Panel A shows ELISA quantification of interleukin-1 beta, interleukin-6 (I L-6), tumor necrosis factor alpha, and interleukin-12 (I L-12) in supernatants from infected wild-type and A k 4 knockout thioglycolate-elicited peritoneal macrophages at 24 hours post-infection. Panel B shows reverse transcription quantitative P C R analysis of C-C motif chemokine ligand 2, C-X-C motif chemokine ligand 1, and C-X-C motif chemokine ligand 3 (C x c l 3) transcript levels in infected wild-type and A k 4 knockout macrophages at 2 hours post-gentamicin treatment. Panel C shows bacterial uptake measured by colony-forming unit assay at 0 hours post-gentamicin (phagocytosis assay). Panel D shows intracellular bacterial burden at 2 hours and 6 hours post-gentamicin (bacterial killing assay). Panel E shows transmission electron microscopy images of intracellular Listeria monocytogenes in wild-type and A k 4 knockout bone marrow-derived macrophages at 24 hours post-infection, with mitochondria indicated by arrows and accompanying quantification. Panel F shows survival curves of wild-type and A k 4 knockout mice following systemic Listeria monocytogenes infection. Panel G shows bacterial loads (colony-forming units) in spleen and liver from infected wild-type and A k 4 knockout mice. Panel H shows survival curves of A k 4 floxed mice and A k 4 floxed L y s M-C r e mice. Panel I shows bacterial loads in spleen and liver of A k 4 floxed and A k 4 floxed LysM-Cre mice. Panel J shows survival curves of A k 4 floxed and A k 4 floxed C D 4-Cre mice. Panel K shows bacterial loads in spleen and liver of A k 4 floxed and A k 4 floxed C D 4-Cre mice.
Ak4 is essential to macrophage-mediated resistance to bacterial infection. WT and Ak4 KO TG-pMacs were infected with Listeria at an MOI of 5 for 1 h, followed by treatment with 250 μg/ml gentamicin. Cells were washed with PBS and maintained in 50 μg/ml gentamicin until harvest. (A) Concentrations of IL-1β, IL-6, TNFα, and IL-12 in the supernatant of Listeria-infected WT and Ak4 KO TG-pMacs at 24 h after infection were measured by ELISA (n = 4–7). (B) Transcript levels of Ccl2, Cxcl1, and Cxcl3 in Listeria-infected WT and Ak4 KO TG-pMacs were assessed by RT-qPCR at 2 h after gentamicin treatment (n = 3). (C and D) Bacterial burden in Listeria-infected WT and Ak4 KO TG-pMacs was quantified by CFU assays at 0 (C), 2, and 6 h (D) after Gem treatment (n = 4 per time point). (E) Enumeration of intracellular Listeria in WT (n = 19) and Ak4 KO (n = 13) BMDMs at 24 h after infection was measured using cryo-EM. Mitochondria were indicated by the red arrow. Scale bar, 2 μm. (F–K) Female and male mice were i.p. infected with 5 × 104 and 5 × 105Listeria, respectively. Survival was monitored daily for 14 days. On day 3 after infection, spleens and livers were harvested, homogenized, and plated on TSA plates to assess bacterial load. (F and G) Survival rate (n = 19 for WT and Ak4 KO) (F) and bacterial burden (n = 7 for WT and Ak4 KO) (G) in the liver and spleen of female mice. (H and I) Survival rate (n = 14 Ak4f/f, n = 20 Ak4f/fLysMCre) (H) and bacterial load (n = 5 for WT and Ak4 KO) (I) in the liver and spleen of female mice. (J and K) Survival rate (n = 18 for Ak4f/f, n = 14 for Ak4f/fCD4Cre) (J) and bacterial load (n = 9 for Ak4f/f, n = 5 for Ak4f/fCD4Cre) (K) in the liver and spleen of male mice. Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t test (A–E, G, I, and K) or log-rank test (F, H, and J). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are representative of three independent experiments, and each point represents data from one mouse with two technical repeats.