Aryl hydrocarbon receptor: current perspectives on key signaling partners and immunoregulatory role in inflammatory diseases.

The aryl hydrocarbon receptor (AhR) is a versatile environmental sensor and transcription factor found throughout the body, responding to a wide range of small molecules originating from the environment, our diets, host microbiomes, and internal metabolic processes. Increasing evidence highlights AhR's role as a critical regulator of numerous biological functions, such as cellular differentiation, immune response, metabolism, and even tumor formation. Typically located in the cytoplasm, AhR moves to the nucleus upon activation by an agonist where it partners with either the aryl hydrocarbon receptor nuclear translocator (ARNT) or hypoxia-inducible factor 1ß (HIF-1ß). This complex then interacts with xenobiotic response elements (XREs) to control the expression of key genes. AhR is notably present in various crucial immune cells, and recent research underscores its significant impact on both innate and adaptive immunity. This review delves into the latest insights on AhR's structure, activating ligands, and its multifaceted roles. We explore the sophisticated molecular pathways through which AhR influences immune and lymphoid cells, emphasizing its emerging importance in managing inflammatory diseases. Furthermore, we discuss the exciting potential of developing targeted therapies that modulate AhR activity, opening new avenues for medical intervention in immune-related conditions.

Soybean oil-based HFD induces gut dysbiosis that leads to steatosis, hepatic inflammation and insulin resistance in mice.

High-fat diets (HFDs) shape the gut microbiome and promote obesity, inflammation, and liver steatosis. Fish and soybean are part of a healthy diet; however, the impact of these fats, in the absence of sucrose, on gut microbial dysbiosis and its association with liver steatosis remains unclear. Here, we investigated the effect of sucrose-free soybean oil-and fish oil-based high fat diets (HFDs) (SF-Soy-HFD and SF-Fish-HFD, respectively) on gut dysbiosis, obesity, steatosis, hepatic inflammation, and insulin resistance. C57BL/6 mice were fed these HFDs for 24 weeks. Both diets had comparable effects on liver and total body weights. But 16S-rRNA sequencing of the gut content revealed induction of gut dysbiosis at different taxonomic levels. The microbial communities were clearly separated, showing differential dysbiosis between the two HFDs. Compared with the SF-Fish-HFD control group, the SF-Soy-HFD group had an increased abundance of Bacteroidetes, Firmicutes, and Deferribacteres, but a lower abundance of Verrucomicrobia. The Clostridia/Bacteroidia (C/B) ratio was higher in the SF-Soy-HFD group (3.11) than in the SF-Fish-HFD group (2.5). Conversely, the Verrucomicrobiacae/S24_7 (also known as Muribaculaceae family) ratio was lower in the SF-Soy-HFD group (0.02) than that in the SF-Fish-HFD group (0.75). The SF-Soy-HFD group had a positive association with S24_7, Clostridiales, Allobaculum, Coriobacteriaceae, Adlercreutzia, Christensenellaceae, Lactococcus, and Oscillospira, but was related to a lower abundance of Akkermansia, which maintains gut barrier integrity. The gut microbiota in the SF-Soy-HFD group had predicted associations with host genes related to fatty liver and inflammatory pathways. Mice fed the SF-Soy-HFD developed liver steatosis and showed increased transcript levels of genes associated with de novo lipogenesis (Acaca, Fasn, Scd1, Elovl6) and cholesterol synthesis (Hmgcr) pathways compared to those in the SF-Fish-HFD-group. No differences were observed in the expression of fat uptake genes (Cd36 and Fabp1). The expression of the fat efflux gene (Mttp) was reduced in the SF-Soy-HFD group. Moreover, hepatic inflammation markers (Tnfa and Il1b) were notably expressed in SF-Soy-HFD-fed mice. In conclusion, SF-Soy-HFD feeding induced gut dysbiosis in mice, leading to steatosis, hepatic inflammation, and impaired glucose homeostasis.

IL-1ß-Induced CXCL10 Expression in THP-1 Monocytic Cells Involves the JNK/c-Jun and NF-κB-Mediated Signaling.

CXCL10 (IP-10) plays a key role in leukocyte homing to the inflamed tissues and its increased levels are associated with the pathophysiology of various inflammatory diseases including obesity and type 2 diabetes. IL-1ß is a key proinflammatory cytokine that is found upregulated in meta-inflammatory conditions and acts as a potent activator, inducing the expression of cytokines/chemokines by immune cells. However, it is unclear whether IL-1ß induces the expression of CXCL10 in monocytic cells. We, therefore, determined the CXCL10 induction using IL-1ß in THP1 monocytic cells and investigated the mechanisms involved. Monocytes (human monocytic THP-1 cells) were stimulated with IL-1ß. CXCL10 gene expression was determined with real-time RT-PCR. CXCL10 protein was determined using ELISA. Signaling pathways were identified by using Western blotting, inhibitors, siRNA transfections, and kinase assay. Our data show that IL-1ß induced the CXCL10 expression at both mRNA and protein levels in monocytic cells (p = 0.0001). Notably, only the JNK inhibitor (SP600125) significantly suppressed the IL-1ß-induced CXCL10 expression, while the inhibitors of MEK1/2 (U0126), ERK1/2 (PD98059), and p38 MAPK (SB203580) had no significant effect. Furthermore, IL-1ß-induced CXCL10 expression was decreased in monocytic cells deficient in JNK/c-Jun. Accordingly, inhibiting the JNK kinase activity markedly reduced the IL-1ß-induced JNK/c-Jun phosphorylation in monocytic cells. NF-κB inhibition by Bay-117085 and resveratrol also suppressed the CXCL10 expression. Our findings provide preliminary evidence that IL-1ß stimulation induces the expression of CXCL10 in monocytic cells which requires signaling via the JNK/c-Jun/NF-κB axis.

Unraveling Verapamil's Multidimensional Role in Diabetes Therapy: From ß-Cell Regeneration to Cholecystokinin Induction in Zebrafish and MIN6 Cell-Line Models.

This study unveils verapamil's compelling cytoprotective and proliferative effects on pancreatic ß-cells amidst diabetic stressors, spotlighting its unforeseen role in augmenting cholecystokinin (CCK) expression. Through rigorous investigations employing MIN6 ß-cells and zebrafish models under type 1 and type 2 diabetic conditions, we demonstrate verapamil's capacity to significantly boost ß-cell proliferation, enhance glucose-stimulated insulin secretion, and fortify cellular resilience. A pivotal revelation of our research is verapamil's induction of CCK, a peptide hormone known for its role in nutrient digestion and insulin secretion, which signifies a novel pathway through which verapamil exerts its therapeutic effects. Furthermore, our mechanistic insights reveal that verapamil orchestrates a broad spectrum of gene and protein expressions pivotal for ß-cell survival and adaptation to immune-metabolic challenges. In vivo validation in a zebrafish larvae model confirms verapamil's efficacy in fostering ß-cell recovery post-metronidazole infliction. Collectively, our findings advocate for verapamil's reevaluation as a multifaceted agent in diabetes therapy, highlighting its novel function in CCK upregulation alongside enhancing ß-cell proliferation, glucose sensing, and oxidative respiration. This research enriches the therapeutic landscape, proposing verapamil not only as a cytoprotector but also as a promoter of ß-cell regeneration, thereby offering fresh avenues for diabetes management strategies aimed at preserving and augmenting ß-cell functionality.

TNF-α/Stearate Induced H3K9/18 Histone Acetylation Amplifies IL-6 Expression in 3T3-L1 Mouse Adipocytes.

Extensive evidence supports the connection between obesity-induced inflammation and the heightened expression of IL-6 adipose tissues. However, the mechanism underlying the IL-6 exacerbation in the adipose tissue remains unclear. There is general agreement that TNF-α and stearate concentrations are mildly elevated in adipose tissue in the state of obesity. We hypothesize that TNF-α and stearate co-treatment induce the increased expression of IL-6 in mouse adipocytes. We therefore aimed to determine IL-6 gene expression and protein production by TNF-α/stearate treated adipocytes and investigated the mechanism involved. To test our hypothesis, 3T3-L1 mouse preadipocytes were treated with TNF-α, stearate, or TNF-α/stearate. IL-6 gene expression was assessed by quantitative real-time qPCR. IL-6 protein production secreted in the cell culture media was determined by ELISA. Acetylation of histone was analyzed by Western blotting. Il6 region-associated histone H3 lysine 9/18 acetylation (H3K9/18Ac) was determined by ChIP-qPCR. 3T3-L1 mouse preadipocytes were co-challenged with TNF-α and stearate for 24 h, which led to significantly increased IL-6 gene expression (81 ± 2.1 Fold) compared to controls stimulated with either TNF-α (38 ± 0.5 Fold; p = 0.002) or stearate (56 ± 2.0 Fold; p = 0.013). As expected, co-treatment of adipocytes with TNF-α and stearate significantly increased protein production (338 ± 11 pg/mL) compared to controls stimulated with either TNF-α (28 ± 0.60 pg/mL; p = 0.001) or stearate (53 ± 0.20 pg/mL, p = 0.0015). Inhibition of histone acetyltransferases (HATs) with anacardic acid or curcumin significantly reduced the IL-6 gene expression and protein production by adipocytes. Conversely, TSA-induced acetylation substituted the stimulatory effect of TNF-α or stearate in their synergistic interaction for driving IL-6 gene expression and protein production. Mechanistically, TNF-α/stearate co-stimulation increased the promoter-associated histone H3 lysine 9/18 acetylation (H3K9/18Ac), rendering a transcriptionally permissive state that favored IL-6 expression at the transcriptional and translational levels. Our data represent a TNF-α/stearate cooperativity model driving IL-6 expression in 3T3-L1 cells via the H3K9/18Ac-dependent mechanism, with implications for adipose IL-6 exacerbations in obesity.

Gut Dysbiosis Shaped by Cocoa Butter-Based Sucrose-Free HFD Leads to Steatohepatitis, and Insulin Resistance in Mice.

BACKGROUND: High-fat diets cause gut dysbiosis and promote triglyceride accumulation, obesity, gut permeability changes, inflammation, and insulin resistance. Both cocoa butter and fish oil are considered to be a part of healthy diets. However, their differential effects on gut microbiome perturbations in mice fed high concentrations of these fats, in the absence of sucrose, remains to be elucidated. The aim of the study was to test whether the sucrose-free cocoa butter-based high-fat diet (C-HFD) feeding in mice leads to gut dysbiosis that associates with a pathologic phenotype marked by hepatic steatosis, low-grade inflammation, perturbed glucose homeostasis, and insulin resistance, compared with control mice fed the fish oil based high-fat diet (F-HFD). RESULTS: C57BL/6 mice (5-6 mice/group) were fed two types of high fat diets (C-HFD and F-HFD) for 24 weeks. No significant difference was found in the liver weight or total body weight between the two groups. The 16S rRNA sequencing of gut bacterial samples displayed gut dysbiosis in C-HFD group, with differentially-altered microbial diversity or relative abundances. Bacteroidetes, Firmicutes, and Proteobacteria were highly abundant in C-HFD group, while the Verrucomicrobia, Saccharibacteria (TM7), Actinobacteria, and Tenericutes were more abundant in F-HFD group. Other taxa in C-HFD group included the Bacteroides, Odoribacter, Sutterella, Firmicutes bacterium (AF12), Anaeroplasma, Roseburia, and Parabacteroides distasonis. An increased Firmicutes/Bacteroidetes (F/B) ratio in C-HFD group, compared with F-HFD group, indicated the gut dysbiosis. These gut bacterial changes in C-HFD group had predicted associations with fatty liver disease and with lipogenic, inflammatory, glucose metabolic, and insulin signaling pathways. Consistent with its microbiome shift, the C-HFD group showed hepatic inflammation and steatosis, high fasting blood glucose, insulin resistance, increased hepatic de novo lipogenesis (Acetyl CoA carboxylases 1 (Acaca), Fatty acid synthase (Fasn), Stearoyl-CoA desaturase-1 (Scd1), Elongation of long-chain fatty acids family member 6 (Elovl6), Peroxisome proliferator-activated receptor-gamma (Pparg) and cholesterol synthesis (ß-(hydroxy ß-methylglutaryl-CoA reductase (Hmgcr). Non-significant differences were observed regarding fatty acid uptake (Cluster of differentiation 36 (CD36), Fatty acid binding protein-1 (Fabp1) and efflux (ATP-binding cassette G1 (Abcg1), Microsomal TG transfer protein (Mttp) in C-HFD group, compared with F-HFD group. The C-HFD group also displayed increased gene expression of inflammatory markers including Tumor necrosis factor alpha (Tnfa), C-C motif chemokine ligand 2 (Ccl2), and Interleukin-12 (Il12), as well as a tendency for liver fibrosis. CONCLUSION: These findings suggest that the sucrose-free C-HFD feeding in mice induces gut dysbiosis which associates with liver inflammation, steatosis, glucose intolerance and insulin resistance.

Neutral Sphingomyelinase 2 Inhibition Limits Hepatic Steatosis and Inflammation.

Non-alcoholic fatty liver disease (NAFLD) is manifested by hepatic steatosis, insulin resistance, hepatocyte death, and systemic inflammation. Obesity induces steatosis and chronic inflammation in the liver. However, the precise mechanism underlying hepatic steatosis in the setting of obesity remains unclear. Here, we report studies that address this question. After 14 weeks on a high-fat diet (HFD) with high sucrose, C57BL/6 mice revealed a phenotype of liver steatosis. Transcriptional profiling analysis of the liver tissues was performed using RNA sequencing (RNA-seq). Our RNA-seq data revealed 692 differentially expressed genes involved in processes of lipid metabolism, oxidative stress, immune responses, and cell proliferation. Notably, the gene encoding neutral sphingomyelinase, SMPD3, was predominantly upregulated in the liver tissues of the mice displaying a phenotype of steatosis. Moreover, nSMase2 activity was elevated in these tissues of the liver. Pharmacological and genetic inhibition of nSMase2 prevented intracellular lipid accumulation and TNFα-induced inflammation in in-vitro HepG2-steatosis cellular model. Furthermore, nSMase2 inhibition ameliorates oxidative damage by rescuing PPARα and preventing cell death associated with high glucose/oleic acid-induced fat accumulation in HepG2 cells. Collectively, our findings highlight the prominent role of nSMase2 in hepatic steatosis, which could serve as a potential therapeutic target for NAFLD and other hepatic steatosis-linked disorders.

The role of TLR2 in exercise-induced immunomodulation in normal weight individuals.

Toll-like receptors (TLRs) have been targeted for therapeutic drug development for several disorders, including cardiovascular diseases (CVD), and diabetes mellitus. Daily levels physical activity (PA) has been purported to influence the systemic circulation of cytokines, affecting the overall activation of TLRs and influencing the inflammatory milieu. Objective and self-reported daily PA was tracked in 69 normal-weight adults. Freedson's cut-offs categorized daily PA intensity into the 25th lowest, medium, and top percentiles. Monocytic TLR2 expression was quantified by flow cytometry in fresh whole blood. Cross-sectional associations between flow cytometry measured TLR2+ subsets and clinical biomarkers were evaluated. PA increased circulation of TLR2+ monocytes. TLR2 expression was adversely corelated with reduced diastolic blood pressure (DBP), triglyceride (TG), and matrix metallopeptidase 9 (MMP9) levels. However, regression analysis indicated that only TG levels were independently linked with TLR2+ subsets in circulation in active participants. Higher daily levels of physical activity are associated with improved cardiovascular blood markers and elevated circulatory monocytic TLR2+ subsets. These findings suggest that TLR2 may play a role in modulating CVD risk factors in individuals leading physically active lifestyles.

Low carbohydrate intake correlates with trends of insulin resistance and metabolic acidosis in healthy lean individuals.

Introduction: Both obesity and a poor diet are considered major risk factors for triggering insulin resistance syndrome (IRS) and the development of type 2 diabetes mellitus (T2DM). Owing to the impact of low-carbohydrate diets, such as the keto diet and the Atkins diet, on weight loss in individuals with obesity, these diets have become an effective strategy for a healthy lifestyle. However, the impact of the ketogenic diet on IRS in healthy individuals of a normal weight has been less well researched. This study presents a cross-sectional observational study that aimed to investigate the effect of low carbohydrate intake in healthy individuals of a normal weight with regard to glucose homeostasis, inflammatory, and metabolic parameters. Methods: The study included 120 participants who were healthy, had a normal weight (BMI 25 kg/m2), and had no history of a major medical condition. Self-reported dietary intake and objective physical activity measured by accelerometry were tracked for 7 days. The participants were divided into three groups according to their dietary intake of carbohydrates: the low-carbohydrate (LC) group (those consuming <45% of their daily energy intake from carbohydrates), the recommended range of carbohydrate (RC) group (those consuming 45-65% of their daily energy intake from carbohydrates), and the high-carbohydrate (HC) group (those consuming more than 65% of their daily energy intake from carbohydrates). Blood samples were collected for the analysis of metabolic markers. HOMA of insulin resistance (HOMA-IR) and HOMA of ß-cell function (HOMA-ß), as well as C-peptide levels, were used for the evaluation of glucose homeostasis. Results: Low carbohydrate intake (<45% of total energy) was found to significantly correlate with dysregulated glucose homeostasis as measured by elevations in HOMA-IR, HOMA-ß% assessment, and C-peptide levels. Low carbohydrate intake was also found to be coupled with lower serum bicarbonate and serum albumin levels, with an increased anion gap indicating metabolic acidosis. The elevation in C-peptide under low carbohydrate intake was found to be positively correlated with the secretion of IRS-related inflammatory markers, including FGF2, IP-10, IL-6, IL-17A, and MDC, but negatively correlated with IL-3. Discussion: Overall, the findings of the study showed that, for the first time, low-carbohydrate intake in healthy individuals of a normal weight might lead to dysfunctional glucose homeostasis, increased metabolic acidosis, and the possibility of triggering inflammation by C-peptide elevation in plasma.

Enhanced Anticancer Activity of Nanoformulation of Dasatinib against Triple-Negative Breast Cancer.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer accounting for around 15% of identified breast cancer cases. TNBC lacks human epidermal growth factor receptor 2 (HER2) amplification, is hormone independent estrogen (ER) and progesterone receptors (PR) negative, and is not reactive to current targeted therapies. Existing treatment relies on chemotherapeutic treatment, but in spite of an initial response to chemotherapy, the inception of resistance and relapse is unfortunately common. Dasatinib is an approved second-generation inhibitor of multiple tyrosine kinases, and literature data strongly support its use in the management of TNBC. However, dasatinib binds to plasma proteins and undergoes extensive metabolism through oxidation and conjugation. To protect dasatinib from fast pharmacokinetic degradation and to prolong its activity, it was encapsulated on poly(styrene-co-maleic acid) (SMA) micelles. The obtained SMA-dasatinib nanoparticles (NPs) were evaluated for their physicochemical properties, in vitro antiproliferative activity in different TNBC cell lines, and in vivo anticancer activity in a syngeneic model of breast cancer. Obtained results showed that SMA-dasatinib is more potent against 4T1 TNBC tumor growth in vivo compared to free drug. This enhanced effect was ascribed to the encapsulation of the drug protecting it from a rapid metabolism. Our finding highlights the often-overlooked value of nanoformulations in protecting its cargo from degradation. Overall, results may provide an alternative therapeutic strategy for TNBC management.

Oral Insulin Delivery Using Poly (Styrene Co-Maleic Acid) Micelles in a Diabetic Mouse Model.

The oral delivery of insulin is a convenient and safe physiological route of administration for management of diabetes mellitus. In this study, we developed a poly-(styrene-co-maleic acid) (SMA) micellar system for oral insulin delivery to overcome the rapid degradation of insulin in the stomach, improve its absorption in the intestine, and provide a physiologically-relevant method of insulin to reach portal circulation. The insulin was encapsulated into SMA micelles in a pH-dependent process. The charge and size of the nanoparticles were determined by dynamic light scattering. The insulin loading of the nanoparticles was measured by HPLC. The transport of the SMA-insulin through biological membranes was assessed in vitro using Caco-2 cells, ex vivo rat intestinal section, and in vivo in a streptozotocin-induced diabetes mouse model. SMA-insulin micelles were negatively charged and had a mean diameter of 179.7 nm. SMA-insulin efficiently stimulated glucose uptake in HepG-2 hepatic cells and was transported across the Caco-2 epithelial cells in vitro by 46% and ex vivo across intestinal epithelium by 22%. The animal studies demonstrated that orally-administered SMA-insulin can produce a hypoglycemic effect up to 3 h after administration of one dose. Overall, our results indicate that SMA micelles are capable of the oral delivery of bioactive compounds like insulin and can be effective tools in the management of diabetes.

Nanotechnology in Insulin Delivery for Management of Diabetes.

Diabetes is a group of diseases characterized by hyperglycemia and originating from the deficiency or resistance to insulin, or both. Ultimately, the most effective treatment for patients with diabetes involves subcutaneous injections of insulin. However, this route of administration is often painful and inconvenient, as most patients will have to selfadminister it at least twice a day for the rest of their lives. Also, infection, insulin precipitation, and either lipoatrophy or lipohypertrophy are frequently observed at the site of injection. To date, several alternative routes of insulin administration have been explored, including nasal, pulmonary and oral. Although the delivery of insulin is an ideal route for diabetic patients, several limitations have to be overcome such as the rapid degradation of insulin in gastric fluid and low oral bioavailability. Numerous strategies have been carried out to improve these limited parameters such as the use of enzyme inhibitors, absorption enhancers, mucoadhesive polymers and chemical modification for receptor-mediated absorption. Also, insulin-loaded nanocarriers bypass several physiological barriers. This current review focuses on the various barriers existing in the delivery of insulin through the oral route and the strategies undertaken so far to overcome those obstacles using nanocarriers as a potential vehicle of insulin.

Raloxifene nano-micelles effect on triple-negative breast cancer is mediated through estrogen receptor-ß and epidermal growth factor receptor.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that differs in progression, recurrence, and prognosis from other forms of breast cancer. The heterogeneity of TNBC has remained a challenge as no targeted therapy is currently available. Previously, we and others have demonstrated that raloxifene, a selective oestrogen receptor modulator, was also acting independently of the oestrogen receptor-α. However, raloxifene is characterised by a low bioavailability in vivo. Thus, we encapsulated raloxifene into a styrene-maleic acid (SMA) micelle to improve its pharmacokinetics. The micellar raloxifene had higher cytotoxicity when compared to the free formulation, promoted a higher cellular uptake and affected critical signalling pathways. Furthermore, SMA-raloxifene reduced TNBC tumour growth more efficiently than free raloxifene. Finally, we showed that this effect was partially mediated through oestrogen receptor-ß. In conclusion, we have provided new insight into the role of raloxifene nanoformulation in improving the management of TNBC.

Curcumin⁻Copper Complex Nanoparticles for the Management of Triple-Negative Breast Cancer.

Breast cancer is the most common cancer diagnosed among females worldwide. Although breast cancer survival has largely improved in the past 30 years, it remains highly heterogeneous in its response to treatment. Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of the estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor-2 (Her2). While TNBC may initially be responsive to chemotherapy, recurrence and subsequent high mortality rates are frequently reported. Studies have shown curcumin and its derivatives to be effective against TNBC cell lines in vitro. To improve its anti-cancer effects, we have synthesized Fe3+⁻curcumin (Fe⁻Cur3) and Cu2+⁻curcumin (CD) complexes and investigated them experimentally. Further, CD was encapsulated into a poly(styrene)-co-maleic acid (SMA) micelle to enhance its stability. We assessed the cytotoxicity of these formulations both in vitro and in vivo. SMA⁻CD demonstrated dose-dependent cytotoxicity and abolished TNBC tumor growth in vivo. The encapsulation of the curcumin⁻copper complex improved its anti-cancer activity without overt adverse effects in a murine model of TNBC. These results provide evidence and insights into the value of nanoformulations in enhancing drug-delivery and increasing the potential therapeutic efficacy of curcumin derivatives.