Cerebral amyloid angiopathy and the immune system.

Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of amyloid protein in the walls of cerebral blood vessels. This deposition of amyloid causes damage to the cerebral vasculature, resulting in blood-brain barrier disruption, cerebral hemorrhage, cognitive decline, and dementia. The role of the immune system in CAA is complex and not fully understood. While the immune system has a clear role in the rare inflammatory variants of CAA (CAA related inflammation and Abeta related angiitis), the more common variants of CAA also have immune system involvement. In a protective role, immune cells may facilitate the clearance of beta-amyloid from the cerebral vasculature. The immune system can also contribute to CAA pathology, promoting vascular injury, blood-brain barrier breakdown, inflammation, and progression of CAA. In this review, we summarize the role of the immune system in CAA, including the potential of immune based treatment strategies to slow vascular disease in CAA and associated cognitive impairment, white matter disease progression, and reduce the risk of cerebral hemorrhage. HIGHLIGHTS: The immune system has a role in cerebral amyloid angiopathy (CAA) which is summarized in this review. There is an inflammatory response to beta-amyloid that may contribute to brain injury and cognitive impairment. Immune cells may facilitate the clearance of beta-amyloid from the cerebral vasculature. Improved understanding of the immune system in CAA may afford novel treatment to improve outcomes in patients with CAA.

Sex Differences in Thrombin Generation in Patients with Acute Ischemic Stroke.

Sex differences in stroke exist, including variation in stroke risk and outcome. Differences in thrombin generation may contribute to this variation between females and males. To examine this, we assessed sex differences in thrombin generation between females and males with acute ischemic stroke and the relationship to blood cell gene expression. In 97 patients with acute ischemic stroke, thrombin generation was measured by thrombin generation assay. Blood cell gene expression was measured by microarray. Differences in thrombin generation between sexes were identified and the relationship to blood cell gene expression examined. Genes associated with sex differences in thrombin generation were analyzed by functional pathway analysis. Females and males had similar overall capacity to generate thrombin. The peak thrombin generated in females was 468.8 nM (SD 91.6), comparable to males (479.3nM;SD 90.8; p = 0.58). Lag time, time to peak thrombin, and endogenous thrombin potential were also similar between females and males. While overall thrombin generation was comparable between females and males with stroke, differences in genes that promote this thrombin generation exist. Females with high peak thrombin had an increase in genes that promote thrombosis, and platelet activation. In contrast, males with high peak thrombin had a decrease in genes involved in thrombus degradation. Females and males with acute ischemic stroke have similar capacity to generate thrombin, however, differences may exist in how this thrombin generation is achieved, with females having increased thrombin signaling, and platelet activation, and males having decreased thrombus degradation. This suggests regulatory differences in thrombosis may exist between females and males that may contribute to sex differences in stroke.

Optical opening of the blood-brain barrier for targeted and ultra-sparse viral infection of cells in mouse cortex.

Adeno-associated viruses (AAVs) are used in a wide array of experimental situations for driving expression of biosensors, recombinases, and opto-/chemo-genetic actuators in the brain. However, conventional approaches for minimally invasive, spatially precise, and ultra-sparse AAV-mediated transduction of cells during imaging experiments have remained a significant challenge. Here, we show that intravenous injection of commercially available AAVs at different doses, combined with laser-based perforation of cortical capillaries through a cranial widow, allows for ultra-sparse, titratable, and micron-level precision for delivery of viral vectors with relatively little inflammation or tissue damage. Further, we show the utility of this approach for eliciting sparse expression of GCaMP6, channelrhodopsin, or fluorescent reporters in neurons and astrocytes within specific functional domains in normal and stroke-damaged cortex. This technique represents a facile approach for targeted delivery of viral vectors that should assist in the study of cell types and circuits in the cortex.

Sex and interferon gamma signaling regulate microglia migration in the adult mouse cortex in vivo.

Although microglia possess the unique ability to migrate, whether mobility is evident in all microglia, is sex dependent, and what molecular mechanisms drive this, is not well understood in the adult brain. Using longitudinal in vivo two-photon imaging of sparsely labeled microglia, we find a relatively small population of microglia (~5%) are mobile under normal conditions. Following injury (microbleed), the fraction of mobile microglia increased in a sex-dependent manner, with male microglia migrating significantly greater distances toward the microbleed relative to their female counterparts. To understand the signaling pathways involved, we interrogated the role of interferon gamma (IFNγ). Our data show that in male mice, stimulating microglia with IFNγ promotes migration whereas inhibiting IFNγ receptor 1 signaling inhibits them. By contrast, female microglia were generally unaffected by these manipulations. These findings highlight the diversity of microglia migratory responses to injury, its dependence on sex and the signaling mechanisms that modulate this behavior.

microRNA as a therapeutic for ischemic stroke.

microRNA (miRNA) are important regulators of gene expression. miRNA have the potential as a treatment to modulate genes, pathways and cells involved in ischemic stroke. In this review, we specifically present miRNA in stroke as a treatment to decrease thrombosis, reduce blood brain barrier (BBB) disruption and hemorrhagic transformation (HT), modulate inflammation, and modify angiogenesis. miRNA as a treatment for stroke is an emerging area with evidence from animal studies demonstrating its potential. While no miRNA is currently approved for human use, several have shown promise in clinical trials to treat medical conditions, such as miR-122 for hepatitis C. The role of miRNA as a treatment for specific applications in ischemic stroke is presented including a discussion of the benefits and barriers of miRNA as a treatment, and directions for future advancement.

Longitudinal functional imaging of VIP interneurons reveals sup-population specific effects of stroke that are rescued with chemogenetic therapy.

Stroke profoundly disrupts cortical excitability which impedes recovery, but how it affects the function of specific inhibitory interneurons, or subpopulations therein, is poorly understood. Interneurons expressing vasoactive intestinal peptide (VIP) represent an intriguing stroke target because they can regulate cortical excitability through disinhibition. Here we chemogenetically augmented VIP interneuron excitability in a murine model of photothrombotic stroke and show that it enhances somatosensory responses and improves recovery of paw function. Using longitudinal calcium imaging, we discovered that stroke primarily disrupts the fidelity (fraction of responsive trials) and predictability of sensory responses within a subset of highly active VIP neurons. Partial recovery of responses occurred largely within these active neurons and was not accompanied by the recruitment of minimally active neurons. Importantly, chemogenetic stimulation preserved sensory response fidelity and predictability in highly active neurons. These findings provide a new depth of understanding into how stroke and prospective therapies (chemogenetics), can influence subpopulations of inhibitory interneurons.

Induction of Systemic Lupus Erythematosus-like Syndrome in BALB/c Mice Leads to Disturbance in Splenic T Cell Subpopulations.

Mechanisms underlying the systemic lupus erythematosus (SLE) have not yet been elucidated. In this study, we evaluated the balance of T cell subsets in BALB/c mice model of SLE induced; using Con A and polyamines as DNA immunogenicity modifiers. BALB/c mice were immunized subcutaneously with 50 µg extracted DNA from cells cultured in different conditions: splenocytes+ polyamines (group P), splenocytes+ Con A (group A), splenocytes+ polyamines+ Con A (group PA) and splenocytes only (control). Anti-double-stranded DNA -(ds-DNA) antibodies, proteinuria, and antinuclear autoantibodies were assessed by enzyme-linked immunosorbent assay, Bradford method, and immunofluorescence respectively. Transcription factors of different T helper subsets were examined by real-time polymerase chain reaction. The serum level of the anti-dsDNA antibody in group PA was higher than that in the other groups (p>0.05). Antinuclear antibody (ANA) titer increased in groups A and PA. Proteinuria level in group PA was significantly higher than that in the control group (p<0.001). Expression of Foxp3 was decreased in group A (p=0.001). Additionally, the ratios of T-bet/GATA3 and T-bet/Foxp3 were also increased in group A. (p>0.05). Our results revealed an increased ratio of Th1 to Th2 and decreased expression of Foxp3 in group A, but group PA manifested more obvious signs of the disease. These results suggest that other mechanisms rather than disturbance in T cells' balance may involve the development of disease symptoms.

Invasion of phagocytic Galectin 3 expressing macrophages in the diabetic brain disrupts vascular repair.

The cellular events that dictate the repair of damaged vessels in the brain, especially in those with vascular risk factors such as diabetes, is poorly understood. Here, we dissected the role of resident microglia and infiltrative macrophages in determining the repair of ruptured cerebral microvessels. Using in vivo time-lapse imaging, gene expression analysis, and immunohistochemistry, we identified a unique population of phagocytic Galectin 3 (Gal3) expressing macrophages, distinct from resident microglia, which infiltrated and aggregated at the site of injury in diabetic mice and were associated with the elimination of microvessels. Depletion of these infiltrative macrophages in diabetic mice attenuated phagocytic activity and prevented the loss of blood vessels after injury. These findings highlight a previously unknown role for infiltrative Gal3 expressing macrophages in promoting vessel elimination after brain injury and provide impetus for future studies to determine whether depleting these cells can facilitate vascular repair in at risk populations.

Caspase-1 inhibition prevents glial inflammasome activation and pyroptosis in models of multiple sclerosis.

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS of unknown cause that remains incurable. Inflammasome-associated caspases mediate the maturation and release of the proinflammatory cytokines IL-1ß and IL-18 and activate the pore-forming protein gasdermin D (GSDMD). Inflammatory programmed cell death, pyroptosis, was recently shown to be mediated by GSDMD. Here, we report molecular evidence for GSDMD-mediated inflammasome activation and pyroptosis in both myeloid cells (macrophages/microglia) and, unexpectedly, in myelin-forming oligodendrocytes (ODCs) in the CNS of patients with MS and in the MS animal model, experimental autoimmune encephalomyelitis (EAE). We observed inflammasome activation and pyroptosis in human microglia and ODCs in vitro after exposure to inflammatory stimuli and demonstrate caspase-1 inhibition by the small-molecule inhibitor VX-765 in both cell types. GSDMD inhibition by siRNA transduction suppressed pyroptosis in human microglia. VX-765 treatment of EAE animals reduced the expression of inflammasome- and pyroptosis-associated proteins in the CNS, prevented axonal injury, and improved neurobehavioral performance. Thus, GSDMD-mediated pyroptosis in select glia cells is a previously unrecognized mechanism of inflammatory demyelination and represents a unique therapeutic opportunity for mitigating the disease process in MS and other CNS inflammatory diseases.

Myeloid-derived Suppressor Cells Elimination by 5-Fluorouracil Increased Dendritic Cell-based Vaccine Function and Improved Immunity in Tumor Mice.

Myeloid-derived suppressor cells (MDSCs) are capable of suppressing the immune response. 5-Fluorouracil (5-FU) compared to other chemotherapy drugs have shown considerable decreases in the number of MDSCs without visible effects on T, B and natural killer cells, as well as dendritic cells (DCs). DC-based vaccines considered to be appropriate candidates for cancer immunotherapy. However, due to the presence of various factors like MDSCs in tumor microenvironment, DC vaccine cannot effectively perform its function. The purpose of this study was to evaluate the effect of low doses of 5-FU on the efficacy of DC-based vaccines in preventing and treating of melanoma tumor model. This research was performed on 28 melanoma tumor bearing C57BL/6 female mice. The mice were randomly divided to 4 groups, group 1 is control population while group 2 and 3 were treated with DC vaccine and 5-FU respectively and group 4 was treated with both DC Vaccine and 5-FU. The mice survival, tumor growth rate, number of MDSC and CD8+/ CD107a+ T cells in mice spleen were evaluated in each group with maximum result in group 4. Our results revealed that combination of DC vaccine and 5-FU reduced number of MDSCs (3%) and also tumor growth rate(10%)(p<0.05) and increased mice survival (70%) and increased CD8+ /CD107a+ T cells (25%). This study have shown that combinational therapy with DC vaccine improved immunity in tumor mice compared to the therapy consisting of DC vaccine or 5-FU only.

Suppressed oligodendrocyte steroidogenesis in multiple sclerosis: Implications for regulation of neuroinflammation.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Neurosteroids are reported to exert anti-inflammatory effects in several neurological disorders. We investigated the expression and actions of the neurosteroid, dehydroepiandrosterone (DHEA), and its more stable 3ß-sulphated ester, DHEA-S, in MS and associated experimental models. CNS tissues from patients with MS and animals with experimental autoimmune encephalomyelitis (EAE) displayed reduced DHEA concentrations, accompanied by diminished expression of the DHEA-synthesizing enzyme CYP17A1 in oligodendrocytes (ODCs), in association with increased expression of inflammatory genes including interferon (IFN)-γ and interleukin (IL)-1ß. CYP17A1 was expressed variably in different human neural cell types but IFN-γ exposure selectively reduced CYP17A1 detection in ODCs. DHEA-S treatment reduced IL-1ß and -6 release from activated human myeloid cells with minimal effect on lymphocyte viability. Animals with EAE receiving DHEA-S treatment showed reduced Il1b and Ifng transcript levels in spinal cord compared to vehicle-treated animals with EAE. DHEA-S treatment also preserved myelin basic protein immunoreactivity and reduced axonal loss in animals with EAE, relative to vehicle-treated EAE animals. Neurobehavioral deficits were reduced in DHEA-S-treated EAE animals compared with vehicle-treated animals with EAE. Thus, CYP17A1 expression in ODCs and its product DHEA were downregulated in the CNS during inflammatory demyelination while DHEA-S provision suppressed neuroinflammation, demyelination, and axonal injury that was evident as improved neurobehavioral performance. These findings indicate that DHEA production is an immunoregulatory pathway within the CNS and its restoration represents a novel treatment approach for neuroinflammatory diseases.

MicroRNA-142 regulates inflammation and T cell differentiation in an animal model of multiple sclerosis.

BACKGROUND: MicroRNAs have emerged as an important class of modulators of gene expression. These molecules influence protein synthesis through translational repression or degradation of mRNA transcripts. Herein, we investigated the potential role of miR-142a isoforms, miR-142a-3p and miR-142a-5p, in the context of autoimmune neuroinflammation. METHODS: The expression levels of two mature isoforms of miR-142 were measured in the brains of patients with multiple sclerosis (MS) and the CNS tissues from mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Expression analyses were also performed in mitogen and antigen-stimulated splenocytes, as well as macrophages and astrocytes using real-time RT-PCR. The role of the mature miRNAs was then investigated in T cell differentiation by transfection of CD4+ T cells, followed by flow cytometric analysis of intracellular cytokines. Luciferase assays using vectors containing the 3'UTR of predicted targets were performed to confirm the interaction of miRNA sequences with transcripts. Expression of targets were then analyzed in activated splenocytes and MS/EAE tissues. RESULTS: Expression of miR-142-5p was significantly increased in the frontal white matter from MS patients compared with white matter from non-MS controls. Likewise, expression levels of miR-142a-5p and miR-142a-3p showed significant upregulation in the spinal cords of EAE mice at days 15 and 25 post disease induction. Splenocytes stimulated with myelin oligodendrocyte glycoprotein (MOG) peptide or anti-CD3/anti-CD28 antibodies showed upregulation of miR-142a-5p and miR-142a-3p isoforms, whereas stimulated bone marrow-derived macrophages and primary astrocytes did not show any significant changes in miRNA expression levels. miR-142a-5p overexpression in activated lymphocytes shifted the pattern of T cell differentiation towards Th1 cells. Luciferase assays revealed SOCS1 and TGFBR1 as direct targets of miR-142a-5p and miR-142a-3p, respectively, and overexpression of miRNA mimic sequences suppressed the expression of these target transcripts in lymphocytes. SOCS1 levels were also diminished in MS white matter and EAE spinal cords. CONCLUSIONS: Our findings suggest that increased expression of miR-142 isoforms might be involved in the pathogenesis of autoimmune neuroinflammation by influencing T cell differentiation, and this effect could be mediated by interaction of miR-142 isoforms with SOCS1 and TGFBR-1 transcripts.

Identification of Toxoplasma gondii protein fractions induce immune response against melanoma in mice.

Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses against tumor cells. We recently found that protein components of Toxoplasma gondii (T. gondii) could mature DCs efficiently. Therefore, in this study, we aimed to find the most effective protein components of T. gondii which are able to mature DCs and consequently instruct immune responses in tumor-bearing mice. Soluble tachyzoite antigens (STAgs) were fractionated by ammonium sulfate precipitation and subsequently by anion-exchange HPLC. Immature DCs (iDCs) were treated by these protein fractions and were monitored for IL-12p70 and IL-10 production. Moreover, the capacity of mature DCs (mDCs) to induce lymphocyte proliferation was investigated. Ultimately, we analyzed the ability of mDCs in instructing immune responses in tumor-bearing mice. We found that ammonium sulfate fraction one (A1) matured-DCs produced higher IL-12 level and IL-12/IL-10 ratio; therefore, this fraction was selected for further fractionation by anion-exchange HPLC. The results showed that anion-exchange HPLC fraction 14 (C14) matured-DCs secrete higher levels of IL-12p70 and IL-12p70/IL-10 ratio. Survival of the mice matured by A1 fraction increased significantly compared to other groups. Moreover, SDS-PAGE electrophoresis showed that different obtained fractions have distinct proteins based on their size. These results demonstrate that two protein fractions of T. gondii are able to mature DCs more efficient.

The potential role of angiogenic factors in rheumatoid arthritis.

Angiogenesis is an important phenomenon in the pathogenesis of some diseases, such as numerous types of tumors and autoimmunity, and also a number of soluble and cell-bound factors may stimulate neovascularization in inflammatory reaction processes. Here, by highlighting the significance of angiogenesis reaction in rheumatoid arthritis (RA), we will mainly focus on the role of various growth factors, cytokines, enzymes, cells, hypoxic conditions and transcription factors in the angiogenic process and we will then explain some therapeutic strategies based on blockage of angiogenesis and modification of the vascular pathology in RA.

Listeria monocytogenes protein fraction induces dendritic cells maturation and T helper 1 immune responses.

Fully mature dendritic cells (DCs) play pivotal role in inducing immune responses and converting naïve T lymphocytes into functional Th1 cells. We aimed to evaluate Listeria Monocytogenes-derived protein fractions to induce DC maturation and stimulating T helper (Th)1 immune responses.In the present study, we fractionated Listeria Monocytogenes-derived proteins by adding of ammonium sulfate in a stepwise manner. DCs were also generated from C57BL/6 mice bone marrow precursor cells. Then, the effects of protein fractions on bone marrow derived DC (BMDC) maturation were evaluated. In addition, we assessed the capacity of activated DCs to induce cytokine production and proliferation of lymphocytes.Listeria-derived protein fractions induced fully mature DCs expressing high costimulatory molecules such as CD80, CD86 and CD40. DCs that were activated by selected F3 fraction had low capacity to uptake exogenous antigens while secreted high levels of Interleukine (IL)-12. Moreover, lymphocytes cultured with activated BMDCs produced high amounts of IFN-γ and showed higher proliferation than control. Listeria derived protein fractions differently influenced DC maturation.In conclusion, Listeria protein activated-BMDCs can be used as a cell based vaccine to induce anti-tumor immune responses.