To investigate TLR2 (Toll-like receptor 2) mRNA expression in ischemic hepatic lobes under the condition of partial hepatic ischemia/reperfusion injury in BALB/c mice and its relationship with liver function impairment. A partial ischemia/reperfusion injury model was established. The portal vein and hepatic artery supply to the median and left lobes of the liver were obstructed by an atraumatic artery micro-clip, with the obstruction lasting for about 60 min. Then reperfusion was fulfilled by removal of the clip. The liver samples were collected at the 4th h after the restoration of blood inflow. Total RNA was extracted from the liver samples and analyzed quantitatively by method of real-time PCR. At the same time, portal vein serum and plasma were taken respectively for further detection of the level of endotoxin, tumor necrosis factor alpha (TNF-alpha) and plasmic alanine aminotransferase (pALT). The results indicated that TLR2 mRNA in ischemic lobe was up-regulated markedly in mice partial liver ischemia/reperfusion injury model compared to that in sham operation group (deltaCt: 1.05 +/- 1.02 vs 5.08 +/- 1.36, P<0.001). The level of portal vein pALT and TNF-alpha increased significantly (112.32 +/- 17.56 pg/ml vs 6.07 +/- 5.33 pg/ml, P<0.01; 890 +/- 127 microm/L vs 30 +/- 5 microm/L, P<0.001) . However, the level of portal vein endotoxin remained below the normal line, suggesting a state of non-endotoxemia. TLR2 mRNA expression in ischemic lobe, as well as portal vein pALT and TNF-alpha, was up-regulated in the model of mice partial ischemia/reperfusion injury, suggesting the involvement of TLR2 in ischemia/reperfusion pathological process.
Liver/*blood supply ; Liver/metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; RNA, Messenger/physiology ; Reperfusion Injury/etiology ; Reperfusion Injury/*metabolism ; Toll-Like Receptor 2/*biosynthesis ; Toll-Like Receptor 2/genetics ; Toll-Like Receptor 2/physiology ; Up-Regulation

BACKGROUNDCornea epithelial cells play early and crucial roles in the initiation of ocular surface responses to pathogens. Participation of toll-like receptor (TLR) 2 and TLR4, which are major forms of fungi receptors, may be involved in Aspergillus fumigatus induced immune responses. The objective of the present study was to examine whether inactive Aspergillus fumigatus conidia induce NF-kappaB activation and production of proinflammatory cytokines, and whether the expression of TLR2 and TLR4 were amplified by conidia in cultured immortalized human corneal epithelial cells (THCEs). This may contribute to our knowledge of the mechanism by which the host cornea can successfully defend against invasive fungi.

METHODSAspergillus fumigatus conidia were used to challenge THCE cells. THCE cells were harvested after 0.5, 1, 2 or 4 hours incubation. Real-time quantitative PCR was performed to determine the expression of TLR2, TLR4, TNF-alpha and IL-8. Western blotting was performed to determine the expression of NF-kappaB. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the expression of TNF-alpha and IL-8. And the release of TNF-alpha and IL-8 in the cell supernatant were also assessed by ELISA with or without pretreatment with TLR2 and TLR4 neutralizing antibodies.

RESULTSAspergillus fumigatus conidia elicited the expression of TLR2, TLR4, TNF-alpha and IL-8 mRNA in THCEs. Exposure of THCE cells to Aspergillus fumigatus conidia resulted in NF-kappaB activation, which increased at 30 minutes (increased from 11.35+/-2.74 in the controls to 19.12+/-3.48, P<0.05) and thereafter increased steadily up to 4 hours after challenge (P<0.01). Concomitant with NF-kappaB activation, secretion of TNF-alpha and IL-8 in conidia-challenged cells was increased in a time-dependent manner. Incubation of THCE cells with TLR2 antibody or TLR4 antibody before conidia challenge resulted in inhibition of conidia-induced TNF-alpha and IL-8 secretion (P<0.05), TLR2 antibody and TLR4 antibody together significantly increased inhibition of the conidia-induced secretion of TNF-alpha and IL-8 from THCE cells (P<0.01).

CONCLUSIONAspergillus fumigatus conidia stimulates THCEs inflammatory response through a pathway dependent on TLR2 and TLR4 signaling.

Aspergillus fumigatus ; immunology ; Cells, Cultured ; Epithelium, Corneal ; cytology ; immunology ; Humans ; Interleukin-8 ; biosynthesis ; NF-kappa B ; metabolism ; Toll-Like Receptor 2 ; physiology ; Toll-Like Receptor 4 ; physiology ; Tumor Necrosis Factor-alpha ; biosynthesis

BACKGROUNDTrichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors (CLR) and toll-like receptors (TLR) are the two major pattern recognition receptors (PRRs) that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively.

METHODSHaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae (1×10(6) and 1.5×10(5) colony-forming unit (CFU) in 2 ml medium, respectively) for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns (PAMPs) and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, surface toll-like receptor (TLR) 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter (FACS) 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment.

RESULTSHaCaT cells constitutively expressed mRNA of membrane-bound TLR1, 2, 4 and 6, Dectin1 and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-1.

CONCLUSIONThe cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and induce a lower immune response of HaCaT cells by limited cytokines secretion.

Cells, Cultured ; Cytokines ; biosynthesis ; Humans ; Keratinocytes ; immunology ; Lectins, C-Type ; genetics ; physiology ; RNA, Messenger ; analysis ; Receptors, Pattern Recognition ; genetics ; physiology ; Toll-Like Receptor 2 ; physiology ; Trichophyton ; immunology

To investigate TLR2 (Toll-like receptor 2) mRNA expression in ischemic hepatic lobes under the condition of partial hepatic ischemia/reperfusion injury in BALB/c mice and its relationship with liver function impairment. A partial ischemia/reperfusion injury model was established. The portal vein and hepatic artery supply to the median and left lobes of the liver were obstructed by an atraumatic artery micro-clip, with the obstruction lasting for about 60 min. Then reperfusion was fulfilled by removal of the clip. The liver samples were collected at the 4th h after the restoration of blood inflow. Total RNA was extracted from the liver samples and analyzed quantitatively by method of real-time PCR. At the same time, portal vein serum and plasma were taken respectively for further detection of the level of endotoxin, tumor necrosis factor alpha (TNF-alpha) and plasmic alanine aminotransferase (pALT). The results indicated that TLR2 mRNA in ischemic lobe was up-regulated markedly in mice partial liver ischemia/reperfusion injury model compared to that in sham operation group (deltaCt: 1.05 +/- 1.02 vs 5.08 +/- 1.36, P<0.001). The level of portal vein pALT and TNF-alpha increased significantly (112.32 +/- 17.56 pg/ml vs 6.07 +/- 5.33 pg/ml, P<0.01; 890 +/- 127 microm/L vs 30 +/- 5 microm/L, P<0.001) . However, the level of portal vein endotoxin remained below the normal line, suggesting a state of non-endotoxemia. TLR2 mRNA expression in ischemic lobe, as well as portal vein pALT and TNF-alpha, was up-regulated in the model of mice partial ischemia/reperfusion injury, suggesting the involvement of TLR2 in ischemia/reperfusion pathological process.
Animals ; Liver ; blood supply ; metabolism ; Male ; Mice ; RNA, Messenger ; biosynthesis ; genetics ; physiology ; Reperfusion Injury ; etiology ; metabolism ; Toll-Like Receptor 2 ; biosynthesis ; genetics ; physiology ; Up-Regulation

OBJECTIVEPrevious studies have shown that bacillus calmette-guerin (BCG) can deviate TH2 response toward TH1 response, resulting in a suppressive effect on the development of asthma/atopy. This study examined the effect of BCG treatment on regulatory T cells in asthmatic mice to investigate the possible mechanism.

METHODSKunming mice were sensitized and challenged with ovalbumin (OVA) to establish asthmatic models. Asthmatic mice were injected intradermally with BCG five days before and after sensitization. After 24 hrs of last challenge, bronchoaveolar lavage fluid (BALF) and peripheral blood were collected . The total cells and eosinophils were counted in the BALF. The percentage of CD4(+) CD25(+) in peripheral blood was detected with flow cytometry. Single spleen cell suspension was prepared and cultured in 1640 medium for 48 hrs and then the cytokine IL-10 level in the supernatant was determined using ELISA. The mice which were challenged with normal saline were used as the Normal control group.

RESULTSThe number of total cells and eosinophils in BALF in asthmatic mice [(27.27 +/- 5.36) x 10(7)/L and (6.59 +/- 1.32) x 10(7)/L respectively] were more than in the Normal control group [(1.52 +/- 0.36) x 10(7)/L and zero respectively] (P < 0.01). The number of total cells and eosinophils in BALF in asthmatic mice were reduced after BCG treatment [(13.71 +/- 3.17) x 10(7)/L and (1.43 +/- 0.37) x 10(7)/L respectively] (P < 0.01). The percentage of CD4(+) CD25(+) in peripheral blood of asthmatic mice [(11.59 +/- 1.33)%] was noticeably lower than that of the Control group [(13.66 +/- 1.68)%] (P < 0.01), but increased significantly in asthmatic mice after BCG treatment [(14.40 +/- 2.70)%] (P < 0.05). The IL-10 level in spleen cell supernatant in the BCG-treated group (7.79 +/- 1.34 pg/mL) also increased compared with that in the untreated asthmatic mice (5.54 +/- 0.66 pg/mL) (P < 0.01).

CONCLUSIONSBCG can markedly inhibit the airway inflammation in asthmatic mice possibly by promoting the production of regulatory T cells.

Animals ; Asthma ; immunology ; therapy ; BCG Vaccine ; therapeutic use ; Bronchoalveolar Lavage Fluid ; cytology ; Interleukin-10 ; analysis ; physiology ; Male ; Mice ; T-Lymphocytes, Regulatory ; immunology ; Toll-Like Receptor 2 ; physiology

Microglial cells were purified from a mixed neuroglia culture prepared from the neonatal chicken brain in vitro, and were infected with the vvMDV YL040920 isolate and an attenuated MDV vaccine strain CVI988/Rispens, respectively. The presence of cytopathic effect (CPE) was examined daily, and the MEQ expression in MDV-infected microglia was detected by immunohistochemistry assay. DNA replication of the MDV meq gene and transcription of the gB gene were determined by real-time quantitative PCR (qPCR) and qRT-PCR, respectively. The transcripts of Toll-like receptor (TLR) mRNA in microglia post MDV infection were quantified by qRT-PCR. The results of this study showed that both vvMDV YL040920 and attenuated vaccine strain CVI988/Rispens could infect microglia and produce characteristic CPE with plaque formation. The plaques were formed due to cells shedding at multi-sites, then quickly expanded and integrated. Furthermore, the MEQ protein was detected in nuclei of YL040920 and CVI988/ Rispens-infected microglia, and MDV meq DNA replication and gB gene transcription in MDV-infected microglia were also confirmed. Although both MDV DNA copies and gB transcripts were increased in the virus-infected microglia, the higher viral DNA load and gB transcript were observed for CVI988/Rispens than for YL040920 in vitro (P < or = 0.05/0.001). The transcriptions of TLR15 and TLR1LB gene were found to be up-regulated in microglia following MDV infection in vitro. Purified microglia infected with YL040920 was observed increased TLR15 and TLR1LB transcripts as early as 1 day post infection (dpi), and reached its peak level at 3 dpi, then decreased mildly at 5 dpi. For CVI988/Rispens, it induced an increase of TLR15 transcript as early as 1 dpi, and rose rapidly at 3 dpi, and then decreased slightly at 5 dpi. At the same time, CVI988/Rispens induced the increase of chTLR1LB transcript at 3 dpi and decreased at 5 dpi. By comparing the TLRs transcription between YL040920 and CVI988/Rispens-infected microglia, it was suggested that vvMDV YL040920 might induce more TLR15 transcript than the attenuated vaccine strain CVI988/Rispens (P < or = 0.01/0.001), while CVI988/Rispens induced more TLR1LB transcript than YL040920 (P < or = 0.001).
Animals ; Brain ; metabolism ; virology ; Chickens ; Gene Expression ; Herpesvirus 2, Gallid ; genetics ; physiology ; Marek Disease ; genetics ; metabolism ; virology ; Microglia ; metabolism ; virology ; Poultry Diseases ; genetics ; metabolism ; virology ; Toll-Like Receptor 1 ; genetics ; metabolism ; Toll-Like Receptors ; genetics ; metabolism ; Transcription, Genetic

To study the preventive effect of sophocarpine (Soc) on dextran sulfate sodium (DSS)-induced colitis in mice, in order to analyze the influence of Soc on toll like receptor 4 (TLR4)/mitogen-activated protein kinases (MAPKs) and janus tyrosine kinase 2 signal transducer and activator of transcription 3 (JAK2/STAT3) signal pathways in mice intestinal tissues. The mice was given 2.5% DSS for 6 days to induce the acute colitis model. The Soc-treated group was intraperitoneally injected with sophocarpine 30 mg · kg(-1) · d(-1) since the day before the experiment to the end. The disease activity index (DAI) was assessed everyday, and the colonic morphology and histological damage were observed with HE staining. The mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were detected by real-time RT-PCR. The changes in key protein kinase p38 mitogen-activated protein kinase (p38MAPK), c-Jun NH2-terminal protein kinase1/2 (JNK1/2), extracellular signal-regulated kinase1/2 (ERK1/2), JAK2, STAT3 in TLR4/MAPKs and JAK2/STAT3 signaling pathways were detected by western blot. The result showed that the model group showed statistical significance in body weight, DAI, colon length and histopathological changes compared with the normal group (P <0.05); however, the Soc-treated group showed significant improvements in the above indexes compared with the model group (P <0.05). TNF-α, IL-1β and IL-6 in the model group was significantly higher than that in the normal group (P <0.05), but lowered in the Soc-treated group to varying degrees (P <0.05). In the normal group, the expressions of TLR4 and the phosphorylation of P38, JNK1/2, JAK2, STAT3 were at low levels; in the model group, the phosphorylation of P38, JNK1/2, JAK2, STAT3 increased; the Soc-treated group showed a decrease in TLR4 expression compared with the model group, with notable declines in the phosphorylation of TLR4, P38, JNK1/2, JAK2, STAT3. These findings indicate that Soc can inhibit TLR4/MAPKs, K2/STAT3 signaling pathway activation, reduce the expression of proinflammatory cytokines TNF-α, IL-1β and IL-6 and relieve inflammatory reactions, so as to effectively prevent experimental colitis.
Alkaloids ; pharmacology ; therapeutic use ; Animals ; Colitis ; drug therapy ; immunology ; pathology ; Cytokines ; genetics ; Janus Kinase 2 ; antagonists & inhibitors ; physiology ; Male ; Mice ; Mice, Inbred BALB C ; Phosphorylation ; STAT3 Transcription Factor ; antagonists & inhibitors ; physiology ; Toll-Like Receptor 4 ; antagonists & inhibitors ; physiology

miR-146a is an immunoregulatory microRNA closely associated with viral infection. This study investigated the expression changes of miR-146a in peripheral blood monocytes of HCV-infected patients and the mechanism by which the THP-1 cells were stimulated with HCV core protein in vitro. It was found that in the peripheral blood monocytes of HCV-infected patients, miR-146a expression was upregulated. After treated by interferon/ribavirin, miR-146a expression was decreased when HCV RNA became undetectable. HCV core could directly stimulate THP-1 cells to produce miR-146a. Silencing TLR2 and MyD88 could significantly inhibit the expression of miR-146a. It was concluded that the expression of miR-146a in peripheral blood monocytes of HCV-infected patients was abnormally increased. The TLR2-MyD88 signaling pathway may take part in the overexpression of miR-146a in monocytes stimulated with HCV core protein.
Adult ; Base Sequence ; Cell Line ; DNA Primers ; Female ; Hepatitis C, Chronic ; blood ; Humans ; Male ; MicroRNAs ; blood ; Middle Aged ; Monocytes ; metabolism ; Myeloid Differentiation Factor 88 ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Toll-Like Receptor 2 ; physiology ; Young Adult

BACKGROUNDSepticemia and inflammation-mediated septic shock caused by Vibrio vulnificus (VV) is strongly associated with chronic liver disease. This study examined the effects of antimicrobial therapy on expression of hepatic toll-like receptors and inflammatory cytokines in rats with alcohol-induced liver disease complicated by VV sepsis.

METHODSMale Sprague-Dawley rats were assigned to the following treatment groups: normal control (N), alcoholic liver disease control (A), antimicrobial-treated alcoholic liver disease control (AA), alcoholic liver disease with VV sepsis (AV), and antimicrobial-treated alcoholic liver disease with VV sepsis (AVA). Alcohol-induced liver disease was observed in all groups except N. Expression of mRNAs encoding hepatic toll-like receptors 2 and 4, myeloid differentiation protein-2, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6 and IL-10 was determined by RT-PCR.

RESULTSmRNAs encoding toll-like receptors 2 and 4 and myeloid differentiation protein-2 were significantly up-regulated in group AV as compared to control groups at 2 - 24 hours of sepsis; peak expression occurred at 12 hours. These mRNAs were also up-regulated in group AVA but to lesser degrees than in group AV at comparable time post-infection. mRNAs encoding TNF-alpha, IL-1beta and IL-6 were significantly elevated in group AV as a function of infection. In group AVA as compared to AV, expression of TNF-alpha and IL-1beta mRNAs was lower at 12 - 24 hours post-infection and expression of IL-6 mRNA was lower at 24 hours post-infection. Compared with control groups, IL-10 mRNA expression in group AV was markedly higher at 12 - 24 hours of sepsis. Expression of IL-10 mRNA was lower in group AVA as compared to AV at 24 hours of sepsis.

CONCLUSIONSAntimicrobial therapy reduces expression of toll-like receptors and cytokines in rats with alcohol-induced liver disease complicated by VV sepsis. Monitoring hepatic toll-like receptor and cytokine expression during antibiotic therapy may be valuable for determining the course of VV sepsis in subjects with liver disease.

Adaptor Proteins, Signal Transducing ; genetics ; Animals ; Anti-Infective Agents ; therapeutic use ; Cytokines ; genetics ; Interleukin-10 ; genetics ; Interleukin-1beta ; genetics ; Interleukin-6 ; genetics ; Liver ; drug effects ; metabolism ; Liver Diseases, Alcoholic ; drug therapy ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sepsis ; drug therapy ; genetics ; microbiology ; Toll-Like Receptor 2 ; genetics ; Toll-Like Receptor 4 ; genetics ; Toll-Like Receptors ; genetics ; Tumor Necrosis Factor-alpha ; genetics ; Vibrio Infections ; drug therapy ; Vibrio vulnificus ; physiology

The purpose of this study was to investigate the expression of IL-16 in the rheumatoid synovium and the role of inflammatory cytokines and Toll-like receptor (TLR) ligands in IL-16 production by fibroblast- like synoviocytes (FLS) of rheumatoid arthritis (RA) patients. Immunohistochemical staining was performed with a monoclonal antibody to IL-16 in synovial tissues from patients with RA and likewise in patients with osteoarthritis (OA). FLS were isolated from RA synovial tissues and stimulated with IL-15, IL-1beta, IFN-gamma, and IL-17. The IL-16 mRNA level was assessed by semiquantitative RT-PCR and real time (RT) PCR and a comparison was made between IL-16 mRNA levels produced by RA-FLS and OA-FLS. Production of IL-16 was identified by a western blot assay, and IL-16 production after stimulation by specific ligands of TLR2 and TLR4 was assessed by RT-PCR. While immunohistochemical staining demonstrated strong expression of IL-16 mRNA in synovial tissues from patients with RA, similar findings were not present in the OA group. Moreover, mRNA expression of IL-16 by RA-FLS increased after treatment with IL-17 but not with IL-15, IL-1beta, and IFN-gamma. Specifically, IL-17 increased IL-16 mRNA level by RA-FLS and peripheral blood mononuclear cells in a dose-dependent manner. However, IL-17 did not stimulate IL-16 production in OA-FLS. Peptidoglycan, a selective TLR2 ligand, also increased production of IL-16 by RA-FLS dose- dependently, whereas LPS, a selective TLR4 ligand, had no such stimulatory effect. The results from our data demonstrate that IL-17 and TLR2 ligands stimulate the production of IL-16 by RA-FLS.
Arthritis, Rheumatoid/*metabolism ; Base Sequence ; Blotting, Western ; DNA Primers ; Humans ; Immunohistochemistry ; Interleukin-16/*biosynthesis/genetics ; Interleukin-17/*physiology ; RNA, Messenger/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Toll-Like Receptor 2/metabolism