Hypoxia - mediated impairment of the mitochondrial respiratory chain 6 inhibits the bactericidal activity of macrophages 7 8 9 10

35 36 In infected tissues oxygen tensions are low. As innate immune cells have to operate under 37 these conditions, we analyzed the ability of macrophages (MΦ) to kill Escherichia coli or 38 Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote 39 intracellular bacterial growth, but impaired the bactericidal activity of the host cells against 40 both pathogens. This correlated with a decreased production of reactive oxygen (ROI) and 41 reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and 42 inducible NO synthase (NOS2) double-deficient MΦ revealed that in E. colior S. aureus43 infected cells the reduced antibacterial activity during hypoxia was either entirely or partially 44 independent of the diminished PHOXand NOS2-activity. Hypoxia impaired the 45 mitochondrial activity of infected MΦ. Inhibition of the mitochondrial respiratory chain 46 activity during normoxia (using rotenone or antimycin A) completely or partially mimicked 47 the defective antibacterial activity observed in hypoxic E. colior S. aureus-infected WT MΦ, 48 respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic 49 PHOXNOS2 MΦ further raised the bacterial burden of the cells, which reached the level 50 measured in hypoxic PHOXNOS2 MΦ cultures. Our data demonstrate that the reduced kill 51 of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOXand NOS252 activity, but partially or completely results from an impaired mitochondrial antibacterial 53 effector function. Since pharmacological inhibition of the respiratory chain raised the 54 generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded 55 as the mechanism underlying the antimicrobial activity of mitochondria. (250 words) 56 57 on O cber 8, 2017 by gest http/iai.asm .rg/ D ow nladed fom Abbreviations: 58 BM, bone marrow 59 cybb, cytchrome b-245 beta chain (gp91) 60 DsRed, red fluorescent protein; 61 GFP, green fluorescent protein; 62 HIF, hypoxia-inducible factor; 63 HPRT, hypoxanthine guanine phosphoribosyl transferase; 64 LB, Luria-Bertani 65