Paeonol Attenuated Vascular Fibrosis Through Regulating Treg/Th17 Balance in a Gut Microbiota-Dependent Manner

Background: Paeonol (Pae) is a natural phenolic compound isolated from Cortex Moutan, which exhibits anti-atherosclerosis (AS) effects. Our previous work demonstrated that gut microbiota plays an important role during AS treatment as it affects the efficacy of Pae. However, the mechanism of Pae in protecting against vascular fibrosis as related to gut microbiota has yet to be elucidated.Objective: To investigate the antifibrosis effect of Pae on AS mice and demonstrate the underlying gut microbiota-dependent mechanism.Methods: ApoE-/- mice were fed with high-fat diet (HFD) to replicate the AS model. H&E and Masson staining were used to observe the plaque formation and collagen deposition. Short-chain fatty acid (SCFA) production was analyzed through LC-MS/MS. The frequency of immune cells in spleen was phenotyped by flow cytometry. The mRNA expression of aortic inflammatory cytokines was detected by qRT-PCR. The protein expression of LOX and fibrosis-related indicators were examined by western blot.Results: Pae restricted the development of AS and collagen deposition. Notably, the antifibrosis effect of Pae was achieved by regulating the gut microbiota. LC-MS/MS data indicated that the level of SCFAs was increased in caecum contents. Additionally, Pae administration selectively upregulated the frequency of regulatory T (Treg) cells as well as downregulated the ratio of T helper type 17 (Th17) cells in the spleen of AS mice, improving the Treg/Th17 balance. In addition, as expected, Pae intervention can significantly downregulate the levels of proinflammatory cytokines IL-1β, IL-6, TNF-α, and IL-17 in the aorta, and upregulate the levels of anti-inflammatory factor IL-10, a marker of Treg cells. Finally, Pae’s intervention in the gut microbiota resulted in the restoration of the balance of Treg/Th17, which indirectly downregulated the protein expression level of LOX and fibrosis-related indicators (MMP-2/9 and collagen I/III).Conclusion: Pae attenuated vascular fibrosis in a gut microbiota-dependent manner. The underlying protective mechanism was associated with the improved Treg/Th17 balance in spleen mediated through the increased microbiota-derived SCFA production. Collectively, our results demonstrated the role of Pae as a potential gut microbiota modulator to prevent and treat AS.

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Bacteriophages immunomodulate the response of monocytes

Experimental Biology and Medicine ◽

10.1177/1535370221995154 ◽

2021 ◽

pp. 153537022199515

Author(s):

Lídia Perea ◽

Lorena Rodríguez-Rubio ◽

Juan C Nieto ◽

Carlos Zamora ◽

Elisabet Cantó ◽

...

Keyword(s):

Cytokine Production ◽

Statistical Significance ◽

Control Sample ◽

Polymyxin B ◽

Dependent Manner ◽

Direct Role ◽

Gram Positive Bacteria ◽

Hla Dr ◽

Tnf Α

Bacteriophages are present in fluids from cirrhosis patients. However, their effect on the immune response is unknown. In this work, we explore the role of phages in the phenotype, function, and cytokine production of monocytes. We stimulated healthy monocytes with five different butanol-purified phage suspensions infective for Gram-negative and Gram-positive bacteria. We studied the expression of the monocyte markers involved in lipopolysaccharide recognition (LPS; CD14), antigen presentation (HLA-DR) and co-stimulation (CD86), and the concentration of induced cytokines (TNF-α, IFN-α, and IL-10) by phages. To confirm the direct role of phages without the interference of contaminating soluble LPS in phage suspensions, polymyxin B was added to the cell cultures. Phagocytosis experiments were assessed by flow cytometry using labeled phage suspensions. We observed that butanol-purified phages reduced the surface levels of CD14 and CD86 in monocytes and increased the secreted levels of TNF-α and IL-10 compared with the control sample containing only butanol buffer. All phage suspensions showed downregulation of HLA-DR expression but only Staphylococcus aureus phage contaminated with Escherichia coli reached statistical significance. The addition of polymyxin B did not restore the monocytic response induced by phages, suggesting that the effect was not caused by the presence of LPS. Monocytes were able to phagocyte phages in a dose- and time-dependent manner. To conclude, the phagocytosis of butanol-purified phages altered the phenotype and cytokine production of monocytes suggesting they become tolerogenic.

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The Anti-Inflammatory Potential of Mimosa caesalpiniifolia Following Experimental Colitis: Role of COX-2 and TNF-Alpha Expression

Drug Research ◽

10.1055/s-0043-119750 ◽

2017 ◽

Vol 68 (04) ◽

pp. 196-204 ◽

Cited By ~ 3

Author(s):

Marcelo Silva ◽

Wagner Vilegas ◽

Marcelo da Silva ◽

Ana Paiotti ◽

Mauricio Pastrelo ◽

...

Keyword(s):

Protective Action ◽

Experimental Colitis ◽

Distal Colon ◽

Tnf Alpha ◽

High Dose ◽

Dependent Manner ◽

Hydroalcoholic Extract ◽

Tnf Α ◽

Cox 2

AbstractThe aim of this study was to evaluate the preventive and/or protective action of Mimosa caesalpiniifolia (M. caesalpiniifolia) following experimental colitis in rats. The rats were randomized into ten groups (n=10 per group), as follows: G1 – Sham group:; G2 – TNBS group; G3, G4 –colitis and treated with hydroalcoholic extract of M. caesalpiniifolia 250 mg/kg/day after and before/after inducing colitis, respectively; G5, G6 – colitis and treated with hydroalcoholic extract of M. caesalpiniifolia at 125 mg/kg/day after and before/after inducing colitis respectively; G7,G8 – colitis and treated with ethylacetate fraction of M. caesalpiniifolia at 50 mg/kg/day after and before/after inducing colitis, respectively; G9,G10 – colitis and treated with ethylacetate fraction of M. caesalpiniifolia at 50 mg/kg/day after and before/after inducing colitis, respectively. Rats treated with hydroalcoholic extract of M. caesalpiniifolia for both doses showed lower tissue damage in the distal colon. Ethylacetate fraction was effective at the highest dose only when administrated after inducing colitis. A downregulation of COX-2 was detected to rats suffering colitis and treated with M. caesalpiniifolia at high dose. On the other hand, TNF-alpha immunoexpression decreased in groups treated with M. caesalpiniifolia at low dose after inducing colitis. In summary, our results suggest that M. caesalpiniifolia attenuated the lesions of the colon, reduced inflammation, and modulates the expression of COX-2 and TNF-α during chronic colitis induced by TNBS when using for therapeutic purposes on a dose-dependent manner.

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Comparative Transcriptome Analysis Reveals the Protective Mechanism of Glycyrrhinic Acid for Deoxynivalenol-Induced Inflammation and Apoptosis in IPEC-J2 Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/5974157 ◽

2020 ◽

Vol 2020 ◽

pp. 1-17

Author(s):

Xiaoxiang Xu ◽

Guorong Yan ◽

Juan Chang ◽

Ping Wang ◽

Qingqiang Yin ◽

...

Keyword(s):

Oxidative Stress ◽

Signaling Pathway ◽

Transcriptome Analysis ◽

Animal Feed ◽

Control Group ◽

Protective Mechanism ◽

Dependent Manner ◽

Gene Expressions ◽

Lactate Dehydrogenase Release ◽

Tnf Α

Deoxynivalenol (DON) is the most common mycotoxin that frequently contaminates human food and animal feed, resulting in intestinal diseases and systemic immunosuppression. Glycyrrhinic acid (GA) exhibits various pharmacological activities. To investigate the protective mechanism of GA for DON-induced inflammation and apoptosis in IPEC-J2 cells, RNA-seq analysis was used in the current study. The IPEC-J2 cells were treated with the control group (CON), 0.5 μg/mL DON, 400 μg/mL GA, and 400 μg/mL GA+0.5 μg/mL DON (GAD) for 6 h. Results showed that 0.5 μg/mL DON exposure for 6 h could induce oxidative stress, inflammation, and apoptosis in IPEC-J2 cells. GA addition could specifically promote the proliferation of DON-induced IPEC-J2 cells in a dose- and time-dependent manner. In addition, GA addition significantly increased Bcl-2 gene expression ( P < 0.05 ) and superoxide dismutase and catalase activities ( P < 0.01 ) and decreased lactate dehydrogenase release, the contents of malonaldehyde, IL-8, and NF-κB ( P < 0.05 ), the relative mRNA abundances of IL-6, IL-8, TNF-α, COX-2, NF-κB, Bax, and caspase 3 ( P < 0.01 ), and the protein expressions of Bax and TNF-α. Moreover, a total of 1576, 289, 1398, and 154 differentially expressed genes were identified in CON vs. DON, CON vs. GA, CON vs. GAD, and DON vs. GAD, respectively. Transcriptome analysis revealed that MAPK, TNF, and NF-κB signaling pathways and some chemokines played significant roles in the regulation of inflammation and apoptosis induced by DON. GA may alleviate DON cytotoxicity via the TNF signaling pathway by downregulating IL-15, CCL5, and other gene expressions. These results indicated that GA could alleviate DON-induced oxidative stress, inflammation, and apoptosis via the TNF signaling pathway in IPEC-J2 cells.

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A Novel Role of SIRT1/ FGF-21 in Taurine Protection Against Cafeteria Diet-Induced Steatohepatitis in Rats

Cellular Physiology and Biochemistry ◽

10.1159/000480649 ◽

2017 ◽

Vol 43 (2) ◽

pp. 644-659 ◽

Cited By ~ 7

Author(s):

Azza H. Abd Elwahab ◽

Basma K. Ramadan ◽

Mona F. Schaalan ◽

Amina M. Tolba

Keyword(s):

Caspase 3 ◽

B Cell Lymphoma ◽

Cafeteria Diet ◽

Control Group ◽

Albino Rats ◽

Protective Mechanism ◽

Alcoholic Fatty Liver ◽

Group I ◽

Tnf Α

Background: Non-alcoholic fatty liver disease (NAFLD) is one of the alarmingly rising clinical problems in the 21st century with no effective drug treatment until now. Taurine is an essential amino acid in humans that proved efficacy as a non-pharmacological therapy in a plethora of diseases; however, its impact on NAFLD remains elusive. The aim of the current study is to evaluate the protective mechanism of taurine in experimental steatohepatitis induced by junk food given as cafeteria-diet (CAF-D) in male albino rats. Methods: Forty adult male albino rats of local strain between 8-10 weeks old, weighing 150 ± 20 g, were divided into four equal groups: Group I (control group), Group II (Taurine group), Group III (CAF-D for 12 weeks) and Group IV (CAF-D +Taurine). CAF-D was given in addition to the standard chow for 12 weeks, where each rat was given one piece of beef burger fried in 15 g of sunflower oil, one teaspoonful of mayonnaise, and one piece of petit pan bread, weighing 60g/ piece. In the serum, liver function tests; ALT, AST, ALP, GGT and the lipid profile; TG, TC, HDL-C added to reduced glutathione (GSH) were assessed colorimetrically, while fibroblast growth factor (FGF)-21, adiponectin & interleukin (IL)-6 via ELISA. The same technique was used for the assays of the hepatic levels of FGF-21, silent information regulator (SIRT1), malondialdehyde (MDA),IL-10, tumor necrosis factor-α (TNF-α) as well as the apoptotic markers; caspase-3 and B-cell lymphoma (Bcl-2). Results: The cafeteria-diet induced steatohepatitis was reflected by significantly increased body and liver weight gain, elevation of liver enzymes; ALT, AST, ALP and GGT added to the dyslipidemic panel, presented as increased TC, TG, LDL-C and decreased HDL-C levels. The steatosis-induced inflammatory milieu, marked by elevated serum levels of FGF-21, IL-6, hepatic TNF-α, as well as reduced IL-10 and adiponectin, was associated with steatosis- induced hepatic oxidative stress, reflected by increased hepatic MDA and decreased GSH levels, along with stimulated caspase-3 and decline in BcL-2 hepatic levels. These pathological disturbances were significantly ameliorated by taurine supplementation and evidenced histopathologically. The cross talk between hepatic FGF-21 and SIRT1 and their association to the induced perturbations are novel findings in this study. Taurine's efficacy in restoration of hepatic structure and function is partially via the increase in SIRT1 and associated reduction of FGF-21. Conclusion: The findings of the current study prove the protective role of taurine in NAFLD via a novel role in the amelioration of FGF-21/ SIRT1 axis, which could be considered a new therapeutic target.

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Simplified Head-to-Tail Cyclic Polypeptides as Biomaterial-Associated Antimicrobials with Endotoxin Neutralizing and Anti-Inflammatory Capabilities

International Journal of Molecular Sciences ◽

10.3390/ijms20235904 ◽

2019 ◽

Vol 20 (23) ◽

pp. 5904 ◽

Cited By ~ 1

Author(s):

Na Dong ◽

Chensi Wang ◽

Xinran Li ◽

Yuming Guo ◽

Xiaoli Li

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Cyclic Peptide ◽

Skin Inflammation ◽

Rational Approach ◽

Inflammatory Factor ◽

Potential Candidate ◽

Dependent Manner ◽

Amphiphilic Peptides ◽

Tnf Α

The therapeutic application of antimicrobial peptides (AMPs), a potential type of peptide-based biomaterial, is impeded by their poor antimicrobial activity and potential cytotoxicity as a lack of understanding of their structure–activity relationships. In order to comprehensively enhance the antibacterial and clinical application potency of AMPs, a rational approach was applied to design amphiphilic peptides, including head-to-tail cyclic, linear and D-proline antimicrobial peptides using the template (IR)nP(IR)nP (n = 1, 2 and 3). Results showed that these amphiphilic peptides demonstrated antimicrobial activity in a size-dependent manner and that cyclic peptide OIR3, which contained three repeating units (IR)3, had greater antimicrobial potency and cell selectivity than liner peptide IR3, DIR3 with D-Pro and gramicidin S (GS). Surface plasmon resonance and endotoxin neutralization assays indicated that OIR3 had significant endotoxin neutralization capabilities, which suggested that the effects of OIR3 were mediated by binding to lipopolysaccharides (LPS). Using fluorescence spectrometry and electron microscopy, we found that OIR3 strongly promoted membrane disruption and thereby induced cell lysis. In addition, an LPS-induced inflammation assay showed that OIR3 inhibited the pro-inflammatory factor TNF-α in RAW264.7 cells. OIR3 was able to reduce oxazolone-induced skin inflammation in allergic dermatitis mouse model via the inhibition of TNF-α, IL-1β and IL-6 mRNA expression. Collectively, the engineered head-to-tail cyclic peptide OIR3 was considerable potential candidate for use as a clinical therapeutic for the treatment of bacterial infections and skin inflammation.

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Continuing Effect of Cytokines and Toll-Like Receptor Agonists on Indoleamine-2,3-Dioxygenase-1 in Human Periodontal Ligament Stem/Stromal Cells

Cells ◽

10.3390/cells9122696 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2696

Author(s):

Christian Behm ◽

Alice Blufstein ◽

Johannes Gahn ◽

Barbara Kubin ◽

Andreas Moritz ◽

...

Keyword(s):

Protein Expression ◽

Enzymatic Activity ◽

Stromal Cells ◽

Periodontal Ligament ◽

Dependent Manner ◽

Toll Like Receptor ◽

Indoleamine 2,3 Dioxygenase ◽

Tnf Α ◽

Inflammatory Stimuli ◽

Ifn Γ

Transplanted mesenchymal stem/stromal cells (MSCs) are a promising and innovative approach in regenerative medicine. Their regenerative potential is partly based upon their immunomodulatory activities. One of the most investigated immunomediators in MSCs, such as in periodontal ligament-derived MSCs (hPDLSCs), is indoleamine-2,3-dioxygenase-1 (IDO-1) which is upregulated by inflammatory stimuli, like cytokines. However, there are no data concerning continuing IDO-1 expression in hPDLSCs after the removal of inflammatory stimuli, such as cytokines and toll-like receptor (TLR) agonist-2 and TLR-3. Hence, primary hPDLSCs were stimulated with interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, TLR-2 agonist Pam3CSK4 or TLR-3 agonist Poly I/C. IDO-1 gene and protein expression and its enzymatic activity were measured up to five days after removing any stimuli. IL-1β- and TNF-α-induced IDO-1 expression and enzymatic activity decreased in a time-dependent manner after cessation of stimulation. IFN-γ caused a long-lasting effect on IDO-1 up to five days after removing IFN-γ. Both, TLR-2 and TLR-3 agonists induced a significant increase in IDO-1 gene expression, but only TLR-3 agonist induced significantly higher IDO-1 protein expression and enzymatic activity in conditioned media (CM). IDO-1 activity of Poly I/C- and Pam3CSK4-treated hPDLSCs was higher at one day after removal of stimuli than immediately after stimulation and declined to basal levels after five days. Among all tested stimuli, only IFN-γ was able to induce long-lasting IDO-1 expression and activity in hPDLSCs. The high plasticity of IDO-1 expression and its enzymatic activity in hPDLSCs due to the variable cytokine and virulence factor milieu and the temporal-dependent responsiveness of hPDLSCs may cause a highly dynamic potential of hPDLSCs to modulate immune responses in periodontal tissues.

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GPER modulates tone and coronary vascular reactivity in male and female rats

Journal of Molecular Endocrinology ◽

10.1530/jme-16-0117 ◽

2017 ◽

Vol 59 (2) ◽

pp. 171-180 ◽

Cited By ~ 9

Author(s):

Angelina Rafaela Debortoli ◽

Wender do Nascimento Rouver ◽

Nathalie Tristão Banhos Delgado ◽

Vinicius Mengal ◽

Erick Roberto Gonçalves Claudio ◽

...

Keyword(s):

Protein Expression ◽

Vascular Reactivity ◽

Coronary Circulation ◽

Superoxide Production ◽

Female Rats ◽

Male Rats ◽

Coronary Perfusion ◽

Dependent Manner ◽

Coronary Tone

Compared with age-matched men, premenopausal women are largely protected from coronary artery disease, a difference that is lost after menopause. The effects of oestrogens are mediated by the activation of nuclear receptors (ERα and ERβ) and by the G protein-coupled oestrogen receptor (GPER). This study aims to evaluate the potential role of GPER in coronary circulation in female and male rats. The baseline coronary perfusion pressure (CPP) and the concentration–response curve with a GPER agonist (G-1) were evaluated in isolated hearts before and after the blockade of GPER. GPER, superoxide dismutase (SOD-2), catalase and gp91phox protein expression were assessed by Western blotting. Superoxide production was evaluated ‘in situ’ via dihydroethidium fluorescence (DHE). GPER blockade significantly increased the CPP in both groups, demonstrating the modulation of coronary tone by GPER. G-1 causes relaxation of the coronary bed in a concentration-dependent manner and was significantly higher in female rats. No differences were detected in GPER, SOD-2 and catalase protein expression. However, gp91phox expression and DHE fluorescence were higher in male rats, indicating elevated superoxide production. Therefore, GPER plays an important role in modulating coronary tone and reactivity in female and male rats. The observed differences in vascular reactivity may be related to the higher superoxide production in male rats. These findings help to elucidate the role of GPER-modulating coronary circulation, providing new information to develop a potential therapeutic target for the treatment of coronary heart disease.

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Endogenous IL-β Selectively Converts T Regulatory Cells Into IL-17 Producers During Antigen Stimulation

Blood ◽

10.1182/blood.v116.21.586.586 ◽

2010 ◽

Vol 116 (21) ◽

pp. 586-586

Author(s):

Lequn Li ◽

Jin sub Kim ◽

Vassiliki A Boussiotis

Keyword(s):

T Cells ◽

T Regulatory Cells ◽

Neutralizing Antibody ◽

Treg Cells ◽

Conversion Process ◽

Regulatory Cells ◽

Antigen Stimulation ◽

Tnf Α ◽

Treg Population

Abstract Abstract 586 A major challenge of the immune system is to fight pathogens and tumor antigens while preserving tolerance to self-antigen. T regulatory cells (Treg) are critical extrinsic regulators of immune tolerance and maintenance of lymphoid homeostasis. Recently it was determined that, when used as cell-based immunosuppressive therapy, Treg have a potent effect in preventing GvHD in patients undergoing allogeneic stem cell transplantation. However, several studies suggest that the Treg phenotype is not at end stage of differentiation. Treg can express and produce effector cytokines including IFN-γ and IL-17 under certain conditions, particularly in the context of inflammatory milieu, suggesting that Treg may convert into inflammatory mediators. IL-1β and TNF-α are critical inflammatory cytokines that have been implicated in GvHD. The precise role and the mechanism(s) via which these cytokines may affect development of GvHD remain unclear. In the presence study, we sought to determine whether IL-1β and TNF-α regulate the properties of Treg and specifically whether these cytokines affect Treg expansion and/or conversion into IL-17 producing cells. CD4+CD25+Treg cells were isolated from B6 mice and were stimulated with anti-CD3-plus-anti-CD28 mAbs in the presence of either media, IL-1β or TNF-α. Addition of either cytokine induced Treg proliferation as determined by CFSE. Assessment of intracellular IL-17 expression by flow cytometry and IL-17 production by ELISA revealed that IL-1β but not TNF-α induced conversion of Treg into IL-17 producing cells, suggesting that conversion was mediated via pathways distinct from those that regulate cell cycle progression. To evaluate conversion of Treg to IL-17 producers during antigen stimulation and to determine the role of IL-1β in this process, we used neutral culture conditions in which no exogenous cytokines were supplied. Treg cells isolated from Foxp3GFP-KI mouse were added to cultures of naive conventional CD4+ T cells (Tc) in the presence of APC and anti-CD3 mAb. We found that these conditions preferentially induced conversion of Treg to IL-17 producing cells. To determine the role of IL-1β in this conversion process, we used IL-1β neutralizing antibody. Addition of anti-IL-1β neutralizing antibody reduced IL-17 production to almost undetectable levels. Because it has exogenous IL-6 can induce IL-17 production by both Treg and Tc, we evaluated whether endogenous IL-6 was involved in the conversion of Treg into IL-17 producing cells in our system. Addition of a combination of IL-6 neutralizing and IL-6 receptor blocking antibodies did not affect IL-17 production, suggesting that the conversion process of Treg into IL-17 producing cells was dependent on endogenous IL-1β rather than IL-6. To determine whether IL-1β was mandatory for this process, we used T cells from IL-1R deficient mice. Individual culture of IL−1R−/− Tc or IL-1R−/− Treg with wild type (wt) APC and co-culture of IL-1R−/− Tc and IL-1R−/− Treg with wt APC did not result in detectable IL-17 production. Similarly, no IL-17 production was observed when wt instead of IL-1R−/− Tc were used. In contrast, substitution of IL-1R−/− Treg with wt Treg resulted in abundant IL-17 production. To investigate the in vivo biological relevance of our findings we adoptively transferred Treg cells from either congenic B6.PL mice or IL-1R1−/− mice into IL-1R1−/− recipients, which were then immunized with KLH in IFA. Three days after immunization both IL-1R−/− Treg and IL-1R−/− Tc cells were incapable of producing detectable levels of IL-17 or expressing RORγt, the key transcriptional factor of IL-17. In contrast, a significant percentage of IL-17 and RORγt positive cells were detected within the adoptively transferred Thy1.1+ Treg population. Mechanistic analysis revealed that IL-1β induced activation of p38 and JNK in Treg and addition of pharmacologic inhibitors specific for these MAPKs abrogated IL-17 production. Our studies reveal that although Treg have primarily immunosuppressive functions they may also facilitate pro-inflammatory responses as they can be converted into IL-17 producing cells by IL-1β. These observations may have significant implications on clinical strategies that employ Treg for control of GvHD and suggest that further intervention might be required to prevent attainment of pro-inflammatory properties by Treg while maintaining their suppressive function. Disclosures: No relevant conflicts of interest to declare.

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Beneficial Effects of Celastrol on Immune Balance by Modulating Gut Microbiota in Dextran Sodium Sulfate-Induced Ulcerative Colitis

10.1101/2021.09.28.462065 ◽

2021 ◽

Author(s):

Desheng Hu ◽

Mingyue Li ◽

Weina Guo ◽

Yalan Dong ◽

Wenzhu Wang ◽

...

Keyword(s):

Ulcerative Colitis ◽

Gut Microbiota ◽

Protective Effect ◽

Intestinal Permeability ◽

Chronic Inflammatory Bowel Disease ◽

Protective Mechanism ◽

Therapeutic Effects ◽

Dependent Manner ◽

Colonic Inflammation ◽

Anti Inflammatory

Ulcerative colitis (UC) is a chronic inflammatory bowel disease caused by multi-factors including colonic inflammation and microbiota dysbiosis. Previous studies have indicated that Celastrol (CSR) has strong anti-inflammatory and immune-inhibitory effects. Here, we investigated the effects of CSR on colonic inflammation and the mucosal immunity in an experimental colitis model, and addressed the mechanism by which CSR preforms the protective effect. We characterized the therapeutic effects and the potential mechanism of CSR in treating UC using histological staining, intestinal permeability assay, cytokine assay, flow cytometry, fecal microbiota transplantation (FMT), 16S rRNA sequencing, untargeted metabolomics, and cell differentiation approaches. CSR administration significantly ameliorated DSS-induced colitis, as evidenced by the recovery of body weight and colon length, decreased disease activity index (DAI) score, as well as decreased intestinal permeability. CSR down-regulated the secretion of proinflammatory cytokines, upregulated the anti-inflammatory mediators, and improved the balances of Treg/Th1 and Treg/Th17 to maintain colonic immune homeostasis. However, the protective effects of CSR disappeared when the antibiotic cocktail was applied to deplete the gut microbiota, and the gut microbiota-mediated effect was confirmed by FMT. Furthermore, CSR treatment increased the gut microbiota diversity and composition, and raised the metabolic productions of pyruvate and adenosine, which probably involve in gut microbiota mediated protective effect. In conclusion, CSR ameliorates colonic inflammation in a gut microbiota-dependent manner. The underlying protective mechanism is associated with the rectified Treg/Th1 and Treg/Th17 balance, and increased pyruvate and adenosine production. The study provided the solid evidence that CSR might be a promising therapeutic drug for UC.

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Dysregulated Macrophage Pro-Inflammatory Cytokine Production and Chronic Colitis in Mice Lacking Both Gab2 and Gab3

Blood ◽

10.1182/blood.v124.21.457.457 ◽

2014 ◽

Vol 124 (21) ◽

pp. 457-457

Author(s):

Tamisha Y. Vaughan-Whitley ◽

Hikaru Nishio ◽

Barry Imhoff ◽

Zhengqi Wang ◽

Silvia T. Bunting ◽

...

Keyword(s):

Bone Marrow ◽

Protein Expression ◽

Knockout Mice ◽

Inflammatory Cytokine ◽

Cytokine Production ◽

Wild Type ◽

Inflammatory Cytokine Production ◽

Tnf Α ◽

Single Knockout

Abstract Macrophages are responsible for protecting the body against foreign invaders. We have been studying the role of Grb2-associated binding proteins (Gabs) in macrophage biology. In mice, Gabs are adaptor proteins that include three family members (Gab1, Gab2, and Gab3) that play critical regulatory roles in modulating cytokine receptor signaling. Gab2 knockout mice have no developmental defects but have impaired allergic responses, osteoclast defects, altered mast cell development, and altered hematopoiesis. Gab3 knockout mice have no defined phenotypes alone and although highly expressed in macrophages, a functional role was not found despite considerable focus on this cell type. Therefore, we set out to determine the combined role of Gab2 and Gab3 to determine whether they performed redundant functions not observable in single knockout mice. To analyze regulation of macrophage cytokine production, a Gab2/3 deficient mouse model was generated on the C57BL/6 background. Bone Marrow Derived Macrophages (BMDM) were expanded from the bone marrow (BM) of wild-type (WT), Gab2 and Gab3 single knockout and Gab2/3 knockout mice and found to similarly co-express CD11b and F4/80. However, Gab2/3 knockout BM produced only 30% of wild-type BMDM numbers. Despite reductions in BMDM absolute numbers, isolated BMDM demonstrated significant induction of pro-inflammatory cytokines TNF-α and IL-12 and anti-inflammatory cytokine IL-10 mRNA at baseline. Interestingly, after LPS stimulation (100ng/ml) we detected much greater induction of TNF-α and IL-12 mRNA and protein expression. Interestingly, despite increased IL-10 mRNA induction in Gab2/3 knockout BMDM, no IL-10 protein expression could be detected by Luminex assay. No changes were observed in production of interferon or STAT1 activation in these BMDM. Studies have shown that rapamycin treatment of macrophages suppresses mTORC1 and subsequently reduces IL-10 production and promotes pro-inflammatory cytokine production. Gab2 is known for its role in regulating the PI3K pathway through interactions with the p85 regulatory subunit of PI3K. Therefore, we also examined whether mTOR activation was effected by Gab2/3 deficiency causing altered cytokine expression. Deletion of Gab2/3 in BMDMs treated with LPS showed an inhibition of 4EBP1 phosphorylation and increased AKT phosphorylation. These results suggest that Gabs may play a critical role in modulating mTOR activation and potentially causing defects in protein translation that reflect in reduced IL-10 cytokine levels in Gab2/3 knockout cells. IL-10 has a critical immunoregulatory role that is dysregulated in patients with inflammatory bowel disease. IL-10 deficient mice develop colitis due to loss of mucosal immune tolerance. Strikingly, as early as two months of age in vivo 12/32 (37.5%) Gab2/3 knockout mice developed rectal prolapse and suffered from diarrhea within a six month period. Histological analysis of isolated colons using a scoring system confirmed spontaneous development of colitis in Gab2/3 knockout mice compared to no phenotypes observed in WT and single knockout controls. To determine whether the BM was directly involved in the disease, BM chimeras were generated using irradiated WT mice as recipients and Gab2/3 knockout mice as donors. Susceptible recipients receiving Gab2/3 knockout BM showed a more invasive colitis phenotype than the spontaneous disease and resulted in forced euthanization due to body weight decreases greater than 25%. Multiple ulcerations were present in most of the colon proximal region, with extensive epithelial damage, transmural inflammation, and in some mice adenocarcinoma. Notably, we did not observe adenocarcinoma in untransplanted Gab2/3 knockout mice, suggesting that epithelial deletion of Gab2/3 may suppress cancer whereas in the bone marrow chimera model, the epithelial cells are WT and can be transformed. Similar phenotypes were also observed in secondary transplant recipients. Lastly, treatment of Gab2/3 knockout mice with dextran-sodium-sulfate (DSS) induced rapid severe colitis that resulted in death of 80% and 40% of Gab2/3 knockout and WT mice respectively. Overall, these observations demonstrate a major redundant role for Gab2 and Gab3 in macrophage immune surveillance required for the prevention of colitis in mice. Disclosures No relevant conflicts of interest to declare.

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