Cytokine Production In Mice Research Articles

Astaxanthin Alleviates Autoimmune Hepatitis by Modulating CD8+ T Cells: Insights From Mass Cytometry and Single-Cell RNA Sequencing Analyses.

Astaxanthin (ASX) is an oxygen-containing non-vitamin A carotenoid pigment. However, the role of ASX in autoimmune hepatitis (AIH) remains unclear. In this study, a mouse model of AIH is established induced by concanavalin A (ConA). Mass cytometry and single-cell RNA sequencing (scRNA-seq) are used to analyze the potential role of ASX in regulating the immune microenvironment of AIH. ASX treatment effectively alleviated liver damage induced by ConA and downregulated pro-inflammatory cytokines production in mice. Mass cytometry and scRNA-seq analyses revealed a significant increase in the number of CD8+ T cells following ASX treatment. Functional markers of CD8+ T cells, such as CD69, MHC II, and PD-1, are significantly downregulated. Additionally, specific CD8+ T cell subclusters (subclusters 4, 13, 24, and 27) are identified, each displaying distinct changes in marker gene expression after ASX treatment. This finding suggests a modulation of CD8+ T cell function by ASX. Finally, the key transcription factors for four subclusters of CD8+ T cells are predicted and constructed a cell-to-cell communication network based on receptor-ligand interactions probability. In conclusion, ASX holds the potential to ameliorate liver damage by regulating the number and function of CD8+ T cells.

Enhancing Therapeutic Efficacy of Cinnamon Essential Oil by Nanoemulsification for Intravaginal Treatment of Candida Vaginitis.

Due to its prevalence, recurrence, and the emergence of drug-resistance, Candida vaginitis significantly impacts the well-being of women. Although cinnamon essential oil (CEO) possesses antifungal activity, its hydrophobic properties limit its clinical application. To overcome this challenge, a nanoemulsification technology was employed to prepare cinnamon essential oil-nanoemulsion (CEO@NE), and its therapeutic efficacy and action mechanism for Candida vaginitis was investigated in vivo and in vitro. CEO@NE, composed of 4% CEO, 78% distilled water, and 18% Tween 80, was prepared by ultrasonic nanoemulsification. The physical properties, anti-Candida activity, cytotoxicity, immunomodulatory potential and storage stability of CEO@NE were explored. Subsequently, the effect of intravaginal CEO@NE treatment on Candida vaginitis was investigated in mice. To comprehend the possible mechanism of CEO@NE, an analysis was conducted to ascertain the production of intracellular reactive oxygen species (ROS) in C. albicans. CEO@NE, with the droplet size less than 100 nm and robust storage stability for up to 8 weeks, exhibited comparable anti-Candida activity with CEO. CEO@NE at the concentration lower than 400 μg/mL had no cytotoxic and immunomodulatory effects on murine splenocytes. Intravaginal treatment of CEO@NE (400 μg/mL, 20 μL/day/mouse for 5 consecutive days) curbed Candida colonization, ameliorated histopathological changes, and suppressed inflammatory cytokine production in mice intravaginally challenged with C. albicans. Notably, this treatment preserved the density of vaginal lactic acid bacteria (LAB) crucial for vaginal health. Co-culturing C. albicans with CEO@NE revealed concentration-dependent augmentation of intracellular ROS generation and ensuing cell death. In addition, co-culturing LPS-stimulated murine splenocytes with CEO@NE yielded a decrease in the generation of cytokines. This discovery provides insight into the conceivable antifungal and anti-inflammatory mechanisms of CEO@NE to tackle Candida vaginitis. CEO@NE offers a promising avenue to address the limitations of current treatments, providing novel strategy for treating Candida vaginitis.

Expression of circadian regulatory genes is dysregulated by increased cytokine production in mice subjected to concomitant intestinal injury and parenteral nutrition.

We have developed a mouse model of Parenteral Nutrition Associated Cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of bile acid and sterol signaling and transport. In the liver, the master circadian gene regulators Bmal/Arntl and Clock drive circadian modulation of hepatic functions, including bile acid synthesis. Once activated, Bmal and Clock are downregulated by several transcription factors including Reverbα (Nr1d1), Dbp (Dbp), Dec1/2 (Bhlhe40/41), Cry1/2 (Cry1/2) and Per1/2 (Per1/2). The aim of this study was to examine the effects of PN on expression of hepatic circadian rhythm (CR) regulatory genes in mice. WT, IL1KO or TNFRKO mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN) and the expression of key CR regulatory transcription factors evaluated. Animals were NPO on a 14 hr light-dark cycle and were administered PN continuously over 24 hrs. Mice were sacrificed, and hepatic tissue obtained at 9-10AM (Zeitgeber Z+3/Z+4 hrs). PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and total bilirubin and the effect of i.p. injection of recombinant IL-1β (200ng/mouse) or TNFα (200ng/mouse) on CR expression was examined after 4 hrs. In the PNAC model, DSS-PN increased serum biomarkers of hepatic injury (ALT, AST, serum bile acids) which was suppressed in both DSS-PN IL1KO and DSS-PN TNFRKO mice. In WT DSS-PN, mRNA expression of Arntl and Dec1 was suppressed corresponding to increased Nr1d1, Per2, Dbp and Dec2. These effects were ameliorated in both DSS-PN IL1KO and DSS-PN TNFRKO groups. Western analysis of the circadian transcription factor network revealed in WT mice DSS-PN significantly suppressed Reverbα, Bmal, Dbp, Per2 and Mtnr1b. With the exception of Dbp, DSS-PN mediated suppression was ameliorated by both IL1KO and TNFRKO. Intraperitoneal injection of IL-1β or TNFα into WT mice increased serum AST and ALT and suppressed mRNA expression of Nr1d1, Arntl and Clock and increased Dbp and Per2. Altered expression of CR-dependent regulatory genes during PNAC accompanies cholestasis and is, in part, due to increased cytokine (IL-1β and TNFα) production. Evaluation of the effects of modulating CR in PNAC thus deserves further investigation.

Early life stress (ELS) induces aortic stiffening and increased cytokine production in mice

ELS exposure (childhood adversity such as household dysfunction, abuse, or neglect) is an independent risk factor for adult-onset cardiovascular disease (CVD). ELS exposure is associated with increased circulating inflammatory markers in humans, as well as elevated aortic inflammatory monocytes in mice. The cellular sources and mechanisms of excessive cytokine production behind these findings are still poorly understood. We hypothesized that mice exposed to ELS, maternal separation with early weaning (MSEW), will have greater arterial stiffening and greater reactive aortic macrophages leading to a larger cytokine response when activated with LPS than normally reared (NR) mice. The MSEW model includes pups separated from the dam 4h/day (postnatal day (PD) 2 to PD5), 8h/day (PD6 to 16), and weaned at PD17. NR pups remain with the dams until weaning at PD21. Pulse wave velocity (PWV), a measure of aortic stiffness, determined at 6 wks of age found that MSEW mice had elevated PWV as compared to NR (NR: 1.2±0.08 mm/sec, MSEW: 2.1±0.04 mm/sec, p<0.0001, n=4,6). In the aorta, total F4/80+ macrophages as well as cells expressing activation markers CD86 and MHCII were elevated in 14 wk old MSEW mice (%F4/80+: NR:2.73±0.34, MSEW 5.04±0.56, p=0.004, n=7,5; %CD86: NR: 0.12±0.03, MSEW: 0.52±0.09, p=0.004, n=7,5; %MHCII: NR: 1.14±0.09, MSEW: 1.92±0.22, p=0.001, n=7,5). MSEW mice showed significantly elevated levels of macrophages (%CD11c-CD11b+F4/80+ of CD45+) in the spleen red pulp (NR: 1.09±0.04, MSEW: 1.28±0.07, P=0.03, n=14,10). However, expression of activation markers CD86 and MHCII as well as TNFα, IL-1b and IL6 in splenic macrophages from 14 wk old MSEW mice did not significantly differ between the MSEW and NR cohorts. Finally, we isolated splenic macrophages, stimulated with LPS (100 ng/mL) for 24 hrs and cytokine secretion measured by cytokine array. The qualitative array showed that the total supernatant of macrophage cultures from MSEW sample had greater IL1-ra, IL-16, G-CSF, CXCL10, macrophage inflammatory protein 1B and 2, and TNFα compared to cultures from NR samples. These results suggest that macrophages of MSEW mice are primed for inflammatory responses and may secrete intracellular cytokines compared to macrophages from NR mice. Elevated PWV is an early indicator for the development of CVD, seen as early as 6 wks in MSEW mice. Taken together these findings suggest a greater propensity for those who have experienced ELS to develop CVD. Future studies will aim to elucidate the role of the elevated macrophage derived cytokines in arterial stiffening. NIH R25 DK 112731, NIH R25 DK 115353, NIH T32 DK 116672 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Recombinant Hexon Protein as a New Bovine Adenovirus Type 3 Subunit Vaccine Candidate.

Bovine adenovirus (BAdV) type 3 causes respiratory and gastroenteric diseases of varying severity in cattle, particularly newborn calves. Trials have been conducted of a vaccination against the diseases caused by BAdV using both modified live-virus and inactivated-virus preparations in cattle, but no commercial BAdV-3 vaccine has yet reached the market. Therefore, there is an urgent need to develop new, safe, and effective vaccines against BAdV-3. Recombinant hexon protein (rhexon) of BAdV-3 was expressed in the E. coli system to evaluate immune response in mice and goats. Antibody responses and cytokine levels were analysed and the effects of administrations of different amounts of recombinant protein compared. Long-term antibody production was evaluated by indirect ELISA, and the total immunoglobulin G secreted by goats and mice immunised with the purified rhexon protein was determined. The immunised mice had a stronger antibody response than the control group at eight weeks post vaccination. The immunised groups also showed significantly higher (P ˂ 0.05) expression of interferon-γ, interleukin 2 (in mice), and interleukin 21 (in goats) at four weeks. Furthermore, vaccination with rhexon was able to induce long-term antibody production for at least 16 weeks in mice and goats. The rhexon protein induced immune responses, especially long-term antibody production and T helper 1 cell cytokine production in mice and goats. The immunogenic properties of this protein make it a promising subunit vaccine antigen.

Polynucleotides Compensate for Impaired T-Dependent Antibody Production Induced in C57Bl/6 Mice by a Nucleotide-Free Diet both in Vivo and in Vitro, but a Mononucleotide-Nucleoside Mixture Is Effective Only In Vivo

Actions of nucleotides on in vitro humoral immune responses were studied in mice fed a nucleotide-free diet, a nucleotide-free diet plus a mononucleotide-nucleoside mixture or a nucleotide-free diet plus yeast RNA (polynucleotides). Cultured spleen cells from mice fed a nucleotide-free diet produced fewer numbers of antibody-secreting cells in response to a T-dependent antigen, compared with those from controls fed nucleotide-supplemented diets. Immunoglobulin M concentrations in these supernatants were significantly lower in cultured cells from mice fed the nucleotide-free diet or the nucleotide-free diet plus the mononucleotide/nucleoside mixture compared with concentrations in cells from mice fed the nucleotide-free diet plus RNA. Concanavalin A-potentiated cytokine (interleukin-4 and interleukin-5) production by purified T helper cells was also lower in cultured cells from mice fed a nucleotide-free diet than in those from mice fed nucleotide-supplemented diets. In vivo supplementation with the mononucleotide/nucleoside mixture restored impaired in vitro antibody and concanavalin A-potentiated cytokine production in mice fed a nucleotide-free diet. However, addition of RNA to the culture enhanced antibody production in spleen cells from mice of all diet groups. Supplementing the culture with RNA did not enhance mitogen-potentiated cytokine production. This in vitro action of RNA was retained after the removal of oligonucleotides (molecular weight <1000), but was reduced by modification of bases and cleavage of phosphodiester bonds of RNA. Thus the in vitro action of RNA is mainly attributed to polynucleotides, indicating their potential role in modulation of local humoral immune responses in the body.

Comparison of cytokine production in mice inoculated with messenger RNA vaccines BNT162b2 and mRNA-1273.

Two messenger RNA (mRNA) vaccines of BNT162b2 and mRNA-1273 were licensed. The most common adverse event is regional pain at the injection site in 80%. As systemic reactions, fatigue and headache were noted in 40%-60% and febrile illness in 10%-40% of the recipients. To investigate the mechanism of adverse events, cytokine profiles were investigated in mice. Muscle tissue and serum samples were obtained on days 0, 1, 3, 5, and 7, and at 2 and 4 weeks after the first dose. The second dose was given 4 weeks after the first dose and samples were obtained. After inoculation with 0.1 mL of mRNA-1273, IFN-γ and IL-2 were detected in muscle tissues and serum samples on day 1 of the second doses, and similar profiles were observed for IL-4, IL-5, and IL-12 production. mRNA-1273 induced higher levels of Th1 and Th2 cytokines. TNF-α was induced in muscle tissues on day 1 of the first dose and enhanced on day 1 of the second dose after inoculation with BNT162b2 and mRNA-1273. IL-6 was also detected in muscle tissue on day 1 of the first dose, but it decreased after day 3, and enhanced production was demonstrated on day 1 of the second dose. Granulocyte colony-stimulating factor in muscle tissues showed a similar profile. The induction of inflammatory cytokines in the mouse model is related to the cause of adverse events in humans, with a higher incidence of adverse events after the second dose.

Virus-like particles containing a prefusion-stabilized F protein induce a balanced immune response and confer protection against respiratory syncytial virus infection in mice.

Respiratory syncytial virus (RSV) is a serious respiratory pathogen in infants and young children worldwide. Currently, no licensed RSV vaccines are available. In this study, we explored stable prefusion conformation virus-like particles (Pre-F VLPs) as RSV vaccine candidates. RSV fusion (F) protein mutants were constructed to form stabilized Pre-F or postfusion (Post-F) configurations. VLPs containing Pre-F or Post-F protein were generated using a recombinant baculovirus (rBV)-insect cell expression system. The assembly and immunological properties of Pre-F or Post-F VLPs were investigated. Pre-F and Post-F VLPs contained antigenic sites Ø and I of pre- and postfusion conformations, respectively. Compared with Post-F VLPs, immunization with Pre-F VLPs elicited upregulation of IFN-γ, IL-2 and IL-10 and downregulation of IL-4 and IL-5 cytokine production in mice. A high percentage of CD25+ Foxp3+ cells or a low percentage of IL-17A-producing cells among CD4+ T cells was observed in the lungs of mice vaccinated with Pre-F VLPs. Importantly, immunization with Pre-F VLPs induced a high level of RSV neutralizing antibody and a balanced immune response, which protected mice against RSV infection without evidence of immunopathology. Our results suggested that Pre-F VLPs generated from rBV-insect cells represent promising RSV vaccine candidates.

Antigenicity and Immunogenicity Analysis of the E. coli Expressed FMDV Structural Proteins; VP1, VP0, VP3 of the South African Territories Type 2 Virus.

An alternative vaccine design approach and diagnostic kits are highly required against the anticipated pandemicity caused by the South African Territories type 2 (SAT2) Foot and Mouth Disease Virus (FMDV). However, the distinct antigenicity and immunogenicity of VP1, VP0, and VP3 of FMDV serotype SAT2 are poorly understood. Similarly, the particular roles of the three structural proteins in novel vaccine design and development remain unexplained. We therefore constructed VP1, VP0, and VP3 encoding gene (SAT2:JX014256 strain) separately fused with His-SUMO (histidine-small ubiquitin-related modifier) inserted into pET-32a cassette to express the three recombinant proteins and separately evaluated their antigenicity and immunogenicity in mice. The fusion protein was successfully expressed and purified by the Ni-NTA resin chromatography. The level of serum antibody, spleen lymphocyte proliferation, and cytokines against the three distinct recombinant proteins were analyzed. Results showed that the anti-FMDV humoral response was triggered by these proteins, and the fusion proteins did enhance the splenocyte immune response in the separately immunized mice. We observed low variations among the three fusion proteins in terms of the antibody and cytokine production in mice. Hence, in this study, results demonstrated that the structural proteins of SAT2 FMDV could be used for the development of immunodiagnostic kits and subunit vaccine designs.

Structure Elucidation of Calyxoside B, a Bipolar Sphingolipid from a Marine Sponge Cladocroce sp. through the Use of Beckmann Rearrangement.

Marine sponges are an excellent source of biologically active secondary metabolites. We focus on deep-sea sponges for our discovery study. A marine sponge Cladocroce sp. exhibited cytotoxic activity in the bioactivity screening. From this sponge a previously unreported cytotoxic glycosphingolipid, calyxoside B, was isolated and the structure of this compound was elucidated by analyses of MS and NMR spectra and chemical derivatization. We converted the ketone in the middle of a long aliphatic chain into an oxime to which was applied Beckmann rearrangement to afford two positional isomers of amides. The products were subjected to acidic hydrolysis followed by LC-MS analysis, permitting us to assign unequivocally the position of the ketone. Calyxoside B shows cytotoxicity against HeLa cells with an IC50 value of 31 µM and also weakly stimulated the production of cytokines in mice.

Interleukin-2-Inducible T-cell Kinase (ITK) signaling regulates non-canonical regulatory T cells

Abstract Regulatory T cells (Tregs) play an indispensable role in maintaining immunological tolerance to self-antigens and foreign antigens in suppressing excessive immune responses deleterious to the host by exerting a suppressive program towards a wide variety of target immune cells. Here we showed that ITK signaling pathways control the increased frequency of “Non-Canonical” CD4+CD25−FoxP3+ Tregs (ncTregs) population as well as canonical Tregs CD4+CD25+FoxP3+ expression significantly. Using several animals models we demonstrated that transplanting ncTregs and cTregs from ITK deficient mice ameliorated WT T cells mediated Graft versus Host Disease (GVHD). Furthermore, our data showed that adaptively transferring ncTregs and cTregs from ITK deficient mice significantly reduces proinflammatory cytokine production and T cell proliferation in GVHD induced recipient mice. Furthermore, our data uncover that adaptatively transferring ncTregs and cTregs from ITK deficient mice to GVHD induced recipient mice reduced tissue damage which induced by WT T cells. Finally, we have recently developed a novel peptide inhibitor which enhance Tregs expression in both mice and human. This novel peptide inhibitor also reduced cytokine production in mice and primary GVHD patient samples. Our data demonstrated that targeting specifically SLP76:ITK signaling could be used as a therapeutic strategy not only limit to GVHD but also treat multiple T cells mediated autoimmune disorders.

New Pathways for the Skin's Stress Response: The Cholinergic Neuropeptide SLURP-1 Can Activate Mast Cells and Alter Cytokine Production in Mice

Background: The alpha7 nicotinic acetylcholine receptor (Chrna7) plays an essential anti-inflammatory role in immune homeostasis and was recently found on mast cells (MC). Psychosocial stress can trigger MC hyperactivation and increases pro-inflammatory cytokines in target tissues such as the skin. If the cholinergic system (CS) and Chrna7 ligands play a role in these cascades is largely unknown.Objective: To elucidate the role of the CS in the response to psychosocial stress using a mouse-model for stress-triggered cutaneous inflammatory circuits.Methods: Key CS markers (ACh, Ch, SLURP-1, SLURP-2, Lynx1, Chrm3, Chrna7, Chrna9, ChAT, VAChT, Oct3, AChE, and BChE) in skin and its MC (sMC), MC activation, immune parameters (TNFα, IL1β, IL10, TGFβ, HIF1α, and STAT3) and oxidative stress were analyzed in skin from 24 h noise-stressed mice and in cultured MC (cMC) from C57BL/6 or Chrna7-Knockout mice.Results: First, Chrna7 and SLURP-1 mRNA were exclusively upregulated in stressed skin. Second, histomorphometry located Chrna7 and SLURP-1 in nerves and sMC and demonstrated upregulated contacts and increased Chrna7+ sMC in stressed skin, while 5 ng/mL SLURP-1 degranulated cMC. Third, IL1β+ sMC were high in stressed skin, and while SLURP-1 alone had no significant effect on cMC cytokines, it upregulated IL1β in cMC from Chrna7-KO and in IL1β-treated wildtype cMC. In addition, HIF1α+ sMC were high in stressed skin and Chrna7-agonist AR-R 17779 induced ROS in cMC while SLURP-1 upregulated TNFα and IL1β in cMC when HIF1α was blocked.Conclusions: These data infer that the CS plays a role in the regulation of stress-sensitive inflammatory responses but may have a surprising pro-inflammatory effect in healthy skin, driving IL1β expression if SLURP-1 is involved.

Jararhagin, a snake venom metalloproteinase, induces mechanical hyperalgesia in mice with the neuroinflammatory contribution of spinal cord microglia and astrocytes

Jararhagin is a hyperalgesic metalloproteinase from Bothrops jararaca venom. In rodents, jararhagin induces nociceptive behaviors that correlate with an increase in peripheral cytokine levels. However, the role of the spinal cord glia in pain processing after peripheral stimulus of jararhagin has not been investigated. Aiming to explore this proposal, mice received intraplantar (i.pl.) injection of jararhagin and the following parameters were evaluated: hyperalgesia, spinal cord TNF-α, IL-1β levels, and CX3CR1, GFAP and p-NFκB activation. The effects of intrathecal (i.t.) injection of TNF-α soluble receptor (etanercept), IL-1 receptor antagonist (IL-1Ra), and inhibitors of NFκB (PDTC), microglia (minocycline) and astrocytes (α-aminoadipate) were investigated. Jararhagin inoculation induced cytokine production (TNF-α and IL-1β) in the spinal cord, which was reduced by treatment with PDTC (40% and 50%, respectively). Jararhagin mechanical hyperalgesia and cytokine production were inhibited by treatment with etanercept (67%), IL-1Ra (60%), PDTC (70%), minocycline (60%) and α-aminoadipate (45%). Furthermore, jararhagin induced an increase in p-NFκB, CX3CR1 and GFAP detection in the spinal cord indicating activation of NFκB, microglia and astrocytes. These results demonstrate for the first time that jararhagin-induced mechanical hyperalgesia is dependent on spinal cord activation of glial cells, consequent NFκB activation, and cytokine production in mice.

Iridin Prevented Against Lipopolysaccharide-Induced Inflammatory Responses of Macrophages via Inactivation of PKM2-Mediated Glycolytic Pathways.

PurposeAbnormal glycolysis of immune cells contributed to the development of inflammatory response. Inhibition of this Warburg phenotype could be a promising strategy for preventing various inflammatory diseases. Iridin (IRD) is a natural isoflavone, and exerts anticancer, antioxidant, and anti-inflammatory effects. However, the underlying mechanism of IRD on acute inflammation remains unknown. In this study, the protective effects of IRD against lipopolysaccharide (LPS)-induced inflammation were investigated in murine macrophage RAW264.7 cells and in mice.MethodsThe inhibition of IRD on NO production in culture medium was detected by Griess assay while the levels of TNF-α, IL-1β, and MCP-1 were detected by ELISA assay. The effects of IRD on OCR and ECAR levels in LPS-treated macrophages were monitored by using Seahorse Analyzer. The apoptosis rate as well as the release of ROS and NO of RAW264.7 cells were analyzed by flow cytometric assay. The protective effects of IRD were investigated on LPS-induced inflammation in mice. The expressions of PKM2 and its downstream (p-JAK1, p-STAT1, p-STAT3, p-p65, iNOS, and COX2) in cells and in lung tissues were detected by Western blotting analysis.ResultsIRD treatment at the concentrations of 12.5–50 μM significantly inhibited the productions of TNF-α, IL-1β, MCP-1, and ROS, and suppressed the levels of glucose uptake and lactic acid in LPS-treated RAW264.7 cells. Oral administration with IRD (20–80 mg/kg) inhibited LPS-induced acute lung injury as well as inflammatory cytokine production in mice. Moreover, IRD targeted pyruvate kinase isozyme type M2 (PKM2) and suppressed its downstream p-JAK1, p-STAT1, p-STAT3, p-p65, iNOS, and COX2, which could be abolished by PKM2 agonist DASA-58 and antioxidant N-acetyl-L-cysteine, but partly be reversed by NF-κB activator CUT129 and JAK1 activator RO8191.ConclusionIRD alleviated LPS-induced inflammation through suppressing PKM2-mediated pathways, and could be a potential candidate for the prevention of inflammatory diseases.

Helminthostachys zeylanica Water Extract Ameliorates Airway Hyperresponsiveness and Eosinophil Infiltration by Reducing Oxidative Stress and Th2 Cytokine Production in a Mouse Asthma Model.

Helminthostachys zeylanica is a traditional folk herb used to improve inflammation and fever in Taiwan. Previous studies showed that H. zeylanica extract could ameliorate lipopolysaccharide-induced acute lung injury in mice. The aim of this study was to investigate whether H. zeylanica water (HZW) and ethyl acetate (HZE) extracts suppressed eosinophil infiltration and airway hyperresponsiveness (AHR) in asthmatic mice, and decreased the inflammatory response and oxidative stress in tracheal epithelial cells. Human tracheal epithelial cells (BEAS-2B cells) were pretreated with various doses of HZW or HZE (1 μg/ml–10 μg/ml), and cell inflammatory responses were induced with IL-4/TNF-α. In addition, female BALB/c mice sensitized with ovalbumin (OVA), to induce asthma, were orally administered with HZW or HZE. The result demonstrated that HZW significantly inhibited the levels of proinflammatory cytokines, chemokines, and reactive oxygen species in activated BEAS-2B cells. HZW also decreased ICAM-1 expression and blocked monocytic cells from adhering to inflammatory BEAS-2B cells in vitro. Surprisingly, HZW was more effective than HZE in suppressing the inflammatory response in BEAS-2B cells. Our results demonstrated that HZW significantly decreased AHR and eosinophil infiltration, and reduced goblet cell hyperplasia in the lungs of asthmatic mice. HZW also inhibited oxidative stress and reduced the levels of Th2 cytokines in bronchoalveolar lavage fluid. Our findings suggest that HZW attenuated the pathological changes and inflammatory response of asthma by suppressing Th2 cytokine production in OVA-sensitized asthmatic mice.

CD204-positive monocytes and macrophages ameliorate septic shock by suppressing proinflammatory cytokine production in mice

Sepsis is defined as a life-threatening multiorgan dysfunction caused by dysregulated inflammatory response to infection. It remains the primary cause of death from infection if not diagnosed and treated promptly. Therefore, a better understanding of the mechanism for resolving inflammation is needed. Monocytes and macrophages play a pivotal role not only in the induction but also in the suppression of inflammation. However, a tissue-resident macrophage subset that regulates a hyperinflammatory state during sepsis has not been explored. Here we show that CD204+ monocytes and/or macrophages rescued mice from endotoxin-induced septic shock. Serum and tissue proinflammatory cytokine levels were significantly upregulated in the absence of these cells. This study provided evidence that CD204+ monocytes and/or macrophages ameliorate septic shock by suppressing proinflammatory cytokine production.

Apamin from bee venom suppresses inflammation in a murine model of gouty arthritis

Ethnopharmacological relevanceBee venom (BV) has been used for the treatment of inflammatory diseases, such as rheumatoid arthritis, and for the relief of pain in traditional oriental medicine. Aim of studyThe aim of this study was to determine the anti-inflammatory effect of BV on monosodium urate (MSU)-induced gouty arthritis in a mouse model. Materials and methodsTo develop a mouse model of acute gouty arthritis, 4 mg 50 μL−1 of MSU crystal suspension was injected intradermally into the right paw. After MSU crystal injection, we evaluated inflammatory cytokine production in mice of the BV-treated (0.5 and 1 mg kg−1 body weight) and apamin (APM)-treated (0.5 and 1 mg kg−1 body weight) groups. The positive control group was administered a colchicine (1 mg kg−1 body weight) injection with MSU crystals. ResultsBV and APM treatment suppressed inflammatory paw edema in MSU-administered mice. It also exerted anti-inflammatory effects in mice with gouty arthritis by inhibiting proinflammatory cytokine production and inflammasome formation. Interestingly, MSU crystal formation was decreased by BV and APM treatment. ConclusionsThese results suggest that the APM from BV might be useful for the treatment of gouty arthritis due to its anti-inflammatory activities.

A Novel C Type CpG Oligodeoxynucleotide Exhibits Immunostimulatory Activity In Vitro and Enhances Antitumor Effect In Vivo.

BackgroundC type CpG oligodeoxynucleotides (CpG-C ODNs), possessing the features of both A type and B type CpG ODNs, exert a variety of immunostimulatory activities and have been demonstrated as an effective antitumor immunotherapy. Based on the structural characteristics, we designed 20 potential ODNs with the aim of synthesizing an optimal, novel CpG-C ODN specific to human and murine Toll-like receptor 9 (TLR9). We also sought to investigate the in vitro immunostimulatory and in vivo antitumor effects of the novel CpG-C ODN.MethodsTwenty potential CpG-C ODNs were screened for their ability to secrete interferon (IFN)-α, and interleukin (IL)-6 and tumor necrosis factor (TNF)-α production for the three most promising sequences were assayed in human peripheral blood mononuclear cells (PBMCs) by enzyme-linked immunosorbent assay (ELISA) or cytometric bead array assay. The functions of human and mouse B cells, and cytokine production in mice induced by the most promising sequence, HP06T07, were determined by flow cytometry and ELISA. Growth and morphology of tumor tissues in in vivo murine models inoculated with CT26 cells were analyzed by a growth inhibition assay and immunohistochemistry, respectively.ResultsAmong the 20 designed ODNs, HP06T07 significantly induced IFN-α, IL-6, and TNF-α secretion, and promoted B-cell activation and proliferation in a dose-dependent manner in human PBMCs and mouse splenocytes in vitro. Intratumoral injection of HP06T07 notably suppressed tumor growth and prolonged survival in the CT26 subcutaneous mouse model in a dose-dependent manner. HP06T07 administered nine times at 2-day intervals (I2) eradicated tumor growth at both primary and distant sites of CT26 tumors. HP06T07 restrained tumor growth by increasing the infiltration of T cells, NK cells, and plasmacytoid dendritic cells (pDCs).ConclusionsHP06T07, a novel CpG-C ODN, shows potent immunostimulatory activity in vitro and suppresses tumor growth in the CT26 subcutaneous mouse model.

Polygonatum kingianum rhizome extract alleviates collagen antibody-induced arthritis by modulating proinflammatory cytokine production in mice

Objective: To evaluate the anti-arthritic effects of Polygonatum kingianum rhizome extract using both in vitro and in vivo models. Methods: Lipopolysaccharide-induced RAW 264.7 macrophages were treated with an ethanol extract of Polygonatum kingianum rhizomes at different concentrations to determine nitric oxide and prostaglandin E2 (PGE2) production. For in vivo study, Polygonatum kingianum ethanol extract was further investigated for its antiinflammatory effect in a mouse model with collagen antibody-induced arthritis. Phytochemical study of Polygonatum kingianum ethanol extract was also performed. Results: Saponins (142 mg/g total yield) was the main component in the Polygonatum kingianum ethanol extract. 5α,8α-ergosterol peroxide, (E,E)-9-oxooctadeca-10,12-dienoic acid and 3-(2’-hydroxy-4’-methoxy-benzyl)-5,7-dihydroxy-8-methyl-chroman-4-one were isolated from the extract. Polygonatum kingianum ethanol extract exhibited potential anti-inflammatory effects by inhibiting nitric oxide and PGE2 production in RAW 264.7 cells in a dose-dependent manner. The level of arthritis in mice with collagen antibody-induced arthritis was significantly reduced (P Conclusions: Polygonatum kingianum ethanol extract has beneficial effects on inflammatory cytokine regulation and PGE2 inhibition in an experimental mouse model with collagen antibody-induced arthritis. The phytochemical screening reveals that the saponin, as the main component, and sterols (daucosterol and 5α,8α-ergosterol peroxide) from Polygonatum kingianum ethanol extract may contribute to its promising in vitro and in vivo anti-inflammatory activities.

Dehydroepiandrosterone resisted E. Coli O157:H7-induced inflammation via blocking the activation of p38 MAPK and NF-κB pathways in mice

Dehydroepiandrosterone (DHEA), a critical metabolite in cholesterol metabolism, can regulate the inflammatory responses in humans or animals. However, the precise mechanisms of these beneficial actions remains poorly understood. Present study aims to clarify the anti-inflammatory function of DHEA and its possible mechanisms in the E. coli O157:H7-stimulated mice. The results indicated that DHEA reduced the mortality of mice and bacterial concentration in the peritoneal fluid in the E. coli-stimulated mice. DHEA increased the spleen index, the activity of lactate dehydrogenase and acid phosphatase; while it decreased the nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity in mice. The mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interferon gamma (IFN-γ) were decreased, whereas the interleukin-4 (IL-4) and interleukin-10 (IL-10) mRNA levels were increased in the E. coli-stimulated mice treated with DHEA. Moreover, DHEA treatment reversed the increasing of IFN-γ/IL-4 ratio in mice caused by E. coli infection. Importantly, DHEA blocked the nuclear translocation of p65 through down-regulation the IκB-α protein phosphorylation level in the mice stimulated with E. coli O157:H7. No statically changes were showed on the phospho (p)-ERK1/2 and p-JNK1/2 protein level, while DHEA significantly suppressed the p-p38 protein level in mice. The above results indicated that DHEA alleviated inflammatory responses by suppressing NO secretion and promoting Th2-associated anti-inflammatory cytokines production in mice; and this action might relate to the blocking of p38 MAPK and NF-κB signaling pathways activation. All the above results provide substantial information for understanding the anti-inflammatory function of DHEA and further support it as a potential immunomodulatory in prevention inflammatory and bacterial infection diseases.