![Ak4 enhances macrophage antibacterial activities by regulating mtDNA synthesis to boost mtROS production. WT and Ak4 KO TG-pMacs were pretreated with DMSO, dNs, Gem, or ddC at the indicated concentration for 1 h, followed by infection with Listeria at an MOI of 5 for 1 h. Cells were then treated with 250 μg/ml gentamicin, washed with PBS, and maintained in 50 μg/ml gentamicin for 6 h prior to analysis. (A and B) MFI of MitoSox and H2DCFDA in WT and Ak4 KO pMacs from Listeria-infected mice for 3 days was analyzed by flow cytometry (n = 6–10). (C–E) WT and Ak4 KO TG-pMacs were pretreated with DMSO, 1 μM MitoPQ (a mitochondria-targeted redox cycling compound [C]), 200 μM MitoTempo (a mitochondria-targeted antioxidant [D]), or 20 mM NAC (a general cellular antioxidant [E]) for 1 h prior to Listeria infection. Intracellular bacterial loads were assessed by plating cell lysates onto TSA plates and counting CFUs at 24 h after plating (n = 3–4). (F) MFI of MitoSox in dNs-, Gem-, and ddC-treated WT TG-pMacs after Listeria infection was analyzed by flow cytometry (n = 4). (G–I) MFI of MitoSox in dNs- (G), Gem- (H), or ddC- (I) treated WT and Ak4 KO TG-pMacs after Listeria infection was analyzed by flow cytometry (n = 4). (J and K) MFI of MitoSox in dNs- (J) or Gem- (K) treated WT and Ak4 KO TG-pMacs after Salmonella infection was analyzed by flow cytometry (n = 4). (L) Mock, Ak4 WT, or kinase-dead Ak4 mutants were transduced into Ak4 KO TG-pMacs using lentiviral vectors. Mock-transduced WT TG-pMacs served as controls. MFI of MitoSox from transduced cells after Listeria infection was measured by flow cytometry (n = 4). Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t test (A and B) or one-way ANOVA (C–L). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are representative of two independent experiments, and each point represents data from one mouse with two technical repeats. MFI, mean fluorescence intensity.](https://cdn.rupress.org/rup/content_public/journal/jem/223/4/10.1084_jem.20250978/1/jem_20250978_fig6.png?Expires=1776437793&Signature=F3aMMDw~vPfifprFiLviaF~s313Z1-2j9XigV5qpxgexXOcbY96BSBIgaWuNI2r0rHCc~uF9oNbo0LFvYhaUSJd2rxGAa6UskNkzJPdFDa4unOD~BXK7Ud5NP0bgI2jN2PPGeBKG8bBo4lxDcfvADoaRviNJIjujp9p8dbPF5VZ86ZxX7qdezzCCHW2Wd94M~rxSBx7iN-TAeRTdTDc1FN7Uu~Jt5zQp-eikv2brwebMMRtw7O543q3jj7EmeDpoj4spCPgaXlsiT6Lzm3a5bdIOf-q0c45bjrxQ05ogPcQ7BJsQiILeAkc2vBBcQZjaFYOu3c1I5SkvYvB8cDsZPQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Panel A shows MitoSOX fluorescence in W T and A k 4 knockout cells. A k 4 deficiency reduces mitochondrial superoxide levels. Panel B shows H 2 D C F D A fluorescence, measuring total cellular reactive oxygen species. A k 4 knockout cells show decreased overall R O S. Panel C shows C F U of Listeria after Mito P Q treatment. Mitochondrial R O S induction alters bacterial burden, differing by genotype. Panel D shows C F U after Mito Tempo treatment. Scavenging mitochondrial R O S modulates Listeria growth. Panel E shows C F U after N A C treatment. General R O S inhibition affects bacterial survival in W T and A k 4 knockout cells. Panel F shows R O S levels during Salmonella infection with inhibitors (d N, gentamicin, d d C). Chemical treatments reduce mitochondrial R O S. Panel G shows R O S levels with oligomycin or oligomycin plus d N. Inhibition of mitochondrial ATP synthase lowers R O S. Panel H shows R O S levels with gentamicin treatment. Antibiotic exposure reduces R O S differently in WT and knockout cells. Panel I shows R O S levels with d d C treatment. Mitochondrial D N A depletion decreases R O S, with genotype differences. Panel J shows R O S levels during Salmonella infection with d N treatment. R O S production is partially reduced in both genotypes. Panel K shows R O S levels with gentamicin during Salmonella infection. Gentamicin significantly suppresses ROS generation. Panel L shows MitoSOX fluorescence in A k 4 knockout cells reconstituted with W T or mutants. Restoring A K 4 rescues mitochondrial ROS levels compared to empty vector.
Ak4 enhances macrophage antibacterial activities by regulating mtDNA synthesis to boost mtROS production. WT and Ak4 KO TG-pMacs were pretreated with DMSO, dNs, Gem, or ddC at the indicated concentration for 1 h, followed by infection with Listeria at an MOI of 5 for 1 h. Cells were then treated with 250 μg/ml gentamicin, washed with PBS, and maintained in 50 μg/ml gentamicin for 6 h prior to analysis. (A and B) MFI of MitoSox and H2DCFDA in WT and Ak4 KO pMacs from Listeria-infected mice for 3 days was analyzed by flow cytometry (n = 6–10). (C–E) WT and Ak4 KO TG-pMacs were pretreated with DMSO, 1 μM MitoPQ (a mitochondria-targeted redox cycling compound [C]), 200 μM MitoTempo (a mitochondria-targeted antioxidant [D]), or 20 mM NAC (a general cellular antioxidant [E]) for 1 h prior to Listeria infection. Intracellular bacterial loads were assessed by plating cell lysates onto TSA plates and counting CFUs at 24 h after plating (n = 3–4). (F) MFI of MitoSox in dNs-, Gem-, and ddC-treated WT TG-pMacs after Listeria infection was analyzed by flow cytometry (n = 4). (G–I) MFI of MitoSox in dNs- (G), Gem- (H), or ddC- (I) treated WT and Ak4 KO TG-pMacs after Listeria infection was analyzed by flow cytometry (n = 4). (J and K) MFI of MitoSox in dNs- (J) or Gem- (K) treated WT and Ak4 KO TG-pMacs after Salmonella infection was analyzed by flow cytometry (n = 4). (L) Mock, Ak4 WT, or kinase-dead Ak4 mutants were transduced into Ak4 KO TG-pMacs using lentiviral vectors. Mock-transduced WT TG-pMacs served as controls. MFI of MitoSox from transduced cells after Listeria infection was measured by flow cytometry (n = 4). Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t test (A and B) or one-way ANOVA (C–L). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are representative of two independent experiments, and each point represents data from one mouse with two technical repeats. MFI, mean fluorescence intensity.