TLR4 and infection: While LPS-prestimulated macrophages are often used to study inflammasome activation, we used unstimulated macrophages in our experiments for several reasons: 1) to examine the role of TLR2 during natural infection in the absence of exogenous stimuli, 2) to mimic the early events of natural infection, and 3) because F. novicida makes an LPS with very low stimulatory activity [18] and TLR4 is not normally activated during infection with Francisella.