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Pancreatic islet transplantation is proposed as a cure for type 1 diabetes mellitus (T1D). Despite its success in optimal regulation of glucose levels, limitations in longevity of islet grafts still require innovative solutions. Inflammatory stress post-transplantation and loss of extracellular matrix attribute to the limited β-cell survival. Pancreatic stellate cells (PSCs), identified as pancreatic-specific stromal cells, have the potential to play a crucial role in preserving islet survival. Our study aimed to determine the effects of PSCs co-cultured with human CM β-cells and human islets under inflammatory stress induced by a cytokine cocktail of IFN-γ, TNF-α and IL-1β. Transwell culture inserts were utilized to assess the paracrine impact of PSCs on β-cells, alongside co-cultures enabling direct interaction between PSCs and human islets. We found that co-culturing PSCs with human CM β-cells and human cadaveric islets had rescuing effects on cytokine-induced stress. Effects were different under normoglycemic and hyperglycemic conditions. PSCs were associated with upregulation of β-cell mitochondrial activity and suppression of inflammatory gene expression. The rescuing effects exist both in indirect and direct co-culture methods. Furthermore, we tested whether PSCs have rescuing effects on human islets in conventional alginate-based microcapsules and in composite microcapsules composed of alginate-pectin collagen type IV, laminin sequence RGD, Nec-1, and amino acid. PSCs partially prevented cytokine-induced stress in both systems, but beneficial effects were stronger in composite capsules. Our findings show novel effects of PSCs on islet health. Islets and PSCs coculturing or co-transplantation might mitigate the inflammation stress and improve islet transplantation outcomes.

The intestinal epithelium is an important gatekeeper of the human body by forming a barrier for the luminal content of the intestine. The barrier function is regulated by a complex crosstalk between different cell types, including cells from the enteric nervous system (ENS). ENS is considered to influence gastrointestinal processes and functions, but its direct effect on epithelial barrier function remains to be confirmed. To investigate the effect of nerve cells on the gut barrier function, an in vitro co-culture system was established in which T84 intestinal epithelial cells and SH-SY5Y nerve cells were seeded in ratios of 29:1 and 14:1. When the epithelial barrier was disrupted with the calcium ionophores A23187, we found that nerve cells exert a protective effect on A23187-induced disruption and that this protective effect is nerve cell concentration-dependent. This was demonstrated by rescuing effects on transepithelial electrical resistance (TEER) and upregulation of tight junction (TJ) protein expression. Furthermore, we studied whether similar rescuing effects could be achieved with the human milk oligosaccharides (hMOs) 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL). Our results illustrate that in the presence of nerve cells 2′-FL and 3-FL do not have any additional rescuing effects, but that these hMOs can substitute the rescuing effects of nerve cells in the absence of nerve cells. Meanwhile, 2′-FL and 3-FL show different regulation effects on TJ expression. These findings provide valuable insights into potential therapeutic strategies for maintaining intestinal barrier integrity.

In this study, an in vitro co-culture model using an electric cell-substrate impedance sensing system (ECIS) for testing the impact of real-time fermentation of non-digestible carbohydrates (NDCs) by the intestinal microbiota on gut barrier function was established. We applied Lactobacillus plantarum WCFS1 as a model intestinal bacterium and alginate-pectin as immobilization polymers as well as a source of NDCs to determine the impact of pectin fermentation on the barrier function of T84 gut epithelial cells. In the first design, L. plantarum WCFS1 was encapsulated in an alginate capsule followed by embedding in an agar layer to mimic a firm mucus layer that might be present in the colon. In this experimental design, the presence of the agar layer interfered with the transepithelial electrical resistance (TEER) measurement of T84 cells. Subsequently, we removed the agar layer and used encapsulated bacteria in an alginate gel and found that the TEER measurement was adequate. The encapsulation of the L. plantarum WCFS1 does avoid direct contact with cells. Also, the encapsulation system allows higher amounts of packing densities of L. plantarum WCFS1 in a limited space which can limit the oxygen concentration within the capsule and therefore create anaerobic conditions. To test this design, T84 cells were co-incubated with L. plantarum alginate-capsules supplemented with graded loads of fermentable pectin (0, 4, and 8 mg/ml per capsule) to investigate the effect of pectin fermentation on gut barrier function. We observed that as the pectin content in the L. plantarum capsules increased, pectin showed a gradually stronger protective effect on the TEER of the gut epithelium. This could partly be explained by enhanced SCFA production as both lactate and acetate were enhanced in L. plantarum containing alginate capsules with 8 mg/ml pectin. Overall, this newly designed in vitro co-culture model allows for studying the impact of bacteria-derived fermentation products but also for studying the direct effects of NDCs on gut barrier function in a relatively high-throughput way.

Subcutaneous polymer scaffolds have shown potential for creating an optimal transplantation site in cellular replacement therapy, e.g., when transplanting insulin-producing cells to cure type 1 diabetes. Imperative for these scaffolds is a high degree of vascularization to guarantee long-term functional cellular survival. In this study, the effect of the nitric oxide (NO) donor S-nitroso-N-acetyl-dl-penicillamine (SNAP) on the vascularization degree of a subcutaneous poly(d,l-lactide-co-ε-caprolactone) (PDLLCL) scaffold was investigated. To this end, scaffolds were implanted under the skin of C57BL/6 mice. Each mouse received a control scaffold and a scaffold containing SNAP. At day 7, 14, and 28, the oxygen percentage within the scaffolds was measured and at day 28, the vascularization degree was determined with lectin infusion and gene expression analysis. We measured lower oxygen percentages within the scaffolds containing the NO-donor up to day 14 compared to the control scaffolds, but no differences were found at day 28. Although blood vessels in the scaffolds were well perfused, no differences between the groups were found in the lectin staining and gene expression of vascular markers, such as CD31, CD105, and VEGFa. To conclude, in this biomaterial setting, addition of a NO-donor did not improve the vascularization degree of the subcutaneous scaffold.

Abstract Human triosephosphate isomerase (HsTIM) is a central glycolytic enzyme and is overexpressed in cancer cells with accelerated glycolysis. Triple-negative breast cancer is highly dependent on glycolysis and is typically treated with a combination of surgery, radiation therapy, and chemotherapy. Deamidated HsTIM was recently proposed as a druggable target. Although thiol-reactive drugs affect cell growth in deamidated HsTIM-complemented cells, the role of this protein as a selective target has not been demonstrated. To delve into the usefulness of deamidated HsTIM as a selective target, we assessed its natural accumulation in breast cancer cells. We found that deamidated HsTIM accumulates in breast cancer cells but not in noncancerous cells. The cancer cells are selectively programmed to undergo cell death with thiol-reactive drugs that induced the production of methylglyoxal (MGO) and advanced glycation-end products (AGEs). In vivo, a thiol-reactive drug effectively inhibits the growth of xenograft tumors with an underlying mechanism involving deamidated HsTIM. Our findings demonstrate the usefulness of deamidated HsTIM as target to develop new therapeutic strategies for the treatment of cancers and other pathologies in which this post translationally modified protein accumulates.

Reducing inflammation by diet is a major goal for prevention or lowering symptoms of a variety of diseases, such as auto-immune reactions and cancers. Natural polysaccharides are increasingly gaining attention due to their potential immunomodulating capacity. Structures of those molecules are highly important for their effects on the innate immune system, cytokine production and secretion, and enzymes in immune cells. Such polysaccharides include β-glucans, pectins, fucoidans, and fructans. To better understand the potential of these immunomodulatory molecules, it is crucial to enhance dedicated research in the area. A bibliometric analysis was performed to set a starting observation point. Major pillars of inflammation, such as pattern recognition receptors (PRRs), enzymatic production of inflammatory molecules, and involvement in specific pathways such as Nuclear-factor kappa-B (NF-kB), involved in cell transcription, survival, and cytokine production, and mitogen-activated protein kinase (MAPK), a regulator of genetic expression, mitosis, and cell differentiation. Therefore, the outcomes from polysaccharide applications in those scenarios are discussed.

Background. Islet transplantation is a promising treatment for type 1 diabetes. It has the potential to improve glycemic control, particularly in patients suffering from hypoglycemic unawareness and glycemic instability. As most islet grafts do not function permanently, efforts are needed to create an accessible and replaceable site, for islet grafts or for insulin-producing cells obtained from replenishable sources. To this end, we designed and tested an artificial, polymeric subcutaneous transplantation site that allows repeated transplantation of islets. Methods. In this study, we developed and compared scaffolds made of poly(D,L,-lactide-co-ε-caprolactone) (PDLLCL) and polycaprolactone (PCL). Efficacy was first tested in mice‚ and then, as a proof of principle for application in a large animal model, the scaffolds were tested in pigs, as their skin structure is similar to that of humans. Results. In mice, islet transplantation in a PCL scaffold expedited return to normoglycemia in comparison to PDLLCL (7.7 ± 3.7 versus 16.8 ± 6.5 d), but it took longer than the kidney capsule control group. PCL also supported porcine functional islet survival in vitro. Subcutaneous implantation of PDLLCL and PCL scaffolds in pigs revealed that PCL scaffolds were more stable and was associated with less infiltration by immune cells than PDLLCL scaffolds. Prevascularized PCL scaffolds were therefore used to demonstrate the functional survival of allogenic islets under the skin of pigs. Conclusions. To conclude, a novel PCL scaffold shows efficacy as a readily accessible and replaceable, subcutaneous transplantation site for islets in mice and demonstrated islet survival after a month in pigs.

Our understanding of microorganisms residing within our gut and their roles in the host metabolism and immunity advanced greatly over the past 20 years. Currently, microbiome studies are shifting from association and correlation studies to studies demonstrating causality of identified microbiome signatures and identification of molecular mechanisms underlying these interactions. This transformation is crucial for the efficient translation into clinical application and development of targeted strategies to beneficially modulate the intestinal microbiota. As mechanistic studies are still quite challenging to perform in humans, the causal role of microbiota is frequently evaluated in animal models that need to be appropriately selected. Here, we provide a comprehensive overview on approaches that can be applied in addressing causality of host-microbe interactions in five major animal model organisms (Caenorhabditis elegans, Drosophila melanogaster, zebrafish, rodents, and pigs). We particularly focused on discussing methods available for studying the causality ranging from the usage of gut microbiota transfer, diverse models of metabolic and immune perturbations involving nutritional and chemical factors, gene modifications and surgically induced models, metabolite profiling up to culture-based approached. Furthermore, we addressed the impact of the gut morphology, physiology as well as diet on the microbiota composition in various models and resulting species specificities. Finally, we conclude this review with the discussion on models that can be applied to study the causal role of the gut microbiota in the context of metabolic syndrome and host immunity. We hope this review will facilitate important considerations for appropriate animal model selection.

Pectin, a plant-derived polysaccharide, possesses immense technological and biological application value. Several variables influence pectin’s physicochemical aspects, resulting in different fermentations, interactions with receptors, and other functional properties. Some of those variables are molecular weight, degree of methylation and blockiness, and monosaccharide composition. Cancer cell cytotoxicity, important fermentation-related byproducts, immunomodulation, and technological application were found in cell culture, animal models, and preclinical and clinical assessments. One of the greater extents of recent pectin technological usage involves nanoencapsulation methods for many different compounds, ranging from chemotherapy and immunotherapy to natural extracts from fruits and other sources. Structural modification (modified pectin) is also utilized to enhance the use of dietary fiber. Although pectin is already recognized as a component of significant importance, there is still a need for a comprehensive review that delves into its intricate relationships with biological effects, which depend on the source and structure of pectin. This review covers all levels of clinical research, including cell culture, animal studies, and clinical trials, to understand how the plant source and pectin structures influence the biological effects in humans and some technological applications of pectin regarding human health.

Mohammadi et al. show that the transplantation of pancreatic islets within mesenchymal-stem cell (MSC) exosome-loaded hybrid alginate microcapsules in diabetic mice lead to diminished immune-based foreign body response and hyperglycemia control. This is due to immunomodulatory behavior of MSC exosomes at the engraftment site where they exert local immunosuppressive effects.

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.

Gastrointestinal immunity: Pectin protection The dietary fiber pectin plays several immunity-related roles in the gastrointestinal tract and could help manage inflammatory conditions according to a review of recent research. Pectin, found in many fruits and vegetables, takes various structural forms that have different impacts on gut health. Martin Beukema and co-workers at University Medical Centre Groningen in The Netherlands reviewed how these pectin structures interact with the gastrointestinal immune barrier. Studies have shown that pectins can strengthen mucous membranes and epithelial tissue, and activate or inhibit immune cells such as macrophages. Pectins also have a positive influence on the diversity and abundance of microbiota that inhabit the gut, for example helping beneficial bacteria to adhere to epithelial cells while blocking the adhesion of pathogens. Beukema and co-workers point to an urgent need for more studies in humans.

Grafting of microencapsulated pancreatic islets has been proposed as an alternative to exogenous insulin for the treatment of type 1 diabetes mellitus. Microencapsulated islets are protected from direct contact with immune cells and larger immune‐active molecules such as immunoglobulins. Unfortunately, many islet cells in the microcapsules are lost in the immediate period after transplantation due to an early host immune response limiting long‐term function of the graft. Gusperimus has shown to reduce the inflammatory responses to grafted encapsulated islets, but it cannot be appropriately used because it is easily hydrolyzed leading to loss of activity. To temporarily modulate the inflammatory response directly after implantation and stabilize gusperimus, squalene‐gusperimus nanoparticles (Sq‐GusNPs) are developed and incorporated in human islets‐containing alginate‐based microcapsules. A prolonged and continuous release of gusperimus is achieved. This offers an anti‐inflammatory microenvironment in the vicinity of the microcapsules and a reduction of cytokine secretion by lipopolysaccharides‐activated human macrophages. Release of gusperimus from Sq‐GusNPs does not affect the in vitro viability or function of human pancreatic islets. The data illustrate that incorporation of Sq‐GusNPs in alginate microcapsules offers an opportunity to temporarily modulate the immediate immune response after the grafting procedure of encapsulated islets cells and reduce loss of islet cells.

Scope: Cost-effective microbial biosynthesis of 2′-fucosyllactose (2′-FL) allows its application in infant formula. The specific effects of 2′-FL on the gastrointestinal immune barrier are still largely unknown. Methods/results: Here, we quantified and compared the effects of HMOs isolated from human milk, 2′-FL/lactose (Lac), and 2′-FL on the expression of the mucus associated genes MUC2, TFF3, RETLB and the Golgi sulfotransferases, CHST5, and GAL3ST5, in human goblet cells. We also determined whether these compounds have protective effects on A23187-induced barrier disruption of human T84 gut epithelial cells in vitro. The impact of isolated HMOs and 2′-FL/Lac on the mRNA expression of the mucus-related genes was minor while pure 2′-FL significantly induced GAL3ST2 and CHST5. Isolated HMOs, 2′-FL/Lac and 2′-FL all prevented A23187-induced barrier disruption in human T84 cells. Conclusion: Our findings indicate that 2′-FL modulates the secretory function of goblet cells and protects gut epithelial cells.

Beyond the problem in public health that protist-generated diseases represent, understanding the variety of mechanisms used by these parasites to interact with the human immune system is of biological and medical relevance. Giardia lamblia is an early divergent eukaryotic microorganism showing remarkable pathogenic strategies for evading the immune system of vertebrates. Among various multifunctional proteins in Giardia, arginine deiminase is considered an enzyme that plays multiple regulatory roles during the life cycle of this parasite. One of its most important roles is the crosstalk between the parasite and host. Such a molecular “chat” is mediated in human cells by membrane receptors called Toll-like receptors (TLRs). Here, we studied the importance of the 3D structure of giardial arginine deiminase (GlADI) to immunomodulate the human immune response through TLRs. We demonstrated the direct effect of GlADI on human TLR signaling. We predicted its mode of interaction with TLRs two and four by using the AlphaFold-predicted structure of GlADI and molecular docking. Furthermore, we showed that the immunomodulatory capacity of this virulent factor of Giardia depends on the maintenance of its 3D structure. Finally, we also showed the influence of this enzyme to exert specific responses on infant-like dendritic cells.

COVID-19 might lead to multi-organ failure and, in some cases, to death. The COVID-19 severity is associated with a “cytokine storm.” Danger-associated molecular patterns (DAMPs) are proinflammatory molecules that can activate pattern recognition receptors, such as toll-like receptors (TLRs). DAMPs and TLRs have not received much attention in COVID-19 but can explain some of the gender-, weight- and age-dependent effects. In females and males, TLRs are differentially expressed, likely contributing to higher COVID-19 severity in males. DAMPs and cytokines associated with COVID-19 mortality are elevated in obese and elderly individuals, which might explain the higher risk for severer COVID-19 in these groups. Adenosine signaling inhibits the TLR/NF-κB pathway and, through this, decreases inflammation and DAMPs’ effects. As vaccines will not be effective in all susceptible individuals and as new vaccine-resistant SARS-CoV-2 mutants might develop, it remains mandatory to find means to dampen COVID-19 disease severity, especially in high-risk groups. We propose that the regulation of DAMPs via adenosine signaling enhancement might be an effective way to lower the severity of COVID-19 and prevent multiple organ failure in the absence of severe side effects.

Cellular overgrowth and fibrosis in the foreign body response can compromise the function of transplanted cells. Here, the authors report on the zwitterionically modified alginates for the encapsulation of cells to reduce cellular overgrowth and demonstrate application in mice, dogs and pigs.

Human milk oligosaccharides (HMOs) have beneficial immune effects but the mechanisms of action are not well-understood. Here we study Toll-like receptor (TLR) signaling of the HMOs 2′-FL, 3-FL, 6′-SL, LNT2, and LNnT and their effect on cytokine production in human macrophages. 3-FL activated TLR2 and LNT2 activated all TLRs in a dose-dependent way. In an inhibition assay, 2′-FL, 6′-SL, and LNnT inhibited TLR5 and 7, while 3-FL inhibited TLR5, 7, and 8. 6′-SL showed a synergistic effect on ssRNA40-induced TLR8 activation. In addition, we measured HMO-induced cytokine production in THP1 macrophages. IL-10 and TNFα were induced by LNT2, and the effects were NF-κB dependent, while the other HMOs had minor effects. The potent effects of LNT2 might be explained by the unique N-acetylglucosamine end that binds to leucine-rich repeats on TLRs. Our data suggest that the effects of HMOs on TLR signaling and immunomodulation of macrophages are HMO-structure dependent.

The diverse and dynamic microbial community of the human gastrointestinal tract plays a vital role in health, with gut microbiota supporting the development and function of the gut immune barrier. Crosstalk between microbiota-gut epithelium and the gut immune system determine the individual health status, and any crosstalk disturbance may lead to chronic intestinal conditions, such as inflammatory bowel diseases (IBD) and celiac disease. Microbiota-derived metabolites are crucial mediators of host-microbial interactions. Some beneficially affect host physiology such as short-chain fatty acids (SCFAs) and secondary bile acids. Also, tryptophan catabolites determine immune responses, such as through binding to the aryl hydrocarbon receptor (AhR). AhR is abundantly present at mucosal surfaces and when activated enhances intestinal epithelial barrier function as well as regulatory immune responses. Exogenous diet-derived indoles (tryptophan) are a major source of endogenous AhR ligand precursors and together with SCFAs and secondary bile acids regulate inflammation by lowering stress in epithelium and gut immunity, and in IBD, AhR expression is downregulated together with tryptophan metabolites. Here, we present an overview of host microbiota-epithelium- gut immunity crosstalk and review how microbial-derived metabolites contribute to host immune homeostasis. Also, we discuss the therapeutic potential of bacterial catabolites for IBD and celiac disease and how essential dietary components such as dietary fibers and bacterial tryptophan catabolites may contribute to intestinal and systemic homeostasis.

The gastrointestinal tract (GIT) is colonized by a broad spectrum of microorganism, of which bacteria are especially recognized for the health effects they exert. However insight in the effector molecules on the bacteria responsible are still largely lacking. Recently, bacterial exopolysaccharides (EPS), which are glycan structures on the bacterial cell wall, have gained considerable attention for their effects on human health. EPS are found to act as polymers that directly influence bacterial interactions with their host, such as bacterial adhesion to the GIT epithelium, their influence on immune responses, and the intestinal microbiota. These effects are highly dependent on the type of EPS structures and might be related to specific monosaccharides or linkages in EPS. Here, current knowledge on structure-specific EPS functions is reviewed in view of possible use of EPS to stimulate intestinal health and the formulation of dietary EPS mixtures or the possible creation of new EPS structures.

Human milk oligosaccharides (hMOs) are important bioactive components in mother’s milk contributing to infant health by supporting colonization and growth of gut microbes. In particular, Bifidobacterium genus is considered to be supported by hMOs. Approximately 200 different hMOs have been discovered and characterized, but only a few abundant hMOs can be produced in sufficient amounts to be applied in infant formula. These hMOs are usually supplied in infant formula as single molecule, and it is unknown which and how individual hMOs support growth of individual gut bacteria. To investigate how individual hMOs influence growth of several relevant intestinal bacteria species, we studied the effects of three hMOs (2′-fucosyllactose, 3-fucosyllactose, and 6′-sialyllactose) and an hMO acid hydrolysate (lacto-N-triose) on three Bifidobacteria and one Faecalibacterium and introduced a co-culture system of two bacterial strains to study possible cross-feeding in presence and absence of hMOs. We observed that in monoculture, Bifidobacterium longum subsp. infantis could grow well on all hMOs but in a structure-dependent way. Faecalibacterium prausnitzii reached a lower cell density on the hMOs in stationary phase compared to glucose, while B. longum subsp. longum and Bifidobacterium adolescentis were not able to grow on the tested hMOs. In a co-culture of B. longum subsp. infantis with F. prausnitzii, different effects were observed with the different hMOs; 6′-sialyllactose, rather than 2′-fucosyllactose, 3-fucosyllactose, and lacto-N-triose, was able to promote the growth of B. longum subsp. infantis. Our observations demonstrate that effects of hMOs on the tested gut microbiota are hMO-specific and provide new means to support growth of these specific beneficial microorganisms in the intestine.

Toll-like receptor 8 (TLR-8) plays a role in the pathogenesis of autoimmune disorders and associated gastrointestinal symptoms that reduce quality of life of patients. Dietary interventions are becoming more accepted as mean to manage onset, progression, and treatment of a broad spectrum of inflammatory conditions. In this study, we assessed the impact of N-glycans derived from bovine lactoferrin (bLF) on the inhibition of TLR-8 activation. We investigated the effects of N-glycans in their native form, as well as in its partially demannosylated and partially desialylated form, on HEK293 cells expressing TLR-8, and in human monocyte-derived dendritic cells (MoDCs). We found that in HEK293 cells, N-glycans strongly inhibited the ssRNA40 induced TLR-8 activation but to a lesser extent the R848 induced TLR-8 activation. The impact was compared with a pharmaceutical agent, i.e., chloroquine (CQN), that is clinically applied to antagonize endosomal TLR- activation. Inhibitory effects of the N-glycans were not influenced by the partially demannosylated or partially desialylated N-glycans. As the difference in charge of the N-glycans did not influence the inhibition capacity of TLR-8, it is possible that the inhibition mediated by the N-glycans is a result of a direct interaction with the receptor rather than a result of pH changes in the endosome. The inhibition of TLR-8 in MoDCs resulted in a significant decrease of IL-6 when cells were treated with the unmodified (0.5-fold, p < 0.0001), partially demannosylated (0.3-fold, p < 0.0001) and partially desialylated (0.4-fold, p < 0.0001) N-glycans. Furthermore, the partially demannosylated and partially desialylated N-glycans showed stronger inhibition of IL-6 production compared with the native N-glycans. This provides evidence that glycan composition plays a role in the immunomodulatory activity of the isolated N-glycans from bLF on MoDCs. Compared to CQN, the N-glycans are specific inhibitors of TLR-8 activation and of IL-6 production in MoDCs. Our findings demonstrate that isolated N-glycans from bLF have attenuating effects on TLR-8 induced immune activation in HEK293 cells and human MoDCs. The inhibitory capacity of N-glycans isolated from bLF onTLR-8 activation may become a food-based strategy to manage autoimmune, infections or other inflammatory disorders.

Lactic acid bacteria (LAB) can support host health and crosstalks between ligands on bacterial cells and Toll-like receptors (TLR) are known as key inducers of these health effects. LAB-secreted bioactive factors also possess health-promoting properties but the underlying mechanisms remain to be identified. Here, we report that secreted factors of different LAB strains induce cytokine (IL-10 and TNF-α) production by THP1-differentiated human macrophages in a species- and strain-dependent manner. LAB-secreted products also drive immune-stimulating and anti-pathogenic M1-macrophage polarization. Moreover, bacterial supernatants induce species- and strain-dependent TLR2 activation. Several bacterial strains such as L. casei CCFM9 and L. brevis CCFM498 cannot activate TLR signaling, but their secreted products strongly activate TLR2. This indicates that LAB-secreted components may further boost the functional effects of bacterial strains. Our results confirm direct interactions of LAB-secreted components with TLRs, and provide novel insights in how LABs may provide immune-modulation.

Previous studies have shown that necrostatin-1 (Nec-1) supplementation improved the viability of murine islets following exposure to nitric oxide, increased the survival of human islets during hypoxic culture, and augmented the maturation of pre-weaned porcine islets (PPIs) after 7 days of tissue culture. A limitation of these studies is that only one concentration of Nec-1 was used, and no studies have determined the optimal dose of Nec-1 for PPIs. Thus, the present study examined the effects of Nec-1 on PPIs at four different doses-0, 25, 50, 100, and 200 μM-after 7 days of tissue culture when supplemented on day 3. PPIs were isolated from pancreata of pre-weaned Yorkshire piglets (8-15 days old) and cultured in a specific islet maturation media added with Nec-1 on day 3 of tissue culture at 4 different doses-0, 25, 50, 100, and 200 μM (n = 6 for each dose). After 7 days of tissue culture, islets were assessed for recovery, viability, endocrine cellular content, GLUT2 expression in beta cells, and insulin secretion after glucose challenge. Nec-1 did not affect the viability of both intact islets and dissociated islets cells during tissue culture regardless of doses. Islets cultured in media supplemented with Nec-1 at 100 μM, but not 25, 50, or 200 μM, had a significantly higher recovery, composition of endocrine cells, GLUT2 expression in beta cells, and insulin secretion capacity than control islets cultured in media without Nec-1 supplementation. Moreover, culturing islets in 200 μM Nec-1 supplemented media not only failed to improve the insulin release but resulted in a lower glucose-induced insulin stimulation index compared to islets cultured in media added with 100 μM Nec-1. Xenotransplantation using porcine islets continues to demonstrate scientific advances to justify this area of research. Our findings indicate that Nec-1 supplementation at 100 μM was most effective to enhance the in vitro maturation of PPIs during tissue culture.

Medical devices for cell therapy can be improved through prevascularization. In this work we study the vascularization of a porous polymer device, previously used by our group for pancreatic islet transplantation with results indicating improved glycemic control. Oxygen partial pressure within such devices was monitored non-invasively using an optical technique. Oxygen-sensitive tubes were fabricated and placed inside devices prior to subcutaneous implantation in nude mice. We tested the hypothesis that vascularization will be enhanced by administration of the pro-angiogenic factor hydrogen sulfide (H 2 S). We found that oxygen dynamics were unique to each implant and that the administration of H 2 S does not result in significant changes in perfusion of the devices as compared with control. These observations suggest that vascular perfusion and density are not necessarily correlated, and that the rate of vascularization was not enhanced by the pro-angiogenic agent.

Pregnancy is associated with adaptations of the immune response and with changes in the gutmicrobiota. We hypothesized the gut microbiota are involved in inducing (part of) the immunological adaptations during pregnancy. To test this hypothesis, we collected feces from pregnant conventional mice before and during pregnancy (days 7, 14, and 18) and microbiota were measured using 16S RNA sequencing. At day 18, mice were sacrificed and splenic (various Th cell populations) and blood immune cells (monocyte subsets) were measured by flow cytometry. The data were compared with splenic and blood immune cell populations from pregnant (day 18) germfree mice and non-pregnant conventional and germfree mice. Finally, the abundances of the individual gut bacteria in the microbiota of each conventional pregnant mouse were correlated to the parameters of the immune response of the same mouse. The microbiota of conventional mice were significantly different at the end of pregnancy (day 18) as compared with pre-pregnancy (Permanova, p < 0.05). The Shannon index was decreased and the Firmicutes/Bacteroidetes ratio was increased (Friedman followed by Dunn's test, p < 0.05), while abundances of various species (such as Allobaculum stercoricanis, Barnesiella intestihominis, and Roseburia faecis) were significantly different at day 18 compared with pre-pregnancy. In pregnant conventional mice, the percentage of Th1 cells was decreased, while the percentages of Treg cells and Th2 cells were or tended to be increased vs. non-pregnant mice. In germfree mice, only the percentage of Th1 cells was decreased in pregnant vs. non-pregnant mice, with no effect of pregnancy on Treg and Th2 cells. The percentages of monocyte subsets were affected by pregnancy similarly in conventional and germfree mice. However, the activation status of monocytes (expression of CD80 and MHCII) was affected by pregnancy mainly in conventional mice, and not in germfree mice. Correlation (Spearman's coefficient) of pregnancy affected microbiota with pregnancy affected immune cells, i.e., immune cells that were only affected differently in conventional mice and germfree mice, showed 4 clusters of bacteria and 4 clusters of immune cells, some of these clusters were correlated with each other. For instance, the microbiota in cluster 1 and 2 (in which there were various short chain fatty acid producing microbiota) are positively correlated with immune cells in cluster B, containing Treg cells and Th2 cells. Microbiota and immune cells are affected by pregnancy in mice. The different immunological adaptations to pregnancy between conventional and germfree mice, such as the increase in Treg and tendency to an increase in Th2 cells in conventional pregnant mice only, may suggest that the microbiota may play a role in adapting the maternal immune response to pregnancy.

Abstract Background The influence of age on basophils is poorly understood, as well as the effect of aging-associated microbiota on basophils. Therefore, we studied the influence of aging and aging-associated microbiota on basophil frequency and phenotype, and differentiation from basophil precursors. Results Basophils became more abundant in bone marrow (BM) and spleens of 19-month-old mice compared with 4-month-old mice. Aged basophils tended to express less CD200R3 and more CD123, both in BM and spleen. Differences in microbiota composition with aging were confirmed by 16S sequencing. Microbiota transfers from young and old mice to germ-free recipients revealed that CD11b tended to be lowered on splenic basophils by aging-associated microbiota. Furthermore, abundance of Alistipes, Oscillibacter, Bacteroidetes RC9 gut group, and S24–7 family positively correlated and CD123 expression, whereas Akkermansia abundance negatively correlated with basophils numbers. Subsequently, we purified FcεRIα+CD11c−CD117− BM-derived basophils and found that those from aged mice expressed lower levels of CD11b upon stimulation. Higher frequencies of IL-4+ basophils were generated from basophil precursors of aged mice, which could be reproduced in basophils derived from germ-free recipients of aging-associated microbiota. Conclusions Collectively, these results show the influence of aging on basophils. Furthermore, this study shows that aging-associated microbiota altered activation of BM-derived basophils in a similar fashion as observed in BM-derived basophils from aged mice.

Abstract Background A dysbiosis in the intestinal microbiome plays a role in the pathogenesis of several immunological diseases. These diseases often show a sex bias, suggesting sex differences in immune responses and in the intestinal microbiome. We hypothesized that sex differences in immune responses are associated with sex differences in microbiota composition. Methods Fecal microbiota composition (MITchip), mRNA expression in intestinal tissue (microarray), and immune cell populations in mesenteric lymph nodes (MLNs) were studied in male and female mice of two mouse strains (C57B1/6OlaHsd and Balb/cOlaHsd). Transcriptomics and microbiota data were combined to identify bacterial species which may potentially be related to sex-specific differences in intestinal immune related genes. Results We found clear sex differences in intestinal microbiota species, diversity, and richness in healthy mice. However, the nature of the sex effects appeared to be determined by the mouse strain as different bacterial species were enriched in males and females of the two strains. For example, Lactobacillus plantarum and Bacteroides distasonis were enriched in B6 females as compared to B6 males, while Bifidobacterium was enriched BALB/c females as compared to BALB/c males. The strain-dependent sex effects were also observed in the expression of immunological genes in the colon. We found that the abundance of various bacteria (e.g., Clostridium leptum et rel.) which were enriched in B6 females positively correlated with the expression of several genes (e.g., Il-2rb, Ccr3, and Cd80) which could be related to immunological functions, such as inflammatory responses and migration of leukocytes. The abundance of several bacteria (e.g., Faecalibacterium prausnitzii et rel. and Coprobacillus et rel.- Clostridium ramosum et rel.) which were enriched in BALB/c males positively correlated to the expression of several genes (e.g., Apoe, Il-1b, and Stat4) related to several immunological functions, such as proliferation and quantity of lymphocytes. The net result was the same, since both mouse strains showed similar sex induced differences in immune cell populations in the MLNs. Conclusions Our data suggests a correlation between microbiota and intestinal immune populations in a sex and strain-specific way. These findings may contribute to the development of more sex and genetic specific treatments for intestinal-related disorders.

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme’s structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman’s method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis.

Particulate β-glucans enhanced NF-κB expression in cell-lines co-expressing Dectin-1A-TLR4 and Dectin-1B-TLR4, while soluble β-glucans only synergistically acted on Dectin-1A-TLR4. This was different with Dectin-1 co-expressing TLR2 and TLR5, which inhibited activation after particulate and soluble β-glucan stimulation. The co-acting effect of extracellular TLR2, TLR4 and TLR5 and Dectin-1 by particulate and soluble β-glucan on cytokine production was confirmed in THP-1 macrophages. Dectin-1 activation by particulate β-glucans during TLR2 and TLR4 but not TLR5 blocking was shown to enhance the pro-inflammatory cytokines IL-6, IL-8 and TNF-α in THP-1 macrophages. This effect was stronger with particulate than with soluble β-glucan. The data demonstrate that β-glucans is an immune regulatory ligand for TLR2 and TLR4.

Introduction: Blood C-reactive protein (CRP) and fecal calprotectin levels are routinely measured as surrogate markers of disease activity in Inflammatory Bowel Disease (IBD), but often do not correlate well with the degree of mucosal inflammation in the intestine as established by endoscopy. Therefore, novel predictive biomarkers are urgently needed that better reflect mucosal disease activity in IBD. The aim of this study was to identify a combination of serum inflammatory biomarkers predictive for endoscopic disease activity.Methods: Serum concentrations of 10 inflammatory biomarkers were analyzed in 118 IBD patients [64 Crohn's disease (CD), 54 ulcerative colitis (UC)] and 20 healthy controls. In a subset of 71 IBD patients, endoscopic disease activity was established. Non-parametric ROC estimation with bootstrap inference was used to establish the best combination of inflammatory biomarkers predicting endoscopic disease activity.Results: Six (6) inflammatory biomarkers (serum amyloid A (SAA), Eotaxin-1, IL-6, IL-8, IL-17A, and TNF-α) showed better prediction of IBD disease activity than routine measures (CRP, fecal calprotectin and HBI/SCCAI scores). The best combination of predictive inflammatory biomarkers consisted of serum SAA, IL-6, IL-8, and Eotaxin-1, showing an optimism-adjusted area under the ROC (AuROC) curve of 0.84 (95% CI: 0.73–0.94, P < 0.0001), which predicted significantly better (P = 0.002) than serum CRP levels with an AuROC of 0.57 (95% CI: 0.43–0.72, P = 0.32).Conclusion: The combination of SAA, IL-6, IL-8, and Eotaxin-1 reliably predicts endoscopic disease activity in IBD and might be valuable for monitoring disease activity and management of the disease.

Intestinal homeostasis underpins the development of type 1 diabetes (T1D), and dietary manipulations to enhance intestinal homeostasis have been proposed to prevent T1D. The current study aimed to investigate the efficacy of supplementing a novel specific low-methoxyl pectin (LMP) dietary fiber in preventing T1D development. Female NOD mice were weaned onto control or 5% (wt/wt) LMP supplemented diets for up to 40 weeks of age, overt diabetes incidence and blood glucose were monitored. Then broad-spectrum antibiotics (ABX) treatment per os for 7 days followed by gut microbiota transfer was performed to demonstrate gut microbiota-dependent effects. Next-generation sequencing was used for analyzing the composition of microbiota in caecum. Concentration of short chain fatty acids were determined by GC-MS. The barrier reinforcing tight junction proteins zonula occludens-2 (ZO-2), claudin-1 and NOD like receptor protein 3 (NLRP3) inflammasome activation were determined by Western blot. The proportion of CD25+Foxp3+CD4+ regulatory T cell (Foxp3+ Treg) in the pancreas, pancreatic and mesenteric lymph nodes was analyzed by flow cytometry. We found that LMP supplementation ameliorated T1D development in non-obese diabetic (NOD) mice, as evidenced by decreasing diabetes incidence and fasting glucose levels in LMP fed NOD mice. Further microbiota analysis revealed that LMP supplementation prevented T1D-associated caecal dysbiosis and selectively enriched caecal bacterial species to produce more SCFAs. The LMP-mediated microbial balance further enhanced caecal barrier function and shaped gut-pancreatic immune environment, as characterized by higher expression of tight junction proteins claudin-1, ZO-2 in caecum, increased Foxp3+ Treg population and decreased NLRP3 inflammasome activation in both caecum and pancreas. The microbiota-dependent beneficial effect of LMP on T1D was further proven by the fact that aberration of caecal microbiota by ABX treatment worsened T1D autoimmunity and could be restored with transfer of feces of LMP-fed NOD mice. These data demonstrate that this novel LMP limits T1D development by inducing caecal homeostasis to shape pancreatic immune environment. This finding opens a realistic option for gut microbiota manipulation and prevention of T1D in humans.

Immunoisolation of pancreatic islets is a technology in which islets are encapsulated in semipermeable but immunoprotective polymeric membranes. The technology allows for successful transplantation of insulin-producing cells in the absence of immunosuppression. Different approaches of immunoisolation are currently under development. These approaches involve intravascular devices that are connected to the bloodstream and extravascular devices that can be distinguished in micro- and macrocapsules and are usually implanted in the peritoneal cavity or under the skin. The technology has been subject of intense fundamental research in the past decade. It has co-evolved with novel replenishable cell sources for cure of diseases such as Type 1 Diabetes Mellitus that need to be protected for the host immune system. Although the devices have shown significant success in animal models and even in human safety studies most technologies still suffer from undesired tissue responses in the host. Here we review the past and current approaches to modulate and reduce tissue responses against extravascular cell-containing micro- and macrocapsules with a focus on rational choices for polymer (combinations). Choices for polymers but also choices for crosslinking agents that induce more stable and biocompatible capsules are discussed. Combining beneficial properties of molecules in diblock polymers or application of these molecules or other anti-biofouling molecules have been reviewed. Emerging are also the principles of polymer brushes that prevent protein and cell-adhesion. Recently also immunomodulating biomaterials that bind to specific immune receptors have entered the field. Several natural and synthetic polymers and even combinations of these polymers have demonstrated significant improvement in outcomes of encapsulated grafts. Adequate polymeric surface properties have been shown to be essential but how the surface should be composed to avoid host responses remains to be identified. Current insight is that optimal biocompatible devices can be created which raises optimism that immunoisolating devices can be created that allows for long term survival of encapsulated replenishable insulin-producing cell sources for treatment of Type 1 Diabetes Mellitus.

Natural polysaccharides with health benefits are characterized by a large structural diversity and differ in building blocks, linkages, and lengths. They contribute to human health by functioning as anti-adhesives preventing pathogen adhesion, stimulate immune maturation and gut barrier function, and serve as fermentable substrates for gut bacteria. Examples of such beneficial carbohydrates include the human milk oligosaccharides (HMOs). Also, specific non-digestible carbohydrates (NDCs), such as galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS) are being produced with this purpose in mind, and are currently added to infant formula to stimulate the healthy development of the newborn. They mimic some functions of HMO, but not all. Therefore, many research efforts focus on identification and production of novel types of NDCs. In this review, we give an overview of the few NDCs currently available [GOS, FOS, polydextrose (PDX)], and outline the potential of alternative oligosaccharides, such as pectins, (arabino)xylo-oligosaccharides, and microbial exopolysaccharides (EPS). Moreover, state-of-the-art techniques to generate novel types of dietary glycans, including sialylated GOS (Sia-GOS) and galactosylated chitin, are presented as a way to obtain novel prebiotic NDCs that help shaping the infant microbiome.

Sufficient dietary fibre intake reduces the risk of several diseases, but the mechanisms linking fibre structure and health effects remain unclear. To analyse the influence of the amount of methyl groups esterified to the backbone, lemon pectins of different degrees of methyl esterification (30, 56, and 74DM) were studied for immune receptor activating potential and epithelial barrier protection. In reporter assays, the pectins demonstrated TLR/MyD88 dependent activation of NF-κB/AP-1, which increased with increasing pectin DM. To analyse the importance of backbone structure, the pectins were enzymatically digested into oligomers, which abrogated TLR activating potential. The 30 and 74DM pectins induced strong protection of the epithelial barrier measured by T84 transepithelial electrical resistance (TEER). These results indicate that activation of immune cells by lemon pectins is TLR dependent, and the intact polymer backbone is indispensable for activation. In addition, DM is a determining factor in activating potential and epithelial barrier protective effects.

Oat β-Glucans were studied for their immunological impact before and after enzymatic digestion in order to enhance the efficacy of oat β-Glucans for application in functional foods. Oat β-Glucan is reported to have minimal impact compared to its fungal counterpart in vitro. Digestion with endo-glucanase enhanced its efficacy towards stimulating MCP-1, RANTES, IL-8, and IL-4 production in human dendritic cells as compared to the nondigested β-Glucan. This effect resulted from an enhanced activation of the Dectin-1 receptor. Our data suggest that the immune-stimulation was dependent on the β-(1-3) linkages and the reduced particle size of digested β-Glucans. Thus, we show that enzymatic pre-digestion of dietary fibres such as oat β-Glucan enhances its impact on specific immune receptors. We also demonstrate that particle size and/or molecular weight of oat β-Glucans and exposure of specific binding sites for the receptors might be important tools for designing efficacious functional feed and food additives.

Background: Non-digestible carbohydrates are added to infant formula to mimic the effects of human milk oligosaccharide by acting as prebiotics and stimulating the immune system. Although not yet used in infant formulas, β-glucans are known to have beneficial health effects, and are therefore of potential interest for supplementation. Methods and results: We investigated the in vitro fermentation of native and endo-1,3(4)-β-glucanase-treated oat β-glucan using pooled fecal inocula of 2- and 8-week-old infants. While native oat β-glucan was not utilized, both inocula specifically utilized oat β-glucan oligomers containing β(1→4)-linkages formed upon enzyme treatment. The fermentation rate was highest in the fecal microbiota of 2-week-old infants, and correlated with a high lactate production. Fermentation of media supplemented with native and enzyme-treated oat β-glucans increased the relative abundance of Enterococcus and attenuated pro-inflammatory cytokine production (IL-1β, IL-6, TNFα) in immature dendritic cells. This attenuating effect was more pronounced after enzyme treatment. This attenuation might result from the enhanced ability of fermented oat β-glucan to stimulate Dectin-1 receptors. Conclusion: Our findings demonstrate that endo-1,3(4)-β-glucanase treatment enhances the fermentability of oat β-glucan and attenuates pro-inflammatory responses. Hence, this study shows that especially enzyme-treated oat β-glucans have a high potential for supplementation of infant formula.

Islet transplantation into subcutaneous polymer scaffolds has shown to successfully induce normoglycemia in type 1 diabetes models. Vascularization of these scaffolds is imperative for optimal control of glucose levels. We studied the effect of the vascular stimulator hydrogen sulfide (H2S) on the degree of vascularization of a scaffold and the role of the immune system in this process. Scaffolds were subcutaneously implanted in immunocompetent C57BL/6 and immunocompromised nude mice. Mice received twice-daily intraperitoneal injections of the fast-releasing H2S donor sodium hydrosulfide (NaHS, 25 or 50 μmol/kg) or saline for 28 days. After 63 days the vascular network was analyzed by histology and gene expression. Here we showed that the vascularization of a subcutaneous scaffold in nude mice was significantly impaired by H2S treatment. Both the CD31 gene and protein expression were reduced in these scaffolds compared to the saline-treated controls. In C57BL/6 mice, the opposite was found, the vascularization of the scaffold was significantly increased by H2S. The mRNA expression of the angiogenesis marker CD105 was significantly increased compared to the controls as well as the number of CD31 positive blood vessels. In conclusion, the immune system plays an important role in the H2S mediated effect on vascularization of subcutaneous scaffolds.

With the increasing resistance of bacteria to current antibiotics, novel compounds are urgently needed to treat bacterial infections. Streptozotocin (STZ) is a natural product that has broad-spectrum antibiotic activity, albeit with limited use because of its toxicity to pancreatic β cells. In an attempt to derivatize STZ through structural modification at the C3 position, we performed the synthesis of three novel STZ analogues by making use of our recently developed regioselective oxidation protocol. Keto-STZ (2) shows the highest inhibition of bacterial growth (minimum inhibitory concentration (MIC) and viability assays), but is also the most cytotoxic compound. Pre-sensitizing the bacteria with GlcNAc increased the antimicrobial effect, but did not result in complete killing. Interestingly, allo-STZ (3) revealed moderate concentration-dependent antimicrobial activity and no cytotoxicity towards β cells, and deoxy-STZ (4) showed no activity at all.