Validation of the use of Akt inhibitors as host-directed therapy in a murine model of Chlamydia trachomatis infection of uterine horn explants

GAMBARTE TUDELA, JULIAN; DAMIANI, MARIA TERESA ; DEL BALZO DIEGO; ALONSO BIVOU, MARIANO; RUSSI ROMINA CECILIA

Congreso; World Microbe Forum.; 2021

As an obligate intracellular bacterium, Chlamydia trachomatis (CT) bears the capacity to produce persistent infections which may lead to therapeutic failure. Newpharmacological approaches, like host-directed therapy, should be explored. Recently, we documented that the AKT/AS160/Rab14 pathway is hijacked by thebacterium to ensure the proper obtention of sphingolipids and, thus, its replication. These results position AKT as a promising host target for CT-specifictreatment. As a first approach, four Akt inhibitors in Phase II clinical trial for cancer treatment were selected. Bacterial invasion upon AKT inhibition was assessedin the HeLa epithelial cell line showing a differential behavior for each inhibitor with a maximum reduction of 50% of infection. CT replication was then evaluatedand, likewise, differential behavior among the inhibitors was observed with a maximum reduction in replication of 80%. In addition, we investigated the effect oncell death and apoptosis of AKT inhibitors in a murine macrophage cell line (RAW 246.7) challenged with CT. A clear increase of early apoptosis was observed forall inhibitors. Macrophage functioning was further evaluated by its capacity to produce and secrete TNFα in response to CT. A significant decrease of TNFαexpression was confirmed for all the inhibitors compared to control. These two results lead us to study the activity of NFκB. Surprisingly, the autocrine TNFαinduced phosphorylation of IκBα, a negative regulator of NFκB, in CT-challenged macrophages was prevented upon the use of any of the inhibitors. We nextevaluated the transcriptional effect of AKT inhibition on NFκB-inducible cytokines (TNFα, IL6, IL1β, IL10) upon CT challenge and/or TNFα autocrine stimulation.For all of the genes assayed, a significant reduction of mARN expression relative to control was detected on CT and TNFα stimulated macrophages upon Aktinhibition. With the aim of validating these results we proceeded to work on a novel murine model of ex vivo infection of uterine horn explants. We confirmedthat the use of the inhibitors profoundly decreased both the bacterial burden and the expression of pro-inflammatory TNFα within the tissue corroborating theresults obtained previously in vitro. This model allows us to monitor early post-infection events and more complex responses executed by innate immunity thatotherwise could not be studied in 2D cellular models and enabled us to validate the proof-of-concept use of AKT inhibitors as CT-specific antimicrobials.