Vitamin B12 attenuates leukocyte inflammatory signature in COVID-19 via methyl-dependent changes in epigenetic markings.

COVID-19 induces chromatin remodeling in host immune cells, and it had previously been shown that vitamin B12 downregulates some inflammatory genes via methyl-dependent epigenetic mechanisms. In this work, whole blood cultures from moderate or severe COVID-19 patients were used to assess the potential of B12 as adjuvant drug. The vitamin normalized the expression of a panel of inflammatory genes still dysregulated in the leukocytes despite glucocorticoid therapy during hospitalization. B12 also increased the flux of the sulfur amino acid pathway, that regulates the bioavailability of methyl. Accordingly, B12-induced downregulation of CCL3 strongly and negatively correlated with the hypermethylation of CpGs in its regulatory regions. Transcriptome analysis revealed that B12 attenuates the effects of COVID-19 on most inflammation-related pathways affected by the disease. As far as we are aware, this is the first study to demonstrate that pharmacological modulation of epigenetic markings in leukocytes favorably regulates central components of COVID-19 physiopathology.

Sichen Formula Ameliorates Lipopolysaccharide-Induced Acute Lung Injury via Blocking the TLR4 Signaling Pathways.

Purpose: Sichen (SC) formula is a classic prescription of Tibetan medicine. Due to its potential anti-inflammatory effect, the SC formula has been clinically used to treat respiratory diseases for many years in the Chinese Tibet region. The present study aimed to investigate the anti-inflammatory effect of SC and explore the underlying mechanisms. Methods: SC formula was characterized by HPLC analysis. The acute lung injury (ALI) mouse model was induced by direct intratracheal lipopolysaccharide (LPS) instillation, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. Meanwhile, RAW264.7 macrophages were stimulated by LPS. The contents of inflammatory mediators in the culture medium were determined by ELISA. Protein levels were determined by immunohistochemical staining or Western blotting. Nuclear localization of NF-κB, AP-1, and IRF3 was performed using immunofluorescence and Western blotting. Results: In the LPS-induced ALI mouse model, SC treatment suppressed the secretion of inflammatory mediators (TNF-α, IL-6, IL-1ß, MCP-1, MIP-1α, and RANTES) in BALF. SC treatment hindered the recruitment of macrophages. SC treatment also inhibited the expression of CD68, p-p65, and TLR4 in the lung tissue. In the LPS-exposed RAW264.7 cells, the cell viability was not changed up to 400 µg/mL of SC. SC concentration-dependently suppressed the production of nitric oxide, prostaglandin E2, TNF-α, IL-6, MCP-1, MIP-1α, and RANTES in LPS-challenged RAW264.7 cells. The expression levels of iNOS, COX-2, p-p38, p-JNK, p-ERK, p-TBK1, p-IKKα/ß, p-IκB, p-p65, p-c-Jun, and p-IRF3 were decreased after SC treatment. Moreover, the nuclear translocation of p65, c-Jun, and IRF3 was also blocked by SC treatment. Conclusion: SC treatment inhibited the inflammatory responses in LPS-induced ALI mouse model/RAW264.7 macrophages. The underlying mechanism of this action may be closely associated with the suppression of TLR4 signaling pathways. These research findings provide further pharmacological justifications for the medicinal use of SC in the management of respiratory diseases.

The improvement of nonalcoholic steatohepatitis by Poria cocos polysaccharides associated with gut microbiota and NF-κB/CCL3/CCR1 axis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) has been linked to inflammation induced by intestinal microbiota. Poria cocos polysaccharides (PCP) possesses anti-inflammation and immunomodulation functions; however, its preventive effects against NASH and potential mechanisms need to be explored. METHODS: The composition of PCP was determined using ion chromatography. C57BL/6 mice were administered the methionine and choline deficient (MCD) diet for 4 weeks to establish the NASH model or methionine-choline-sufficient (MCS) diet to serve as the control. Mice were assigned to the MCS group, MCD group, low-dose PCP (LP) group, and high-dose PCP (HP) group, and were administered the corresponding medications via gavage. Serum biochemical index analysis and liver histopathology examination were performed to verify the successful establishment of NASH model and to evaluate the efficacy of PCP. The composition of intestinal bacteria was profiled through 16S rRNA gene sequencing. Hepatic RNA sequencing (RNA-Seq) was performed to explore the potential mechanisms, which were further confirmed using qPCR, western blot, and immunohistochemistry. RESULTS: PCP consists of glucose, galactose, mannose, D-glucosamine hydrochloride, xylose, arabinose, and fucose. PCP could significantly alleviate symptoms of NASH, including histological liver damage, impaired hepatic function, and increased oxidative stress. Meanwhile, HP could reshape the composition of intestinal bacteria by significantly increasing the relative abundance of Faecalibaculum and decreasing the level of endotoxin load derived from gut bacteria. PCP could also downregulate the expression of pathways associated with immunity and inflammation, including the chemokine signaling pathway, Toll-like receptor signaling pathway, and NF-kappa B signaling pathway. The expression levels of CCL3 and CCR1 (involved in the chemokine signaling pathway), Tlr4, Cd11b, and NF-κb (involved in the NF-kappa B signaling pathway), and Tnf-α (involved in the TNF signaling pathway) were significantly reduced in the HP group compared to the MCD group. CONCLUSIONS: PCP could prevent the development of NASH, which may be associated with the modulation of intestinal microbiota and the downregulation of the NF-κB/CCL3/CCR1 axis.

The analgesic properties of Yu-Xue-Bi tablets in the inflammatory pain mice: By the inhibition of CCL3-mediated macrophage transmigration into the spinal cord.

ETHNOPHARMACOLOGICAL RELEVANCE: Until now, inflammatory pain, especially ones with central sensitization in the spinal cord, is far from effectively treated. Yu-Xue-Bi Tablets (YXB) is a patented medicine, which has been widely applied for inflammatory pain. However, its therapeutic characteristics and mechanism remain unknown. AIM OF THE STUDY: This study is designed to evaluate the analgesic characteristics and explore the underlying mechanism of YXB in the inflammatory pain model induced by Complete Freund's Adjuvant (CFA). MATERIALS AND METHODS: The analgesic effects were measured by Von Frey test. The expression of calcitonin gene-related peptide (CGRP) was quantified by immunofluorescence. The expression of immune factors was analyzed via Luminex assay. The further quantifications of C-C Motif chemokine ligand 3 (CCL3) were verified by Enzyme-linked immunosorbent assay (ELISA). The transmigration of macrophage and activation of microglia were evaluated by immunofluorescence. Spinal injections of purified CCL3, CCR1 antagonist (J113863) and CCR5 antagonist (Maraviroc) were used to clarify roles of CCL3 assumed in the pharmacological mechanism of YXB. RESULTS: In CFA mice, YXB ameliorated the mechanical allodynia in dose and time dependent way, suppressed the central sensitization in dose dependent way. In the L5 spinal cord, YXB downregulated the expression of macrophage M1 pro-inflammatory factors TNFRI and CCL3, inhibited the transmigration of circulating macrophage and the activation of microglia. Purified CCL3 led to the transmigration of macrophage, activation of microglia, central sensitization, and mechanical allodynia in the Sham mice. Inhibitors of CCR1 and CCR5 attenuated above symptoms in CFA mice. Purified CCL3 blocked YXB mediated down regulation of CCL3, inhibition of macrophage transmigration, but not activation of microglia. CONCLUSION: YXB exerts the analgesic effects by inhibiting CCL3-mediated peripheral macrophage transmigrate into spinal cord. This study provided a novel approach for inflammatory pain treatment and new insight into the pharmacological action of YXB.

DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B.

OBJECTIVE: To explore the molecular bases of Chinese medicine (CM) syndrome classification in chronic hepatitis B (CHB) patients in terms of DNA methylation, transcription and cytokines. METHODS: Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients (DNA methylation: 15 cases; serum cytokines: 62 cases) with different CM syndromes, including dampness and heat of Gan (Liver) and gallbladder (CHB1, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan stagnation and Pi (Spleen) deficiency (CHB2, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan and Shen (Kidney) yin deficiency (CHB3, DNA methylation: 5 cases, serum cytokines: 16 cases), CHB with hidden symptoms (HS, serum cytokines:16 cases) and healthy controls (DNA methylation: 6 cases). DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples. Genome-wide DNA methylation was detected using Human Methylation 450K Assay and furthered verified using pyrosequencing. Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay (ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively. RESULTS: Totally 28,667 loci, covering 18,403 genes were differently methylated among CHB1, CHB2 and CHB3 (P<0.05 and |Δß value| > 0.17). Further validation showed that compared with HS, the hg19 CHR6: 29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1 (P<0.05). However, they remained unaltered in CHB2 (P>0.05). Levels of Interleukin (IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups (P<0.05). Levels of macrophage inflammatory protein (MIP)-1α and MIP-1ß were higher in CHB3 than other groups and leukemia inhibitory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups (P<0.05). IL-12, MIP-1α and MIP-1ß concentrations were positively correlated with human leukocyte antigen F (HLA-F) mRNA expression (R2=0.238, P<0.05; R2=0.224, P<0.05; R=0.447, P<0.01; respectively). Furthermore, combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1, CHB2 and HS. CONCLUSIONS: Demethylation of CpG loci in 5' UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12, MIP-1α and MIP-1ß levels, indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB. REGISTRATION NO: ChiCTR-RCS-13004001.

DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B / 中国结合医学杂志

OBJECTIVE@#To explore the molecular bases of Chinese medicine (CM) syndrome classification in chronic hepatitis B (CHB) patients in terms of DNA methylation, transcription and cytokines.@*METHODS@#Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients (DNA methylation: 15 cases; serum cytokines: 62 cases) with different CM syndromes, including dampness and heat of Gan (Liver) and gallbladder (CHB1, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan stagnation and Pi (Spleen) deficiency (CHB2, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan and Shen (Kidney) yin deficiency (CHB3, DNA methylation: 5 cases, serum cytokines: 16 cases), CHB with hidden symptoms (HS, serum cytokines:16 cases) and healthy controls (DNA methylation: 6 cases). DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples. Genome-wide DNA methylation was detected using Human Methylation 450K Assay and furthered verified using pyrosequencing. Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay (ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively.@*RESULTS@#Totally 28,667 loci, covering 18,403 genes were differently methylated among CHB1, CHB2 and CHB3 (P<0.05 and |Δβ value| > 0.17). Further validation showed that compared with HS, the hg19 CHR6: 29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1 (P<0.05). However, they remained unaltered in CHB2 (P>0.05). Levels of Interleukin (IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups (P<0.05). Levels of macrophage inflammatory protein (MIP)-1α and MIP-1β were higher in CHB3 than other groups and leukemia inhibitory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups (P<0.05). IL-12, MIP-1α and MIP-1β concentrations were positively correlated with human leukocyte antigen F (HLA-F) mRNA expression (R2=0.238, P<0.05; R2=0.224, P<0.05; R=0.447, P<0.01; respectively). Furthermore, combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1, CHB2 and HS.@*CONCLUSIONS@#Demethylation of CpG loci in 5' UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12, MIP-1α and MIP-1β levels, indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB.@*REGISTRATION NO@#ChiCTR-RCS-13004001.

Biomarkers for febrile urinary tract infection in children.

BACKGROUND: The sensitivity and specificity of the leukocyte esterase test for the diagnosis of urinary tract infection (UTI) are suboptimal. Recent studies have identified markers that appear to more accurately differentiate children with and without UTI. The objective of this study was to determine the accuracy of these markers, which included CCL3, IL-8, CXCL1, TNF-alpha, IL-6, IFN-gamma, IL-17, IL-9, IL-2, and NGAL, in the diagnosis of UTI. METHODS: This was a prospective cross-sectional study to compare inflammatory proteins between urine samples from febrile children with a UTI, matched febrile controls without a UTI, and asymptomatic healthy controls. RESULTS: We included 192 children (75 with febrile UTI, 69 febrile controls, and 48 asymptomatic healthy controls). Urinary proteins that best discriminated between febrile children with and without UTI were NGAL, a protein that exerts a local bacteriostatic role in the urinary tract through iron chelation; CCL3, a chemokine involved in leukocyte recruitment; and IL-8, a cytokine involved in neutrophil recruitment. Levels of these proteins were generally undetectable in asymptomatic healthy children. CONCLUSIONS: NGAL, CCL3, and IL-8 may be useful in the early diagnosis of UTI. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01391793) A higher resolution version of the Graphical abstract is available as Supplementary information.

Antiinflammatory properties of antiLy6G antibody disappear during magnetic field exposure in rats with carrageenan induced acute paw inflammation.

Antiinflammatory properties of pulsed magnetic field (PMF) treatments or administration of antiLy6G antibody have been previously reported. In this study, we hypothesized that, the combination of PMF treatments and antiLy6G administration may synergistically potentiate their antiinflammatory actions. The effects of the combination of PMF treatments and antiLy6G administration were investigated by examining the inflammatory signs, histopathological properties of the inflamed site, and measuring the macrophage inflammatory protein-1 alpha (MIP-1α/CCL3) and myeloperoxidase (MPO) levels of inflamed paw tissues in rats with carrageenan-induced acute paw inflammation. In this present study, PMF treatments alone or administration of antiLy6G alone ameliorated the acute inflammation. However, their combination exacerbated the inflammatory signs, hyperalgesia, allodynia, edema and fever, and aggravated the inflammatory conditions by excessive infiltration of inflammatory cells to the inflamed site. These opposing effects of the combined treatments may correlate with enhanced levels of MIP-1α and MPO in inflamed paws. Present results indicated that the combination of the PMF treatments and antiLy6G administration may not provide additional benefits and may actually cause an aggravation of the acute inflammatory process. Findings may also suggest that during neutrophil or immune cell-targeted treatments for inflammatory states, magnetic field exposure may cause unexpected negative consequences.

Inflammatory response under zinc deficiency is exacerbated by dysfunction of the T helper type 2 lymphocyte-M2 macrophage pathway.

Nutritional zinc deficiency leads to immune dysfunction and aggravates inflammation. However, the underlying mechanism remains unknown. In this study, the relationship between macrophage subtypes (M1 and M2) and helper T lymphocytes (Th1 and Th2) was investigated using the spleen from rats fed zinc-deficient or standard diet. In experiment I, 5-week-old male Sprague-Dawley rats were fed a zinc-deficient diet (without zinc additives) or a standard diet (containing 0·01% zinc) for 6 weeks. In experiment II, the rats were divided into four groups: one group was fed a standard diet for 6 weeks; two groups were fed zinc-deficient diets and were injected three times a week with either saline or interleukin-4 (IL-4) (zinc-deficient/IL-4 i.p.); a fourth group (zinc-deficient/standard) was fed a zinc-deficient diet for 6 weeks followed by a standard diet for 4 weeks. In experiment I; GATA-binding protein 3 (GATA-3) protein level, M2 macrophage, CD3+  CD8+ cells, and IL-4/IL-13-positive cells significantly decreased in the spleens of the zinc-deficient group. Additionally, IL-1ß and macrophage inflammatory protein-1α (MIP-1α) mRNA levels significantly increased in the splenic macrophages of the zinc-deficient group. In experiment II; M2 macrophages, CD3+  CD8+ cells, IL-4/IL-13-positive cells, and GATA-3 protein levels significantly increased in the spleens of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Furthermore, IL-1ß and MIP-1α mRNA levels decreased in the splenic macrophages of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Zinc deficiency-induced aggravated inflammation is related to Th2 lymphocytes and followed by the association with loss of GATA-3, IL-4 and anti-inflammatory M2 macrophages. Importantly, IL-4 injection or zinc supplementation can reverse the effects of zinc deficiency on immune function.

Soybean oil containing ginseng saponins as adjuvants promotes production of cytokines and enhances immune responses to foot-and-mouth disease vaccine.

In the present study, the adjuvant effect of soybean oil containing ginseng root saponins (SO-GS-R) on the immune response to foot-and-mouth disease vaccine (FMDV) in mice was investigated. When immunized with FMDV antigen emulsified in an SO-GS-R formulation, mice generated remarkably higher serum antibody and cytokine responses than mice immunized with FMDV antigen alone. To elucidate the mechanisms underlying the adjuvant effect of SO-GS-R, we measured cytokines in serum and muscle tissue after intramuscular injection of SO-GS-R. The results showed that injection of SO-GS-R significantly increased the levels of IL-1ß, IL-5, IL-6, G-CSF, KC, MCP-1, MIP-1α, and MIP-1ß in both serum and muscle. These results suggested that SO-GS-R recruits neutrophils, eosinophils, T cells and macrophages, causing immune cell recruitment at the injection site, driving antigen-presenting cells to actively participate in the onset of immunity, and amplifying the immune responses. Considering its adjuvant activity and plant-derived properties, SO-GS-R should be further studied for its adjuvant effect on vaccines used in food animals.

Pharmacodynamics and proteomic analysis of acalabrutinib therapy: similarity of on-target effects to ibrutinib and rationale for combination therapy.

Acalabrutinib, a highly selective Bruton's tyrosine kinase inhibitor, is associated with high overall response rates and durable remission in previously treated chronic lymphocytic leukemia (CLL); however, complete remissions were limited. To elucidate on-target and pharmacodynamic effects of acalabrutinib, we evaluated several laboratory endpoints, including proteomic changes, chemokine modulation and impact on cell migration. Pharmacological profiling of samples from acalabrutinib-treated CLL patients was used to identify strategies for achieving deeper responses, and to identify additive/synergistic combination regimens. Peripheral blood samples from 21 patients with relapsed/refractory CLL in acalabrutinib phase I (100-400 mg/day) and II (100 mg BID) clinical trials were collected prior to and on days 8 and 28 after treatment initiation and evaluated for plasma chemokines, reverse phase protein array, immunoblotting and pseudoemperipolesis. The on-target pharmacodynamic profile of acalabrutinib in CLL lymphocytes was comparable to ibrutinib in measures of acalabrutinib-mediated changes in CCL3/CCL4 chemokine production, migration assays and changes in B-cell receptor signaling pathway proteins and other downstream survival proteins. Among several CLL-targeted agents, venetoclax, when combined with acalabrutinib, showed optimal complementary activity in vitro, ex vivo and in vivo in TCL-1 adoptive transfer mouse model system of CLL. These findings support selective targeting and combinatorial potential of acalabrutinib.

Dachaihu decoction ameliorates pancreatic fibrosis by inhibiting macrophage infiltration in chronic pancreatitis.

AIM: To explore the role of macrophages in chronic pancreatitis (CP) and the effect of Dachaihu decoction (DCHD) on pancreatic fibrosis in mice. METHODS: KunMing mice were randomly divided into a control group, CP group, and DCHD group. In the CP and DCHD groups, mice were intraperitoneally injected with 20% L-arginine (3 g/kg twice 1 d/wk for 6 wk). Mice in the DCHD group were administered DCHD intragastrically at a dose of 14 g/kg/d 1 wk after CP induction. At 2 wk, 4 wk and 6 wk post-modeling, the morphology of the pancreas was observed using hematoxylin and eosin, and Masson staining. Interleukin-6 (IL-6) serum levels were assayed using an enzyme-linked immunosorbent assay. Double immunofluorescence staining was performed to observe the co-expression of F4/80 and IL-6 in the pancreas. Inflammatory factors including monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α) and IL-6 were determined using real time-polymerase chain reaction. Western blot analysis was used to detect fibronectin levels in the pancreas. RESULTS: Compared with the control group, mice with 20% L-arginine-induced CP had obvious macrophage infiltration and a higher level of fibrosis. IL-6 serum concentrations were significantly increased. Double immunofluorescence staining showed that IL-6 and F4/80 were co-expressed in the pancreas. With the administration of DCHD, the infiltration of macrophages and degree of fibrosis in the pancreas were significantly attenuated; IL-6, MCP-1 and MIP-1α mRNA, and fibronectin levels were reduced. CONCLUSION: The dominant role of macrophages in the development of CP was mainly related to IL-6 production. DCHD was effective in ameliorating pancreatic fibrosis by inhibiting macrophage infiltration and inflammatory factor secretion in the pancreas.

Cardioprotective effect of Malva sylvestris L. in myocardial ischemic/reprefused rats.

PURPOSE: The present investigation evaluated the cardioprotective effect of Malva sylvestris L. (MS) on myocardial ischemic/reperfusion (MI/R) in rats. METHODS: All animals were divided into four groups: the sham operated group, ischemia/reperfusion group (MI/R), and the MS (250 and 500mg/kg) treated groups, who received MS 250 and 500mg/kg intragastrically for 15 consecutive days, respectively. At the end of the protocol, concentrations of aspartate transaminase (AST), creatine kinase-MB fraction (CK-MB) and lactate dehydrogenase (LDH) were estimated in serum and the concentrations of other parameters, such as C-reactive protein, macrophage inflammatory protein 1 alpha (MIP-1α), and nitric oxide (NO) were also estimated in the blood. Tissue homogenate concentrations of inflammatory cytokines, such as tumour necrosis factor-α (TNF-α), interlukin-1ß (IL-1ß), IL-10 and IL-6 as well as oxidative stress parameters, such as lipid peroxidation, catalase, and superoxide dismutase were estimated in MI/R rats. RESULT: Significant decreases (p<0.01) in AST, LDH, and CK-MB levels were observed in the MS-treated group compared with those in the MI/R group. C-reactive protein and MIP-1α levels decreased in the MS-treated group compared with those in the MI/R group. Plasma NO level was significantly enhanced in the MS-treated group than in the MI/R group. Moreover, treatment with MS significantly reduced TNF-α, IL-1ß, and IL-6 levels and increased IL-10 levels in the MS group compared with the MI/R group. Treatment with MS also attenuated the altered oxidative stress parameters in MI/R rats. CONCLUSION: The present results indicate the cardioprotective effects of MS of reducing oxidative stress and the inflammatory response in MI/R rats.

N-butyldeoxynojirimycin treatment restores the innate fear response and improves learning in mucopolysaccharidosis IIIA mice.

UNLABELLED: Mucopolysaccharidosis IIIA is a heritable neurodegenerative disorder resulting from the dysfunction of the lysosomal hydrolase sulphamidase. This leads to the primary accumulation of the complex carbohydrate heparan sulphate in a wide range of tissues and the secondary neuronal storage of gangliosides GM2 and GM3 in the brain. GM2 storage is associated with CNS deterioration in the GM2 gangliosidosis group of lysosomal storage disorders and may also contribute to MPS CNS disease. N-butyldeoxynojirimycin, an inhibitor of ceramide glucosyltransferase activity and therefore of ganglioside synthesis, was administered to MPS IIIA mice both prior to maximal GM2 and GM3 accumulation (early treatment) and after the maximum level of ganglioside had accumulated in the brain (late treatment) to determine if behaviour was altered by ganglioside level. Ceramide glucosyltransferase activity was decreased in both treatment groups; however, brain ganglioside levels were only decreased in the late treatment group. Learning in the water cross maze was improved in both groups and the innate fear response was also restored in both groups. A reduction in the expression of inflammatory gene Ccl3 was observed in the early treatment group, while IL1ß expression was reduced in both treatment groups. Thus, it appears that NB-DNJ elicits a transient decrease in brain ganglioside levels, some modulation of inflammatory cytokines and a functional improvement in behaviour that can be elicited both before and after overt neurological changes manifest. SYNOPSIS: NB-DNJ improves learning and restores the innate fear response in MPS IIIA mice by decreasing ceramide glucosyltransferase activity and transiently reducing ganglioside storage and/or modulating inflammatory signals.

Induction of Canonical Wnt Signaling by the Alarmins S100A8/A9 in Murine Knee Joints: Implications for Osteoarthritis.

OBJECTIVE: Both alarmins S100A8/A9 and canonical Wnt signaling have been found to play active roles in the development of experimental osteoarthritis (OA). However, what activates canonical Wnt signaling remains unknown. This study was undertaken to investigate whether S100A8 induces canonical Wnt signaling and whether S100 proteins exert their effects via activation of Wnt signaling. METHODS: Expression of the genes for S100A8/A9 and Wnt signaling pathway members was measured in an experimental OA model. Selected Wnt signaling pathway members were overexpressed, and levels of S100A8/A9 were measured. Activation of canonical Wnt signaling was determined after injection of S100A8 into naive joints and induction of collagenase-induced OA in S100A9-deficient mice. Expression of Wnt signaling pathway members was tested in macrophages and fibroblasts after S100A8 stimulation. Canonical Wnt signaling was inhibited in vivo to determine if the effects of S100A8 injections were dependent on Wnt signaling. RESULTS: The alarmins S100A8/A9 and members of the Wnt signaling pathway showed coinciding expression in synovial tissue in an experimental OA model. Synovial overexpression of selected Wnt signaling pathway members did not result in increased expression of S100 proteins. In contrast, intraarticular injection of S100A8 increased canonical Wnt signaling, whereas canonical Wnt signaling was decreased after induction of experimental OA in S100A9-deficient mice. S100A8 stimulation of macrophages, but not fibroblasts, resulted in increased expression of canonical Wnt signaling members. Overexpression of Dkk-1 to inhibit canonical Wnt signaling decreased the induction of matrix metalloproteinase 3, interleukin-6, and macrophage inflammatory protein 1α after injection of S100A8. CONCLUSION: Our findings indicate that the alarmin S100A8 induces canonical Wnt signaling in macrophages and murine knee joints. The effects of S100A8 are partially dependent on activation of canonical Wnt signaling.

Intrathecal curcumin attenuates pain hypersensitivity and decreases spinal neuroinflammation in rat model of monoarthritis.

Curcumin is a major component of turmeric and reportedly has anti-inflammatory and anti-oxidant effects. Neuroinflammation has been recognized to play an important role in the pathogenesis of various diseases in the central nervous system. Here we investigated the anti-nociceptive and anti-neuroinflammatory effect of curcumin on arthritic pain in rats. We found that repeated oral treatment with curcumin, either before or after complete Freund's adjuvant (CFA) injection, dose-dependently attenuated CFA-induced mechanical allodynia and thermal hyperalgesia, but had no effect on joint edema. Repeated intrathecal injection of curcumin reversed CFA-induced pain hypersensitivity. Furthermore, such a curcumin treatment reduced CFA-induced activation of glial cells and production of inflammatory mediators [interleukin-1ß (IL-1ß), monocyte chemoattractant protein-1 (MCP-1), and monocyte inflammatory protein-1 (MIP-1α)] in the spinal cord. Curcumin also decreased lipopolysaccharide-induced production of IL-1ß, tumor necrosis factor-α, MCP-1, and MIP-1α in cultured astrocytes and microglia. Our results suggest that intrathecal curcumin attenuates arthritic pain by inhibiting glial activation and the production of inflammatory mediators in the spinal cord, suggesting a new application of curcumin for the treatment of arthritic pain.

Lycium barbarum polysaccharide attenuates type II collagen-induced arthritis in mice.

No curative treatment is yet available for rheumatoid arthritis (RA), wherein chronic synovitis progresses to cartilage and bone destruction. Considering the recently recognized anti-inflammatory properties of Lycium barbarum polysaccharide (LBP; a derivative of the goji berry), we established the collagen type II-induced arthritis (CIA) mouse model to investigate the potential therapeutic effects and mechanisms of LBP. The CIA-induced changes and LBP-related effects were assessed by micro-computed tomography measurement of bone volume/tissue volume and by ELISA and western blotting detection of inflammatory mediators and matrix metalloproteinases (MMPs). The CIA mice showed substantial bone damage, bone loss, and increased concentrations of TNF-α, IL-6, IL-17, PGE2, MIP-1, anti-type II collagen IgG, MMP-1, and MMP-3. LBP treatments produced significant dose-dependent improvements in CIA-induced bone damage and bone loss, and significantly reduced CIA-stimulated expression of the inflammatory mediators and MMPs. Thus, LBP therapy can preserve bone integrity in CIA mice, possibly through down-regulation of inflammatory mediators.

Fish-oil-derived n-3 PUFAs reduce inflammatory and chemotactic adipokine-mediated cross-talk between co-cultured murine splenic CD8+ T cells and adipocytes.

BACKGROUND: Obese adipose tissue (AT) inflammation is characterized by dysregulated adipokine production and immune cell accumulation. Cluster of differentiation (CD) 8+ T cell AT infiltration represents a critical step that precedes macrophage infiltration. n-3 (ω-3) Polyunsaturated fatty acids (PUFAs) exert anti-inflammatory effects in obese AT, thereby disrupting AT inflammatory paracrine signaling. OBJECTIVE: We assessed the effect of n-3 PUFAs on paracrine interactions between adipocytes and primary CD8+ T cells co-cultured at the cellular ratio observed in obese AT. METHODS: C57BL/6 mice were fed either a 3% menhaden fish-oil + 7% safflower oil (FO) diet (wt:wt) or an isocaloric 10% safflower oil (wt:wt) control (CON) for 3 wk, and splenic CD8+ T cells were isolated by positive selection (via magnetic microbeads) and co-cultured with 3T3-L1 adipocytes. Co-cultures were unstimulated (cells alone), T cell receptor stimulated, or lipopolysaccharide (LPS) stimulated for 24 h. RESULTS: In LPS-stimulated co-cultures, FO reduced secreted protein concentrations of interleukin (IL)-6 (-42.6%), tumor necrosis factor α (-67%), macrophage inflammatory protein (MIP) 1α (-52%), MIP-1ß (-62%), monocyte chemotactic protein (MCP) 1 (-23%), and MCP-3 (-19%) vs. CON, which coincided with a 74% reduction in macrophage chemotaxis toward secreted chemotaxins in LPS-stimulated FO-enriched co-culture-conditioned media. FO increased mRNA expression of the inflammatory signaling negative regulators monocyte chemoattractant 1-induced protein (Mcpip; +9.3-fold) and suppressor of cytokine signaling 3 (Socs3; +1.7-fold), whereas FO reduced activation of inflammatory transcription factors nuclear transcription factor κB (NF-κB) p65 and signal transducer and activator of transcription 3 (STAT3) by 27% and 33%, respectively. Finally, mRNA expression of the inflammasome components Caspase1 (-36.4%), Nod-like receptor family pyrin domain containing 3 (Nlrp3; -99%), and Il1b (-68.8%) were decreased by FO compared with CON (P ≤ 0.05). CONCLUSION: FO exerted an anti-inflammatory and antichemotactic effect on the cross-talk between CD8+ T cells and adipocytes and has implications in mitigating macrophage-centered AT-driven components of the obese phenotype.

The impact of dietary fibers on dendritic cell responses in vitro is dependent on the differential effects of the fibers on intestinal epithelial cells.

SCOPE: In the present study, the direct interaction of commonly consumed fibers with epithelial or dendritic cells (DCs) was studied. METHODS AND RESULTS: The fibers were characterized for their sugar composition and chain length profile. When in direct contact, fibers activate DCs only mildly. This was different when DCs and fibers were co-cultured together with supernatants from human epithelial cells (Caco spent medium). Caco spent medium enhanced the production of IL-12, IL-1Ra, IL-6, IL-8, TNF-α, MCP-1 (monocyte chemotactic protein), and MIP-1α but this was strongly attenuated by the dietary fibers. This attenuating effect on proinflammatory cytokines was dependent on the interaction of the fibers with Toll-like receptors as it was reduced by Pepinh-myd88. The interaction of galacto-oligosaccharides, chicory inulin, wheat arabinoxylan, barley ß-glucan with epithelial cells and DCs led to changes in the production of the Th1 cytokines in autologous T cells, while chicory inulin, and barley ß-glucan reduced the Th2 cytokine IL-6. The Treg-promoting cytokine IL-10 was induced by galacto-oligosaccharides whereas chicory inulin decreased the IL-10 production. CONCLUSIONS: Our results suggest that dietary fibers can modulate the host immune system not only by the recognized mechanism of effects on microbiota but also by direct interaction with the consumer's mucosa. This modulation is dietary fiber type dependent.

Combination of macrophage inflammatory protein 1 alpha with existing therapies to enhance the antitumor effects on murine hepatoma.

Existing therapies such as irradiation or sorafenib have limited success in the treatment of hepatocellular carcinoma (HCC) due to tumor recurrence and metastasis. Therefore, combination with other therapeutics is often considered. Macrophage inflammatory protein-1 alpha (MIP-1α) is a member of a family of chemoattractant cytokines that can induce the migration of monocytes, which in turn can play a role in fighting tumors. This study investigated whether intravenous injection of MIP-1α in conjunction with irradiation or sorafenib could enhance the antitumor effects on murine hepatoma. An HCa-I tumor was grown on the right thigh of each C3H/HeN mouse. Mice were then treated with 10 Gy of irradiation, sorafenib, or a combination of MIP-1α with either irradiation or sorafenib, and antitumor and antimetastatic effects were then investigated. To understand the mechanisms, changes in the level of immunological markers were also evaluated. Combination treatment of MIP-1α with irradiation or sorafenib resulted in a significant enhancement of antitumor effects, prevention of lung metastasis and increase in host survival. This was achieved by significantly increasing the levels of the immunological markers: Cluster Differentiation (CD) 8, CD107A and CD11C. We conclude that a combination treatment of MIP-1α with irradiation or sorafenib would be a useful strategy for management of hepatoma.