Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Facilitate Tubular Epithelial Cell Dedifferentiation and Growth via Hepatocyte Growth Factor Induction

During acute kidney injury (AKI), tubular cell dedifferentiation initiates cell regeneration; hepatocyte growth factor (HGF) is involved in modulating cell dedifferentiation. Mesenchymal stem cell (MSC)-derived microvesicles (MVs) deliver RNA into injured tubular cells and alter their gene expression, thus regenerating these cells. We boldly speculated that MVs might induce HGF synthesis via RNA transfer, thereby facilitating tubular cell dedifferentiation and regeneration. In a rat model of unilateral AKI, the administration of MVs promoted kidney recovery. One of the mechanisms of action is the acceleration of tubular cell dedifferentiation and growth. Both in vivo and in vitro, rat HGF expression in damaged rat tubular cells was greatly enhanced by MV treatment. In addition, human HGF mRNA present in MVs was delivered into rat tubular cells and translated into the HGF protein as another mechanism of HGF induction. RNase treatment abrogated all MV effects. In the in vitro experimental setting, the conditioned medium of MV-treated injured tubular cells, which contains a higher concentration of HGF, strongly stimulated cell dedifferentiation and growth, as well as Erk1/2 signaling activation. Intriguingly, these effects were completely abrogated by either c-Met inhibitor or MEK inhibitor, suggesting that HGF induction is a crucial contributor to the acceleration of cell dedifferentiation and growth. All these findings indicate that MV-induced HGF synthesis in damaged tubular cells via RNA transfer facilitates cell dedifferentiation and growth, which are important regenerative mechanisms.     

Heightened pro-inflammatory effect of preeclamptic placental microvesicles on peripheral blood immune cells in humans

Normal pregnancy is associated with the presence of circulating placental microvesicles (MVs). Increased MV shedding and altered immune activation are seen in patients with preeclampsia, suggesting that placental MVs may play a role in the pathophysiology of this disease. Therefore, the aim of this study was to investigate the activation of peripheral blood mononuclear cells (PBMCs) by MVs shed by first-trimester, normal term, and preeclamptic term placenta. First-trimester and preeclamptic term, but not normal term, placental-derived MVs activated PBMCs, as evidenced by elevated IL1B. Significant changes were also seen with several other cytokines and chemokines, and in general when compared to normal term MVs, preeclamptic MVs induced a greater pro-inflammatory response in PBMCs. Pretreatment of PBMCs with first-trimester or normal term placental MVs resulted in a dampened IL1B response to a subsequent lipopolysaccharide (LPS) challenge. In contrast, treatment of PBMCs with preeclamptic term placental MVs exacerbated the LPS response. This was also the case for several other cytokines and chemokines. These studies suggest that placental MVs can modulate basal peripheral immune cell activation and responsiveness to LPS during normal pregnancy, and that in preeclampsia this effect is exacerbated.     

The role of microvesicles in tissue repair

Microvesicles (MVs) are released by almost all cells in resting and activated conditions. First described several years ago, it is only recently that their mechanisms of action are being elucidated, and their potential role in health and disease is drawing increasing attention. The main function of MVs is signaling through specific interactions with target cells and the transferring of gene products. Gaining further insights into the molecular specificity of MVs has allowed identification of the cellular source and may provide new diagnostic tools in the future. Indeed, an increasing body of evidence indicates that MVs are capable of mediating tissue repair in models of acute kidney and liver injury. In this review, we will discuss the mechanisms through which MVs from stem cells may act on target cells and may modify the response to injury. Furthermore, MVs from inflammatory cells are suspected to be involved in various diseases, such as cardiovascular and renal diseases, pathological pregnancy, tumors and sepsis. MVs are no doubt also involved in modulating immunity, and future studies will clarify their functional role in negatively modulating the cell response. Their role in physiological and pathological processes is increasingly appreciated. Depending on the cell source and the condition, MVs may be either beneficial or detrimental to the host. The recognition of their pathogenetic role may suggest new approaches to future therapies.     

The role of microvesicles in tissue repair

Microvesicles (MVs) are released by almost all cells in resting and activated conditions. First described several years ago, it is only recently that their mechanisms of action are being elucidated, and their potential role in health and disease is drawing increasing attention. The main function of MVs is signaling through specific interactions with target cells and the transferring of gene products. Gaining further insights into the molecular specificity of MVs has allowed identification of the cellular source and may provide new diagnostic tools in the future. Indeed, an increasing body of evidence indicates that MVs are capable of mediating tissue repair in models of acute kidney and liver injury. In this review, we will discuss the mechanisms through which MVs from stem cells may act on target cells and may modify the response to injury. Furthermore, MVs from inflammatory cells are suspected to be involved in various diseases, such as cardiovascular and renal diseases, pathological pregnancy, tumors and sepsis. MVs are no doubt also involved in modulating immunity, and future studies will clarify their functional role in negatively modulating the cell response. Their role in physiological and pathological processes is increasingly appreciated. Depending on the cell source and the condition, MVs may be either beneficial or detrimental to the host. The recognition of their pathogenetic role may suggest new approaches to future therapies.     

dsRNA induces apoptosis through an atypical death complex associating TLR3 to caspase-8

Toll-like receptor 3 (TLR3) is a pattern-recognition receptor known to initiate an innate immune response when stimulated by double-stranded RNA (dsRNA). Components of TLR3 signaling, including TIR domain-containing adapter inducing IFN-α (TRIF), have been demonstrated to contribute to dsRNA-induced cell death through caspase-8 and receptor interacting protein (RIP)1 in various human cancer cells. We provide here a detailed analysis of the caspase-8 activating machinery triggered in response to Poly(I:C) dsRNA. Engagement of TLR3 by dsRNA in both type I and type II lung cancer cells induces the formation of an atypical caspase-8-containing complex that is devoid of classical death receptors of the TNFR superfamily, but instead is physically associated to TLR3. The recruitment of caspase-8 to TLR3 requires RIP1, and is negatively modulated by cellular inhibitor of apoptosis protein (cIAP)2-TNF receptor-associated factor (TRAF)2-TNFR-associated death domain (TRADD) ubiquitin ligase complex, which regulates RIP1 ubiquitination. Intriguingly, unlike Fas- or TRAILR-dependent death signaling, caspase-8 recruitment and activation within the TLR3 death-signaling complex appears not to be stringently dependent on Fas-associated with death domain (FADD). Our findings uncover a novel aspect of the molecular mechanisms involved during apoptosis induced by the innate immune receptor TLR3 in cancer cells.     

Bruton's tyrosine kinase mediates NF-kappa B activation and B cell survival by B cell-activating factor receptor of the TNF-R family

Loss of Bruton's tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-kappaB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-kappaB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-kappaB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IkappaB kinase gamma-containing complexes and defective IkappaBalpha degradation. In addition, Btk-deficient B cells produce reduced levels of NF-kappaB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-kappaB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-kappaB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-kappaB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-kappaB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.     

Niche origin of mesenchymal stem cells derived microvesicles determines opposing effects on NSCLC: Primary versus metastatic

Novel therapeutic approaches that address the malignant cells in their stroma microenvironment are urgently needed in lung cancer. The stroma resident mesenchymal stem cells (MSCs) interact with cancer cells in diverse ways including microvesicles (MVs) that transfer proteins and RNA species thereby modulating recipient cells' phenotype. Previously, we have demonstrated that MSCs' secretome from the primary non-small cell lung cancer (NSCLC) niche (lung) and metastatic niche (bone marrow (BM)) demonstrate opposite effects on NSCLC cells in a translation initiation (TI) dependent manner. Here, we examined the effect of MVs secreted from BM-MSCs' or lung-MSCs (healthy, NSCLC) to NSCLC phenotype. Briefly, NSCLC cell lines treated with Lung or BM-MSCs' MVs were assayed for viability (WST-1), cell count/death (trypan), migration (scratch), TI status and MAPKs activation (immunoblotting). Corresponding to previous published trends, Lung-MSCs' MVs promoted NSCLC cells' assayed traits whereas, BM-MSCs' MVs suppressed them. Activation of MAPKs and autophagy was registered in lung-MSCs MVs treated NSCLC cell lines only. Furthermore, lung-MSCs' MVs' treated NSCLC cells demonstrated an early (5 min) activation of MAPKs and TI factors (peIF4E/peIF4GI) not evident in BM-MSCs MVs treated cells. These observations depict a role for MSCs'-MVs in NSCLC phenotype design and display distinct differences between the primary and metastatic niches that correspond to disease progression.In conclusion, the systemic nature of MVs marks them as attractive therapeutic markers/targets and we propose that identification of specific cargoes/signals that differentiate between MSCs MVs of primary and metastatic niches may introduce fresh therapeutic approaches.     

Paramyxovirus Activation and Inhibition of Innate Immune Responses

Paramyxoviruses represent a remarkably diverse family of enveloped nonsegmented negative-strand RNA viruses, some of which are the most ubiquitous disease-causing viruses of humans and animals. This review focuses on paramyxovirus activation of innate immune pathways, the mechanisms by which these RNA viruses counteract these pathways, and the innate response to paramyxovirus infection of dendritic cells (DC). Paramyxoviruses are potent activators of extracellular complement pathways, a first line of defense that viruses must face during natural infections. We discuss mechanisms by which these viruses activate and combat complement to delay neutralization. Once cells are infected, virus replication drives type I interferon (IFN) synthesis that has the potential to induce a large number of antiviral genes. Here we describe four approaches by which paramyxoviruses limit IFN induction: by limiting synthesis of IFN-inducing aberrant viral RNAs, through targeted inhibition of RNA sensors, by providing viral decoy substrates for cellular kinase complexes, and through direct blocking of the IFN promoter. In addition, paramyxoviruses have evolved diverse mechanisms to disrupt IFN signaling pathways. We describe three general mechanisms, including targeted proteolysis of signaling factors, sequestering cellular factors, and upregulation of cellular inhibitors. DC are exceptional cells with the capacity to generate adaptive immunity through the coupling of innate immune signals and T cell activation. We discuss the importance of innate responses in DC following paramyxovirus infection and their consequences for the ability to mount and maintain antiviral T cells.     

The extrinsic RNA-sensing pathway for adjuvant immunotherapy of cancer

Infection with RNA viruses presents a typical pattern of virus products, double-stranded RNA (dsRNA), and induces the maturation of antigen-presenting dendritic cell (mDC). There are several dsRNA sensors that are differentially distributed on the cell membrane and in the cytoplasm and are variably expressed depending on the cell type. Among these sensors, TLR3 links to the adaptor TICAM-1 (TRIF), which is characterized by its unique multipronged signaling cascades for cytokine/chemokine production, apoptosis and autophagy in both immune and tumor cells. In the context of mDC maturation, various cellular events are further induced in response to dsRNA; these include cross-priming followed by CD8+ CTL induction, NK activation and proliferation of CD4+ T cells including Th1, Th2, Treg and Th17 cells. In this review, we focus on the potential role of dsRNA in modulating the inflammatory milieu around mDCs and tumor-associated antigens to drive specific cellular effectors against the tumor.     

Oligonucleotide motifs that disappear during the evolution of influenza virus in humans increase alpha interferon secretion by plasmacytoid dendritic cells

CpG motifs in an A/U context have been preferentially eliminated from classical H1N1 influenza virus genomes during virus evolution in humans. The hypothesis of the current work is that CpG motifs in a uracil context represent sequence patterns with the capacity to induce an immune response, and the avoidance of this immunostimulatory signal is the reason for the observed preferential decline. To analyze the immunogenicity of these domains, we used plasmacytoid dendritic cells (pDCs). pDCs express pattern recognition receptors, including Toll-like receptor 7 (TLR7), which recognizes guanosine- and uridine-rich viral single-stranded RNA (ssRNA), including influenza virus ssRNA. The signaling through TLR7 results in the induction of inflammatory cytokines and type I interferon (IFN-I), an essential process for the induction of specific adaptive immune responses and for mounting a robust antiviral response mediated by IFN-α. Secretion of IFN-α is also linked to the activation of other immune cells, potentially amplifying the effect of an initial IFN-α secretion. We therefore also examined the role of IFN-α-driven activation of NK cells as another source of selective pressure on the viral genome. We found direct evidence that CpG RNA motifs in a U-rich context control pDC activation and IFN-α-driven activation of NK cells, likely through TLR7. These data provide a potential explanation for the loss of CpG motifs from avian influenza viruses as they adapt to mammalian hosts. The selective decrease of CpG motifs surrounded by U/A may be a viral strategy to avoid immune recognition, a strategy likely shared by highly expressed human immune genes.     

Microvesicles Derived from Adult Human Bone Marrow and Tissue Specific Mesenchymal Stem Cells Shuttle Selected Pattern of miRNAs

Background

Cell-derived microvesicles (MVs) have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs) and liver resident stem cells (HLSCs) were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs).

Methodology/Principal Findings

MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation.

Conclusions

This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem cells may, at least in part, depend on MV-shuttled miRNAs. Data generated from this study, stimulate further functional investigations on the predicted target genes and pathways involved in the biological effect of human adult stem cells.     

Single-cell RNA sequencing reveals the sustained immune cell dysfunction in the pathogenesis of sepsis secondary to bacterial pneumonia

Sepsis is a leading cause of mortality in intensive care unit worldwide, it's accompanied by immune cell dysfunction induced by multiple factors. However, little is known about the specific alterations in immune cells in the dynamic pathogenesis of sepsis secondary to bacterial pneumonia. Here, we used single cell RNA sequencing (scRNA-seq) to profile peripheral blood mononuclear cells (PBMCs) in a healthy control and two patients with sepsis secondary to bacterial pneumonia, including acute, stable and recovery stage. We analyzed the quantity and function of immune cells. During disease course, interferon gamma response was upregulated; T/NK cell subtypes presented activation and exhaustion properties, which might be driven by monocytes through IL-1β signaling pathways; The proportion of plasma cells was increased, which might be driven by NK cells through IFN signaling pathways; Additionally, interferon gamma response was upregulated to a greater degree in sepsis secondary to pneumonia induced by SARS-COV-2 compared with that induced by influenza virus and bacteria.     

IL-4-dependent Th2 collateral priming to inhaled antigens independent of Toll-like receptor 4 and myeloid differentiation factor 88

Allergic asthma is an inflammatory lung disease thought to be initiated and directed by type 2 helper T cells responding to environmental Ags. The mechanisms by which allergens induce Th2-adaptive immune responses are not well understood, although it is now clear that innate immune signals are required to promote DC activation and Th2 sensitization to inhaled proteins. However, the effect of ongoing Th2 inflammation, as seen in chronic asthma, on naive lymphocyte activation has not been explored. It has been noted that patients with atopic disorders demonstrate an increased risk of developing sensitivities to new allergens. This suggests that signals from an adaptive immune response may facilitate sensitization to new Ags. We used a Th2-adoptive transfer murine model of asthma to identify a novel mechanism, termed "collateral priming," in which naive CD4(+) T cells are activated by adaptive rather than innate immune signals. Th2 priming to newly encountered Ags was dependent on the production of IL-4 by the transferred Th2 population but was independent of Toll-like receptor 4 signaling and the myeloid differentiation factor 88 Toll-like receptor signaling pathway. These results identify a novel mechanism of T cell priming in which an Ag-specific adaptive immune response initiates distinct Ag-specific T cell responses in the absence of classical innate immune system triggering signals.     

Circulating membrane-derived microvesicles in redox biology

Microparticles or microvesicles (MVs) are subcellular membrane blebs shed from all cells in response to various stimuli. MVs carry a battery of signaling molecules, many of them related to redox-regulated processes. The role of MVs, either as a cause or as a result of cellular redox signaling, has been increasingly recognized over the past decade. This is in part due to advances in flow cytometry and its detection of MVs. Notably, recent studies have shown that circulating MVs from platelets and endothelial cells drive reactive species-dependent angiogenesis; circulating MVs in cancer alter the microenvironment and enhance invasion through horizontal transfer of mutated proteins and nucleic acids and harbor redox-regulated matrix metalloproteinases and procoagulative surface molecules; and circulating MVs from red blood cells and other cells modulate cell–cell interactions through scavenging or production of nitric oxide and other free radicals. Although our recognition of MVs in redox-related processes is growing, especially in the vascular biology field, much remains unknown regarding the various biologic and pathologic functions of MVs. Like reactive oxygen and nitrogen species, MVs were originally believed to have a solely pathological role in biology. And like our understanding of reactive species, it is now clear that MVs also play an important role in normal growth, development, and homeostasis. We are just beginning to understand how MVs are involved in various biological processes—developmental, homeostatic, and pathological—and the role of MVs in redox signaling is a rich and exciting area of investigation.     

Control of Drosophila Blood Cell Activation via Toll Signaling in the Fat Body

The Toll signaling pathway, first discovered in Drosophila, has a well-established role in immune responses in insects as well as in mammals. In Drosophila, the Toll-dependent induction of antimicrobial peptide production has been intensely studied as a model for innate immune responses in general. Besides this humoral immune response, Toll signaling is also known to activate blood cells in a reaction that is similar to the cellular immune response to parasite infections, but the mechanisms of this response are poorly understood. Here we have studied this response in detail, and found that Toll signaling in several different tissues can activate a cellular immune defense, and that this response does not require Toll signaling in the blood cells themselves. Like in the humoral immune response, we show that Toll signaling in the fat body (analogous to the liver in vertebrates) is of major importance in the Toll-dependent activation of blood cells. However, this Toll-dependent mechanism of blood cell activation contributes very little to the immune response against the parasitoid wasp, Leptopilina boulardi, probably because the wasp is able to suppress Toll induction. Other redundant pathways may be more important in the defense against this pathogen.     

Microvesicles as promising biological tools for diagnosis and therapy

ABSTRACT

Introduction: Shed by most cells, in response to a myriad of stimuli, extracellular vesicles (EVs) carry proteins, lipids, and various nucleic acids. EVs encompass diverse subpopulations differing for biogenesis and content. Among these, microvesicles (MVs) derived from plasma membrane, are key regulators of physiopathological cellular processes including cancer, inflammation and infection. This review is unique in that it focuses specifically on the MVs as a mediator of information transfer. In fact, few proteomic studies have rigorously distinguished MVs from exosomes.

Areas covered: Aim of this review is to discuss the proteomic analyses of the MVs. Many studies have examined mixed populations containing both exosomes and MVs. We discuss MVs’ role in cell-specific interactions. We also show their emerging roles in therapy and diagnosis.

Expert commentary: We see MVs as therapeutic tools for potential use in precision medicine. They may also have potential for allowing the identification of new biomarkers. MVs represent an invaluable tool for studying the cell of origin, which they closely represent, but it is critical to build a repository with data from MVs to deepen our understanding of their molecular repertoire and biological functions.     

Proteins Regulating Microvesicle Biogenesis and Multidrug Resistance in Cancer

Microvesicles (MV) are emerging as important mediators of intercellular communication. While MVs are important signaling vectors for many physiological processes, they are also implicated in cancer pathology and progression. Cellular activation is perhaps the most widely reported initiator of MV biogenesis, however, the precise mechanism remains undefined. Uncovering the proteins involved in regulating MV biogenesis is of interest given their role in the dissemination of deleterious cancer traits. MVs shed from drug‐resistant cancer cells transfer multidrug resistance (MDR) proteins to drug‐sensitive cells and confer the MDR phenotype in a matter of hours. MDR is attributed to the overexpression of ABC transporters, primarily P‐glycoprotein and MRP1. Their expression and functionality is dependent on a number of proteins. In particular, FERM domain proteins have been implicated in supporting the functionality of efflux transporters in drug‐resistant cells and in recipient cells during intercellular transfer by vesicles. Herein, the most recent research on the proteins involved in MV biogenesis and in the dissemination of MV‐mediated MDR are discussed. Attention is drawn to unanswered questions in the literature that may prove to be of benefit in ongoing efforts to improve clinical response to chemotherapy and circumventing MDR.     

Microvesicles Derived from Human Wharton's Jelly Mesenchymal Stem Cells Promote Human Renal Cancer Cell Growth and Aggressiveness through Induction of Hepatocyte Growth Factor

In our previous study, microvesicles (MVs) released from human Wharton''s jelly mesenchymal stem cells (hWJ-MSCs) retard the growth of bladder cancer cells. We would like to know if MVs have a similar effect on human renal cell carcinoma (RCC). By use of cell culture and the BALB/c nu/nu mice xeno-graft model, the influence of MVs upon the growth and aggressiveness of RCC (786-0) was assessed. Cell counting kit-8 (CCK-8) assay, incidence of tumor, tumor size, Ki-67 or TUNEL staining was used to evaluate tumor cell growth in vitro or in vivo. Flow cytometry assay (in vitro) or examination of cyclin D1 expression (in vivo) was carried out to determine the alteration of cell cycle. The aggressiveness was analyzed by Wound Healing Assay (in vitro) or MMP-2 and MMP-9 expression (in vivo). AKT/p-AKT, ERK1/2/p-ERK1/2 or HGF/c-MET expression was detected by real-time PCR or western blot. Our data demonstrated that MVs promote the growth and aggressiveness of RCC both in vitro and in vivo. In addition, MVs facilitated the progression of cell cycle from G_0/1 to S. HGF expression in RCC was greatly induced by MVs, associated with activation of AKT and ERK1/2 signaling pathways. RNase pre-treatment abrogated all effects of MVs. In summary, induction of HGF synthesis via RNA transferred by MVs activating AKT and ERK1/2 signaling is one of crucial contributors to the pro-tumor effect.     

Membrane vesicles are immunogenic facsimiles of Salmonella typhimurium that potently activate dendritic cells, prime B and T cell responses, and stimulate protective immunity in vivo

Gram-negative bacteria produce membrane vesicles (MVs) from their outer membrane during growth, although the mechanism for MV production and the advantage that MVs provide for bacterial survival in vivo remain unknown. MVs function as an alternate secretion pathway for Gram-negative bacteria; therefore, MV production in vivo may be one method by which bacteria interact with eukaryotic cells. However, the interactions between MVs and cells of the innate and adaptive immune systems have not been studied extensively. In this study, we demonstrate that MVs from Salmonella typhimurium potently stimulated professional APCs in vitro. Similar to levels induced by bacterial cells, MV-stimulated macrophages and dendritic cells displayed increased surface expression of MHC-II and CD86 and enhanced production of the proinflammatory mediators NO, TNF-alpha, and IL-12. MV-mediated dendritic cell stimulation occurred by TLR4-dependent and -independent signals, indicating the stimulatory properties of Salmonella MVs, which contain LPS, do not strictly rely on signaling through TLR4. In addition to their strong proinflammatory properties, MVs contained Ags recognized by Salmonella-specific B cells and CD4(+) T cells; MV-vaccinated mice generated Salmonella-specific Ig and CD4(+) T cell responses in vivo and were significantly protected from infectious challenge with live Salmonella. Our findings demonstrate that MVs possess important inflammatory properties as well as B and T cell Ags known to influence the development of Salmonella-specific immunity to infection in vivo. Our findings also reveal MVs are a functional nonviable complex vaccine for Salmonella by their ability to prime protective B and T cell responses in vivo.