Bone marrow dendritic cells deficient for CD40 and IL-23p19 are tolerogenic in vitro

Objective(s): In addition to pro-inflammatory role, dendritic cells (DCs) can also be anti-inflammatory when they acquire tolerogenic phenotype. In this study we tested the role of CD40 and IL-23p19 in antigen presenting function of bone marrow-derived DCs (BMDCs) by comparing BMDCs derived from CD40 knockout (CD40KO-DCs) and IL-23p19 (IL-23p19KO-DCs) knockout mice with those from C57BL/6 mice (Cont-DCs). We have focused on CD40 and IL-23, as these molecules have well established pro-inflammatory roles in a number of autoimmune and inflammatory diseases. Materials and Methods: The expression of maturation markers MHC II and co-stimulatory molecules CD40, CD80, and CD86 was analyzed by flow cytometry, while the expression of CD40 and IL-23p19 was measured by RT-PCR. The capacity of BMDCs to activate CD4+ T cells was evaluated by 3H-thymidine incorporation, and the capacity of BMDCs to uptake antigen was evaluated using fluorescent OVA and flow cytometry. Results: The lack of CD40 or IL-23p19 had no effect on uptake of FITC-OVA by the DCs, confirming their immature phenotype. Moreover, CD40KO-DCs had significantly reduced capacity to stimulate proliferation of CD4+ T cells. CD4+ T cells activated by CD40KO-DCs and IL-23p19KO-DCs produced significantly less IFN-γ (P-value ≤0.05), while CD4+ T cells stimulated by IL-23p19KO-DCs produced less GM-CSF and more IL-10 than Cont-DCs. Conclusion: This study shows that CD40KO-DCs and IL-23p19KO-DCs have a marked tolerogenic potency in vitro. Future in vivo studies should determine if and to what extent DCs lacking CD40 or IL-23 have a potential to be useful in therapy of autoimmune inflammation

CNS derived Extracellular Vesicles as Biomarkers in Multiple Sclerosis (MS)

This project aims to develop biomarkers for the diagnosis and treatment of multiple sclerosis (MS), a clinically heterogenous disease resulting from demyelination of the central nervous system. Extracellular vesicles (EVs) have shown potential as such a biomarker. EVs were isolated from the plasma and cerebrospinal fluid (CSF) of patients with suspected MS and patients with headaches. Participants are recruited before starting treatment for their condition and controls were only included if the cause of headache was determined to be non-inflammatory. Proteomic analysis will be conducted on the plasma and CSF before isolating the EVs, as well as the isolated plasma and CSF derived EVs themselves. Currently, 7 MS patient’s samples and 7 control headache patients have enrolled. Proteomic bulk analysis will be conducted in January on the samples providing immediate and consistent comparison points. Preliminary analysis has shown a higher concentration of EVs in patients in MS compared to those with headaches consistent with the literature, and initial target molecules such as L-tryptophan have been found to be downregulated. This should be consistent with the bulk analysis. Recruitment was delayed as a result of the COVID-19 pandemic, however, as this study is the first to look at both plasma and CSF derived EVs at the diagnostic phase of MS, the results should reveal target molecules for further assessment with a larger sample size. Although the focus of the field of extracellular vesicles in MS has centered around treatment delivery mechanisms, this study should help show their potential as diagnostic biomarkers

Interaction of RNA-binding protein HuR and miR-466i regulates GM-CSF expression.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by T helper 17 (Th17) cells plays an essential role in autoimmune diseases. Transcriptional regulation of Th17 cell differentiation has been extensively studied, but post-transcriptional regulation of Th17 cell differentiation has remained less well characterized. The RNA-binding protein HuR functions to promote the stability of target mRNAs via binding the AU-rich elements of the 3\u27 untranslated region (3\u27UTR) of numerous pro-inflammatory cytokines including IL-4, IL-13, IL-17 and TNF-α. However, whether HuR regulates GM-CSF expression in Th17 cells has not been fully investigated. Here we showed that HuR conditional knockout (KO) Th17 cells have decreased GM-CSF mRNA in comparison with wild-type (WT) Th17 cells, and that HuR binds directly to GM-CSF mRNA 3\u27UTR. Interestingly, HuR deficiency increased the levels of certain microRNA expression in Th17 cells; for example, miR-466i functioned to mediate GM-CSF and IL-17 mRNA decay, which was confirmed by in vitro luciferase assay. Furthermore, we found that HuR promoted Mxi1 expression to inhibit certain miRNA expression. Taken together, these findings indicate that interaction of HuR and miR-466i orchestrates GM-CSF expression in Th17 cells

Distinct Role of IL-27 in Immature and LPS-Induced Mature Dendritic Cell-Mediated Development of CD4+ CD127+3G11+ Regulatory T Cell Subset

Interleukin-27 (IL-27) plays an important role in regulation of anti-inflammatory responses and autoimmunity; however, the molecular mechanisms of IL-27 in modulation of immune tolerance and autoimmunity have not been fully elucidated. Dendritic cells (DCs) play a central role in regulating immune responses mediated by innate and adaptive immune systems, but regulatory mechanisms of DCs in CD4+ T cell-mediated immune responses have not yet been elucidated. Here we show that IL-27 treated mature DCs induced by LPS inhibit immune tolerance mediated by LPS-stimulated DCs. IL-27 treatment facilitates development of the CD4+ CD127+3G11+ regulatory T cell subset in vitro and in vivo. By contrast, IL-27 treated immature DCs fail to modulate development of the CD4+CD127+3G11+ regulatory T cell sub-population in vitro and in vivo. Our results suggest that IL-27 may break immune tolerance induced by LPS-stimulated mature DCs through modulating development of a specific CD4+ regulatory T cell subset mediated by 3G11 and CD127. Our data reveal a new cellular regulatory mechanism of IL-27 that targets DC-mediated immune responses in autoimmune diseases such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE)

Potential roles of extracellular vesicles in the pathophysiology, diagnosis, and treatment of autoimmune diseases.

Since extracellular vesicles (EVs) were discovered in 1983 in sheep reticulocytes samples, they have gradually attracted scientific attention and become a topic of great interest in the life sciences field. EVs are small membrane particles, released by virtually every cell that carries a variety of functional molecules. Their main function is to deliver messages to the surrounding area in both physiological and pathological conditions. Initially, they were thought to be either cell debris, signs of cell death, or unspecific structures. However, accumulating evidence support a theory that EVs are a universal mechanism of communication. Thanks to their biological characteristics and functions, EVs are likely to represent a promising strategy for obtaining pathogen information, identifying therapeutic targets and selecting specific biomarkers for a variety of diseases, such as autoimmune diseases. In this review, we provide a brief overview of recent progress in the study of the biology and functions of EVs. We also discuss their roles in diagnosis and therapy, with particular emphasis on autoimmune diseases

Dimethyl fumarate suppresses granulocyte macrophage colony-stimulating factor-producing Th1 cells in CNS neuroinflammation.

Objective To study the immunomodulatory effect of dimethyl fumarate (DF) on granulocyte macrophage colony-stimulating factor (GM-CSF) production in CD4+ T cells in experimental autoimmune encephalomyelitis (EAE) and human peripheral blood mononuclear cells (PBMCs). Methods We collected splenocytes and CD4+ T cells from C57BL/6 wild-type and interferon (IFN)-γ–deficient mice. For human PBMCs, venous blood was collected from healthy donors, and PBMCs were collected using the Percoll gradient method. Cells were cultured with anti-CD3/28 in the presence/absence of DF for 3 to 5 days. Cells were stained and analyzed by flow cytometry. Cytokines were measured by ELISA in cell supernatants. For in vivo experiments, EAE was induced by myelin oligodendrocyte glycoprotein35–55 and mice were treated with oral DF or vehicle daily. Results DF acts directly on CD4+ T cells and suppresses GM-CSF–producing Th1 not Th17 or single GM-CSF+ T cells in EAE. In addition, GM-CSF suppression depends on the IFN-γ pathway. We also show that DF specifically suppresses Th1 and GM-CSF–producing Th1 cells in PBMCs from healthy donors. Conclusions We suggest that DF exclusively suppresses GM-CSF–producing Th1 cells in both animal and human CD4+ T cells through an IFN-γ–dependent pathway. These findings indicate that DF has a better therapeutic effect on patients with Th1-dominant immunophenotype. However, future longitudinal study to validate this finding in MS is needed

Therapeutic effect of baicalin on experimental autoimmune encephalomyelitis is mediated by SOCS3 regulatory pathway

Natural compounds derived from medicinal plants have long been considered a rich source of novel therapeutic agents. Baicalin (Ba) is a bioactive flavonoid compound derived from the root of Scutellaria baicalensis, an herb widely used in traditional medicine for the treatment of various inflammatory diseases. In this study, we investigate the effects and mechanism of action of Ba in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Ba treatment effectively ameliorated clinical disease severity in myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced EAE, and reduced inflammation and demyelination of the central nervous system (CNS). Ba reduced infiltration of immune cells into the CNS, inhibited expression of proinflammatory molecules and chemokines, and prevented Th1 and Th17 cell differentiation via STAT/NF B signaling pathways. Further, we showed that SOCS3 induction is essential to the effects of Ba, given that the inhibitory effect of Ba on pathogenic Th17 responses was largely abolished when SOCS3 signaling was knocked down. Taken together, our findings demonstrate that Ba has significant potential as a novel anti-inflammatory agent for therapy of autoimmune diseases such as MS

Combination Therapy With Fingolimod and Neural Stem Cells Promotes Functional Myelination in vivo Through a Non-immunomodulatory Mechanism

Myelination, which occurs predominantly postnatally and continues throughout life, is important for proper neurologic function of the mammalian central nervous system (CNS). We have previously demonstrated that the combination therapy of fingolimod (FTY720) and transplanted neural stem cells (NSCs) had a significantly enhanced therapeutic effect on the chronic stage of experimental autoimmune encephalomyelitis, an animal model of CNS autoimmunity, compared to using either one of them alone. However, reduced disease severity may be secondary to the immunomodulatory effects of FTY720 and NSCs, while whether this therapy directly affects myelinogenesis remains unknown. To investigate this important question, we used three myelination models under minimal or non-inflammatory microenvironments. Our results showed that FTY720 drives NSCs to differentiate into oligodendrocytes and promotes myelination in an ex vivo brain slice culture model, and in the developing CNS of healthy postnatal mice in vivo. Elevated levels of neurotrophic factors, e.g., brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, were observed in the CNS of the treated infant mice. Further, FTY720 and NSCs efficiently prolonged the survival and improved sensorimotor function of shiverer mice. Together, these data demonstrate a direct effect of FTY720, beyond its known immunomodulatory capacity, in NSC differentiation and myelin development as a novel mechanism underlying its therapeutic effect in demyelinating diseases

Response of Astrocyte Subpopulations Following Spinal Cord Injury.

There is growing appreciation for astrocyte heterogeneity both across and within central nervous system (CNS) regions, as well as between intact and diseased states. Recent work identified multiple astrocyte subpopulations in mature brain. Interestingly, one subpopulation (Population C) was shown to possess significantly enhanced synaptogenic properties in vitro, as compared with other astrocyte subpopulations of adult cortex and spinal cord. Following spinal cord injury (SCI), damaged neurons lose synaptic connections with neuronal partners, resulting in persistent functional loss. We determined whether SCI induces an enhanced synaptomodulatory astrocyte phenotype by shifting toward a greater proportion of Population C cells and/or increasing expression of relevant synapse formation-associated genes within one or more astrocyte subpopulations. Using flow cytometry and RNAscope in situ hybridization, we found that astrocyte subpopulation distribution in the spinal cord did not change to a selectively synaptogenic phenotype following mouse cervical hemisection-type SCI. We also found that spinal cord astrocytes expressed synapse formation-associated genes to a similar degree across subpopulations, as well as in an unchanged manner between uninjured and SCI conditions. Finally, we confirmed these astrocyte subpopulations are also present in the human spinal cord in a similar distribution as mouse, suggesting possible conservation of spinal cord astrocyte heterogeneity across species