Potential Use of Extracellular Vesicles Generated by Microbubble-Assisted Ultrasound as Drug Nanocarriers for Cancer Treatment.

Extracellular vesicles (EVs)-carrying biomolecules derived from parental cells have achieved substantial scientific interest for their potential use as drug nanocarriers. Ultrasound (US) in combination with microbubbles (MB) have been shown to trigger the release of EVs from cancer cells. In the current study, the use of microbubbles-assisted ultrasound (USMB) to generate EVs containing drug cargo was investigated. The model drug, CellTracker™ green fluorescent dye (CTG) or bovine serum albumin conjugated with fluorescein isothiocyanate (BSA FITC) was loaded into primary human endothelial cells in vitro using USMB. We found that USMB loaded CTG and BSA FITC into human endothelial cells (HUVECs) and triggered the release of EVs containing these compounds in the cell supernatant within 2 h after treatment. The amount of EV released seemed to be correlated with the increase of US acoustic pressure. Co-culturing these EVs resulted in uptake by the recipient tumour cells within 4 h. In conclusion, USMB was able to load the model drugs into endothelial cells and simultaneously trigger the release of EVs-carrying model drugs, highlighting the potential of EVs as drug nanocarriers for future drug delivery in cancer.

Clinical efficacy of leflunomide in primary Sjogren's syndrome is associated with regulation of T-cell activity and upregulation of IL-7 receptor α expression.

OBJECTIVES: To investigate whether the immunomodulatory capacities of leflunomide are associated with clinical efficacy in the treatment of primary Sjögren's syndrome (SS) in a phase II pilot study. METHODS: Peripheral blood mononuclear cells from 13 primary SS patients were obtained at baseline and after 24 weeks of leflunomide treatment. Ex-vivo production of interleukin (IL) 1ß and tumour necrosis factor α (TNFα) and of interferon (IFN), IL-4, as well as TNFα ELISA measured production on T-cell and monocyte stimulation. In addition, the authors investigated the ability of leflunomide to influence systemic levels of inflammatory cytokines, as well as T-cell activation markers and the expression of IL-7 receptor α by flow cytometry. Correlations between changes in cytokine levels and changes in clinical response parameters were studied. RESULTS: Ex-vivo production of IL-1ß and TNFα was decreased at 24 weeks in the whole patient group, whereas IFN and IL-4 production were not significantly changed. However, a significant decrease in T-cell-stimulated IFN and TNFα production was observed in clinical responders, but not in non-responders. Moreover, significant correlations were found between increased sialometry values and decreased IFN and TNFα production. In addition, leflunomide reduced levels of inflammatory serum cytokines and CD40L expression, whereas it upregulated IL-7Rα expression on CD4 T cells with persistent serum IL-7 concentrations. CONCLUSIONS: Leflunomide treatment suppressed cytokine release from circulating immune cells. Inhibition of T-helper 1 cell cytokine production was related to clinical efficacy. This suggests that selective T-cell targeting might be a relevant therapeutic strategy in primary SS, possibly enhancing clinical efficacy and safety.

Numbers of CD25+Foxp3+ T cells that lack the IL-7 receptor are increased intra-articularly and have impaired suppressive function in RA patients.

OBJECTIVES: To investigate numbers and function of CD25(+) regulatory T cells (Tregs) that lack IL-7 receptor (CD127) expression in RA. METHODS: Numbers of CD4 T cells expressing either CD25 or CD127, and those co-expressing or lacking both CD25 and CD127 were assessed in peripheral blood (PB) of RA patients and healthy controls, and in paired samples of SF and PB from RA patients. All T-cell subsets were analysed for FoxP3 expression. The anergic state and the capacity to suppress CD127(+) proliferating responder T cells were determined. RESULTS: Numbers of CD127(-) T cells and CD25(+) Tregs in PB of RA patients were not different from controls but significantly increased in SF compared with PB. CD25(+) and CD127(-) T cells showed comparable FoxP3 expression. CD127(+) T cells hardly expressed FoxP3. PB CD25(+)CD127(-) T cells identified a subset that consisted for 75% of FoxP3(+) cells. SF CD25(+)CD127(-) T-cell number was increased; however, in SF fewer of these cells were FoxP3(+). CD25(+)CD127(-) T cells were anergic, and in controls potent suppressors of CD127(+) proliferating T cells, but in RA patients these cells showed impaired suppression. In line with this, IL-7 had an increased capacity to activate total CD4 T cells from SF as compared with PB despite increased numbers of CD25(+)CD127(-) in SF. CONCLUSIONS: These data demonstrate improved identification of FoxP3(+) T cells in RA patients by the absence of CD127 in addition to CD25 expression. Increased numbers of CD25(+)CD127(-) T cells are found in inflamed RA joints, but they have an impaired suppressive function, which could contribute to the persistent arthritis in these patients.

Tumor Drug Distribution after Local Drug Delivery by Hyperthermia, In Vivo.

Tumor drug distribution and concentration are important factors for effective tumor treatment. A promising method to enhance the distribution and the concentration of the drug in the tumor is to encapsulate the drug in a temperature sensitive liposome. The aim of this study was to investigate the tumor drug distribution after treatment with various injected doses of different liposomal formulations of doxorubicin, ThermoDox (temperature sensitive liposomes) and DOXIL (non-temperature sensitive liposomes), and free doxorubicin at macroscopic and microscopic levels. Only ThermoDox treatment was combined with hyperthermia. Experiments were performed in mice bearing a human fibrosarcoma. At low and intermediate doses, the largest growth delay was obtained with ThermoDox, and at the largest dose, the largest growth delay was obtained with DOXIL. On histology, tumor areas with increased doxorubicin concentration correlated with decreased cell proliferation, and substantial variations in doxorubicin heterogeneity were observed. ThermoDox treatment resulted in higher tissue drug levels than DOXIL and free doxorubicin for the same dose. A relation with the distance to the vasculature was shown, but vessel perfusion was not always sufficient to determine doxorubicin delivery. Our results indicate that tumor drug distribution is an important factor for effective tumor treatment and that its dependence on delivery formulation merits further systemic investigation.

MicroRNA-130a Contributes to Type-2 Classical DC-activation in Sjögren's Syndrome by Targeting Mitogen- and Stress-Activated Protein Kinase-1.

Objectives: Considering the critical role of microRNAs (miRNAs) in regulation of cell activation, we investigated their role in circulating type-2 conventional dendritic cells (cDC2s) of patients with primary Sjögren's syndrome (pSS) compared to healthy controls (HC). Methods: CD1c-expressing cDC2s were isolated from peripheral blood. A discovery cohort (15 pSS, 6 HC) was used to screen the expression of 758 miRNAs and a replication cohort (15 pSS, 11 HC) was used to confirm differential expression of 18 identified targets. Novel targets for two replicated miRNAs were identified by SILAC in HEK-293T cells and validated in primary cDC2s. Differences in cytokine production between pSS and HC cDC2s were evaluated by intracellular flow-cytometry. cDC2s were cultured in the presence of MSK1-inhibitors to investigate their effect on cytokine production. Results: Expression of miR-130a and miR-708 was significantly decreased in cDC2s from pSS patients compared to HC in both cohorts, and both miRNAs were downregulated upon stimulation via endosomal TLRs. Upstream mediator of cytokine production MSK1 was identified as a novel target of miR-130a and overexpression of miR-130a reduced MSK1 expression in cDC2s. pSS cDC2s showed higher MSK1 expression and an increased fraction of IL-12 and TNF-α-producing cells. MSK1-inhibition reduced cDC2 activation and production of IL-12, TNF-α, and IL-6. Conclusions: The decreased expression of miR-130a and miR-708 in pSS cDC2s seems to reflect cell activation. miR-130a targets MSK1, which regulates pro-inflammatory cytokine production, and we provide proof-of-concept for MSK1-inhibition as a therapeutic avenue to impede cDC2 activity in pSS.

Synovial T cell hyporesponsiveness to myeloid dendritic cells is reversed by preventing PD-1/PD-L1 interactions.

INTRODUCTION: The aim of this study was to investigate PD-1/PD-L1 involvement in the hyporesponsiveness of rheumatoid arthritis (RA) synovial fluid (SF) CD4 T cells upon stimulation by thymic stromal lymphopoietin (TSLP)-primed CD1c myeloid dendritic cells (mDCs). METHODS: Expression of PD-1 on naïve (Tn), central memory (Tcm) and effector memory (Tem) CD4 T cell subsets was assessed by flow cytometry. PD-L1 expression and its regulation upon TSLP stimulation of mDCs from peripheral blood (PB) and SF of RA patients were investigated by quantitative RT-PCR and flow cytometry. The involvement of PD-1/PD-L1 interactions in SF T cell hyporesponsiveness upon (TSLP-primed) mDC activation was determined by cell culture in the presence of PD-1 blocking antibodies, with or without interleukin 7 (IL-7) as a recognized suppressor of PD-1 expression. RESULTS: PD-1 expression was increased on CD4 T cells derived from SF compared with PB of RA patients. TSLP increased PD-L1 mRNA expression in both PB and SF mDCs. PD-L1 protein expression was increased on SF mDCs compared with PB mDCs and was associated with T cell hyporesponsiveness. Blockade of PD-1, as well as IL-7 stimulation, during cocultures of memory T cells and (TSLP-primed) mDCs from RA patients significantly recovered T cell proliferation. CONCLUSION: SF T cell hyporesponsiveness upon (TSLP-primed) mDC stimulation in RA joints is partially dependent on PD-1/PD-L1 interactions, as PD-1 and PD-L1 are both highly expressed on SF T cells and mDCs, respectively, and inhibiting PD-1 availability restores T cell proliferation. The potential of IL-7 to robustly reverse this hyporesponsiveness suggests that such proinflammatory cytokines in RA joints strongly contribute to memory T cell activation.

Thymic stromal lymphopoietin, a novel proinflammatory mediator in rheumatoid arthritis that potently activates CD1c+ myeloid dendritic cells to attract and stimulate T cells.

OBJECTIVE: To determine the levels of thymic stromal lymphopoietin (TSLP) and the numbers of TSLP receptor (TSLPR)-expressing CD1c+ (blood dendritic cell antigen 1-positive) myeloid dendritic cells (MDCs) in the joints as compared with the peripheral blood (PB) of patients with rheumatoid arthritis (RA), as well as to determine the capacity of TSLP to induce MDC-dependent T cell activation. METHODS: TSLP levels were measured in synovial fluid (SF) samples from patients with RA and those with osteoarthritis (OA). MDC numbers in PB and SF samples from RA patients and TSLPR expression on these cells were assessed by fluorescence-activated cell sorter analysis. PB and SF MDCs from RA patients were stimulated with TSLP, and cytokine production was measured by multiplex immunoassay. TSLP-primed MDCs were cocultured with autologous CD4+ T cells in the absence of additional stimuli, and subsequently, cell proliferation and cytokine production were measured. RESULTS: TSLP levels were significantly increased in SF samples from RA versus OA patients. The numbers of TSLPR-expressing MDCs in the SF of RA patients were significantly increased as compared to those in the PB, and SF MDCs displayed increased levels of TSLPR. TSLP selectively stimulated the production of thymus and activation-regulated chemokine and macrophage inflammatory protein 1α by CD1c+ MDCs. TSLP-primed MDCs from PB and SF potently stimulated the proliferation of autologous CD4+ T cells as compared to unstimulated MDCs. Enhanced proliferation was associated with increased production of interferon-γ, interleukin-17 (IL-17), and IL-4. CONCLUSION: These data support an inflammatory mechanism by which increased intraarticular TSLP in RA potently activates TSLPR-expressing CD1c+ MDCs in the joints to secrete chemokines, causing chemotaxis and subsequent activation of CD4+ T cells. In addition to the demonstrated inflammatory potential of TSLP in experimental arthritis, this suggests that TSLP and TSLPR-expressing MDCs could both play a pivotal role in the immunopathology of RA.

Interleukin-7 and Toll-like receptor 7 induce synergistic B cell and T cell activation.

OBJECTIVES: To investigate the potential synergy of IL-7-driven T cell-dependent and TLR7-mediated B cell activation and to assess the additive effects of monocyte/macrophages in this respect. METHODS: Isolated CD19 B cells and CD4 T cells from healthy donors were co-cultured with TLR7 agonist (TLR7A, Gardiquimod), IL-7, or their combination with or without CD14 monocytes/macrophages (T/B/mono; 1 : 1 : 0,1). Proliferation was measured using 3H-thymidine incorporation and Ki67 expression. Activation marker (CD19, HLA-DR, CD25) expression was measured by FACS analysis. Immunoglobulins were measured by ELISA and release of cytokines was measured by Luminex assay. RESULTS: TLR7-induced B cell activation was not associated with T cell activation. IL-7-induced T cell activation alone and together with TLR7A synergistically increased numbers of both proliferating (Ki67+) B cells and T cells, which was further increased in the presence of monocytes/macrophages. This was associated by up regulation of activation markers on B cells and T cells. Additive or synergistic induction of production of immunoglobulins by TLR7 and IL-7 was associated by synergistic induction of T cell cytokines (IFNγ, IL-17A, IL-22), which was only evident in the presence of monocytes/macrophages. CONCLUSIONS: IL-7-induced CD4 T cell activation and TLR7-induced B cell activation synergistically induce T helper cell cytokine and B cell immunoglobulin production, which is critically dependent on monocytes/macrophages. Our results indicate that previously described increased expression of IL-7 and TLR7 together with increased numbers of macrophages at sites of inflammation in autoimmune diseases like RA and pSS significantly contributes to enhanced lymphocyte activation.

Intra-articular CD1c-expressing myeloid dendritic cells from rheumatoid arthritis patients express a unique set of T cell-attracting chemokines and spontaneously induce Th1, Th17 and Th2 cell activity.

INTRODUCTION: Myeloid dendritic cells (mDCs) are potent T cell-activating antigen-presenting cells that have been suggested to play a crucial role in the regulation of immune responses in many disease states, including rheumatoid arthritis (RA). Despite this, studies that have reported on the capacity of naturally occurring circulating mDCs to regulate T cell activation in RA are still lacking. This study aimed to evaluate the phenotypic and functional properties of naturally occurring CD1c (BDCA-1)+ mDCs from synovial fluid (SF) compared to those from peripheral blood (PB) of RA patients. METHODS: CD1c+ mDC numbers and expression of costimulatory molecules were assessed by fluorescence-activated cell sorting (FACS) analysis in SF and PB from RA patients. Ex vivo secretion of 45 inflammatory mediators by mDCs from SF and PB of RA patients was determined by multiplex immunoassay. The capacity of mDCs from SF to activate autologous CD4+ T cells was measured. RESULTS: CD1c+ mDC numbers were significantly increased in SF versus PB of RA patients (mean 4.7% vs. 0.6%). mDCs from SF showed increased expression of antigen-presenting (human leukocyte antigen (HLA) class II, CD1c) and costimulatory molecules (CD80, CD86 and CD40). Numerous cytokines were equally abundantly produced by mDCs from both PB and SF (including IL-12, IL-23, IL-13, IL-21). SF mDCs secreted higher levels of interferon γ-inducible protein-10 (IP-10), monokine induced by interferon γ (MIG) and, thymus and activation-regulated chemokine (TARC), but lower macrophage-derived chemokine (MDC) levels compared to mDCs from PB. mDCs from SF displayed a strongly increased capacity to induce proliferation of CD4+ T cells associated with a strongly augmented IFNγ, IL-17, and IL-4 production. CONCLUSIONS: This study suggests that increased numbers of CD1c+ mDCs in SF are involved in the inflammatory cascade intra-articularly by the secretion of specific T cell-attracting chemokines and the activation of self-reactive T cells.