Gene expression profiles of RAW264.7 macrophages stimulated with preparations of LPS differing in isolation and purity.

Lipopolysaccharide is a major component of the cell wall of Gram-negative bacteria and a potent stimulator of innate immune response via TLR4. Studies on the LPS action both in vivo and in vitro have used different preparations of LPS, including ultra-pure LPS (LIST) and a less pure but less expensive form (Sigma) isolated from Escherichia coli serotype O111:B4. The difference between the effects of these compounds has not been well studied although this information is important in understanding TLR stimulation. In this study, we compared response of RAW264.7 macrophage cells treated LIST or Sigma LPS for 6 h and 24 h. Gene expression data were analyzed to identify specific genes and pathways that are in common and unique to the two LPS preparations. Seven hundred fifty-five genes were differentially expressed at 6 h in response to Sigma LPS and 973 were differentially expressed following LIST LPS treatment, with 503 in common. At 24 h, Sigma LPS induced or repressed 901 genes while 1646 genes were differentially regulated by LIST LPS treatment; 701 genes were shared by two forms of LPS. Although considerably more genes were differentially expressed in response to LIST LPS, similar molecular pathways and transcriptional networks were activated by the two LPS preparations. We also treated bone marrow-derived macrophages (BMMs) from three strains of mice with different concentrations of LIST and Sigma LPS and showed that BMMs produced more IL-6 and TNF-α in response to LIST LPS at low LPS concentrations but, at higher LPS concentrations, more cytokines were produced in response to stimulation by Sigma LPS. Together, these findings suggest that, despite activation of similar molecular pathways by LIST and Sigma LPS preparations, residual protein impurities in the Sigma LPS preparation may nevertheless influence the transcriptional profile attributed to TLR4 stimulation.

Duke Scholars

Author David Stephen Pisetsky Medicine, Rheumatology and Immunology