Interferon-ß modulates microglial polarization to ameliorate delayed tPA-exacerbated brain injury in ischemic stroke.

Tissue plasminogen activator (tPA) is the only FDA-approved drug for the treatment of ischemic stroke. Delayed tPA administration is associated with increased risks of blood-brain barrier (BBB) disruption and hemorrhagic transformation. Studies have shown that interferon beta (IFNß) or type I IFN receptor (IFNAR1) signaling confers protection against ischemic stroke in preclinical models. In addition, we have previously demonstrated that IFNß can be co-administered with tPA to alleviate delayed tPA-induced adverse effects in ischemic stroke. In this study, we investigated the time limit of IFNß treatment on the extension of tPA therapeutic window and assessed the effect of IFNß on modulating microglia (MG) phenotypes in ischemic stroke with delayed tPA treatment. Mice were subjected to 40 minutes transient middle cerebral artery occlusion (MCAO) followed by delayed tPA treatment in the presence or absence of IFNß at 3h, 4.5h or 6h post-reperfusion. In addition, mice with MG-specific IFNAR1 knockdown were generated to validate the effects of IFNß on modulating MG phenotypes, ameliorating brain injury, and lessening BBB disruption in delayed tPA-treated MCAO mice. Our results showed that IFNß extended tPA therapeutic window to 4.5h post-reperfusion in MCAO mice, and that was accompanied with attenuated brain injury and lessened BBB disruption. Mechanistically, our findings revealed that IFNß modulated MG polarization, leading to the suppression of inflammatory MG and the promotion of anti-inflammatory MG, in delayed tPA-treated MCAO mice. Notably, these effects were abolished in MG-specific IFNAR1 knockdown MCAO mice. Furthermore, the protective effect of IFNß on the amelioration of delayed tPA-exacerbated ischemic brain injury was also abolished in these mice. Finally, we identified that IFNß-mediated modulation of MG phenotypes played a role in maintaining BBB integrity, because the knockdown of IFNAR1 in MG partly reversed the protective effect of IFNß on lessening BBB disruption in delayed tPA-treated MCAO mice. In summary, our study reveals a novel function of IFNß in modulating MG phenotypes, and that may subsequently confer protection against delayed tPA-exacerbated brain injury in ischemic stroke.

4-Ethylguaiacol Modulates Neuroinflammation and Promotes Heme Oxygenase-1 Expression to Ameliorate Brain Injury in Ischemic Stroke.

Ischemic stroke is caused by a sudden reduction in cerebral blood flow that subsequently induces a complex cascade of pathophysiological responses, leading to brain inflammation and irreversible infarction. 4-ethylguaiacol (4-EG) is reported to suppress inflammatory immune responses. However, whether 4-EG exerts anti-inflammatory effects in ischemic stroke remains unexplored. We evaluated the therapeutic potential of 4-EG and examined the cellular and molecular mechanisms underlying the protective effects of 4-EG in ischemic stroke. The effect of 4-EG in ischemic stroke was determined by using a transient middle cerebral artery occlusion (MCAO) animal model followed by exploring the infarct size, neurological deficits, microglia activation, inflammatory cytokine production, blood-brain barrier (BBB) disruption, brain endothelial cell adhesion molecule expression, and microglial heme oxygenase-1 (HO-1) expression. Nrf2-/- and HO-1 inhibitor ZnPP-treated mice were also subjected to MCAO to evaluate the role of the Nrf2/HO-1 pathway in 4-EG-mediated protection in ischemic stroke. We found that 4-EG attenuated infarct size and neurological deficits, and lessened BBB disruption in ischemic stroke. Further investigation revealed that 4-EG suppressed microglial activation, peripheral inflammatory immune cell infiltration, and brain endothelial cell adhesion molecule upregulation in the ischemic brain. Finally, we identified that the protective effect of 4-EG in ischemic stroke was abolished in Nrf2-/- and ZnPP-treated MCAO mice. Our results identified that 4-EG confers protection against ischemic stroke and reveal that the protective effect of 4-EG in ischemic stroke is mediated through the induction of the Nrf2/HO1 pathway. Thus, our findings suggest that 4-EG could be developed as a novel therapeutic agent for the treatment of ischemic stroke.

Immunoresponsive gene 1 modulates the severity of brain injury in cerebral ischaemia.

Inflammatory stimuli induce immunoresponsive gene 1 expression that in turn catalyses the production of itaconate through diverting cis-aconitate away from the tricarboxylic acid cycle. The immunoregulatory effect of the immunoresponsive gene 1/itaconate axis has been recently documented in lipopolysaccharide-activated mouse and human macrophages. In addition, dimethyl itaconate, an itaconate derivative, was reported to ameliorate disease severity in the animal models of psoriasis and multiple sclerosis. Currently, whether immunoresponsive gene 1/itaconate axis exerts a modulatory effect in ischaemic stroke remains unexplored. In this study, we investigated whether immunoresponsive gene 1 plays a role in modulating ischaemic brain injury. In addition, the molecular mechanism underlying the protective effects of immunoresponsive gene 1 in ischaemic stroke was elucidated. Our results showed that immunoresponsive gene 1 was highly induced in the ischaemic brain following ischaemic injury. Interestingly, we found that IRG1-/- stroke animals exhibited exacerbated brain injury, displayed with enlarged cerebral infarct, compared to wild-type stroke controls. Furthermore, IRG1-/- stroke animals presented aggravated blood-brain barrier disruption, associated with augmented Evans blue leakage and increased immune cell infiltrates in the ischaemic brain. Moreover, IRG1-/- stroke animals displayed elevated microglia activation, demonstrated with increased CD68, CD86 and Iba1 expression. Further analysis revealed that immunoresponsive gene 1 was induced in microglia after ischaemic stroke, and deficiency in immunoresponsive gene 1 resulted in repressed microglial heme oxygenase-1 expression and exacerbated ischaemic brain injury. Notably, the administration of dimethyl itaconate to compensate for the deficiency of immunoresponsive gene 1/itaconate axis led to enhanced microglial heme oxygenase-1 expression, alleviated ischaemic brain injury, improved motor function and decreased mortality in IRG1-/- stroke animals. In summary, we demonstrate for the first time that the induction of immunoresponsive gene 1 in microglia following ischaemic stroke serves as an endogenous protective mechanism to restrain brain injury through heme oxygenase-1 up-regulation. Thus, our findings suggest that targeting immunoresponsive gene 1 may represent a novel therapeutic approach for the treatment of ischaemic stroke.

4-Ethylguaiacol modulates neuroinflammation and Th1/Th17 differentiation to ameliorate disease severity in experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis (MS) is a progressive autoimmune disease characterized by the accumulation of pathogenic inflammatory immune cells in the central nervous system (CNS) that subsequently causes focal inflammation, demyelination, axonal injury, and neuronal damage. Experimental autoimmune encephalomyelitis (EAE) is a well-established murine model that mimics the key features of MS. Presently, the dietary consumption of foods rich in phenols has been reported to offer numerous health benefits, including anti-inflammatory activity. One such compound, 4-ethylguaiacol (4-EG), found in various foods, is known to attenuate inflammatory immune responses. However, whether 4-EG exerts anti-inflammatory effects on modulating the CNS inflammatory immune responses remains unknown. Thus, in this study, we assessed the therapeutic effect of 4-EG in EAE using both chronic and relapsing-remitting animal models and investigated the immunomodulatory effects of 4-EG on neuroinflammation and Th1/Th17 differentiation in EAE. METHODS: Chronic C57BL/6 EAE and relapsing-remitting SJL/J EAE were induced followed by 4-EG treatment. The effects of 4-EG on disease progression, peripheral Th1/Th17 differentiation, CNS Th1/Th17 infiltration, microglia (MG) activation, and blood-brain barrier (BBB) disruption in EAE were evaluated. In addition, the expression of MMP9, MMP3, HO-1, and Nrf2 was assessed in the CNS of C57BL/6 EAE mice. RESULTS: Our results showed that 4-EG not only ameliorated disease severity in C57BL/6 chronic EAE but also mitigated disease progression in SJL/J relapsing-remitting EAE. Further investigations of the cellular and molecular mechanisms revealed that 4-EG suppressed MG activation, mitigated BBB disruption, repressed MMP3/MMP9 production, and inhibited Th1 and Th17 infiltration in the CNS of EAE. Furthermore, 4-EG suppressed Th1 and Th17 differentiation in the periphery of EAE and in vitro Th1 and Th17 cultures. Finally, we found 4-EG induced HO-1 expression in the CNS of EAE in vivo as well as in MG, BV2 cells, and macrophages in vitro. CONCLUSIONS: Our work demonstrates that 4-EG confers protection against autoimmune disease EAE through modulating neuroinflammation and inhibiting Th1 and Th17 differentiation, suggesting 4-EG, a natural compound, could be potentially developed as a therapeutic agent for the treatment of MS/EAE.

Interferon-ß alleviates delayed tPA-induced adverse effects via modulation of MMP3/9 production in ischemic stroke.

Tissue plasminogen activator (tPA) is the only US Food and Drug Administration (FDA)-approved drug for ischemic stroke. However, delayed tPA administration is associated with increased risk of blood-brain barrier (BBB) disruption and hemorrhagic transformation (HT). Interferon-ß (IFNß), an FDA-approved drug for the treatment of multiple sclerosis, is a cytokine with immunomodulatory properties. Previous studies, including ours, demonstrated that IFNß or type I IFN receptor signaling conferred protection against ischemic stroke in preclinical models, suggesting IFNß might have translational therapeutic potential for the treatment of ischemic stroke. Currently, whether IFNß could be coadministered with tPA to alleviate delayed tPA-induced adverse effects remains unknown. To elucidate that, IFNß was coadministered with delayed tPA to ischemic stroke animals, and the severity and pathology of ischemic brain injury were assessed. We found delayed tPA treatment exacerbated ischemic brain injury, manifested by aggravated BBB disruption and HT. Notably, IFNß ameliorated delayed tPA-exacerbated brain injury and alleviated adverse effects. Mechanistic studies revealed IFNß suppressed tPA-enhanced neuroinflammation and MMP3/9 production in the ischemic brain. Furthermore, we identified IFNß suppressed MMP9 production in microglia and attenuated tight junction protein degradation in brain endothelial cells. Moreover, we observed that peripheral immune cells may participate to a lesser extent in delayed tPA-exacerbated brain injury during the early phase of ischemic stroke. In conclusion, we provide the first evidence that IFNß can be coadministered with tPA to mitigate delayed tPA-induced adverse effects of BBB disruption and HT that could potentially extend the tPA therapeutic window for the treatment of ischemic stroke.

Dimethyl itaconate, an itaconate derivative, exhibits immunomodulatory effects on neuroinflammation in experimental autoimmune encephalomyelitis.

BACKGROUND: Inflammatory stimuli induce immunoresponsive gene 1 (IRG1) expression that in turn catalyzes the production of itaconate from the tricarboxylic acid cycle. Itaconate has recently emerged as a regulator of immune cell functions, especially in macrophages. Studies show that itaconate is required for the activation of anti-inflammatory transcription factor Nrf2 by LPS in mouse and human macrophages, and LPS-activated IRG1-/- macrophages that lack endogenous itaconate production exhibit augmented inflammatory responses. Moreover, dimethyl itaconate (DMI), an itaconate derivative, inhibits IL-17-induced IκBς activation in keratinocytes and modulates IL-17-IκBς pathway-mediated skin inflammation in an animal model of psoriasis. Currently, the effect of itaconate on regulating macrophage functions and peripheral inflammatory immune responses is well established. However, its effect on microglia (MG) and CNS inflammatory immune responses remains unexplored. Thus, we investigated whether itaconate possesses an immunomodulatory effect on regulating MG activation and CNS inflammation in animal models of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). METHODS: Chronic C57BL/6 EAE was induced followed by DMI treatment. The effect of DMI on disease severity, blood-brain barrier (BBB) disruption, MG activation, peripheral Th1/Th17 differentiation, and the CNS infiltration of Th1/Th17 cells in EAE was determined. Primary MG was cultured to study the effect of DMI on MG activation. Relapsing-remitting SJL/J EAE was induced to assess the therapeutic effect of DMI. RESULTS: Our results show DMI ameliorated disease severity in the chronic C57BL/6 EAE model. Further analysis of the cellular and molecular mechanisms revealed that DMI mitigated BBB disruption, inhibited MMP3/MMP9 production, suppressed microglia activation, inhibited peripheral Th1/Th17 differentiation, and repressed the CNS infiltration of Th1 and Th17 cells. Strikingly, DMI also exhibited a therapeutic effect on alleviating severity of relapse in the relapsing-remitting SJL/J EAE model. CONCLUSIONS: We demonstrate that DMI suppresses neuroinflammation and ameliorates disease severity in EAE through multiple cellular and molecular mechanisms, suggesting that DMI can be developed as a novel therapeutic agent for the treatment of MS/EAE through its immunomodulatory and anti-inflammatory properties.

Isolation of Mouse Cerebral Microvasculature for Molecular and Single-Cell Analysis.

Brain microvasculature forms a specialized structure, the blood-brain barrier (BBB), to maintain homeostasis and integrity of the central nervous system (CNS). The BBB dysfunction is emerging as a critical contributor to multiple neurological disorders, including stroke, traumatic brain injury, autoimmune multiple sclerosis, and neurodegenerative diseases. The brain microvasculature exhibits highly cellular and regional heterogeneity to accommodate dynamic changes of microenvironment during homeostasis and diseases. Thus, investigating the underlying mechanisms that contribute to molecular or cellular changes of the BBB is a significant challenge. Here, we describe an optimized protocol to purify microvessels from the mouse cerebral cortex using mechanical homogenization and density-gradient centrifugation, while maintaining the structural integrity and functional activity of the BBB. We show that the isolated microvessel fragments consist of BBB cell populations, including endothelial cells, astrocyte end-feet, pericytes, as well as tight junction proteins that seal endothelial cells. Furthermore, we describe the procedures to generate single-cell suspensions from isolated microvessel fragments. We demonstrate that cells in the single-cell suspensions are highly viable and suitable for single-cell RNA-sequencing analysis. This protocol does not require transgenic mice and cell sorting equipment to isolate fluorescence-labeled endothelial cells. The optimized procedures can be applied to different disease models to generate viable cells for single-cell analysis to uncover transcriptional or epigenetic landscapes of BBB component cells.

Dithiolethione ACDT suppresses neuroinflammation and ameliorates disease severity in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disorder characterized by the central nervous system (CNS) infiltration of myelin-specific pathogenic T cells followed by brain inflammation in association with demyelination. Similarly, experimental autoimmune encephalomyelitis (EAE), the animal model of MS, also exhibits increased CNS infiltration of pathogenic T cells, including Th1 and Th17, leading to detrimental effects of neuroinflammation and demyelination. We previously reported that 3H-1,2-dithiole-3-thione (D3T), the structurally-simplest of the sulfur-containing dithiolethiones, exerted a promising therapeutic effect in EAE. In the current study we report that 5-Amino-3-thioxo-3H-(1,2)dithiole-4-carboxylic acid ethyl ester (ACDT), a substituted derivative of D3T, exhibits anti-inflammatory properties in EAE. ACDT, administered post immunization, delayed disease onset and reduced disease severity in chronic C57BL/6 EAE, and ACDT, administered during disease remission, suppressed disease relapse in relapsing-remitting SJL/J EAE. Further analysis of the cellular and molecular mechanisms underlying the protective effects of ACDT in EAE revealed that ACDT inhibited pathogenic T cell infiltration, suppressed microglia activation, repressed neurotoxic A1 astrocyte generation, lessened blood-brain barrier disruption, and diminished MMP3/9 production in the CNS of EAE. In summary, we demonstrate that ACDT suppresses neuroinflammation and ameliorates disease severity in EAE through multiple cellular mechanisms. Our findings suggest the potential of developing ACDT as a novel therapeutic agent for the treatment of MS/EAE.

3H-1,2-Dithiole-3-thione as a novel therapeutic agent for the treatment of ischemic stroke through Nrf2 defense pathway.

Cerebral ischemic stroke accounts for more than 80% of all stroke cases. During cerebral ischemia, reactive oxygen species produced in brain tissue induce oxidative stress and inflammatory responses. D3T, the simplest compound of the cyclic, sulfur-containing dithiolethiones, is found in cruciferous vegetables and has been reported to induce antioxidant genes and glutathione biosynthesis through activation of Nrf2. In addition to antioxidant activity, D3T was also reported to possess anti-inflammatory effects. In this study, we evaluated the therapeutic potential of D3T for the treatment of ischemic stroke and investigated the mechanisms underlying the protective effects of D3T in ischemic stroke. Mice subjected to transient middle cerebral artery occlusion/reperfusion (tMCAO/R) were administered with vehicle or D3T to evaluate the effect of D3T in cerebral brain injury. We observed D3T reduced infarct size, decreased brain edema, lessened blood-brain barrier disruption, and ameliorated neurological deficits. Further investigation revealed D3T suppressed microglia (MG) activation and inhibited peripheral inflammatory immune cell infiltration of CNS in the ischemic brain. The protective effect of D3T in ischemic stroke is mediated through Nrf2 induction as D3T-attenuated brain injury was abolished in Nrf2 deficient mice subjected to tMCAO/R. In addition, in vitro results indicate the induction of Nrf2 by D3T is required for its suppressive effect on MG activation and cytokine production. In summary, we demonstrate for the first time that D3T confers protection against ischemic stroke, which is mediated through suppression of MG activation and inhibition of CNS peripheral cell infiltration, and that the protective effect of D3T in ischemic stroke is dependent on the activation of Nrf2.

3H-1,2-dithiole-3-thione as a novel therapeutic agent for the treatment of experimental autoimmune encephalomyelitis.

3H-1,2-dithiole-3-thione (D3T), the simplest member of the sulfur-containing dithiolethiones, is found in cruciferous vegetables, and has been previously reported to be a potent inducer of antioxidant genes and glutathione biosynthesis by activation of the transcription factor Nrf2. D3T is a cancer chemopreventive agent and possesses anti-inflammatory properties. Although D3T has been shown to protect against neoplasia, the effect of D3T in the autoimmune inflammatory disease multiple sclerosis/experimental autoimmune encephalomyelitis (EAE) is unknown. The present study is the first report of the therapeutic effect of D3T in EAE. Our results show D3T, administered post immunization, not only delays disease onset but also dramatically reduces disease severity in EAE. Strikingly, D3T, administered post disease onset of EAE, effectively prevents disease progression and exacerbation. Mechanistic studies revealed that D3T suppresses dendritic cell activation and cytokine production, inhibits pathogenic Th1 and Th17 differentiation, represses microglia activation and inflammatory cytokine expression, and promotes microglia phase II enzyme induction. In summary, these results indicate that D3T affects both innate and adaptive immune cells, and the protective effect of D3T in EAE might be attributed to its effects on modulating dendritic cell and microglia activation and pathogenic Th1/Th17 cell differentiation.

Interferon-ß Modulates Inflammatory Response in Cerebral Ischemia.

BACKGROUND: Stroke is a leading cause of death in the world. In >80% of strokes, the initial acute phase of ischemic injury is due to the occlusion of a blood vessel resulting in severe focal hypoperfusion, excitotoxicity, and oxidative damage. Interferon-ß (IFNß), a cytokine with immunomodulatory properties, was approved by the US Food and Drug Administration for the treatment of relapsing-remitting multiple sclerosis for more than a decade. Its anti-inflammatory properties and well-characterized safety profile suggest that IFNß has therapeutic potential for the treatment of ischemic stroke. METHODS AND RESULTS: We investigated the therapeutic effect of IFNß in the mouse model of transient middle cerebral artery occlusion/reperfusion. We found that IFNß not only reduced infarct size in ischemic brains but also lessened neurological deficits in ischemic stroke animals. Further, multiple molecular mechanisms by which IFNß modulates ischemic brain inflammation were identified. IFNß reduced central nervous system infiltration of monocytes/macrophages, neutrophils, CD4(+) T cells, and γδ T cells; inhibited the production of inflammatory mediators; suppressed the expression of adhesion molecules on brain endothelial cells; and repressed microglia activation in the ischemic brain. CONCLUSIONS: Our results demonstrate that IFNß exerts a protective effect against ischemic stroke through its anti-inflammatory properties and suggest that IFNß is a potential therapeutic agent, targeting the reperfusion damage subsequent to the treatment with tissue plasminogen activator.

Differential effects of IFN-ß on IL-12, IL-23, and IL-10 expression in TLR-stimulated dendritic cells.

MS is an autoimmune disease characterized by immune cell infiltration in the CNS, leading to cumulative disability. IFN-ß, used clinically in RR-MS reduces lesion formation and rates of relapse. Although the molecular mechanisms are not entirely elucidated, myeloid cells appear to be a major target for the therapeutic effects of IFN-ß. DCs have a critical role in experimental models of MS through their effect on encephalitogenic Th1/Th17 cell differentiation and expansion. Here we focused on the effects of IFN-ß on DC expression of cytokines involved in the control of Th1/Th17 differentiation and expansion. Administration of IFN-ß to mice immunized with MOG35-55 inhibited IL-12 and IL-23 expression in splenic DC and reduced in vivo differentiation of Th1/Th17 cells. IFN-ß affected cytokine expression in TLR-stimulated DC in a similar manner in vitro, inhibiting IL-12 and IL-23 and stimulating IL-10 at both mRNA and protein levels, by signaling through IFNAR. We investigated the role of the signaling molecules STAT1/STAT2, IRF-1 and IRF-7, and of the PI3K→GSK3 pathway. IFN-ß inhibition of the IL-12 subunits p40 and p35 was mediated through STAT1/STAT2, whereas inhibition of IL-23 was STAT1 dependent, and the stimulatory effect on IL-10 expression was mediated through STAT2. IFN-ß induces IRF-7 and, to a lesser degree, IRF-1. However, neither IRF mediated the effects of IFN-ß on IL-12, IL-23, or IL-10. We found that the PI3K pathway mediated IL-12 inhibition but did not interfere with the inhibition of IL-23 or stimulation of IL-10.

Histologic analysis of neural elements in the human sacroiliac joint.

STUDY DESIGN: The posterior ligament of the human sacroiliac joint was examined for nerves and nerve endings using histologic and immunohistochemical techniques. OBJECTIVE: To identify nerve fibers and mechanoreceptors in the posterior ligament. SUMMARY OF BACKGROUND DATA: According to the findings of previous studies, the human sacroiliac joint receives myelinated and unmyelinated axons that presumably conduct pain and proprioceptive impulses derived from mechanoreceptors and free nerve endings in the human sacroiliac joint. METHODS: Tissue obtained from six patients was stained with gold chloride and that obtained from six additional patients was stained using antibodies specific for substance P and protein gene product 9.5. RESULTS: The staining of joint tissue using the gold chloride technique showed myelinated and unmyelinated nerve fibers, two morphotypes of paciniform encapsulated mechanoreceptors, and a single nonpaciniform mechanoreceptor. Analysis using immunohistochemical staining for protein gene product 9.5 did not unequivocally show axons, nerve fascicles, or mechanoreceptors. Similarly, analysis based on immunohistochemical staining for substance P, one of several neurotransmitters known to signal pain from the periphery, showed reactive elements that may have been nerves, but because of background staining, could not be positively identified as such. CONCLUSIONS: The presence of nerve fibers and mechanoreceptors in the sacroiliac ligament demonstrates that the central nervous system receives information, certainly proprioceptive, and possibly pain from the sacroiliac joint. Although it is not known how the central nervous system uses such information, it seems reasonable to speculate that the proprioceptive information is used to optimize upper body balance at this joint. In addition, because the staining techniques used generally to show nerves and nerve elements in periarticular connective tissue are nonspecific, the distinction between neural and nonneural should be made on the basis of both morphologic and staining characteristics.