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

Food processing : The influence of the maillard reaction on immunogenicity and allergenicity of food proteins

The majority of foods that are consumed in our developed society have been processed. Processing promotes a non-enzymatic reaction between proteins and sugars, the Maillard reaction (MR). Maillard reaction products (MRPs) contribute to the taste, smell and color of many food products, and thus influence consumers’ choices. However, in recent years, MRPs have been linked to the increasing prevalence of diet- and inflammation-related non-communicable diseases including food allergy. Although during the last years a better understanding of immunogenicity of MRPs has been achieved, still only little is known about the structural/chemical characteristics predisposing MRPs to interact with antigen presenting cells (APCs). This report provides a comprehensive review of recent studies on the influence of the Maillard reaction on the immunogenicity and allergenicity of food proteins

No glycation required : Interference of casein in age receptor binding tests

For the determination of the binding of heated cow’s milk whey proteins such as β-lactoglobulin to the receptors expressed on immune cells, inhibition ELISA with the soluble form of the receptor for advanced glycation end products (sRAGE) and scavenger receptor class B (CD36) has been successfully used in the past. However, binding to heated and glycated caseins in this read-out system has not been tested. In this study, inhibition ELISA was applied to measure the binding of cow’s milk casein alone, as well as all milk proteins together, which underwent differential heat treatment, to sRAGE and CD36, and we compared those results to a dot blot read out. Moreover, binding to sRAGE and CD36 of differentially heated milk protein was measured before and after in vitro digestion. Casein showed binding to sRAGE and CD36, independent from the heat treatment, in ELISA, while the dot blot showed only binding to high-temperature-heated milk protein, indicating that the binding is not related to processing but to the physicochemical characteristics of the casein. This binding decreased after passage of casein through the intestinal phase

Immunomodulation by Processed Animal Feed : The Role of Maillard Reaction Products and Advanced Glycation End-Products (AGEs)

The immune system provides host protection to infection with pathogenic organisms, while at the same time providing tolerance upon exposure to harmless antigens. Thus, an impaired immune function is associated with increased susceptibility to infections with increased disease severity and thereby necessitating the therapeutic use of antibiotics. Livestock performance and feed efficiency, in addition to their health status, are dependent on the microbial load of their gut, the barrier function of the intestinal epithelium and the activity of the mucosal immune system, all of which can be modulated by dietary components. The majority of feeds that are consumed in pets and livestock have been processed. Processing promotes a non-enzymatic reaction between proteins and sugars called Maillard reaction (MR). Maillard reaction products (MRPs) and advanced Maillard reaction products (AGEs) determine taste, smell, and color of many food products therefore the MR is highly relevant for the feed industry. MRPs interact with different types of immune receptors, including the receptor for advanced glycation end products (RAGE) and immunomodulatory potential of feed proteins can be modified by Maillard reaction. This MR has become an important concern since MRPs/AGEs have been shown to contribute to increasing prevalence of diet-related chronic inflammatory states in the gut with negative health consequences and performance. The immunomodulatory effects of dietary MRPs and AGEs in livestock and pet animals are far less well-described, but widely considered to be similar to the relevant concepts and mechanisms obtained in the human field. This review will highlight immunological mechanisms underlying initiation of the innate and adaptive immune responses by MRPs/AGEs present in animal feeds, which are currently not completely understood. Bridging this knowledge gap, and taking advantage of progress in the human field, will significantly improve nutritional quality of feed and increase the prevention of diet-mediated inflammation in animals

The effect of soy processing on its allergenicity : Discrepancy between IgE binding and basophil stimulation tests

Presently, many studies assess allergenicity via IgE immunoblotting and IgE binding tests; however, IgE detection does not always signal the manifestation of a clinical allergy. However, the capacity of food allergens to trigger basophils makes it possible to use the in vitro functional basophil activation test (BAT) to assess allergenicity. The effect of the Maillard reaction (MR) on the allergenic potential of processed soy proteins was evaluated by two IgE binding tests (Competitive ELISA and Inhibition ImmunoCAP), a Western Blot and a functional BAT; with the aim to analyze whether the sIgE binding results correspond to the functional assay results. The results between the IgE binding tests and the functional assay were in-line for 2 of the 6 studied patients. For one patient there was no correlation between any of the results. For the raw soy protein extract heated with glucose for 10 and 30 min (SH SPE + Glu and LH SPE + Glu, respectively), the results were in-line for only 3 out of the 6 patients. Thus, the present study shows a discrepancy between IgE binding tests and basophil stimulation when assessing the effect of soy processing on its allergenicity. Since IgE-binding capacity does not always correlate to IgE cross-linking capacity, the conclusions of the allergenic potential based on the IgE binding tests alone should be drawn with care and further studies on this matter would benefit from the inclusion of a functional assay such as the BAT

Immunological Characterization of Dutch Sesame Seed-Allergic Patients

Background: Sesame seed is an allergen of growing importance worldwide. However, knowledge of the clinically relevant sesame allergen and its cross-reactivity with homologous allergens is limited. The aim of this study was the immunological characterization of Dutch sesame seed-allergic patients and evaluation of cross-reactivity between sesame seed, tree nut and pollen allergens using different sources of allergen extracts. Methods: Six patients with a medical history of sesame seed allergy were included, i.e. 5 with an anaphylactic reaction and 1 with an oral allergy syndrome (OAS). The immunological background of the sesame seed and tree nut IgE sensitization was characterized with Western blotting and a basophil activation test (BAT). The major sesame allergen was identified by nanoLC-MS/MS. Cross-reactivity was measured using an immuno-inhibition assay with the Phadia ImmunoCAP system. Results: Oleosin was identified as the major allergen for the 5 patients with an anaphylactic reaction to sesame seed, but no cross-reactivity between sesame and tree nut proteins was observed. For the patient with OAS, IgE specific to oleosin was not detected but cross-reactivity between sesame seed and tree nut proteins was observed. The BAT and ImmunoCAP inhibition test added value to the clinical and immunological characterization of sesame seed-sensitized patients, distinguishing relevant and non-relevant sensitizations. Conclusions: Our immunological approach enabled us to fully characterize the sensitization pattern of 6 sesame seed-allergic patients. The different protein composition of commercially available allergen extracts influences the outcomes of the immunological assays and thus also the diagnosis to a large extent.</p

Immunomodulation by Processed Animal Feed : The Role of Maillard Reaction Products and Advanced Glycation End-Products (AGEs)

The immune system provides host protection to infection with pathogenic organisms, while at the same time providing tolerance upon exposure to harmless antigens. Thus, an impaired immune function is associated with increased susceptibility to infections with increased disease severity and thereby necessitating the therapeutic use of antibiotics. Livestock performance and feed efficiency, in addition to their health status, are dependent on the microbial load of their gut, the barrier function of the intestinal epithelium and the activity of the mucosal immune system, all of which can be modulated by dietary components. The majority of feeds that are consumed in pets and livestock have been processed. Processing promotes a non-enzymatic reaction between proteins and sugars called Maillard reaction (MR). Maillard reaction products (MRPs) and advanced Maillard reaction products (AGEs) determine taste, smell, and color of many food products therefore the MR is highly relevant for the feed industry. MRPs interact with different types of immune receptors, including the receptor for advanced glycation end products (RAGE) and immunomodulatory potential of feed proteins can be modified by Maillard reaction. This MR has become an important concern since MRPs/AGEs have been shown to contribute to increasing prevalence of diet-related chronic inflammatory states in the gut with negative health consequences and performance. The immunomodulatory effects of dietary MRPs and AGEs in livestock and pet animals are far less well-described, but widely considered to be similar to the relevant concepts and mechanisms obtained in the human field. This review will highlight immunological mechanisms underlying initiation of the innate and adaptive immune responses by MRPs/AGEs present in animal feeds, which are currently not completely understood. Bridging this knowledge gap, and taking advantage of progress in the human field, will significantly improve nutritional quality of feed and increase the prevention of diet-mediated inflammation in animals.</p

Hydrophobicity drives receptor-mediated uptake of heat-processed proteins by THP-1 macrophages and dendritic cells, but not cytokine responses

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 β-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.</p

Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins.

SCOPE: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. METHODS AND RESULTS: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. CONCLUSION: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