Leukemia Cells Attenuate the Response of Leukemia-Associated Macrophages to LPS By Down Regulation of TLR4

Tumor-associated macrophages (TAMs) play important roles on tumor progress in solid tumors. Two therapeutic methods targeting TAMs, i.e. depleting TAMs and ¡°re-educating¡± TAMs to anti-tumoral M1 phenotype, have been proposed. LPS, which can stimulate macrophages to M1 phenotype, was suggested for anti-tumoral therapy. Recently, we reported leukemia-associated macrophages (LAMs), which showed leukemia-promoting effects, in leukemic microenvironment. In this study, we investigate whether LAMs can be re-educated by LPS in vitro.

THP-1 cells were treated with PMA for 48 hours for differentiation to macrophages. Then macrophages were treated with LPS for 24hrs directly (M¦Õ-LPS) or after cocultured with K562 or Jurkat cells for 48hrs (LAM-LPS). BrdU incorporation assay and TUNEL assay were used to detect the proliferation and apoptosis respectively. Significant decrease of BrdU positive rate was detected in M¦Õ-LPS while little change was detected in LAM-LPS. The TUNEL positive rates of both M¦Õ-LPS and LAM-LPS decreased. The expression of polarization related genes was studied by real time PCR. Increase of most M1 phenotype genes was detected and little difference of most M2 phenotype genes was detected in M¦Õ-LPS, whereas little difference of both M1 and M2 phenotype genes was detected in TAM-LPS. To further investigate the mechanism, Western blot was used to detect the M1 polarization determining transcription factor STAT1 among different groups. Drastic increase of p-STAT1 was detected in M¦Õ-LPS while little change was detected in LAM-LPS. These data suggested that LAMs had attenuated response to LPS. MAPK signal pathways were also examined by Western blot analysis. ERK, JNK and P38 pathway were significantly activated in M¦Õ-LPS, which was not observed in LAM-LPS. In order to further investigate the mechanism, real time PCR was used to detect the expression of LPS receptor complex: TLR4, CD14 and MD2. The results showed that TLR4 was decreased in LAM cocultured with both leukemia cells when macrophages without coculture or LPS treatment were set as control. Decrease of TLR4 was also observed in M¦Õ-LPS and LAM-LPS.

Our results suggested that leukemia cells attenuate the response of LAMs to LPS by down regulation of TLR4.