Inflammatory Cytokine Gene Research Articles

Plasmodium falciparum malaria drives epigenetic reprogramming of human monocytes toward a regulatory phenotype.

In malaria-naïve children and adults, Plasmodium falciparum-infected red blood cells (Pf-iRBCs) trigger fever and other symptoms of systemic inflammation. However, in endemic areas where individuals experience repeated Pf infections over many years, the risk of Pf-iRBC-triggered inflammatory symptoms decreases with cumulative Pf exposure. The molecular mechanisms underlying these clinical observations remain unclear. Age-stratified analyses of uninfected, asymptomatic Malian individuals before the malaria season revealed that monocytes of adults produced lower levels of inflammatory cytokines (IL-1β, IL-6 and TNF) in response to Pf-iRBC stimulation compared to monocytes of Malian children and malaria-naïve U.S. adults. Moreover, monocytes of Malian children produced lower levels of IL-1β and IL-6 following Pf-iRBC stimulation compared to 4–6-month-old infants. Accordingly, monocytes of Malian adults produced more IL-10 and expressed higher levels of the regulatory molecules CD163, CD206, Arginase-1 and TGM2. These observations were recapitulated in an in vitro system of monocyte to macrophage differentiation wherein macrophages re-exposed to Pf-iRBCs exhibited attenuated inflammatory cytokine responses and a corresponding decrease in the epigenetic marker of active gene transcription, H3K4me3, at inflammatory cytokine gene loci. Together these data indicate that Pf induces epigenetic reprogramming of monocytes/macrophages toward a regulatory phenotype that attenuates inflammatory responses during subsequent Pf exposure.Trial Registration: ClinicalTrials.gov NCT01322581.

Metformin attenuates diabetic renal injury via the AMPK-autophagy axis.

Diabetic nephropathy (DN) is a clinical condition characterized by kidney damage that is observed in patients with diabetes. DN is the main cause of end-stage renal disease (ESRD), which is the final stage of chronic kidney disease. Increasing evidence suggests that metformin, a characteristic oral hypoglycemic drug used for treating diabetes, exerts beneficial effects on various medical conditions and diseases, including cancer, cardiovascular diseases and thyroid-related disorders. However, the impact of metformin on DN remains unknown. The present study investigated whether metformin could attenuate the inflammatory response, fibrosis and increased oxidative stress observed during DN in diabetic/dyslipidemic (db/db) mice. The kidneys of the mice (12-16 weeks) were isolated for immunohistochemistry and western blotting. The results demonstrated that metformin significantly reduced the oxidative damage and fibrosis in the kidneys of db/db mice. Furthermore, metformin treatment significantly inhibited the generation of inflammatory cytokines, including TNF-α and IL-1β in db/db mice. These effects were induced by the activation of the AMP-activated protein kinase (AMPK) pathway, which was mediated by increased phosphorylation of AMPK and mammalian target of rapamycin (mTOR), resulting in autophagy and the simultaneous decrease in reactive oxygen species production, cell apoptosis and inflammatory response. These findings suggested that metformin may reduce DN damage via regulation of the AMPK-mTOR-autophagy axis and indicated that metformin may be considered as a potential target in the treatment of DN.

Equine Genital Squamous Cell Carcinoma Associated with EcPV2 Infection: RANKL Pathway Correlated to Inflammation and Wnt Signaling Activation.

Simple SummaryEquine genital squamous cell carcinomas (egSCCs) associated with papilloma virus (PV) infection have been recently proposed as model for human PV-induced SCC. In both species, PV mucosal infections often induce cervical, oropharyngeal, penile, anal, vaginal, and vulvar cancer. The aim of this study was to clarify the molecular mechanisms behind egSCCs associated with equine papillomavirus 2 (EcPV2) infection investigating receptor activator of nuclear factor-kappa B ligand (RANKL), Wnt, and interleukin (IL)17 signaling pathways. RANKL has been recently demonstrated to play a crucial role in several human tumors, associated with a poor prognosis and metastatic spread; novel targeted therapies through RANKL silencing monoclonal antibodies have been undertaken. EcPV2-E6 DNA was checked, and viral presence was confirmed in 91% of cases, whereas oncogene expression was 60.8% for E6 and 34.7% for E2. RANKL, NFKBp50, NFKBp65, IL6, IL17, IL23p19, IL8, IL12p35, IL12p40, BCATN1, FOSL1, and LEF1 gene expression showed a significant upregulation in tumor samples compared to healthy tissues. Our results describe an inflammatory environment characterized by the increased expression of several cytokines and the activation of RANKL/RANK, IL17A, and canonical and non-canonical Wnt signaling pathways. These results may be helpful to identify new targets for immunotherapy strategies confirming egSCCs as a model for the human disease.Equine genital squamous cell carcinomas (egSCCs) are among the most common equine tumors after sarcoids, severely impairing animal health and welfare. Equus caballus papillomavirus type 2 (EcPV2) infection is often related to these tumors. The aim of this study was to clarify the molecular mechanisms behind egSCCs associated with EcPV2 infection, investigating receptor activator of nuclear factor-kappa B ligand (RANKL) signaling in NF-kB pathway, together with the Wnt and IL17 signaling pathways. We analyzed the innate immune response through gene expression evaluation of key cytokines and transcription factors. Moreover, Ki67 index was assessed with immunohistochemistry. EcPV2-E6 DNA was checked, and viral presence was confirmed in 21 positive out to 23 cases (91%). Oncogene expression was confirmed in 14 cases (60.8%) for E6 and in 8 (34.7%) for E2. RANKL, nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB)-p50, NFKBp65, interleukin (IL)-6, IL17, IL23p19, IL8, IL12p35, IL12p40, β-catenin (BCATN1), FOS like 1 (FOSL1), and lymphoid enhancer binding factor 1 (LEF1) showed a significant upregulation in tumor samples compared to healthy tissues. Our results describe an inflammatory environment characterized by the activation of RANKL/RANK and IL17 with the relative downstream pathways, and a positive modulation of inflammatory cytokines genes such as IL6 and IL8. Moreover, the increase of BCATN1, FOSL1, and LEF1 gene expression suggests an activation of both canonical and non-canonical Wnt signaling pathway that could be critical for carcinogenesis and tumor progression.

Intense Pulsed Light Attenuates UV-Induced Hyperimmune Response and Pigmentation in Human Skin Cells.

The skin of an organism is affected by various environmental factors and fights against aging stress via mechanical and biochemical responses. Photoaging induced by ultraviolet B (UVB) irradiation is common and is the most vital factor in the senescence phenotype of skin, and so, suppression of UVB stress-induced damage is critical. To lessen the UVB-induced hyperimmune response and hyperpigmentation, we investigated the ameliorative effects of intense pulsed light (IPL) treatment on the photoaged phenotype of skin cells. Normal human epidermal keratinocytes and human epidermal melanocytes were exposed to 20 mJ/cm2 of UVB. After UVB irradiation, the cells were treated with green (525–530 nm) and yellow (585–592 nm) IPL at various time points prior to the harvest step. Subsequently, various signs of excessive immune response, including expression of proinflammatory and melanogenic genes and proteins, cellular oxidative stress level, and antioxidative enzyme activity, were examined. We found that IPL treatment reduced excessive cutaneous immune reactions by suppressing UVB-induced proinflammatory cytokine expression. IPL treatment prevented hyperpigmentation, and combined treatment with green and yellow IPL synergistically attenuated both processes. IPL treatment may exert protective effects against UVB injury in skin cells by attenuating inflammatory cytokine and melanogenic gene overexpression, possibly by reducing intracellular oxidative stress. IPL treatment also preserves antioxidative enzyme activity under UVB irradiation. This study suggests that IPL treatment is a useful strategy against photoaging, and provides evidence supporting clinical approaches with non-invasive light therapy.

Elevated Extracellular cGMP Produced after Exposure to Enterotoxigenic Escherichia coli Heat-Stable Toxin Induces Epithelial IL-33 Release and Alters Intestinal Immunity.

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies identified heat-stable enterotoxin (ST)-producing ETEC as a prevalent diarrheal pathogen in children younger than 5 years. While many studies have evaluated the interaction of ETEC heat-labile enterotoxin (LT) with host epithelium and immunity, few investigations have attempted similar studies with ST. To further understand ST pathogenesis, we examined the impact of ST on cGMP localization, epithelial cell cytokine production, and antibody development following immunization. In addition to robust intracellular cGMP in T84 cells in the presence of phosphodiesterase inhibitors (PDEis) that prevent the breakdown of cyclic nucleotides, we found that prolonged ST intoxication induced extracellular cGMP accumulation in the presence or absence of PDEis. Further, ST intoxication induced luminal cGMP in vivo in mice, suggesting that secreted cGMP may have other cellular functions. Using transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR), we demonstrated that ST intoxication, or treatment with the clinically used ST mimic linaclotide, altered inflammatory cytokine gene expression, including the interleukin 1 (IL-1) family member IL-33, which could also be induced by cell-permeative 8-Br-cGMP. Finally, when present during immunization, ST suppressed induction of antibodies to specific antigens. In conclusion, our studies indicate that ST modulates epithelial cell physiology and the interplay between the epithelial and immune compartments.

Effects of sulfated polysaccharides isolated from Codium fragile on inflammatory cytokine gene expression and Edwardsiella tarda infection in rockfish, Sebastes schlegelii

Sulfated polysaccharides (SPs) derived from Codium fragile (sponge seaweed) can regulate cytokine expression in mammalian macrophages, NK cell lines and olive flounder head kidney primary cells in vitro. In this study, we found that SPs from C. fragile exhibited anti-bacterial activities against fish pathogenic bacteria including Streptococcus parauberis, Lactococcus garvieae, Aeromonas salmonicida and Edwardsiella tarda at a minimum inhibitory concentration of 2 mg/mL, but not against S. iniae or Vibrio anguillarum. Immunostimulatory effects of SPs from C. fragile on rockfish (Sebastes schlegelii) were evaluated by analyzing mRNA expression levels of inflammatory cytokines (interleukin (IL)-1β, IL-8, IL-6 and tumor necrosis factor (TNF)-α) and anti-inflammatory cytokines (IL-10) both in vitro and in vivo. Results revealed that expression levels of all genes tested were upregulated in rockfish head kidney and spleen cells by SPs from C. fragile in a dose/time-dependent manner in vitro. By contrast, expression levels of these genes were significantly (p < 0.05) downregulated in the head kidney and spleen of rockfish in vivo at 1 and 3 days post intraperitoneal injection of SPs from C. fragile. In the liver, these genes were downregulated on day 1, but upregulated on day 3. Treatment with SPs downregulated the expression of these genes in spleen, but upregulated IL-10 gene expression in the intestine and liver. Meanwhile, when fish were fed with crude SPs for 4 weeks and challenged with E. tarda, infected fish started to die starting from 2 days after immune challenge. The cumulative mortality of the 0.1% group was significantly lower (p < 0.05) than that of the control group without feeding with SPs. Expression levels of IL-1β and IL-6 genes were significantly (p < 0.05) upregulated in head kidney of the 0.5% group on day 1 while IL-1β gene expression was downregulated on day 3 in the liver. These results indicate that SPs from C. fragile can regulate the immune gene expression in rockfish and that a diet containing 0.1% crude SPs can reduce the mortality of rockfish caused by E. tarda infection.

Possible Dietary Effects of Insect-Based Diets across Zebrafish (Danio rerio) Generations: A Multidisciplinary Study on the Larval Phase.

Simple SummaryFish meal and fish oil represent the optimal ingredients for aquafeed formulation. However, their partial or complete substitution with more sustainable alternatives, like insects, is required for a further development of the aquaculture sector. Nutritional programming through parental feeding may enhance the ability of the progeny to utilize insect-based diets. In the present study, five experimental diets characterized by increasing fish meal substitution levels with full-fat Black Soldier Fly (Hermetia illucens; BSF) prepupae meal (0%, 25%, 50%, 75% and 100%), used for zebrafish broodstock rearing, were provided to the progeny (first filial generation, F1). The effects of BSF-based diets on F1 zebrafish larvae were investigated through a multidisciplinary approach. No significant differences among the experimental groups were observed in terms of growth, hepatic lipid accumulation and gut health. Furthermore, increasing fish meal substitution levels with BSF prepupae meal resulted in a positive modulation of both stress and immune response. Results demonstrated that nutritional programming via broodstock nutrition should be considered a valuable solution to increase the use of insect meal in aquafeeds formulation and improve fish culture sustainability.Insects represent a valuable and sustainable alternative ingredient for aquafeed formulation. However, insect-based diets have often highlighted controversial results in different fish species, especially when high inclusion levels were used. Several studies have demonstrated that nutritional programming through parental feeding may allow the production of fish better adapted to use sub-optimal aquafeed ingredients. To date, this approach has never been explored on insect-based diets. In the present study, five experimental diets characterized by increasing fish meal substitution levels with full-fat Black Soldier Fly (Hermetia illucens; BSF) prepupae meal (0%, 25%, 50%, 75% and 100%) were used to investigate the effects of programming via broodstock nutrition on F1 zebrafish larvae development. The responses of offspring were assayed through biometric, gas chromatographic, histological, and molecular analyses. The results evidenced that the same BSF-based diets provided to adults were able to affect F1 zebrafish larvae fatty acid composition without impairing growth performances, hepatic lipid accumulation and gut health. Groups challenged with higher BSF inclusion with respect to fish meal (50%, 75% and 100%) showed a significant downregulation of stress response markers and a positive modulation of inflammatory cytokines gene expression. The present study evidences that nutritional programming through parental feeding may make it possible to extend the fish meal substitution level with BSF prepupae meal in the diet up to almost 100% without incurring the well-known negative side effects of BSF-based diets.

Acinetobacter baumannii LOS Regulate the Expression of Inflammatory Cytokine Genes and Proteins in Human Mast Cells.

Herein, we investigated the effect of bacterial lipooligosaccharides (LOS), from Acinetobacter baumannii, on the expression of pro-inflammatory genes that play an essential role in bacterial clearance. LAD2 human mast cells were stimulated with LOS derived from two strains of A. baumannii—ATCC 19606 and MDRA T14. LOS exposure induced the expression of genes for pro-inflammatory mediators, including TNF-α, IL-8, LTC4S, CCL4, and TLR4. The mRNA expression levels of a majority of the pro-inflammatory genes, except TLR4, in A. baumannii-LOS stimulated mast cells were increased. Moreover, co-culture of neutrophils with the supernatant obtained from LOS (ATCC 19606 and MDRA T14)-induced LAD2 cells increased the transmigration of neutrophils, which plays a critical role in the early protection against bacterial infections. The results of the present study suggest that LOS could be involved in the pathogenicity of A. baumannii by inducing inflammatory responses via mast cells and that IL-8 is involved in recruiting neutrophils in response to bacterial invasion.

NLRP3 inflammasome mediated pyroptosis is involved in cadmium exposure-induced neuroinflammation through the IL-1β/IkB-α-NF-κB-NLRP3 feedback loop in swine

Cadmium (Cd) chloride, as widely distributed toxic environmental pollutants by using in industry, severely imperils animal and human health. Pyroptosis is a Cas1-dependent pro-inflammatory programmed cell death and involves in various types of diseases. Nevertheless, the mechanism of pyroptosis and Cd-induced neurotoxicity remains obscure. To investigate the specific molecular mechanisms of Cd-induced neurotoxicity, 10 weaned piglets were randomly divided into 2 groups treated with 0 and 20 mg/kg CdCl2 in the diet for 40 days. The levels of pyroptosis, mitochondrial and inflammation-related genes were validated by qRT-PCR and WB in vivo. Our results revealed that Cd caused cerebral histopathology lesions, inducing cerebral pyroptosis and the mass generation of inflammatory cytokines, as indicated by the increased NLRP3 inflammasome activation (NLRP3, Cas1 and ASC) and the upregulation of inflammation factors IL-2, IL-6, IL-7 and inhibition of IL-10. Subsequently, further research indicated that Cd triggered pyroptosis via activating the TRAF6-IkB-α-NF-κB pathway, which interfered with the phosphorylation and ubiquitination of IkB-α. Furthermore, Cd caused mitochondrial dysfunction and fragmentation by inhibiting the AMPK-PGC-1α-NRF1/2 signaling pathway and reduced the expression of mitochondrial-related regulatory factors OPA1, TFAM and mtDNA, resulting in the increase of NLRP3 inflammasome. Besides, we found eight hub genes (IKK, IKB-α, NLRP3, TRAF6, NF-κB, AMPK, TNFα and PGC-1α), mainly related to the interaction between the NF-κB pathway and NLRP3 inflammasome. Overall, these results demonstrated that Cd could promote the IL-1β/IkB-α-NF-κB-NLRP3 inflammasome activation positive feedback loop to result in neuroinflammation in swine, which provided new insights in understanding Cd-induced toxicity.

Tribulus terrestris L. Extract Protects against Lipopolysaccharide-Induced Inflammation in RAW 264.7 Macrophage and Zebrafish via Inhibition of Akt/MAPKs and NF-κB/iNOS-NO Signaling Pathways.

Inflammation response is a regulated cellular process and excessive inflammation has been recognized in numerous diseases, such as cardiovascular disease, neurodegenerative disease, inflammatory bowel disease, and cancer. Tribulus terrestris L. (TT), also known as Bai Jili in Chinese, has been applied in traditional Chinese medicine for thousands of years while its anti-inflammatory activity and underlying mechanism are not fully elucidated. Here, we hypothesize Tribulus terrestris L. extract (BJL) which presents anti-inflammatory effect, and the action mechanism was also investigated. We employed the transgenic zebrafish line Tg(MPO:GFP), which expresses green fluorescence protein (GFP) in neutrophils, and mice macrophage RAW 264.7 cells as the in vivo and in vitro model to evaluate the anti-inflammatory effect of BJL, respectively. The production of nitric oxide (NO) was measured by Griess reagent. The mRNA expression levels of inflammatory cytokines and inducible nitric oxide synthase (iNOS) were measured by real-time PCR, and the intracellular total or phosphorylated protein levels of NF-κB, Akt, and MAPKs including MEK, ERK, p38, and JNK were detected by western blot. We found that BJL significantly inhibited fin transection or lipopolysaccharide- (LPS-) induced neutrophil migration and aggregation in zebrafish in vivo. In mice macrophage RAW 264.7 cells, BJL ameliorated LPS-triggered excessive release of NO and transcription of inflammatory cytokine genes including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). BJL also reduced the LPS-induced elevations of intracellular iNOS and nuclear factor kappa B (NF-κB) which mediate the cellular NO and inflammatory cytokine productions, respectively. Moreover, LPS dramatically increased the phosphorylation of Akt and MAPKs including MEK, ERK, p38, and JNK in RAW 264.7 cells, while cotreatment BJL with LPS suppressed their phosphorylation. Taken together, our data suggested that BJL presented potent anti-inflammatory effect and the underlying mechanism was closely related to the inhibition of Akt/MAPKs and NF-κB/iNOS-NO signaling pathways.

Anti-cancer and anti-inflammatory effects elicited by short chain fatty acids produced by Escherichia coli isolated from healthy human gut microbiota

BackgroundExtracellular metabolites of short chain fatty acids (SCFA) excreted by gut microbiota have been reported to play an important role in the regulation of intestinal homeostasis. Apart from supplying energy, SCFA also elicit immune stimulation in animal and human cells. Therefore, an attempt was conducted to isolate SCFA producing bacteria from healthy human microbiota. The anti-cancer and anti-inflammatory effects of extracellular metabolites and individual SFCA were further investigated by using breast, colon cancer and macrophage cells. Toxin, inflammatory and anti-inflammatory cytokine gene expressions were investigated by RT-qPCR analyses in this study.ResultsEscherichia coli KUB-36 was selected in this study since it has the capability to produce seven SCFA extracellularly. It produced acetic acid as the main SCFA. It is a non-exotoxin producer and hence, it is a safe gut microbiota. The IC50 values indicated that the E. coli KUB-36 metabolites treatment elicited more potent cytotoxicity effect on MCF7 breast cancer cell as compared to colon cancer and leukemia cancer cells but exhibited little cytotoxic effects on normal breast cell. Furthermore, E. coli KUB-36 metabolites and individual SCFA could affect inflammatory responses in lipopolysaccharide-induced THP-1 macrophage cells since they suppressed inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α well as compared to the control, whilst inducing anti-inflammatory cytokine IL-10 expression.ConclusionSCFA producing E. coli KUB-36 possessed vast potential as a beneficial gut microbe since it is a non-exotoxin producer that exhibited beneficial cytotoxic effects on cancer cells and elicited anti-inflammatory activity simultaneously. However, the probiotic characteristic of E. coli KUB-36 should be further elucidated using in vivo animal models.

Long-term exposure to 2-amino-3-methylimidazo[4,5-f]quinoline can trigger a potential risk of Parkinson's disease

Humans are exposed to heterocyclic amines (HCAs) from a wide range of sources, such as protein-rich thermally processed foods, cigarette smoke, contaminated river water, the atmosphere, soil, and forest fire ash. Although the carcinogenic and mutagenic hazards of HCAs have been widely studied, the potential neurotoxicity of these compounds still needs to be further elucidated. Here, we studied the neurotoxicity of the HCA 2-amino-3-methylimidazole[4,5-f]quinoline (IQ) in vivo by utilizing a zebrafish model. After 35 days of exposure at 8, 80, and 800 ng/mL, zebrafish exploratory behavior and locomotor activity were significantly inhibited, and light/dark preference behaviors were also disturbed. Moreover, the expression of Parkinson's disease (PD)-related genes and proteins, dopamine-related genes, neuroplasticity-related genes, antioxidant enzyme genes and inflammatory cytokine genes in the zebrafish brain was significantly affected. The numbers of NeuN neurons in the midbrain were decreased in exposed zebrafish, while the numbers of apoptotic cells were increased. In summary, our research suggests that IQ is neurotoxic and significantly associated with PD and that long-term exposure to IQ may contribute to PD risk. This risk may be related to IQ-mediated effects on mitochondrial homeostasis and induction of oxidative stress and inflammation.

LINC00261 relieves the progression of sepsis-induced acute kidney injury by inhibiting NF-κB activation through targeting the miR-654-5p/SOCS3 axis.

Sepsis is a life-threatening disease, which can cause the dysfunction of multiple organs, including kidney. Recently, a number of studies found that the long non-coding RNA (lncRNA) is closely associated with the development and progression of sepsis; however, the role of long intergenic non-protein coding RNA 261 (LINC00261) in sepsis-induced acute kidney injury is poorly understood. In this study, we found the expression of LINC00261 was significantly decreased in the serum of patients with sepsis than healthy controls. A similar result was also observed in the mouse model of sepsis induced by lipopolysaccharide (LPS). Further investigations revealed that overexpression of LINC00261 improved the viability, suppressed the apoptosis and reduced the generation of inflammatory cytokines in LPS-treated HK-2 cells. Mechanistically, we confirmed that LINC00261 could function as a sponge to combine with microRNA-654-5p (miR-654-5p) which inhibits nuclear factor-κB (NF-κB) activity by targeting suppressor of cytokine signaling 3 (SOCS3). In conclusion, our results demonstrate that LINC00261 may regulate the progression of sepsis-induced acute kidney injury via the miR-654-5p/SOCS3/NF-κB pathway and therefore provides a new insight into the treatment of this disease.

Down-regulation of lncRNA SNHG5 relieves sepsis-induced acute kidney injury by regulating the miR-374a-3p/TLR4/NF-κB pathway.

Sepsis is an acute systemic infectious disease engendered by infectious factors, which can cause the dysfunction of multiple organs, including acute kidney injury (AKI). Recently, more and more researchers are focussing on long noncoding RNA (lncRNA) that is closely associated with the development and progression of various diseases; however, the role and mechanism of lncRNA in sepsis-induced AKI are not fully understood. Here, we found a significant increase in the expression of lncRNA small nuclear RNA host gene 5 (SNHG5) in the serum of patients with sepsis than healthy controls. Similar results were obtained from mouse model of sepsis. Further investigations revealed that knockdown of SNHG5 improves the viability and reduces the rate of apoptosis and the generation of inflammatory cytokines in HK-2 and TCMK-1 cells treated with lipopolysaccharide. Mechanistically, we showed that SNHG5 can combine with microRNA-374a-3p (miR-374a-3p), which inhibits nuclear factor-κB (NF-κB) activity by targeting TLR4. In conclusion, our results demonstrate that SNHG5 may regulate sepsis-induced AKI via the miR-374a-3p/TLR4/NF-κB pathway, therefore providing a new insight into the treatment of this disease.

Associations between Inflammatory Cytokine Gene Polymorphisms and Susceptibilities to Intracranial Aneurysm in Chinese Population

Intracranial aneurysm (IA) is a complex disease caused by genetic and environmental factors. Evidence indicates that inflammation plays an important role in IA occurrence. We aimed to explore the associations between inflammatory cytokine gene polymorphisms and IA in a Chinese population. This study enrolled 768 participants of Han ethnicity, including 384 patients with IA and 384 healthy individuals. Sixteen single nucleotide polymorphisms (SNPs) of IL1, IL6, IL12, and TNF-α genes were genotyped using the Sequenom MassARRAY platform. Univariate and multivariate logistic regression analyses were used to analyze the associations. We found IL12B rs3181216 was significantly associated with IA in the recessive and additive models (OR = 0.46, 95% CI = 0.23–0.89, P = 0.022; OR = 0.74, 95% CI = 0.56–0.98, P = 0.034, respectively). TNF-α rs1799964 was associated with IA in dominant and additive models (OR = 0.67, 95% CI = 0.46–0.98, P = 0.041; OR = 0.71, 95% CI = 0.51–0.98, P = 0.034, respectively). IL1A rs17561 was associated with single IA susceptibility (dominant model: OR = 0.52, 95% CI = 0.31–0.85, P = 0.040). The IL12B rs3181216 polymorphism was associated with single IA susceptibility in the recessive model (OR = 0.41, 95% CI = 0.18–0.93, P = 0.033). The IL12B rs2195940 polymorphism was associated with multiple IAs susceptibility (dominant model: OR = 0.28, 95% CI = 0.09–0.89, P = 0.031; additive model: OR = 0.28, 95% CI = 0.09–0.90, P = 0.032). TNF-α rs1799964 was associated with multiple IAs susceptibility in the dominant model (OR = 0.54, 95% CI = 0.30–0.97, P = 0.040). No associations were found between other polymorphisms and IA susceptibility. Therefore, IL1A, IL12B, and TNF-α gene polymorphisms are associated with IA susceptibility in a Chinese population.

Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening

BackgroundIn-feed antibiotics are being phased out in livestock production worldwide. Alternatives to antibiotics are urgently needed to maintain animal health and production performance. Host defense peptides (HDPs) are known for their broad-spectrum antimicrobial and immunomodulatory capabilities. Enhancing the synthesis of endogenous HDPs represents a promising antibiotic alternative strategy to disease control and prevention.MethodsTo identify natural products with an ability to stimulate the synthesis of endogenous HDPs, we performed a high-throughput screening of 1261 natural products using a newly-established stable luciferase reporter cell line known as IPEC-J2/pBD3-luc. The ability of the hit compounds to induce HDP genes in porcine IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and jejunal explants were verified using RT-qPCR. Augmentation of the antibacterial activity of porcine 3D4/31 macrophages against a Gram-negative bacterium (enterotoxigenic E. coli) and a Gram-positive bacterium (Staphylococcus aureus) were further confirmed with four selected HDP-inducing compounds.ResultsA total of 48 natural products with a minimum Z-score of 2.0 were identified after high-throughput screening, with 21 compounds giving at least 2-fold increase in luciferase activity in a follow-up dose-response experiment. Xanthohumol and deoxyshikonin were further found to be the most potent in inducing pBD3 mRNA expression, showing a minimum 10-fold increase in IPEC-J2, 3D4/31 cells, and jejunal explants. Other compounds such as isorhapontigenin and calycosin also enhanced pBD3 mRNA expression by at least 10-fold in both IPEC-J2 cells and jejunal explants, but not 3D4/31 cells. In addition to pBD3, other porcine HDP genes such as pBD2, PG1-5, and pEP2C were induced to different magnitudes by xanthohumol, deoxyshikonin, isorhapontigenin, and calycosin, although clear gene- and cell type-specific patterns of regulation were observed. Desirably, these four compounds had a minimum effect on the expression of several representative inflammatory cytokine genes. Furthermore, when used at HDP-inducing concentrations, these compounds showed no obvious direct antibacterial activity, but significantly augmented the antibacterial activity of 3D4/31 macrophages (P < 0.05) against both Gram-negative and Gram-positive bacteria.ConclusionsOur results indicate that these newly-identified natural HDP-inducing compounds have the potential to be developed as novel alternatives to antibiotics for prophylactic and therapeutic treatment of infectious diseases in livestock production.

Association Study between Proinflammatory Cytokine Gene Polymorphism and Risk of Recurrent Spontaneous Abortion

To investigate the correlation between inflammatory cytokine gene polymorphisms and the risk of recurrent miscarriage. A total of 120 patients with recurrent spontaneous abortion in our hospital between January 2019 and December 2020 were selected as the recurrent spontaneous abortion group and 120 healthy pregnant women with early pregnancy who visited the hospital during the same period were used as the control group and the genotype of interleukin-18 was detected by single nucleotide polymorphism typing technique and the validation method was Hardy Weinberg’s law of genetic equilibrium was used to verify the genotype of interleukin-18 and the relationship between interleukin-18 genotype and the risk of recurrent spontaneous abortion was analyzed by logistic method. The results showed that the level of interleukin-18 in the recurrent spontaneous abortion group was significantly higher than that in the control group (p 0.05); the interleukin-18 (rs1946519) and interleukin- 18(rs360717) in the control group The actual genotype frequencies of interleukin-18(rs1946519) and interleukin-18 (rs360717) in the control group were also not significantly different from the theoretical values (test values were 0.474 and 0.153, p>0.05); the genotypes of the rs1946519 and rs360717 loci showed significant differences and the recurrent spontaneous abortion group had significantly more A alleles than the control group (p<0.05); rs1946519 and rs360717 locus mutant cases showed significant differences in serum interleukin-17 levels, with AA and TT genotypes showing higher concentrations than other genotypes, respectively (p<0.05); the relative risk of recurrent spontaneous abortion risk in rs1946519 AG and AA gene carriers was 2.468 and 2.468 times higher than that of the GG allele, respectively. The relative risk of recurrent spontaneous abortion in rs360717 TC and TT gene carriers was 1.973 and 1.545 times higher than that of CC allele, respectively and all of them had higher risk of recurrent spontaneous abortion (p<0.05). The rs1946519 polymorphic allele G and rs360717 allele C mutations of interleukin-18 affect the serum hormone levels of patients and lead to an increased risk of recurrent spontaneous abortion in pregnant women. Therefore, interleukin-18 gene polymorphisms may be an important basis for the pathogenesis of recurrent spontaneous abortion and its treatment.

Effect of δ-Tocopherol on Mice Adipose Tissues and Mice Adipocytes Induced Inflammation.

The study aim was to evaluate the potential anti-inflammatory effects of vitamin E analogs, especially α-tocopherol and δ-tocopherol. We used male C57BL/6JJcl mice, which were divided into four groups: the control (C), high-fat and high-sucrose diet (H), high-fat and high-sucrose diet+α-tocopherol (Ha) and high-fat and high-sucrose diet+δ-tocopherol (Hd) groups. The mice were fed for 16 weeks. To the high-fat and high-sucrose diet, 800 mg/kg of α-tocopherol or δ-tocopherol was added more. The final body weight was significantly higher in the H group than in the C group. On the other hand, the final body weight was drastically lower in the Ha group and Hd group than in the H group. However, the energy intake was not significantly different among all groups. Therefore, we assumed that α-tocopherol and δ-tocopherol have potential anti-obesity effect. Besides, inflammatory cytokine gene expression was significantly higher in the epididymal fat of the H group than in the C group. These results showed that inflammation was induced by epididymal fat of mice fed a high-fat and high-sucrose diet for 16 weeks. Unfortunately, addition of α-tocopherol or δ-tocopherol to the diet did not restrain inflammation of epididymal fat. Investigation of the anti-inflammatory effects of α-tocopherol or δ-tocopherol in co-cultured 3T3-L1 cells and RAW264.7 cells showed that δ-tocopherol inhibited increased gene expression of the inflammatory cytokines, IL-1β, IL-6, and iNOS. These results suggest that an anti-inflammatory effect in the δ-tocopherol is stronger than that in the α-tocopherol in vitro. We intend to perform an experiment by in vivo sequentially in the future.

Gut microbiota shape the inflammatory response in mice with an epithelial defect

ABSTRACT Intestinal epithelial cell endoplasmic reticulum (ER) stress has been implicated in intestinal inflammation. It remains unclear whether ER stress is an initiator of or a response to inflammation. Winnie mice, carrying a Muc2 gene mutation resulting in intestinal goblet cell ER stress, develop spontaneous colitis with a depleted mucus barrier and increased bacterial translocation. This study aims to determine whether the microbiota was required for the development of Winnie colitis, and whether protein misfolding itself can initiate inflammation directly in absence of the microbiota. To assess the role of microbiota in driving Winnie colitis, WT and Winnie mice on the same background were rederived into the germ-free facility and housed in the Trexler-type soft-sided isolators. The colitis phenotype of these mice was assessed and compared to WT and Winnie mice housed within a specific pathogen-free facility. We found that Winnie colitis was substantially reduced but not abolished under germ-free conditions. Expression of inflammatory cytokine genes was reduced but several chemokines remained elevated in absence of microbiota. Concomitantly, ER stress was also diminished, although mucin misfolding persisted. RNA-Seq revealed that Winnie differentiated colon organoids have decreased expression of the negative regulators of the inflammatory response compared to WT. This data along with the increase in Mip2a chemokine expression, suggests that the epithelial cells in the Winnie mice are more responsive to stimuli. Moreover, the data demonstrate that intestinal epithelial intrinsic protein misfolding can prime an inflammatory response without initiating the unfolded protein response in the absence of the microbiota. However, the microbiota is necessary for the amplification of colitis in Winnie mice. Genetic predisposition to mucin misfolding in secretory cells initiates mild inflammatory signals. However, the inflammatory signal sets a forward-feeding cycle establishing progressive inflammation in the presence of microbiota. Abbreviations: Endoplasmic Reticulum: ER; Mucin-2: Muc-2; GF: Germ-Free; Inflammatory Bowel Disease: IBD

The Genetic Ablation of TNF-α Attenuates Wnt-Signaling and Adiposity in High Fat Diet-Induced Obese Mice

Abstract A chronic low-grade inflammation is considered as a consequence of obesity, and linked with multiple complications. However, it is under-investigated how inflammatory cytokines mediate adipogenesis. This study investigated the role of Tumor Necrosis Factor α (TNF-α) on adipogenes is over high-fat diet feeding. Three groups of wild type or TNF-α-/-mice with the same C57BL/6 genetic background were utilized in this study: wild type fed with a low-fat diet (WT-LFD), wild type fed with a high-fat diet (WT-HFD), and TNF-α-/- fed with a HFD (TNF-HFD). After 16-wk feeding, inflammatory cytokine, Wnt pathway and adipogenesis-related genes were analyzed. HFD feeding increased body weight in both WT-HFD and TNF-HFD groups, but genetic ablation of TNF-α attenuated HFD-induced obesity. In visceral adipose tissues, HFD elevated Wnt/β-catenin signaling, indicated by decreased phospho-GSK3β and active β-catenin, two key components within the Wnt pathway, and dysregulated adipogenesis, indicated by reduced PPARγ/CEBPα expressions. Whereas, the deletion of TNF-α suppressed Wnt-signaling, and restored expressions of adipogenes is-related genes, which were otherwise decreased in the HFD-induced obese animals. These findings demonstrated a critical role of TNF-α in the regulation of Wnt-signaling and adiposity in mice over a HFD feeding, indicating HFD-induced adipocyte dysfunction could be mitigated by targeting TNF-α and Wnt-signaling.