Figure 3

Absence of TLR4, but not TLR2, impairs the macrophage cytokine response to B. pseudomallei. Bone marrow harvested from wild type, TLR2^-/-, TLR4^-/-, or TLR2/4^-/- mice was cultured in the presence of L929 cell conditioned media for 5–10 days to promote differentiation of macrophages before plating and stimulating with media alone, Pam3CSK4 1000 ng/mL, E. coli 0111:B4 LPS 100 ng/mL, heat-killed BP-1 at a bacteria to cell ratio of 100, BP-1 LPS 1000 ng/mL, K96243 LPS 1000 ng/mL, or BP-1 lipid A 1000 ng/mL. Supernatants were harvested after 24 hours and (A) TNF-α or (B) IL-10 production was measured by ELISA. Data plotted are means ± standard deviations of quadruplicate samples. * indicates p < 0.05 and § indicates p = 0.001 compared with wild type cells stimulated with the same ligand, using analysis of variance followed by the Bonferroni post-test. Other comparisons are not shown for clarity. The TNF-α data displayed are from one of six independently performed experiments stimulating various combinations of wild type, TLR2^-/-, TLR4^-/-, or TLR2/4^-/- macrophages with these ligands, and measuring TNF-α. In one of three experiments comparing cytokine responses from TLR2^-/- macrophages to wild type macrophages, production of TNF-α in response to heat-killed Bp was not significantly increased. The IL-10 data displayed are from one of five independently performed experiments stimulating various combinations of wild type, TLR2^-/-, TLR4^-/-, or TLR2/4^-/- macrophages with these ligands, and measuring IL-10. In two of three experiments comparing cytokine responses from TLR2^-/- macrophages to wild type macrophages, production of IL-10 in response to heat-killed Bp was not significantly increased.