Endoplasmic reticulum stress response in immune cells contributes to experimental autoimmune encephalomyelitis pathogenesis in rats.

We examined the role of endoplasmic reticulum (ER) stress and the ensuing unfolded protein response (UPR) in the development of the central nervous system (CNS)-directed immune response in the rat model of experimental autoimmune encephalomyelitis (EAE). The induction of EAE with syngeneic spinal cord homogenate in complete Freund's adjuvant (CFA) caused a time-dependent increase in the expression of ER stress/UPR markers glucose-regulated protein 78 (GRP78), X-box-binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and phosphorylated eukaryotic initiation factor 2α (eIF2α) in the draining lymph nodes of both EAE-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rats. However, the increase in ER stress markers was more pronounced in AO rats. CFA alone also induced ER stress, but the effect was weaker and less sustained compared to full immunization. The ultrastructural analysis of DA lymph node tissue by electron microscopy revealed ER dilatation in lymphocytes, macrophages, and plasma cells, while immunoblot analysis of CD3-sorted lymph node cells demonstrated the increase in ER stress/UPR markers in both CD3+ (T cell) and CD3- (non-T) cell compartments. A positive correlation was observed between the levels of ER stress/UPR markers in the CNS-infiltrated mononuclear cells and the clinical activity of the disease. Finally, the reduction of EAE clinical signs by ER stress inhibitor ursodeoxycholic acid was associated with the decrease in the expression of mRNA encoding pro-inflammatory cytokines TNF and IL-1ß, and encephalitogenic T cell cytokines IFN-γ and IL-17. Collectively, our data indicate that ER stress response in immune cells might be an important pathogenetic factor and a valid therapeutic target in the inflammatory damage of the CNS.

The presence of Mott cells in the lymph nodes of rats with experimental autoimmune encephalomyelitis.

Mott cells are plasma cells that have multiple spherical Russell bodies packed in their cytoplasm. Russell bodies are dilated endoplasmic reticulum cisternae filled with aggregates of immunoglobulins that are neither secreted nor degraded. Mott cells were observed in our study by light and electron microscope in the lymph nodes of rats with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Mott cells were detected on hematoxylin and eosin (HE)-stained lymph node sections as vacuolated cells with eccentrically positioned nuclei and large number of faint blue spherical inclusions in the cytoplasm. Electron microscopic investigation revealed the presence of Russell bodies of the "medusa" form inside Mott cells in lymph node ultra-thin sections of EAE animals. Mott cells expressed the plasma cell marker CD138 and either kappa or lambda immunoglobulin light chains, indicating their origin from polyclonally activated B cells. Finally, Mott cells were associated with active EAE, as they were not found in the lymph nodes of EAE-resistant Albino Oxford rats. The presence of Russell bodies implies an excessive production of immunoglobulins in EAE, thus further emphasizing the role of B cells, and among them Mott cells, in the pathogenesis of this animal model of multiple sclerosis.

Effects of metformin and simvastatin treatment on ultrastructural features of liver macrophages in HFD mice.

Type 2 diabetes is a major health burden to the society. Macrophages and liver inflammation emerged as important factors in its development. We investigated ultrastructural changes in the liver, with a special emphasis on macrophages in high fat diet (HFD) fed C57BL/6 J mice treated with metformin or simvastatin, two drugs that are used frequently in diabetes. Both metformin and simvastatin reduced the liver damage in HFD fed animals, manifested as the prevention of nonalcoholic steatohepatitis development and reduced activation and number of macrophages in the liver, as well as the percentage of these cells with lipid droplets in the cytoplasm compared to untreated HFD animals. In contrast with untreated HFD-fed animals, lipid droplets were not observed in lysosomes of macrophages in HFD animals treated with metformin and simvastatin. These findings provide new insight into the effects of metformin and simvastatin on the liver in this experimental model of type 2 diabetes and provide further rationale for implementation of statins in the therapeutic regimens in this disease.

MAP kinase-dependent autophagy controls phorbol myristate acetate-induced macrophage differentiation of HL-60 leukemia cells.

We investigated the mechanisms and the role of autophagy in the differentiation of HL-60 human acute myeloid leukemia cells induced by protein kinase C (PKC) activator phorbol myristate acetate (PMA). PMA-triggered differentiation of HL-60 cells into macrophage-like cells was confirmed by cell-cycle arrest accompanied by elevated expression of macrophage markers CD11b, CD13, CD14, CD45, EGR1, CSF1R, and IL-8. The induction of autophagy was demonstrated by the increase in intracellular acidification, accumulation/punctuation of autophagosome marker LC3-II, and the increase in autophagic flux. PMA also increased nuclear translocation of autophagy transcription factors TFEB, FOXO1, and FOXO3, as well as the expression of several autophagy-related (ATG) genes in HL-60 cells. PMA failed to activate autophagy inducer AMP-activated protein kinase (AMPK) and inhibit autophagy suppressor mechanistic target of rapamycin complex 1 (mTORC1). On the other hand, it readily stimulated the phosphorylation of mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) via a protein kinase C-dependent mechanism. Pharmacological or genetic inhibition of ERK or JNK suppressed PMA-triggered nuclear translocation of TFEB and FOXO1/3, ATG expression, dissociation of pro-autophagic beclin-1 from its inhibitor BCL2, autophagy induction, and differentiation of HL-60 cells into macrophage-like cells. Pharmacological or genetic inhibition of autophagy also blocked PMA-induced macrophage differentiation of HL-60 cells. Therefore, MAP kinases ERK and JNK control PMA-induced macrophage differentiation of HL-60 leukemia cells through AMPK/mTORC1-independent, TFEB/FOXO-mediated transcriptional and beclin-1-dependent post-translational activation of autophagy.

Transcriptional block of AMPK-induced autophagy promotes glutamate excitotoxicity in nutrient-deprived SH-SY5Y neuroblastoma cells.

We investigated the role of autophagy, a controlled lysosomal degradation of cellular macromolecules and organelles, in glutamate excitotoxicity during nutrient deprivation in vitro. The incubation in low-glucose serum/amino acid-free cell culture medium synergized with glutamate in increasing AMP/ATP ratio and causing excitotoxic necrosis in SH-SY5Y human neuroblastoma cells. Glutamate suppressed starvation-triggered autophagy, as confirmed by diminished intracellular acidification, lower LC3 punctuation and LC3-I conversion to autophagosome-associated LC3-II, reduced expression of proautophagic beclin-1 and ATG5, increase of the selective autophagic target NBR1, and decreased number of autophagic vesicles. Similar results were observed in PC12 rat pheochromocytoma cells. Both glutamate-mediated excitotoxicity and autophagy inhibition in starved SH-SY5Y cells were reverted by NMDA antagonist memantine and mimicked by NMDA agonists D-aspartate and ibotenate. Glutamate reduced starvation-triggered phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) without affecting the activity of mammalian target of rapamycin complex 1, a major negative regulator of autophagy. This was associated with reduced mRNA levels of autophagy transcriptional activators (FOXO3, ATF4) and molecules involved in autophagy initiation (ULK1, ATG13, FIP200), autophagosome nucleation/elongation (ATG14, beclin-1, ATG5), and autophagic cargo delivery to autophagosomes (SQSTM1). Glutamate-mediated transcriptional repression of autophagy was alleviated by overexpression of constitutively active AMPK. Genetic or pharmacological AMPK activation by AMPK overexpression or metformin, as well as genetic or pharmacological autophagy induction by TFEB overexpression or lithium chloride, reduced the sensitivity of nutrient-deprived SH-SY5Y cells to glutamate excitotoxicity. These data indicate that transcriptional inhibition of AMPK-dependent cytoprotective autophagy is involved in glutamate-mediated excitotoxicity during nutrient deprivation in vitro.

Comparative analysis of cell death mechanisms induced by lysosomal autophagy inhibitors.

We performed a comparative analysis of molecular cytotoxic mechanisms of lysosomal autophagy inhibitors bafilomycin A1, chloroquine, and ammonium chloride in B16 mouse melanoma cells. All agents caused oxidative stress, mitochondrial depolarization, and caspase-dependent apoptotic death, which was not affected by genetic inactivation of autophagy. Cathepsin inhibition reduced only the cytotoxicity of chloroquine, indicating its ability to cause lysosomal membrane permeabilization. Bafilomycin reduced the mRNA levels of anti-apoptotic Bcl-2, while chloroquine and ammonium chloride increased the mRNA expression of pro-apoptotic Pten and Puma, as well as anti-apoptotic Bcl-xL. Ammonium chloride additionally increased the mRNA expression of pro-apoptotic Bim and p53. All three agents decreased the activity of mechanistic target of rapamycin (mTOR) and increased the activation of p38 mitogen-activated protein kinase (MAPK). Chloroquine and ammonium chloride additionally stimulated the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), respectively, while only bafilomycin increased the phosphorylation of the energy sensor AMP-activated protein kinase (AMPK). mTOR activator leucine did not affect the cytotoxicity of lysosomal inhibitors. p38 MAPK inhibitor SB203580 reduced the cytotoxicity of bafilomycin but increased that of chloroquine and ammonium chloride. The pharmacological inhibition of ERK1/2, JNK, and AMPK potentiated the cytotoxicity of chloroquine, ammonium chloride, and bafilomycin, respectively. The observed mechanistic differences were associated with antagonistic interactions of lysosomal inhibitors in B16 cell killing. In conclusion, all investigated lysosomal inhibitors cause autophagy-independent mitochondrial dysfunction and apoptotic death, but differ in the ability to affect lysosomal permeabilization, balance between pro- and anti-apoptotic molecules of Bcl-2 family, and MAPK/AMPK signaling.

Graphene quantum dots inhibit T cell-mediated neuroinflammation in rats.

We investigated the therapeutic capacity of nano-sized graphene sheets, called graphene quantum dots (GQD), in experimental autoimmune encephalomyelitis (EAE), an animal model of immune-mediated central nervous system (CNS) damage. Intraperitoneally administered GQD (10 mg/kg/day) accumulated in the lymph node and CNS cells of Dark Agouti rats in which EAE was induced by immunization with spinal cord homogenate in complete Freund's adjuvant. GQD significantly reduced clinical signs of EAE when applied throughout the course of the disease (day 0-32), while the protection was less pronounced if the treatment was limited to the induction (day 0-7 post-immunization) or effector (from day 8 onwards) phase of the disease. GQD treatment diminished immune infiltration, demyelination, axonal damage, and apoptotic death in the CNS of EAE animals. GQD also reduced the numbers of interferon-γ-expressing T helper (Th)1 cells, as well as the expression of Th1 transcription factor T-bet and proinflammatory cytokines tumor necrosis factor, interleukin-1, and granulocyte-macrophage colony-stimulating factor in the lymph nodes and CNS immune infitrates. The protective effect of GQD in EAE was associated with the activation of p38 and p42/44 mitogen-activated protein kinases (MAPK) and Akt in the lymph nodes and/or CNS. Finally, GQD protected oligodendrocytes and neurons from T cell-mediated damage in the in vitro conditions. Collectively, these data demonstrate the ability of GQD to gain access to both immune and CNS cells during neuroinflammation, and to alleviate immune-mediated CNS damage by modulating MAPK/Akt signaling and encephalitogenic Th1 immune response.

Metformin exacerbates and simvastatin attenuates myelin damage in high fat diet-fed C57BL/6 J mice.

Diabetic neuropathy is one of the most deleterious complications of diabetes mellitus in humans. High fat diet (HFD)-fed C57BL/6 J mice are a widely used animal model for type 2 diabetes mellitus and metabolic syndrome. We investigated the effects of metformin and simvastatin on the ultrastructural characteristics of sciatic nerve fibers in these mice. Metformin treatment increased the number of structural defects of the myelin sheet surrounding these fibers in already affected nerves of HFD fed mice, and simvastatin treatment reduced these numbers to the levels seen in control mice. These results warrant further research on the effects of metformin and statins in patients developing diabetic neuropathy and advise caution when deciding about optimal treatment modalities in these patients.

Aggressive human neuroblastomas show a massive increase in the numbers of autophagic vacuoles and damaged mitochondria.

Autophagy is activated in cancer cells in response to multiple stresses and has been demonstrated to promote tumor cell survival and drug resistance in neuroblastoma (NB). This study was conducted to analyze the ultrastructural features of peripheral neuroblastic tumors (pNTs) and identify the relation of the types of NTs, the proliferation rate, and MYCN gene amplification with a number of autophagic vacuoles. Our results indicate that aggressive human NBs show a massive increase in the number of autophagic vacuoles associated with proliferation rate and that alteration of the mitochondria might be an important factor for the induction of autophagy in NTs.

Neuroprotective arylpiperazine dopaminergic/serotonergic ligands suppress experimental autoimmune encephalomyelitis in rats.

Arylpiperazine-based dopaminergic/serotonergic ligands exert neuroprotective activity. We examined the effect of arylpiperazine D2 /5-HT1A ligands, N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl}-phenyl]-picolinamide (6a) and N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide (6b), in experimental autoimmune encephalomyelitis (EAE), a model of neuroinflammation. Both compounds (10 mg/kg i.p.) reduced EAE clinical signs in spinal cord homogenate-immunized Dark Agouti rats. Compound 6b was more efficient in delaying the disease onset and reducing the maximal clinical score, which correlated with its higher affinity for D2 and 5-HT1A receptors. The protection was retained if treatment was limited to the effector (from day 8 onwards), but not the induction phase (day 0-7) of EAE. Compound 6b reduced CNS immune infiltration and expression of mRNA encoding the proinflammatory cytokines tumor necrosis factor, IL-6, IL-1, and GM-CSF, TH 1 cytokine IFN-γ, TH 17 cytokine IL-17, as well as the signature transcription factors of TH 1 (T-bet) and TH 17 (RORγt) cells. Arylpiperazine treatment reduced apoptosis and increased the activation of anti-apoptotic mediators Akt and p70S6 kinase in the CNS of EAE animals. The in vitro treatment with 6b protected oligodendrocyte cell line OLN-93 and neuronal cell line PC12 from mitogen-activated normal T cells or myelin basic protein-activated encephalitogenic T cells. In conclusion, arylpiperazine dopaminergic/serotonergic ligands suppress EAE through a direct neuroprotective action and decrease in CNS inflammation. Arylpiperazine dopaminergic/serotonergic ligands reduce neurological symptoms of acute autoimmune encephalomyelitis in rats without affecting the activation of autoreactive immune response, through mechanisms involving a decrease in CNS immune infiltration, as well as direct protection of CNS from immune-mediated damage. These data indicate potential usefulness of arylpiperazine-based compounds in the treatment of neuroinflammatory disorders such as multiple sclerosis.

Intraspinal Delivery of Polyethylene Glycol-coated Gold Nanoparticles Promotes Functional Recovery After Spinal Cord Injury.

Failure of the mammalian central nervous system (CNS) to regenerate effectively after injury leads to mostly irreversible functional impairment. Gold nanoparticles (AuNPs) are promising candidates for drug delivery in combination with tissue-compatible reagents, such as polyethylene glycol (PEG). PEG administration in CNS injury models has received interest for potential therapy, but toxicity and low bioavailability prevents clinical application. Here we show that intraspinal delivery of PEG-functionalized 40-nm-AuNPs at early stages after mouse spinal cord injury is beneficial for recovery. Positive outcome of hind limb motor function was accompanied by attenuated inflammatory response, enhanced motor neuron survival, and increased myelination of spared or regrown/sprouted axons. No adverse effects, such as body weight loss, ill health, or increased mortality were observed. We propose that PEG-AuNPs represent a favorable drug-delivery platform with therapeutic potential that could be further enhanced if PEG-AuNPs are used as carriers of regeneration-promoting molecules.

Large graphene quantum dots alleviate immune-mediated liver damage.

We investigated the effect of large (40 nm) graphene quantum dots (GQDs) in concanavalin A (Con A; 12 mg/kg i.v.)-induced mouse hepatitis, a T cell-mediated liver injury resembling fulminant hepatitis in humans. Intravenously injected GQDs (50 mg/kg) accumulated in liver and reduced Con A-mediated liver damage, as demonstrated by histopathological analysis and a decrease in liver lipid peroxidation and serum levels of liver transaminases. The cleavage of apoptotic markers caspase-3/PARP and mRNA levels of proapoptotic mediators Puma, Noxa, Bax, Bak1, Bim, Apaf1, and p21, as well as LC3-I conversion to autophagosome-associated LC3-II and expression of autophagy-related (Atg) genes Atg4b, Atg7, Atg12, and beclin-1, were attenuated by GQDs, indicating a decrease in both apoptosis and autophagy in the liver tissue. This was associated with the reduced liver infiltration of immune cells, particularly the T cells producing proinflammatory cytokine IFN-γ, and a decrease in IFN-γ serum levels. In the spleen of GQD-exposed mice, mRNA expression of IFN-γ and its transcription factor T-bet was reduced, while that of the IL-33 ligand ST2 was increased. The hepatoprotective effect of GQDs was less pronounced in ST2-deficient mice, indicating that it might depend on ST2 upregulation. In vitro, GQDs inhibited splenocyte IFN-γ production, reduced the activation of extracellular signal-regulated kinase in macrophage and T cell lines, inhibited macrophage production of the free radical nitric oxide, and reduced its cytotoxicity toward hepatocyte cell line HepG2. Therefore, GQDs alleviate immune-mediated fulminant hepatitis by interfering with T cell and macrophage activation and possibly by exerting a direct hepatoprotective effect.

Aberrant tissue positioning of metallophilic macrophages in the thymus of XCL1-deficient mice.

Metallophilic macrophages hold a strategic position within the thymic tissue and play a considerable function in thymic physiology. The development and positioning of these cells within thymic tissue are regulated by complex molecular mechanisms involving different cytokine/chemokine axes. Herein, we studied the role of XCL1 signaling in these processes. We show that in the XCL1-deficient thymus numerous metallophilic macrophages are aberrantly positioned in the thymic cortex, instead of their normal location in the cortico-medullary zone. Still, these cells retain their normal appearance: very large size with prominent, ramifying cytoplasmic prolongations. This shows that XCL1 signaling is not involved in morphological development, but rather in correct positioning of metallophilic macrophages within the thymic tissue. In contrast to thymic metallophilic macrophages, the positioning of splenic marginal metallophilic macrophages is not affected by XCL1-deficiency.

Autophagy inhibition uncovers the neurotoxic action of the antipsychotic drug olanzapine.

We investigated the role of autophagy, a controlled cellular self-digestion process, in regulating survival of neurons exposed to atypical antipsychotic olanzapine. Olanzapine induced autophagy in human SH-SY5Y neuronal cell line, as confirmed by the increase in autophagic flux and presence of autophagic vesicles, fusion of autophagosomes with lysosomes, and increase in the expression of autophagy-related (ATG) genes ATG4B, ATG5, and ATG7. The production of reactive oxygen species, but not modulation of the main autophagy repressor MTOR or its upstream regulators AMP-activated protein kinase and AKT1, was responsible for olanzapine-triggered autophagy. Olanzapine-mediated oxidative stress also induced mitochondrial depolarization and damage, and the autophagic clearance of dysfunctional mitochondria was confirmed by electron microscopy, colocalization of autophagosome-associated MAP1LC3B (LC3B henceforth) and mitochondria, and mitochondrial association with the autophagic cargo receptor SQSTM1/p62. While olanzapine-triggered mitochondrial damage was not overtly toxic to SH-SY5Y cells, their death was readily initiated upon the inhibition of autophagy with pharmacological inhibitors, RNA interference knockdown of BECN1 and LC3B, or biological free radical nitric oxide. The treatment of mice with olanzapine for 14 d increased the brain levels of autophagosome-associated LC3B-II and mRNA encoding Atg4b, Atg5, Atg7, Atg12, Gabarap, and Becn1. The administration of the autophagy inhibitor chloroquine significantly increased the expression of proapoptotic genes (Trp53, Bax, Bak1, Pmaip1, Bcl2l11, Cdkn1a, and Cdkn1b) and DNA fragmentation in the frontal brain region of olanzapine-exposed animals. These data indicate that olanzapine-triggered autophagy protects neurons from otherwise fatal mitochondrial damage, and that inhibition of autophagy might unmask the neurotoxic action of the drug.

Short-term exposure to UV-A, UV-B, and UV-C irradiation induces alteration in cytoskeleton and autophagy in human keratinocytes.

Ultraviolet radiation (UV) induces a series of morphological and ultrastructural alterations in human epidermis. Alterations observed in irradiated keratinocytes in morphological studies done before were cell retraction with loss of intercellular interactions, surface blebbing, and eventually cell death by apoptosis. The aim of this study was to investigate effect of UV-A, UV-B, and UV-C irradiation on the cytoskeleton of human keratinocytes. Keratinocytes were obtained by exfoliative scrubbing procedure from buccal mucosa of healthy individuals, and treated with UV-A, UV-B, and UV-C radiation. Afterward, treated cell were labeled with anti-alfa-tubulin and anti-human-cytokeratin and analyzed by light and confocal microscopy. The intensity of the cytokeratin labeling was found to be much higher in all irradiated cells than in control cells as observed by light microscope and measured with the Image J program. This measurement showed that with the decrease in the wavelengths of UV irradiation the intensity of the labeling of cells increases. Although the authors expected to find the collapse of microtubules toward the cell nucleus or their rearrangement in UV-treated cells, these alterations were not verified on cell smears labeled with anti-alfa tubulin observed by confocal microscope. When they used electron microscopy to examine in more detail the morphology of irradiated cells, they did not find apoptotic cells, but found features of autophagy in UV-treated keratinocytes. The authors assume that autophagy found as a result of UV radiation of human keratinocytes acts as a cytoprotective mechanism against UV-induced apoptosis.