Zymosan Research Articles

Protection by selective mTORC2 inhibition of Zymosan-induced hypotension and systemic inflammation mediated via IKKα/IκB-α/NF-κB activation

Non-septic shock is a serious condition leading to multiple organ dysfunction. Although targeting the mammalian target of the rapamycin complex 1 (mTORC1) signaling pathway exerts potent anti-inflammatory activity, little is known about mTORC2’s contribution to non-septic shock. Thus, our research aims to investigate mTORC2's contribution and associated changes of IκB kinase (IKKα)/inhibitor κB (IκB-α)/nuclear factor-ĸB (NF-κB) pathway on Zymosan (ZYM)-induced non-septic rat model using the novel mTORC2 selective inhibitor JR-AB2–011. Rats were given saline (4 ml/kg), dimethylsulfoxide (DMSO) (4 ml/kg), ZYM (500 mg/kg), and (or) JR-AB2–011 (1 mg/kg). Mean arterial pressure (MAP) and heart rate (HR) of rats were recorded. JR-AB2–011 reversed both ZYM-induced reduction in MAP and increase in HR. Protein expression and/or phosphorylation of rictor, protein kinase B (Akt), IκB-α, IKKα, NF-κB p65, inducible nitric oxide synthase (iNOS), nitrotyrosine, cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, besides prostaglandin (PG) E2 levels were measured. The enhanced expression of the proteins mentioned above has been inhibited by JR-AB2–011. These data suggest mTORC2’s promising role in ZYM-induced hypotension and systemic inflammation mediated via IKKα/IκB-α/NF-κB pathway.

Evaluation of Anti-Inflammatory Activity of the New Cardiotonic Steroid γ-Benzylidene Digoxin 8 (BD-8) in Mice.

Cardiotonic steroids are known to bind to Na+/K+-ATPase and regulate several biological processes, including the immune response. The synthetic cardiotonic steroid γ-Benzylidene Digoxin 8 (BD-8) is emerging as a promising immunomodulatory molecule, although it has remained largely unexplored. Therefore, we tested the immunomodulatory potential of BD-8 both in vitro and in vivo. Hence, primary mouse macrophages were incubated with combinations of BD-8 and the pro-inflammatory fungal protein zymosan (ZYM). Nitric oxide (NO) production was determined by Griess reagent and cytokines production was assessed by enzyme-linked immunosorbent assay. Inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), p-nuclear factor kappa B p65 (NF-κB p65), p-extracellular signal-regulated kinase (p-ERK), and p-p38 were evaluated by flow cytometry. Macrophages exposed to BD-8 displayed reduced phagocytic activity, NO levels, and production of the proinflammatory cytokine IL-1β induced by ZYM. Furthermore, BD-8 diminished the expression of iNOS and phosphorylation of NF-κB p65, ERK, and p38. Additionally, BD-8 exhibited anti-inflammatory capacity in vivo in a carrageenan-induced mouse paw edema model. Taken together, these findings demonstrate the anti-inflammatory activity of BD-8 and further reinforce the potential of cardiotonic steroids and their derivatives as immunomodulatory molecules.

Whole-blood culture-derived cytokine combinations for the diagnosis of tuberculosis.

The diagnosis of tuberculosis (TB) disease and TB infection (TBI) remains a challenge, and there is a need for non-invasive and blood-based methods to differentiate TB from conditions mimicking TB (CMTB), TBI, and healthy controls (HC). We aimed to determine whether combination of cytokines and established biomarkers could discriminate between 1) TB and CMTB 2) TB and TBI 3) TBI and HC. We used hemoglobin, total white blood cell count, neutrophils, monocytes, C-reactive protein, and ten Meso Scale Discovery analyzed cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon (IFN)-ɣ, and tumor necrosis factor (TNF)-α) in TruCulture whole blood tubes stimulated by lipopolysaccharides (LPS), zymosan (ZYM), anti-CD3/28 (CD3), and unstimulated (Null) to develop three index tests able to differentiate TB from CMTB and TBI, and TBI from HC. In 52 persons with CMTB (n=9), TB (n=23), TBI (n=10), and HC (n=10), a combination of cytokines (LPS-IFN-ɣ, ZYM-IFN-ɣ, ZYM-TNF-α, ZYM-IL-1β, LPS-IL-4, and ZYM-IL-6) and neutrophil count could differentiate TB from CMTB with a sensitivity of 52.2% (95% CI: 30.9%-73.4%) and a specificity of 100 % (66.4%-100%). Null- IFN-ɣ, Null-IL-8, CD3-IL-6, CD3-IL-8, CD3-IL-13, and ZYM IL-1b discriminated TB from TBI with a sensitivity of 73.9% (56.5% - 91.3%) and a specificity of 100% (69.2-100). Cytokines and established biomarkers failed to differentiate TBI from HC with ≥ 98% specificity. Selected cytokines may serve as blood-based add-on tests to detect TB in a low-endemic setting, although these results need to be validated.

Immunomodulatory zymosan/ι-carrageenan/ agarose hydrogel for targeting M2 to M1 macrophages (antitumoral).

Several studies have been performed on the immunomodulatory effects of yeast β-(1,3) glucan, but there is no proper evaluation of the thermal and immunomodulating properties of zymosan (ZM). Thermogravimetry analysis indicated a 54% weight loss of ZM at 270 °C. Circular dichroism showed absorption peaks in the region of 250 to 400 nm, suggesting a helical coil β-sheet configuration. XRD showed a broad peak at 2θ of 20.38°, indicating the crystalline nature, and the size was found to be 23 nm. ZM is biocompatible and showed no toxicity against L929 and RAW 264.7 cell lines (cell viability > 90%). Immunomodulatory studies with PCR showed upregulation of M1 genes in human differentiated THP-1 macrophage cell lines, which were responsible for antitumor properties. The uptake of ZM particles inside the differentiated THP-1 macrophages and Raw 264.7 cells was confirmed (Video clip). ZM particle uptake via Dectin-1 was identified by competitive receptor blocking. Seaweed derived carrageenan/ZM/agarose hydrogel was successfully prepared (@5 : 5 wt%) and was seen to support the growth of L929 cells (1 × 105 cells per mL) and have a higher swelling (≈250-280%). This study indicates that ZM-based hydrogel could be a potential drug carrier (Rifampicin and Levofloxacin) for targeting tumour-associated macrophages (M2).

The NR4A Orphan Receptor Modulator C-DIM12 Selectively Alters Inflammatory Mediators in Myeloid Cells

Orphan nuclear receptor subfamily 4A (NR4A) are key regulators of inflammatory responses, largely by their interactions with NF-κB. Over the last decade, several NR4A modulators have been developed, and they are showing potential as therapeutics, although their widespread use in laboratory settings is limited. Here, we have examined, using myeloid cell line THP-1, whether the NR4A modulator 3-[(4-Chlorophenyl)-(1H-indol-3-yl)methyl]-1H-indole (C-DIM12) can alter the inflammatory outcome of six inflammatory ligands: lipopolysaccharide (LPS), tumour necrosis factor alpha (TNFα), interleukin-1 beta (IL-1β), flagellin (FL), lipoteichoic acid (LTA), and zymosan (ZY). We demonstrate that C-DIM12 (10 µM) selectively alters the secretion of inflammatory chemokine MCP-1 following exposure to distinct inflammatory ligands in a concentration-dependent manner. Furthermore, data obtained from THP-1 Lucia cell experiments show that 10 µM C-DIM12, and not 1 µM C-DIM12, can significantly attenuate the increased NF-κB transcriptional activity observed following the exposure to several inflammatory ligands (LPS, FL, TNFα, LTA, and ZY). Lastly, experimental analysis confirms that the cellular action(s) of C-DIM12 is independent of changes in metabolic parameters. Thus, these data contribute to the understanding of how the NR4A modulator C-DIM12 alters inflammatory responses in a myeloid cell following exposure to multiple ligands.

The Impact of Avpr1a Expression and Host-Microbiome Interactions on Visceral Hypersensitivity

Visceral hypersensitivity (VH) is commonly cited as a major driver of chronic abdominal pain in "functional" gastrointestinal disorders (e.g., irritable bowel syndrome) where persistent and/or recurrent abdominal pain is the primary unifying symptom regardless of any alterations in bowel habits. The complexity of VH is in part influenced by genetic factors and individual differences in gut microbiome composition, yet specific mechanisms that generate VH remain incompletely understood. Correspondingly, current treatments to primarily focus on symptom management rather than targeting physiological mechanisms responsible for generating VH. We have begun to examine the role of genetic susceptibility and microbiome response dynamics in VH development using a preclinical model of intracolonic zymosan (ZYM) administration in which there are strain differences to VH susceptibility. Preliminary data reveals differential susceptibility between ZYM-induced VH in two closely related C57BL/6 sub strains, one from Taconic Biosciences (C57BL/6NTac) and the other from Jackson Laboratory (C57BL/6J). We have identified a VH candidate gene that encodes the arginine-vasopressin receptor 1A (AVPR1A) protein. We have further observed dynamic strain differences in the location and composition of the gut microbiome in response to ZYM corresponding to VH susceptibility. Ongoing studies are focused on teasing apart the potential bidirectional relationship(s) between genetic susceptibility and host-microbiome interactions in the etiology of VH. Identifying underlying mechanisms that drive VH would provide novel targets for pharmacological intervention and decrease reliance on opioids, which are prescribed at a significantly higher rate to patients who report abdominal pain with no accompanying structural disease. Grant support from R21 NS104789/NS/NINDS (KMB), R03 NS096454/NS/NINDS (KMB), Rita Allen Foundation Award in Pain (KMB), P20GM103418 (EEY and KMB), and a K-INBRE recruitment startup package.

Agaricus blazei Murill polysaccharides alleviate oxidative stress and inflammatory responses against liver and lung injury

As a kind of well-known edible mushroom, the medicinal values of Agaricus blazei Murill also have been well acknowledged. There is no efficient treatment strategy for multiple organ dysfunction syndrome (MODS) so far, a crucial cause of clinical death. The present work was designed to evaluate the beneficially protective effects of Agaricus blazei Murill polysaccharides (ABMP) on liver and lung against zymosan (ZY)-induced MODS related damages. The MODS mice were successfully established by given one-time injection of zymosan solution (500 mg/kg) intraperitoneally. The in vivo results demonstrated that ABMP could improve the 10-day survival rate, ease abnormal physiological behavior and alleviate tissues oxidative stress in MODS mice. Besides, ABMP showed potential effects on attenuating the functional impairments of liver and lung. Meanwhile, ABMP effectively inhibited the activation of NF-κB signaling pathway and reduced the expressions of inflammatory cytokines, including TNF-α, IL-1β, IL-6, COX-2 and PGE-2. Additionally, the structural characterizations showed that the significant function of ABMP was possibly related with the β-pyranose configuration with a triple-helical structure composing of glucose mainly with the proper molecular weights of 12.26 kDa. These findings demonstrated that ABMP exhibited potential protections against liver and lung damages in the ZY-induced MODS mice, providing more theoretical basis and reference significance for the exploration of biological activity of A. blazei Murill.

Defective Interferon-Gamma Production Is Common in Chronic Pulmonary Aspergillosis.

Immune defects in chronic pulmonary aspergillosis (CPA) are poorly characterized. We compared peripheral blood cytokine profiles in patients with CPA versus healthy controls and explored the relationship with disease severity. Interferon-gamma (IFNγ), interleukin (IL)-17, tumor necrosis factor-α, IL-6, IL-12, and IL-10 were measured after in vitro stimulation of whole blood with lipopolysaccharide (LPS), phytohemagglutinin, β-glucan, zymosan (ZYM), IL-12 or IL-18, and combinations. Clinical parameters and mortality were correlated with cytokine production. Cytokine profiles were evaluated in 133 patients (57.1% male, mean age 61 years). In comparison to controls, patients with CPA had significantly reduced production of IFNγ in response to stimulation with β-glucan + IL-12 (312 vs 988 pg/mL), LPS + IL-12 (252 vs 1033 pg/mL), ZYM + IL-12 (996 vs 2347 pg/mL), and IL-18 + IL-12 (7193 vs 12330 pg/mL). Age >60 (hazard ratio [HR], 1.71; 95% confidence interval [CI], 1.00-2.91; P = .05) and chronic obstructive pulmonary disease (HR, 1.69; 95% CI, 1.03-2.78; P = .039) were associated with worse survival, whereas high IFNγ production in response to beta-glucan + IL-12 stimulation (HR, 0.48; 95% CI, .25-0.92; P = .026) was associated with reduced mortality. Patients with CPA show impaired IFNγ production in peripheral blood in response to stimuli. Defective IFNγ production ability correlates with worse outcomes. Immunotherapy with IFNγ could be beneficial for patients showing impaired IFNγ production in CPA.

Targeted volatolomics of human monocytes: Comparison of 2D‐GC/TOF‐MS and 1D‐GC/Orbitrap‐MS methods

Blood is a complex biological matrix providing valuable information on nutritional, metabolic, and immune status. The detection of blood biomarkers requires sensitive analytical methods because analytes are at very low concentrations. Peripheral blood monocytes play a crucial role in inflammatory processes, and the metabolites released by monocytes during these processes might serve as important signalling molecules and biomarkers of particular physiological states. Headspace solid-phase microextraction (HS-SPME) combined with two different mass spectrometric platforms, two-dimensional (2D) gas chromatography coupled to time-of-flight mass spectrometry (2D-GC/TOF-MS) and one-dimensional gas chromatography coupled to Orbitrap mass spectrometry (GC/Orbitrap‐MS), were applied for the investigation of volatile organic compounds (VOCs) produced by human peripheral blood monocytes. An optimized method was subsequently applied for the characterization of changes in VOCs induced by lipopolysaccharides (LPS) and zymosan (ZYM) stimulation. Overall, the 2D‐GC/TOF‐MS and the 1D‐GC/Orbitrap‐MS analyses each yielded about 4000 and 400 peaks per sample, respectively. In total, 91 VOCs belonging to eight different chemical classes were identified. The samples were collected in two fractions, conditioned media for monitoring extracellularly secreted molecules and cell pellet samples to determine the intracellular composition of VOCs. Alcohols, ketones, and hydrocarbons were the main chemical classes of the metabolic profile identified in cell fractions. Aldehydes, acids and cyclic compounds were characteristic of the conditioned media fraction. Here we demonstrate that HS-SPME-2D‐GC/TOF‐MS is more suitable for the identification of specific VOC profiles produced by human monocytes than 1D‐GC/Orbitrap‐MS. We define the signature of VOCs occurring early after monocyte activation and characterise the signalling compounds released by immune cells into media.

Polymorphonuclear Myeloid-Derived Cells That Contribute to the Immune Paralysis Are Generated in the Early Phase of Sepsis via PD-1/PD-L1 Pathway.

Immune paralysis is a protracted state of immune suppression following the early/acute inflammatory phase of sepsis. CD11b+ Gr-1+ cells induced during sepsis are heterogeneous myeloid-derived cells (MDCs). This study investigated the contribution of MDCs to immune paralysis. Treatment of mice with zymosan (ZM) induced a marked increase in the total number of splenocytes with an increase in the proportion of Gr-1hi cells and a decrease in the proportion of T cells on day 7; levels of these cells eventually return to levels similar to those of control mice on day 21. T-cell activation and gamma interferon (IFN-γ) expression by CD8+ T cells were clearly impaired in ZM-treated mice on day 21 (d21-ZM mice). Gr-1hi cells showed a CD11b+ Ly6Ghi polymorphonuclear phenotype. Injection of lipopolysaccharide (LPS) into d21-ZM mice impaired interleukin 6 (IL-6) production in serum, accompanied by accumulation of CD11b+ Gr-1hi cells in the peripheral blood. Transfer of Gr-1hi cells from d21-ZM mice into intact mice impaired IL-6 production, but similar transfer of Gr-1hi cells from PD-1/PD-L1-deficient d21-ZM mice showed no such suppressive effect. Conversely, either depletion of Gr-1hi cells by treatment with anti-Gr-1 monoclonal antibody (MAb) or neutralization of the PD-1/PD-L1 pathway by anti-PD-1 and anti-PD-L1 MAbs during the induction phase of sepsis ameliorated ZM-induced immune suppression. Our results suggest that the PD-1/PD-L1-mediated generation of Gr-1hi cells in the early phase of sepsis is required for the late phase of immune paralysis.

Protective effects of sulfated polysaccharides from Lentinula edodes on the lung and liver of MODS mice.

In this work, the effects of sulfated polysaccharides from Lentinula edodes (SPLE) on zymosan (ZYM)-induced multiple organ dysfunction syndrome (MODS) mice were investigated. Using the MODS mice model, biochemical works have already shown that in mice treated with SPLE, the lung parameters of GGT, C3 and hs-CRP were down-regulated and the hepatic parameters of TC, TG, ALT and AST, HDLC, LDL-C and VLDL-C were improved, the serum levels of CK, Cr and Amy were decreased, and the levels of inflammatory factors such as TNF-α, IL-1β, IL-6 and IL-10 were also reduced, the activity of antioxidant enzymes SOD and CAT enhanced, and the content of MDA was reduced. In addition, histopathology of the lung and liver confirmed the beneficial effects of SPLE on MODS mice, indicating that SPLE played a role in protecting the organ function of MODS mice. In addition, SPLE was characterized as a sulfated β-glucan linked by β-type glycosidic bonds. These conclusions indicated that SPLE had effective antioxidant and anti-inflammatory activities, and could be used as a functional food and medicine to prevent MODS.

Zymosan promotes proliferation, Candida albicans adhesion and IL-1\u03b2 production of oral squamous cell carcinoma in vitro

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck squamous cell carcinoma (HNSCC), and the effect of zymosan (ZYM), a component of the yeast cell wall, on oral cancer remains unclear. The CCK-8 proliferation assay was performed to evaluate the effect of ZYM on the proliferation of the OSCC cell lines WSU-HN4, WSU-HN6 and CAL27, and the potential mechanism was explored by quantitative real-time PCR, immunofluorescence assay and western blot. A cell adhesion assay was conducted to determine the adhesion of Candida albicans to OSCC cells, and the expression of related genes, including TLR2, MyD88, NLRP3, ASC, Caspase-1 and IL-1β, and proteins, including TLR2, MyD88, NF-κB p65, p-NF-κB p65 and E-cadherin was determined. Additionally, the pro-inflammatory cytokines including IL-6, IL-8, TNF-α and IL-1β produced by OSCC cells were detected using a chemiluminescence immunoassay (CLIA). In the current study, the CCK-8 assay showed that ZYM promoted the proliferation of WSU-HN4, WSU-HN6 and CAL27 cells via the TLR2/MyD88 pathway. The cell adhesion assay showed that the number of C. albicans cells per field significantly increased in ZYM-treated OSCC cells compared to controls. When treated with ZYM, OSCC cells secreted significantly more pro-inflammatory cytokine IL-1β, which could enhance inflammation in oral cancer microenvironment. In conclusion, ZYM from the fungal cell wall promotes the proliferation, C. albicans adhesion and IL-1β production in OSCC, as demonstrated by in vitro experiments.

Synthesis, Characterization, and Biological Activity of Aminated Zymosan.

Zymosan (ZM), a naturally occurring insoluble macromolecule obtained from the cell wall of Saccharomyces cerevisiae, is used as a functional food (as dietary fiber), phagocytic stimulus, and immune potentiator. The present study aimed to increase its solubility and evaluate its immunological application. ZM was converted into soluble 6-amino-6-deoxy-β-(1-3)-glucan of a molecular weight of 296 kDa by reduction. Detailed structural characterization of aminated ZM was determined by Fourier transform infrared spectroscopy and two-dimensional NMR analysis (2D, COSY, TOCSY, ROSEY, NOSEY, and HSQC). Aminated ZM was biocompatible with Raw 264.7 macrophage cell lines up to a concentration of 100 μg/mL. Rhodamine tagging revealed that the aminated ZM microparticles were found localized within the nucleus of Raw 264.7 cells. Both native and aminated ZM showed a similar expression pattern of inflammatory genes in Raw 264.7.

NF‐κB activation mediates LPS‐or zymosan‐induced hypotension and inflammation reversed by BAY61‐3606, a selective Syk inhibitor, in rat models of septic and non‐septic shock

We have previously demonstrated that the activation of the spleen tyrosine kinase (Syk)/inhibitory-κB (IκB)-α/nuclear factor-κB (NF-κB) p65 signalling pathway contributes to hypotension and inflammatory response in a rat models of zymosan (ZYM)-induced non-septic shock. The purpose of this study was to further examine the possible mechanism underlying the effect of inhibition of Syk by BAY61-3606 via NF-κB activity at the level of nuclear translocation regarding the production of vasodilator and proinflammatory mediators in lipopolysaccharide (LPS) (septic)- and ZYM (non-septic)-induced shock. Administration of LPS (10mg/kg, ip) or ZYM (500mg/kg, ip) to male Wistar rats decreased mean arterial pressure and increased heart rate that was associated with an increase in the activities of cyclooxygenase and nitric oxide synthase, tumour necrosis factor-α, and interleukin-8 levels, and NF-κB activation and nuclear translocation in sera and/or cardiovascular and renal tissues. BAY61-3606 (3mg/kg, ip), the selective Syk inhibitor, given 1hour after LPS- or ZYM injection reversed all the above-mentioned effects. These results suggest that Syk contributes to the LPS- or ZYM-induced hypotension and inflammation associated with transactivation of NF-κB in septic and non-septic shock.

The effect of hyperbaric oxygen on the function of peritoneal macrophages in septic mice

Objective To investigate the changes of peritoneal macrophage function in sepsis mice after hyperbaric oxygen (HO) therapy. Methods One hundred C57BL/6 mice were randomly(random number) divided into four groups in equal number (n=25): control group (normal saline), sepsis group,hyperbaric oxygen group, sepsis+ hyperbaric oxygen group; the sepstic mice were treated by intra-peritoneal injection with zymosan; The mice of hyperbaric oxygen group and sepsis+ hyperbaric oxygen group received hyperbaric oxygen therapy (2 atm, 2 h, 100% O2). The number of surviving mice was obsevred at 72 h after the intra-peritoneal injection with zymosan; The percentage of M1 macrophage was detected by flow cytometry. The expression of mRNA of inducible nitric oxide synthase (iNOS) in peritoneal macrophage was detected with RT-PCR. The levels of IL-12 and IL-10 in peripheral blood were measured by ELISA. Results Compared with sepsis group, the number of surviving mice of sepsis+ hyperbaric oxygen group was significantly higher (9 vs. 16, P<0.01). The percentages of M1 macrophage in sepsis+ hyperbaric oxygen group was significantly lower than that in sepsis group [(4.75±0.14)% vs. (2.25±0.16)%, F=803.438, P<0.01]. The expression of mRNA of inducible nitric oxide synthase(iNOS) in sepsis+ hyperbaric oxygen group were significantly lower than that in sepsis group [(39.62±1.74) vs. (48.32±3.34), F=31.992, P<0.01]. The level of IL-12 in peripheral blood in sepsis+ hyperbaric oxygen group was significantly lower than that in sepsis group [(242.62±19.10) pg/mL vs. (159.51±6.35)pg/mL, F=102.282, P<0.01]. The level of IL-10 was significantly higher than that in sepsis group [(521.26±6.3) pg/mL vs. (188.83±8.53) pg/mL, F=5 896.006, P<0.01]. Conclusions Hyperbaric oxygen therapy had effective effect on increasing the number of surviving mice of sepsis group, reducing the level of IL-12 in peripheral blood and rising the level of IL-10 in peripheral blood. By inhibiting the expression of M1 macrophage and iNOS, hyperbaric oxygen could reduce the excessive inflammatory response and play a role in the protection of mice. Key words: Hyperbaric oxygen; Sepsis; Peritoneal macrophages; Inducible nitric oxide synthase; Interleukin-12; Interleukin-10

Zymosan attenuates melanoma growth progression, increases splenocyte proliferation and induces TLR-2/4 and TNF-\u03b1 expression in mice

BackgroundMelanoma is one of the most common types of skin malignancies. Since current therapies are suboptimal, considerable interest has focused on novel natural-based treatments. Toll-like receptors (TLRs) play an important role in evoking innate immunity against cancer cells. Zymosan, a known TLR-2 agonist, is a glucan derived from yeast cell walls with promising immunomodulatory effects. The aim of this study was to evaluate whether Saccharomyces cerevisiae-derived zymosan-modulated skin melanoma progression by regulation of TLR-2 and TLR-4 expression in peritoneal macrophages and serum TNF-α level.MethodsMale C57BL/6 mice were divided into four groups: i) zymosan-treated (Z), ii) Melanoma-bearing mice (M), iii) Melanoma-bearing mice treated with zymosan (ZM) and iv) a healthy control group (negative control). 15 days after melanoma induction, mice were injected i.p. with zymosan (10 μg) daily for 4 consecutive days. Mice were CO2-euthanized and serum TNF-α level, TLR-2 and TLR-4 expression in peritoneal macrophages and tumor growth measured. Splenocytes were treated ex-vivo with zymosan to determine viability and proliferation.ResultsTumor weight significantly decreased following therapeutic dosing with zymosan (P < 0.05). This was associated with zymosan-induced upregulation of TLR-2, TLR-4 and TNF-α mRNA in peritoneal macrophages and enhanced serum TNF-α levels (P < 0.05). Splenocyte number and viability were increased in a concentration-dependent manner by zymosan.ConclusionsOur study suggests that zymosan-induced upregulation of TLR-2, TLR-4 and TNF-α gene expression and of TNF-α release; together with increased level of lymphocyte proliferation may play a role in the inhibition of melanoma progression.

The role of Syk/IĸB-α/NF-ĸB pathway activation in the reversal effect of BAY 61-3606, a selective Syk inhibitor, on hypotension and inflammation in a rat model of zymosan-induced non-septic shock.

Spleen tyrosine kinase (Syk), a non-receptor tyrosine kinase, plays an important role in allergic diseases and inflammation. Syk triggers several intracellular signalling cascades including Toll-like receptor signalling to activate inflammatory responses following fungal infection but the role of this enzyme in zymosan (ZYM)-induced non-septic shock and its impacts on hypotension and inflammation in rats is not well understood. This study was conducted to determine the effects of Syk inhibition on ZYM-induced alterations in the expression and/or activities of Syk, inhibitor ĸB (IĸB)-α, and nuclear factor-ĸB (NF-ĸB) p65. We also examined the effect of Syk inhibition on inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and tumour necrosis factor (TNF)-α, and activity of myeloperoxidase (MPO) that contribute to hypotension and inflammation. Administration of ZYM (500mg/kg, ip) to male Wistar rats decreased blood pressure and increased heart rate. These changes were associated with increased expression and/or activities of Syk, NF-κB p65, iNOS and COX-2 and decreased expression of IκB-α with enhanced levels of nitrite, nitrotyrosine, 6-keto-PGF1α , and TNF-α and activity of MPO in renal, cardiac and vascular tissues. ZYM administration also elevated serum and tissue nitrite levels. The selective Syk inhibitor BAY 61-3606 (3mg/kg, ip) given 1hour after ZYM injection reversed all of these changes induced by ZYM. These results suggest that Syk/IĸB-α/NF-ĸB pathway activation contributes to hypotension and inflammation caused by the production of vasodilator and proinflammatory mediators in the zymosan-induced non-septic shock model.

Tylvalosin Induces Apoptosis of Porcine Neutrophils and Macrophages, Efferocytosis by Porcine Macrophages and Modulation of Lipid Mediators of Inflammation in vitro: A New Class of Anti‐Microbial with Anti‐inflammatory and Pro‐resolution Properties?

Mucosal inflammatory diseases cause great economic losses in the livestock industry. Previous research has shown that some macrolides effectively modulate bacterial‐induced inflammation in animal models. Tylvalosin (TYL) is a new macrolide antibiotic, meant to treat porcine respiratory diseases, in which inflammation is a major component of pathophysiology. The immune modulating effects of tylvalosin are unknown. We hypothesized that TYL may promote the resolution of inflammation in pigs, hence making it an appealing anti‐infective therapeutic.AimsThe aims of the study were to characterize the effects of this macrolide on (1) apoptosis (2) necrosis and reactive oxygen species (ROS) production (3) cytokine, chemokine and lipid mediators of inflammation – leukotriene B4 (LTB−4), prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) (4) phagocytosis and efferocytosis of neutrophils (PMNs) by macrophages.MethodsPorcine blood was collected from the jugular vein for isolation of PMNs and monocytes. Isolated monocytes were grown and matured (verified by esterase staining) over the course of 7 days. Leukocytes were treated with vehicle, TYL, lincomyosin or positive controls: LPS, staurosporine or calcium ionophore. (1) Apoptosis was measured by western blot for cleaved‐caspase 3, TUNEL staining for nicked DNA, and cell death ELISA (2) lactate dehydrogenase (LDH) assay for necrosis detection was used to assess cell death. ROS levels were assessed using bioassays. (3) Multiplex‐bead based assays and qPCR were employed to assess macrophage cytokine and chemokine levels. Reverse phase‐HPLC and liquid chromatography tandem mass spectrometry were used to assess PMN lipid mediator secretions (4) Macrophage phagocytosis and efferocytosis were determined using zymosan (ZYM) phagocytosis assays, TUNEL staining, and myeloperoxidase (MPO) assay.Results(1) Porcine PMN and macrophage apoptosis were increased in cells exposed to TYL in a time and dose dependent manner (P&lt;0.05), but not in the other groups. (2) Tylvalosin had not effect on LDH or ROS levels in macrophages or PMNs. (3) Tylvalosin reduced CXCL8 mRNA transcription and protein secretion in macrophages. IL‐1α but not IL‐1β protein secretion was inhibited. TYL reduced levels of pro‐inflammatory LTB4 and PGE2, while inducing the production of pro‐resolving LXA4 in TYL‐treated porcine PMNs (4) Treated and untreated macrophages had similar levels of phagocytic uptake of ZYM particles. In contrast, MPO revealed a dose dependent increase in efferocytosis of PMN's in macrophages upon TYL treatment (P&lt;0.05).ConclusionsThese findings suggest that TYL promotes apoptosis and efferocytosis of porcine leukocytes, while having no significant effect on necrosis or ROS production. The effect appears to be, at least in part drug‐selective, and does not affect phagocytic function. TYL inhibits LTB4 and PGE‐2, while enhancing the production of LXA4. Together, these results suggest that tylvalosin may have anti‐inflammatory and pro‐resolving properties in porcine tissues.Support or Funding InformationECO Animal Health, Natural Sciences and Engineering Research Council &amp; University of Calgary

Combination therapy of molecular hydrogen and hyperoxia improves survival rate and organ damage in a zymosan-induced generalized inflammation model.

Multiple organ dysfunction syndrome (MODS) is a leading cause of mortality in critically ill patients. Hyperoxia treatment may be beneficial to critically ill patients. However, the clinical use of hyperoxia is hindered as it may exacerbate organ injury by increasing reactive oxygen species (ROS). Hydrogen gas (H2) exerts a therapeutic antioxidative effect by selectively reducing ROS. Combination therapy of H2 and hyperoxia has previously been shown to significantly improve survival rate and organ damage extent in mice with polymicrobial sepsis. The aim of the present study was to investigate whether combination therapy with H2 and hyperoxia could improve survival rate and organ damage in a zymosan (ZY)-induced generalized inflammation model. The results showed that the inhalation of H2 (2%) or hyperoxia (98%) alone improved the 14-day survival rate of ZY-challenged mice from 20 to 70 or 60%, respectively. However, combination therapy with H2 and hyperoxia could increase the 14-day survival rate of ZY-challenged mice to 100%. Furthermore, ZY-challenged mice showed significant multiple organ damage characterized by increased serum levels of aspartate transaminase, alanine transaminase, blood urea nitrogen and creatinine, as well as lung, liver and kidney histopathological scores at 24 h after ZY injection. These symptoms where attenuated by H2 or hyperoxia alone; however, combination therapy with H2 and hyperoxia had a more marked beneficial effect against lung, liver and kidney damage in ZY-challenged mice. In addition, the beneficial effects of this combination therapy on ZY-induced organ damage were associated with decreased serum levels of the oxidative product 8-iso-prostaglandin F2α, increased activity of superoxide dismutase and reduced levels of the proinflammatory cytokines high-mobility group box 1 and tumor necrosis factor-α. In conclusion, combination therapy with H2 and hyperoxia provides enhanced therapeutic efficacy against multiple organ damage in a ZY-induced generalized inflammation model, suggesting the potential applicability of H2 and hyperoxia in the therapy of conditions associated with inflammation-related MODS.

The effects of agmatine on acute peritoneal inflammatory injury and neutrophil infiltration induced by zymosan in mice

To investigate the protective effect of agmatine (AGM) against peritoneal inflammatory response and neutrophil (PMN) infiltration induced by zymosan (ZYM) in mice. Thirty-six adult male C57BL/6 mice were randomly divided into sham group, model group, and AGM treatment group. Peritonitis model was reproduced by intra-peritoneal injection of 1 mg/mL ZYM (0.5 mL), while equivalent phosphate buffer saline (PBS) was given to sham group. 200 mg/kg AGM was injected into peritoneal cavity after ZYM challenge in AGM treatment group. Six mice in each group were sacrificed at 2 hours and 6 hours, respectively, after reproduction of the model. Blood sample and peritoneal lavage fluid (PLF) were collected. The levels of keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), tumor necrosis factor-α (TNF-α), interleukins-6 (IL-6) in serum and PLF were determined by enzyme linked immunosorbent assay (ELISA). The number of leukocytes and PMN in PLF were determined by hemocytometer and flow cytometry, respectively. Compared with sham group, all serum and PLF levels of KC, MIP-2, TNF-α and IL-6 were greatly elevated at 2 hours after ZYM injection in model group, while AGM treatment could dramatically reduce the levels of the above-mentioned cytokines in serum and PLF as compared with those of the model group [serum KC (ng/L): 990.7±137.9 vs. 2 053.2±262.7, MIP-2 (ng/L): 642.2±124.4 vs. 1 369.7±146.5, TNF-α (ng/L): 608.6±38.1 vs. 1 044.7±101.0, IL-6 (ng/L): 1 058.2±129.1 vs. 1 443.3±190.1; PLF KC (ng/L): 7 462.3±839.6 vs. 12 723.5±1 515.7, MIP-2 (ng/L): 1 570.8±193.4 vs. 3 471.4±384.7, TNF-α (ng/L): 1 115.8±156.7 vs. 1 499.2±231.2, IL-6 (ng/L): 2 646.5±223.2 vs. 3 126.7±291.4; all P < 0.05]. The expressions of KC, MIP-2 and TNF-α at 6 hours were significantly lower than those at 2 hours in model group and AGM treatment group, but IL-6 levels were further increased. The levels of KC and MIP-2 in serum and PLF at 6 hours were decreased to the levels of sham group. At 6 hours after the reproduction of the model, the number of total inflammatory cells and PMN of PLF in the model group was significantly higher than those of the sham group. In contrast, AGM notably lowered the number of inflammatory cells and PMN in peritoneal fluid after ZYM attack [total inflammatory cells (×109/L): 14.7±1.1 vs. 2.0±0.4, 10.1±1.2 vs. 14.7±1.1; PMN (×109/L): 11.37±1.22 vs. 0.18±0.05, 7.69±0.57 vs. 11.37±1.22, all P < 0.05]. AGM can effectively alleviate acute peritoneal inflammatory injury induced by ZYM, mainly through reducing the secretion of inflammatory mediators and chemokines, and inhibiting the infiltration of leukocytes and neutrophils.