SummaryThe blood-brain barrier (BBB) plays a crucial role in brain homeostasis, thereby maintaining the brain environment precise for optimal neuronal function. Its dysfunction is an intriguing complication of systemic lupus erythematosus (SLE). SLE is a systemic autoimmune disorder where neurological complications occur in 5-50% of cases and is associated with impaired BBB integrity. Complement activation occurs in SLE and is an important part of the clinical profile. Our earlier studies demonstrated that C5a generated by complement activation caused the loss of brain endothelial layer integrity in rodents. The goal of the current study was to determine the translational potential of these studies to a human system. To assess this, we used a two dimensional in vitro BBB model constructed using primary human brain microvascular endothelial cells and astroglial cells, which closely emulates the in vivo BBB allowing the assessment of BBB integrity. Increased permeability monitored by changes in transendothelial electrical resistance and cytoskeletal remodelling caused by actin fiber rearrangement were observed when the cells were exposed to lupus serum and C5a, similar to the observations in mice. In addition, our data show that C5a/C5aR1 signalling alters nuclear factor-jB translocation into nucleus and regulates the expression of the tight junction proteins, claudin-5 and zonula occludens 1 in this setting. Our results demonstrate for the first time that C5a regulates BBB integrity in a neuroinflammatory setting where it affects both endothelial and astroglial cells. In addition, we also demonstrate that our previous findings in a mouse model, were emulated in human cells in vitro, bringing the studies one step closer to understanding the translational potential of C5a/C5aR1 blockade as a promising therapeutic strategy in SLE and other neurodegenerative diseases.
Galectins are a family of β-galactoside-binding lectins that are important modulators of homeostasis in the central nervous system (CNS). Galectin-1 is a pivotal regulator of microglia activation that alters the immune balance from neurodegeneration to neuroprotection and could have therapeutic relevance in HIV associated neurocognitive disorders (HAND). We have previously shown that galectin-1 treatment decreased oxidative stress in microglia and hypothesize that the mechanism underlying this phenomenon is the cross regulatory interactions between Nitric oxide (NO) and Arginase I activity in microglia. We induced microglial activation and examined the effect of galectin-1 on the expression of various M1/M2 microglial phenotypic markers. Since, TNF-α is associated with activation of microglial cells involved in pathogenesis of neurodegenerative diseases, we treated HIV transfected human microglial cell cultures (CHME-5/HIV) with TNF-α followed by treatment with galectin-1, to examine the galectin-1 mediated neuro-modulatory response. Our results show that treatment of CHME-5/HIV microglia with galectin-1 reduced TNF-α induced oxidative stress by ~40%, and also significantly reduced iNOS gene expression and NO production while correspondingly increasing arginase-1, cationic amino acid transporter (CAT-1) gene expression and arginase activity. Galectin-1 treatment results in shifting microglia polarization from M1 toward the beneficial M2 phenotype which may prevent neurodegeneration and promote neuroprotection. Thus, our data suggests that galectin-1 treatment reduces neuroinflammation in the CNS microenvironment via the modulation of the NO-arginase network in microglia and thus could play a neuroprotective role in HAND. Further, the therapeutic potential of galectin-1 could be enhanced by conjugation of galectin-1 onto gold nanoparticles (Au-NP), resulting in a nanogold-galectin-1 (Au-Gal-1) multivalent complex that will have more clinical translational efficacy than free galectin-1 by virtue of increasing the payload influx.
Asthma is a chronic inflammatory respiratory disease characterized by airway inflammation, airway hyperresponsiveness and reversible airway obstruction. Understanding the mechanisms that underlie the various endotypes of asthma could lead to novel and more personalized therapies for individuals with asthma. Using a tissue inhibitor of metalloproteinases 1 (TIMP-1) knockout murine allergic asthma model, we previously showed that TIMP-1 deficiency results in an asthma phenotype, exhibiting airway hyperreactivity, enhanced eosinophilic inflammation and T helper type 2 cytokine gene and protein expression following sensitization with ovalbumin. In the current study, we compared the expression of Galectins and other key cytokines in a murine allergic asthma model using wild-type and TIMP-1 knockout mice. We also examined the effects of Galectin-3 (Gal-3) inhibition on a non-T helper type 2 cytokine interleukin-17 (IL-17) to evaluate the relationship between Gal-3 and the IL-17 axis in allergic asthma. Our results showed a significant increase in Gal-3, IL-17 and transforming growth factor-β gene expression in lung tissue isolated from an allergic asthma murine model using TIMP-1 knockout. Gal-3 gene and protein expression levels were also significantly higher in lung tissue from an allergic asthma murine model using TIMP-1 knockout. Our data show that Gal-3 may regulate the IL-17 axis and play a pivotal role in the modulation of inflammation during experimental allergic asthma.
Methamphetamine (Meth) abuse can lead to the breakdown of the blood-brain-barrier (BBB) integrity leading to compromised CNS function. The role of Galectins in the angiogenesis process in tumor-associated endothelial cells (EC) is well established; however no data are available on the expression of Galectins in normal human brain microvascular endothelial cells and their potential role in maintaining BBB integrity. We evaluated the basal gene/protein expression levels of Galectin-1, -3 and -9 in normal primary human brain microvascular endothelial cells (BMVEC) that constitute the BBB and examined whether Meth altered Galectin expression in these cells, and if Galectin-1 treatment impacted the integrity of an in-vitro BBB. Our results showed that BMVEC expressed significantly higher levels of Galectin-1 as compared to Galectin-3 and -9. Meth treatment increased Galectin-1 expression in BMVEC. Meth induced decrease in TJ proteins ZO-1, Claudin-3 and adhesion molecule ICAM-1 was reversed by Galectin-1. Our data suggests that Galectin-1 is involved in BBB remodeling and can increase levels of TJ proteins ZO-1 and Claudin-3 and adhesion molecule ICAM-1 which helps maintain BBB tightness thus playing a neuroprotective role. Galectin-1 is thus an important regulator of immune balance from neurodegeneration to neuroprotection, which makes it an important therapeutic agent/target in the treatment of drug addiction and other neurological conditions.
Methamphetamine (METH) is a drug of abuse, the acute and chronic use of which induces neurotoxic responses in the human brain, ultimately leading to neurocognitive disorders. Our goals were to understand the impact of METH on microglial mitochondrial respiration and to determine whether METH induces the activation of the mitochondrial-dependent intrinsic apoptosis pathway in microglia. We assessed the expression of pro- apoptosis genes using qPCR of RNA extracted from a human microglial cell line (HTHU). We examined the apoptosis-inducing effects of METH on microglial cells using digital holographic microscopy (DHM) to quantify real-time apoptotic volume decrease (AVD) in microglia in a noninvasive manner. METH treatment significantly increased AVD, activated Caspase 3/7, increased the gene expression levels of the pro- apoptosis proteins, APAF-1 and BAX, and decreased mitochondrial DNA content. Using immunofluorescence analysis, we found that METH increased the expression of the mitochondrial proteins cytochrome c and MCL-1, supporting the activation of mitochondrion-dependent (intrinsic) apoptosis pathway. Cellular bio-energetic flux analysis by Agilent Seahorse XF Analyzer revealed that METH treatment increased both oxidative and glycolytic respiration after 3 h, which was sustained for at least 24 h. Several events, such as oxidative stress, neuro-inflammatory responses, and mitochondrial dysfunction, may converge to mediate METH-induced apoptosis of microglia that may contribute to neurotoxicity of the CNS. Our study has important implications for therapeutic strategies aimed at preserving mitochondrial function in METH abusing patients.
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