme-linked immunosorbent assay (ELISA) was used to detect levels of interleukin (IL)-1ß, IL-18, IL-2, IL-4, IL-6 and tumor necrosis factor alpha (TNF-α), and flow cytometry was adopted to determine T-helper (Th)1 and Th2 cell percentage to assess the extent of pyroptosis and Th1/Th2 imbalance. RESULTS: The uterine of the model group exhibited pathological alterations and higher degree of cell apoptosis. Compared with the control rats, model group showed lower protein levels of p-p50/p50 (p < 0.001), p-NF-κB/NF-κB (p < 0.001), p-IκB (p < 0.001), and TLR4 protein (p < 0.001) and mRNA (p < 0.001). Elevated levels of caspase-1 (p < 0.001), caspase-5 (p < 0.001), IL-1ß (p < 0.001), IL-18 (p < 0.001), IL-2 (p < 0.01), TNF-α (p < 0.05) and Th1/Th2 (p < 0.001) as well as reduced levels of IL-4 (p < 0.05) and IL-6 (p < 0.01) were observed in the model group, which could however be reversed by metronidazole (p < 0.01) or doxycycline (p < 0.01), with a more significant effect detected if a combination of the two drugs was administered (p < 0.01). CONCLUSIONS: The combination of doxycycline and metronidazole protects against rat endometritis by inhibiting TLR4/NF-κB pathway-mediated inflammation and suppressing pyroptosis.

Lactoferrin ameliorated obesity-induced endothelial dysfunction by inhibiting the Tak1/IL-18/eNOS pathway between PVAT and vascular endothelium.

Vascular endothelial dysfunction (ED) is one of the mechanisms underlying obesity-related hypertension. Perivascular adipose tissue (PVAT) surrounds blood vessels and influences the vascular endothelium function. Previous studies have demonstrated the antihypertensive effects of lactoferrin (LF) and its hydrolysates through various mechanisms. However, the effect of LF on ED and PVAT has not yet been investigated. In this study, we examined the influence of LF on ED and PVAT using high-fat diet mice as well as MAEC cells and 3T3-L1 adipocytes. Finally, LF supplementation decreases the systolic blood pressure (SBP), serum adhesion molecule (ICAM-1 and VCAM-1), and aorta ROS levels, and improves endothelium-dependent relaxation function in high-fat diet mice. Moreover, LF supplementation down-regulates the Tak1/IL-18/eNOS pathway between PVAT and aorta and enhances the NO generation in high-fat diet mice. In addition, we observe that LF decreases the expression levels of IL-18 and p-Tak1 in 3T3-L1 adipocytes, but fails to influence the eNOS and p-eNOS expression levels in MAEC cells. Finally, the significant associations between LF and IL-18 and SBP and hypertension risk are also observed in obesity children only. These findings provide evidence that the Tak1/IL-18/eNOS pathway between the aorta and PVAT is important in obesity-related ED, and LF may improve ED or even hypertension by down-regulating this pathway.

Identification of ANXA3 as a biomarker associated with pyroptosis in ischemic stroke.

BACKGROUND: Pyroptosis plays an important role in the pathological process of ischemic stroke (IS). However, the exact mechanism of pyroptosis remains unclear. This paper aims to reveal the key molecular markers associated with pyroptosis in IS. METHODS: We used random forest learning, gene set variation analysis, and Pearson correlation analysis to screen for biomarkers associated with pyroptosis in IS. Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen and glucose deprivation/reoxygenation (OGD/R) models were constructed in vitro and in vivo. Cells were transfected with an Annexin A3 silencing (si-ANXA3) plasmid to observe the effects of ANXA3 on OGD/R + lipopolysaccharides (LPS)-induced pyroptosis. qRT‒PCR and western blotting were used to detect the expression of potential biomarkers and pyroptotic pathways. RESULTS: Samples from a total of 170 IS patients and 109 healthy individuals were obtained from 5 gene expression omnibus databases. Thirty important genes were analyzed by random forest learning from the differentially expressed genes. Then, we investigated the relationship between the above genes and the pyroptosis score, obtaining three potential biomarkers (ANXA3, ANKRD22, ADM). ANXA3 and ADM were upregulated in the MCAO/R model, and the fold difference in ANXA3 expression was greater. Pyroptosis-related factors (NLRP3, NLRC4, AIM2, GSDMD-N, caspase-8, pro-caspase-1, cleaved caspase-1, IL-1ß, and IL-18) were upregulated in the MCAO/R model. Silencing ANXA3 alleviated the expression of pyroptosis-related factors (NLRC4, AIM2, GSDMD-N, caspase-8, pro-caspase-1, cleaved caspase-1, and IL-18) induced by OGD/R + LPS or MCAO/R. CONCLUSION: This study identified ANXA3 as a possible pyroptosis-related gene marker in IS through bioinformatics and experiments. ANXA3 could inhibit pyroptosis through the NLRC4/AIM2 axis.