Cytokines In Serum Of Mice Research Articles

Fritillaria cirrhosa D. Don Alleviates Inflammatory Progression and Suppresses M1 Polarization of Macrophages in Chronic Obstructive Pulmonary Disease

Introduction: Chronic obstructive pulmonary disease (COPD) is a progressive and largely irreversible disease. Current therapeutic approaches for COPD are limited in terms of slowing disease progression and suppressing pulmonary inflammation. Therefore, this study aimed to identify a method for alleviating inflammation in COPD. Methods: A COPD-like mouse model was established and treated with or without Fritillaria cirrhosa D. Don (hereinafter referred to as Fritillaria). The expression levels of inflammatory cytokines in mouse serum were detected by using enzyme-linked immunosorbent assay (ELISA). Additionally, lung tissue was analyzed by hematoxylin-eosin staining and immunohistochemistry analysis, respectively. MLE-12 cells were exposed to cigarette smoke extract (CSE) and treated with or without Fritillaria. The MTT assay was conducted to assess cell viability. The activation of NF-κB p65 was determined by Western blotting (WB). Finally, flow cytometry was applied to analyze the M1 macrophage percentage. Results: The results displayed that Fritillaria downregulated the levels of IL-1β, IL-6, IL-8, and TNF-α in the COPD-like mouse serum and MLE-12 cells. Fritillaria alleviated the inflammatory response in lung tissue of COPD-like mice. The cell viability of MLE-12 cells considerably decreased when exposed to CSE, which could be restored by adding Fritillaria. The Fritillaria reduced the activation of the pro-inflammatory factor NF-κB p65 and inhibited M1 polarization of macrophages, thereby mitigating the inflammatory response. Conclusion: In conclusion, Fritillaria exhibits beneficial effects in suppressing pulmonary infection-related inflammation in both the COPD-like mouse model and in vitro cell experiments.

Anti-inflammatory effect of the combined treatment of LMT-28 and kaempferol in a collagen-induced arthritis mouse model.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and swelling. Several studies have demonstrated that RA fibroblast-like synovial cells (RA-FLS) play an important role in RA pathogenesis. Activated RA-FLS contribute to synovial inflammation by secreting inflammatory cytokines including interleukin (IL)-1β, IL-6 and tumor necrosis factor-α. LMT-28 is derivative of oxazolidone and exerts anti-inflammatory effects on RA via IL-6 signaling pathway regulation. LMT-28 also regulates T cell differentiation in RA condition. However, the effect of LMT-28 on the migration and invasion of RA-FLS remains unknown. Kaempferol has been reported to have pharmacological effects on various diseases, such as inflammatory diseases, autoimmune diseases, and cancer. Additionally, kaempferol has been reported to inhibit RA-FLS migration and invasion, but it is not known about the therapeutic mechanism including molecular mechanism such as receptor. The present study aimed to investigate the synergistic effects of the combined treatment of LMT-28 and kaempferol on RA-FLS activation and RA pathogenesis in mouse model. LMT-28 and kaempferol co-administration inhibited RA disease severity and histological collapse in the joint tissues of CIA mice, as well as downregulated the levels of pro-inflammatory cytokines in mouse serum. Additionally, the combined treatment inhibited excessive differentiation of T helper 17 cells and osteoclasts. Furthermore, compared with single treatments, combined treatment showed enhanced inhibitory effects on the hyperactivation of IL-6-induced signaling pathway in RA-FLS. Combined treatment also inhibited RA-FLS cell proliferation, migration, and invasion and suppressed the expression of matrix metalloproteinase in RA-FLS. Furthermore, we confirmed that the combined treatment inhibited chondrocyte proliferation, migration, and invasion. In conclusion, our results suggest that the combined treatment of LMT-28 and kaempferol exerts a synergistic effect on the RA development via the regulation of IL-6-induced hyperactivation of RA-FLS. Furthermore, this study suggests that combination therapies can be an effective therapeutic option for arthritis.

Effect of per os administration of fucoxanthin alcoholic and oil extracts to Balb/c mice on biochemical parameters and serum cytokine level

Microalgae are a source of various biologically active substances, including carotenoids. One of the carotenoids of diatom microalgae is fucoxanthin, which has antioxidant, antimicrobial, antitumour and anti-inflammatory properties that can be used in the treatment of a wide range of human and animal pathologies. The aim of the study was to investigate the effect of alcohol and oil extract of fucoxanthin on biochemical parameters and levels of serum cytokines in mice during intragastric administration. Material and methods. Fucoxanthin extracts from the microalga Nanofrustulum shiloi were used. Mice of Balb/c strain were intragastrically administrated with 0.5 ml of oil (500 μg/ml) or alcoholic extract of fucoxanthin (187.5 μg/ml), mice in the comparison group were given 0.5 ml of olive oil or 45% ethyl alcohol once a day within 5 days. On the 8th day, blood serum was obtained and the level of albumin, triglycerides, cholesterol, low (LDL) and high density lipoproteins (HDL), urea, uric acid, creatinine, aspartate (AST) and alanine aminotransferases (ALT), lactate, stable nitric oxide (NO) metabolites (nitrites), cytokines (IL-1β, IL-6, IL-10, TNF-α, IFN-γ) was determined using ELISA with photometric detection. Results and discussion. In the comparison group, ethyl alcohol promoted an increase in serum triglyceride, cholesterol, LDL, HDL, ALT, AST, nitrite, IL-10, IFN-γ and TNF-α content compared to intact animals. Fucoxanthin alcoholic extract administration increased the level of LDL, HDL, uric acid, TNF-α and decreased albumin, cholesterol, uric acid, creatinine, ALT, AST, IL-6 content compared to control. The olive oil group showed a decrease in serum triglycerides, cholesterol, urea, uric acid, ALT and AST content but an increase in LDL, HDL, nitrite level compared to the control. Administration of fucoxanthin oil extract to animals decreased albumin, triglycerides, cholesterol, HDL, uric acid, ALT, AST, lactate, IL-6, IL-10 compared to control. Conclusions. Thus, it was discovered that fucoxanthin in Balb/c mice contributes to a decrease in hepatic synthetic function, as evidenced by the change in the concentration of albumin, triglycerides, uric acid), influences energy metabolism (creatinine, lactate), cell membrane stability (cholesterol, LDL, HDL), serum transaminase activity, pro- and anti-inflammatory cytokines content.

Pharmacokinetic Study of rhIL-18BP and Its Effect on Radiation-Induced Cytokine Changes in Mouse Serum and Intestine

Administration of recombinant human IL-18 binding protein (rhIL-18BP), a natural antagonist of IL-18, significantly increased mouse survival after lethal doses of irradiation. To further understand the roles of IL-18BP in radiation mitigation, we studied the pharmacokinetic (PK) parameters of rhIL-18BP, and the serum and intestinal cytokine changes in CD2F1 mice treated with vehicle or rhIL-18BP after 9.0 Gy total body irradiation (TBI). For the PK study, non-compartmental pharmacokinetic analysis was performed using PKsolver. Serum and intestine specimens were collected to measure 44-cytokine levels. Principal component analysis showed a clear separation of the non-irradiated samples from the irradiated samples; and partial separation with or without rhIL-18BP treatment. Cytokine clusters that were significantly correlated in the serum or intestine, respectively were identified. On the individual cytokine levels, serum and intestinal cytokines that were significantly changed by irradiation and rhIL-18BP treatment were identified. Finally, cytokines that were significantly correlated between their serum and intestinal levels were identified. The current study established the PK parameters of rhIL-18BP in mice, identified significantly changed cytokines in mouse serum and intestine after radiation exposure and rhIL-18BP treatment. Current data provide critical insights into IL-18BP's mechanism of action as a radiation mitigator.

Adipose-derived mesenchymal stem cells suppress ozone-mediated airway inflammation in a mouse model of chronic obstructive pulmonary disease

Objective and designOzone exposure is an important risk factor for Chronic Obstructive Pulmonary Disease (COPD) which is a global public health concern. Until now, there is no effective approach to reverse airflow limitation and accelerated loss of lung function completely. Here, we delineate the efficacy of mouse allogeneic adipose-derived mesenchymal stem cells (mASCs) in the treatment of COPD mice by intratracheal and intravenous administration. MethodsIn this study, we established ozone-exposed COPD model in mice and were administered intratracheally or intravenously with mASCs which were extracted, cultured, and identified in vitro. ResultsWe observed that exposure to ozone resulted in a marked lung neutrophilia with high levels of inflammatory cell counts, enhanced expression of cytokines IL-1β and TNF-α, reduced expression of IL-10, lung function and airspace enlargement. mASCs intratracheal administration rescured the lung neutrophilia, lung function and emphysema-like phenotype. Similar results were observed in mice with mASCs intravenous administration. But the altered levels of serum cytokines in mice with mASCs intratracheal administration appears more robust than those in mice with mASCs intravenous administration. ConclusionsCollectively, these data indicate that intratracheal administration of mASCs appears more effective in treating ozone-induced COPD compared to intravenous administration of mASCs, although the two approaches can be comparable in safety. mASCs are expected to become a new potential intervention strategy for COPD.

Blood Bacteria-Free DNA in Septic Mice Enhances LPS-Induced Inflammation in Mice through Macrophage Response.

Although bacteria-free DNA in blood during systemic infection is mainly derived from bacterial death, translocation of the DNA from the gut into the blood circulation (gut translocation) is also possible. Hence, several mouse models with experiments on macrophages were conducted to explore the sources, influences, and impacts of bacteria-free DNA in sepsis. First, bacteria-free DNA and bacteriome in blood were demonstrated in cecal ligation and puncture (CLP) sepsis mice. Second, administration of bacterial lysate (a source of bacterial DNA) in dextran sulfate solution (DSS)-induced mucositis mice elevated blood bacteria-free DNA without bacteremia supported gut translocation of free DNA. The absence of blood bacteria-free DNA in DSS mice without bacterial lysate implies an impact of the abundance of bacterial DNA in intestinal contents on the translocation of free DNA. Third, higher serum cytokines in mice after injection of combined bacterial DNA with lipopolysaccharide (LPS), when compared to LPS injection alone, supported an influence of blood bacteria-free DNA on systemic inflammation. The synergistic effects of free DNA and LPS on macrophage pro-inflammatory responses, as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10), pro-inflammatory genes (NFκB, iNOS, and IL-1β), and profound energy alteration (enhanced glycolysis with reduced mitochondrial functions), which was neutralized by TLR-9 inhibition (chloroquine), were demonstrated. In conclusion, the presence of bacteria-free DNA in sepsis mice is partly due to gut translocation of bacteria-free DNA into the systemic circulation, which would enhance sepsis severity. Inhibition of the responses against bacterial DNA by TLR-9 inhibition could attenuate LPS-DNA synergy in macrophages and might help improve sepsis hyper-inflammation in some situations.

Lingonberry Fruit Ethanol Extract Ameliorates DSS-Induced Ulcerative Colitis In Vivo and In Vitro

Ulcerative colitis (UC) is an inflammatory chronic intestinal disease with pathological characteristics, including imbalanced immune function and the overexpression of inflammatory cytokines and mediators. Inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6) were oversecreted in UC condition. Cyclooxygenase (COX)-2 and prostaglandin (PG)E2 were also overexpressed in colon tissue. Lingonberry (LB) (Vaccinium vitis-idaea L.) possesses pharmacological activities, including anti-oxidant, anti-cancer, and anti-obesity effects. To explore LB’s effects on UC, BALB/c mice were administered with 3% (w/v) dextran sulphate sodium (DSS) and LB extract (70% ethanol) orally for nine days. The severity of UC was measured by the change in body weight and colon length. To evaluate LB’s regulatory effect on inflammatory cytokines, the enzyme-linked immunosorbent assay (ELISA) kit was used to measure the inflammatory cytokines in mouse serum. Mouse peritoneal microphages were used to detect LB’s anti-inflammatory effect. The results showed that LB treatment ameliorated less weight loss and longer colon length compared to the DSS-treated group. LB treatment also ameliorated the secretion of inflammatory cytokines. These results indicated that LB has potential as an herbal medicine to treat UC.

Protective Immunity Against Neospora caninum Infection Induced by 14-3-3 Protein in Mice.

Neospora caninum is an apicomplexan parasite that infects many mammals and remains a threatening disease worldwide because of the lack of effective drugs and vaccines. Our previous studies demonstrated that N. caninum 14-3-3 protein (Nc14-3-3), which is included in N. caninum extracellular vesicles (NEVs), can induce effective immune responses and stimulate cytokine expression in mouse peritoneal macrophages. However, whether Nc14-3-3 has a protective effect and its mechanisms are poorly understood. Here, we evaluated the immune responses and protective effects of Nc14-3-3 against exposure to 2 × 107 Nc-1 tachyzoites. Antibody (IgG, IgGl, and IgG2a) levels and Th1-type (IFN-γ and IL-12) and Th2-type (IL-4 and IL-10) cytokines in mouse serum, survival rates, survival times, and parasite burdens were detected. In the present study, the immunostimulatory effect of Nc14-3-3 was confirmed, as it triggered Th1-type cytokine (IFN-γ and IL-12) production in mouse serum 2 weeks after the final immunization. Moreover, the immunization of C57BL/6 mice with Nc14-3-3 induced high IgG antibody levels and significant increases in CD8+ T lymphocytes in the spleens of mice, indicating that the cellular immune response was significantly stimulated. Mouse survival rates and times were significantly prolonged after immunization; the survival rates were 40% for Nc14-3-3 immunization and 60% for NEV immunization, while mice that received GST, PBS, or blank control all died at 13, 9, or 8 days, respectively, after intraperitoneal N. caninum challenge. In addition, qPCR analysis indicated that there was a reduced parasite burden and diminished pathological changes in the mice immunized with Nc14-3-3. Our data demonstrate that vaccination of mice with Nc14-3-3 elicits both cellular and humoral immune responses and provides partial protection against acute neosporosis. Thus, Nc14-3-3 could be an effective antigen candidate for vaccine development for neosporosis.

Fine particulate matter (PM2.5) promotes IgE-mediated mast cell activation through ROS/Gadd45b/JNK axis

BackgroundMast cells play an important role in allergic responses and persistently exposure to environmental fine particulate matter (PM2.5) exacerbates allergic diseases,but the details remained elucidative. ObjectivesTo investigate the effect of PM2.5 on IgE-mediated mast cell responses through an IgE-mediated mouse model and mast cell activation. MethodsThe β-hexosaminidase release and a BALB/c model of passive cutaneous anaphylaxis (PCA) was used to test IgE-mediated mast cells activation in vitro and in vivo. RNA-Seq technique was conducted to study the gene expression profile. Reactive oxygen species (ROS) production was measured by flow-cytometry. RT-PCR,WB and ELISA were performed to examine targeting molecules expression. ResultsPM2.5 facilitated IgE-mediated degranulation and increased cytokines expression in mast cells. Meanwhile, the Evan’s blue extravasation as well as serum cytokines in mice was increased after treatment with PM2.5. Furthermore, PM2.5 treatment dramatically increased the expression of Gadd45b which is an oxidative stress molecule that directly activates down-stream pathway, such as MEKK4/JNK. PM2.5 treatment activated MEKK4, JNK1/2 but not ERK1/2 and p38. Meanwhile, Knockdown of Gadd45b significantly attenuated PM2.5-mediated JNK1/2 activation and expression of cytokines. In addition, a JNK1/2-specific inhibitor SP600125 blocked IgE-mediated mast cell activation and cytokine release in PCA model mice. Moreover, PM2.5 treatment increased the ROS level and ROS inhibitor dramatically blocked the PM2.5-induced ROS production and reversed the PM2.5-mediated gene expression in the mitochondrial respiratory chain. ConclusionsPM2.5 regulates ROS production through Gadd45b/MEKK4/JNK pathway, facilitating IgE-mediated mast cell activation.

Regulatory effect of glutathione on treg/Th17 cell balance in allergic rhinitis patients through inhibiting intracellular autophagy

Background Glutathione is a potential therapy for systemic lupus erythematosus, but its role in allergic rhinitis (AR) has not been determined. This report probed into the actions of glutathione in AR, so as to supplement evidence for a therapeutical countermeasure for AR. Methods In this study, peripheral blood mononuclear cells (PBMCs) of patients were extracted and processed with glutathione. PBMCs and nasal mucosa tissues were collected from AR mouse models treated with or without glutathione. The proportions of Th17/Treg cell markers and autophagy-related molecules in the nasal mucosa, PBMCs or Th17/Treg cells were assessed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB) or flow cytometry analysis, and serum contents of related factors were analyzed by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was applied to observe the thickness of mouse mucosa. Results IL-17A, RORγt, Beclin1 and LC3-II/LC3-I levels were increased in AR patients, while Foxp3 and P62 were decreased. The serum contents of IL-17A and eosinophil cationic protein (ECP) in AR patients were elevated, but IL-10 level was reduced. In PBMCs of AR patients, the levels of IL-17A and LC3-II were increased, and the levels of Foxp3 and P62 were decreased, while these changes could be reversed by glutathione. In AR mouse models, glutathione could balance Th17/Treg cells, reduce autophagy, correct the levels of related cytokines in mouse serum, and shrunk mucosa thickness. Conclusion Glutathione could rescue the imbalance of Treg/Th17 cells by suppressing intracellular autophagy, which might be beneficial to the treatment of AR patients.

Nemonoxacin Has Immunoprotective Effects on Reducing Mortality in Lipopolysaccharide-Induced Mouse Sepsis Model.

Nemonoxacin is a novel non-fluorinated quinolone. The effect of nemonoxacin on modulation of host immune response is not known. We sought to determine whether nemonoxacin has immunoprotective effects on lipopolysaccharide (LPS)-induced mouse sepsis model. Therefore, mice were challenged with lethal dose LPS (12.5mg/kg) only or LPS with multi-dose nemonoxacin (40mg/kg q12h) by intraperitoneal injection, and the results showed nemonoxacin could significantly reduce mortality from 80 to 30% in this model. The effect of nemonoxacin on immune cells in vivo and in vitro was also investigated. Mice were treated with sublethal LPS (5mg/kg) or LPS + nemonoxacin, the myeloid cell subsets in mouse spleen were analyzed by flow cytometry, and cytokines in mouse serum were measured by ELISA. Additionally, mouse macrophage RAW264.7 cells were treated with LPS or LPS + nemonoxacin to investigate the immune modulatory effect of nemonoxacin in vitro, and the level of cytokines in cell culture supernatant was determined by ELISA. Analysis of myeloid cell subsets in the spleen showed nemonoxacin pretreatment could significantly inhibit LPS-induced proliferation of macrophages and dendritic cells but have no effect on neutrophils. Nemonoxacin could significantly reduce the expression of pro-inflammatory cytokines IL-6 and TNF-α while increase anti-inflammatory cytokine IL-10 expression, which were induced by LPS in vivo and in vitro. Finally, the immunomodulation of nemonoxacin in macrophage phagocytosis was also examined. The results displayed nemonoxacin pretreatment could significantly enhance the phagocytic function of macrophage. In conclusion, nemonoxacin has immune modulatory and protective effect on LPS-induced inflammation in vivo and in vitro.

THU0219 FIRST-INHUMAN STUDY OF SAFETY, PHARMACOKINETICS AND PHARMACODYNAMICS OF IRAK1/4 INHIBITOR R835 IN HEALTHY SUBJECTS

Background:Toll-Like Receptors (TLR) and Interleukin-1 Receptors (IL-1R) play a critical role in the innate immune response as microbial and tissue damage sensors, providing a bridge between the innate and adaptive immunity. Interleukin receptor associated kinases (IRAK) 1 and 4 are serine/threonine kinases that are essential for signaling downstream of most TLRs and IL-1Rs and the resulting production of pro-inflammatory cytokines. Suppression of TLR and IL-1R signaling through inhibition of IRAK1/4 kinases is a promising therapeutic approach for the treatment of inflammatory and autoimmune diseases. We have identified a potent and selective IRAK1/4 inhibitor (R835) that showed dose-dependent inhibition of lipopolysaccharide (LPS, a TLR4 agonist), and IL-1β induced serum cytokines in mice. R835 prevented disease onset and progression in multiple rodent models of inflammatory diseases, including arthritis and lupus models.Objectives:The aim of this FIH study was to characterize the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of R835 after single or multiple dose oral administrations.Methods:This study was a randomized, placebo-controlled, double-blind Phase 1 study in healthy subjects in three parts: single ascending doses (20 mg-1920 mg, Part A) with food effect in a separate cohort (480 mg), multiple ascending doses (120 mg and 960 mg, BID, Part B) with a caffeine interaction (960 mg cohort), and an intravenous LPS challenge test at 240 mg oral dose of R835 (Part C).Results:Single doses of up to 480 mg R835 in organic solution, single doses of up to 1920 mg R835 as capsule, multiple doses of 120 mg R835 Q12H (organic solution), and 960 mg R835 Q12H (capsule) were safe and well tolerated. All R835 related adverse events (AEs) were mild in intensity and reversible, and mostly associated with the higher doses of R835 in the organic solution. The most common AEs were headache and gastrointestinal disturbance. The PK of R835 was linear and dose proportional in exposure over the dose range studied. A nominal level of accumulation in plasma achieved rapidly upon repeated BID administrations with steady-state essentially attained in 2 days. A high-fat meal with the capsule formulation resulted in slow rate of absorption but had no effect on the extent of absorption. There was no effect of R835 on metabolism of caffeine (P450 CYP1A2 prototype substrate). In the LPS challenge test, R835 profoundly inhibited the acute release of cytokines, including TNF-α, IL-6, IL-8, MIP1α and MIP1β, but had no impact on CRP release.Conclusion:R835 was well tolerated after single or multiple dose administrations. The most common AEs were headache and gastrointestinal disturbance. For both of the formulations tested, the PK of R835 was linear and exposure was dose proportional with rapid steady-state attainment following BID administration. There was no drug-drug interaction by use of caffeine as the protype substrate. R835 inhibited the LPS induced release of cytokines in the serum, including TNF-α, IL-6, IL-8, MIP1α and MIP1β, mirroring preclinical data in mice. The desirable PK and safety profile combined with proof of mechanism, as demonstrated by inhibition of cytokine release, support progression of R835 into Phase II clinical development as an agent for the treatment of inflammatory and autoimmune diseases.Disclosure of Interests: :Lucy Yan Shareholder of: Amgen, Rigel, Employee of: Amgen, Rigel, Sandra Tong Shareholder of: Rigel, Employee of: Rigel, Anthony Absalom: None declared, Izaak den Daas: None declared, Gary Park Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vanessa Taylor Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Donna Chow Shareholder of: Rigel, Employee of: Rigel, Meng Lee Shareholder of: Rigel, Employee of: Rigel, Hanzhe Zheng Shareholder of: Rigel, Employee of: Rigel, Andrew Chow Shareholder of: Rigel, Employee of: Rigel

3\u2032-Sialyllactose prebiotics prevents skin inflammation via regulatory T cell differentiation in atopic dermatitis mouse models

3′-Sialyllactose (3′-SL), a natural prebiotic, maintains immune homeostasis and exerts anti-inflammatory and anti-arthritic effects. Although regulatory T cells (Tregs) prevent excessive inflammation and maintain immune tolerance, the effect of 3′-SL on Treg regulation is unclear. This study aimed to investigate the effect of 3′-SL on Treg responses in atopic dermatitis (AD) pathogenesis. Oral administration of 3′-SL reduced AD-like symptoms such as ear, epidermal, and dermal thickness in repeated topical application of house dust mites (HDM) and 2,4-dinitrochlorobenzene (DNCB). 3′-SL inhibited IgE, IL-1β, IL-6, and TNF-α secretion and markedly downregulated AD-related cytokines including IL-4, IL-5, IL-6, IL-13, IL-17, IFN-γ, TNF-α, and Tslp through regulation of NF-κB in ear tissue. Additionally, in vitro assessment of Treg differentiation revealed that 3′-SL directly induced TGF-β-mediated Treg differentiation. Furthermore, 3′-SL administration also ameliorated sensitization and elicitation of AD pathogenesis by suppressing mast cell infiltration and production of IgE and pro-inflammatory cytokines in mouse serum by mediating the Treg response. Furthermore, Bifidobacterium population was also increased by 3′-SL administration as prebiotics. Our data collectively show that 3′-SL has therapeutic effects against AD progression by inducing Treg differentiation, downregulating AD-related cytokines, and increasing the Bifidobacterium population.

Liu Shen Wan inhibits influenza a virus and excessive virus-induced inflammatory response via suppression of TLR4/NF-κB signaling pathway in vitro and in vivo

Ethnopharmacological relevanceLiu Shen Wan (LSW), first prescribed in “Lei Yunshang Song Fen Tang Fang”, traditional Chinese medicine (TCM), is used to cure influenza, tonsillitis, pharyngitis and mumps for more than one hundred years. Aim of studyLSW was proved extensive pharmacological properties, for instance, anti-inflammatory, anticancer, antiviral, analgesic, antibacterial and immunomodulatory activities. Nevertheless, the mechanism of this process and the evaluation of this product is still ambiguous. Hence, the study was designed to investigate the antiviral and anti-inflammatory activities of LSW against the influenza virus in vitro and vivo. Materials and methodsThe antiviral activities of LSW were assayed in virus-infected cells and mice. To study the antiviral effects of LSW against influenza A/PR/8/34 virus (PR8), we employed CPE inhibition assay with different concentrations of LSW at different times of infection in vitro. The mice were intranasally infected with virus to induce viral pneumonia, then treated with different doses of LSW. The death protection of the mice, the lung index, virus titer and pathological changes in the lung tissue of mice were investigated to estimate the anti-virus effect of LSW. Moreover, RT-qPCR was used to determine the mRNA expression of TNF-α, IL-1β, IL-6, and IFN-γ in the A549 cells and the supernatant of lung tissues, and the concentrations of these four cytokines in serum of mice were determined with ELISA. Western blot was used to determine the expression of TLR4, p-NF-κB p65, NF-κB p65, p-IκBα and IκBα in the A549 cells and lung tissues, which are the key targets of TLR4/NF-κB pathway. Moreover, the immunohistochemical assay was used to determine the expression of the NF-κB p65 in the mice lungs. ResultsLSW could significantly inhibit influenza virus at different stages of viral replication (at the process of the pre-, post-, and co-virus infection) in vitro. And LSW (100 mg/kg and 50 mg/kg) could effectively increase the survival time of mice. The virus titres, lung index, pathological changes in the mice lungs also decreased. Moreover, LSW could significantly reduce the contents of IL-1β, TNF-α, IFN-γ and IL-6 in the infected cells and the infected-mice. In addition, LSW could significantly reduce the expression of TLR4, p-NF-κB p65, NF-κB p65 and p-IκBα, while increase the IκBα in the infected cells and in the lung of mice. ConclusionsLSW could significantly not only inhibit virus replication and proliferation in vitro, but also ameliorate pneumonia damage in vivo. The antiviral effect was attributed to down-regulating the expression of inflammatory cytokines induced by influenza virus via regulating the activity of TLR4/NF-кB signaling pathway.

Abstract LB-C16: The percentage of tumor infiltrating lymphocytes in a panel of solid cancer PDX models correlates with a specific human cytokine pattern in peripheral blood of the tumor bearing animal

Abstract In the advent of immune modulatory compounds being part of most of the oncology drug development pipelines, the available preclinical models have to be characterized with respect to their interaction with the tumor microenvironment in general and with the immune cells of the host specifically. In the current study we analyzed 31 PDX models covering a broad range of solid cancer entities with regard to the phenotypic characteristics in a humanized environment. Well established PDX models from six different entities (breast, colon, renal, pancreatic, NSCL cancer and glioblastoma) were implanted subcutaneously into immune-compromised mice substituted with CD34+ hematopoietic stem cells (hu-mice). Tumor characterization included: tumor growth curves, take rates, human immune cell infiltrates of tumor (=TILs), peripheral blood and bone marrow (determined by flow cytometry, FC, and IHC) and secretion of human as well as murine cytokines in mouse serum (bead array: human 40plex (Biorad, #171AK99MR2; murine 23plex (Biorad, #M60009RDPD). The panel of 31 different PDX models of solid cancer displayed a distinct TIL and cytokine profile per model. The analysis of TILs revealed a large bandwidth of immune cell infiltration in the respective model ranging from 94% huCD45+ cells (renal cancer RXF 2773) to 3.54% huCD45+ (NSCLC LXFA 1041). Of note, the percentage and phenotype of the TILs was not related to the percentage and phenotype of the human immune cells in the BM or peripheral blood. In a next step, we categorized the PDX models into hot (>5% TILs =huCD45+) and cold (<5% TILs=huCD45+) tumors and compared the two groups with regard to the different read-outs. Differences between the two groups were found with regard to their sensitivity towards checkpoint inhibitor treatment (>5% TILS were more sensitive), the human cytokine profile and their CD4/CD8. A trend towards a higher mutational burden was observe in the group with >5%TILs. No difference between the two categories was determined in TIL composition (despite CD4/CD8 ratio), tumor growth behavior and secretion of mouse cytokines. Taken together, the characterization of PDX models with respect to their TIL composition facilitates model selection for innovative drugs in the immune-oncology field. The possibility to discriminate hot vs cold tumors by a minimal-invasive method like serum analysis of cytokine expression profile as potential biomarker will help to characterize the complete PDX collection of more than 500 models but will as well serve as additional read-out in preclinical I-O studies. Citation Format: Eva Oswald, Anna Edinger, Anne-Lise Peille, Hagen Klett, Julia Schueler. The percentage of tumor infiltrating lymphocytes in a panel of solid cancer PDX models correlates with a specific human cytokine pattern in peripheral blood of the tumor bearing animal [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C16. doi:10.1158/1535-7163.TARG-19-LB-C16

Macrophages protect against loss of adipose tissue during cancer cachexia.

BackgroundCancer cachexia represents a central obstacle in medical oncology as it is associated with poor therapy response and reduced overall survival. Systemic inflammation is considered to be a key driver of cancer cachexia; however, clinical studies with anti‐inflammatory drugs failed to show distinct cachexia‐inhibiting effects. To address this contradiction, we investigated the functional importance of innate immune cells for hepatocellular carcinoma (HCC)‐associated cachexia.MethodsA transgenic HCC mouse model was intercrossed with mice harbouring a defect in myeloid cell‐mediated inflammation. Body composition of mice was analysed via nuclear magnetic resonance spectroscopy and microcomputed tomography. Quantitative PCR was used to determine adipose tissue browning and polarization of adipose tissue macrophages. The activation state of distinct areas of the hypothalamus was analysed via immunofluorescence. Multispectral immunofluorescence imaging and immunoblot were applied to characterize sympathetic neurons and macrophages in visceral adipose tissue. Quantification of pro‐inflammatory cytokines in mouse serum was performed with a multiplex immunoassay. Visceral adipose tissue of HCC patients was quantified via the L3 index of computed tomography scans obtained during routine clinical care.ResultsWe identified robust cachexia in the HCC mouse model as evidenced by a marked loss of visceral fat and lean mass. Computed tomography‐based analyses demonstrated that a subgroup of human HCC patients displays reduced visceral fat mass, complementing the murine data. While the myeloid cell‐mediated inflammation defect resulted in reduced expression of pro‐inflammatory cytokines in the serum of HCC‐bearing mice, this unexpectedly did not translate into diminished but rather enhanced cachexia‐associated fat loss. Defective myeloid cell‐mediated inflammation was associated with decreased macrophage abundance in visceral adipose tissue, suggesting a role for local macrophages in the regulation of cancer‐induced fat loss.ConclusionsMyeloid cell‐mediated inflammation displays a rather unexpected beneficial function in a murine HCC model. These results demonstrate that immune cells are capable of protecting the host against cancer‐induced tissue wasting, adding a further layer of complexity to the pathogenesis of cachexia and providing a potential explanation for the contradictory results of clinical studies with anti‐inflammatory drugs.

A study of the effect of complexes of 3d-metal ions with gluconic acid on synthesis of cytokines in experimental immunodeficiency

Aim. Evaluation of the influence of synthesized gluconates of 3d-metals on production of cytokines in blood serum of mice with experimental immunodeficiency.
 Materials and Methods. The effect of complex compounds of bivalent 3d-metals (Mn, Fe, Co, Cu, Zn) with glucuronic acid on production of cytokines: interleukin (IL)-1β, IL-6, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) in blood serum of mice with experimental immunodeficiency induced by a single intraperitoneal administration of cyclophosphamide at the dose of 50 mg/kg was studied. 3d-Metal gluconates (10-2mol/l) were orally introduced for comparison with immunostimulatory drug «Licopid®» and calcium gluconate (doses were calculated according to the instructions) daily for two weeks, starting from the second day after the injection of cyclophosphamide. Levels of cytokines were determined by immunoenzyme analysis.
 Results. On the 16th day after the induction of immunodeficiency, levels of cytokines in blood serum of mice decreased: IL-1β – by 75.0%, IL-6 – by 65.2%, IFN-γ – by 61.6%, TNF-α –by 55.6% (p<0.05). Stimulatory effect of gluconates of 3d-metals on synthesis of cytokines depended on the used metal: the effect of MnGl, FeGl, CoGl, CuGl was lower than of licopid. ZnGl produced a weaker effect than licopid only on synthesis of IL-6, and equal effect on secretion of IL-1β and IFN-γ, and the highest effect on TNF-α (p<0.05 for all comparisons). Calcium gluconate did not produce any significant effect on the content of cytokines.
 Conclusion. The obtained results show a significant role of 3d-metals in maintenance of immune homeostasis, which, taking into account the literature data, may be explained by their action through activation of the nuclear transcription factor NF-κB which controls expression of cytokines.

The inhibition of malignant melanoma cell invasion of bone by the TLR7 agonist R848 is dependent upon pro-inflammatory cytokines produced by bone marrow macrophages.

Distant metastasis remarkably worsens the prognoses of malignant melanoma patients. Toll-like receptors (TLRs) recognize molecules derived from many types of pathogens and activate the innate intravital immune system. In this study, we examined the effects of R848, a TLR7 ligand, on bone invasion by malignant melanoma cells. Mice underwent transplantation with cells of a malignant melanoma cell line B16F10, and were also administered R848 every three days. Hindlimbs were obtained 13 days after transplantation and invasion of bone marrow by B16F10 cells was evaluated. ELISA was used to determine the concentrations of cytokines in mouse serum and in the culture medium from bone marrow macrophages (BMMs) in the presence or absence of R848. In addition, MTS assays were used to examine the effects of media from BMM cultures on the proliferation of B16F10 cells. The rate of infiltration by B16F10 cells and the area of invasion were significantly reduced with R848 administration. Furthermore, serum levels of IL-6, IL-12, and IFN-γ were significantly increased in mice administered R848, with the same trend observed in the culture medium of BMMs treated with R848. In addition, B16F10 cell proliferation was suppressed by the addition of medium from cultured BMMs treated with R848. Neutralization by antibodies against IL-6, IL-12, and IFN-γ abrogated the suppression of proliferation of B16F10 cells by culture medium from BMMs treated with R848. Our results suggest that R848 drives the production of IL-6, IL-12, and IFN-γ in BMMs, which reduces proliferation and bone invasion by B16F10 cells.

Anti-inflammatory activity of a thermophilic serine protease inhibitor from extremophile Pyrobaculum neutrophilum

Serine protease inhibitors (serpins) are a superfamily of proteins involved in many important biological processes, including inflammation. Serpins dysfunction-related diseases are mainly treated by augmentation therapy using serpins purified from human plasma. Pnserpin from hyperthermophilic archaeon Pyrobaculum neutrophilum showed protease inhibition activity and high stability. In this study, we examined the anti-inflammatory activity of Pnserpin using xylene-induced acute inflammatory model of mouse ear swelling and lipopolysaccharide (LPS)-induced murine RAW 264.7 macrophages cellular model. The inhibition of mouse ear swelling and the production of pro-inflammatory cytokines in mouse serum or in macrophages cell were used to evaluate the anti-inflammatory effect of Pnserpin. Our results showed that Pnserpin could inhibit the xylene-induced mouse ear swelling and suppress the production of pro-inflammatory cytokines in mouse serum and in LPS-induced RAW264.7 cells. This study indicated that Pnserpin might have anti-inflammatory effect in vivo and in vitro.

Anti-inflammatory action of the ethanolic extract from Sargassum serratifolium on lipopolysaccharide-stimulated mouse peritoneal macrophages and identification of active components

Sargassum, a genus of brown algae (Phaeophyceae) in the Sargassaceae family, comprises approximately 400 species in the world. Among them, Sargassum serratifolium has been reported to have high level of meroterpenoids, which have antioxidant, anti-inflammatory, and neuroprotective activities. This study investigated the anti-inflammatory mechanism of ethanolic extract of S. serratifolium (ESS) in lipopolysaccharide (LPS)-stimulated peritoneal macrophages and identified anti-inflammatory compounds in ESS. ESS inhibited LPS-induced nitric oxide (NO) and prostaglandin E2 and the expression of inducible nitric oxide synthase and cyclooxygenase-2. ESS also reduced the release of pro-inflammatory cytokines in a dose-dependent manner. LPS-induced nuclear factor-κB transcriptional activity and translocation into the nucleus were significantly inhibited by ESS treatment through the prevention of the degradation of inhibitor κB-α. Furthermore, ESS inhibited the production of pro-inflammatory cytokines in mouse serum. The main anti-inflammatory components in ESS were identified as sargahydroquinoic acid, sargachromenol, and sargaquinoic acid based on the inhibition of NO production. Our results indicate that ESS can be used as a potential source of therapeutic agents for the treatment of inflammatory diseases.