Direct Binding of Bovine IgG-Containing Immune Complexes to Human Monocytes and Their Putative Role in Innate Immune Training

Bovine milk IgG (bIgG) was shown to bind to and neutralize the human respiratory synovial virus (RSV). In animal models, adding bIgG prevented experimental RSV infection and increased the number of activated T cells. This enhanced activation of RSV-specific T cells may be explained by receptor-mediated uptake and antigen presentation after binding of bIgG-RSV immune complexes (ICs) with FcγRs (primarily CD32) on human immune cells. This indirect effect of bIgG ICs on activation of RSV-specific T cells was confirmed previously in human T cell cultures. However, the direct binding of ICs to antigen-presenting cells has not been addressed. As bovine IgG can induce innate immune training, we hypothesized that this effect could be caused more efficiently by ICs. Therefore, we characterized the expression of CD16, CD32, and CD64 on (peripheral blood mononuclear cells (PBMCs), determined the optimal conditions to form ICs of bIgG with the RSV preF protein, and demonstrated the direct binding of these ICs to human CD14 + monocytes. Similarly, bIgG complexed with a murine anti-bIgG mAb also bound efficiently to the monocytes. To evaluate whether the ICs could induce innate immune training more efficiently than bIgG itself, the resulted ICs, as well as bIgG, were used in an in vitro innate immune training model. Training with the ICs containing bIgG and RSV preF protein-but not the bIgG alone-induced significantly higher TNF-α production upon LPS and R848 stimulation. However, the preF protein itself nonsignificantly increased cytokine production as well. This may be explained by its tropism to the insulin-like growth factor receptor 1 (IGFR1), as IGF has been reported to induce innate immune training. Even so, these data suggest a role for IgG-containing ICs in inducing innate immune training after re-exposure to pathogens. However, as ICs of bIgG with a mouse anti-bIgG mAb did not induce this effect, further research is needed to confirm the putative role of bIgG ICs in enhancing innate immune responses in vivo

Educating the human immune system: direct and indirect effects of bovine milk components

In this thesis, we aimed to evaluate the immunomodulatory effects of (selected) bovine milk components (and metabolites induced by them) on the immune function of humans. Chapter 1 gives an introduction to the topic and provides an outline of fundamental aspects of the immune system that are referred to in later chapters. In Chapter 2, we summarized and addressed the primary components of bovine milk that have the potential to induce epigenetic changes to exert their immune-supportive effects during childhood. We reviewed the proposed mechanisms, including innate immune training that induces epigenetic modification. Through these mechanisms, the components may exert an effect on the immune system with implications for allergies and asthma. Living in a farm environment and raw bovine milk bioactive components were addressed as contributing factors that may reduce allergies in infancy and beyond. HMOs and bovine milk oligosaccharides (mainly 3'-Sialyllactose) serve as substrates for the SCFA-producing microbiota. SCFAs are potent immune modulators and have significant roles in maintaining homeostasis and steering the response of the immune cells to the environment. In Chapter 3, we showed that butyrate and propionate had inhibitory effects on the activation of myeloid cells and lymphocytes, whereas acetate had a more selective impact on the immune cells. Production of inflammatory cytokines was suppressed in monocytes, mDCs, and pDCs, as well as T lymphocytes. SCFAs could not train the monocytes for enhanced response to secondary TLR stimulation in vitro but instead induced a tolerance-like phenotype. We attempted to explain the observed effects according to the differential expression of relevant SCFA receptors and transporters. Bovine milk IgG (bIgG) binds to human pathogens such as RSV and, via the Fc portion, interacts with the FcγRs on human immune cells. The relevance of bIgG-containing immune complexes (ICs) on activation of CD32 was studied in Chapter 4, where we could establish a method to show the direct binding of bIgG ICs to immune cells. It was demonstrated that ICs containing bIgG are directly bound to human CD14+. Subsequently, we could show the role of bIgG ICs on induction of trained immunity after binding to monocytes while contrary to previous reports, (monomeric) bIgG alone did not have similar effects suggestive of the presence of other contributing factors. Human infection challenge models are used as an alternative to field trials to study the immune-supportive effects of dietary components. In Chapter 5, we found that ingestion of a dairy product (WPC) in a human challenge model did not influence the responsiveness of myeloid cells from healthy volunteers to ex vivo stimulation with TLR ligands. It also did not change the gene expression pattern of the PBMCs isolated from the same donors. Although the model was utilized successfully before, the study product did not exert beneficial effects. We speculated that the study population might be a critical factor for no apparent impact. In Chapter 6, we focused on optimizing an E. coli infection challenge model in humans to study the protective effects of dietary components and the correlates of protection at rechallenge. Primary infection with even low doses (1E6 CFU) of E. coli protected the subjects against reinfection with a high dose (1E10 CFU) of the same pathogen. Following the primary infection, serum anti-CFAII IgG levels were raised, and monocytes and mDCs responded more strongly to ex vivo stimulation. The latter may indicate the occurrence of trained immunity in vivo. Throughout Chapter 7, we reviewed and discussed the most important findings of our research and placed the findings in a broader context by relating them to the most recent published literature. In addition, we identified subjects for future study perspectives to follow the work done in this project. Immunomodulation by nutrition or supplementation of the food with potent immunomodulatory components can provide immune support for the immune system in individuals with an immature or impaired immune system. To substantiate the dietary components' beneficial effects and define the supporting mechanisms involved, we studied bovine IgG and metabolites induced by milk oligosaccharides to substantiate their health-promoting and immune-mediated effects

The in vitro gastrointestinal digestion-associated protein corona of polystyrene nano- and microplastics increases their uptake by human THP-1-derived macrophages

Abstract Background Micro- and nanoplastics (MNPs) represent one of the most widespread environmental pollutants of the twenty-first century to which all humans are orally exposed. Upon ingestion, MNPs pass harsh biochemical conditions within the gastrointestinal tract, causing a unique protein corona on the MNP surface. Little is known about the digestion-associated protein corona and its impact on the cellular uptake of MNPs. Here, we systematically studied the influence of gastrointestinal digestion on the cellular uptake of neutral and charged polystyrene MNPs using THP-1-derived macrophages. Results The protein corona composition was quantified using LC‒MS–MS-based proteomics, and the cellular uptake of MNPs was determined using flow cytometry and confocal microscopy. Gastrointestinal digestion resulted in a distinct protein corona on MNPs that was retained in serum-containing cell culture medium. Digestion increased the uptake of uncharged MNPs below 500 nm by 4.0–6.1-fold but did not affect the uptake of larger sized or charged MNPs. Forty proteins showed a good correlation between protein abundance and MNP uptake, including coagulation factors, apolipoproteins and vitronectin. Conclusion This study provides quantitative data on the presence of gastrointestinal proteins on MNPs and relates this to cellular uptake, underpinning the need to include the protein corona in hazard assessment of MNPs. Graphical abstrac

Direct Binding of Bovine IgG-Containing Immune Complexes to Human Monocytes and Their Putative Role in Innate Immune Training

Bovine milk IgG (bIgG) was shown to bind to and neutralize the human respiratory synovial virus (RSV). In animal models, adding bIgG prevented experimental RSV infection and increased the number of activated T cells. This enhanced activation of RSV-specific T cells may be explained by receptor-mediated uptake and antigen presentation after binding of bIgG-RSV immune complexes (ICs) with FcγRs (primarily CD32) on human immune cells. This indirect effect of bIgG ICs on activation of RSV-specific T cells was confirmed previously in human T cell cultures. However, the direct binding of ICs to antigen-presenting cells has not been addressed. As bovine IgG can induce innate immune training, we hypothesized that this effect could be caused more efficiently by ICs. Therefore, we characterized the expression of CD16, CD32, and CD64 on (peripheral blood mononuclear cells (PBMCs), determined the optimal conditions to form ICs of bIgG with the RSV preF protein, and demonstrated the direct binding of these ICs to human CD14 + monocytes. Similarly, bIgG complexed with a murine anti-bIgG mAb also bound efficiently to the monocytes. To evaluate whether the ICs could induce innate immune training more efficiently than bIgG itself, the resulted ICs, as well as bIgG, were used in an in vitro innate immune training model. Training with the ICs containing bIgG and RSV preF protein-but not the bIgG alone-induced significantly higher TNF-α production upon LPS and R848 stimulation. However, the preF protein itself nonsignificantly increased cytokine production as well. This may be explained by its tropism to the insulin-like growth factor receptor 1 (IGFR1), as IGF has been reported to induce innate immune training. Even so, these data suggest a role for IgG-containing ICs in inducing innate immune training after re-exposure to pathogens. However, as ICs of bIgG with a mouse anti-bIgG mAb did not induce this effect, further research is needed to confirm the putative role of bIgG ICs in enhancing innate immune responses in vivo

Extracellular flux analyses reveal differences in mitochondrial PBMC metabolism between high-fit and low-fit females

Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) can possibly serve as a cellular metabolic read-out for lifestyle factors and lifestyle interventions. However, the impact of PBMC composition on PBMC metabolism is not yet clear, neither is the differential impact of a longer-term lifestyle factor versus a short-term lifestyle intervention. We investigated the effect of aerobic fitness level and a recent exercise bout on PBMC metabolism in females. PBMCs from 31 young female adults divided into a high-fit (V˙O2peak ≥ 47 mL/kg/min, n = 15) and low-fit (V˙O2peak ≤ 37 mL/kg/min, n = 16) groups were isolated at baseline and overnight after a single bout of exercise (60 min, 70% V˙O2peak). Oxygen consumption rate (OCR) and glycolytic rate (GR) were measured using extracellular flux (XF) assays and PBMC subsets were characterized using fluorescence-activated cell sorting (FACS). Basal OCR, FCCP-induced OCR, spare respiratory capacity, ATP-linked OCR, and proton leak were significantly higher in high-fit than in low-fit females (all P 0.05). A recent exercise bout did not significantly affect GR or OCR parameters (all P > 0.05). The overall PBMC composition was similar between high-fit and low-fit females. Mitochondrial PBMC function was significantly higher in PBMCs from high-fit than from lowfit females, which was unrelated to PBMC composition and not impacted by a recent bout of exercise. Our study reveals a link between PBMC metabolism and levels of aerobic fitness, increasing the relevance of PBMC metabolism as a marker to study the impact of lifestyle factors on human health

The Impact of Milk and Its Components on Epigenetic Programming of Immune Function in Early Life and Beyond: Implications for Allergy and Asthma

Specific and adequate nutrition during pregnancy and early life is an important factor in avoiding non-communicable diseases such as obesity, type 2 diabetes, cardiovascular disease, cancers, and chronic allergic diseases. Although epidemiologic and experimental studies have shown that nutrition is important at all stages of life, it is especially important in prenatal and the first few years of life. During the last decade, there has been a growing interest in the potential role of epigenetic mechanisms in the increasing health problems associated with allergic disease. Epigenetics involves several mechanisms including DNA methylation, histone modifications, and microRNAs which can modify the expression of genes. In this study, we focus on the effects of maternal nutrition during pregnancy, the effects of the bioactive components in human and bovine milk, and the environmental factors that can affect early life (i.e., farming, milk processing, and bacterial exposure), and which contribute to the epigenetic mechanisms underlying the persistent programming of immune functions and allergic diseases. This knowledge will help to improve approaches to nutrition in early life and help prevent allergies in the future

Dietary Intervention with Whey Protein Concentrate Does Not Affect Toll-like Receptor Responses and Gene Expression Patterns in Peripheral Blood Mononuclear Cells of Healthy Volunteers

Bovine milk contains bioactive proteins, carbohydrates, and phospholipids with immunomodulatory properties impacting human immunity, potentially contributing to resistance to infections and allergies through diverse mechanisms. One such mechanism is the enhancing of the innate immune response to secondary pathogen-related stimuli, termed innate immune training. Although in vitro studies demonstrate that milk immunoglobulin G (IgG) can train human monocytes, evidence for in vivo immune training is limited. To explore the potential of bovine IgG for inducing innate immune training in vivo, this human study utilized an IgG-rich whey protein concentrate (WPC). Healthy male volunteers were assigned to a high dose WPC, low dose WPC, or placebo group. Blood was collected pre- and post-two weeks of WPC consumption. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with TLR ligands, evaluating IL-6 and TNF-α production by monocytes, myeloid DCs, and plasmacytoid DCs. Additionally, RNA was isolated for differential gene expression (DGE) analysis. Results indicated that the two-week WPC intervention did not influence the ex vivo response of studied cells to TLR agonists. Furthermore, PBMC gene expression patterns showed no significant differences between the placebo and high dose WPC groups. The data suggests that oral WPC ingestion did not enhance immune responses in young, healthy male participants

The Impact of Milk and Its Components on Epigenetic Programming of Immune Function in Early Life and Beyond : Implications for Allergy and Asthma

Specific and adequate nutrition during pregnancy and early life is an important factor in avoiding non-communicable diseases such as obesity, type 2 diabetes, cardiovascular disease, cancers, and chronic allergic diseases. Although epidemiologic and experimental studies have shown that nutrition is important at all stages of life, it is especially important in prenatal and the first few years of life. During the last decade, there has been a growing interest in the potential role of epigenetic mechanisms in the increasing health problems associated with allergic disease. Epigenetics involves several mechanisms including DNA methylation, histone modifications, and microRNAs which can modify the expression of genes. In this study, we focus on the effects of maternal nutrition during pregnancy, the effects of the bioactive components in human and bovine milk, and the environmental factors that can affect early life (i.e., farming, milk processing, and bacterial exposure), and which contribute to the epigenetic mechanisms underlying the persistent programming of immune functions and allergic diseases. This knowledge will help to improve approaches to nutrition in early life and help prevent allergies in the future.</p

A Double‐Blind, Randomized Intervention Study on the Effect of a Whey Protein Concentrate on E. coli‐Induced Diarrhea in a Human Infection Model

Infectious diseases are a major cause of morbidity and mortality worldwide. Nutritional interventions may enhance resistance to infectious diseases or help to reduce clinical symptoms. Here, we investigated whether a whey protein concentrate (WPC) could decrease diarrheagenic Escherichia coli‐induced changes in reported stool frequency and gastrointestinal complaints in a double‐blind, parallel 4‐week intervention study. Subjects were randomly assigned to a whey hydrolysate placebo group, a low‐dose WPC group or a high‐dose WPC group. After 2 weeks of consumption, subjects (n = 121) were orally infected with a high dose of live but attenuated diarrheagenic E. coli (strain E1392/75‐2A; 1E10 colony‐forming units). Subjects recorded information on stool consistency and the frequency and severity of symptoms in an online diary. The primary outcome parameters were a change in stool frequency (stools per day) and a change in Gastrointestinal Symptom Rating Scale (GSRS) diarrhea score between the first and second days after infection. Neither dose of the whey protein concentrate in the dietary treatment affected the E. coli‐induced increase in stool frequency or GSRS diarrhea score compared to placebo treatment. The composition of the microbiota shifted between the start of the study and after two weeks of consumption of the products, but no differences between the intervention groups were observed, possibly due to dietary guidelines that subjects had to adhere to during the study. In conclusion, consumption of the whey protein concentrate by healthy adults did not reduce diarrhea scores in an E. coli infection model compared to a whey hydrolysate placebo control

Low doses of diarrhoeagenic E. coli induce enhanced monocyte and mDC responses and prevent development of symptoms after homologous rechallenge

The experimental challenge with attenuated enterotoxigenic E. coli strain E1392/75-2A prevents diarrhea upon a secondary challenge with the same bacteria. A dose-response pilot study was performed to investigate which immunological factors are associated with this protection. Healthy subjects were inoculated with increasing E. coli doses of 1E6-1E10 CFU, and three weeks later, all participants were rechallenged with the highest dose (1E10 CFU). Gastrointestinal discomfort symptoms were recorded, and stool and blood samples were analyzed. After the primary challenge, stool frequency, diarrhea symptom scores, and E. coli-specific serum IgG (IgG-CFA/II) titer increased in a dose-dependent manner. Fecal calprotectin and serum IgG-CFA/II response after primary challenge were delayed in the lower dose groups. Even though stool frequency after the secondary challenge was inversely related to the primary inoculation dose, all E. coli doses protected against clinical symptoms upon rechallenge. Ex vivo stimulation of PBMCs with E. coli just before the second challenge resulted in increased numbers of IL-6+/TNF-α+ monocytes and mDCs than before the primary challenge, without dose-dependency. These data demonstrate that primary E. coli infection with as few as 1E6 CFU protects against a high-dose secondary challenge with a homologous attenuated strain. Increased serum IgG-CFA/II levels and E. coli-induced mDC and monocyte responses after primary challenge suggest that protection against secondary E. coli challenges is associated with adaptive as well as innate immune responses