Identification of an Interferon Signaling Pathway Linking Membrane Cholesterol Accessibility to Host Defense Against Pathogens

Interferon-γ (IFN-γ) is a multipotent cytokine that is critical to the host innate immune defense against bacterial infection, and functions through transcriptional induction of hundreds of IFN-γ stimulated genes (γ-ISGs). However, the antibacterial roles of many γ-ISGs remain poorly defined. Here, I describe my efforts to characterize mechanisms by which specific γ-ISGs confer cell-intrinsic immunity against bacterial pathogens. Unexpectedly, I found that IFN-γ-activated macrophages secreted a soluble product that potently inhibited infection of the Gram-positive intracellular pathogen Listeria monocytogenes. To identify this factor, I first created a cDNA lentiviral library of more than 400 highly representative γ-ISGs and carried out a gain-of-function flow cytometry screen to determine the effect of each gene on infection. The results of this screen identified eight genes that potently reduce infection. Next, I determined whether these γ-ISGs produced a soluble molecule that could suppress L. monocytogenes infection in trans. Notably, conditioned media from Cholesterol 25-Hydroxylase (CH25H)-expressing cells potently enhanced bacterial resistance of naive cells, suggesting it may be responsible for the previously observed activity in macrophages. Indeed, Ch25h-/- macrophages failed to produce a soluble antibacterial metabolite, while the enzymatic product of CH25H, 25-Hydroxycholesterol (25HC), potently inhibited L. monocytogenes infection. I demonstrated that 25HC inhibits infection of non-phagocytic cells from diverse tissue lineages, and that administration of 25HC to mice significantly reduced bacterial burden in an oral gavage model. Furthermore, I found that 25HC blocks L. monocytogenes cell-to-cell spread by attenuating the formation of plasma membrane protrusions. In collaboration with the post-doctoral fellow Kristen Johnson, I have used toxin-based biosensors to determine that activation of acyl-CoA: cholesterol acyltransferase (ACAT) rapidly mobilized a specific pool of cholesterol termed “accessible cholesterol” from the plasma membrane (PM) to the ER. Importantly, the antibacterial function of 25HC was dependent on this ability to deplete PM accessible cholesterol. Together, these studies have uncovered a heretofore unknown mechanism by which IFN-mediated reorganization of the PM restricts dissemination of intracellular pathogens.