Human casein alpha s1 (CSN1S1) skews in vitro differentiation of monocytes towards macrophages.

BACKGROUND: The milk-derived protein human Casein alpha s1 (CSN1S1) has recently been detected in blood cells and was shown to possess proinflammatory properties. In the present study, we investigated the effect of CSN1S1 on the differentiation of monocytes. METHODS: Primary human monocytes were stimulated with recombinant CSN1S1 and compared to cells stimulated with GM-CSF/IL-4 or M-CSF/IFNγ. Morphological changes were assessed by microscopy and quantification of surface markers of differentiation by FACS analysis. Phagocytic activity of CSN1S1 stimulated cells was measured by quantification of zymosan labeled particle uptake. The role of mitogen activated protein kinases for CSN1S1-induced differentiation of monocytes and proinflammatory cytokine expression was assessed by supplementation of specific inhibitors. RESULTS: CSN1S1 at a concentration of 10 µg/ml resulted in morphological changes (irregular shape, pseudopodia) and aggregation of cells, comparable to changes observed in M-CSF/IFNγ differentiated macrophages. Surface marker expression was altered after 24 h with an upregulation of CD14 (mean 2.5 fold) and CD64 (1.9 fold) in CSN1S1 stimulated cells. CSN1S1 treated cells showed a characteristic surface marker pattern for macrophages after 120 h of incubation (CD14high, CD64high, CD83low, CD1alow) comparable to changes observed in M-CSF/IFNγ treated monocytes. Furthermore, phagocytic activity was increased 1.4 and 1.9 fold following stimulation with 10 µg/ml CSN1S1 after 24 and 48 h, respectively. Early GM-CSF, but not GM-CSF/IL-4 induced differentiation of monocytes towards dendritic cells (DC) was inhibited by addition of CSN1S1. Finally, CSN1S1 induced upregulation of CD14 was impeded by inhibition of ERK1/2, while inhibition of the mitogen activated protein kinases JNK and p38 did not influence cellular differentiation. However, JNK and p38 inhibitors impeded CSN1S1 induced secretion of the proinflammatory cytokines IL-1b or IL-6. CONCLUSIONS: CSN1S1 skews in vitro differentiation of monocytes towards a macrophage-like phenotype. Data is accumulating that functions of CSN1S1 are beyond nutritional properties and include immunomodulatory effects.

Ser71 of αS1-Casein is Phosphorylated in Breast Milk-Evidence from Targeted Mass Analysis.

SCOPE: The casein phosphoproteins in mother's milk supply calcium and phosphate ions and make them biologically available to the newborn. Human αS1-casein is of particular interest being also an autoantigen and proinflammatory cytokine. Phosphorylation of αS1-casein by casein kinase 2 completely abolishes binding to toll-like receptor 4 and proinflammatory effects. It is, however, not known, which amino acids are affected. Therefore, breast milk samples were analyzed in an effort to detect the phosphorylation sites of αS1-casein. METHODS AND RESULTS: Breast milk samples were tryptically digested. Target tandem MS analysis confirmed the known phosphorylation sites S33 and S41; evidence for pS89 was found in some samples. Experimental support for the presence of pS31 and pS34 was weak. Phosphorylation of a new site in αS1-casein, S71, was reproducibly measured in all samples, albeit at much lower intensity than pS33 and pS41. CONCLUSION: Phospho-occupancy rates varied greatly and could not be confidently correlated to other parameters within the cohort of 20 donors. The new phosphosite S71 is located in the neighborhood of the serine-rich region and may contribute to the cluster of high charge density at normal milk pH, likely exerting an influence on protein tertiary structure and thus function.

Quantification of αS1-casein in breast milk using a targeted mass spectrometry-based approach.

The caseins comprise a milk protein fraction of high nutritional value and, as more recently discovered, of immunologic relevance. In particular, αS1-casein (CSN1S1) is of interest being a potential autoantigen. So far, the concentration of caseins in human milk was primarily determined by indirect methods. The aim of this study was to directly measure the CSN1S1 content in breast milk using mass spectrometry (MS). The quantification was based on tryptic CSN1S1 peptides with the best response in liquid chromatography (LC)-MS/MS analysis. Targeted experiments allowed both specific and sensitive detection at the low fmol level. For this pilot study, twenty breast milk samples of the first week post-partum were analyzed and contained between 3 and 540µg/ml CSN1S1. Limitations of CSN1S1 quantification are discussed.

Development of a surface display ELISA to detect anti-IgG antibodies against bovine αS1-casein in human sera.

The aim of the present study was to develop a surface display ELISA (SD-ELISA) for IgG-serum reaction against bovine casein αS1 (CSN1S1). In a SD-ELISA, the antigen is displayed on the surface of Escherichia coli using the autodisplay technology and whole cells of E. coli are used to coat the microplates for serum testing. After establishing the setup of the SD-ELISA with polyclonal rabbit antiserum against bovine CSN1S1, the SD-ELISA was validated with 20 human sera, of which 10 sera were proven to have an IgG-mediated reaction against bovine CSN1S1 and 10 sera were shown to be negative for this reaction. Receiver operating characteristics (ROC) analysis revealed sensitivity of 100% and a specificity of 100% at a cut-off value of 0.133. Furthermore, human serum of 48 patients with known reactivity against human CSN1S1 (31 positive and 17 negative) was examined by the newly developed SD-ELISA to exclude cross-reactivity. Twenty human sera showed an IgG-mediated reaction against bovine CSN1S1. Eleven of these sera were positive for the reactivity against human CSN1S1, and nine were negative. In conclusion it was demonstrated that the performance of SD-ELISA is comparable to established ELISA without loss in sensitivity or specificity. Based on the advantages of this method - in particular no need for time-consuming and expensive antigen production and purification - the SD-ELISA is a potent alternative to convenient methods for identification and especially high-throughput screening of new antigens in the field of food allergies.