Species-dependent impact of immunosuppressive squalene-gusperimus nanoparticles and adipose-derived stem cells on isolated human and rat pancreatic islets.

Transplantation of pancreatic islets is a promising approach to controlling glucose levels in type 1 diabetes mellitus (T1DM), but islet survival is still limited. To overcome this, islet co-culture with mesenchymal stromal cells (MSCs) together with safe immunosuppressive agents like squalene-gusperimus nanoparticles (Sq-GusNPs) may be applied. This could support islet survival and engraftment. Here, we studied how Sq-GusNPs and adipose-derived stem cells (ASCs) influence islets response under pro-inflammatory conditions. Through qRT-PCR, we studied the expression of specific genes at 24 hours in human and rat islets and ASCs in co-culture under indirect contact with or without treatment with Sq-GusNPs. We characterized how the response of islets and ASCs starts at molecular level before impaired viability or function is observed and how this response differs between species. Human islets and ASCs responses showed to be principally influenced by NF-κB activation, whereas rat islet and ASCs responses showed to be principally mediated by nitrosative stress. Rat islets showed tolerance to inflammatory conditions due to IL-1Ra secretion which was also observed in rat ASCs. Human islets induced the expression of cytokines and chemokines with pro-angiogenic, tissue repair, and anti-apoptotic properties in human ASCs under basal conditions. This expression was not inhibited by Sq-GusNPs. Our results showed a clear difference in the response elicited by human and rat islets and ASCs in front of an inflammatory stimulus and Sq-GusNPs. Our data support the use of ASCs and Sq-GusNP to facilitate engraftment of islets for T1DM treatment.

The level and distribution of methyl-esters influence the impact of pectin on intestinal T cells, microbiota, and Ahr activation.

Pectins are dietary fibres that modulate T cell immunity, microbiota composition, and fermentation profiles, but how this is influenced by the degree of methyl-esterification (DM) and degree-of-blockiness (DB) of pectin is unknown. Here, we demonstrate that supplementation of DM19(high-DB), DM49(low-DB) and DM43(high-DB) pectins at a low dose increased the frequencies of intestinal T-helper (Th)1 and Th2 cells after 1 week of pectin supplementation in mice, whereas DM18(low-DB) did not. After 4 weeks of supplementation with those pectins, Th1 and Th2 frequencies returned to control levels, whereas Rorγt+ regulatory T-cell frequencies increased. These structure-dependent effects could derive from induced shifts in microbiota composition that differed between DM18(low-DB) pectin and the other pectins. T-cell-modulating effects were not short-chain-fatty acid-dependent, but rather through an increase in Aryl-hydrocarbon-receptor-activating components. Thus, pectins with a specific combination of DM and DB have an impact on intestinal T cell-immunity in mice, when supplemented at a low dose.

In vitro determination of the immunosuppressive effect, internalization, and release mechanism of squalene-gusperimus nanoparticles for managing inflammatory responses.

Gusperimus is an anti-inflammatory drug that has shown to be effective in managing autoimmunity and preventing graft rejection. This is unstable and easily broken down into cytotoxic components. We encapsulated gusperimus binding it covalently to squalene obtaining squalene-gusperimus nanoparticles (Sq-GusNPs). These nanoparticles enhanced the immunosuppressive effect of gusperimus in both mouse macrophages and T cells. The half-maximal inhibitory concentration in macrophages was 9-fold lower for Sq-GusNPs compared with the free drug. The anti-inflammatory effect of the Sq-GusNPs was maintained over time without cytotoxicity. By studying nanoparticles uptake by cells with flow cytometry, we demonstrated that Sq-GusNPs are endocytosed by macrophages after binding to low-density lipoprotein receptors (LDLR). In presence of cathepsin B or D release of gusperimus is increased demonstrating the participation of proteases in the release process. Our approach may allow the application of Sq-GusNPs for effective management of inflammatory disorders including autoimmunity and graft rejection.

Pectin limits epithelial barrier disruption by Citrobacter rodentium through anti-microbial effects.

SCOPE: C. rodentium is the murine equivalent of Enteropathogenic Escherichia. coli (EPEC) and Enterohemorrhagic Escherichia coli (EHEC) which induce damage to the intestinal epithelial barrier that results in diarrhea and intestinal inflammation. Dietary fibre intake can be an effective approach to limit epithelial damage by these enteric pathogens. Therefore, the protective effect of dietary fibre pectin against dysfunction of epithelial barrier integrity upon C. rodentium infection was investigated. METHODS AND RESULTS: Pectins that structurally differed in the degree and distribution of methylesters were tested on barrier protective effects on epithelial cells against C. rodentium by measuring transepithelial electrical resistance and lucifer yellow fluxes. All three pectins protected the epithelial barrier from C. rodentium induced damage in a structure-independent manner. These barrier protective effects were also independent of pectin-induced TLR2 activation. Furthermore, the pectins induced anti-adhesive effects on C. rodentium by interacting with C. rodentium and not with epithelial cells. This may be explained by antimicrobial effects of pectins on C. rodentium and not on other enteric bacteria including Lactobacillus plantarum and E. coli. A competition ELISA for binding of C. rodentium to pectin supported this finding as it showed that pectin interacts strongly with C. rodentium, whereas it interacts weakly or not with L. plantarum or E. coli. CONCLUSION: These findings demonstrate that pectin protects the epithelial barrier from C. rodentium induced damage by inducing anti-microbial effects.

The impact of the level and distribution of methyl-esters of pectins on TLR2-1 dependent anti-inflammatory responses.

Pectins have anti-inflammatory effects via Toll-like receptor (TLR) inhibition in a degree of methyl-esterification-(DM)-dependent manner. However, pectins also vary in distribution of methyl-esters over the galacturonic-acid (GalA) backbone (Degree of Blockiness - DB) and impact of this on anti-inflammatory capacity is unknown. Pectins mainly inhibit TLR2-1 but magnitude depends on both DM and DB. Low DM pectins (DM18/19) with both low (DB86) and high DB (DB94) strongly inhibit TLR2-1. However, pectins with intermediate DM (DM43/DM49) and high DB (DB60), but not with low DB (DB33), inhibit TLR2-1 as strongly as low DM. High DM pectins (DM84/88) with DB71 and DB91 do not inhibit TLR2-1 strongly. Pectin-binding to TLR2 was confirmed by capture-ELISA. In human macrophages, low DM and intermediate DM pectins with high DB inhibited TLR2-1 induced IL-6 secretion. Both high number and blockwise distribution of non-esterified GalA in pectins are responsible for the anti-inflammatory effects via inhibition of TLR2-1.

Design and characterization of Squalene-Gusperimus nanoparticles for modulation of innate immunity.

Immunosuppressive drugs are widely used for the treatment of autoimmune diseases and to prevent rejection in organ transplantation. Gusperimus is a relatively safe immunosuppressive drug with low cytotoxicity and reversible side effects. It is highly hydrophilic and unstable. Therefore, it requires administration in high doses which increases its side effects. To overcome this, here we encapsulated gusperimus as squalene-gusperimus nanoparticles (Sq-GusNPs). These nanoparticles (NPs) were obtained from nanoassembly of the squalene gusperimus (Sq-Gus) bioconjugate in water, which was synthesized starting from squalene. The size, charge, and dispersity of the Sq-GusNPs were optimized using the response surface methodology (RSM). The colloidal stability of the Sq-GusNPs was tested using an experimental block design at different storage temperatures after preparing them at different pH conditions. Sq-GusNPs showed to be colloidally stable, non-cytotoxic, readily taken up by cells, and with an anti-inflammatory effect sustained over time. We demonstrate that gusperimus was stabilized through its conjugation with squalene and subsequent formation of NPs allowing its controlled release. Overall, the Sq-GusNPs have the potential to be used as an alternative in approaches for the treatment of different pathologies where a controlled release of gusperimus could be required.

Mitochondrial function in immune cells in health and disease.

One of the main functions of mitochondria is production of ATP for cellular energy needs, however, it becomes more recognized that mitochondria are involved in differentiation and activation processes of immune cells. Upon activation, immune cells have a high need for energy. Immune cells have different strategies to generate this energy. In pro-inflammatory cells, such as activated monocytes and activated T and B cells, the energy is generated by increasing glycolysis, while in regulatory cells, such as regulatory T cells or M2 macrophages, energy is generated by increasing mitochondrial function and beta-oxidation. Except for being important for energy supply during activation, mitochondria also induce immune responses. During an infection, they release mitochondrial danger associated molecules (DAMPs) that resemble structures of bacterial derived pathogen associated molecular patterns (PAMPs). Such mitochondrial DAMPS are for instance mitochondrial DNA with hypomethylated CpG motifs or a specific lipid that is only present in prokaryotic bacteria and mitochondria, i.e. cardiolipin. Via release of such DAMPs, mitochondria guide the immune response towards an inflammatory response against pathogens. This is an important mechanism in early detection of an infection and in stimulating and sustaining immune responses to fight infections. However, mitochondrial DAMPs may also have a negative impact. If mitochondrial DAMPs are released by damaged cells, without the presence of an infection, such as after a trauma, mitochondrial DAMPs may induce an undesired inflammatory response, resulting in tissue damage and organ dysfunction. Thus, immune cells have developed mechanisms to prevent such undesired immune activation by mitochondrial components. In the present narrative review, we will describe the current view of mitochondria in regulation of immune responses. We will also discuss the current knowledge on disturbed mitochondrial function in immune cells in various immunological diseases.

In utero sFlt-1 exposure differentially affects gene expression patterns in fetal liver.

The soluble fms-like tyrosine kinase factor 1 (sFlt-1) is a major contributor to antiangiogenesis during preeclampsia. However, little is known about the effects of sFlt-1 on fetal health. In this study we aim to evaluate the effects of the sFlt-1 concentration during pregnancy on fetal liver physiology. We used adenoviral gene delivery in Sprague-Dawley dams (seven females, 10 weeks old) during mid-gestation (gestational day 8) with adenovirus overexpressing sFlt-1, and age-matched controls (six females, 10 weeks old) with empty adenoviral virus in order to quantify the sFlt-1 concentrations in pregnant dams. Dams exposed to adenoviral sFlt-1 delivery were subdivided into a low (n=4) and high sFlt-1 (n=3) group based on host response to the virus. One-way analysis of variance showed that fetuses (five per dam) exposed to high sFlt-1 concentrations in utero show fetal growth restriction (1.84±0.043 g high sFlt-1 v. 2.32±0.036 g control; mean (M)±s.e.m.; P<0.001), without hypertension or proteinuria in the dams. In continuation, the microarray analysis of the fetal liver of the high sFlt-1 group showed significant enrichment of key genes for fatty acid metabolism and Ppara targets. In addition, using pyrosequencing, we found that the Ppara enrichment in the high sFlt-1 group is accompanied by decreased methylation of its promoter (1.89±0.097 mean % methylation in high sFlt-1 v. 2.26±0.095 mean % methylation in control, M±s.e.m., P<0.02). Our data show that high sFlt-1 concentrations during pregnancy have detrimental effects on the fatty acid metabolism genes and the Ppara targets in the fetal liver.

The role of autoimmunity in women with type 1 diabetes and adverse pregnancy outcome: A missing link.

The incidence of pregnancy complications in women with type 1 Diabetes Mellitus (T1D) is greater than in healthy pregnant women. This has mostly been attributed to hyperglycemia. However, despite the implementation of stricter guidelines regarding glycemic control, pregnancy complications remain more common in women with T1D. This may suggest that other etiological factors are involved. We suggest that the immune response may play a role, since the immune response has to adapt during pregnancy in order to facilitate implantation, placental and fetal development, and aberrant immunological adaptations to pregnancy are involved in various pregnancy complications. Since T1D is an autoimmune disorder, the question rises whether the immune response of women with T1D is able to adapt properly during pregnancy. Here we review the current proof and views on the role of aberrant immunological adaptations in pregnancy complications and whether such aberrant adaptations could be involved in the pregnancy complications of T1D patients.

The influence of maternal obesity on macrophage subsets in the human decidua.

Obesity is seen as a low grade inflammatory state, and is associated with adverse pregnancy outcomes. Disturbed macrophage characteristics might be essential in obesity associated pregnancy pathology via effects on the regulation of angiogenesis and placental development. This study aims to address the effects of maternal obesity on macrophage subsets in the decidua of women with term uncomplicated pregnancies. Macrophages were isolated from the decidua basalis and decidua parietalis of women with pre-gravid BMI < 25 (control) and BMI > 30 (obese). Macrophages were characterized and quantified using multi-color flow cytometry. Placentas of 10 obese and 10 control women after an uncomplicated term pregnancy were included. The decidua parietalis, but not decidua basalis, showed significantly lower levels of M1-type (HLA-DR+, CD163-) macrophages (p < 0.05) in obese women (4,3% of total macrophages) compared to control women (5,3% of total macrophages). The lower levels of M1 macrophages, considered to be pro-inflammatory, might indicate a mechanism to compensate for the pro-inflammatory environment in obese women to ensure healthy pregnancy outcomes.

Extracellular ATP and adenosine: The Yin and Yang in immune responses?

Extracellular adenosine 5'-triphosphate (ATP) and adenosine molecules are intimately involved in immune responses. ATP is mostly a pro-inflammatory molecule and is released during hypoxic condition and by necrotic cells, as well as by activated immune cells and endothelial cells. However, under certain conditions, for instance at low concentrations or at prolonged exposure, ATP may also have anti-inflammatory properties. Extracellular ATP can activate both P2X and P2Y purinergic receptors. Extracellular ATP can be hydrolyzed into adenosine in a two-step enzymatic process involving the ectonucleotidases CD39 (ecto-apyrase) and CD73. These enzymes are expressed by many cell types, including endothelial cells and immune cells. The counterpart of ATP is adenosine, which is produced by breakdown of intra- or extracellular ATP. Adenosine has mainly anti-inflammatory effects by binding to the adenosine, or P1, receptors (A1, A2A, A2B, and A3). These receptors are also expressed in many cells, including immune cells. The final effect of ATP and adenosine in immune responses depends on the fine regulatory balance between the 2 molecules. In the present review, we will discuss the current knowledge on the role of these 2 molecules in the immune responses.

Maternal monocytes in pregnancy and preeclampsia in humans and in rats.

Monocytes are short-lived cells, arising from the bone marrow and maturing in the circulation. They play an important role in immune responses and are thought to be important for healthy pregnancy. In humans, 3 subpopulations of monocytes have been identified: classical, intermediate and non-classical monocytes. These subpopulations have different functions and phenotypical characteristics. Healthy pregnancy is characterized by a pro-inflammatory condition, with increased numbers of monocytes and monocyte activation as well as with increased numbers of intermediate monocytes and decreased numbers of classical monocytes. This may suggest monocyte maturation. Preeclampsia is an important pregnancy complication characterized by hypertension and proteinuria developing in the second half of pregnancy. The pathophysiology of preeclampsia is associated with further activation of the inflammatory response, further activation of monocytes and further monocyte maturation. In the present review we focus on the role of monocyte activation and maturation in healthy and preeclamptic pregnancy.

Immunomodulating properties of protein hydrolysates for application in cow's milk allergy.

Cow's milk proteins cause allergic symptoms in 2-3% of all infants. In these individuals, the tolerogenic state of the intestinal immune system is broken, which can lead to sensitization against antigens and eventually to allergic responses. Although a true treatment for food allergy is not available, symptoms can be avoided by providing the infants with hydrolyzed proteins. Hydrolyzed proteins are proteins that are enzymatically degraded. They lack typical allergenic IgE-binding epitopes but are also thought to play a pertinent role in other mechanisms inducing hypoallergenic effects. This review discusses the mechanisms and evidence for immunomodulating properties of cow's milk hydrolysates. Hydrolysates are found to strengthen the epithelial barrier, modulate T-cell differentiation, and decrease inflammation. Some studies suggest a role for hydrolysates in manipulating pathogen recognition receptors signaling as underlying mechanism. Peptides from hydrolysates have been shown to bind to TLR2 and TLR4 and influence cytokine production in epithelial cells and macrophages. Current insight suggests that hydrolysates may actively participate in modulating the immune responses in subjects with cow's milk allergy and those at risk to develop cow's milk allergy. However, more research is required to design effective and reproducible means to develop targeting strategies to modulate the immune response.

Impaired trophoblast invasion and increased numbers of immune cells at day 18 of pregnancy in the mesometrial triangle of type 1 diabetic rats.

INTRODUCTION: Type 1 diabetes (T1D) is associated with adverse pregnancy outcome, usually attributed to hyperglycemia. Recently, we showed that pregnancy outcome in normoglycemic T1D rats was characterized by decreased fetal and placental weight, suggesting impaired placental development. In the present study, we tested the hypothesis that trophoblast invasion and spiral artery (SA) remodeling is impaired in T1D rats ant that this is associated with aberrant local presence of NK cells and macrophages in the mesometrial triangle (MT). METHODS: Placentae with MT from pregnant biobreeding diabetes-prone (BBDP; T1D model) rats, control biobreeding diabetes-resistant (BBDR) and Wistar-rats were dissected at day 18 of gestation and stained for trophoblast invasion, SA remodeling, uNK cells and macrophages. RESULTS: Interstitial trophoblast invasion and SA remodeling was impaired in BBDP-rats vs. control rats, coinciding with increased presence of NK cells and an increased iNOS+/CD206+ ratio of macrophages. DISCUSSION: Decreased fetal and placental weight in BBDP-rats was associated with diminished interstitial trophoblast invasion and less optimal SA remodeling, increased numbers of NK cells and increased iNOS+/CD206+ macrophage ratio in the MT of BBDP-rats. CONCLUSIONS: The impaired trophoblast invasion and SA remodeling may be due to an aberrant local immune-response and may result in damage to the fetal placenta and insufficient supply of nutrients towards the fetus with eventually decreased fetal weight as a consequence.

Extracellular ATP decreases trophoblast invasion, spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats.

INTRODUCTION: Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant rats induced preeclampsia-like symptoms such as albuminuria and placental ischemia. We hypothesized that ATP inhibits trophoblast invasion and spiral artery remodeling and affects macrophages and natural killer (NK) cells present in the rat mesometrial triangle. METHODS: Pregnant rats were infused with ATP or saline (control) on day 14 of pregnancy. Rats were sacrificed on day 15, 17 or 20 of pregnancy and placentas with mesometrial triangle were collected. Sections were stained for trophoblast cells, α-smooth muscle actin (spiral artery remodeling), NK cells and various macrophage populations. Expression of various cytokines in the mesometrial triangle was analyzed using real-time RT-PCR. RESULTS: ATP infusion decreased interstitial trophoblast invasion on day 17 and spiral artery remodeling on day 17 and 20, increased activated tartrate resistant acid phosphatase (TRAP)-positive macrophages on day 15, decreased NK cells on day 17 and 20, and decreased inducible nitric oxide synthase (iNOS)-positive and CD206-positive macrophages and TNF-α and IL-33 expression at the end of pregnancy (day 20). DISCUSSION: Interstitial trophoblast invasion and spiral artery remodeling in the rat mesometrial triangle were decreased by infusion of ATP. These ATP-induced modifications were preceded by an increase in activated TRAP-positive macrophages and coincided with NK cell numbers, suggesting that they are involved. CONCLUSION: Trophoblast invasion and spiral artery remodeling may be inhibited by ATP-induced activated macrophages and decreased NK cells in the mesometrial triangle in rat pregnancy.

The effect of Porphyromonas gingivalis lipopolysaccharide on pregnancy in the rat.

OBJECTIVE: Periodontitis, mostly associated with Porphyromonas gingivalis, has frequently been related to adverse pregnancy outcomes. We therefore investigated whether lipopolysaccharides of P. gingivalis (Pg-LPS) induced pregnancy complications in the rat. METHODS: Experiment 1: pregnant rats (day 14) received increasing Pg-LPS doses (0.0-50.0 µg kg(-1) bw; n = 2/3 p per dose). Maternal intra-aortic blood pressure, urinary albumin excretion, placental and foetal weight and foetal resorptions were documented. Experiment 2: 10.0 µg kg(-1) bw (which induced the highest blood pressure together with decreased foetal weight in experiment 1) or saline was infused in pregnant and non-pregnant rats (n = 7/9 p per group). Parameters of experiment 1 and numbers of peripheral leucocytes as well as signs of inflammation in the kidney and placenta were evaluated. RESULTS: Pg-LPS infusion in pregnant rats increased maternal systolic blood pressure, reduced placental weight (dose dependently) and decreased foetal weight and induced foetal resorptions. It, however, did not induce proteinuria or a generalised inflammatory response. No effects of Pg-LPS were seen in non-pregnant rats. CONCLUSION: Pg-LPS increased maternal blood pressure, induced placental and foetal growth restriction, and increased foetal resorptions, without inducing proteinuria and inflammation. Pg-LPS may therefore play a role in pregnancy complications induced by periodontitis.

Altered expression of immune-associated genes in first-trimester human decidua of pregnancies later complicated with hypertension or foetal growth restriction.

During pregnancy the maternal immune system has to coordinate uterine spiral-artery remodelling, trophoblast invasion, and acceptance of the semi-allogenic fetus simultaneously. As dysregulation of the immune system is associated with adverse pregnancy outcomes, we analysed first-trimester deciduas of pregnancies for immune parameters in later complicated pregnancies. Higher IL6 and macrophage mRNA expression, and lower ratios of regulatory macrophages were found in first-trimester deciduas of pregnancies later complicated with pregnancy-induced hypertension. Lower Gata3 (Th2) mRNA expression was found in deciduas of pregnancies with later foetal growth restriction. Our results suggest that adverse pregnancy outcomes are associated with immunological disturbances in first-trimester deciduas.

Hydrogen sulfide producing enzymes in pregnancy and preeclampsia.

Preeclampsia, a human pregnancy specific disorder is characterized by an anti-angiogenic state. As hydrogen sulfide (H(2)S) has pro-angiogenic and anti-oxidative characteristics, we hypothesized that H(2)S levels could play a role in the pathogenesis of preeclampsia and studied the placental expression of the H(2)S-producing enzymes cystathionine-γ-lyase (CSE) and cystathionine-ß-synthase (CBS). CBS and CSE protein are expressed in the fetal-placental endothelium and CBS only in Hofbauer cells. CBS mRNA expression is decreased (p = 0.002) in early-onset preeclampsia, while CSE mRNA is unchanged. Thus, down regulation of CBS during early-onset preeclampsia may result in less H(2)S-production and may aid in the anti-angiogenic state.