Pro-inflammatory Cytokines IL-1α Research Articles

Suppression of Toll-like receptor 2-mediated proinflammatory responses by Mycobacterium tuberculosis protein Rv3529c.

Microorganisms are known to devise various strategies to thwart protective responses by the host. One such strategy is to incorporate sequences and domains in their genes/proteins that have similarity to various domains of the host proteins. In this study, we report that Mycobacterium tuberculosis protein Rv3529c exhibits significant similarity to the death domain of the TLR pathway adaptor protein MyD88. Incubation of macrophages with Rv3529c specifically inhibited TLR2-mediated proinflammatory responses. This included attenuated oxidative burst, reduced phosphorylation of MAPK-ERK, reduced activation of transcription factor NF-κB and reduced secretion of proinflammatory cytokines IFN-γ, IL-6, and IL-17A with a concomitant increased secretion of suppressor cytokines IL-10 and TGF-β. Importantly, Rv3529c significantly inhibited TLR2-induced association of MyD88 with IRAK1 by competitively binding with IRAK1. Further, Rv3529c mediated inhibition of apoptosis and phagosome-lysosome fusion. Lastly, incubation of macrophages with Rv3529c increased bacterial burden inside macrophages. The data presented show another strategy evolved by M. tuberculosis toward immune evasion that centers on incorporating sequences in proteins that are similar to crucial proteins in the innate immune system of the host.

Correlation between individual inflammation genetic profile and platelet rich plasma efficacy in hair follicle regeneration: a pilot study reveals prognostic value of IL-1a polymorphism.

Hair loss generates severe psychosocial implications. To date, exploring the prognostic factors of possible clinical benefit of autologous blood concentrate platelet rich plasma (PRP) was failed. The aim of our pilot study was to explore the correlation between the individual inflammation genetic profile and PRP efficacy in the treatment of hair follicle regeneration. 41 volunteers (25 men, 16 women) took part in this retrospective study. All the patients were scheduled for 4 sessions of PRP application with intervals of 40-60 days. All the patients were checked up at 6 weekly intervals for 6 months and, then, at the end of the first year. A panel of 5 polymorphisms on 4 genes (IL-1a, IL-1b, IL-6, and IL-10) implicated in the individual genetic inflammation profile were performed. A significant increase rate in hair density was noticed after the third month of treatment in 32/41 (78%) of the subjects. We found an interesting association between the pro-inflammatory cytokine IL-1α polymorphism C>A (rs17561) and responders to PRP treatment. The cases carrying C/C genotype (coding for Ser114) were 21 (66%) in responders and only 2 (22%) in non-responders (p<0.05). In addition, about IL-1a, the frequency of G/G genotype in responder patients was over two times lower in responder (31%) than in non-responder patients (78%). Our pilot study demonstrated a correlation between the individual genetic inflammatory profile and the efficacy of the PRP treatment in males. On the contrary, in females, it showed a negative correlation. IL-1a could be used as a prognostic value for PRP efficacy. Also, these results provide preliminary evidence that may encourage the design of controlled clinical trials to properly test this modus operandi on a large number of subjects.

Curcumin alleviates IL-17A-mediated p53-PAI-1 expression in bleomycin-induced alveolar basal epithelial cells.

Bleomycin-mediated inflammatory pathway is known to play an important role in the up regulation of oxidative stress. IL-17A is a pro-inflammatory cytokine involved in the modulation of fibrosis. The complex underlying mechanism for the said phenomenon remains unclear. This newly defined investigation was designed to understand the changes associated with 1L-17A mediated up-regulation of p53 and PAI-1 expression and the role of curcumin in attenuating this process. A549 cells were treated with bleomycin (BLM) and IL-17A to induce the inflammatory response in vitro. Curcumin, a known anti-inflammatory bioactive compound was administered as an intervention. Cytotoxicity in the treatment groups was assessed using Methyl thiazolyl tetrazolium (MTT) assay. Cell migration was evaluated using scratch assay. Protein expressions were studied using Western blot analysis for the downstream effector molecules of IL-17A mediated inflammatory pathways. In MTT assay, BLM treatment showed cytotoxicty upto 88% at a concentration of 1000 μM after 48 h of treatment. Cell migration assay results revealed that curcumin blocked the migration of cells to the area of the scratch. BLM treatment to the cells significantly induced the expression of pro-inflammatory cytokine IL-17A, which in turn modulated p53-PAI-1 expression. Bioactive compound curcumin showed anti-inflammatory and anti-apoptotic activity. Curcumin also regulated the BLM and IL-17A mediated changes in p53-PAI-1 expression. Curcumin has the ability to regulate inflammatory cytokines during BLM-induced injury and their effect on p53-PAI-1 expression. It can be used as a potential anti-inflammatory and anti-fibrinolytic component for intervening the epithelial cell damage.Very little information is provided till date on the inflammatory mechanism controlling the fibrinolytic system in acute lung injury (ALI). Damage to alveolar epithelial cells during ALI is important in the development of pulmonary fibrosis (PF). Most forms of ALI are characterized by defective alveolar fibrinolysis, inflammation, and fibrotic lesions. Recent reports show that alveolar epithelial cells express uPA, uPAR, and p53-mediated changes inhibit epithelial cell viability contributing to ALI. Thus, the roles of pulmonary epithelial cells in the inflammatory cascades activated after noninfectious injury, and the key signaling mediators of this process were actively investigated in this study. This investigation revealed that curcumin is an effective inhibitor of BLM-induced inflammation, apoptosis, and migration of basal alveolar epithelial cells. These results throw an insight into the possibility of developing curcumin as a novel therapeutic for ALI.

Curcumin affords neuroprotection and inhibits α-synuclein aggregation in lipopolysaccharide-induced Parkinson's disease model.

Parkinson's disease (PD) pathology is characterized by the abnormal accumulation and aggregation of the pre-synaptic protein α-synuclein in the dopaminergic neurons as Lewy bodies (LBs). Curcumin, which plays a neuroprotective role in various animal models of PD, was found to directly modulate the aggregation of α-synuclein in in vitro as well as in in vivo studies. While curcumin has been shown to exhibit strong anti-oxidant and anti-inflammatory properties, there are a number of other possible mechanisms by which curcumin may alter α-synuclein aggregation which still remains obscure. Therefore, the present study was designed to understand such concealed mechanisms behind neuroprotective effects of curcumin. An animal model of PD was established by injecting lipopolysaccharide (LPS, 5µg/5µl PBS) into the substantia nigra (SN) of rats which was followed by curcumin administration (40mg/kg b.wt (i.p.)) daily for a period of 21 days. Modulatory functions of curcumin were evident from the inhibition of astrocytic activation (GFAP) by immunofluorescence and NADPH oxidase complex activation by RT-PCR. Curcumin supplementation prevented the LPS-induced upregulation in the protein activity of transcription factor NFκB, proinflammatory cytokines (TNF-α, IL-1β, and IL-1α), inducible nitric oxide synthase (iNOS) as well as the regulating molecules of the intrinsic apoptotic pathway (Bax, Bcl-2, Caspase 3 and Caspase 9) by ELISA. Curcumin also resulted in significant improvement in the glutathione system (GSH, GSSG and redox ratio) and prevented iron deposition in the dopaminergic neurons as depicted from atomic absorption spectroscopy (AAS) and Prussian blue staining, respectively. Curcumin also prevented α-synuclein aggregates in the dopaminergic neurons as observed from gene as well as protein activity of α-synuclein using RT-PCR and IHC. Collectively, our results suggest that curcumin can be further pursued as a candidate drug in the molecules targeted therapy for PD and other related synucleopathies.

Layer-by-Layer Engineered Microbicide Drug Delivery System Targeting HIV-1 gp120: Physicochemical and Biological Properties.

The purpose of this study was to engineer a model anti-HIV microbicide (tenofovir) drug delivery system targeting HIV-1 envelope glycoprotein gp120 (HIV-1 g120) for the prevention of HIV sexual transmission. HIV-1 g120 and mannose responsive particles (MRP) were prepared through the layer-by-layer coating of calcium carbonate (CaCO3) with concanavalin A (Con A) and glycogen. MRP average particle size ranged from 881.7 ± 15.45 nm to 1130 ± 15.72 nm, depending on the number of Con A layers. Tenofovir encapsulation efficiency in CaCO3 was 74.4% with drug loading of 16.3% (w/w). MRP was non-cytotoxic to Lactobacillus crispatus, human vaginal keratinocytes (VK2), and murine macrophage RAW 264.7 cells and did not induce any significant proinflammatory nitric oxide release. Overall, compared to control, no statistically significant increase in proinflammatory cytokine IL-1α, IL-1β, IL-6, MKC, IL-7, and interferon-γ-inducible protein 10 (IP10) levels was observed. Drug release profiles in the presence of methyl α-d-mannopyranoside and recombinant HIV-1 envelope glycoprotein gp120 followed Hixson-Crowell and Hopfenberg kinetic models, indicative of a surface-eroding system. The one Con A layer containing system was found to be the most sensitive (∼2-fold increase in drug release vs control SFS:VFS) at the lowest HIV gp120 concentration tested (25 μg/mL). Percent mucoadhesion, tested ex vivo on porcine vaginal tissue, ranged from 10% to 21%, depending on the number of Con A layers in the formulation. Collectively, these data suggested that the proposed HIV-1 g120 targeting, using MRP, potentially represent a safe and effective template for vaginal microbicide drug delivery, if future preclinical studies are conclusive.

Dextromethorphan Exhibits Anti-inflammatory and Immunomodulatory Effects in a Murine Model of Collagen-Induced Arthritis and in Human Rheumatoid Arthritis

Dextromethorphan (d-3-methoxy-17-methylmorphinan, DXM) is a commonly used antitussive with a favorable safety profile. Previous studies have demonstrated that DXM has anti-inflammatory and immunomodulatory properties; however, the effect of DXM in rheumatoid arthritis (RA) remains unknown. Herein, we found that DXM treatment attenuated arthritis severity and proinflammatory cytokine expression levels, including TNF-α, IL-6, and IL-17A, in paw tissues of CIA mice. DXM treatment also reduced serum TNF-α, IL-6, and IL-17A levels of CIA mice and patients with RA. DXM further decreased the production of anti-CII IgG, IFN-γ, and IL-17A in collagen-reactive CD4+ T cells extracted from the lymph nodes of CIA mice. In vitro incubation of bone marrow–derived dendritic cells with DXM limited CD4+ T-cell proliferation and inflammatory cytokine secretion. In conclusion, our results showed that DXM attenuated arthritis symptoms in CIA mice and significantly reduced proinflammatory cytokines in patients with RA, suggesting that it can be used as an anti-arthritic agent.

T helper type 17 cells in immune-mediated glomerular disease.

CD4+ T cells are important drivers of tissue damage in immune-mediated renal diseases, such as anti-glomerular basement membrane glomerulonephritis, anti-neutrophil cytoplasmic antibody-associated glomerulonephritis, and lupus nephritis. The discovery of a distinct, IL-17-producing CD4+ T-cell lineage termed T helper type 17 (TH17) cells has markedly advanced current understanding of the pathogenic mechanisms of organ-specific immunity and the pathways that lead to target organ damage. TH17 cells are characterized by the expression of the transcription factor RORγt, the production of the pro-inflammatory cytokines IL-17A, IL-17F, IL-22, and high expression of the chemokine receptor C-C-motif chemokine receptor 6 (CCR6). An emerging body of evidence from experimental models and human studies supports a key role for these cells in the development of renal damage, and has led to the identification of targets to inhibit the production of TH17 cells in the intestine, their migration, or their actions within the kidney. Here, we describe the identification, regulation, and function of TH17 cells and their associated pathways in immune-mediated kidney diseases, with a particular focus on the mechanisms underlying renal tissue injury. We also discuss the rationale for the translation of these findings into new therapeutic approaches in patients with autoimmune kidney disease.

Involvement of NLRP10 in IL-1α induction of oral epithelial cells by periodontal pathogens

This study investigated the pathogenesis of periodontitis and the role of nucleotide-binding oligomerization domain-like receptor protein 10 (NLRP10). The human oral epithelial cell line HOK-16B was infected with two periodontal pathogens, Tannerella forsythia and Fusobacterium nucleatum, at various MOIs. RT-PCR and immunoblotting demonstrated that infection increased mRNA and protein expression of NLRP10, respectively. The siRNA-mediated NLRP10 knockdown significantly reduced IL-1α expression and secretion. Both bacteria induced phosphorylation of ERK, JNK and p38 MAP kinases in HOK-16B cells. NLRP10 knockdown impaired ERK phosphorylation only. ERK inhibition significantly decreased the expression of T. forsythia- and F. nucleatum-induced IL-1α. Our data suggest that NLRP10 is involved in activating the ERK signalling pathway in HOK-16B cells infected with T. forsythia and F. nucleatum. This pathway likely augments the pro-inflammatory cytokine IL-1α levels, which may play a critical role in periodontitis.

Large numbers of interleukins-22- and -17A-producing T helper cells in cholangiocarcinoma related to liver fluke infection.

Cholangiocarcinoma (CCA) associated with liver fluke infection involves inflammatory and immune processes; however, whether these involve the proinflammatory cytokine IL-17A and proliferative cytokine IL-22 remains unclear. Here, numbers of IL-22- and IL-17A-producing Th cells and cytokine concentrations in 30 patients with CCA and long-term liver fluke infection, 40 patients with liver-fluke infection but not CCA, and 16 healthy controls were compared. Analyses were performed using immunohistochemistry, flow cytometry, ELISA and RT-PCR. Immunohistochemical staining showed weaker expression of IL-22 and IL-17A in patients with CCA with than in those without liver fluke infection (P < 0.01). Flow cytometry revealed significantly greater median proportions of IL-22-producing T helper cells in patients with CCA (2.2%) than in those without it (0.69%) or controls (0.4%, P < 0.001). Similar results were obtained for IL-17A-producing T helper cells. ELISA revealed plasma concentrations of IL-22 were 1.3-fold higher in patients with CCA than in those without it and 4.6-fold higher than in controls (P < 0.001). Plasma concentrations of IL-17A were 2.5-fold higher in patients with CCA than in those without it, and 21-fold higher than in controls (P < 0.001). Amounts of IL-22 and IL-17A mRNAs in blood were significantly higher in patients with CCA than in the other two groups. Proportions of CD4+ CD45RO+ T cells producing IL-22 correlated with proportions producing IL-17A (r = 0.759; P < 0.001), and plasma concentrations of IL-22 correlated with those of IL-17A (r = 0.726; P < 0.001). These results suggest that both IL-17A and IL-22 affect development of CCA related to liver fluke infection.

Abstract 2698: IL-17A alone or in combination with IL-4/IFN-γ induces Duox2/DuoxA2 expression and reactive oxygen production in human colon and pancreatic cancer cells

Abstract NADPH oxidase (NOX)-derived reactive oxygen species (ROS) contribute significantly to inflammation-associated carcinogenesis. Expression of dual oxidase 2 (Duox2), one of seven members of the NOX gene family, is up-regulated in inflammatory bowel disease, chronic pancreatitis, and in many human malignancies including carcinomas of the colon, pancreas, prostate, lung, and breast. Previously, we demonstrated that Stat1 and/or NF-kB play a critical role in modulating the enhanced expression of Duox2, and its cognate maturation factor DuoxA2, by the pro-inflammatory mediators IFN-γ and lipopolysaccharide in human pancreatic cancer cells. This cytokine-mediated increase in Duox2 expression was responsible for a significant increase in tumor cell H2O2 production, DNA damage, and HIF-1α and VEGFA upregulation in pancreatic cancer cell lines. Using the T84 human colon cancer line, and BxPC-3, AsPC-1, and HTB134 pancreatic cancer cell lines, we now report that the pro-inflammatory cytokine IL-17A, pivotal in the adaptive immune response against various pathogens, and in the promotion of colon and pancreatic cancer progression, strongly induces Duox2/DuoxA2 mRNA and protein expression (alone or in combination with IL-4 or IFN-γ). In BxPC-3 cells, IL-17A enhances the expression of Duox2/DuoxA2 in a time- and concentration-dependent manner, which leads to a significant increase in the generation of extracellular H2O2. When IL-17A is combined with IL-4 in either T-84 or AsPc-1 lines, Duox2-mediated ROS formation is sufficient to produce a DNA damage response, indicated by the strong induction of γH2AX expression; moreover, in AsPc-1 cells, Duox2-specific siRNA significantly attenuated cytokine-induced ROS production and γH2AX phosphorylation. IL-17A in combination with IL-4 also significantly increases Stat3 phosphorylation and promotes proliferation in AsPC-1 cells. Ongoing cellular experiments are focused on whether the cytokine-mediated upregulation of Duox2/DuoxA2 is responsible for Stat3 activation and cell growth promotion in the K-ras mutated AsPC-1 cells. Bio-informatic analysis with the TCGA data set, as well as RT-PCR using surgically-resected human colon cancer samples compared to adjacent, uninvolved colonic epithelium revealed that Duox2 was significantly upregulated in human colon cancers. These studies, in concert with our previous data demonstrating high-level Duox protein expression in pancreatitis, pancreatic intra-epithelial neoplasia (PanIN), and frank pancreatic cancer, strongly suggest that ROS, produced as a consequence of cytokine-related Duox2 up-regulation, may be associated with the development of inflammation-related gastrointestinal malignancies. Citation Format: James H. Doroshow, Yongzhong Wu, Jiamo Lu, Smitha Antony, Jennifer Meitzler, Agnes Juhasz, Guojian Jiang, Iris Dahan, Mariam Konate, Krishnendu Roy. IL-17A alone or in combination with IL-4/IFN-γ induces Duox2/DuoxA2 expression and reactive oxygen production in human colon and pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2698. doi:10.1158/1538-7445.AM2017-2698

1,25-Dihydroxyvitamin D3 suppressed experimental autoimmune encephalomyelitis through both immunomodulation and oligodendrocyte maturation

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has recently been found to have the anti-inflammatory potential to suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis; however, its direct effect on neural cells is not clear. In the current study we show that 1,25(OH)2D3 treatment effectively suppressed clinical signs of ongoing EAE and reduced inflammation and demyelination scores in the central nervous system (CNS). The treatment significantly decreased production/expression of pro-inflammatory cytokines IFN-γ, GM-CSF and IL-17A, while it increased anti-inflammatory cytokines IL-4 and IL-10. Further, 1,25(OH)2D3 treatment effectively elevated the numbers of neural stem cells, oligodendrocyte precursor cells, as well as oligodendrocytes in disease lesions in the CNS. These results, together with its in vitro effect of inducing oligodendrocyte differentiation as shown in our previous findings, demonstrate that 1,25(OH)2D3 suppressed EAE not only by its immunomodulatory capacity, but also by its effect on oligodendrocyte differentiation and maturation, and thus has potential for remyelination and neural repair.

Ciproxifan improves cholinergic transmission, attenuates neuroinflammation and oxidative stress but does not reduce amyloid level in transgenic mice

AimThe present study is aimed to investigate the ability of ciproxifan, a histamine H3 receptor antagonist to inhibit β-amyloid (Aβ)-induced neurotoxicity in SK-N-SH cells and APP transgenic mouse model. Materials and methodsIn vitro studies was designed to evaluate the neuroprotective effects of ciproxifan in Aβ25–35 - induced SK-N-SH cells. For the in vivo study, ciproxifan (1 and 3mg/kg, i.p.) was administrated to transgenic mice for 15days and behaviour was assessed using the radial arm maze (RAM). Brain tissues were collected to measure Aβ levels (Aβ1–40 and Aβ1–42), acetylcholine (ACh), acetylcholinesterase (AChE), nitric oxide (NO), lipid peroxidation (LPO), antioxidant activities, cyclooxygenases (COX) and cytokines (IL-1α, IL-1β and IL-6), while plasma was collected to measure TGF-1β. ResultsThe in vitro studies demonstrated neuroprotective effect of ciproxifan by increasing cell viability and inhibiting reactive oxygen species (ROS) in Aβ25–35-induced SK-N-SH cells. Ciproxifan significantly improved the behavioural parameters in RAM. Ciproxifan however, did not alter the Aβ levels in APP transgenic mice. Ciproxifan increased ACh and showed anti-oxidant properties by reducing NO and LPO levels as well as enhancing antioxidant levels. The neuroinflammatory analysis showed that ciproxifan reduced both COX-1 and COX-2 activities, decreased the level of pro-inflammatory cytokines IL-1α, IL-1β and IL-6 and increased the level of anti-inflammatory cytokine TGF-1β. ConclusionThis present study provides scientific evidence of the use of ciproxifan via antioxidant and cholinergic pathways in the management of AD.

A 2-oxa-spiro[5.4]decane scaffold displays neurotrophic, neurogenic and anti-neuroinflammatory activities with high potential for development as a versatile CNS therapeutic

Following our recent discovery of a new scaffold exhibiting significant neurotrophic and neurogenic activities, a structurally tweaked analogue, embodying a 2-oxa-spiro [5.4]decane framework, has been conceptualised and found to be more potent and versatile. It exhibits enhanced neurotrophic and neurogenic action in in vitro, ex vivo and in vivo models and also shows robust neuroprotection in mouse acute cerebral stroke model. The observed attributes are traceable to the predominant activation of the TrkB-PI3K-AKT-CREB pathway. In addition, it also exhibits remarkable anti-neuroinflammatory activity by concurrently down-regulating pro-inflammatory cytokines IL-1α and IL-6, thereby providing a unique molecule with a trinity of neuroactivities, i.e. neurotrophic, neurogenic and anti-inflammatory. The new chemical entity disclosed here has the potential to be advanced as a versatile therapeutic molecule to treat stroke, depression, and possibly other neuropsychiatric disorders associated with attenuated neurotrophic/ neurogenic activity, together with heightened neuroinflammation.

‘The role of phagocytic NADPH oxidase and innate immune cells in the immune activation in toluene diisocyanate (TDI)-induced lung inflammation’

Abstract Innate lymphoid cells (ILCs) have been known to play an important role in development of lymphoid tissues and in the initiation of inflammation at barrier surfaces in response to infection or tissue damage. Isocyanates are low-molecular-weight compounds noted for inducing an oxidant stress-dependent pulmonary inflammation, which is the leading cause of occupational asthma. Our previous studies indicated that phagocytic NADPH oxidase is an essential regulator in TDI-induced airway inflammation characterized by a server lung inflammation with both Th17 and Th2 immune components. In this study, we hypothesized that ILCs activation may play an important role in phagocytic NADPH oxidase-mediated downstream immune mechanism in TDI-induced airway inflammation. The wild type (WT) and phagocytic NADPH oxidase deficient (Ncf1−/−) mice were sensitized and challenged with 3% TDI to induce lung inflammation. Our results showed that type 2 inflammatory cytokines, including IL-33 and IL-5, increased in both WT and Ncf1−/−mice but more significantly in TDI-induced lung inflammation. Moreover, the pro-inflammatory cytokine IL-1b and IL-17A production was only induced by TDI-exposure in WT but not in Ncf1−/− mice. We also found that the population of lineage negative and CD45 positive cells was identified in inflammation lung tissue. Comparison of ILCs population between TDI-induced WT mice and Ncf1−/− mice showed that the number of ILC2s and ILC3s were significantly higher in WT mice. These results suggested that ROS may modulate the activation of ILC2s, ILC3s and may determine the Th2 and Th17 cytokines production in the TDI-induced lung inflammation.

Itk independent development of natural Th17 cells and IL17A-driven hypersensitivity pneumonitis

Abstract The tyrosine kinase, Itk, plays a critical role in T cell receptor signaling, and we have previously shown that Itk is required for the development of Th17 cells and their production of the pro-inflammatory cytokine IL17A. IL17A has been implicated in numerous pulmonary inflammatory diseases. We have previously shown that Itk−/− mice do not develop allergic airway disease coupled with protected reduced production of IL17A in the lungs in a model of allergic asthma. Farmer’s lung is a subset of hypersensitivity pneumonitis caused by repeated exposure to the bacteria Saccharopolyspora rectivirgula (SR). This IL17A-driven disease is characterized by an influx of neutrophils and T lymphocytes. Here we show that a population of natural Th17 cell develop in the absence of Itk. Furthermore, exposure to SR drives robust IL17A response produced by CD4+ T cells even in the absence of Itk, accompanied by the development of pulmonary inflammation. These studies suggest that TCR signaling through Itk differentially regulates the development of conventional and natural Th17 cells. Understanding how Itk modulates the Th17 cytokine responses will allow us to better understand the precise role of Itk in the production of IL17A in airway inflammation.

Oral administration of methysticin improves cognitive deficits in a mouse model of Alzheimer's disease

IntroductionThere is increasing evidence for the involvement of chronic inflammation and oxidative stress in the pathogenesis of Alzheimer's disease (AD). Nuclear factor erythroid 2-related factor 2 (Nrf2) is an anti-inflammatory transcription factor that regulates the oxidative stress defense. Our previous experiments demonstrated that kavalactones protect neuronal cells against Amyloid β (Aβ)-induced oxidative stress in vitro by Nrf2 pathway activation. Here, we tested an in vivo kavalactone treatment in a mouse model of AD. MethodsThe kavalactone methysticin was administered once a week for a period of 6 months to 6 month old transgenic APP/Psen1 mice by oral gavage. Nrf2 pathway activation was measured by methysticin treatment of ARE-luciferase mice, by qPCR of Nrf2-target genes and immunohistochemical detection of Nrf2. Aβ burden was analyzed by CongoRed staining, immunofluorescent detection and ELISA. Neuroinflammation was assessed by immunohistochemical stainings for microglia and astrocytes. Pro-inflammatory cytokines in the hippocampus was determined by Luminex multi-plex assays. The hippocampal oxidative damage was detected by oxyblot technique and immunohistochemical staining against DT3 and 4-HNE. The cognitive ability of mice was evaluated using Morris water maze. ResultsMethysticin treatment activated the Nrf2 pathway in the hippocampus and cortex of mice. The Aβ deposition in brains of methysticin-treated APP/Psen1 mice was not altered compared to untreated mice. However, methysticin treatment significantly reduced microgliosis, astrogliosis and secretion of the pro-inflammatory cytokines TNF-α and IL-17A. In addition, the oxidative damage of hippocampi from APP/Psen1 mice was reduced by methysticin treatment. Most importantly, methysticin treatment significantly attenuated the long-term memory decline of APP/Psen1 mice. ConclusionIn summary, these findings show that methysticin administration activates the Nrf2 pathway and reduces neuroinflammation, hippocampal oxidative damage and memory loss in a mouse model of AD. Therefore, kavalactones might be suitable candidates to serve as lead compounds for the development of a new class of neuroprotective drugs.

Assessment of Th17/Treg cells and Th cytokines in an improved immune thrombocytopenia mouse model

ABSTRACTObjectives: The improved passive immune thrombocytopenia (ITP) mouse model has been extensively utilized for the study of ITP. However, how closely this model matches the human inflammation state and immune background is unclear. Our study aimed to explore the profile of Th cytokines and Th17/Treg cells in the model.Methods: We induced the ITP mouse model by dose-escalation injection of MWReg30. The serum levels of cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-17A, and TGF-β1) were measured by enzyme-linked immunosorbent assay and the frequency of Th17 and Treg cells was measured by flow cytometry. The mRNA expression of Foxp3 and RORrt was measured by real-time PCR.Results: The serum levels of cytokines IFN-γ, TGF-β1, IL-4, and IL-10 were significantly lower in ITP mice. The secretion of serum proinflammatory cytokines IL-2 and IL-17A and the percentage of Th17 cells showed no statistically significant increase. In ITP mice the frequency of Treg cells and mRNA expression of Foxp3 was significantly lower in splenocytes.Conclusion: Our data suggest that the improved passive ITP mouse model does not mimic the autoimmune inflammatory process of human ITP. Compared with human ITP, this model has a similar change in frequency of Treg cells, which may directly or indirectly result from antibody-mediated platelet destruction due to attenuated release of TGF-β.

Resveratrol ameliorates the chemical and microbial induction of inflammation and insulin resistance in human placenta, adipose tissue and skeletal muscle.

Gestational diabetes mellitus (GDM), which complicates up to 20% of all pregnancies, is associated with low-grade maternal inflammation and peripheral insulin resistance. Sterile inflammation and infection are key mediators of this inflammation and peripheral insulin resistance. Resveratrol, a stilbene-type phytophenol, has been implicated to exert beneficial properties including potent anti-inflammatory and antidiabetic effects in non-pregnant humans and experimental animal models of GDM. However, studies showing the effects of resveratrol on inflammation and insulin resistance associated with GDM in human tissues have been limited. In this study, human placenta, adipose (omental and subcutaneous) tissue and skeletal muscle were stimulated with pro-inflammatory cytokines TNF-α and IL-1β, the bacterial product lipopolysaccharide (LPS) and the synthetic viral dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) to induce a GDM-like model. Treatment with resveratrol significantly reduced the expression and secretion of pro-inflammatory cytokines IL-6, IL-1α, IL-1β and pro-inflammatory chemokines IL-8 and MCP-1 in human placenta and omental and subcutaneous adipose tissue. Resveratrol also significantly restored the defects in the insulin signalling pathway and glucose uptake induced by TNF-α, LPS and poly(I:C). Collectively, these findings suggest that resveratrol reduces inflammation and insulin resistance induced by chemical and microbial products. Resveratrol may be a useful preventative therapeutic for pregnancies complicated by inflammation and insulin resistance, like GDM.

Immunodepletion and Hypoxia Preconditioning of Mouse Compact Bone Cells as a Novel Protocol to Isolate Highly Immunosuppressive Mesenchymal Stem Cells

Compact bones (CB) are major reservoirs of mouse mesenchymal stem cells (mMSC). Here, we established a protocol to isolate MSC from CB and tested their immunosuppressive potential. Collagenase type II digestion of BM-flushed CB from C57B/6 mice was performed to liberate mMSC precursors from bone surfaces to establish nondepleted mMSC. CB cells were also immunodepleted based on the expression of CD45 (leukocytes) and TER119 (erythroid cells) to eliminate hematopoietic cells. CD45-TER119- CB cells were subsequently used to generate depleted mMSC. CB nondepleted and depleted mMSC progenitors were cultured under hypoxic conditions to establish primary mMSC cultures. CB depleted mMSC compared to nondepleted mMSC showed greater cell numbers at subculturing and had increased functional ability to differentiate into adipocytes and osteoblasts. CB depleted mMSC had high purity and expressed key mMSC markers (>85% Sca-1, CD29, CD90) with no mature hematopoietic contaminating cells (<5% CD45, CD11b) when subcultured to passage 5 (P5). Nondepleted mMSC cultures, however, were less pure and heterogenous with <72% Sca-1+, CD29+, and CD90+ cells at early passages (P1 or P2), along with high percentages of contaminating CD11b+ (35.6%) and CD45+ (39.2%) cells that persisted in culture long term. Depleted and nondepleted mMSC nevertheless exhibited similar potency to suppress total (CD3+), CD4+ and CD8+ T cell proliferation, in a dendritic cell allostimulatory one-way mixed lymphocyte reaction. CB depleted mMSC, pretreated with proinflammatory cytokines IFN-γ, TNF-α, and IL-17A, showed superior suppression of CD8+ T cell, but not CD4+ T cell proliferation, relative to untreated-mMSC. In conclusion, CB depleted mMSC established under hypoxic conditions and treated with selective cytokines represent a novel source of potent immunosuppressive MSC. As these cells have enhanced immune modulatory function, they may represent a superior product for use in clinical allotransplantation.

Caspase1 Inhibitor Ac-YVAD-CMK Prevents and Treats the Acute Graft Versus Host Disease in Mice

To explore the effect of Caspase 1 inhibitor Ac-YVAD-CMK on acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation(allo-HSCT) and its mechanism. Experiments were divided randomly into 3 groups: allogeneic hematopoietic stem cell transplantation combined with splenic cell infusion group (TS group, n=12), allogeneic hematopoietic stem cell transplantation combined with splenic cell infusion plus injection of low dose Caspase 1 inhibitor group (TS+low dose of C group, n=16) and plus high dose Caspase1 inhibitor (TS+high dose of C group, n=19). The body weight of mice in each group was dynamically detected, and the clinical manifestation of GVHD and score of aGVHD were determined, and the chimerism rate of mice was detected after transplantation for 14 days. Th1, Th2 and Th17 cells in peripheral blood were examined by flow cytometry. Peripheral proinflammatory cytokines IL-1β, IFN-γ, IL-1α and IL-18 were examined by enzyme-linked immunosorbent assay(ELISA). The tissues sections of GVHD target organs (liver, lung, colon and skin) were stained with HE for histopathologic examination and histopathologic score. Ac-YVAD-CMK could alleviate murine aGVHD and pathological injury, decreare the incidence and severity of aGVHD in recipient mice. The detection of Th cell subsets in peripheral blood by flow cytometry showed that compared with TS group, the Th1 cell ratio in TS+low dose of C and TS+high dose of C groups was significantly reduced (P<0.05), while the Th2 and Th17 cell ratio was significantly enhanced (P<0.05) in TS+low dose of high dose of C groups. The detection of peripheral inflamematory cytokines by ELISA demonstrated that the inflammatory cytokines including IL-1β,IFN-γ,IL-18 and IL-1α were reduced significantly (P<0.05). Ac-YVAD-CMK can improve aGVHD by inhibiting Caspase 1 and reducing the release of some inflammatory cytokines, thereby alleviated the aGVHD pathological damage.