The lung-resident alveolar macrophages (AMs) are the initial host cells infected by inhaled Mycobacterium tuberculosis (Mtb). While traditionally perceived as a conducive environment for Mtb replication and spread, our recent studies using fluorescent Mtb fitness reporter strains and single-cell RNA-sequencing (scRNA-seq) have revealed a more intricate reality. We discovered two primary AM populations in the Mtb-infected lung: SiglecF+ CD38+ pro-inflammatory AMs, which house stressed bacteria and express high levels of Nos2, and SiglecF+ CD38- AMs, which lack Nos2 expression and accommodate replicative bacteria. Intriguingly, our preliminary findings indicate that CD38- AMs, which exhibit limited Mtb containment capabilities, are the predominant infected AM population during the early infection stages. Moreover, intranasal BCG vaccination prior to Mtb exposure significantly reduces the number of Mtb-infected host cells and markedly increases the proportion of CD38+ infected AMs. This suggests a critical role for the CD38+ AM phenotype in triggering a protective immune response following vaccination. Although these AM subsets appear transcriptionally indistinguishable in naive mice, they demonstrate distinct chromatin organization, suggesting that epigenetic factors dictate their response to infection. Our hypothesis is that a higher prevalence of CD38+ AMs within the lung airways leads to improved early inhibition of Mtb proliferation and spread, thereby unveiling a viable avenue for immune mediate control of tuberculosis infection. Specific Aim 1: Elucidate the host molecular mechanisms contributing to the limited response of CD38- AMs to Mtb infection. To decipher the host molecular mechanisms underlying the blunted response of CD38- AMs to Mtb infection, we will employ an integrated multi-omics approach to analyze the changes in chromatin organization and transcription factor activity post-Mtb infection. We will then manipulate the AM immune environment through intranasal BCG vaccination to identify those epigenetic changes that are associated with long-term protection against Mtb infection. Specific Aim 2: Implement CRISPR-based genetic perturbation approaches in macrophages to evaluate the role of selected host responses in Mtb infection. Initially, we will use a CRISPR-based genetic perturbation approach to validate the role of candidate host genes, identified in our existing datasets, in generating pro-inflammatory macrophage responses that control Mtb infection. We will then extend this approach to functionally validate additional targets emerging from Specific Aim 1.