Identifying the role of SPTLC2 in alveolar macrophage population maintenance during Mycobacterium tuberculosis infection

Washington University, Saint Louis MO

Investigators

Abstract

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), the disease responsible for the most deaths by an infectious pathogen worldwide. The host immune response plays a critical role in determining infection outcome, however the factors contributing to a protective response versus progression to active disease are incompletely understood. During Mtb infection, alveolar macrophages (AMs) are the first cells to confront the bacteria. AMs phagocytose Mtb but cannot control the bacteria, ultimately dying and spreading Mtb to recruited immune cells. Among these recruited cells are bone marrow (BM)-derived monocytes that can differentiate into lung interstitial macrophages or pre-AMs, with the latter migrating into the alveoli and maturing to replenish the AM population. Recent work identified that SPTLC2, an enzymatic subunit required for de novo sphingolipid synthesis, is required in innate immune cells during Mtb infection. Mice deleting Sptlc2 in innate immune cells (Sptlc2fl/fl-LysM-Cre) or lung macrophages and dendritic cells specifically (Sptlc2fl/fl-CD11c-Cre) fail to replenish the AM population during Mtb infection, have increased susceptibility to infection relative to Sptlc2fl/fl control mice, and develop a Pulmonary Alveolar Proteinosis (PAP)-like pathology. We determined by single-cell RNA sequencing that monocyte-derived AM precursors that delete Sptlc2 fail to become AMs, instead differentiating into interstitial and other monocyte-derived macrophages, however preliminary data suggests these cells do not have impaired capacity to differentiate into AMs. Investigation of potential causes for failed AM repopulation identified that LPL-/- mice, which lack actin bundling protein L-plastin, also fail to maintain the AM population during Mtb infection. L-plastin is essential for perinatal pre-AM alveolar localization, so these data implicate a role for actin in AM population maintenance during Mtb infection. Sphingolipid species produced downstream of SPTLC2 include membrane lipid raft components upon which actin nucleates and polymerizes, but it is not understood how Sptlc2 deletion alters the sphingolipid profile of CD11c-expressing cells during Mtb infection or by what mechanism this impairs AM repopulation. This proposal will test the hypothesis that SPTLC2-deficiency limits the sphingolipid profile of monocyte-derived AM precursors during Mtb infection, impairing actin polymerization and preventing their migration into the alveolar space. Aim 1 will establish if actin-mediated migration of monocyte-derived AM precursors into the alveoli is Sptlc2-dependent by investigating migration capacity and recruited cell fate, and by determining if SPTLC2 is required for actin polymerization at sphingolipid- containing rafts in AM precursor membranes. Aim 2 will identify the functional role of Sptlc2 in AM repopulation by profiling how loss of SPTLC2 and Mtb infection alters AM and AM-precursor sphingolipid profiles and identifying if sphingolipids can be reconstituted by supplementation in Sptlc2-deleting cells. These studies will elucidate fundamental aspects of immune homeostasis during Mtb infection.