The Role Of Borrelia Burgdorferi Virulence Factor BmpA In The Stimulated THP-1 Cells Producing Chemokines

Objectives:1. To test the hypothesis that the recombinant Borrelia burgdorferi membrane protein A (rBmpA) will stimulate the immune cells (THP-1 cells selected as experimental subjects in this experiment) producing proinflammatory chemokine storm. Using the antibody protein microarray technology for high-throughput detection of the secretion of 38 chemokines in the human THP-1 cell culture supernatant, which stimulated by rBmpA.2. According to the microarray results from the first part of experiment with research status, the chemokines (CXCL13, CXCL5 and CCL1) were detected. To explore the mechanism of Lyme arthritis stimulated by rBmpA in the cellular level by the use of the ELISA to detect the secretion of the chemokines.3. According to the microarray results from the first part of experiment with research status, the chemokines (CXCL13, CXCL5 and CCL1) were detected. To explore the mechanism of Lyme arthritis stimulated by rBmpA in the cellular level by the use of the Quantificational Real Time PCR to detect the expression of the chemokines.4. To investigated the inhibition effort of Isoforskolin (ISOF) in Lyme inflammation caused by rBmpA.Methods:1. THP-1 cells cultured in 96-well plates, matured into macrophages by treatment with a phorbol ester (PMA) and were stimulated by rBmpA through 48 hours. Cell culture supernatants were collected at 24-hour and 48-hour time point. The concentrations of the chemokine CXCL13, CXCL5 and CCL1 in cell supernatants were detected by ELISA.2. THP-1 cells cultured in 96-well plates, matured into macrophages by treatment with PMA and were stimulated by rBmpA through 48 hours. The cell lysates were collected in 24-hour and 48-hour time points by using Trizol reagent. The expression levels of CXCL13, CXCR5 and CCL1 gene were detected by QRT-PCR.3. THP-1 cells cultured in 96-well plates, matured into macrophages by treatment with PMA and were affected by rBmpA and ISOF through 72 hours. The cell lysates were collected in 24-hour,48-hour and 72-hour time points by using Trizol reagent. The expression levels of IL-6 and TNF-a gene were detected by QRT-PCR.Results:1. The results of the antibody protein microarray technology for high-throughput: rBmpA can stimulate 14 kinds of chemokines in the human THP-1 cell culture supernatant. In this paper 3 kinds of chemokines (CXCL13, CXCL5 and CCL1) were detected to explore the mechanism of Lyme arthritis caused by rBmpA.2. The concentrations of the chemokine CXCL13, CXCL5 and CCL1 in cell supernatants increased in the rBmpA group, compared with the control group, the difference was statistically significant (p<0.05); and the concentrations of the 3 chemokines in cell supernatants increased much in the higher concentration group of the rBmpA.3. The expression of chemokine CXCL13, CXCL5 and CCL1 gene in the cell lysates increased in the rBmpA group, compared with the control group, the difference was statistically significant (p<0.05); and the expression of 3 chemokines in the cell lysates increased much in the higher concentration group of the rBmpA.4. Compared with rBmpA group, the gene expression of IL-6 and TNF-a were significantly reduced in the rBmpA+ISOF group.Conclusions:1.14 kinds of chemokines in the human THP-1 cell culture supernatant were stimulated by rBmpA.2. rBmpA was up-regulated the concentrations of the chemokine CXCL13, CXCL5 and CCL1 in cell supernatants.3. rBmpA was up-regulated the chemokine CXCL13, CXCL5 and CCL1 gene in the cell lysates.4. ISOF could significantly inhibit the inflammatory cytokines (IL-6 and TNF-a) in THP-1 cells by treatment with rBmpA.