Investigation of the relationship between Community-acquired respiratory distress syndrome toxin and the High-mobility group box protein 1-Toll-like receptors-Myeloid differentiation factor 88 signaling pathway in Mycoplasma pneumoniae pneumonia

Background: In recent years, reports of refractory Mycoplasma pneumoniae pneumonia (RMPP) have gradually increased, including reports on how these conditions threaten the lives of children. However, the specific mechanism of Mycoplasma pneumoniae pneumonia (MPP) remains unclear. This study aimed to investigate the relationship between community-acquired respiratory distress syndrome toxin (CARDS TX) and High-mobility group box protein 1-Toll-like receptors-Myeloid differentiation factor 88 (HMGB1-TLRs-MyD88) in MPP and to examine the immune pathogenesis of Mycoplasma pneumoniae infection. Methods: Children who were diagnosed with MPP and examined by bronchoscopy were included in the MPP group. Additionally, children who underwent bronchoscopy because of bronchial foreign bodies in the same period were included in the control group. Gene expression of CARDS TX, HMGB1, Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), MyD88, and cluster of differentiation 14 (CD14) in bronchoalveolar lavage fluid (BALF) were detected using real-time reverse transcription-polymerase chain reaction. Correlations between CARDS TX and HMGB1-TLRs-MyD88 were analyzed. Results: CARDS TX, HMGB1, TLR2, MyD88, and CD14 mRNA expression in BALF in the MPP group was significantly higher than that in the control group (all P< 0.05). CARDS TX mRNA expression was positively correlated with HMGB1, TLR2, MyD88, and CD14 mRNA expression (all P< 0.05). Furthermore, HMGB1 mRNA expression was positively correlated with TLR2, MyD88, and CD14 mRNA expression (all P< 0.05). Conclusions: CARDS TX may participate in the immune pathogenesis of MPP through the HMGB1-TLRs/CD14-MyD88 pathway.

Investigation of The Relationship Between CARDS TX and The HMGB1-TLRs-MyD88 Signaling Pathway in MPP

Background: This study aimed to investigate the relationship between community-acquired respiratory distress syndrome toxin (CARDS TX) and HMGB1-TLRs-MyD88 in Mycoplasma pneumoniae pneumonia (MPP) and to examine the immune pathogenesis of Mycoplasma pneumoniae infection. Methods: Children who were diagnosed with MPP and examined by bronchoscopy were included in the MPP group. Additionally, children who underwent bronchoscopy because of bronchial foreign bodies in the same period were included in the control group. Gene expression of CARDS TX, high-mobility group box protein 1 (HMGB1), Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and cluster of differentiation 14 (CD14) in bronchoalveolar lavage fluid (BALF) were detected using real-time reverse transcription-polymerase chain reaction. Correlations between CARDS TX and HMGB1-TLRs-MyD88 were analyzed. Results: CARDS TX, HMGB1, TLR2, MyD88, and CD14 mRNA expression in BALF in the MPP group was significantly higher than that in the control group (all P< 0.05). CARDS TX mRNA expression was positively correlated with HMGB1, TLR2, MyD88, and CD14 mRNA expression (all P< 0.05). Furthermore, HMGB1 mRNA expression was positively correlated with TLR2, MyD88, and CD14 mRNA expression (all P< 0.05). Conclusions: CARDS TX may participate in the immune pathogenesis of MPP through the HMGB1-TLRs/CD14-MyD88 pathway.

165 THE EFFECT OF shRNA TARGETING CLUSTER OF DIFFERENTIATION ANTIGEN 14 ON GENE EXPRESSION OF TNF-α, TLR4, AND IL-6 IN LIPOPOLYSACCHARIDE-INDUCED BUFFALO PERIPHERAL BLOOD MONOCYTE/MACROPHAGE

Cluster of differentiation antigen 14 (CD14) plays a crucial role in the inflammatory response to lipopolysaccharide (LPS), which interacts with TLR4 and MD-2 to enable cell activation, leading to inflammation. Several studies have proved that upstream inhibition of bacterial LPS/toll-like receptor 4 (TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. In this study, to explore the effect of CD14 down-regulation on TLR4 signal conductive-related genes expression after stimulation by LPS, five CD14 shRNA (319/421/755/970/1041) sequences and a negative control sequence (NC-1864) were synthesised and used to construct lentiviral recombinant plasmid pSicoR-GFP-shRNA. Lentiviral recombinant plasmids of pSicoR-GFP-shRNA and fusion expression vector of pDsRed-N1-buffalo CD14 were co-transfected into HEK293 using liposome. At 72 h after transfection, the expression of exogenous buffalo CD14 mRNA was reduced at different level for all shRNA plasmids, in which shRNA-1041 had the highest interfering efficiency by RT-qPCR and fluorescence-activated cell sorting analysis. Then, buffalo peripheral blood monocyte/macrophage was purified and infected by the CD14 shRNA lentivirus. After 7 days of infection, the cells were stimulated by 1 µg mL–1 LPS for 3 h, then the mRNA expression level of CD14, TLR4, IL-6, and TNF-α transcripts in the cells were detected by the RT-qPCR method. After stimulation by LPS, the expression of endogenous CD14 was significantly reduced by CD14 shRNA-1041, the mRNA expression level of TLR4, IL-6, and TNF-α genes was also significantly down-regulated in comparison with control group (P ≤ 0.01). In conclusion, the selected CD14 shRNA-1041 cannot only inhibit the expression of endogenous CD14 mRNA in buffalo peripheral blood monocyte/macrophage, but also downregulate the mRNA expression of CD14, TLR4, IL-6, and TNF-α. The above results demonstrate that knockdown of endogenous CD14 has obvious coordination effects on the signal conductive function of TLR4 after stimulating by LPS, and shRNA technology will provide a new way to prevent endotoxin-related diseases in livestock. This work was supported by the National Transgenic Project (2009ZX08007-009B), Guangxi natural science funding (2012GXNSFCB053002), and funding of State Key Laboratory of Subtropical Bioresource Conservation and Utilisation (KSL-CUSAb-2012-02).

Decrease in TSH Receptor Autoantibodies during Antithyroid Treatment: Relationship with a Long Noncoding Heg RNA and Cdk1 mRNA in Mononuclear Cells

We have previously shown that a long noncoding RNA transcript Heg is negatively correlated with TSH receptor autoantibodies (TRAb) in patients with untreated Graves' disease and with CD14 mRNA in treated patients and controls. Thus patients with high concentrations of Heg RNA have low levels of TRAb or CD14 mRNA, respectively. Here we show that an additional factor, gene expression of Cdk1 in mononuclear cells, is positively related to concentrations of TRAb in patients with untreated Graves' disease. Cdk1 mRNA is very important for regulation of cell cycle activity. It is well known that TRAb decrease significantly during treatment with antithyroid drugs. This decrease during treatment cannot be explained by Heg RNA, which remains unchanged. Cdk1 mRNA decreased significantly during treatment to values below values obtained in normal subjects. Thus both Heg RNA and Cdk1 mRNA may influence the level of TSH receptor autoantibodies but by different mechanisms.

Enhancement of the endotoxin recognition pathway by ventilation with a large tidal volume in rabbits

Ventilation with a small tidal volume (Vt) is associated with better clinical outcomes than with a large Vt, particularly in critical settings, including acute lung injury. To determine whether Vt influences the lipopolysaccaharide (LPS) recognition pathway, we studied CD14 expression in rabbit lungs and the release of TNF-α by cultured alveolar macrophages after 240 min of ventilation with a large (20 ml/kg) vs. a small (5 ml/kg) Vt. We also applied small or large Vt to lungs instilled with 50 μg/kg of LPS. The alveolar macrophages collected after large Vt ventilation revealed a 20-fold increase in LPS-induced TNF-α release compared with those collected after small Vt ventilation, whereas TNF-α was undetectable without LPS stimulation. In animals ventilated with a large Vt, the expression of CD14 mRNA in whole lung homogenates and the expression of CD14 protein on alveolar macrophages, assessed by immunohistochemistry, were both significantly increased in the absence of LPS stimulation. A large Vt applied to LPS-instilled lungs increased the pulmonary albumin permeability and TNF-α release into the plasma. These results suggest that mechanical stress caused by a large Vt sensitizes the lungs to endotoxin, a phenomenon that may occur partially via the upregulation of CD14.