Cell migration has two opposite faces: although necessary for physiological processes such as immune responses, it can also have detrimental effects by enabling metastatic cells to invade new organs. ...In vivo, migration occurs in complex environments and often requires a high cellular deformability, a property limited by the cell nucleus. Here we show that dendritic cells, the sentinels of the immune system, possess a mechanism to pass through micrometric constrictions. This mechanism is based on a rapid Arp2/3-dependent actin nucleation around the nucleus that disrupts the nuclear lamina, the main structure limiting nuclear deformability. The cells' requirement for Arp2/3 to pass through constrictions can be relieved when nuclear stiffness is decreased by suppressing lamin A/C expression. We propose a new role for Arp2/3 in three-dimensional cell migration, allowing fast-moving cells such as leukocytes to rapidly and efficiently migrate through narrow gaps, a process probably important for their function.
The NLRP3 inflammasome has been implicated in the pathogenesis of a wide variety of human diseases. A few compounds have been developed to inhibit NLRP3 inflammasome activation, but compounds ...directly and specifically targeting NLRP3 are still not available, so it is unclear whether NLRP3 itself can be targeted to prevent or treat diseases. Here we show that the compound CY-09 specifically blocks NLRP3 inflammasome activation. CY-09 directly binds to the ATP-binding motif of NLRP3 NACHT domain and inhibits NLRP3 ATPase activity, resulting in the suppression of NLRP3 inflammasome assembly and activation. Importantly, treatment with CY-09 shows remarkable therapeutic effects on mouse models of cryopyrin-associated autoinflammatory syndrome (CAPS) and type 2 diabetes. Furthermore, CY-09 is active ex vivo for monocytes from healthy individuals or synovial fluid cells from patients with gout. Thus, our results provide a selective and direct small-molecule inhibitor for NLRP3 and indicate that NLRP3 can be targeted in vivo to combat NLRP3-driven diseases.
Osteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. ...Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in communication among joint tissue cells.
Human synovial fibroblasts (SFB) and articular chondrocytes were obtained from normal knee joints. Exosomes isolated from conditioned medium of SFB were analyzed for size, numbers, markers and function. Normal articular chondrocytes were treated with exosomes from SFB, and Interleukin-1β (IL-1β) stimulated SFB. OA-related genes expression was quantified using real-time PCR. To analyze exosome effects on cartilage tissue, we performed glycosaminoglycan release assay. Angiogenic activity of these exosomes was tested in migration and tube formation assays. Cytokines and miRNAs in exosomes were analyzed by Bio-Plex multiplex assay and NanoString analysis.
Exosomes from IL-1β stimulated SFB significantly up-regulated MMP-13 and ADAMTS-5 expression in articular chondrocytes, and down-regulated COL2A1 and ACAN compared with SFB derived exosomes. Migration and tube formation activity were significantly higher in human umbilical vein endothelial cells (HUVECs) treated with the exosomes from IL-1β stimulated SFB, which also induced significantly more proteoglycan release from cartilage explants. Inflammatory cytokines, IL-6, MMP-3 and VEGF in exosomes were only detectable at low level. IL-1β, TNFα MMP-9 and MMP-13 were not detectable in exosomes. NanoString analysis showed that levels of 50 miRNAs were differentially expressed in exosomes from IL-1β stimulated SFB compared to non-stimulated SFB.
Exosomes from IL-1β stimulated SFB induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints.
STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following ...stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.
Abstract
The purpose of our study was to better understand the effects of mitochondrial-division inhibitor 1 (Mdivi-1) on mitochondrial fission, mitochondrial biogenesis, electron transport ...activities and cellular protection. In recent years, researchers have found excessive mitochondrial fragmentation and reduced fusion in a large number of diseases with mitochondrial dysfunction. Therefore, several groups have developed mitochondrial division inhibitors. Among these, Mdivi-1 was extensively studied and was found to reduce dynamin-related protein 1 (Drp1) levels and excessive mitochondrial fission, enhance mitochondrial fusion activity and protect cells. However, a recent study by Bordt et al. (1) questioned earlier findings of the beneficial, inhibiting effects of Mdivi-1. In the current study, we studied the protective effects of Mdivi-1 by studying the following: mRNA and protein levels of electron transport chain (ETC) genes; mitochondrial dynamics and biogenesis genes; enzymatic activities of ETC complexes I, II, III and IV; the mitochondrial network; mitochondrial size & number; Drp1 GTPase enzymatic activity and mitochondrial respiration (1) in N2a cells treated with Mdivi-1, (2) overexpressed with full-length Drp1 + Mdivi-1-treated N2a cells and (3) Drp1 RNA silenced+Mdivi-1-treated N2a cells. We found reduced levels of the fission genes Drp1 and Fis1 levels; increased levels of the fusion genes Mfn1, Mfn2 and Opa1; and the biogenesis genes PGC1α, nuclear respiration factor 1, nuclear respiratory factor 2 and transcription factor A, mitochondrial. Increased levels mRNA and protein levels were found in ETC genes of complexes I, II and IV genes. Immunoblotting data agreed with mRNA changes. Transmission electron microscopy analysis revealed reduced numbers of mitochondria and increased length of mitochondria (1) in N2a cells treated with Mdivi-1, (2) cells overexpressed with full-length Drp1 + Mdivi-1-treated N2a cells and (3) Drp1 RNA silenced+Mdivi-1-treated N2a cells. Immunofluorescence analysis revealed that mitochondrial network was increased. Increased levels of complex I, II and IV enzymatic activities were found in all three groups of cells treated with low concentration of Mdivi-1. Mitochondrial function was increased and GTPase Drp1 activity was decreased in all three groups of N2a cells. These observations strongly suggest that Mdivi-1 is a Drp1 inhibitor and directly reduces mitochondrial fragmentation and further, Mdivi-1 is a promising molecule to treat human diseases with ETC complexes, I, II and IV.
The cell-to-cell transmission of viral resistance is a potential mechanism for amplifying the interferon-induced antiviral response. In this study, we report that interferon-α (IFN-α) induced the ...transfer of resistance to hepatitis B virus (HBV) from nonpermissive liver nonparenchymal cells (LNPCs) to permissive hepatocytes via exosomes. Exosomes from IFN-α-treated LNPCs were rich in molecules with antiviral activity. Moreover, exosomes from LNPCs were internalized by hepatocytes, which mediated the intercellular transfer of antiviral molecules. Finally, we found that exosomes also contributed to the antiviral response of IFN-α to mouse hepatitis virus A59 and adenovirus in mice. Thus, we propose an antiviral mechanism of IFN-α activity that involves the induction and intercellular transfer of antiviral molecules via exosomes.
Aerobic glycolysis regulates T cell function. However, whether and how primary cancer alters T cell glycolytic metabolism and affects tumor immunity in cancer patients remains a question. Here we ...found that ovarian cancers imposed glucose restriction on T cells and dampened their function via maintaining high expression of microRNAs miR-101 and miR-26a, which constrained expression of the methyltransferase EZH2. EZH2 activated the Notch pathway by suppressing Notch repressors Numb and Fbxw7 via trimethylation of histone H3 at Lys27 and, consequently, stimulated T cell polyfunctional cytokine expression and promoted their survival via Bcl-2 signaling. Moreover, small hairpin RNA-mediated knockdown of human EZH2 in T cells elicited poor antitumor immunity. EZH2(+)CD8(+) T cells were associated with improved survival in patients. Together, these data unveil a metabolic target and mechanism of cancer immune evasion.
The immune synapse is an exquisitely evolved means of communication between T cells and antigen-presenting cells (APCs) during antigen recognition. Recent evidence points to the transfer of RNA via ...exosomes as a novel mode of intercellular communication. Here we show that exosomes of T, B and dendritic immune cells contain microRNA (miRNA) repertoires that differ from those of their parent cells. We investigate whether miRNAs are exchanged during cognate immune interactions, and demonstrate the existence of antigen-driven unidirectional transfer of miRNAs from the T cell to the APC, mediated by the delivery of CD63+ exosomes on immune synapse formation. Inhibition of exosome production by targeting neutral sphingomyelinase-2 impairs transfer of miRNAs to APCs. Moreover, miRNAs transferred during immune synapsis are able to modulate gene expression in recipient cells. Thus, our results support a mechanism of cellular communication involving antigen-dependent, unidirectional intercellular transfer of miRNAs by exosomes during immune synapsis.
Staphylococcus aureus is both a prominent cause of nosocomial infections with significant morbidity and mortality and a commensal with nasal carriage in around 30% of the population. The rapid spread ...of multi‐resistant strains necessitates novel therapeutic strategies, a challenging task because the species S. aureus and the host response against it are highly variable. In a prospective study among 2023 surgical and non‐surgical patients, 12 patients developed S. aureus bacteremia. They were analysed in detail using a personalized approach. For each patient, the extracellular proteins of the infecting S. aureus strain were identified and the developing antibody response was assessed on 2‐D immunoblots. S. aureus carriers showed clear evidence of strain‐specific pre‐immunization. In all immune‐competent bacteremia patients, antibody binding increased strongly, in most cases already at diagnosis. In endogenous infections, the pattern of antibody binding was similar to the pre‐infection pattern. In exogenous infections, in contrast, the pre‐infection pattern was radically altered with the acquisition of new specificities. These were characteristic for individual patients. Nevertheless, a common signature of 11 conserved S. aureus proteins, recognized in at least half of the bacteremic patients, was identified. All patients mounted a dynamic antibody response to a subset of these proteins.
Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are complex chronic inflammatory conditions of the gastrointestinal tract that are driven by perturbed ...cytokine pathways. Anti-tumor necrosis factor-α (TNF) antibodies are mainstay therapies for IBD. However, up to 40% of patients are nonresponsive to anti-TNF agents, which makes the identification of alternative therapeutic targets a priority. Here we show that, relative to healthy controls, inflamed intestinal tissues from patients with IBD express high amounts of the cytokine oncostatin M (OSM) and its receptor (OSMR), which correlate closely with histopathological disease severity. The OSMR is expressed in nonhematopoietic, nonepithelial intestinal stromal cells, which respond to OSM by producing various proinflammatory molecules, including interleukin (IL)-6, the leukocyte adhesion factor ICAM1, and chemokines that attract neutrophils, monocytes, and T cells. In an animal model of anti-TNF-resistant intestinal inflammation, genetic deletion or pharmacological blockade of OSM significantly attenuates colitis. Furthermore, according to an analysis of more than 200 patients with IBD, including two cohorts from phase 3 clinical trials of infliximab and golimumab, high pretreatment expression of OSM is strongly associated with failure of anti-TNF therapy. OSM is thus a potential biomarker and therapeutic target for IBD, and has particular relevance for anti-TNF-resistant patients.
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