This study investigated the functions and regulatory mechanism of Portulacae Herba and its chemical components on the Toll-like receptor 4(TLR4)/myeloid differentiation primary response 88(MyD88)/nuclear factor kappa B(NF-κB) inflammatory signaling pathway in the colon tissue of mice with dextran sodium sulfate(DSS)-induced ulcerative colitis(UC). A total of 35 mice were randomly divided into groups, including a blank group, a model group, a mesalazine group(0. 5 g·kg~(-1)), and low, medium,and high dose alkaloids from Portulacae Herba groups(9, 18, 36 mg·kg~(-1)), and a combination treatment group, with 5 mice in each group. The blank group was given purified water, while the other groups were continuously given a 3% DSS solution for 7 days to induce the UC model. From day 8 onwards, the treatment group received oral gavage according to the prescribed doses for 14 days. The overall condition, body weight, stool characteristics, and presence of blood in the stool were recorded daily. After the experiment, the disease activity index(DAI) was assessed for each group, and colon length was measured. Histopathological changes in colon tissue were examined using hematoxylin-eosin(HE) staining. The levels of pro-inflammatory cytokines, tumor necrosis factor-α(TNF-α),and interleukin-1β( IL-1β) in serum were measured by enzyme-linked immunosorbent assay( ELISA). The protein and m RNA expression of TLR4, MyD88, and NF-κB in colon tissue were measured using Western blot and quantitative real-time PCR(qPCR).Compared to the blank group, the model group showed a significant decrease in body weight, a notable increase in DAI scores, a significant shortening of colon length, and evident histopathological damage. The levels of inflammatory cytokines TNF-α and IL-1β in the serum were significantly elevated, and the protein and m RNA expression of TLR4, MyD88, and NF-κB in colon tissue were significantly up-regulated. In contrast, the alkaloids from Portulacae Herba treatment groups significantly improved symptoms and reduced body weight loss in mice, decreased DAI scores, alleviated colon shortening, lowered serum levels of TNF-α and IL-1β,significantly down-regulated the expression levels of TLR4, MyD88, and NF-κB proteins and genes in colon tissue, as well as reduced histopathological damage. Therefore, the study suggests that alkaloids from Portulacae Herba can alleviate intestinal inflammation damage in DSS-induced UC mice, with its mechanism involving the TLR4/MyD88/NF-κB signaling pathway.
Animals ; Colitis, Ulcerative/immunology* ; Toll-Like Receptor 4/immunology* ; Myeloid Differentiation Factor 88/metabolism* ; Mice ; NF-kappa B/metabolism* ; Signal Transduction/drug effects* ; Male ; Alkaloids/administration & dosage* ; Drugs, Chinese Herbal/administration & dosage* ; Humans ; Female ; Colon/metabolism* ; Disease Models, Animal

Two novel diketopiperazines (1 and 5), along with ten known compounds (2-4, 6-12) demonstrating significant skin inflammation inhibition, were isolated from a marine-derived fungus identified as Aspergillus sp. FAZW0001. The structural elucidation and configurational reassessments of compounds 1-5 were established through comprehensive spectral analyses, with their absolute configurations determined via single crystal X-ray diffraction using Cu Kα radiation, Marfey's method, and comparison between experimental and calculated electronic circular dichroism (ECD) spectra. Compounds 1, 2, and 8 exhibited significant anti-inflammatory activities in Propionibacterium acnes (P. acnes)-induced human monocyte cell lines. Compound 8 demonstrated the ability to down-regulate interleukin-1β (IL-1β) expression by inhibiting Toll-like receptor 2 (TLR2) expression and modulating the activation of myeloid differentiation factor 88 (MyD88), mitogen-activated protein kinase (MAPK), and nuclear factor κB (NF-κB) signaling pathways, thus reducing the cellular inflammatory response induced by P. acnes. Additionally, compound 8 showed the capacity to suppress mitochondrial reactive oxygen species (ROS) production and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation, thereby reducing IL-1β maturation and secretion. A three-dimensional quantitative structure-activity relationships (3D-QSAR) model was applied to compounds 5-12 to analyze their anti-inflammatory structure-activity relationships.
Humans ; Aspergillus/chemistry* ; Diketopiperazines/isolation & purification* ; Anti-Inflammatory Agents/isolation & purification* ; Interleukin-1beta/genetics* ; Toll-Like Receptor 2/immunology* ; Propionibacterium acnes/drug effects* ; NF-kappa B/genetics* ; Molecular Structure ; Myeloid Differentiation Factor 88/immunology* ; Monocytes/immunology* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Cell Line

OBJECTIVETo investigate the expression of Toll-like receptor (TLR) mRNA in enterovirus 71(EV-A71) infected human Jurkat T cells and clarify the role of TLRs in the pathogenesis of EV-A71 infection-induced inflammation.

METHODSEV-A71 strains were isolated from feces of children patients with hand, foot and mouth disease in 2014 by Shenzhen Center for Disease Control and Prevention. Human Jurkat T cells were infected with 200 μl EV-A71 at 10(3) cell culture infective dose 50%(CCID50)/ml. The expression of TLR1-TLR10 mRNA in human Jurkat T cells was assessed at different exposure time by RT-PCR. Levels of TLR7 mRNA expression were detected by real-time PCR, and levels of myeloid differentiation factor 88 (MyD88) by western blot. The cytokine secretion of interleukin (IL)-6, IL-8 and Tumor Necrosis Factor α (TNF-α) was analyzed by ELISA assay.

RESULTSThe relative expression level of TLR7 mRNA in human Jurkat T cells were 1.26 ± 0.15, 1.75 ± 0.20, 2.26 ± 0.23 and 3.74 ± 0.62 in 6, 12, 24 and 48 h after EV-A71 infection, which the differences were significant with mock-infected group(t values were -2.96, -6.38, -9.57, -7.71; P<0.05). Western blot showed that the protein expression levels of MyD88 had increased 1.34 times and 2.17 times in 24 h and 48 h after EV-A71 infection compared with mock-infected group. After infected for 24 h and 48 h, the levels of IL-6 were (302.86 ± 38.11), (179.70 ± 14.50) pg/ml, which were significantly higher than mock-infected group (176.42 ± 9.60), (179.70 ± 14.50) pg/ml (t values were -5.57, -18.54, P<0.05). The levels of TNF-α in EV-A71 infected group (100.81 ± 9.81) pg/ml was higher than that in mock-infected group (56.19 ± 6.94) pg/ml, and the difference was significant (t=-6.43, P=0.003).

CONCLUSIONTLR7 is the main pattern recognition receptor responsible for EV-A71 recognition in immune cells, which then leads to the activation of TLR7 downstream signaling and the production of proinflammatory cytokines.

Blotting, Western ; Cell Line ; Enterovirus A, Human ; pathogenicity ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Myeloid Differentiation Factor 88 ; metabolism ; Real-Time Polymerase Chain Reaction ; T-Lymphocytes ; immunology ; virology ; Toll-Like Receptor 7 ; immunology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the role of miR-21 in airway immunologic dysfunction induced by cold air irritation.

METHODSImmortalized human airway epithelial cell lines BEAS-2B and 16HBE cells were cultured in air-liquid phases. The differential expressions of endogenous miR-21, miR-164, and miR-155 in the cells induced by cold air exposure for different time were detected by real-time PCR. The reporter plasmid containing wild-type or mutated 3'UTR of TLR-4 were constructed and co-transfected into BEAS-2B cells or 16HBE cells together with miR-21 mimic, miR-21 mimic control, miR-21 inhibitor, or miR-21 inhibitor control. Following the transfection, dual luciferase reporter assay was performed to verify the action of miR-21 on TLR-4. miR-21 mimic, miR-21 mimic control, miR-21 inhibitor, and miR-21 inhibitor control were transfected via lipofectamine 2000 in BEAS-2B or 16HBE cells that were subsequently exposed to a temperature at 37 degrees celsius; or cold irritation (30 degrees celsius;), and the protein levels of TLR-4/MyD88 were detected by Western blotting.

RESULTSCold irritation caused a time- dependent up-regulation of miR-21 in both BEAS-2B and 16HBE cells (P<0.05) without obviously affecting the expressions of miR-164 and miR-155. Dual luciferase reporter assay demonstrated a direct combination of miR-21 and its target protein TLR-4. The synthesis levels of TLR-4/MyD88 protein were decreased in miR-21 mimic group even at a routine culture temperature (P<0.05), as also seen in cells with cold irritation (P<0.05). Treatment with the miR-21 inhibitor partially attenuated cold irritation-induced down-regulation of TLR-4/MyD88 protein (P<0.05).

CONCLUSIONCold air irritation-induced airway immunologic dysfunction is probably associated with TLR-4/MyD88 down-regulation by an increased endogenic miR-21.

3' Untranslated Regions ; Blotting, Western ; Cell Line ; Cold Temperature ; Down-Regulation ; Epithelial Cells ; immunology ; metabolism ; Humans ; Luciferases ; MicroRNAs ; metabolism ; Myeloid Differentiation Factor 88 ; metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Toll-Like Receptor 4 ; metabolism ; Transfection ; Up-Regulation

Endotoxic responses to bacterial lipopolysaccharide (LPS) are triggered by Toll-like receptor 4 (TLR4) and involve the production of inflammatory mediators, including interleukin-6 (IL-6), by macrophages. The detailed mechanism of IL-6 production by macrophages in response to LPS has remained unclear, however. We now show that LPS induces IL-6 synthesis in mouse peritoneal macrophages via the leukotriene B4 receptor BLT2. Our results suggest that TLR4-MyD88 signaling functions upstream of BLT2 and that the generation of reactive oxygen species (ROS) by NADPH oxidase 1 (Nox1) and consequent activation of the transcription factor nuclear factor (NF)-kappaB function downstream of BLT2 in this response. These results suggest that a TLR4-MyD88-BLT2-Nox1-ROS-NF-kappaB pathway contributes to the synthesis of IL-6 in LPS-stimulated mouse macrophages.
Animals ; Cell Line ; Interleukin-6/*biosynthesis ; Leukotriene B4/metabolism ; Ligands ; Lipopolysaccharides/immunology ; Macrophages/immunology/*metabolism ; Macrophages, Peritoneal/immunology/metabolism ; Mice ; Myeloid Differentiation Factor 88/*metabolism ; NADH, NADPH Oxidoreductases/metabolism ; NF-kappa B/metabolism ; Reactive Oxygen Species/metabolism ; Receptors, Leukotriene B4/*metabolism ; Signal Transduction

Toxoplasma gondii is an intracellular protozoan parasite that causes a Th1 cellular immunity. Our previous study showed that T. gondii lysate antigen (TLA) treatment in S180 tumor-bearing mice resulted in tumor reduction by suppressing CD31 expression, a marker of angiogenesis. In the present study, to investigate tumor suppressive effect of TLA under the absence of T lymphocytes, athymic nude mice were compared with euthymic mice in the anti-tumorigenic effect triggered by TLA in CT26 tumors. According to the results, intratumorally injected TLA reduced tumor growth and TIMP-1 level, a metastatic marker, in both euthymic and athymic mice. TLA treatment led to a sharp increase in IL-12 expression in serum cytokine profiling of athymic mice, and increased MyD88 signals in macrophages derived from the bone marrow, implying the activation of innate immunity. The selective induction of IL-12 by TLA treatment had an anti-tumorigenic effect.
Animals ; Antigens, Protozoan/*immunology ; Disease Models, Animal ; Immunity, Innate ; Immunotherapy/*methods ; Interleukin-12/*blood ; Macrophages/immunology ; Mice, Inbred BALB C ; Mice, Nude ; Myeloid Differentiation Factor 88/analysis ; Neoplasms/pathology/*therapy ; Toxoplasma/*immunology ; Treatment Outcome

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.
Acute-Phase Proteins/metabolism ; Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; Antigens, CD14/metabolism ; Bone Marrow Cells/cytology/drug effects ; CD8-Positive T-Lymphocytes/*immunology ; Carrier Proteins/metabolism ; Cell Differentiation/drug effects ; Cell Nucleus/drug effects/metabolism ; Cell Proliferation/drug effects ; Cytokines/biosynthesis ; Dendritic Cells/cytology/drug effects/enzymology/*immunology ; Enzyme Activation/drug effects ; Lymphocyte Activation/*drug effects ; Membrane Glycoproteins/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinases/metabolism ; Myeloid Differentiation Factor 88/metabolism ; NF-kappa B/metabolism ; Neoplasms/immunology/*pathology ; Pectins/*pharmacology ; Phenotype ; Protein Transport/drug effects ; Receptors, Chemokine/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes, Cytotoxic/cytology/drug effects ; Toll-Like Receptor 4/*agonists/metabolism

BACKGROUNDSmoking causes frequent asthma attacks, leading to a rapid decline in lung function in patients with asthma, and it can also reduce the therapeutic effect of glucocorticoids in patients with asthma. Therefore, the present study aimed to investigate the effect of cigarette smoke on the expression of myeloid differentiation factor 88 (MyD88) in marrow dendritic cells (DCs) in asthmatic rats, and to explore the molecular mechanism of cigarette smoke exposure on asthma by DCs.

METHODSForty Wistar rats were randomly divided into the following groups: control, smoke exposure, asthma, and asthma combined with smoke exposure. The animal model was established, and then rat bone marrow-derived DCs were collected. Additionally, rat spleen lymphocytes and bone marrow-derived DCs were cultured together for mixed lymphocyte responses. Interferon (IFN)-gamma and interleukin (IL)-4, IL-10, and IL-12 expressions were determined by enzyme-linked immunosorbent assay (ELISA). MyD88 expression was determined by Western blotting. The proliferation of lymphocytes was examined with methyl thiazolyl tetrazolium (MTT) colorimetric assay.

RESULTSMyD88 expression was decreased in the asthma combined with smoke exposure group compared to the asthma group (P < 0.01), and IL-10 and IL-12 expressions were decreased in the asthma combined with smoke exposure group compared to control group (P < 0.01). In addition, DCs stimulating activity on allogeneic lymphocytes were significantly decreased in the smoke exposure combined with asthma group compared to the control and asthma groups (P < 0.01). After allogeneic mixed lymphocyte responses, IL-4 expression was increased and IFN-gamma was decreased in the asthma group and the asthma combined with smoke exposure group compared to control group (P < 0.01). IL-4 expression was increased and IFN-gamma was decreased in the asthma combined with smoke exposure group compared to the asthma group (P < 0.01). The study also showed that MyD88 expression was positively correlated with IL-12 and IFN-gamma expressions and the activity of lymphocytes (P < 0.01), and negatively correlated with IL-4 expression (P < 0.01).

CONCLUSIONSSmoking aggravates asthma by weankening immunological mechanism. MyD88-dependent pathways may play a role in the immunological balance and activation of lymphocytes.

Animals ; Asthma ; immunology ; metabolism ; Bone Marrow Cells ; cytology ; metabolism ; Dendritic Cells ; drug effects ; metabolism ; Lymphocyte Activation ; drug effects ; Male ; Myeloid Differentiation Factor 88 ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Smoking ; adverse effects

OBJECTIVEA great deal of clinical evidence and epidemiologic data suggest that Kawasaki disease (KD) is correlated with an acute regulating imbalance of immunology. Lots of evidences in the past suggested that nuclear transcription factor-kappaB and preinflammation factors were up-regulated significantly in patients with KD. But the causative factors are still unknown. Toll-like receptors (TLRs) is a type I trans-membrane protein which could recognize ligands of pathogen microbes, activate the nuclear transcription factor-kappaB and promote gene transcription of pre-inflammation factors and co-stimulatory molecules on cell surface. Aberrant activation of signal pathway of TLRs could interfere with autoimmune tolerance and cause autoimmune diseases. The study was designed to investigate the role of signal transduction of TLRs on immunological pathogenesis of KD.

METHODSSixteen children with KD and 16 age-matched health children were studied. Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the levels of TLRs 1 - 10, MD-2, MyD88, IL-1beta, IL-6 and IL-8 mRNA expressions in peripheral blood mononuclear cells, and expressions of TLRs 2, 4 and co-stimulatory molecules such as CD80 and CD86 in monocyte/macrophage were analyzed by flow cytometry.

RESULTS(1) Compared with control group, the protein and mRNA levels of TLR4 in KD group were up-regulated significantly [(Real-time PCR: 325.22 +/- 50.34 vs. 2.20 +/- 0.23, P < 0.01); (flow cytometry: 15.96% +/- 5.94% vs. 3.21% +/- 0.62%, P < 0.01)], the difference being not significant as to other TLRs. (2) Transcriptional levels of MD-2 and Myd88 were significantly up-regulated in acute phase of KD (P < 0.01), and down-regulated after the treatment with intravenous gamma globulin therapy. (3) Expressions of co-stimulatory molecules and cytokines in monocyte/macrophage during acute phase of KD were higher than those of control group (P < 0.01).

CONCLUSIONExpressions of TLRs 4, MD-2 and Myd88 were up-regulated during acute phase in KD, suggesting that aberrant activation of TLRs 4 might be one of the initiating factors of immune aberrance in KD.

Case-Control Studies ; Child, Preschool ; Female ; Flow Cytometry ; Humans ; Immunoglobulins, Intravenous ; immunology ; therapeutic use ; Immunologic Factors ; immunology ; therapeutic use ; Infant ; Leukocytes, Mononuclear ; drug effects ; immunology ; metabolism ; Male ; Mucocutaneous Lymph Node Syndrome ; drug therapy ; immunology ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; RNA, Messenger ; Reverse Transcriptase Polymerase Chain Reaction ; Toll-Like Receptor 4 ; genetics ; metabolism ; Toll-Like Receptors ; genetics ; metabolism ; Up-Regulation ; drug effects