Zheng-Hao Piao

Thioredoxin-Interacting Protein Regulates Hematopoietic Stem Cell Quiescence and Mobilization under Stress Conditions

Hematopoietic stem cells (HSCs) are maintained in a quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signals. The mechanisms regulating the quiescence and mobilization of HSCs, however, remain unclear. In this study, we report that the expression of thioredoxin-interacting protein (TXNIP) is decreased during HSC activation. In Txnip−/− mice, the long-term reconstituting HSC population is decreased and exhausted, and its capacity to repopulate is rapidly lost. These effects are associated with hyperactive Wnt signaling, an active cell cycle, and reduced p21 expression under conditions of stress. TXNIP deficiency reduced the CXCL12- and osteopontin-mediated interaction between HSCs and the bone marrow, and impaired homing and retention in the osteoblastic niche, resulting in mobilized HSCs. Therefore, we propose that TXNIP is essential for maintaining HSC quiescence and the interaction between HSCs and the BM niche.

TOX regulates the differentiation of human natural killer cells from hematopoietic stem cells in vitro.

Natural killer (NK) cells act important roles in innate immunity and adaptive immunity. However, the mechanisms governing NK cell development have not been clearly elucidated. Previous studies have shown that an HMG (high-mobility group) protein, TOX, is important for regulating the differentiation program of developing T cells in mice. In this study, we examined the role of TOX in differentiation of human NK cells. Knockdown of TOX in differentiating cells decreased the NK cell population identified by expression of NK surface markers and receptors. In addition, over-expression of TOX enhanced the differentiation of NK cells which give rise to a population showing effector functions of mature NK cells. Moreover, TOX influenced expression of T-bet (T-box expressed in T cells, also as known as Tbx21) during NK cell development. Overall, these results suggest that TOX is required for IL-15-mediated NK cell differentiation and affected expression of T-bet that plays critical roles in NK differentiation and maturation.

Suppressor of cytokine signaling 2 regulates IL-15-primed human NK cell function via control of phosphorylated Pyk2.

NK cells are capable of killing virus-infected or tumor cells and producing IFN-γ. Resting NK cells, however, have only minimal cytolytic activity and secrete a low level of IFN-γ. The cytokine IL-15 can promote the expression of effector functions by resting NK cells. In this study, we demonstrate that suppressor of cytokine signaling 2 (SOCS2) has a novel role in IL-15–primed human NK cell function. SOCS2 expression was upregulated in NK cells following stimulation with IL-15. During IL-15–mediated NK cell priming, SOCS2 interacted with phosphorylated proline-rich tyrosine kinase 2 (Pyk2) at tyrosine 402 (p-Pyk2Tyr402) and induced the proteasome-mediated degradation of p-Pyk2Tyr402 via ubiquitination. Knockdown of SOCS2 resulted in the accumulation of p-Pyk2Tyr402 and blocked NK cell effector functions. In addition, NK cell cytolytic activity and IFN-γ production were inhibited by overexpression of the wild-type of Pyk2 but not by the overexpression of tyrosine 402 mutant of Pyk2. These results suggest that SOCS2 regulates human NK cell effector functions via control of phosphorylated Pyk2 depending on IL-15 existence.

RasGRP1 Is Required for Human NK Cell Function

Cross-linking of NK activating receptors activates phospholipase-γ and subsequently induces diacylglycerol and Ca2+ as second messengers of signal transduction. Previous studies reported that Ras guanyl nucleotide-releasing protein (RasGRP) 1, which is activated by diacylglycerol and Ca2+, is crucial for TCR-mediated Ras-ERK activation. We now report that RasGRP1, which can also be detected in human NK cells, plays an essential role in NK cell effector functions. To examine the role of RasGRP1 in NK cell functions, the expression of RasGRP1 was suppressed using RNA interference. Knockdown of RasGRP1 significantly blocked ITAM-dependent cytokine production as well as NK cytotoxicity. Biochemically, RasGRP1-knockdown NK cells showed markedly decreased ability to activate Ras, ERK, and JNK. Activation of the Ras-MAPK pathway was independently shown to be indispensable for NK cell effector functions via the use of specific pharmacological inhibitors. Our results reveal that RasGRP1 is required for the activation of the Ras-MAPK pathway leading to NK cell effector functions. Moreover, our data suggest that RasGRP1 might act as an important bridge between phospholipase-γ activation and NK cell effector functions via the Ras-MAPK pathway.

Pseudomonas aeruginosa eliminates natural killer cells via phagocytosis-induced apoptosis.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes the relapse of illness in immunocompromised patients, leading to prolonged hospitalization, increased medical expense, and death. In this report, we show that PA invades natural killer (NK) cells and induces phagocytosis-induced cell death (PICD) of lymphocytes. In vivo tumor metastasis was augmented by PA infection, with a significant reduction in NK cell number. Adoptive transfer of NK cells mitigated PA-induced metastasis. Internalization of PA into NK cells was observed by transmission electron microscopy. In addition, PA invaded NK cells via phosphoinositide 3-kinase (PI3K) activation, and the phagocytic event led to caspase 9-dependent apoptosis of NK cells. PA-mediated NK cell apoptosis was dependent on activation of mitogen-activated protein (MAP) kinase and the generation of reactive oxygen species (ROS). These data suggest that the phagocytosis of PA by NK cells is a critical event that affects the relapse of diseases in immunocompromised patients, such as those with cancer, and provides important insights into the interactions between PA and NK cells.

Osteopontin promotes the development of natural killer cells from hematopoietic stem cells.

The detailed mechanisms driving the development of natural killer (NK) cells from hematopoietic stem cells remain to be clearly elucidated. Here, we show that osteopontin (OPN) is a key factor for NK development. OPN‐deficient mice evidenced severe impairments of NK development in bone marrow (BM) and spleen in which the NK populations that express CD122 and NK cell receptors were reduced. However, the absence of intrinsic OPN expression did not affect NK development, whereas the absence of OPN in the microenvironment caused a significant reduction in NK population. The expression of OPN was induced by interleukin (IL)‐15 in BM stromal cells, and the defect in NK differentiation in IL‐15−/− hematopoietic precursor cells (HPC) was recovered by addition of recombinant OPN, suggesting that the microenvironmental OPN may be a key factor in IL‐15‐mediated NK differentiation. In addition, OPN‐driven NK maturation was reduced in T‐bet‐deficient HPC, suggesting that T‐bet is required for OPN‐mediated NK development. Collectively, these results show that paracrine OPN signaling drives NK‐lineage commitment, thus ultimately promoting NK cell development.

YC-1 enhances natural killer cell differentiation from hematopoietic stem cells

NK cells play crucial roles in innate immunity and adaptive immunity. The detailed mechanisms, however, governing NK cell development remains unclear. In this study, we report that YC-1 significantly enhances NK cell populations differentiated from human umbilical cord blood hematopoietic stem cells (HSCs). NK cells increased by YC-1 display both phenotypic and functional features of fully mature NK (mNK) cells, but YC-1 does not affect the activation of mNK cells. YC-1 did not affect cGMP production and phosphorylation of STAT-5 which is essential for IL-15R signaling. On the other hand, YC-1 increased p38 MAPK phosphorylation during NK cell differentiation. Furthermore, p38 inhibitor SB203580 inhibited the differentiation of NK cells enhanced by YC-1. Taken together, these data suggest that YC-1 enhances NK cell differentiation through the activation of p38 MAPK which is involved in NK cell differentiation.

TXNIP regulates germinal center generation by suppressing BCL-6 expression

The detailed mechanism driving the germinal center (GC) reaction to B cell lymphomagenesis has not been clarified. Thioredoxin interacting protein (TXNIP), also known as vitamin D3 up-regulated protein 1 which is an important tumor repressor, is involved in stress responses, redox regulation, and cellular proliferation. Here, we report that TXNIP has a potential role in the formation of GC in peripheral lymphoid organs where B lymphocytes divide rapidly. First, we compared changes in GC from wild type mice and Txnip(-/-) mice. After immunization, Txnip(-/-) mice exhibited higher expression level of BCL-6 and larger percentage of GC B cells with the reduction in antibody production and plasma cell numbers. In addition, Txnip(-/-) spleens had a much larger population which expressed Ki-67, a marker of cell proliferation, in the red pulp border than WT spleens. Furthermore, the expression of BCL-6 was decreased in TXNIP overexpressing cells and elevated in TXNIP deficient cells. Taken together, we conclude that TXNIP may contribute to the formation of GCs after immunization. During this process, TXNIP suppresses BCL-6 expression.

IL-22 producing NKp46+ innate lymphoid cells can differentiate from hematopoietic precursor cells

The IL-22 NKp46(+) innate lymphoid cells, NCR22 cells, are very important for the early host defense against microbial pathogens. We show here that NCR22 cells were differentiated from Lin(-)CD127(+)CD117(+) cells that were derived from hematopoietic precursor cells (HPCs) of mouse bone marrow cells. The combination of low concentrations of IL-23 and IL-15 induced differentiation of NCR22 cells from Lin(-)CD127(+)CD117(+) cells. NCR22 cells expressed a large amount of IL-22 and RORγt, and they had poor cytolytic activity and produced little IFN-γ. Lin(-)CD127(+)CD117(+) cells were very similar to intestinal lamina propria LTi-like cells; both cells dominantly expressed RORγt and IL-22. Meanwhile, Lin(-)CD127(-)CD117(+) cells that were also derived from HPCs did not express RORγt and IL-22, and they developed into conventional NK cells, not into NCR22 cells. These findings revealed that NCR22 cells can be differentiated from Lin(-)CD127(+)CD117(+) cells which are derived from HPCs.