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Investigations into the immunological response of proteins is often masked by lipopolysaccharide (LPS) contamination. We report an optimized Triton X-114 (TX-114) based LPS extraction method for β-lactoglobulin (BLG) and soy protein extract suitable for cell-based immunological assays. Optimization of an existing TX-114 based phase LPS extraction method resulted in >99% reduction of LPS levels. However, remaining TX-114 was found to interfere with LPS and protein concentration assays and decreased viability of THP-1 macrophages and HEK-Blue 293 cells. Upon screening a range of TX-114 extraction procedures, TX-114-binding beads were found to most effectively lower TX-114 levels without affecting protein structural properties. LPS-purified proteins showed reduced capacity to activate TLR4 compared to non-treated proteins. LPS-purified BLG did not induce secretion of pro-inflammatory cytokines from THP-1 macrophages, as non-treated protein did, showing that LPS contamination masks the immunomodulatory effect of BLG. Both HEK293 cells expressing TLR4 and differentiated THP-1 macrophages were shown as a relevant model to screen the protein preparations for biological effects of LPS contamination. The reported TX-114 assisted LPS-removal from protein preparations followed by bead based removal of TX-114 allows evaluation of natively folded protein preparations for their immunological potential in cell-based studies.

Respiratory infections place a heavy burden on the health care system, particularly in the winter months. Individuals with a vulnerable immune system, such as very young children and the elderly, and those with an immune deficiency, are at increased risk of contracting a respiratory infection. Most respiratory infections are relatively mild and affect the upper respiratory tract only, but other infections can be more serious. These can lead to pneumonia and be life-threatening in vulnerable groups. Rather than focus entirely on treating the symptoms of infectious disease, optimizing immune responsiveness to the pathogens causing these infections may help steer towards a more favorable outcome. Nutrition may have a role in such prevention through different immune supporting mechanisms. Nutrition contributes to the normal functioning of the immune system, with various nutrients acting as energy sources and building blocks during the immune response. Many micronutrients (vitamins and minerals) act as regulators of molecular responses of immune cells to infection. It is well described that chronic undernutrition as well as specific micronutrient deficiencies impair many aspects of the immune response and make individuals more susceptible to infectious diseases, especially in the respiratory and gastrointestinal tracts. In addition, other dietary components such as proteins, pre-, pro- and synbiotics, and also animal- and plant-derived bioactive components can further support the immune system. Both the innate and adaptive defense systems contribute to active antiviral respiratory tract immunity. The initial response to viral airway infections is through recognition by the innate immune system of viral components leading to activation of adaptive immune cells in the form of cytotoxic T cells, the production of neutralizing antibodies and the induction of memory T and B cell responses. The aim of this review is to describe the effects of a range different dietary components on anti-infective innate as well as adaptive immune responses and to propose mechanisms by which they may interact with the immune system in the respiratory tract.

Beta-glucans enable functional reprogramming of innate immune cells, a process defined as “trained immunity”, which results in enhanced host responsiveness against primary (training) and/or secondary infections (resilience). Trained immunity holds great promise for promoting immune responses in groups that are at risk ( e.g. elderly and patients). In this study, we modified an existing in vitro model for trained immunity by actively inducing monocyte-to-macrophage differentiation using M-CSF and applying continuous exposure. This model reflects mucosal exposure to β-glucans and was used to study the training effects of a variety of soluble or non-soluble β-glucans derived from different sources including oat, mushrooms and yeast. In addition, trained immunity effects were related to pattern recognition receptor usage, to which end, we analyzed β-glucan-mediated Dectin-1 activation. We demonstrated that β-glucans, with different sources and solubilities, induced training and/or resilience effects. Notably, trained immunity significantly correlated with Dectin-1 receptor activation, yet Dectin-1 receptor activation did not perform as a sole predictor for β-glucan-mediated trained immunity. The model, as validated in this study, adds on to the existing in vitro model by specifically investigating macrophage responses and can be applied to select non-digestible dietary polysaccharides and other components for their potential to induce trained immunity.

Although an impact of processing on immunogenicity of food proteins has clearly been demonstrated, the underlying mechanisms are still unclear. We applied 3 different processing methods: wet heating (60 °C) and low- or high-temperature (50 °C or 130 °C, respectively) dry-heating in absence or presence of reducing sugars, to [beta]-lactoglobulin (BLG), lysozyme and thyroglobulin, which represent dietary proteins with different pI or molecular weight. Uptake of the soluble fraction of the samples was tested in two types of, genetically homogeneous, antigen-presenting cells (macrophages and dendritic cells derived from THP-1 monocytes). This revealed a strong correlation between the uptake of the different protein samples by macrophages and dendritic cells, and confirmed the key role of hydrophobicity, over aggregation, in determining the uptake. Several uptake routes were shown to contribute to the uptake of BLG by macrophages. However, cytokine responses following exposure of macrophages to BLG samples were not related to the levels of uptake. Together, our results demonstrate that heat-treatment-induced increased hydrophobicity is the prime driving factor in uptake, but not in cytokine production, by THP-1 macrophages.

Background There is increasing evidence that dietary fat, especially saturated fat, promotes the translocation of lipopolysaccharide (LPS) via chylomicron production in the gut. Chylomicrons can subsequently transport LPS to other parts of the body, where they can induce low-grade chronic inflammation that is linked to various metabolic and gut-related diseases. To identify promising (food) compounds that can prevent or ameliorate LPS-related low-grade inflammation, we developed and optimized a bicameral in vitro model for dietary fat-induced LPS translocation that closely mimics the in vivo situation and facilitates high-throughput screening. Methods Caco-2 cells were cultured in monolayers and differentiated to a small intestinal phenotype in 21 days. Thereafter, optimal conditions for fat-induced chylomicron production were determined by apical exposure of Caco-2 cells to a dilution range of in vitro digested palm oil and sunflower oil, optionally preceded by a 1-week apical FBS deprivation (cultured without apical fetal bovine serum). Chylomicron production was assessed by measuring basolateral levels of the chylomicron-related marker apolipoprotein B. Next, LPS was coincubated at various concentrations with the digested oils, and fat-induced LPS translocation to the basolateral side was assessed. Results We found that dietary fat-induced LPS translocation in Caco-2 cells was optimal after apical exposure to digested oils at a 1:50 dilution in combination with 750 ng/mL LPS, preceded by 1 week of apical FBS deprivation. Coincubation with the chylomicron blocker Pluronic L81 confirmed that fat-induced LPS translocation is mediated via chylomicron production in this Caco-2 cell model. Conclusion We developed a robust Caco-2 cell model for dietary fat-induced LPS translocation that can be used for high-throughput screening of (food) compounds that can reduce LPS-related low-grade inflammation.

More understanding of the risk-benefit effect of the glycoalkaloid tomatine is required to be able to estimate the role it might play in our diet. In this work, we focused on effects towards intestinal epithelial cells based on a Caco-2 model in order to analyze the influence on the cell monolayer integrity and on the expression levels of genes involved in cholesterol/sterol biosynthesis (LDLR), lipid metabolism (NR2F2), glucose and amino acid uptake (SGLT1, PAT1), cell cycle (PCNA, CDKN1A), apoptosis (CASP-3, BMF, KLF6), tight junctions (CLDN4, OCLN2) and cytokine-mediated signaling (IL-8, IL1β, TSLP, TNF-α). Furthermore, since the bioactivity of the compound might vary in the presence of a food matrix and following digestion, the influence of both pure tomatine and in vitro digested tomatine with and without tomato fruit matrix was studied. The obtained results suggested that concentrations <20 µg/mL of tomatine, either undigested or in vitro digested, do not compromise the viability of Caco-2 cells and stimulate cytokine expression. This effect of tomatine, in vitro digested tomatine or in vitro digested tomatine with tomato matrix differs slightly, probably due to variations of bioactivity or bioavailability of the tomatine. The results lead to the hypothesis that tomatine acts as hormetic compound that can induce beneficial or risk toxic effects whether used in low or high dose.

Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies.

Anti-cancer T-cell responses are often halted due to the immune-suppressive micro-environment, in part related to tumor-associated macrophages. In the current study, we assessed indigestible β-glucans (oatβG, curdlan, grifolan, schizophyllan, lentinan, yeast whole glucan particles (yWGP), zymosan and two additional yeast-derived β-glucans a and b) for their physicochemical properties as well as their effects on the plasticity of human monocyte-derived macrophages that were polarized with IL-4 to immune-suppressive macrophages. Beta-glucans were LPS/LTA free, and tested for solubility, molecular masses, protein and monosaccharide contents. Curdlan, yeast-b and zymosan re-polarized M(IL-4) macrophages towards an M1-like phenotype, in particular showing enhanced gene expression of CCR7, ICAM1 and CD80, and secretion of TNF-α and IL-6. Notably, differential gene expression, pathway analysis as well as protein expressions demonstrated that M(IL-4) macrophages treated with curdlan, yeast-b or zymosan demonstrated enhanced production of chemo-attractants, such as CCL3, CCL4, and CXCL8, which contribute to recruitment of monocytes and neutrophils. The secretion of chemo-attractants was confirmed when using patient-derived melanoma-infiltrating immune cells. Taken together, the bacterial-derived curdlan as well as the yeast-derived β-glucans yeast-b and zymosan have the unique ability to preferentially skew macrophages towards a chemo-attractant-producing phenotype that may aid in anti-cancer immune responses.

The aim of this study was to investigate the effects of three Lactobacillus plantarum strains on in-vivo small intestinal barrier function and gut mucosal gene transcription in human subjects. The strains were selected for their differential effects on TLR signalling and tight junction protein rearrangement, which may lead to beneficial effects in a stressed human gut mucosa. Ten healthy volunteers participated in four different intervention periods: 7-day oral intake of either L. plantarum WCFS1, CIP104448, TIFN101 or placebo, proceeded by a 4 weeks wash-out period. Lactulose-rhamnose ratio (an indicator of small intestinal permeability) increased after intake of indomethacin, which was given as an artificial stressor of the gut mucosal barrier (mean ratio 0.06 ± 0.04 to 0.10 ± 0.06, p  = 0.001), but was not significantly affected by the bacterial interventions. However, analysis in small intestinal biopsies, obtained by gastroduodenoscopy, demonstrated that particularly L. plantarum TIFN101 modulated gene transcription pathways related to cell-cell adhesion with high turnover of genes involved in tight- and adhesion junction protein synthesis and degradation ( e.g. actinin alpha-4, metalloproteinase-2). These effects were less pronounced for L. plantarum WCFS1 and CIP104448. In conclusion, L. plantarum TIFN101 induced the most pronounced probiotic properties with specific gene transcriptional effects on repair processes in the compromised intestine of healthy subjects.

Adoptive transfer of T cells gene-engineered with antigen-specific T cell receptors (TCRs) has proven its feasibility and therapeutic potential in the treatment of malignant tumors. To ensure further clinical development of TCR gene therapy, it is necessary to target immunogenic epitopes that are related to oncogenesis and selectively expressed by tumor tissue, and implement strategies that result in optimal T cell fitness. In addition, in particular for the treatment of solid tumors, it is equally necessary to include strategies that counteract the immune-suppressive nature of the tumor micro-environment. Here, we will provide an overview of the current status of TCR gene therapy, and redefine the following three challenges of improvement: \"choice of target antigen\"; \"fitness of T cells\"; and \"sensitization of tumor milieu.\" We will categorize and discuss potential strategies to address each of these challenges, and argue that advancement of clinical TCR gene therapy critically depends on developments toward each of the three challenges.