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Abstract
BackgroundThe 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.MethodsPrimary 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.ResultsCSN1S1 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.ConclusionsCSN1S1 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.
Highlights
The milk-derived protein human Casein alpha s1 (CSN1S1) has recently been detected in blood cells and was shown to possess proinflammatory properties
We included primary cells stimulated with Granulocyte-macrophage colonystimulating factor (GM-CSF) or GM-CSF/IL4, and MCSF or Macrophage colony-stimulating factor (M-CSF)/IFNγ for comparison to CSN1S1, as the latter stimulants are known to mediate in vitro differentiation towards the respective cell types [18,19]
These modifications observed in M-CSF or M-CSF/IFNγ stimulated cells were similar to changes observed in cells stimulated with CSN1S1, which formed aggregates and developed pseudopodia
Summary
The milk-derived protein human Casein alpha s1 (CSN1S1) has recently been detected in blood cells and was shown to possess proinflammatory properties. Human milk contains numerous proteins with properties beyond nutritional function [1]. Caseins are a main protein constituent of human milk and casein fragments exert a number of biological effects including the modulation of leukocyte adhesion [2], chemotactic properties [3,4,5,6], and effects on monocytic cells, like for instance increased expression of IL-1β [13]. Initial events in many inflammatory conditions crucially involve macrophages [14]. Macrophages usually originate from monocytes that are produced in the bone marrow and reach target tissues via systemic circulation [15]. We investigated the effect of CSN1S1 on monocytes and possible effects on cellular differentiation in vitro
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