Lactoferrin is required for early B cell development in C57BL/6 mice
Journal of Hematology & Oncology volume 14, Article number: 58 (2021)
Lactoferrin (Lf) is widely distributed in mammalian milk, various tissues, and their exocrine fluids and has many physiological functions, such as bacteriostasis, antivirus, and immunoregulation. Here, we provide evidence that lactoferrin is required for early stages of B cell development in mice. Lactoferrin-deficient (Lf−/−) C57BL/6 mice showed systematic reduction in total B cells, which was attributed to the arrest of early B cell development from pre-pro-B to pro-B stage. Although the Lf−/− B cell “seeds” generated greater pro-B cells comparing to wild type (WT) littermates, the Lf−/− mice bone marrow had less stromal cells, and lower CXCL12 expression, produced a less favorable “microenvironment” for early B cell development. The underlying mechanism was mediated through ERK and AKT signalings and an abnormality in the transcription factors related to early differentiation of B cells. The Lf−/− mice also displayed abnormal antibody production in T cell-dependent and T cell-independent immunization experiments. In a pristane-induced lupus model, Lf−/− mice had more serious symptoms than WT mice, whereas lactoferrin treatment alleviated these symptoms. This study demonstrates a novel role of lactoferrin in early B cell development, suggesting a potential benefit for using lactoferrin in B cell-related diseases.
To the editor,
Lactoferrin (LF) is widely existed in mammalian milk, neutrophils particles and various tissues and their exudates. It plays a protective role of the body through a variety of functions, such as anti-infection, anti-oxidation, and immune modulation [1, 2]. By constructing a lactoferrin gene knockout (Lf−/−) C57BL/6 mice [3, 4], here we investigated the effect of lactoferrin deficiency on B cell development.
Proportion of CD45+, T, and dendritic cells remained fairly consistent, but proportion of total B cells of Lf−/− mice were significantly lower than that of WT controls (Fig. 1a, Additional file 2 Fig. S1). Lactoferrin deficiency did not cause an increase of B cell apoptosis (Fig. 1b). Proportion of hematopoietic progenitor cells displayed little difference between WT and Lf−/− (Fig. 1c, d). Proportion of pro-B, pre-B and immature B cells in bone marrow of Lf−/− mice were all significantly lower, whereas proportion of pre-pro-B cells was higher than that of WT (Fig. 1e, f), implying that lactoferrin deficiency inhibited the transition from pre-pro-B to pro-B stage. mRNA expression levels of lactoferrin are dynamic in developing B cells, peaking at the pre-pro-B stage (Fig. 1g).
Contrary to our expectations, the capability of pre-pro-B to generate pro-B cells was significantly higher in Lf−/− group than WT both in vitro (Fig. 1h, i) and in vivo (Fig. 1j, Additional file 3 Fig. S2), implying that Lf−/− “seeds” generated more pro-B cells comparing to WT “seeds”. We analyzed the global transcriptome change of pre-pro-B and pro-B cells from WT and Lf−/− mice. GO, KEGG, and GSEA  reveal the signaling differences between them (Additional file 4 Fig. S3, Additional file 5 Fig. S4). Transcription factors Pu.1, Bcl11a, E2A, Ebf1, and Pax5 regulate the differentiation of common lymphoid progenitor (CLP) cells into B cell lineage . Expression levels of Pax5, Ebf1 and Tcf3 in Lf−/− pre-pro-B and pro-B cells were higher than WT (Additional file 4 Fig. S3F, Additional file 5 Fig. S4E).
In another side, in vivo bone marrow transplantation experiment revealed that lactoferrin deficiency in bone marrow microenvironment retards the transition from pre-pro-B to pro-B stage (Fig. 1k). So, the dysregulation of early B cell development in lactoferrin-deficient mice is both cell autonomous and is associated with bone marrow microenvironment.
Proportion of stromal cells in Lf−/− bone marrow were decreased significantly comparing to WT (Fig. 2a). CXCL12 expressions were significantly lower in Lf−/− bone marrow stromal cells but not the IL-7 expressions (Fig. 2b–d). Lactoferrin can modulate Cxcl12 expression in mouse stromal cell OP9 (Additional file 6 Fig. S5A, B); and CXCL12 protein promoted early B cell differentiation (Fig. 2e). CXCL12 modulate the differentiation of lymphocytes by binding to its receptor CXCR4 . Expression of CXCR4 in B cells isolated from Lf−/− mice bone marrow was higher than WT (Fig. 2f, Additional file 6 Fig. S5C). CXCR4 inhibitor suppressed B cells differentiation, while CXCL12 can promote it (Fig. 2g). The CXCL12-CXCR4 axis transmits signals through MAPK and AKT signalings . CXCR4 inhibitor reduced the phosphorylation levels of AKT and ERK, whereas CXCL12 increased them (Fig. 2h, Additional file 6 Fig. S5D). AKT or ERK inhibitors suppressed B cell differentiation, while CXCL12 can reverse their effects (Fig. 2i). So, Lf−/− mouse bone marrow stromal cells were not conducive to the early B cell development compared with WT controls (Fig. 2j).
Dysregulation of difference type B cells were observed in the spleen of Lf−/− mice comparing to WT (Additional file 7 Fig. S6A, B). Under the stimulation of TD and TI antigens, lactoferrin deficiency led to a decrease in proportion of total, follicular and T1 B cells (Additional file 7 Fig. S6C). The TNP-specific IgM and IgG2a, 2b responses were higher in Lf−/− mice (Additional file 7 Fig. S6D, E).
Systemic lupus erythematosus (SLE) is associated with a number of immunomodulatory abnormalities including B cell dysfunction . In a pristane-induced SLE mouse model, the degree of injury to the glomerular filtration barrier, kidney damage, glomerular deposits of IgG antibodies, serum levels of IgM and IgG2b, amounts of anti-dsDNA total IgM and IgG in WT mice was lower than that in Lf−/− mice, and oral lactoferrin treatment alleviated the symptoms (Additional file 8 Fig S7). Lactoferrin plays a protective role in the SLE model.
In conclusion, this study demonstrates that lactoferrin is required for the early development of B cells in C57BL/6 mice by regulating the microenvironment of bone marrow stroma through CXCL12 release. Lf−/− mice had more severe symptoms in a SLE model, which can be alleviated by oral administration of lactoferrin, and that was related to the dysfunction of B cells induced by lactoferrin deficiency. Lactoferrin may be applied in preventative medicine or nutrition supplies for B cell-related diseases.
Availability of data and materials
All data generated or analyzed during this study are included in this published article. The RNA-seq raw expression files and details have been deposited in NCBI GEO under accession number GSE163097.
Common lymphoid progenitor
Common myeloid progenitor
Enzyme-linked immunosorbent assay
Hematopoietic stem cell
Systemic lupus erythematosus
T cell independent
T cell dependent
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We thank Professors Wen Zhou, Jie Zhou for providing reagents and key suggestions.
National Natural Science Foundation of China (81672889, 81874170, 82073261, 82060042, 32000665, 81660273), China 111 Project (111-2-12).
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This study was approved by the ethical review committees of the Central South University.
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Representative flow analysis diagrams for analysis of the different hematopoietic cells.
Representative flow analysis diagrams of in vivo bone marrow transplantation experiment.
Lactoferrin deficiency alters genes expression profile and key pathways in pre-pro-B cells.
Lactoferrin deficiency alters genes expression profile and key pathways in pro-B cells.
Effect of lactoferrin deficiency on Cxcl2 expression.
Lactoferrin deficiency affects the proportion of splenic B cells subclasses and antibody production in B cells.
Lactoferrin deficiency promotes the progression of SLE in mice.
Information on the antibodies used in FACS or mice experiments.
Antibodies information for ELISA.
Primer sequences used for qPCR.
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Wei, L., Liu, C., Wang, J. et al. Lactoferrin is required for early B cell development in C57BL/6 mice. J Hematol Oncol 14, 58 (2021). https://doi.org/10.1186/s13045-021-01074-6