To investigate the expression of Toll-like receptor (TLR) 2 and 4 mRNA in local tissues of model of oropharyngeal candidiasis in mice and to explore the potential role of TLR2 and TLR4 in earlier period of immune response, a murine model of oropharyngeal candidiasis inoculated by cotton wool balls saturated with Candida albicans was established. Mice were sacrificed at the indicated time points and the oropharyngeal tissues were excised. The expression of TLR2 and TLR4 mRNA was detected by RT-PCR. The results showed that low level of TLR2/4 mRNA could be detected in oropharyngeal tissues, but they were markedly up-regulated 6 h after inoculation, peaking after 12-24 h. Tissue TLR4 mRNA was gradually down-regulated 24-48 h, while TLR2 mRNA levels remained high up to the 72nd h. These data suggested that oropharyngeal infection of Candida albicans could result in up-regulation of TLR2/4 mRNA expression in local tissues, which might play important roles in earlier period of immune response.
Candidiasis/metabolism ; Candidiasis, Oral/*metabolism ; Mouth Mucosa/*metabolism ; Pharyngitis/metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Random Allocation ; Toll-Like Receptor 2/*biosynthesis ; Toll-Like Receptor 2/genetics ; Toll-Like Receptor 4/*biosynthesis ; Toll-Like Receptor 4/genetics

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

To investigate the expression of Toll-like receptor (TLR) 2 and 4 mRNA in local tissues of model of oropharyngeal candidiasis in mice and to explore the potential role of TLR2 and TLR4 in earlier period of immune response, a murine model of oropharyngeal candidiasis inoculated by cotton wool balls saturated with Candida albicans was established. Mice were sacrificed at the indicated time points and the oropharyngeal tissues were excised. The expression of TLR2 and TLR4 mRNA was detected by RT-PCR. The results showed that low level of TLR2/4 mRNA could be detected in oropharyngeal tissues, but they were markedly up-regulated 6 h after inoculation, peaking after 12-24 h. Tissue TLR4 mRNA was gradually down-regulated 24-48 h, while TLR2 mRNA levels remained high up to the 72nd h. These data suggested that oropharyngeal infection of Candida albicans could result in up-regulation of TLR2/4 mRNA expression in local tissues, which might play important roles in earlier period of immune response.
Animals ; Candidiasis ; metabolism ; Candidiasis, Oral ; metabolism ; Female ; Male ; Mice ; Mouth Mucosa ; metabolism ; Pharyngitis ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Toll-Like Receptor 2 ; biosynthesis ; genetics ; Toll-Like Receptor 4 ; biosynthesis ; genetics

OBJECTIVETo investigate the effects of tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on the expression of pattern recognition receptors (PRRs) on the surface of mouse alveolar macrophages.

METHODSAlveolar macrophages from mouse were cultured in DMEM supplemented with 10% (V/V) endotoxin-free calf serum. After the alveolar macrophages were stimulated with TNF alpha and IFN gamma (concentration, 20 ng/ml) for 3 h, 6 h and 12 h, the expression of PRRs, including cluster of differentiation 14 (CD14), scavenger receptor (SR), toll-like receptor 4 (TLR4), TLR2 and TLR9 mRNA and proteins were examined by RT-PCR and immunohistochemistry.

RESULTSThe expressions of CD14, TLR2 and TLR9 receptors, which were related with cellular activation, were up-regulated by the stimulation of TNF alpha and IFN gamma (P < 0.05), while SR, which was related with cellular defense action, was down-regulated (P < 0.05). Although the expression of TLR4 was up-regulated, there was no statistical significance (P > 0.05).

CONCLUSIONSThe cytokines such as TNF alpha and IFN gamma could also produce feedback regulation on the expression of PRRs at the levels of genes and proteins. Such regulation on the PRRs expression would be significant for further amplification of inflammation cascade and eventually leading to uncontrolled inflammation.

Animals ; Cells, Cultured ; Interferon-gamma ; pharmacology ; Lipopolysaccharide Receptors ; biosynthesis ; genetics ; Macrophages, Alveolar ; metabolism ; Mice ; RNA, Messenger ; genetics ; Receptors, Pattern Recognition ; biosynthesis ; Toll-Like Receptor 2 ; biosynthesis ; genetics ; Toll-Like Receptor 4 ; biosynthesis ; genetics ; Toll-Like Receptor 9 ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; pharmacology

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

Toll-like receptors (TLRs) family may play important roles in inflammatory bowel disease. This study examined the expression of TLR2, TLR4 and TLR9 in the colonic tissues of patients with ulcerative colitis (UC) and explored their roles in the pathogenesis of UC. Colonic biopsies were taken from the colon of 30 patients with mild or moderate UC (at active phase) and 10 healthy controls during colonoscopy. TLR2, TLR4 and TLR9 protein expression levels were immunohistochemically detected. The mRNA expression levels of TLR2, TLR4 and TLR9 were assessed by reverse transcription polymerase chain reaction (RT-PCR). The disease activity index (DAI), colonoscopic and histologic grades and fecal microbial flora were determined. Histological examination showed that the intestinal mucous membrane of UC patients underwent acute inflammation changes. Immunohistochemistry exhibited that the expression levels of TLR2, TLR4 and TLR9 in colon epithelia and inflammatory cells were higher in UC patients than in control group (P<0.01). The mRNA expression levels of TLR2, TLR4 and TLR9 were increased in UC patients but were not detected in the normal controls. Expression levels of TLR2, TLR4 and TLR9 were positively correlated, and bore close correlation with DAI, colonoscopic and histologic grades and fecal microbial flora. An important mechanism of UC might be that abnormal activation of mucosal immunity by intestinal dysbacteriosis caused dysregulation of TLRS that mediates innate immunity.
Colitis, Ulcerative ; genetics ; metabolism ; pathology ; Colon ; metabolism ; microbiology ; Colonoscopy ; Feces ; microbiology ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; Intestinal Mucosa ; metabolism ; microbiology ; Male ; Reverse Transcriptase Polymerase Chain Reaction ; Severity of Illness Index ; Toll-Like Receptor 2 ; biosynthesis ; genetics ; Toll-Like Receptor 4 ; biosynthesis ; genetics ; Toll-Like Receptor 9 ; biosynthesis ; genetics

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

OBJECTIVETo study changes of TLR2 signaling pathway expression in Kupffer cells during the process of hepatic ischemia/reperfusion in a mice model and the mechanism of TLR2 signaling pathway participating in hepatic ischemia/reperfusion injury.

METHODSBALB/c mice were divided into 3 groups: sham operation (SH), ischemia/reperfusion (I/R) and GdCl3 treatment (Gd) groups. After 4 h of reperfusion, the expression of TLR2 mRNA and membrane TLR2 protein were analyzed in ischemic lobes of the livers, and in Kupffer cells isolated from ischemic lobes. The expression of NF-kappaB in ischemic lobes was also examined. Levels of endotoxin, ALT and TNFalpha were measured at the same time point.

RESULTSThe expressions of TLR2 mRNA and protein in both ischemic hepatic lobes and Kupffer cells isolated from ischemic lobes were increased in the I/R group compared to those in the SH group, as well as the expression of NF-kappaB in ischemic lobes, which was down regulated by intravenous GdCl3 treatment. Levels of ALT and TNFalpha in the portal vein were higher in the I/R group than in the SH group, which also were decreased with treatment of GdCl3. The level of endotoxin in the three groups remained constant.

CONCLUSIONTLR2 signaling pathway in Kupffer cells is activated during the process of hepatic ischemic/reperfusion injury. The activation of TLR2 signaling pathway in Kupffer cells may play a role in this process.

Animals ; Kupffer Cells ; metabolism ; Liver ; blood supply ; Male ; Mice ; Mice, Inbred BALB C ; Reperfusion Injury ; metabolism ; Signal Transduction ; Toll-Like Receptor 2 ; biosynthesis ; genetics

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

OBJECTIVETo study the effects of heat-killed Penicillium marneffei (PM) on the expressions of toll-like receptor-4 (TLR-4), toll-like receptor-2 (TLR-2) and dendritic cell associated C-type lectin-1 (Dectin-1)and the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). in mouse peritoneal macrophages.

METHODSMouse peritoneal macrophages were cultured in the presence of heat-killed yeast-phase PM for 24 h, and the average fluorescence intensity of TLR-2, TLR-4, and Dectin-1 in the macrophages was detected using flow cytometry. Fluorescent staining of the macrophages was performed to observe the fluorescence of TLR-2, TLR-4, and Dectin-1 with confocal microscopy. TNF-alpha mRNA in the cell culture supernatant was measured with real-time PCR, and TNF-alpha protein detected using enzyme-linked immunosorbent assay (ELISA).

RESULTSThe average fluorescence intensity of TLR-2, TLR-4 and Dectin-1 in the macrophages was increased in response to a 24-h PM stimulation, and the stimulated macrophages produced large amounts of TNF-alpha.

CONCLUSIONPM up-regulates the expression of TLR-2, TLR-4 and Dectin-1 in mouse peritoneal macrophages, and their expressions are directly associated with macrophage activation.

Animals ; Cells, Cultured ; Lectins, C-Type ; Macrophages, Peritoneal ; cytology ; immunology ; metabolism ; Male ; Membrane Proteins ; biosynthesis ; Mice ; Mice, Inbred BALB C ; Nerve Tissue Proteins ; biosynthesis ; Penicillium ; immunology ; Toll-Like Receptor 2 ; biosynthesis ; Toll-Like Receptor 4 ; biosynthesis ; Tumor Necrosis Factor-alpha ; biosynthesis