Xiuyuan Sun

Reelin promotes the adhesion and drug resistance of multiple myeloma cells via integrin β1 signaling and STAT3

Reelin is an extracellular matrix (ECM) protein that is essential for neuron migration and positioning. The expression of reelin in multiple myeloma (MM) cells and its association with cell adhesion and survival were investigated. Overexpression, siRNA knockdown, and the addition of recombinant protein of reelin were used to examine the function of reelin in MM cells. Clinically, high expression of reelin was negatively associated with progression-free survival and overall survival. Functionally, reelin promoted the adhesion of MM cells to fibronectin via activation of α5β1 integrin. The resulting phosphorylation of Focal Adhesion Kinase (FAK) led to the activation of Src/Syk/STAT3 and Akt, crucial signaling molecules involved in enhancing cell adhesion and protecting cells from drug-induced cell apoptosis. These findings indicate reelins important role in the activation of integrin-β1 and STAT3/Akt pathways in multiple myeloma and highlight the therapeutic potential of targeting reelin/integrin/FAK axis.

Changes of Cytokines during a Spaceflight Analog - a 45-Day Head-Down Bed Rest

Spaceflight is associated with deregulation in the immune system. Head-down bed rest (HDBR) at -6° is believed to be the most practical model for examining multi-system responses to microgravity in humans during spaceflight. In the present study, a 45-day HDBR was performed to investigate the alterations in human immune cell distributions and their functions in response to various stimuli. The effect of countermeasure, Rhodiola rosea (RR) treatment, was also examined. A significant decrease of interferon-γ (IFN-γ) and interleukin-17 (IL-17) productions by activated T cells, increase of IL-1β and IL-18 by activated B and myeloid cells were observed during HDBR. The upregulation of serum cortisol was correlated with the changes of IL-1 family cytokines. In addition, a significant increase of memory T and B cell and regulatory T cells (Treg) were also detected. The uptake of RR further decreased IFN-γ level and slowed down the upregulation of IL-1 family cytokines. These data suggest that for prolonged HDBR and spaceflight, the decreased protective T cell immunity and enhanced proinflammatory cytokines should be closely monitored. The treatment with RR may play an important role in suppressing proinflammatory cytokines but not in boosting protective T cell immunity.

Homeostatic Properties and Phenotypic Maturation of Murine CD4+ Pre-Thymic Emigrants in the Thymus

After a tightly regulated developmental program in the thymus, “mature” single positive (SP) thymocytes leave the thymus and enter the periphery. These newly arrived recent thymic emigrants (RTEs) are phenotypically and functionally immature, and will complete a dynamic maturation in the peripheral lymphoid organs before being licensed to be resident naïve T cells. To study the early events occurring in the RTE maturation process, we identified the phenotype of CD4+ pre-RTEs, a population of CD4+ SP thymocytes that have acquired the thymus egress capability. Compared to peripheral naïve T cells, CD4+ pre-RTEs displayed superior survival capability in lymphoreplete mice and faster proliferation under lymphopenic condition. The differences in Bcl2/Bim expression and/or heightened IL-7 signaling pathway may account for the pre-RTEs’ better responsiveness to homeostatic signals. Qa2, the expression of which indicates the phenotypic maturation of SPs and RTEs, was found to be upregulated in CD4+ pre-RTEs in thymic perivascular space. Migratory dendritic cells that surround this region contribute to Qa2 expression in pre-RTEs. The dendritic cell-driven Qa2 induction of CD4+ pre-RTEs is independent of MHC class II and Aire molecules.

Trx1/TrxR1 system regulates post-selected DP thymocytes survival by modulating ASK1-JNK/p38 MAPK activities

A key process in the development of T lymphocyte in the thymus is T‐cell receptor (TCR) selection. It is controlled by complex signaling pathways that contain redox‐sensitive molecules. However, the redox status early after TCR selection and how redox regulators promote the survival of post‐selected DP thymocytes has not been directly addressed. The present study demonstrated that the transition from pre‐ to post‐selected double‐positive (DP) stages was accompanied with an increase of reactive oxygen species (ROS) and a transient surge in the expression of a variety of redox regulators. Among them, the thioredoxin (Trx)1/thioredoxin reductase (TrxR)1 system was found to be critically involved in the regulation of cell survival of DP thymocytes, especially that of post‐selected CD69+ subset, as its inhibition caused a specific reduction of these cells both in vitro and in vivo, most likely owing to increased apoptosis. Suppression of the glutathione‐dependent redox system, on the other hand, showed no obvious impact. Biochemically, treatment of DP thymcoytes with TrxR1 inhibitor alone or in conjunction with anti‐CD3 resulted in enhanced phosphorylation of redox‐sensitive ASK‐1, JNK and p38 MAPK, and upregulated expression of Bim. Taken together, the data presented here suggest that the timely upregulation of Trx1/TrxR1 and the active control of intracellular redox status is critical for the survival of thymocytes during and short after positive selection.

Retention and tolerance of autoreactive CD4(+) recent thymic emigrants in the liver.

Mechanisms of peripheral tolerance play a critical role in preventing T cells that escape from negative selection in the thymus from initiating autoimmune reactions. To investigate the site of peripheral tolerance induction, we examined migration and activation of recent thymic emigrants (RTEs) in liver, spleen, lymph node and peripheral blood. We show that a fraction of RTE precursors were retained in the liver independent of the secondary lymphoid organs. Compared to RTEs from the lymph nodes, RTEs from the liver proliferated more and many exhibited an activated phenotype with the capability of producing IL-10 upon activation. Liver RTEs also responded poorly to interleukin (IL)-7 and were more prone to apoptosis. Following transfer into RAG(-/-) recipients, liver RTEs induced more severe inflammation and T cell infiltration in the lung and colon. The extrathymic expression of MHC and Aire is required for the acquisition of tolerogenic phenotype of newly generated thymic emigrants in the liver. These results suggest that the liver is the first checkpoint in the periphery to filter, retain, and enforce tolerance to autoreactive CD4(+) thymic emigrants that escape from negative selection.

Critical role of SP thymocyte motility in regulation of thymic output in neonatal Aire -/- mice

Autoimmune regulator (Aire) is essential in the perinatal period to prevent the multiorgan autoimmunity. Here we show that Aire-regulated single positive thymocyte trafficking in neonatal period is critical for thymic egress. Reduced thymic emigration was found in Aire−/− mice during neonatal period, leading to enhanced homeostatic expansion of peripheral T cells as early as 2 weeks of age. In neonatal Aire−/− mice, thymic expression of CCR7 ligands were dramatically reduced, resulting in decreased thymocyte motility and thymocyte emigration. This reduction of thymic egress in Aire−/− mice was alleviated beyond 3 weeks of age by an early upregulation of S1P1 signaling. As the numbers and quality of thymic emigrants are essential for the establishment and maintenance of peripheral tolerance, the reduced thymic emigration during neonatal period may deteriorate autoimmunity caused by the emigration of autoreactive T cells.

Simulated microgravity disrupts intestinal homeostasis and increases colitis susceptibility

The immune systems can be altered by spaceflight in many aspects, but microgravity‐related mucosal immune changes and its clinical significance have not been well studied. The purpose of this study was to investigate whether simulated microgravity influences the intestinal homeostasis and increases the susceptibility to colon inflammation. The hindlimb unloading (HU) mouse model was used to simulate the microgravity condition. Three percent dextran sulfate sodium (DSS) was given to mice to induce colitis. Compared to ground control (Ctrl) mice, the HU ones revealed an impaired intestinal homeostasis and increased susceptibility to DSS‐induced colitis. This includes an early‐onset, 4‐fold expansion of segmented filamentous bacteria (SFB), more than 2‐fold decrease in regulatory T (Treg) cell numbers and IL‐10 production, ~2‐fold increase in colonic IL‐1β expression, 2‐fold increase in circulating neutrophils, and colonic neutrophil infiltration. The application of antibiotics ameliorated the Treg and IL‐10 reductions but did not significantly dampen neutrophilia and elevated expression of colonic IL‐1β. These results indicate that the intestinal microflora and innate immune system both respond to simulated microgravity and together, contribute to the proinflammatory shift in the gut microenvironment. The data also emphasize the necessity for evaluating the susceptibility to inflammatory bowel diseases (IBDs) in distant space travels.—Li, P., Shi, J., Zhang, P., Wang, K., Li, J., Liu, H., Zhou, Y., Xu, X., Hao, J., Sun, X., Pang, X., Li, Y., Wu, H., Chen, X., Ge, Q. Simulated microgravity disrupts intestinal homeostasis and increases colitis susceptibility. FASEB J. 29, 3263‐3273 (2015). www.fasebj.org

Redox balance of mouse medullary CD4 single-positive thymocytes

After positive selection, the newly differentiated single‐positive (SP) thymocytes undergo negative selection to eliminate autoreactive T cells, functional maturation to acquire immunocompetence and egress capability. To investigate whether the intracellular reduction/oxidation (redox) balance has an important role on SP maturation, the levels of intracellular reactive oxygen species (ROS) and the expression of proteins that regulate ROS were compared among the four subsets of mouse TCRαβ+CD4+CD8− thymocytes (SP1–SP4) that represent sequential stages of SP differentiation program. A gradual increase of ROS and a gradual decrease of thioredoxin were revealed along the SP maturation process. The high ROS level at the mature SP stage did not result from a specific enrichment at this stage of natural regulatory T cells and SP thymocytes undergoing negative selection (Helios positive). An increase of ROS in the most mature SP4 cells resulted in enhanced cytokine production upon stimulation, whereas an early increase of ROS in the immature SP1 thymocytes resulted in enhanced apoptosis. Aire−/− mice that have defects in negative selection and a developmental blockage at the SP3–SP4 transition showed significantly less ROS in SP3 thymocytes. Thymic epithelial cells that have been shown to promote SP maturation in vitro also increased the ROS level of SP thymocytes. These results suggest that ROS may be involved in promoting the functional maturation of CD4+ SPs and thymic medullary microenvironment contributes to the pro‐oxidant shift of SP thymocytes.

Liver sinusoidal endothelial cells induce tolerance of autoreactive CD4 + recent thymic emigrants

The liver is a unique lymphoid organ whose microenvironment is biased towards tolerance induction. We previously found that a proportion of CD4+ autoreactive recent thymic emigrants (RTEs) retained in the liver after thymic egress and acquired IL-10 producing capability. To investigate the tolerance of these liver persisting CD4+ RTEs in more detail and to study the liver stromal cell types that facilitate the tolerogenic changes in young T cells, the phenotype and function of liver RTEs were further characterized and the impact of liver sinusoidal endothelial cells (LSECs) and Kupffer cells on RTEs were examined using an in vitro co-culture system. More than 70% of CD4+ CD44hi RTEs in the liver acquired Foxp3-LAG3+ CD49b− regulatory phenotype and function. But higher ratio of apoptosis with enhanced FasL and Bim expression was also found in these CD4+ liver RTEs when compared to those in the lymph nodes and spleen. LSECs played an important role in RTEs’ acquisition of tolerogenic and regulatory phenotype. These results indicate an important role of liver microenvironment in enforcing peripheral tolerance to CD4+ thymic emigrants against self- and gut-derived antigens.

Toll-like receptor 4-mediated signaling regulates IL-7-driven proliferation and differentiation of B-cell precursors

Lipopolysaccharide (LPS) is known to be a potent activator of mature B cells by signaling through Toll-like receptor 4 (TLR4). Its impact on early B-cell development, however, is not well defined. When comparing to C3H/HeN mice, TLR4-mutant C3H/HeJ mice showed an increase in the number of pro-B and pre-B cells in the bone marrow. When cultured in the presence of IL-7, the proliferation of pro-B and large pre-B cells was significantly inhibited by LPS, possibly due to reduced IL-7 receptor-α (IL-7Rα) expression. Meanwhile, the generation of IgM+/IgD+ B cells was greatly enhanced in IL-7 cultures of pro-B and pre-B cells. Consistent with these results, treatment with LPS facilitated the progression of adoptively transferred B220+IgM−IgD− precursors into IgD+ cells. Overall, these data suggest that LPS has a profound influence on early B-cell development, which may contribute to the deregulated B-cell development under physiological and pathological conditions such as bacterial infections.