Characterization and biodistribution of under-employed gene therapy vector AAV7.

IMPORTANCE: The use of adeno-associated viruses (AAVs) as gene delivery vectors has vast potential for the treatment of many severe human diseases. Over one hundred naturally existing AAV capsid variants have been described and classified into phylogenetic clades based on their sequences. AAV8, AAV9, AAVrh.10, and other intensively studied capsids have been propelled into pre-clinical and clinical use, and more recently, marketed products; however, less-studied capsids may also have desirable properties (e.g., potency differences, tissue tropism, reduced immunogenicity, etc.) that have yet to be thoroughly described. These data will help build a broader structure-function knowledge base in the field, present capsid engineering opportunities, and enable the use of novel capsids with unique properties.

The presence of CpGs in AAV gene therapy vectors induces a plasmacytoid dendritic cell-like population very early after administration.

AAV-mediated gene transfer is a promising platform still plagued by potential host-derived, antagonistic immune responses to therapeutic components. CpG-mediated TLR9 stimulation activates innate immune cells and leads to cognate T cell activation and suppression of transgene expression. Here, we demonstrate that CpG depletion increased expression of an antibody transgene product by 2-3-fold as early as 24 h post-vector administration in mice. No significant differences were noted in anti-transgene product/ anti-AAV capsid antibody production or cytotoxic gene induction. Instead, CpG depletion significantly reduced the presence of a pDC-like myeloid cell population, which was able to directly bind the antibody transgene product via Fc-FcγR interactions. Thus, we extend the mechanisms of TLR9-mediated antagonism of transgene expression in AAV gene therapy to include the actions of a previously unreported pDC-like cell population.

Mesenchymal stem cells differentially modulate effector CD8+ T cell subsets and exacerbate experimental autoimmune encephalomyelitis.

Mesenchymal stem cells (MSC) have emerged as a promising candidate for inflammatory suppression and disease amelioration, especially of neuro-inflammatory diseases such as multiple sclerosis (MS). Auto-reactive CD4+ and CD8+ T cells acquire pathogenic IFNγ-producing- (Type I) and IL-17A-producing- (Type 17) effector phenotypes in MS and its animal model experimental autoimmune encephalomyelitis (EAE). Although MSC have been extensively demonstrated to suppress pathogenic effector CD4+ T cells and CD4+ T cell-mediated EAE, surprisingly few studies have addressed their modulation of effector CD8+ T cells represented in MS or their impact on CD8+ T cell-mediated EAE. We find that MSC differentially modulate CD8+ T cell development depending on effector T cell subtype. MSC drive activated low-IFNγ producers toward an enhanced high-IFNγ Tc1-like phenotype but strongly inhibit the production of IL-17A and Tc17 polarization in vitro. These observations are underscored by differential MSC modulation of T cell activation, proliferation, and signature transcription factor up-regulation. In addition, effector CD8+ T cells co-cultured with MSC exhibited increased production of IL-2, a molecule known to enhance IFNγ, yet suppress IL-17A, production. Based on these in vitro effects on CD8+ T cells, we next evaluated their impact on the severity of EAE. To better evaluate CD8+ T cells, we immunized mice with MOG37-50 , which is a CD8-targeted epitope. Our results revealed a worsening of disease, consistent with their in vitro stimulation of Tc1 cells. These findings highlight the emerging duality of MSC in immune modulation and provide implications for their future use in immune-related diseases.

KLF4 modulates expression of IL-6 in dendritic cells via both promoter activation and epigenetic modification.

The initiation and maintenance of the immune response require a coordinated regulation of signal transduction pathways. Identifying the mechanisms by which these pathways are controlled and modulated is a significant goal of immunology. In the present report, we show a novel role for the zinc finger transcription factor Kruppel-like factor 4 (KLF4) in the modulation of the inflammatory immune response via its regulation of IL-6. We analyzed the role of KLF4 in the production of IL-6 by dendritic cells. Our data indicate that KLF4 can act in a dual function manner. It acts as a transcription factor in that it can bind to and activate the IL-6 promoter at specific binding sites. KLF4 also has a role in the chromatin remodeling of the IL-6 promoter in that cells deficient in KLF4 exhibited a relative hypoacetylation. These results indicate a molecular role for KLF4 in modulating the intensity of the inflammatory response and help to explain its pleiotropic role in different settings.

Structural mechanics of the mosquito heart and its function in bidirectional hemolymph transport.

The insect circulatory system transports nutrients, signaling molecules, wastes and immune factors to all areas of the body. The primary organ driving circulation is the dorsal vessel, which consists of an abdominal heart and a thoracic aorta. Here, we present qualitative and quantitative data characterizing the heart of the mosquito, Anopheles gambiae. Visual observation showed that the heart of resting mosquitoes contracts at a rate of 1.37 Hz (82 beats per minute) and switches contraction direction, with 72% of contractions occurring in the anterograde direction (toward the head) and 28% of contractions occurring in the retrograde direction (toward the tip of the abdomen). The heart is tethered to the midline of the abdominal tergum by six complete and three incomplete pairs of alary muscles, and propels hemolymph at an average velocity of 8 mm s(-1) by sequentially contracting muscle fibers oriented in a helical twist with respect to the lumen of the vessel. Hemolymph enters the heart through six pairs of incurrent abdominal ostia and one pair of ostia located at the thoraco-abdominal junction that receive hemolymph from the abdominal hemocoel and thoracic venous channels, respectively. The vessel expels hemolymph through distal excurrent openings located at the anterior end of the aorta and the posterior end of the heart. In conclusion, this study presents a comprehensive revision and expansion of our knowledge of the mosquito heart and for the first time quantifies hemolymph flow in an insect while observing dorsal vessel contractions.

MYMD-1, a novel alkaloid compound, ameliorates the course of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease that remains in need of effective therapies. Plant-derived medicines have appealing properties for the treatment of autoimmune diseases. MYMD-1 is a synthetic plant alkaloid that has been shown to ameliorate the course of autoimmune thyroiditis. The goal of the present study was to determine whether MYMD-1 would produce similar beneficial effects in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin oligodendrocyte glycoprotein. MYMD-1 improved the course of EAE and suppressed activation of effector T cells without causing global immunosuppression or toxicity. These results suggest that MYMD-1 may be of interest for evaluating for the treatment of autoimmune diseases.

Frontline Science: Induction of experimental autoimmune encephalomyelitis mobilizes Th17-promoting myeloid derived suppressor cells to the lung.

Myeloid-derived suppressor cells (MDSCs) are a diverse group of cells that are recognized for their remarkable suppressive effects on pro-inflammatory T cells. The pleiotropic nature of these cells, however, has been demonstrated by their differential effects on immune responses in different settings. Our and others' work has demonstrated suppressive effects of these cells. We previously demonstrated that these cells were mobilized to the lungs during experimental autoimmune encephalomyelitis (EAE), which is a murine model of multiple sclerosis, and potently inhibited CD8+ T cell responses against influenza infection. Interestingly, they appeared to have a lesser effect on CD4+ T cells, and in fact, others have demonstrated that spleen-derived MDSCs could actually promote Th17 differentiation. We sought to determine the role of lung-derived MDSCs on EAE pathogenesis, as excursion through the lungs by pathologic CNS-Ag targeted T cells was shown to be critical for EAE induction. Our results indicate a robust accumulation of granulocytic MDSCs in the lungs of mice during EAE, which could promote Th17 polarization, and which coincided with the trafficking of autoimmune-targeted T cells through the lungs. These studies underscore the pleiotropic effect of MDSCs on T cells and their potential pro-inflammatory phenotypes in neuro-inflammatory disease. Understanding both the intrinsic multifunctional nature of these cells and the ability to influence organ-specific targets such as the CNS from remote organs such as lungs will help to elucidate both mechanisms of disease and possible new therapeutic approaches.

The Dynamics of the Gut Microbiome in Multiple Sclerosis in Relation to Disease.

Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease of unknown etiology, although genetic components and environmental triggers are thought to collude to commence pathogenesis. Numerous investigations are now demonstrating the role of the gut microbiota in neuroinflammation and how alterations in its content may be associated with MS disease. This article explores the studies using MS rodent models to determine the roles of gut bacteria in neuroinflammatory disease, evaluate the evidence linking gut bacterial dysbiosis and MS, and give insight into potential MS therapies targeting the gut microbiota currently under investigation.

Gemcitabine directly inhibits effector CD4 T cell activation and prevents experimental autoimmune encephalomyelitis.

Pro-inflammatory T cells are critical to the pathogenesis of multiple sclerosis (MS). We investigated the potential for the anti-proliferative, pro-apoptotic drug gemcitabine to affect development of MS-relevant effector TH1, TH17, and Treg cells. Gemcitabine directly suppressed proliferation, activation, and induced apoptosis of all effector subsets in subtype and dose-dependent fashion. This drug also prevented development of disease in the MS model experimental autoimmune encephalomyelitis (EAE) and significantly reduced the abundance of TH1 and TH17 cells. Our results indicate that pathogenic CD4+ T cells may be viable targets by gemcitabine for therapeutic benefit in MS.

CNS-targeted autoimmunity leads to increased influenza mortality in mice.

The discovery that central nervous system (CNS)-targeted autoreactive T cells required a process of licensing in the lung revealed an unexpected relationship between these organs. The clinical and immunological significance of this finding is bidirectional in that it showed not only a mechanism by which T cells become pathogenic before entering the CNS, but also the potential for this process to influence lung immunity as well. Epidemiological studies have shown that people with multiple sclerosis (MS) suffer from increased morbidity and mortality from infectious diseases, independent of immunosuppressive therapies. Respiratory infections account for a large percentage of deaths of people with MS. In this study, to investigate the mechanisms responsible for this enhanced susceptibility, we established a comorbid model system in which mice with experimental autoimmune encephalomyelitis (EAE) were administered a sublethal dose of influenza. Whereas mice with either EAE alone or influenza alone survived, 70% of comorbid mice died as a result of uncontrolled viral replication. Immunological analyses revealed that the induction of EAE led to a surprising alteration of the lung milieu, converting an effective stimulatory influenza-reactive environment into a suppressive one. These results provide mechanistic information that may help to explain the unexpected immunological interactions.

Emerging Concepts on the Gut Microbiome and Multiple Sclerosis.

Microbiota of the human body perform fundamental tasks that contribute to normal development, health, and homeostasis and are intimately associated with numerous organ systems, including the gut. Microbes begin gut inhabitance immediately following birth and promote proper gut epithelial construction and function, metabolism and nutrition, and immune system development. Inappropriate immune recognition of self-tissue can lead to autoimmune disease, including conditions such as multiple sclerosis (MS), in which the immune system recognizes and attacks central nervous system tissue. Preclinical studies have demonstrated a requirement of gut microbiota for neuroinflammatory autoimmune disease in animal models, and a growing number of clinical investigations are finding associations between MS status and the composition of the gut microbiota. In this review, we examine current undertakings into better understanding the role of gut bacteria and their phages in MS development, review associations of the gut microbiota makeup and MS, and discuss potential mechanisms by which the gut microbiota may be manipulated for therapeutic benefit.

Disparate Effects of Mesenchymal Stem Cells in Experimental Autoimmune Encephalomyelitis and Cuprizone-Induced Demyelination.

Mesenchymal stem cells (MSCs) are pleiotropic cells with potential therapeutic benefits for a wide range of diseases. Because of their immunomodulatory properties they have been utilized to treat autoimmune diseases such as multiple sclerosis (MS), which is characterized by demyelination. The microenvironment surrounding MSCs is thought to affect their differentiation and phenotype, which could in turn affect the efficacy. We thus sought to dissect the potential for differential impact of MSCs on central nervous system (CNS) disease in T cell mediated and non-T cell mediated settings using the MOG35-55 experimental autoimmune encephalomyelitis (EAE) and cuprizone-mediated demyelination models, respectively. As the pathogeneses of MS and EAE are thought to be mediated by IFNγ-producing (TH1) and IL-17A-producing (TH17) effector CD4+ T cells, we investigated the effect of MSCs on the development of these two key pathogenic cell groups. Although MSCs suppressed the activation and effector function of TH17 cells, they did not affect TH1 activation, but enhanced TH1 effector function and ultimately produced no effect on EAE. In the non- T cell mediated cuprizone model of demyelination, MSC administration had a positive effect, with an overall increase in myelin abundance in the brain of MSC-treated mice compared to controls. These results highlight the potential variability of MSCs as a biologic therapeutic tool in the treatment of autoimmune disease and the need for further investigation into the multifaceted functions of MSCs in diverse microenvironments and the mechanisms behind the diversity.

Mesenchymal stem cells: Emerging mechanisms of immunomodulation and therapy.

Mesenchymal stem cells (MSCs) are a pleiotropic population of cells that are self-renewing and capable of differentiating into canonical cells of the mesenchyme, including adipocytes, chondrocytes, and osteocytes. They employ multi-faceted approaches to maintain bone marrow niche homeostasis and promote wound healing during injury. Biomedical research has long sought to exploit their pleiotropic properties as a basis for cell therapy for a variety of diseases and to facilitate hematopoietic stem cell establishment and stromal reconstruction in bone marrow transplantation. Early results demonstrated their usage as safe, and there was little host response to these cells. The discovery of their immunosuppressive functions ushered in a new interest in MSCs as a promising therapeutic tool to suppress inflammation and down-regulate pathogenic immune responses in graft-versus-host and autoimmune diseases such as multiple sclerosis, autoimmune diabetes, and rheumatoid arthritis. MSCs produce a large number of soluble and membrane-bound factors, some of which inhibit immune responses. However, the full range of MSC-mediated immune-modulation remains incompletely understood, as emerging reports also reveal that MSCs can adopt an immunogenic phenotype, stimulate immune cells, and yield seemingly contradictory results in experimental animal models of inflammatory disease. The present review describes the large body of literature that has been accumulated on the fascinating biology of MSCs and their complex effects on immune responses.