FoxO1 controls the expansion of pre-B cells by regulating the expression of interleukin 7 receptor α chain and its signal pathway.

Forkhead box O1 (FoxO1) has a crucial role in the early B cell development. To understand the functional importance of FoxO1 gene in the early B cell expansion, we established a FoxO1 knockdown model using 70Z/3 pre-B cell line. The FoxO1 knockdown 70Z/3 cells (70Z/3-KD cells) showed the down-regulated expression of interleukin 7 receptor α chain (IL-7Rα). Moreover, the signaling via IL-7Rα was significantly attenuated in the 70Z/3-KD cells, and this alteration was fully rescued by re-expression of FoxO1 gene. Compared to the mock cells, loss of FoxO1 reduced the growth rates in the 70Z/3-KD cells, and was fully rescued by reintroduction of FoxO1 gene. The expansion of pre-B cells (CD45R+CD43- fraction) was also reduced by the knockdown of FoxO1 gene. Indeed, FoxO1 induces accumulation in the p27-mediated G0/G1 phase arrest in 70Z/3 cells. FoxO1 bound to the Il7ra locus specifically and regulate the IL-7Rα transcription. In conclusion, FoxO1 regulates the expansion of pre-B cells by regulating the expression of IL-7Rα and its signal transduction.

8:2 Fluorotelomer alcohol causes G1 cell cycle arrest and blocks granulocytic differentiation in HL-60 cells.

Fluorotelomer alcohols (FTOHs) are fluorinated intermediates used in manufacturing specialty polymer and surfactants, with 8:2 FTOH the homologue of largest production. FTOHs were found to pose acute toxicity, hepatotoxicity, nephrotoxicity, developmental toxicity and endocrine-disrupting risks, whereas research regarding immunotoxicity and its underlying mechanism, especially on specific immune cells is limited. Here, we investigated the immunotoxicity of 8:2 FTOH on immature immune cells in an in vitro system. We observed that exposure of HL-60 cells, a human promyelocytic leukemic cell line, to 8:2 FTOH reduced cell viability in a dose- and time-dependent manner. In addition, 8:2 FTOH exposure caused G1 cell cycle arrest in HL-60 cells, while it showed no effect on apoptosis. Exposure to 8:2 FTOH inhibited the mRNA expression of cell cycle-related genes, including CCNA1, CCNA2, CCND1, and CCNE2. Moreover, exposure to 8:2 FTOH inhibited the mRNA expression of granulocytic differentiation-related genes of CD11b, CSF3R, PU.1, and C/EPBε in HL-60 cells . Furthermore, 8:2 FTOH exhibited no effect on intracellular ROS level, while hydralazine hydrochloride (Hyd), one reactive carbonyl species (RCS) scavenger, partially blocked 8:2 FTOH-caused cytotoxicity in HL-60 cells. Overall, the results obtained in the study show that 8:2 FTOH poses immunotoxicity in immature immune cells and RCS may partially underline its mechanism.

CD9+ Regulatory B Cells Induce T Cell Apoptosis via IL-10 and Are Reduced in Severe Asthmatic Patients.

CD9 was recently identified as a marker of murine IL-10-competent regulatory B cells. Functional impairments or defects in CD9+ IL-10-secreting regulatory B cells are associated with enhanced asthma-like inflammation and airway hyperresponsiveness. In mouse models, all asthma-related features can be abrogated by CD9+ B cell adoptive transfer. We aimed herein to decipher the profiles, features, and molecular mechanisms of the regulatory properties of CD9+ B cells in human and mouse. The profile of CD9+ B cells was analyzed using blood from severe asthmatic patients and normal and asthmatic mice by flow cytometry. The regulatory effects of mouse CD9+ B cells on effector T cell death, cell cycle arrest, apoptosis, and mitochondrial depolarization were determined using yellow dye, propidium iodide, Annexin V, and JC-1 staining. MAPK phosphorylation was analyzed by western blotting. Patients with severe asthma and asthmatic mice both harbored less CD19+CD9+ B cells, although these cells displayed no defect in their capacity to induce T cell apoptosis. Molecular mechanisms of regulation of CD9+ B cells characterized in mouse showed that they induced effector T cell cycle arrest in sub G0/G1, leading to apoptosis in an IL-10-dependent manner. This process occurred through MAPK phosphorylation and activation of both the intrinsic and extrinsic pathways. This study characterizes the molecular mechanisms underlying the regulation of CD9+ B cells to induce effector T cell apoptosis in mice and humans via IL-10 secretion. Defects in CD9+ B cells in blood from patients with severe asthma reveal new insights into the lack of regulation of inflammation in these patients.

A TLR9-dependent checkpoint governs B cell responses to DNA-containing antigens.

Mature B cell pools retain a substantial proportion of polyreactive and self-reactive clonotypes, suggesting that activation checkpoints exist to reduce the initiation of autoreactive B cell responses. Here, we have described a relationship among the B cell receptor (BCR), TLR9, and cytokine signals that regulate B cell responses to DNA-containing antigens. In both mouse and human B cells, BCR ligands that deliver a TLR9 agonist induce an initial proliferative burst that is followed by apoptotic death. The latter mechanism involves p38-dependent G1 cell-cycle arrest and subsequent intrinsic mitochondrial apoptosis and is shared by all preimmune murine B cell subsets and CD27- human B cells. Survival or costimulatory signals rescue B cells from this fate, but the outcome varies depending on the signals involved. B lymphocyte stimulator (BLyS) engenders survival and antibody secretion, whereas CD40 costimulation with IL-21 or IFN-γ promotes a T-bet+ B cell phenotype. Finally, in vivo immunization studies revealed that when protein antigens are conjugated with DNA, the humoral immune response is blunted and acquires features associated with T-bet+ B cell differentiation. We propose that this mechanism integrating BCR, TLR9, and cytokine signals provides a peripheral checkpoint for DNA-containing antigens that, if circumvented by survival and differentiative cues, yields B cells with the autoimmune-associated T-bet+ phenotype.

Binding of HLA-G to ITIM-bearing Ig-like transcript 2 receptor suppresses B cell responses.

Inhibition of B cells constitutes a rational approach for treating B cell-mediated disorders. We demonstrate in this article that the engagement of the surface Ig-like transcript 2 (ILT2) inhibitory receptor with its preferential ligand HLA-G is critical to inhibit B cell functions. Indeed, ILT2-HLA-G interaction impedes both naive and memory B cell functions in vitro and in vivo. Particularly, HLA-G inhibits B cell proliferation, differentiation, and Ig secretion in both T cell-dependent and -independent models of B cell activation. HLA-G mediates phenotypic and functional downregulation of CXCR4 and CXCR5 chemokine receptors on germinal center B cells. In-depth analysis of the molecular mechanisms mediated by ILT2-HLA-G interaction showed a G0/G1 cell cycle arrest through dephosphorylation of AKT, GSK-3ß, c-Raf, and Foxo proteins. Crucially, we provide in vivo evidence that HLA-G acts as a negative B cell regulator in modulating B cell Ab secretion in a xenograft mouse model. This B cell regulatory mechanism involving ILT2-HLA-G interaction brings important insight to design future B cell-targeted therapies aimed at reducing inappropriate immune reaction in allotransplantation and autoimmune diseases.

Jurkat cell proliferation is suppressed by Chlamydia (Chlamydophila) pneumoniae infection accompanied with attenuation of phosphorylation at Thr389 of host cellular p70S6K.

Chlamydia (Chlamydophila) pneumoniae infects T lymphocytes and multiplies within them. Our previous studies have indicated that C. pneumoniae infection suppresses proliferation of peripheral blood mononuclear cells stimulated with Staphylococcus-enterotoxin B; however, the mechanism of suppression was unclear. In this study, we explored the molecular mechanism involved in C. pneumoniae infection by using human acute T cell leukemia cell line, Jurkat E6-1. Proliferation of Jurkat cells was suppressed in an m.o.i.-dependent manner by C. pneumoniae infection. The suppression by the infection was particularly evident during the initial 24h of the infection, and down modulation of cyclin D3 protein levels were observed at the same time period by immunoblot analysis. The suppression of the Jurkat cell proliferation and the down modulation of cyclin D3 protein level were only induced by viable C. pneumoniae infection, not by exposure to UV-killed or heat-killed C. pneumoniae. Phosphorylations at Thr308 and Ser473 of AKT were induced by C. pneumoniae infection; however, phosphorylation at Thr389 of the downstream kinase, p70S6K was inhibited by unidentified mechanism associated with C. pneumoniae infection. Taking into account that G1 arrest of the C. pneumoniae infected Jurkat cells were not observed and that p70S6K is one of the most important regulators of protein synthesis, it was suggested that the suppression of Jurkat cell proliferation by C. pneumoniae was at least in part mediated by down modulation of protein synthesis through attenuation of Thr389 phosphorylation of p70S6K.