Gene expression analysis of mouse alveolar macrophages and whole-blood from mice with contained Mycobacterium tuberculosis infection

The aim of this study was to measure the impact of contained infection with Mycobacterium tuberculosis (CMTB) on the immune response of alveolar macrophages (AMs) to intracellular Mtb infection in vivo and on the circulatory cellularity. We characterized the transcriptional profile of murine AMs in CMTB and control mice by sorting cells from whole-lung lysates and performing RNA-sequencing (samples 1-6). We also characterized the transcriptional profile of murine Mtb-infected AMs after aerosol infection by sorting cells from bronchoalveolar lavage fluid and performing low-input RNA-sequencing (samples 7-12). In order to characterize changes in chromatin accessibility induced by CMTB, we performed ATAC-seq on AMs isolated from control and CMTB mice by BAL (samples 13-18). We also generated whole-blood transcriptomes from mice prior to and following the establishment of CMTB (samples 19-28) in order to characterize the circulatory cellularity. Overall design: To establish the model of CMTB, we infected mice intradermally in the ear with 10,000 CFU of a commonly used virulent strain of Mtb, H37Rv. Within 5 days the bacteria trafficked to the ipsilateral superficial cervical lymph nodes where the bacterial burden was relatively stable for at least one year with minimal dissemination to the spleen and no dissemination to the lung. We characterized the effect of CMTB on the basal state of AMs by performing RNA-seq and ATAC-seq analyses on AMs from CMTB and control mice prior to aerosol challenge. In addition, we performed whole-blood RNA-seq analysis of mice prior to and following the establishment of CMTB (day 42) in order to search for changes in the circulatory cellularity. To characterize the impact of CMTB on the early macrophage transcriptional response to Mtb infection, we infected mice with ~3000 CFU of mEmerald-H37Rv, and isolated AMs (CD45+, Zombie Violet-, CD3-/CD19-, Siglec-F+, CD11bmid, and CD64+) from BAL by fluorescence activated cell sorting at 24 hours following infection. Three populations were sorted for analysis by RNA-seq: Mtb-infected AMs (mEmerald+), bystander AMs (mEmerald-), and AMs from naïve mice.