Visualizing Sphingosine-1-Phosphate Receptor 1(S1P1) Signaling During Central Nervous System De- and Remyelination.

Multiple sclerosis (MS) is an inflammatory-demyelinating disease of the central nervous system (CNS) mediated by aberrant auto-reactive immune responses. The current immune-modulatory therapies are unable to protect and repair immune-mediated neural tissue damage. One of the therapeutic targets in MS is the sphingosine-1-phosphate (S1P) pathway which signals via sphingosine-1-phosphate receptors 1-5 (S1P1-5). S1P receptors are expressed predominantly on immune and CNS cells. Considering the potential neuroprotective properties of S1P signaling, we utilized S1P1-GFP (Green fluorescent protein) reporter mice in the cuprizone-induced demyelination model to investigate in vivo S1P - S1P1 signaling in the CNS. We observed S1P1 signaling in a subset of neural stem cells in the subventricular zone (SVZ) during demyelination. During remyelination, S1P1 signaling is expressed in oligodendrocyte progenitor cells in the SVZ and mature oligodendrocytes in the medial corpus callosum (MCC). In the cuprizone model, we did not observe S1P1 signaling in neurons and astrocytes. We also observed ß-arrestin-dependent S1P1 signaling in lymphocytes during demyelination and CNS inflammation. Our findings reveal ß-arrestin-dependent S1P1 signaling in oligodendrocyte lineage cells implying a role of S1P1 signaling in remyelination.

Current strategies employed in the manipulation of gene expression for clinical purposes.

Abnormal gene expression level or expression of genes containing deleterious mutations are two of the main determinants which lead to genetic disease. To obtain a therapeutic effect and thus to cure genetic diseases, it is crucial to regulate the host's gene expression and restore it to physiological conditions. With this purpose, several molecular tools have been developed and are currently tested in clinical trials. Genome editing nucleases are a class of molecular tools routinely used in laboratories to rewire host's gene expression. Genome editing nucleases include different categories of enzymes: meganucleses (MNs), zinc finger nucleases (ZFNs), clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR associated protein (Cas) and transcription activator-like effector nuclease (TALENs). Transposable elements are also a category of molecular tools which includes different members, for example Sleeping Beauty (SB), PiggyBac (PB), Tol2 and TcBuster. Transposons have been used for genetic studies and can serve as gene delivery tools. Molecular tools to rewire host's gene expression also include episomes, which are divided into different categories depending on their molecular structure. Finally, RNA interference is commonly used to regulate gene expression through the administration of small interfering RNA (siRNA), short hairpin RNA (shRNA) and bi-functional shRNA molecules. In this review, we will describe the different molecular tools that can be used to regulate gene expression and discuss their potential for clinical applications. These molecular tools are delivered into the host's cells in the form of DNA, RNA or protein using vectors that can be grouped into physical or biochemical categories. In this review we will also illustrate the different types of payloads that can be used, and we will discuss recent developments in viral and non-viral vector technology.

Pericytes regulate vascular immune homeostasis in the CNS.

Pericytes regulate the development of organ-specific characteristics of the brain vasculature such as the blood-brain barrier (BBB) and astrocytic end-feet. Whether pericytes are involved in the control of leukocyte trafficking in the adult central nervous system (CNS), a process tightly regulated by CNS vasculature, remains elusive. Using adult pericyte-deficient mice (Pdgfbret/ret ), we show that pericytes limit leukocyte infiltration into the CNS during homeostasis and autoimmune neuroinflammation. The permissiveness of the vasculature toward leukocyte trafficking in Pdgfbret/ret mice inversely correlates with vessel pericyte coverage. Upon induction of experimental autoimmune encephalomyelitis (EAE), pericyte-deficient mice die of severe atypical EAE, which can be reversed with fingolimod, indicating that the mortality is due to the massive influx of immune cells into the brain. Additionally, administration of anti-VCAM-1 and anti-ICAM-1 antibodies reduces leukocyte infiltration and diminishes the severity of atypical EAE symptoms of Pdgfbret/ret mice, indicating that the proinflammatory endothelium due to absence of pericytes facilitates exaggerated neuroinflammation. Furthermore, we show that the presence of myelin peptide-specific peripheral T cells in Pdgfbret/ret ;2D2tg mice leads to the development of spontaneous neurological symptoms paralleled by the massive influx of leukocytes into the brain. These findings indicate that intrinsic changes within brain vasculature can promote the development of a neuroinflammatory disorder.

Myeloid sphingosine-1-phosphate receptor 1 is important for CNS autoimmunity and neuroinflammation.

The critical role of sphingosine-1-phosphate (S1P) signaling in lymphocyte trafficking is well recognized, however, the contribution of myeloid cell-S1P signaling in neuroimmunity is less well understood. We previously reported that C57BL/6J mice harboring phosphorylation defective S1P receptor 1 (S1P1) (with mutated serines in the carboxyl terminus, leading to impaired receptor internalization) [S1P1(S5A)] developed severe, TH17-dominant experimental autoimmune encephalomyelitis. In this study, we demonstrate that S1P1-mediated TH17 polarization is not an intrinsic T cell effect, but dependent on sustained S1P1 signaling in myeloid cells. First, utilizing the S1P1(S5A) mice in the EAE model, we observed that S1P1 activated and enhanced antigen presentation function in myeloid cells. Second, sequential phosphorylation of STAT3 occurred in dendritic cells, monocytes, and macrophages/microglia during neuroinflammation. Third, we show that pro-inflammatory (CD45hiCD11b+Ly6Chi) monocytes contribute to TH17 differentiation and neuroinflammation by regulating IL-6 expression. Finally, results from experiments utilizing myeloid cell-specific S1P1 overexpression (S1pr1f/stop/f:LysMCre) mice demonstrate that myeloid cell S1P1 directly contributes to severity of neuroinflammation. These findings reveal the critical contribution of myeloid-S1P1 signaling in CNS autoimmunity.

Cholera toxin enhances interleukin-17A production in both CD4+ and CD8+ cells via a cAMP/protein kinase A-mediated interleukin-17A promoter activation.

Cholera toxin (CT) is a bacterial component that increases intracellular cAMP levels in host cells and suppresses T-cell activation. Recently, CT was reported to induce T helper type 17-skewing dendritic cells and activate interleukin-17A (IL-17A) production in CD4+ T cells through a cAMP-dependent pathway. However, the underlying mechanism by which cAMP regulates IL-17A production in T cells is not completely defined. In this study, we took advantage of a small molecule protein kinase A (PKA) inhibitor (H89) and different cAMP analogues: a PKA-specific activator (N6-benzoyl-adenosine-cAMP), an exchange protein activated by cAMP-specific activator (Rp-8-chlorophenylthio-2'-O-methyl cAMP), and a PKA inhibitor (Rp-8-bromo-cAMP), to elucidate the signalling cascade of cAMP in IL-17A regulation in T cells. We found that CT induced IL-17A production and IL-17A promoter activity in activated CD4+ T cells through a cAMP/PKA pathway. Moreover, this regulation was via cAMP-response element binding protein (CREB) -mediated transcriptional activation by using the transfection of an IL-17A promoter-luciferase reporter construct and CREB small interfering RNA in Jurkat cells. Also, we showed that CREB bound to the CRE motif located at -183 of the IL-17A promoter in vitro. Most interestingly, not only in CD4+ T cells, CT also enhanced cAMP/PKA-dependent IL-17A production and CREB phosphorylation in CD8+ T cells. In conclusion, our data suggest that CT induces an IL-17A-dominated immune microenvironment through the cAMP/PKA/CREB signalling pathway. Our study also highlights the potentials of CT and cAMP in modulating T helper type 17 responses in vivo.

Effects of sphingosine-1-phosphate receptor 1 phosphorylation in response to FTY720 during neuroinflammation.

Fingolimod (FTY720, Gilenya), a sphingosine-1-phosphate receptor (S1PR) modulator, is one of the first-line immunomodulatory therapies for treatment of relapsing-remitting multiple sclerosis (MS). Human S1PR1 variants have been reported to have functional heterogeneity in vitro, suggesting that S1PR1 function may influence FTY720 efficacy. In this study, we examined the influence of S1PR1 phosphorylation on response to FTY720 in neuroinflammation. We found that mice carrying a phosphorylation-defective S1pr1 gene [S1PR1(S5A) mice] were refractory to FTY720 treatment in MOG35-55-immunized and Th17-mediated experimental autoimmune encephalomyelitis (EAE) models. Long-term treatment with FTY720 induced significant lymphopenia and suppressed Th17 response in the peripheral immune system via downregulating STAT3 phosphorylation in both WT and S1PR1(S5A) mice. However, FTY720 did not effectively prevent neuroinflammation in the S1PR1(S5A) EAE mice as a result of encephalitogenic cells expressing C-C chemokine receptor 6 (CCR6). Combined treatment with FTY720 and anti-CCR6 delayed disease progression in S1PR1(S5A) EAE mice, suggesting that CCR6-mediated cell trafficking can overcome the effects of FTY720. This work may have translational relevance regarding FTY720 efficacy in MS patients and suggests that cell type-specific therapies may enhance therapeutic efficacy in MS.

Sphingosine-1-Phosphate (S1P) and S1P Signaling Pathway: Therapeutic Targets in Autoimmunity and Inflammation.

Sphingosine-1-phosphate (S1P) and S1P receptors (S1PR) are ubiquitously expressed. S1P-S1PR signaling has been well characterized in immune trafficking and activation in innate and adaptive immune systems. However, the full extent of its involvement in the pathogenesis of autoimmune diseases is not well understood. FTY720 (fingolimod), a non-selective S1PR modulator, significantly decreased annualized relapse rates in relapsing-remitting multiple sclerosis (MS). FTY720, which primarily targets S1P receptor 1 as a functional antagonist, arrests lymphocyte egress from secondary lymphoid tissues and reduces neuroinflammation in the central nervous system (CNS). Recent studies suggest that FTY720 also decreases astrogliosis and promotes oligodendrocyte differentiation within the CNS and may have therapeutic benefit to prevent brain atrophy. Since S1P signaling is involved in multiple immune functions, therapies targeting S1P axis may be applicable to treat autoimmune diseases other than MS. Currently, over a dozen selective S1PR and S1P pathway modulators with potentially superior therapeutic efficacy and better side-effect profiles are in the pipeline of drug development. Furthermore, newly characterized molecules such as apolipoprotein M (ApoM) (S1P chaperon) and SPNS2 (S1P transporter) are also potential targets for treatment of autoimmune diseases. Finally, the application of therapies targeting S1P and S1P signaling pathways may be expanded to treat several other immune-mediated disorders (such as post-infectious diseases, post-stroke and post-stroke dementia) and inflammatory conditions beyond their application in primary autoimmune diseases.

Polygonum multiflorum Decreases Airway Allergic Symptoms in a Murine Model of Asthma.

The root of Polygonum multiflorum (also called He-Shou-Wu in Chinese) is a common herb and medicinal food in Asia used for its anti-aging properties. Our study investigated the therapeutic potential of an extract of the root of Polygonum multiflorum (PME) in allergic asthma by using a mouse model. Feeding of 0.5 and 1 mg/mouse PME inhibited ovalbumin (OVA)-induced allergic asthma symptoms, including airway inflammation, mucus production, and airway hyper-responsiveness (AHR), in a dose-dependent manner. To discern PME's mechanism of action, we examined the profile and cytokine production of inflammatory cells in bronchial alveolar lavage fluid (BALF). We found that eosinophils, the main inflammatory cell infiltrate in the lung of OVA-immunized mice, significantly decreased after PME treatment. Th2 cytokine levels, including interleukin (IL)-4, IL-5, IL-13, eotaxin, and the proinflammatory cytokine tumor necrosis factor (TNF)-[Formula: see text], decreased in PME-treated mice. Elevated mRNA expression of Th2 transcription factor GATA-3 in the lung tissue was also inhibited after oral feeding of PME in OVA-immunized mice. Thus, we conclude that PME produces anti-asthma activity through the inhibition of Th2 cell activation.

Mechanisms of Cholera Toxin in the Modulation of TH17 Responses.

Numerous studies have shown that TH17 cells and their signature cytokine IL-17A are critical to host defense against various bacterial and fungal infections. The protective responses mediated by TH17 cells and IL-17A include the recruitment of neutrophils, release of antimicrobial peptides and chemokines, and enhanced tight junction of epithelial cells. Due to the importance of TH17 cells in infections, efforts have been made to develop TH17-based vaccines. The goal of vaccination is to establish a protective immunological memory. Most currently approved vaccines are antibody-based and have limited protection against stereotypically different strains. Studies show that T-cell-based vaccines may overcome this limitation and protect hosts against infection of different strains. Two main strategies are used to develop TH17 vaccines: identification of TH17-specific antigens and TH17-skewing adjuvants. Studies have revealed that cholera toxin (CT) induces a potent Th17 response following vaccination. Antigen vaccination along with CT induces a robust TH17 response, which is sometimes accompanied by TH1 responses. Due to the toxicity of CT, it is hard to apply CT in a clinical setting. Thus, understanding how CT modulates TH17 responses may lead to the development of successful TH17-based vaccines.

Cholera toxin directly enhances IL-17A production from human CD4+ T cells.

The significance of Th17 cells and IL-17A signaling in host defense and disease development has been demonstrated in various infection and autoimmune models. Additionally, the generation of Th17 cells is highly influenced by microbes. However, the specific bacterial components capable of shaping Th17 responses have not been well defined. The goals of this study were to understand how a bacterial toxin, cholera toxin (CT), modulates Th17-dominated response in isolated human CD4(+) T cells, and what are the mechanisms associated with this modulation. CD4(+) cells isolated from human peripheral blood were treated with CT. The levels of cytokine production and specific Th cell responses were determined by ELISA, Luminex assay, and flow cytometry. Along with the decreased production of other proinflammatory cytokines (IFN-γ, TNF-α, and IL-2), we found that CT could directly enhance the IL-17A production through a cAMP-dependent pathway. This enhancement is specific for IL-17A but not for IL-17F, IL-22, and CCL20. Interestingly, CT could increase IL-17A production only from Th17-committed cells, such as CCR6(+)CD4(+) T cells and in vitro-differentiated Th17 cells. Furthermore, we also demonstrated that this direct effect occurs at a transcriptional level because CT stimulates the reporter activity in Jurkat and primary CD4(+) T cells transfected with the IL-17A promoter-reporter construct. This study shows that CT has the capacity to directly shape Th17 responses in the absence of APCs. Our findings highlight the potentials of bacterial toxins in the regulation of human Th17 responses.

IL-17A and Th17 cells in lung inflammation: an update on the role of Th17 cell differentiation and IL-17R signaling in host defense against infection.

The significance of Th17 cells and interleukin- (IL-)17A signaling in host defense and disease development has been demonstrated in various infection and autoimmune models. Numerous studies have indicated that Th17 cells and its signature cytokine IL-17A are critical to the airway's immune response against various bacteria and fungal infection. Cytokines such as IL-23, which are involved in Th17 differentiation, play a critical role in controlling Klebsiella pneumonia (K. pneumonia) infection. IL-17A acts on nonimmune cells in infected tissues to strengthen innate immunity by inducing the expression of antimicrobial proteins, cytokines, and chemokines. Mice deficient in IL-17 receptor (IL-17R) expression are susceptible to infection by various pathogens. In this review, we summarize the recent advances in unraveling the mechanism behind Th17 cell differentiation, IL-17A/IL-17R signaling, and also the importance of IL-17A in pulmonary infection.

Evaluation of HUD-funded lead hazard control treatments at 6 years post-intervention.

The Evaluation of the HUD Lead-Based Paint Hazard Control Grant Program (Evaluation) was a HUD-funded study of the effectiveness of lead hazard control (LHC) treatments conducted by 14 grantees in communities across the country. A stratified random sampling scheme was used to select treated units at four grantee sites for continued environmental assessment at 6 years post-intervention. The study compared the relative effectiveness after 6 years of the different classes of interventions used by the grantees, after controlling for such factors as housing conditions and characteristics and resident and neighborhood characteristics. Geometric mean dust-lead levels on floors and window sills were 11% and 23% lower, respectively, at 6 years post-intervention than at any preceding point following the intervention. Although geometric mean window trough dust-lead levels were slightly higher at 6 years post-intervention than at other post-intervention time periods, they were still over 75% lower than before intervention. Treatment at more-intensive levels was associated with lower window sill and window trough dust-lead levels; however, statistical modeling found no significant difference in floor dust-lead loadings over time between the levels of treatment; however, significant differences in window sill and window trough dust-lead levels between treatment levels were evident. Findings from the 6-Year Extension study indicate that across all grantees and treatment strategies the treatments applied were effective at significantly reducing environmental lead levels on floors, window sills, and window troughs at least 6 years following the intervention.