The influence of association between aging and reduced protein intake on some immunomodulatory aspects of bone marrow mesenchymal stem cells: an experimental study.

PURPOSE: Dietary protein deficiency is common in the elderly, compromising hematopoiesis and the immune response, and may cause a greater susceptibility to infections. Mesenchymal stem cells (MSCs) have immunomodulatory properties and are essential to hematopoiesis. Therefore, this study aimed to investigate, in an aging model subjected to malnutrition due a reduced protein intake, aspects related to the immunomodulatory capacity of MSCs. METHODS: Male C57BL/6 mice from young and elderly groups were fed with normoproteic or hypoproteic diets (12% and 2% of protein, respectively) and nutritional, biochemical and hematological parameters were evaluated. MSCs from bone marrow were isolated, characterized and their secretory parameters evaluated, along with gene expression. Additionally, the effects of aging and protein malnutrition on MSC immunomodulatory properties were assessed. RESULTS: Malnourished mice lost weight and demonstrated anemia, leukopenia, and bone marrow hypoplasia. MSCs from elderly animals from both groups showed reduced CD73 expression and higher senescence rate; also, the malnourished state affected CD73 expression in young animals. The production of IL-1ß and IL-6 by MSCs was affected by aging and malnutrition, but the IL-10 production not. Aging also increased the expression of NFκB, reducing the expression of STAT-3. However, MSCs from malnourished groups, regardless of age, showed decreased TGF-ß and PGE2 production. Evaluation of the immunomodulatory capacity of MSCs revealed that aging and malnutrition affected, mainly in lymphocytes, the production of IFN-γ and IL-10. CONCLUSION: Aging and reduced protein intake are factors that, alone or together, influence the immunomodulatory properties of MSCs and provide basic knowledge that can be further investigated to explore whether MSCs' therapeutic potential may be affected.

Effects of different branched-chain amino acids supplementation protocols on the inflammatory response of LPS-stimulated RAW 264.7 macrophages.

Although branched-chain amino acids (BCAA) are commonly used as a strategy to recover nutritional status of critically ill patients, recent findings on their role as immunonutrients have been associated with unfavorable outcomes, especially in obese patients. The present study aimed to explore the effects of different BCAA supplementation protocols in the inflammatory response of LPS-stimulated RAW 264.7 macrophages. Cell cultures were divided into five groups, with and without BCAA supplementation, (2 mmol/L of each amino acid). Then, cell cultures followed three different treatment protocols, consisting of a pretreatment (PT), an acute treatment (AT), and a chronic treatment (CT) with BCAA and LPS stimulation (1 µg/mL). Cell viability was analyzed by MTT assay, NO production was assessed by the Griess reaction and IL-6, IL-10, TNF-α and PGE2 synthesis, was evaluated by ELISA. BCAA significantly increased cell viability in AT and CT protocols, and NO and IL-10 synthesis in all treatment protocols. IL-6 synthesis was only increased in PT and CT protocols. TNF-α and PGE2 synthesis were not altered in any of the protocols and groups. BCAA supplementation was able to increase both pro and anti-inflammatory mediators synthesis by RAW 264.7 macrophages, which was influenced by the protocol applied. Moreover, these parameters were significantly increased by isoleucine supplementation, highlighting a potential research field for future studies.

Development of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia coli compatible with industrial scale and with no antibiotics in the culture medium.

The granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that has important clinical applications for treating neutropenia. Nartograstim is a recombinant variant of human G-CSF. Nartograstim has been produced in Escherichia coli as inclusion bodies (IB) and presents higher stability and biological activity than the wild type of human G-CSF because of its mutations. We developed a production process of nartograstim in a 10-L bioreactor using auto-induction or chemically defined medium. After cell lysis, centrifugation, IB washing, and IB solubilization, the following three refolding methods were evaluated: diafiltration, dialysis, and direct dilution in two refolding buffers. Western blot and SDS-PAGE confirmed the identity of 18.8-kDa bands as nartograstim in both cultures. The auto-induction medium produced 1.17 g/L and chemically defined medium produced 0.95 g/L. The dilution method yielded the highest percentage of refolding (99%). After refolding, many contaminant proteins precipitated during pH adjustment to 5.2, increasing purity from 50 to 78%. After applying the supernatant to cation exchange chromatography (CEC), nartograstim recovery was low and the purity was 87%. However, when the refolding solution was applied to anion exchange chromatography followed by CEC, 91%-98% purity and 2.2% recovery were obtained. The purification process described in this work can be used to obtain nartograstim with high purity, structural integrity, and the expected biological activity. KEY POINTS: • Few papers report the final recovery of the purification process from inclusion bodies. • The process developed led to high purity and reasonable recovery compared to literature. • Nartograstim biological activity was demonstrated in mice using a neutropenia model.

Increased adiposity by feeding growing rats a high-fat diet results in iron decompartmentalisation.

The present study reports the effects of a high-fat (HF) diet of over 8 weeks on the Fe status of growing rats. Tissue Fe levels were analysed by atomic absorption spectrophotometry, and whole-body adiposity was measured by dual-energy X-ray absorptiometry. Histopathology and morphometry of adipose tissue were performed. Liver homogenates were used for measuring ferroportin-1 protein levels by immunoblotting, and transcript levels were used for Fe genes measured by real-time PCR. Tissue Fe pools were fit to a compartmental biokinetic model in which Fe was assessed using fourteen compartments and twenty-seven transfer constants (kj,i from tissue 'i' to tissue 'j') adapted from the International Commission on Radiological Protection (ICRP) 69. Ten kj,i were calculated from the experimental data using non-linear regression, and seventeen were estimated by allometry according to the formula ${k_{i,j}} = a \times {M^b}$. Validation of the model was carried out by comparing predicted and analysed Fe pool sizes in erythrocytes, the liver and the spleen. Body adiposity was negatively associated with serum Fe levels and positively associated with liver Fe stores. An inferred increase in Fe transfer from bone marrow to the liver paralleled higher hepatic Fe concentrations and ferritin heavy-chain mRNA levels in the HF diet-fed animals, suggesting that liver Fe accumulation occurred at least in part due to a favoured liver erythrocyte uptake. If this feeding condition was to be prolonged, impaired Fe decompartmentalisation may occur, ultimately resulting in dysmetabolic Fe overload.

Exogenous glutamine impairs neutrophils migration into infections sites elicited by lipopolysaccharide by a multistep mechanism.

Glutamine (GLN) is the most abundant free amino acid in the body, and is considered as a conditionally essential amino acid under stress conditions, acting as an important modulator of the immune response. We here investigated the role of exogenous GLN treatment on leukocyte migration after the onset of endotoxemia and the intracellular mechanisms of GLN actions on neutrophils. Two in vivo models of endotoxemia caused by lipopolysaccharide of Escherichia coli (LPS) injection were carried out in male outbred Balb/C mice 2-3 months old, as follow: (1) LPS (50 µg/kg) was intravenously injected 1 h prior to intravenous injection of GLN (0.75 mg/kg) and samples were collected 2 h later to investigate the role of GLN on the acute lung inflammation; (2) LPS (1 mg/kg) was intraperitoneally injected 1 h prior to intravenous injection of GLN (0.75 mg/kg) and samples were collected 18 h later to measure the effects of GLN on local and later phases of inflammation in the peritoneum. Results showed that GLN administration reduced the number of neutrophils in the inflamed lungs, partially recovery of the reduced number of leukocytes in the blood; reduced adhesion molecules on lung endothelium and on circulating neutrophils. Moreover, GLN treatment diminished the number of neutrophils, levels of chemotactic cytokine CXCL2 in the inflamed peritoneum, and neutrophils collected from the peritoneum of GLN-treated mice presented lower levels of Rho, Rac, and JNK. Together, our data show novel mechanisms involved in the actions of GLN on neutrophils migration.

A review of select minerals influencing the haematopoietic process.

Micronutrients are indispensable for adequate metabolism, such as biochemical function and cell production. The production of blood cells is named haematopoiesis and this process is highly consuming due to the rapid turnover of the haematopoietic system and consequent demand for nutrients. It is well established that micronutrients are relevant to blood cell production, although some of the mechanisms of how micronutrients modulate haematopoiesis remain unknown. The aim of the present review is to summarise the effect of Fe, Mn, Ca, Mg, Na, K, Co, iodine, P, Se, Cu, Li and Zn on haematopoiesis. This review deals specifically with the physiological requirements of selected micronutrients to haematopoiesis, showing various studies related to the physiological requirements, deficiency or excess of these minerals on haematopoiesis. The literature selected includes studies in animal models and human subjects. In circumstances where these minerals have not been studied for a given condition, no information was used. All the selected minerals have an important role in haematopoiesis by influencing the quality and quantity of blood cell production. In addition, it is highly recommended that the established nutrition recommendations for these minerals be followed, because cases of excess or deficient mineral intake can affect the haematopoiesis process.

Effects of glutamine, taurine and their association on inflammatory pathway markers in macrophages.

The immune system is essential for the control and elimination of infections, and macrophages are cells that act as important players in orchestrating the various parts of the inflammatory/immune response. Amino acids play important role in mediating functionality of the inflammatory response, especially mediating macrophages functions and cytokines production. We investigated the influence of glutamine, taurine and their association on the modulation of inflammatory pathway markers in macrophages. The RAW 264.7 macrophage cell line was cultivated in the presence of glutamine and taurine and proliferation rates, cell viability, cell cycle phases, IL-1α, IL-6, IL-10 and TNF-α as well as H2O2 production and the expression of the transcription factor, NFκB, and its inhibitor, IκBα, were evaluated. Our results showed an increase in viable cells and increased proliferation rates of cells treated with glutamine concentrations over 2 mM, as well as cells treated with both glutamine and taurine. The cell cycle showed a higher percentage of cells in the phases S, G2 and M when they were treated with 2 or 10 mM glutamine, or with glutamine and taurine in cells stimulated with lipopolysaccharide. The pNFκB/NFκB showed reduced ratio expression when cells were treated with 10 mM of glutamine or with glutamine in association with taurine. These conditions also resulted in reduced TNF-α, IL-1α and H2O2 production, and higher production of IL-10. These findings demonstrate that glutamine and taurine are able to modulate macrophages inflammatory pathways, and that taurine can potentiate the effects of glutamine, illustrating their immunomodulatory properties.

Evidence of bone marrow downregulation in brain-dead rats.

Experimental findings support the evidence of a persistent leucopenia triggered by brain death (BD). This study aimed to investigate leucocyte behaviour in bone marrow and blood after BD in rats. BD was induced using intracranial balloon catheter inflation. Sham-operated (SH) rats were trepanned only. Thereafter bone marrow cells were harvested every six hours from the femoral cavity and used for total and differential counts. They were analysed further by flow cytometry to characterize lymphocyte subsets, granulocyte adhesion molecules expression and apoptosis/necrosis [annexin V/propidium iodide (PI) protocol]. BD rats exhibited a reduction in bone marrow cells due to a reduction in lymphocytes (40%) and segmented cells (45%). Bone marrow lymphocyte subsets were similar in BD and SH rats (CD3, P = 0.1; CD4, P = 0.4; CD3/CD4, P = 0.4; CD5, P = 0.4, CD3/CD5, P = 0.2; CD8, P = 0.8). Expression of L-selectin and beta2 -integrins on granulocytes did not differ (CD11a, P = 0.9; CD11b/c, P = 0.7; CD62L, P = 0.1). There were no differences in the percentage of apoptosis and necrosis (Annexin V, P = 0.73; PI, P = 0.21; Annexin V/PI, P = 0.29). In conclusion, data presented suggest that the downregulation of the bone marrow is triggered by brain death itself, and it is not related to changes in lymphocyte subsets, granulocyte adhesion molecules expression or apoptosis and necrosis.

The effects of protein malnutrition on the TNF-RI and NF-κB expression via the TNF-α signaling pathway.

Malnutrition is a nutritional condition that can affect many aspects of the immunological response, including by decreasing cell migration and stimulating phagocytosis; the bactericidal response; changes in reactive oxygen and nitrogen species production; and the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α). This cytokine is primarily produced by macrophages and is associated with a wide range of biological activities, including inflammatory processes, growth, differentiation, and apoptosis. TNF-α acts through the activation of TNF receptors, and mainly receptor I (TNF-RI), which is responsible for most of the effects of TNF-α. This activation triggers a series of intracellular events that result in the activation of the transcription factor NF-κB. In this study, we evaluated the expression of the transcription factor NF-κB, mediated by TNF-α through TNF-RI, in a protein malnutrition (PM) model. Adult male BALB/c mice were submitted to PM, and after loss of approximately 20% of their body weight, their peritoneal macrophages were collected and cultivated with or without TNF-α. The expression of TNF-RI and proteins in its signaling pathway (TRADD, TRAF, RIP, IKK, IKB-α, pIKB-α, NF-κB, and pNF-κB) were evaluated, as well as cytokine production (IL-1α, IL-1ß, IL-6, and IL-12). The compiled results highlight that the malnourished animals presented anemia, leukopenia, and decreased peritoneal cellularity. TNF-RI expression was reduced in the malnourished animals, and NF-κB phosphorylation was also reduced, in association with reduced production of IL-1ß and IL-12. In this study, we observed aspects related to the innate immune response, and the outcome data allowed us to conclude that nutritional status interferes with the macrophage activation and the response capabilities of these cells.

7,12-Dimethylbenz(a)anthracene-induced myelotoxicity differs in mice selected for high or low acute inflammatory response: relationship with aryl hydrocarbon receptor polymorphism.

Polycyclic aromatic hydrocarbons, such as 7,12-dimethylbenz(a)anthracene (DMBA), are environmental pollutants that exert multiple toxic and carcinogenic effects. Studies showed that these effects are mediated by activation of the aryl hydrocarbon receptor (AhR) and modulated by allelic variants of Ahr gene. Here, we investigated the effects of DMBA treatment in the inflammatory response and bone marrow (BM) hematopoietic function of maximal acute inflammatory response (AIRmax) and minimal acute inflammatory response (AIRmin) heterogeneous mouse lines selected for high and low acute inflammatory responsiveness, respectively. The phenotypic selection resulted in the segregation of the Ahr(d) and Ahr(b1) alleles that confer low and high receptor ligand-binding affinity, respectively, in AIRmax and AIRmin mice. We observed a reduction in BM mature granulocyte population in AIRmin mice 24 hours after DMBA treatment while both blast and immature myeloid cells were increased. Proliferation and differentiation of BM myeloid cells in response to in vitro granulocyte-macrophage colony-stimulating factor stimulus were impaired in AIRmin-treated mice. These DMBA effects on myeloid BM cells (BMCs) affected the in vivo leukocyte migration to an inflammatory site induced by polyacrylamide beads (Biogel P-100, Bio-Rad, France) injection in AIRmin mice. On the other hand, these alterations were not observed in DMBA-treated AIRmax mice. These data indicate that DMBA affects myeloid cell differentiation and inflammatory response and Ahr(b1) allele in the genetic background of AIRmin mice contributes to this effect.

Yerba Mate (Ilex paraguariensis) modulates NF-kappaB pathway and AKT expression in the liver of rats fed on a high-fat diet.

To investigate the effect of Yerba Mate (YM) aqueous extract intake on the NF-kB pathway and AKT expression in the liver, muscle, and adipose tissue of rats submitted to a high-fat diet (HFD). Male Wistar rats were fed a control (CON) (n = 24) or a HFD (n = 24) for 12 weeks. Afterwards, rats received YM daily (1 g/kg body weight) for 4 weeks. Intake of YM aqueous extract reduced body weight gain (p < 0.05) and total blood cholesterol (p < 0.05) in the HFD group in comparison to the non-treated HFD group. HFD group demonstrated an increased glycemic response at 5 and 10 min after insulin injection. YM decreased the ratio between phosphorylated and total kinase inhibitor of κB (IKK), increased the ratio of phosphorylated to total form of protein kinase B (AKT) and reduced NF-κB phosphorylation in the liver of the HFD group. Our data suggest a beneficial role of YM in improving metabolic dysfunctions induced by HFD.

Reduced protein intake and aging affects the sustainment of hematopoiesis by impairing bone marrow mesenchymal stem cells.

Protein malnourishment (PM) is common among the elderly, but how aging and PM impact hematopoiesis is not fully understood. This study aimed to assess how aging and PM affect the hematopoietic regulatory function of bone marrow (BM) mesenchymal stem cells (MSCs). Young and aged male C57BL/6J mice were fed with normoproteic or hypoproteic diets and had their nutritional, biochemical, and hematological parameters evaluated. BM MSCs were characterized and had their secretome, gene expression, autophagy, reactive oxygen species production (ROS), and DNA double-stranded breaks evaluated. The modulation of hematopoiesis by MSCs was assayed using in vitro and in vivo models. Lastly, BM invasiveness and mice survival were evaluated after being challenged with leukemic cells of the C1498 cell line. Aging and PM alter biochemical parameters, changing the peripheral blood and BM immunophenotype. MSC autophagy was affected by aging and the frequencies for ROS and DNA double-stranded breaks. Regarding the MSCs' secretome, PM and aging affected CXCL12, IL-6, and IL-11 production. Aging and PM up-regulated Akt1 and PPAR-γ while down-regulating Cdh2 and Angpt-1 in MSCs. Aged MSCs increased C1498 cell proliferation while reducing their colony-forming potential. PM and aging lowered mice survival, and malnourishment accumulated C1498 cells at the BM. Finally, aged and/or PM MSCs up-regulated Sox2, Nanog, Pou5f1, and Akt1 expression while down-regulating Cdkn1a in C1498 cells. Together, aging and PM can induce cell-intrinsic shifts in BM MSCs, creating an environment that alters the regulation of hematopoietic populations and favoring the development of malignant cells.

Modulation of the nuclear factor-kappa B (NF-κB) signalling pathway by glutamine in peritoneal macrophages of a murine model of protein malnutrition.

BACKGROUND AND AIMS: Protein malnutrition affects resistance to infection by impairing the inflammatory response, modifying the function of effector cells, such as macrophages. Recent studies have revealed that glutamine-a non-essential amino acid, which could become conditionally essential in some situations like trauma, infection, post-surgery and sepsis-is able to modulate the synthesis of cytokines. The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappa B (NF-κB) signalling pathway of peritoneal macrophages from malnourished mice. METHODS: Two-month-old male Balb/c mice were submitted to protein-energy malnutrition (n = 10) with a low-protein diet containing 2 % protein, whereas control mice (n = 10) were fed a 12 % protein-containing diet. The haemogram and analysis of plasma glutamine and corticosterone were evaluated. Peritoneal macrophages were pre-treated in vitro with glutamine (0, 0.6, 2 and 10 mmol/L) for 24 h and then stimulated with 1.25 µg LPS for 30 min, and the synthesis of TNF-α and IL-1α and the expression of proteins related to the NF-κB pathway were evaluated. RESULTS: Malnourished animals had anaemia, leucopoenia, lower plasma glutamine and increased corticosterone levels. TNF-α production of macrophages stimulated with LPS was significantly lower in cells from malnourished animals when cultivated in supraphysiological (2 and 10 mmol/L) concentrations of glutamine. Further, glutamine has a dose-dependent effect on the activation of macrophages, in both groups, when stimulated with LPS, inducing a decrease in TNF-α and IL-1α production and negatively modulating the NF-κB signalling pathway. CONCLUSIONS: These data lead us to infer that the protein malnutrition state interferes with the activation of macrophages and that higher glutamine concentrations, in vitro, have the capacity to act negatively in the NF-κB signalling pathway.

Isocaloric intake of a high-fat diet promotes insulin resistance and inflammation in Wistar rats.

The aim of this study was to investigate the effect of isocaloric intake from a high-fat diet (HFD) on insulin resistance and inflammation in rats. Male Wistar rats were fed on an HFD (n = 12) or control diet (n = 12) for 12 weeks. Subsequently, all animals were euthanized, and blood glucose, insulin, free fatty acids, C-reactive protein, lipid profile, cytokines and hepatic-enzyme activity were determined. Carcass chemical composition was also analyzed. During the first and the twelfth weeks of the experimental protocol, the oral glucose tolerance test and insulin tolerance test were performed and demonstrated insulin resistance (P < 0.05) in the HFD group. Although food intake (g) was lower (P < 0.05) in the HFD group compared with the control group, the concentration of total cholesterol, low-density lipoprotein, C-reactive protein and liver weight were all significantly higher. The kinase inhibitor of κB, c-Jun N-terminal kinase and protein kinase B expressions were determined in the liver and skeletal muscle. After an insulin stimulus, the HFD group demonstrated decreased (P = 0.05) hepatic protein kinase B expression, whereas the kinase inhibitor of κB phospho/total ratio was elevated in the HFD muscle (P = 0.02). In conclusion, the isocaloric intake from the HFD induced insulin resistance, associated with impaired insulin signalling in the liver and an inflammatory response in the muscle.

The Effects of Yerba Maté (Ilex Paraguariensis) consumption on IL-1, IL-6, TNF-α and IL-10 production by bone marrow cells in wistar rats fed a high-fat diet.

An excessive consumption of a high-fat diet (HFD) results in becoming overweight or obese, which triggers a chronic inflammatory condition that is associated with a high white blood cell count. Because of the potential for yerba maté (Ilex paraguariensis) (YM) to impact obesity, this study aimed to investigate the effects of YM consumption on the hematological response and on the production of interleukin (IL)-1α, IL-6, tumor necrosis factor (TNF)-α, and IL-10 by bone marrow cells from Wistar rats fed a HFD. Male Wistar rats were fed a control (CON) or HFD diet for twelve weeks. At the end of this period, the rats received YM (1 g/kg/day body weight) for 4 weeks. After euthanasia, hemograms and myelograms were evaluated, while the bone marrow cells were cultured in the presence or absence of lipopolysaccharide (LPS) to evaluate the production of IL-1α, IL-6, TNF-α, and IL-10. The consumption of YM reduced the body weight, the body adiposity, and the cholesterol levels in HFD-fed rats. Bone marrow cells from the HFD group produced more IL-1α, IL-6, and TNF-α, and less IL-10, when compared to cells from the control group, and YM consumption reduced the IL-1α, IL-6, and TNF-α production by the cells. However, cells from the HFD rats that were stimulated with LPS increased their IL-1α, IL-6, and TNF-α production, but YM consumption did not change this result. In summary, the consumption of YM affects the production of IL-1α, IL-6, and TNF-α by bone marrow cells, promotes weight loss, decreases the number of white blood cells, and significantly improves serum cholesterol level in HFD-fed rats. However, the bone marrow cells from the HFD+YM-fed rats challenged with LPS did not show improvement in the inflammatory response compared to the cells from animals fed only a HFD that were also challenged with LPS.

Nutrient-adjusted high-fat diet is associated with absence of periepididymal adipose tissue inflammation: is there a link with adequate micronutrient levels?

The aim of this study was to investigate the real impact of dietary lipids on metabolic and inflammatory response in rat white adipose tissue. Male healthy Wistar rats were fed ad libitum with a control diet (CON, n=12) or with an adjusted high-fat diet (HFD, n=12) for 12 weeks. Oral glucose and insulin tolerance tests were performed during the last week of the protocol. Plasma fatty acid, lipid profile, body adiposity, and carcass chemical composition were analyzed. Plasma concentration of leptin, adiponectin, C-reactive protein (CRP), TNF-α, IL-6, and monocyte chemotactic protein (MCP-1) was measured. Periepididymal adipose tissue was employed to evaluate TNF-α, MCP-1, and adiponectin gene expression as well as NF-κB pathway and AKT proteins. Isocaloric intake of the adjusted HFD did not induce hyperphagia, but promoted an increase in periepididymal (HFD = 2.94 ± 0.77 vs. CON = 1.99 ± 0.26 g/100 g body weight, p = 0.01) and retroperitoneal adiposity (HFD = 3.11 ± 0.81 vs. CON = 2.08 ± 0.39 g/100 g body weight, p = 0.01) and total body lipid content (HFD = 105.3 ± 20.8 vs. CON = 80.5 ± 7.6 g carcass, p = 0.03). Compared with control rats, HFD rats developed glucose intolerance (p=0.01), dyslipidemia (p = 0.02) and exhibited higher C-reactive protein levels in response to the HFD (HFD = 1002 ± 168 vs. CON = 611 ± 260 ng/mL, p = 0.01). The adjusted HFD did not affect adipokine gene expression or proteins involved in inflammatory signaling, but decreased AKT phosphorylation after insulin stimulation in periepididymal adipose tissue (p = 0.01). In this study, nutrient-adjusted HFD did not induce periepididymal adipose tissue inflammation in rats, suggesting that the composition of HFD differently modulates inflammation in rats, and adequate micronutrient levels may also influence inflammatory pathways.

The effect of mate tea (Ilex paraguariensis) on metabolic and inflammatory parameters in high-fat diet-fed Wistar rats.

This study investigated the effects of mate tea (Ilex paraguariensis) aqueous extract consumption on metabolic indicators and inflammatory response of peritoneal macrophages in rats fed a high-fat diet (HFD). Male Wistar rats were fed a control diet or a HFD for 12 weeks. At the end of this period, rats received, or not, daily doses of yerba maté for 4 weeks. The consumption of yerba maté promoted weight loss, attenuated the HFD-detrimental effects on adiposity and insulin sensitivity and decreased blood levels of the inflammatory biomarkers (p < 0.05). Concerning peritoneal macrophages, mate tea consumption decreased the production of interleukin (IL)-6, but did not influence the production of IL-1ß, tumour necrosis factor-α and nitric oxide; cytokine mRNA expression; or the activation of the nuclear factor-κB signalling pathway. In summary, the consumption of mate tea had no consistent effect in the inflammatory response of peritoneal macrophages, but reduced cardiometabolic risk markers.

Delphinidin-3-O-glucoside in vitro suppresses NF-κB and changes the secretome of mesenchymal stem cells affecting macrophage activation.

OBJECTIVE: Anthocyanins are polyphenols that are promising chemopreventive agents. They stand out for their anti-inflammatory properties, with specific modulatory actions on the immune system. Additionally, regarding the immune system, a group of cells identified as mesenchymal stem cells (MSCs) have been attracting attention, mainly because of their capacity to migrate to sites of inflammation and produce potent immunomodulatory effects. Considering the ability of these cells to act on the immune system, as well as the properties of anthocyanins, especially delphinidin, in modulating the immune system, the aim of this study was to investigate the effects of delphinidin in influencing some immunoregulatory properties of MSCs. METHODS: MSCs were cultivated in the presence of delphinidin 3-O-ß-d-glycoside and cell viability, the cell cycle and the production of soluble factors (interleukin [IL]-1ß, IL-6, IL-10, transforming growth factor [TGF]-ß, prostaglandin E2 [PGE2] and nitric oxide [NO]) were evaluated, as was the expression of the transcription factors nuclear factor (NF)-κB and STAT3. Additionally, the effects of conditioned media from MSCs on macrophage activation were assessed. RESULTS: Delphinidin at 50 µM does not affect cell viability. In association with lipopolysaccharide, delphinidin was able to induce MSC proliferation. Additionally, delphinidin modulated the MSC immune response, showing increased levels of anti-inflammatory cytokines such as IL-10 and TGF-ß as well as lower expression of NF-κB. Furthermore, conditioned media from MSCs inhibited macrophage metabolism, reducing the production of IL-1ß, IL-12, and TNF-α and increasing IL-10. CONCLUSIONS: Overall, this work showed that delphinidin can modify the immunomodulatory properties of MSCs, increasing the IL-10 production by macrophages.

Protein restriction impairs the response activation/responsivity of MAPK signaling pathway of hematopoietic stem cells.

Protein restriction (PR) leads to bone marrow hypoplasia with changes in stromal cellularity components of the extracellular matrix in hematopoietic stem cells (HSCs). However, the underlying signaling mechanisms are poorly understood. We hypothesize that PR impairs the HSC mitogen-activated protein kinase (MAPK) signaling pathway response activation. Our aim is to evaluate the activation of MAPK and interleukin-3 (IL-3) proteins in HSC to explain PR-induced bone marrow hypoplasia, which causes altered proliferation and differentiation. C57BL/6 male mice were subjected to a low-protein diet (2% protein) or normoproteic (12% protein). PKC, PLCγ2, CaMKII, AKT, STAT3/5, ERK1/2, JNK, and p38d phosphorylation were evaluated by flow cytometry, and GATA1/2, PU.1, C/EBPα, NF-E2, and Ikz-3 genes (mRNAs) assessed by quantitative real-time-polymerase chain reaction. Pathway proteins, such as PLCγ2, JAK2, STAT3/5, PKC, and RAS do not respond to the IL-3 stimulus in PR, leading to lower activation of ERK1/2 and Ca2+ signaling pathways, consequently lowering the production of hematopoietic transcription factors. Colony forming units granulocyte-macrophage and colony forming units macrophage formation are impaired in PR even after being stimulated with IL-3. Long-term hematopoietic stem cells, short-term hematopoietic stem cells, granulocyte myeloid progenitor, and megakaryocyte-erythroid progenitor cells were significantly reduced in PR animals. This study shows for the first time that activation of MAPK pathway key proteins in HSCs is impaired in cases of PR. Several pathway proteins, such as PLCγ2, JAK2, STAT3, PKC, and RAS do not respond to IL-3 stimulation, leading to lower activation of extracellular signal-regulated protein kinase 1/2 and consequently lower production of hematopoietic transcription factors GATA1/2, PU.1, C/EBPa, NF-E2, and Ikz3. These changes result in a reduction in colony-forming units, proliferation, and differentiation, leading to hypocellularity.

The interaction between aging and protein malnutrition modulates peritoneal macrophage function: An experimental study in male mice.

Malnutrition is considered one of the most common problems in the elderly population worldwide and can significantly interfere in health evolution in these individuals, predisposing them to increased infection susceptibility. The immune response triggered by infections comprises several mechanisms, and macrophages play important roles in this response. This study aimed to evaluate mechanisms related to macrophage function in a model of protein malnutrition in the elderly. Two age groups (young: 3-5 months and elderly: 18-19 months) male C57BL/6NTac mice were subjected to protein malnutrition with a low-protein diet (2 %). The nutritional status, hemogram and number of peritoneal cells were affected by both age and nutritional status. Additionally, the spreading capacity as well as the phagocytic and fungicidal activity of peritoneal macrophages were affected by the nutritional status and age of the animal. Interestingly, the percentages of F4/80+/CD11b+ and CD86+ cells were reduced mostly in elderly animals, while the TLR-4+ population was more affected by nutritional status than by age. The production of pro-inflammatory cytokines such as TNF-α, IL-1α, and IL-6 was also influenced by nutritional status and/or by age, and malnourished animals of advanced age produced higher amounts of the anti-inflammatory cytokine IL-10. Furthermore, the phosphorylation ratio of the transcription factor NFκB (pNFκB/NFκB) was directly affected by the nutritional status, independently of age. Thus, these results allow us to conclude that aging and protein malnutrition compromise macrophage function, likely affecting their immune function, and in aged protein-malnourished animals, this impairment tends to be more pronounced.