critical role in anti-chlamydial defense. The loss of Irga6 renders cells incapable of capturing C. trachomatis in an autolysosome, thus enabling pathogen survival. Irga6 is important in the induction of autophagy independent of C. trachomatis infection Is IFNc capable of activating autophagy in MEFs and is Irga6 required for IFNc-induced autophagy To answer these questions, we used immunoblots to monitor the formation of early autophagosomal precursors and newly formed autophagosomes by following changes in LC3 expression. LC3 exists in two forms: the cytosolic LC3-I form, which has a molecular weight of approximately 18 kDa and the membrane-bound LC3-II form, with a molecular weight of 16 kDa. LC3-II 
is bound to the Control of Ctr via Irga6 5 Control of Ctr via Irga6 6 Control of Ctr via Irga6 membrane of nascent autophagosomes and correlates with the amount of LC3-positive 22761436 autophagosomes. In WT cells, LC3II levels increased in response to 23300835 IFNc stimulation. Notably, levels of IFNc-induced LC3-II were comparable to those induced by rapamycin, a conventional inducer of autophagy. Infection with C. trachomatis for 4 h or 8 h did not influence LC3-II expression. However, IFNc treatment of 193022-04-7 infected WT MEFs induced an upregulation of LC3-II. In general, levels of LC3 protein were substantially lower in Irga62/ 2 cells as compared to WT MEFs. Moreover, IFNc or Rapa treatment had a minimal stimulatory effect on LC3 expression and/or processing in Irga62/2 cells. C. muridarum infection effectively suppressed an increase in LC3 levels upon IFNc treatment. Similarly, no increase in LC3 levels upon IFNc treatment in C. muridarum infected Irga62/2 cells was observed. Atg52/2 cells did not process LC3, as expected for cells that are unable to form autophagosomes. To confirm the importance of Irga6 in autophagy, we quantified the number of autophagosomes in the cytoplasm. In contrast to control untreated WT cells, numbers of GFP-LC3decorated structures per cell in infected and uninfected WT cells exposed to either IFNc or Rapa were increased. In agreement with our immunoblot analysis, IFNc or Rapa induction did not increase the amount of autophagosomes and we observed only low numbers of decorated vesicles in Irga62/2 cells. These data further support a pivotal role for autophagy in C. trachomatis elimination and indicate a role for Irga6 as regulator of IFNc-induced autophagy. Control of Ctr via Irga6 In summary, our data clearly demonstrates the ability of IFNc to induce autophagy in nonphagocytic MEFs. More importantly, low LC3 expression and small numbers of autophagosomes in Irga62/2 cells indicate a role for Irga6 in the regulation of the IFNc-induced autophagic pathway. Our results reveal Irga6 as an important factor in assigning chlamydial inclusions for degradation via autophagy. Discussion Here we elucidate the mechanisms underlying IFNc/IRGsinduced immunity to C. trachomatis in murine cells, demonstrating a pivotal role for Irga6 in mediating host resistance to infection via the induction of autophagy. We show that C. trachomatis, but not C. muridarum, inclusions strongly colocalized with 4 IRGs in response to IFNc, whereas Irgb6 colocalized in the presence or absence of the cytokine. IFNc also induced accumulation of the autophagosomal membrane marker LC3, and the lysosomal component LAMP1, suggesting a rerouting of C. trachomatis phagosomes to autolysosomes. However, autophagydeficient MEFs and WT cells with an impairment in lysosomal
