Fig 4. B. burgdorferi–induced Irf4 expression modulates the induction of pro-inflammatory cytokines in previously experienced macrophages.

(A) Motifs associated with the expression of genes specifically up-regulated in memory macrophages compared to acutely stimulated cells. (B) Venn diagram showing the genes that are co- or differentially regulated by the transcription factors, SpiB, MAFK, and MZF1. The only gene regulated by the 3 transcription factors is Irf4. (C) Irf4 gene expression in acute, memory, and non-stimulated macrophages determined by real-time PCR relative to Rpl19. The results represent 4 independent mice. *, Student t test, p < 0.05. (D) Expression levels of Irf4 in peripheral blood of infected mice over time, as determined by real-time PCR relative to Rpl19. Groups of 3–6 mice were employed for each time point. *, Student t test, p < 0.05. (E) Irf4 expression of 21-day-infected hearts vs. uninfected controls by real-time PCR relative to Rpl19. (F) Log₂ fold induction of Irf4 in infected hearts at different time points of infection. Groups of 3–6 mice were employed for each time point. *, Student t test, p < 0.05. (G) Phagocytosis by BMMs infected with lentivirus containing shIrf4 (red histogram) or the control vector, pLKO (black histogram). The gray histogram represents a 4°C control. The cells were coinfected with lentivirus containing 2 different shRNA sequences (TRCN0000081548; TRCN0000081549). The average level of silencing of the Irf4 was calculated to be at 23 ± 0.05%. (H) TNF and IL-6 induction by B. burgdorferi in Irf4-silenced and control BMMs. The cells were stimulated for 20 hours, followed by the measurement of the cytokines in the supernatants by ELISA. The data underlying the graphs in Fig 4 can be found in S4 Data, GSE125503, and GSE152168. BMM, bone marrow-derived macrophage; GFP, green fluorescent protein; IL, interleukin; PCR, polymerase chain reaction; shRNA, short hairpin RNA; TNF, tumor necrosis factor.