Brucellasuppress innate immunity by down-regulating STING expression in macrophages

Abstract

Author summary: Cytosolic pattern recognition receptors, such as the nucleotide-activated STING 39 molecule, play a critical role in the innate immune system by detecting the presence of intracellular 40 invaders. Brucella bacterial species establish chronic infections in macrophages despite initially 41 activating STING. STING does participate in the control of Brucella infection, as mice or cells lacking 42 STING show a higher burden of Brucella infection. However, we have found that early following 43 infection, Brucella upregulates a microRNA, miR-24, that targets the STING messenger RNA, resulting in 44 lower STING levels. Dead bacteria or bacteria lacking a functional type IV secretion system were 45 defective at upregulating miR-24 and STING suppression, suggesting an active bacteria-driven process. 46Failure to upregulate miR-24 and suppress STING greatly compromised the capacity for Brucella to 47 replicate inside macrophages. Thus, although Brucella initially activate STING during infection, the 48 ensuing STING downregulation serves as a "damage control" mechanism, enabling intracellular 49 infection. Viruses have long been known to target immune sensors such as STING. Our results indicate 50 that intracellular bacterial pathogens also directly target innate immune receptors to enhance their 51 infectious success. 52 confirm the RNAseq data, and determine whether other Brucella species down-regulate STING, v-raf/v-104 myc immortalized murine bone marrow-derived macrophages (17) were uninfected or infected with 105 different Brucella species for 24 hours and STING (Tmem173) mRNA levels assessed via RT-qPCR ( Figure 106 2A). B. melitensis, B. abortus and B. suis are human pathogens and primarily infect ruminants, cattle and 107 swine, respectively. B. neotomae has been isolated from wood rats and voles but also has been isolated 108 in human neurobrucellosis (18). The four species of Brucella significantly down-regulated STING mRNA 109 7 compared to uninfected macrophages. STING protein levels also decreased in cells infected for 24 hours 110 with Brucella (Figure 2B). In macrophages infected with B. melitensis, Tmem173 mRNA down-regulation 111 was evident by 4h post-infection (Figure 2C). 112Tmem173 down-regulation required live bacteria, consistent with an active bacteria-driven process 113 ( Figure 3A). Brucella with mutations in the type IV secretion system (deletion of the critical VirB2 114 subunit (19)) displayed an intermediate phenotype with only modest downregulation of Tmem173, 115suggesting an intact type IV secretion system (T4SS) is required for full STING suppression (Figure 3B). 116Regarding mechanism of suppression, one straightforward possibility was that Brucella infection 117 suppresses the activity of transcription factors required for Tmem173 promoter activity. To address this 118 idea, we utilized a murine STING-promoter driven luciferase reporter (Figure 3C). Brucella infection 119 reliably increased the activity of the STING promoter-driven construct, to a comparable extent as...