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

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.

Ser71 of αS1‐Casein is Phosphorylated in Breast Milk—Evidence from Targeted Mass Analysis

“…Human αS1‐casein is a low abundant member of the soluble casein fraction and an autoantigen . The protein is able to induce proinflammatory cytokine expression in monocytic cells by toll‐like receptor 4 (TLR‐4)‐signaling, it leads to in vitro differentiation of monocytes towards macrophages, and it upregulates the production of GM‐CSF via p38 MAP kinase in human monocytes . More recently our group has also shown that the phosphorylation of αS1‐casein by casein kinase (CK) 2 completely abolished binding to TLR‐4 and proinflammatory cytokine induction.…”

Human casein alpha s1 induces proinflammatory cytokine expression in monocytic cells by TLR4 signaling

“…Human milk contains numerous proteins that interact with the immune system . Particularly, proteins of the casein family and digested casein fragments are multifunctional and possess the ability to chemoattract immune cells such as monocytes , act as chaperones similar to heat shock proteins , stimulate cellular immune functions such as macrophage phagocytosis , act as antioxidants, and interact with opioid receptors .…”

“…To our knowledge, functional analyses of differently P‐CSN1S1 variants have not been described so far. However, unphosphorylated human CSN1S1 was recently shown to exert proinflammatory effects on monocytic cells, such as the potent induction of IL‐1β, GM‐CSF, IL‐6 , and to induce differentiation of monocytes toward macrophages . Furthermore, breastfed individuals display sustained CSN1S1‐IgG production into adulthood .…”

Caseins as Regulators of Hematopoiesis

“…Interestingly, this group found that αS1-casein was linked to specific receptors for the protein in most of the macrophages analyzed and not only that but also that it could induce the production of IL-1 and IL-6, which positions it as an excellent immunomodulatory protein [48]. This same group subsequently demonstrated that αS1-casein in human milk has the ability to differentiate human monocytes from macrophages, and they also increase the phagocytic capacity of the monocytes in vitro once stimulated with the protein [49]. On the other hand, the Santiago-Osorio group showed that the IP inoculation of BALB/c mice with sodium caseinate induces in vivo proliferation of the myeloid lineage in the bone marrow.…”

“…αS1-casein activates the secretion of the proinflammatory cytokines GM-CSF (granulocyte-macrophage colony-stimulating factor), IL1-β (interleukin 1β), IL-6 (interleukin 6), and chemokine IL-8 (interleukin 8) in human monocytes via the mitogen-activated protein kinase p38 (MAPK-p38) signaling pathway [24,49].…”