MCP-1 Gene Expression Research Articles

Medin Induces Pro-Inflammatory Activation of Human Brain Vascular Smooth Muscle Cells.

Medin is one of the most common amyloidogenic proteins and accumulates in the vasculature with aging. Vascular medin accumulation is associated with Alzheimer's disease, vascular dementia and aortic aneurysms. Medin impairs smooth muscle-dependent vasodilation in isolated human brain cerebral arteries. The role of medin in vascular smooth muscle (VSMC) activation is unknown. We aim to evaluate the effects of medin on human brain VSMC activation. VSMCs were exposed to physiologic doses of medin (0.5, 1 and 5 µM) without or with small molecule nuclear factor-κB (NFκB) inhibitor RO106-9920 (10 µM) for 20 hours. Polymerase chain reaction, Western blot/enzyme-linked immunosorbent assay were used to quantify gene and protein expressions/secretions, respectively, of pro-inflammatory factors (interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1) and structural and enzyme proteins associated with VSMC phenotypic transformation (smooth muscle actin alpha 2 (ACTA2), myosin heavy chain 11 (MYH11) and NADPH oxidase 4 (NOX4)). Medin exposure increased VSMC gene expression and protein secretion of IL-6, IL-8 and MCP-1 (protein secretion 46.0±12.8x, 20.2±4.1x and 8.7±3.1x, respectively, medin 5 µM versus vehicle, all p<0.05). There was no change in gene or protein expressions of ACTA2, MYH11 and NOX4. Co-treatment with RO106-9920 reduced medin-induced increases in IL-6 and IL-8 and a trend towards reduced MCP-1 secretion. Medin induced pro-inflammatory activation of human brain VSMCs that is mediated, at least in part, by NFκB. Acute medin treatment did not alter structural proteins involved in VSMC phenotypic transformation. The findings support medin as a potential novel mediator of and therapeutic target for vascular aging pathology.

Metformin Improves the Function of Abdominal Aortic Aneurysm Patient-Derived Aortic Smooth Muscle Cells

Type 2 diabetes mellitus (T2DM) is a cardiovascular risk factor. Paradoxically, a decreased risk of abdominal aortic aneurysm (AAA) presence and growth rate is described among patients with T2DM, associated with metformin use. This study aimed to investigate the effect of metformin on AAA patient derived aortic smooth muscle cell (SMC) function. Aortic biopsies were obtained from patients with AAA (n = 21) and controls (n = 17) during surgery. The SMCs of non-pathological aortic controls, non-diabetic patients with AAA, and diabetic patients with AAA were cultured from explants and treated with or without metformin. The SMC contractility was measured upon ionomycin stimulation, as well as metabolic activity, proliferation, and migration. Then, mRNA and protein expression of markers for contraction, metabolic activity, proliferation, and inflammation were measured. The mRNA expression of KLF4 and GYS1, genes involved in metabolic activity, differed between the SMCs from non-diabetic and diabetic patients with AAA before metformin stimulation (p < .041). However, the effect of metformin on the various SMC functions was similar between non-diabetic and diabetic patients with AAA. Upon stimulation, metformin increased the contractility of AAA patient SMCs (p = .001). The mRNA expression of smoothelin, a marker for the contractile phenotype, increased in the SMCs of patients with AAA after treatment with metformin (p = .006). An increase in metabolic activity (p < .001) and a decrease in proliferation (p < .001) and migration were found in the SMCs of controls and patients with AAA with metformin. Increased mRNA expression of PPARγ, a nuclear receptor involved in mitochondrial biogenesis (p < .009), and a decrease in gene expression of Ki-67, a marker for proliferation (p < .005), were observed. Gene expression of inflammation markers MCP-1 and IL-6, and protein expression of NF-κB p65 decreased after treatment with metformin in patients with AAA. This study found that metformin increases contractility and metabolic activity, and reduces proliferation, migration, and inflammation in aortic SMCs in vitro.

The adipocyte apolipoprotein M is negatively associated with inflammation

Obesity is associated with the development of local adipose tissue (AT) and systemic inflammation. Most adipokines are upregulated with obesity and have pro-inflammatory properties. Few are downregulated and possess beneficial anti-inflammatory effects. The apolipoprotein M (APOM) is an adipokine whose expression is low during obesity and associated with a metabolically healthy AT. Here, the role of adipose-derived APOM on obesity-associated AT inflammation was investigated by measuring the expression of pro-inflammatory genes in human and mouse models. In 300 individuals with obesity, AT APOM mRNA level was negatively associated with plasma hs-CRP. The inflammatory profile was assessed in Apom−/− and WT mice fed a normal chow diet (NCD), or a high-fat diet (HFD) to induce AT inflammation. After HFD, mice had a higher inflammatory profile in AT and liver, and a 50% lower Apom gene expression compared with NCD-fed mice. Apom deficiency was associated with a higher inflammatory signature in AT compared with WT mice but not in the liver. Adeno-associated viruses encoding human APOM were used to induce APOM overexpression: in vivo, in WT mice AT prior to HFD; in vitro, in human adipocytes which conditioned media was applied to ThP-1 macrophages. The murine AT overexpressing APOM gene had a reduced inflammatory profile. The macrophages treated with APOM-enriched media from adipocytes exhibited lower IL6 and MCP1 gene expression compared with macrophages treated with control media, independently of S1P. Our study highlights the protective role of adipocyte APOM against obesity-induced AT inflammation.

Gingival fibroblasts produce paracrine signals that affect osteoclastogenesis in vitro

In periodontitis, gingival fibroblasts (GF) appear to produce a multitude of paracrine factors. However, the influence of GF-derived soluble factors on osteoclastogenesis remains unclear. In this case study, production of paracrine factors by GF was assessed under inflammatory and non-inflammatory conditions, as well as their effect on osteoclastogenesis. Human primary GF were cultured in a transwell system and primed with a cocktail of IL-1β, IL-6 and TNF-α to mimic inflammation. GF were co-cultured directly and indirectly with human peripheral blood mononuclear cells (PBMC). Cytokines and chemokines in supernatants (flow cytometry based multiplex assay), osteoclastogenesis (TRAcP staining) and gene expression (qPCR) were quantified on days 7 and 21.Results from this case study showed that GF communicated via soluble factors with PBMC resulting in a two-fold induction of osteoclasts. Reversely, PBMC induced gene expression of IL-6, OPG and MCP-1 by GF. Remarkably, after priming of GF with cytokines, this communication was impaired and resulted in fewer osteoclasts. This could be partly explained by an increase in IL-10 expression and a decrease in MCP-1 expression. Intriguingly, the short priming of GF resulted in significantly higher expression of inflammatory cytokines that was sustained at both 7 and 21 days.GF appear to produce paracrine factors capable of stimulating osteoclastogenesis in the absence of physical cell-cell interactions. GF cultured in the presence of PBMC or osteoclasts had a remarkably inflammatory phenotype. Given profound expression of both pro- and anti-inflammatory cytokines after the inflammatory stimulus, it is probably the effector hierarchy that leads to fewer osteoclasts.

Laminin 332 functionalized surface improve implant roughness and oral keratinocyte bioactivity

ObjectiveThe biological seal (BS) at the implant-tissue interface is essential for the success of dental implants (DIs), and the absence of a proper BS can lead to peri-implantitis. The basement membrane (BM) and junctional epithelium are critical for sealing the peri-implant mucosa, and laminin 332 is an important protein in binding the epithelium to the implant surface. The aim of this study was to evaluate the response of oral keratinocytes to titanium dental implant surfaces biofunctionalized with laminin 332. DesignThe dental implant surface was treated with a piranha solution to create hydroxyl (OH) groups, facilitating biofunctionalization with laminin 332. The modified surface underwent scanning electron microscopy, surface roughness evaluation, and chemical composition analysis. Human keratinocytes from the Cal-27 line were then cultured on the modified implants for 24 and 48 h to assess viability, morphology, cytokine secretion, and mRNA expression of tissue repair-associated genes. ResultsThe results showed that laminin 332 biofunctionalization of the implant surface resulted in lower values of Ra, Rq and positive surface roughness parameters Rsk, Rku and Rv. The elemental composition showed an increase in nitrogen and carbon content corresponding to protein binding. The biofunctionalized surfaces did not affect cell viability and promoted cytokine secretion (IL-1a and IL-8) and a significant increase (p < 0.05) in MCP-1, EGF, FGF, TGF and VEGF gene expression compared to the control. ConclusionIn conclusion, laminin 332 coating Ti implants was shown to be effective in promoting keratinocyte adhesion, spreading, and viability. This approach could be an alternative way to improve biocompatibility.

Optimization of Naringenin-loaded nanoparticles for targeting of Vanin-1, iNOS, and MCP-1 signaling pathway in HFD-induced obesity

Naringenin, a natural dihydrochalcone flavonoid, exhibits diverse pharmacological properties. This study investigates the hypolipidemic effects of Nar-NPs on obese mice. The characteristics of Nar-NPs, including morphology, particle size, zeta potential, UV–vis, and FT-IR spectra, were examined. The anti-obesity properties of Nar-NPs were evaluated in obese rats, considering LD50, 1/20 LD50, and 1/50 LD50 for treatment preparation. Results indicated that synthesized Nar-NPs were uniform, spherical, and well-dispersed, with a size of 130.06 ± 1.98 nm and with a zeta potential of −25.6 ± 0.8 mV. Nar-NPs exhibited enhancement in the cumulative release of naringenin (56.87 ± 2.45 %) as compared to pure naringenin suspension 87.83 ± 1.84 % in 24 h of the study. The LD50 of Nar-NPs was determined as 412.5 mg/kg.b.w. HFD induced elevated glycemic, oxidative stress, and inflammatory biomarkers while reducing HDL-C, GSH, and superoxide dismutase (SOD) levels. Administration of Nar-NPs significantly mitigated body weight, glucose, insulin, leptin, TC, TG, SREBP1c, pAMPK, PPAR-α, as well as vanin-1, MCP-1, and iNOS mRNA gene expression. Histological investigations supported the biochemical and PCR findings. In a nutshell, the study suggests that the Nar-NPs could serve as a promising and viable pharmacological strategy for the treatment of obesity-related disorders.

Anti-inflammatory Effects of Food Ingredients on Mice Adipose Tissues and Adipocytes.

In 2021, we published three papers related to the anti-inflammatory effects of food ingredients. The present paper reports the effects of vitamin E homologs and sweet basil powder. In these papers, we investigated whether inflammation occurs in the adipose tissue of mice fed a high-fat and high-sucrose diet for 16 weeks. Inflammatory cytokine gene expression was significantly higher in the epididymal fat of the high-fat and high-sucrose diet group than in that of the control diet group. However, the addition of α-tocopherol or δ-tocopherol to the diet could not restrain the inflammation of mice epididymal fats. Thereafter, we investigated the anti-inflammatory effects of α- and δ-tocopherols using the co-cultured cells. Consequently, we clarified that δ-tocopherol inhibited the increase in the gene expressions of inflammatory cytokines. We also examined the effect of sweet basil powder on a similar obese mice model. The final body weight in the high-fat and high-sucrose group that received sweet basil powder was significantly lower than that in the high-fat and high-sucrose diet group. Liver weights were also significantly lower in the high-fat and high-sucrose diet group that received sweet basil powder than in the high-fat and high-sucrose diet group, although adipose tissue weights were unchanged in both groups. Furthermore, sweet basil powder tended to inhibit in lipid synthesis in the mice livers. Therefore, we suggested that sweet basil powder inhibited fatty acid synthesis in mice livers, thereby suppressing liver enlargement, and resulting in body weight loss. Moreover, the gene expression of MCP-1 in the adipose tissue of mice fed a high-fat and high-sucrose diet added with sweet basil powder was significantly lower than that of mice fed a high-fat and high-sucrose diet for 12 weeks. Therefore, sweet basil powder inhibited inflammation onset in the adipose tissue of mice. Taken together, the results suggested that food ingredients, especially vitamin E homologs and sweet basil powder, have anti-inflammatory effects on mice adipose tissue and mice adipocyte-induced inflammation.

Discovery of a First-in-Class GPR183 Antagonist for the Potential Treatment of Rheumatoid Arthritis.

GPR183 is required for humoral immune responses, and its polymorphisms have been associated with inflammatory autoimmune diseases. Despite increasing attention to GPR183 as a potential therapeutic target for autoimmune diseases, relatively few antagonists have been reported, and none of them have progressed to the clinical stage. In this study, we discovered a highly potent GPR183 antagonist, compound 32, with good aqueous solubility, excellent selectivity, and pharmacokinetic properties. Meanwhile, compound 32 showed exceptional efficacy for rheumatoid arthritis (RA) disease in a mouse collagen-induced arthritis (CIA) model, with an efficacious dose of 0.1 mg/kg. Functionally, compound 32 significantly reduced the swelling of paws and joints, the gene expression of proinflammatory cytokines, MCP-1, MMPs, and VEGF, inflammatory cell infiltration, cartilage damage, pannus formation, and bone erosion in the joints of CIA mice in a dose-dependent manner. Hence, these findings suggest compound 32 as a valuable molecule for further development.

Ficolin-2 amplifies inflammation in macrophage-smooth muscle cell cross-talk and increases monocyte transmigration by mechanisms involving IL-1β and IL-6

Ficolin-2, recently identified in atherosclerotic plaques, has been correlated with future acute cardiovascular events, but its role remains unknown. We hypothesize that it could influence plaque vulnerability by interfering in the cross-talk between macrophages (MØ) and smooth muscle cells (SMC). To examine its role and mechanism of action, we exposed an in-vitro co-culture system of SMC and MØ to ficolin-2 (10 µg/mL) and then performed cytokine array, protease array, ELISA, qPCR, Western Blot, and monocyte transmigration assay. Carotid plaque samples from atherosclerotic patients with high plasma levels of ficolin-2 were analyzed by immunofluorescence. We show that ficolin-2: (i) promotes a pro-inflammatory phenotype in SMC following interaction with MØ by elevating the gene expression of MCP-1, upregulating gene and protein expression of IL-6 and TLR4, and by activating ERK/MAPK and NF-KB signaling pathways; (ii) increased IL-1β, IL-6, and MIP-1β in MØ beyond the level induced by cellular interaction with SMC; (iii) elevated the secretion of IL-1β, IL-6, and CCL4 in the conditioned medium; (iv) enhanced monocyte transmigration and (v) in atherosclerotic plaques from patients with high plasma levels of ficolin-2, we observed co-localization of ficolin-2 with SMC marker αSMA and the cytokines IL-1β and IL-6. These findings shed light on previously unknown mechanisms underlying ficolin-2–dependent pathological inflammation in atherosclerotic plaques.

Dienogest does not augment the gene expression of adhesion molecules, MCP-1, and monocyte adherence in human endothelial cells

Objective Medroxyprogesterone acetate (MPA) may increase the risk of atherosclerosis during hormone replacement therapy (HRT); therefore, the effect of progestogens other than MPA on atherosclerotic lesions requires evaluation. Adhesion of monocytes to vascular endothelial cells is an important early step in atherosclerosis progression. MCP-1 is a key chemokine that promotes monocyte migration and adhesion to vascular endothelial cells. In this study, we investigated the effects of dienogest (DNG), an alternative progestogen, on monocyte adhesion and cytokine expression in human umbilical vein endothelial cells (HUVECs). Study Design HUVECs were treated with DNG, natural progesterone, or MPA, followed by interleukin (IL)-1β stimulation. The mRNA expression of adhesion molecules (E-selectin and ICAM-1) and cytokines (MCP-1 and IL-6) was examined using real-time PCR. A flow chamber system was used to examine the effect of DNG on the adhesion of U937 monocytic cells to monolayer HUVECs. Results Unlike MPA, DNG did not alter the mRNA expression of E-selectin, ICAM-1, MCP-1, and IL-6 in HUVECs. Moreover, it did not increase the number of monocytes adhering to HUVECs in the flow chamber system. However, MPA treatment significantly enhanced monocyte adhesion to HUVECs (p < 0.05). Conclusions DNG had no effect on the mRNA expression of adhesion molecules and cytokines in HUVECs, as well as the monocyte adhesion to HUVECs, suggesting that DNG can be explored as an alternative to MPA for HRT.

Inhibitory Effects of Simvastatin on IL-33-Induced MCP-1 via the Suppression of the JNK Pathway in Human Vascular Endothelial Cells.

An alarmin, interleukin (IL)-33 is a danger signal that causes inflammation, inducing chemotactic proteins such as monocyte chemoattractant protein (MCP)-1 in various cells. As statins have pleiotropic actions including anti-inflammatory properties, we investigated the effects of simvastatin on IL-33-induced MCP-1 expression in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated with IL-33 in the presence or absence of simvastatin. Gene expression and protein secretion of MCP-1, phosphorylation of mitogen-activated protein kinase (MAPK), nuclear translocation of phosphorylated c-Jun, and human monocyte migration were investigated. Immunocytochemical staining and Western immunoblot analysis revealed that IL-33 augmented MCP-1 protein expression in HUVECs. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) showed that IL-33 significantly increased MCP-1 mRNA and protein secretion, which were suppressed by c-jun N-terminal kinase (JNK) inhibitor SP600125 and p38 MAPK inhibitor SB203580. Simvastatin inhibited IL-33-induced MCP-1 mRNA, protein secretion, phosphorylation of JNK and c-Jun. Additionally, the IL-33-induced nuclear translocation of phosphorylated c-Jun and THP-1 monocyte migration were also blocked by simvastatin. This study demonstrated that IL-33 induces MCP-1 expression via the JNK and p38 MAPK pathways in HUVECs, and that simvastatin inhibits MCP-1 production by selectively suppressing JNK. Simvastatin may inhibit the progression of IL-33-induced inflammation via suppressing JNK to prevent MCP-1 production.

Growth hormone-releasing hormone antagonist MIA-602 inhibits inflammation induced by SARS-CoV-2 spike protein and bacterial lipopolysaccharide synergism in macrophages and human peripheral blood mononuclear cells.

COVID-19 is characterized by an excessive inflammatory response and macrophage hyperactivation, leading, in severe cases, to alveolar epithelial injury and acute respiratory distress syndrome. Recent studies have reported that SARS-CoV-2 spike (S) protein interacts with bacterial lipopolysaccharide (LPS) to boost inflammatory responses in vitro, in macrophages and peripheral blood mononuclear cells (PBMCs), and in vivo. The hypothalamic hormone growth hormone-releasing hormone (GHRH), in addition to promoting pituitary GH release, exerts many peripheral functions, acting as a growth factor in both malignant and non-malignant cells. GHRH antagonists, in turn, display potent antitumor effects and antinflammatory activities in different cell types, including lung and endothelial cells. However, to date, the antinflammatory role of GHRH antagonists in COVID-19 remains unexplored. Here, we examined the ability of GHRH antagonist MIA-602 to reduce inflammation in human THP-1-derived macrophages and PBMCs stimulated with S protein and LPS combination. Western blot and immunofluorescence analysis revealed the presence of GHRH receptor and its splice variant SV1 in both THP-1 cells and PBMCs. Exposure of THP-1 cells to S protein and LPS combination increased the mRNA levels and protein secretion of TNF-α and IL-1β, as well as IL-8 and MCP-1 gene expression, an effect hampered by MIA-602. Similarly, MIA-602 hindered TNF-α and IL-1β secretion in PBMCs and reduced MCP-1 mRNA levels. Mechanistically, MIA-602 blunted the S protein and LPS-induced activation of inflammatory pathways in THP-1 cells, such as NF-κB, STAT3, MAPK ERK1/2 and JNK. MIA-602 also attenuated oxidative stress in PBMCs, by decreasing ROS production, iNOS and COX-2 protein levels, and MMP9 activity. Finally, MIA-602 prevented the effect of S protein and LPS synergism on NF-кB nuclear translocation and activity. Overall, these findings demonstrate a novel antinflammatory role for GHRH antagonists of MIA class and suggest their potential development for the treatment of inflammatory diseases, such as COVID-19 and related comorbidities.

#3566 ACSS2 AGGRAVATES RENAL TUBULAR INFLAMMATION BY INDUCING THE DYSREGULATION OF FATTY ACID METABOLISM AND MITOCHONDRIAL OXIDATIVE STRESS IN DIABETIC MICE

Abstract Background and Aims Treatment options for diabetic nephropathy (DN) are currently limited. Chronic renal inflammation has been widely recognized as a major promoter of DN. The tubular epithelial cells are a major source of IL-1β in diabetic db/db mice. Inhibition of Acetyl CoA Synthase 2 (ACSS2) has been shown to promote high-fat diet-induced liver injuries, but the underlying mechanism of action of ACSS2 in diabetic renal injuries has not been elucidated. Method Streptozotocin (STZ) was intraperitoneally injected to establish the diabetic mouse model. We investigated the effects of ACSS2 on diabetes-induced renal tubular injury in transgenic mice with global acss2 knockout (acss2−/−). PAS staining was carried out to evaluate pathological injury of kidneys in mice. The renal tissue immunostaining was conducted to detect IL-1β expression and macrophage distribution in kidney tissues of mice. HK-2 cell culture model was achieved by high glucose stimulation in vitro. Real-time PCR analysis was applied to evaluate gene expression of inflammatory factors IL-1β, TNF-α, and MCP-1. Western Blot and immunofluorescence staining were conducted to the activation of the NLRP3 pathway and mitochondrial reactive oxygen species (ROS). Results ACSS2 was upregulated in STZ-induced diabetic renal tubular cells, which is significantly co-expressed in IL-1β-positive renal tubular cells of STZ-induced diabetic mice. The mice with acss2−/- exhibited decreased renal tubular inflammation and inhibited NOD-like receptor protein 3 (NLRP3) inflammasome activation as evidenced by elevated protein levels of NLRP3 and cleaved caspase-1 when compared with diabetic wild-type mice. The selective inhibition of ACSS2 protected HK-2 cells in vitro when exposed to a high glucose environment by downregulating NLRP3 inflammasome and ameliorating mitochondrial oxidative stress. Mechanistically, we showed that mitochondrial dysfunction preceded inflammatory response in vitro. The elimination of mitochondrial ROS using mitochondria-targeted antioxidants (mito-TEMPO) reversed HG-mediated NLRP3 inflammation activation in HK-2 cells, which recapitalized the phenotype observed in acss2-/- mice. We also found that cytoplasm ACSS2, which was increased after HG stimulation, induced fatty acid synthase (FASN) mediated overproduction of free fatty acid. Downregulating FASN using FASN-small interfering RNA restored mitochondrial function, and suppressed NLRP3 inflammasome activation. Conclusion In conclusion, diabetes-induced ACSS2 upregulation led to subsequent FASN-mediated dysregulation of fatty acid metabolism and mitochondrial ROS overproduction, eventually causing NLRP3 inflammasome activation and renal inflammation. Therefore, our findings established the role of ACSS2 in diabetes-induced inflammation of renal tubular epithelium and unveiled the underlying mechanism of its action. ACSS2 appears as a promising target for treating diabetic renal tubular epithelial inflammation.

Elastin-targeted nanoparticles delivering doxycycline mitigate cytokine storm and reduce immune cell infiltration in LPS-mediated lung inflammation.

Cytokine storm invoked during acute and chronic lung injury promotes alveolar damage and remodeling. The current study shows that degraded elastin-targeted nanoparticles releasing doxycycline (Doxy NPs) are potent in mitigating cytokines storm, migration of immune cells in the lungs, and inhibiting inflammasome pathways in the LPS mouse model. Cytokine storm and lung injury were induced using LPS and elastase in C57BL/6 mice (rodent model for emphysema). The mice were then treated with I.V. Doxy NPs, blank NPs, or Doxy a day before LPS administration. Cytokine levels, immune cell population, and MMP activity were analyzed in broncheo-alveolar lavage fluid (BALF) 4 hours after LPS administration. Additionally, gene expression of IL-6, IL-1beta, MCP-1, NLRP3, Caspase 1 and MMPs were investigated in alveolar cells on day 3 after LPS administration. Doxycycline NPs but not Doxycycline significantly decreased IL-6, TNF-α, IL-23 and were significantly more effective in decreasing the percentage of immune cells in the BALF. This is the first in-vivo study to demonstrate that Doxycycline can effectively inhibit inflammasome pathways in the lungs. IV administration of elastin antibody conjugated Doxycycline-loaded albumin NPs can effectively modulate the local immune environment in the lungs, which is not achieved by IV Doxycycline even at 100-fold higher dose. This novel method of drug delivery can effectively lead to the repurposing of traditional Doxycycline as a potential adjunct treatment for managing the cytokine storm in the lungs in COPD and viral infections.

Extracellular vesicles derived from the intestinal microbiota of patients with CGD-associated inflammatory bowel disease have pro-inflammatory effects

BackgroundChronic granulomatous disease (CGD) is an inborn error of immunity characterized by mutations in any one of the 6 subunits forming the nicotinamide-adenine dinucleotide phosphate oxidase complex 2 leading to defective phagocyte-derived ROS production. As a result, patients with CGD have recurrent infections and increased inflammation and 50% of patients with CGD have inflammatory bowel disease (CGD-IBD). Although we have previously demonstrated that patients with CGD-IBD have intestinal dysbiosis, its functional impact is poorly understood. Interestingly, intestinal microbes can produce gut microbiota-derived extracellular vesicles (GMEVs) which can cross the intestinal barrier and have systemic and/or end-organ immunomodulatory effects. Yet, the role of GMEVs in patients with CGD has never been evaluated. ObjectiveTo investigate the impact of GMEVs isolated from patients with CGD, with or without CGD-IBD, on intestinal permeability and inflammatory responses using wild type and NOX2-deficient (CYBB-/-) models. MethodsGMEVs were isolated and characterized from fecal samples obtained from 11 patients without CGD-IBD and 11 patients with active CGD-IBD recruited at the National Institutes of Health. Stimulation with GMEVs was performed on HT29, THP1 and CYBB-/- THP1 cell lines, HT29/THP1 co-culture models, and induced pluripotent stem cell-derived human intestinal organoids. TNF-α, IL-6, IL-8, IL-1β, IP-10, GM-CSF and MCP1 gene expression and production were assessed by RT-qPCR and ELISA, respectively. ResultsOur data showed that stimulation with GMEVs from patients with active CGD-IBD increased intestinal permeability compared to cells stimulated with GMEVs from patients without CGD-IBD. In HT29 cells, increased intestinal permeability was associated with increased gene expression and production of IL-8 and IL-1β. IL-8 was similarly increased in intestinal organoids. In THP1 cells, stimulation led to increased gene expression and production of TNF-α, IL-6 and IP-10, while stimulation in CYBB-/- THP1 cells increased TNF-α and MCP1 gene expression. Finally, stimulation of HT29/THP1 cell co-cultures resulted in increased gene expression of TNF-α, IL-6 and GM-CSF. ConclusionsOur results suggest that GMEVs isolated from patients with CGD-IBD may induce pro-inflammatory immune responses in patients with CGD, thereby contributing to their underlying inflammatory pathology. These findings bring forward GMEVs as a possible new therapeutic target for CGD-IBD. [Display omitted]

Role of altered cortisol production in the development of sarcopenia

Skeletal muscle mass and function is decreased as we age, a condition known as sarcopenia. Ageing is associated with increases in minimum and mean cortisol plasma levels and shortening of the evening cortisol quiescent period, suggesting impaired circadian function and higher basal levels with increasing age (Stamou et al, 2023). It has also been shown that the deleterious effects of bed rest on human skeletal muscle are exacerbated by hypercortisolemia (Fitts et al, 2007).The aim of this study was to examine the direct effects of increasing levels of cortisol on muscle cells. C2C12 myoblasts were fused to myotubes by serum deprivation for up to 7 days. Cellular viability was assessed using light-microscopy to identify any gross changes in cell morphology at 3, 24 and 48 hours following treatment with a range of cortisol concentrations in the physiological range (10 – 0.1uM). Indices of muscle atrophy and evidence of altered levels of inflammatory cytokines was determined by mRNA analyses and release of cytokines/chemokines by myotubes was assessed using bead based multiplex technology (Luminex immunoassay).Cortisol treatment had no significant effect on C2C12 myotube viability at any concentration or time point, or on gene expression of IL-6, RANTES (CCL5) or MCP-1 (CCL2) compared with control untreated myotubes. No increase in the release of cytokines and chemokines from C2C12 myotubes was seen following treatment with cortisol and in some instances a significant decrease in cytokine/chemokine production was seen, particularly following treatment with higher concentrations of cortisol when compared with control values (eg, 10uM cortisol, 24 hours post-treatment: IL-6: 1.6 +/- 1.6% CXCL5: 17.4 +/- 1.8%; KC (CXCL1): 11.6 +/-10.3%. These data are in contrast to previous work from our group which revealed an increase in the release of IL-6, CXCL-1 (KC), CCL2 (MCP-1) and CCL5 (RANTES) from C2C12 myotubes following treatment of myotubes 25ng/ml TNF-α (Lightfoot et al, 2015).In summary, increased levels of cortisol do not appear to have any direct deleterious effects on myotube viability or production of pro-inflammatory cytokines. However, this does not exclude any indirect effects of cortisol on muscle including the effects on other organs indirectly affecting muscle function. It may also be that cortisol has a deleterious effect when muscle is under stressful conditions and this possibility will also be examined. Fitts et al (2007). doi: 10.1152/ajpcell.00573.2006 ; Lightfoot et al. (2015) doi: 10.1016/j.redox.2015.08.007 ; Stamou et al (2023) doi.org/10.1016/j.maturitas.2022.10.006 . The authors would like to thank the EU/BBSRC and CIMA for generous funding. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dihydrotestosterone, and Not Testosterone, Enhances the LPS-Induced Inflammatory Cytokine Gene Expression in Human Adipocytes

The development of obesity-related complications lies in the low-grade inflammatory state consequent to adipocyte dysfunction. The direct involvement of sex hormones in adipose tissue inflammation has been previously suggested, but the evidence is scarce. In this study, we evaluated the effects of sex steroids on the in-vitroexpression of inflammatory mediators in human-derived adipocytes before and after lipopolysaccharide (LPS) exposure. Human adipocytes were differentiated from the vascular stromal fraction of adipose tissue samples of subjects undergoing abdominoplasty. We evaluated MCP-1, IL-1β, IL-6, and TNF-α gene expression in the presence of the main sex steroids, testosterone (T), and 17β-estradiol (E). Furthermore, we analyzed the effects of adipocytes exposure to the non-aromatizable androgen dihydrotestosterone (DHT), together with the effects of adipocytes pre-incubation with the aromatase inhibitor anastrozole alone (A), and in combination with T (A/T) before incubation with LPS. DHT, but not T, significantly enhanced the LPSinduction of MCP-1, IL-1β, IL-6, and TNF-α. Intriguingly, the exposure of adipocytes with A/T dramatically increased the LPS-induced expression of all considered inflammatory cytokines, even more than a hundred-fold. DHT and A/T dramatically enhance LPS-induced inflammatory cytokine expression in human-derived adipocytes. These results confirm the involvement of sex hormones in adipose tissue inflammation, suggesting a specific role for non-aromatizable androgens as the amplificatory sex hormones of the inflammatory response.

Cardiomyocyte-specific deletion of TLR4 attenuates angiotensin II-induced hypertension and cardiac remodeling.

Toll-like receptor 4 (TLR4) is an integral factor in the initiation of the innate immune response and plays an important role in cardiovascular diseases such as hypertension and myocardial infarction. Previous studies from our lab demonstrated that central TLR4 blockade reduced cardiac TLR4 expression, attenuated hypertension, and improved cardiac function. However, the contribution of cardiac specific TLR4 to the development of hypertension and cardiac remodeling is unknown. Therefore, we hypothesized that cardiomyocyte specific knockdown of TLR4 would have beneficial effects on hypertension, cardiac hypertrophy, and remodeling. To test this hypothesis, cardiomyocyte-specific TLR4 knockdown (cTLR4KO) mice were generated by crossing floxed TLR4 mice with Myh6-Cre mice, and subjected to angiotensin II (Ang II, 1 µg/kg/min or vehicle for 14 days) hypertension model. Blood pressure measurements using radio telemetry revealed no differences in baseline mean arterial pressure between control littermates and cTLR4KO mice (103 ± 2 vs. 105 ± 3 mmHg, p > 0.05). Ang II-induced hypertension (132 ± 2 vs. 151 ± 3 mmHg, p < 0.01) was attenuated and cardiac hypertrophy (heart/body weight; 4.7 vs. 5.8 mg/g, p < 0.01) was prevented in cTLR4KO mice when compared with control mice. In addition, the level of myocardial fibrosis was significantly reduced, and the cardiac function was improved in cTLR4KO mice infused with Ang II. Furthermore, cardiac inflammation, as evidenced by elevated gene expression of TNF, IL-6, and MCP-1 in the left ventricle, was attenuated in cTLR4KO mice infused with Ang II. Together, this data revealed a protective role for cardiomyocyte-specific deletion of TLR4 against Ang II-induced hypertension and cardiac dysfunction through inhibition of proinflammatory cytokines.

Endothelial cell CD36 mediates stroke-induced brain injury via BBB dysfunction and monocyte infiltration in normal and obese conditions

CD36 expressed in multiple cell types regulates inflammation, vascular function, and innate immunity. Specifically, CD36 in microvascular endothelial cells (ECs) signals to elicit inflammation and causes EC death. This study investigated roles for EC-CD36 on acute stroke pathology in normal and obese conditions. Obesity induced by a high-fat diet (HD) selectively increased CD36 expression in ECs, not in monocytes/macrophages, in the post-ischemic brain. Mice deficient CD36 in ECs (ECCD36−/−) showed reduced injury size and vascular permeability in normal conditions. While control mice fed a HD developed obesity and aggravated stroke injury, ECCD36−/− mice were resistant to develop an obesity phenotype. Subjecting ECCD36−/− mice to stroke resulted in reduced injury size and BBB disruption. Moreover, the mice had reduced MCP-1 and CCR2 gene expression, resulting in reduced monocyte trafficking with improved survival and acute motor function. Reduced MCP-1 and CCR2 expression was still evident in ECCD36−/− mice subjected to severe stroke, suggesting that monocyte trafficking is an infarct-independent metabolic effect associated with specific EC-CD36 deletion. Our findings demonstrate the importance of EC-CD36 in developing vascular comorbidities and suggest that targeting EC-CD36 is a potential preventative strategy to normalize vascular risk factors, leading to improved acute stroke outcomes.

L-fucose reduces gut inflammation due to T-regulatory response in Muc2 null mice.

Fucose, the terminal glycan of the intestinal glycoprotein Mucin2, was shown to have an anti-inflammatory effect in mouse colitis models and modulate immune response due to macrophage polarization changes. In this study we evaluated the effect of 0.05% L-fucose supplementation of drinking water on immune parameters in the intestine of homozygous mutant Muc2-/-, compared to Muc2+/+ mice. To get into innate and adaptive immunity mechanisms of gut inflammation, we tested PrkdcSCIDMuc2-/- strain, Muc2 knockout on SCID background, that is characterized by lack of lymphocytes, in comparison with PrkdcSCID mice. We evaluated intestinal cytokine profiling, macrophage and eosinophil infiltration, and expression of Nos2 and Arg1 markers of macrophage activation in all strains. Markers of Th1, Treg and Th17 cells (Tbx21, Foxp3, and Rorc expression) were evaluated in Muc2-/- and Muc2+/+ mice. Both Muc2-/- and PrkdcSCIDMuc2-/- mice demonstrated increased numbers of macrophages, eosinophils, elevated levels of TNFa, GM-CSF, and IL-10 cytokines. In Muc2-/- mice we observed a wide range of pro-inflammatory cytokines elevated, such as IFN-gamma, IL-1b, IL-12p70, IL-6, M-CSF, G-CSF, IL-17, MCP-1, RANTES, MIP1b, MIP2. Muc2-/- mice demonstrated increase of Nos2, Tbx21 and Foxp3 genes mRNA, while in PrkdcSCIDMuc2-/- mice Arg1 expression was increased. We found that in Muc2-/- mice L-fucose reduced macrophage infiltration and IL-1a, TNFa, IFNgamma, IL-6, MCP-1, RANTES, MIP1b levels, decreased Nos2 expression, and induced the expression of Treg marker Foxp3 gene. On the contrary, in PrkdcSCIDMuc2-/- mice L-fucose had no effect on macrophage and eosinophil numbers, but increased TNFa, GM-CSF, IL-12p70, IL-6, IL-15, IL-10, MCP1, G-CSF, IL-3 levels and Nos2 gene expression, and decreased Arg1 gene expression. We demonstrated that anti-inflammatory effect of L-fucose observed in Muc2-/- mice is not reproduced in PrkdcSCIDMuc2-/-, which lack lymphocytes. We conclude that activation of Treg cells is a key event that leads to resolution of inflammation upon L-fucose supplementation in Muc2-/- mice.