Expanded NK cells from umbilical cord blood and adult peripheral blood combined with daratumumab are effective against tumor cells from multiple myeloma patients.

In this study we evaluated the potential of expanded NK cells (eNKs) from two sources combined with the mAbs daratumumab and pembrolizumab to target primary multiple myeloma (MM) cells ex vivo. In order to ascertain the best source of NK cells, we expanded and activated NK cells from peripheral blood (PB) of healthy adult donors and from umbilical cord blood (UCB). The resulting expanded NK (eNK) cells express CD16, necessary for carrying out antibody-dependent cellular cytotoxicity (ADCC). Cytotoxicity assays were performed on bone marrow aspirates of 18 MM patients and 4 patients with monoclonal gammopathy of undetermined significance (MGUS). Expression levels of PD-1 on eNKs and PD-L1 on MM and MGUS cells were also quantified. Results indicate that most eNKs obtained using our expansion protocol express a low percentage of PD-1+ cells. UCB eNKs were highly cytotoxic against MM cells and addition of daratumumab or pembrolizumab did not further increase their cytotoxicity. PB eNKs, while effective against MM cells, were significantly more cytotoxic when combined with daratumumab. In a minority of cases, eNK cells showed a detectable population of PD1+ cells. This correlated with low cytotoxic activity, particularly in UCB eNKs. Addition of pembrolizumab did not restore their activity. Results indicate that UCB eNKs are to be preferentially used against MM in the absence of daratumumab while PB eNKs have significant cytotoxic advantage when combined with this mAb.

Glucocorticoids Regulate Bone Marrow B Lymphopoiesis After Stroke.

RATIONALE: After a stroke, patients frequently experience altered systemic immunity resulting in peripheral immunosuppression and higher susceptibility to infections, which is at least partly attributed to lymphopenia. The mechanisms that profoundly change the systemic leukocyte repertoire after stroke are incompletely understood. Emerging evidence indicates that stroke alters hematopoietic output of the bone marrow. OBJECTIVE: To explore the mechanisms that lead to defects of B lymphopoiesis after ischemic stroke. METHODS AND RESULTS: We here report that ischemic stroke triggers brain-bone marrow communication via hormonal long-range signals that regulate hematopoietic B lineage decisions. Bone marrow fluorescence-activated cell sorter analyses and serial intravital microscopy indicate that transient middle cerebral artery occlusion in mice arrests B-cell development beginning at the pro-B-cell stage. This phenotype was not rescued in Myd88-/- and TLR4-/- mice with disrupted TLR (Toll-like receptor) signaling or after blockage of peripheral sympathetic nerves. Mechanistically, we identified stroke-induced glucocorticoid release as the main instigator of B lymphopoiesis defects. B-cell lineage-specific deletion of the GR (glucocorticoid receptor) in CD19-Cre loxP Nr3c1 mice attenuated lymphocytopenia after transient middle cerebral artery. In 20 patients with acute stroke, increased cortisol levels inversely correlated with blood lymphocyte numbers. CONCLUSIONS: Our data demonstrate that the hypothalamic-pituitary-adrenal axis mediates B lymphopoiesis defects after ischemic stroke.

miR-125b controls monocyte adaptation to inflammation through mitochondrial metabolism and dynamics.

Metabolic changes drive monocyte differentiation and fate. Although abnormal mitochondria metabolism and innate immune responses participate in the pathogenesis of many inflammatory disorders, molecular events regulating mitochondrial activity to control life and death in monocytes remain poorly understood. We show here that, in human monocytes, microRNA-125b (miR-125b) attenuates the mitochondrial respiration through the silencing of the BH3-only proapoptotic protein BIK and promotes the elongation of the mitochondrial network through the targeting of the mitochondrial fission process 1 protein MTP18, leading to apoptosis. Proinflammatory activation of monocyte-derived macrophages is associated with a concomitant increase in miR-125b expression and decrease in BIK and MTP18 expression, which lead to reduced oxidative phosphorylation and enhanced mitochondrial fusion. In a chronic inflammatory systemic disorder, CD14+ blood monocytes display reduced miR-125b expression as compared with healthy controls, inversely correlated with BIK and MTP18 messenger RNA expression. Our findings not only identify BIK and MTP18 as novel targets for miR-125b that control mitochondrial metabolism and dynamics, respectively, but also reveal a novel function for miR-125b in regulating metabolic adaptation of monocytes to inflammation. Together, these data unravel new molecular mechanisms for a proapoptotic role of miR-125b in monocytes and identify potential targets for interfering with excessive inflammatory activation of monocytes in inflammatory disorders.

Inhibition of Inflammation and Bone Erosion by RNA Interference-Mediated Silencing of Heterogeneous Nuclear RNP A2/B1 in Two Experimental Models of Rheumatoid Arthritis.

OBJECTIVE: The nuclear protein heterogeneous nuclear RNP A2/B1 (hnRNP A2/B1) is involved in posttranscriptional regulation of gene expression. It is constitutively expressed in lymphoid organs and highly up-regulated in the synovial tissue of patients with rheumatoid arthritis (RA), who may also generate autoantibodies to this protein. This study was undertaken to investigate the potential involvement of hnRNP A2/B1 in the pathogenesis of autoimmune arthritis, by silencing hnRNP A2/B1 expression in 2 animal models of RA. METHODS: Collagen-induced arthritis (CIA) and the K/BxN serum-transfer model were used as animal models of RA. Efficient silencing of hnRNP A2/B1 was achieved using a liposome-based carrier system for delivery of small interfering RNAs. Expression of hnRNP A2/B1 was analyzed by flow cytometry, reverse transcription-quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. The number of osteoclasts was determined by tartrate-resistant acid phosphatase staining. Cytokine levels and anticollagen antibody levels were measured by enzyme-linked immunosorbent assay. RESULTS: Efficient silencing of hnRNP A2/B1 was achieved in all lymphoid organs. In both experimental models, the incidence and severity of arthritis were largely reduced and bone erosion was not detectable as compared to the control groups. Down-modulation of hnRNP A2/B1 significantly interfered with the production of proinflammatory cytokines from monocyte/macrophages, but not from T cells. Consistent with these findings, production of T cell cytokines was not impaired when cells were restimulated in vitro with type II collagen. Furthermore, levels of anticollagen antibodies were not affected by hnRNP A2/B1 silencing. CONCLUSION: Our findings suggest that hnRNP A2/B1 has an important role in regulation of the innate immune system, especially at the level of monocyte/macrophage activation. Therefore, down-modulation of hnRNP A2/B1 seems to affect primarily the effector phase of autoimmune arthritis.

Nicotinamide phosphoribosyltransferase/visfatin expression by inflammatory monocytes mediates arthritis pathogenesis.

OBJECTIVES: Nicotinamide phosphoribosyltransferase (NAMPT)/pre-B-cell colony-enhancing factor/visfatin exerts multiple functions and has been implicated in the pathogenesis of rheumatoid arthritis. To gain insight into its role in arthritis and given that NAMPT is identified as a novel mediator of innate immunity, we addressed the function of monocyte-derived NAMPT in experimental arthritis by selective gene knockdown in inflammatory monocytes. METHODS: siRNA uptake and NAMPT expression were determined in Ly6Chigh and Ly6Clow monocyte subsets following intravenous injection of siRNA against NAMPT (siNAMPT) or non-targeting siRNA (siCT) formulated with the DMAPAP cationic liposome into mice. Mice with established collagen-induced arthritis (CIA) were treated weekly after disease onset with siNAMPT or siCT and clinical features were assessed. T-helper cell frequencies, cytokine production and percentage of IL-6-producing Ly6Chigh monocytes were analysed. Using a co-culture system consisting of purified CD14 monocytes and autologous CD4 T cells, NAMPT and cytokine production, and the percentage of IL-17-producing CD4 T cells, were determined following transfection of CD14 monocytes with siCT or siNAMPT. RESULTS: On intravenous injection, siRNA was preferentially engulfed by Ly6Chigh monocytes, and siRNA-mediated silencing of NAMPT expression in Ly6Chigh monocytes inhibited CIA progression. This effect was associated with reduced IL-6 production by Ly6Chigh monocytes, reduced proportion of Th17 cells and autoantibody titers, and decreased activation and infiltration of monocytes/macrophages and neutrophils in arthritic joints. Moreover, NAMPT-RNAi-silenced CD14 monocytes were found to reduce the percentage of IL-17-producing CD4 T cells in vitro. CONCLUSIONS: Our results show that the expression of NAMPT in Ly6Chigh monocytes promotes many downstream effects involved in inflammatory arthritis and demonstrate the utility of targeting disease-causing genes, such as NAMPT, in Ly6Chigh monocytes for therapeutic intervention in arthritis.

MicroRNAs as new player in rheumatoid arthritis.

MicroRNAs (miRNAs) are small noncoding RNA molecules that negatively regulate gene expression at the post-transcriptional level. Currently, there are 939 mature human miRNA sequences listed in the Sanger updated miRNA registry. There are approximately 1500 predicted miRNAs in the human genome that may regulate the expression of one third of our genes. By controlling the accumulation of the target protein(s) in cells, these regulatory RNA molecules participate in key functions in many physiological networks and their deregulation has been implicated in the pathogenesis of serious human disorders, such as cancer and infection. The implication of miRNAs in immune-mediated disorders such as rheumatoid arthritis (RA) has recently emerged suggesting that miRNA-based therapeutic approaches may have a promising potential in these diseases. Here, we provide an overview of the state-of-the-art on miRNAs in RA, focusing on both systemic and local features of the pathology.

In vivo RNAi-mediated silencing of TAK1 decreases inflammatory Th1 and Th17 cells through targeting of myeloid cells.

Cells from the mononuclear phagocyte system (MPS) act as systemic and local amplifiers that contribute to the progression of chronic inflammatory disorders. Transforming growth factor-ß-activated kinase 1 (TAK1) is a pivotal upstream mitogen-activated protein kinase-kinase-kinase acting as a mediator of cytokine expression. It remains critical to determine in vivo the implication of TAK1 in controlling the innate immune system. Here, we describe a vehicle tailored to selectively deliver siRNAs into MPS cells after intravenous administration, and validate in vivo the potential of the RNAi-mediated TAK1 knock down for immunomodulation. In a mouse model of immune-mediated inflammatory disorder, we show that anti-TAK1 siRNA lipoplexes efficiently alleviate inflammation, severely impair the downstream c-Jun N-terminal kinase and nuclear factor-κB signaling pathways, and decrease the expression of proinflammatory mediators. Importantly, the systemic TAK1 gene silencing decreases the frequency of Th1 and Th17 cells, both mediating autoimmunity in experimental arthritis, demonstrating the immunomodulatory potential of TAK1. Finally, in vitro inhibition of TAK1 in myeloid cells decreases interferon-γ-producing T cells, suggesting that a delivery system able to target MPS cells and to silence TAK1 impacts on pathogenic T effector cells in autoimmunity.

Cationic liposome formulations for RNAi-based validation of therapeutic targets in rheumatoid arthritis.

Several molecules have been identified as critical mediators of chronic inflammation in immune system-mediated disorders such as rheumatoid arthritis (RA), and biological therapies targeting these molecules have been developed during the past two decades. Compared with conventional therapies, anti-TNF biotherapies have greatly improved the treatment of patients with RA, and several biological agents with distinct mechanisms of action are under development. Despite significant advances in this field, unmet medical needs remain. RA is the prototype disease for the evaluation of targeted therapies, and various novel genes have been described as being critically involved in disease pathogenesis. Thus, a novel area of research has recently emerged in the field of RA therapy, involving the genetic screening and validation of novel candidates in vivo using RNAi. Among the vehicles for the efficient targeting of macrophages, which play a critical role in disease chronicity, cationic liposomes represent the most promising option for the safe and specific use of RNAi in vivo. This review discusses the role of cationic liposomes as a mechanism for the systemic administration of siRNAs in the validation of RA therapeutic targets.