Abstract

Mycobacterium tuberculosis (Mtb) remains the top infectious cause of death globally, with an estimated 1.6 million deaths in 2021. About 10% of infected, immunocompetent adults will develop active disease, and the host factors that determine whether the immune response to infection is protective remain incompletely understood. Early events in innate immunity carried out by lung monocytes, macrophages, and dendritic cells are critical for the establishment of a protective immune response, and the pathways in these cells that contribute to protection are of interest to the field. This dissertation covers investigations of three such pathways, sphingolipid biosynthesis in alveolar macrophages and their precursors, BHLHE40-mediated transcriptional regulation of innate immune differentiation and polarization, and autophagy in lung macrophages and dendritic cells during innate-adaptive immune transition. We identified that SPTLC2, a subunit of the enzyme that catalyzes the first and rate-limiting step of sphingolipid synthesis, is required in innate immune cells during Mtb infection. We infected mice with an innate immune cell specific deletion of the gene that encodes SPTLC2 (Sptlc2����/����-Lysm- Cre) and found that they succumb to Mtb infection with a median survival of 93 days, much earlier than Sptlc2����/���� control mice. Infection of the Sptlc2����/����-Lysm-Cre mice is characterized by higher bacterial burden, the development of a striking glossy lung gross pathology, and transcriptional signatures of hypoxia relative to control mice, all of which were observed later than 28 days-post-infection (dpi). In contrast to many other mice with susceptibility to Mtb infection, there are no changes in cellular inflammation of lymphocytes, granulocytes, or dendritic cells that precede the loss of control of bacterial growth. However, beginning by 14 dpi, there is a significant reduction in the abundance of alveolar macrophages (AMs) in the lungs of Sptlc2����/����-Lysm-Cre mice relative to control mice. To investigate the mechanism of the loss of AMs, we performed single-cell RNA sequencing and noted no difference in markers of AM self-renewal or cell death between Sptlc2����/����- Lysm-Cre mice and controls. Instead, we observed a defect in the generation of AM precursors from recruited monocytes in the absence of SPTLC2. The glossy gross pathology, presence of hypoxia, and specific loss of AMs are reminiscent of pulmonary alveolar proteinosis, a disease in which surfactant proteins and lipids that are homeostatically digested by AMs accumulate in the alveolar space. We detected elevated levels of surfactant proteins A and D in the lungs of Sptlc2����/����-Lysm- Cre mice by 56 dpi, confirming the presence of proteinosis. In experiments to confirm that the role for SPTLC2 is intrinsic to the CD11c-expressing AMs, we found that Sptlc2����/����-Cd11c-Cre mice are similarly susceptible to Mtb infection and phenocopy the loss of AMs, glossy lung pathology, and proteinosis observed in the Sptlc2����/����-Lysm-Cre system. These studies establish that SPTLC2 is required in CD11c-expressing cells to maintain an AM population, avoid proteinosis, and survive during Mtb infection in mice. Mtb has been shown to use neutrophils as a replicative niche, and dendritic cells and macrophages both play an important role in initiating adaptive immunity and controlling bacterial replication upon adaptive immune help. We have previously shown that loss of BHLHE40, a transcription factor that is known to regulate pro- and anti-inflammatory immune responses, in CD11c+ cells leads to a neutrophil driven innate immune response characterized by a loss of dendritic cells during Mtb infection and results in early death in mice. Furthermore, BHLHE40 expression is driven by GM-CSF, a cytokine abundant in the lungs during infection, and in vitro studies using GM- CSF cultures revealed that loss of BHLHE40 skewed cytokine responses toward an alternatively activated phenotype upon stimulation with Mtb antigen. The cell types and pathways leading to these phenotypes remained unclear. We turned to single-cell RNA sequencing (scRNAseq) of the GM-CSF culture system to better understand the transcriptional pathways leading to these phenotypes. Using GM-CSF cultured bone marrow from BHLHE40 wild-type or knockout mice, we performed scRNAseq and revealed a previously unappreciated complexity of macrophages, dendritic cells, neutrophils, and a mix of common and cell-type specific precursors. Analysis of the merged data demonstrated a striking loss of DCs and DC precursors in the absence of BHLHE40. RNA velocity analysis revealed a disruption in the ability of DC precursors to differentiate into DCs associated with upregulation of C/EBP�� transcripts. C/EBP�� is a transcription factor that must be downregulated for differentiation into DCs. BHLHE40 binds directly upstream of the Cebpb locus, suggesting that BHLHE40 may directly repress C/EBP�� expression during DC differentiation. Additionally, there is a shift in the macrophage cluster in the absence of BHLHE40. Use of a scoring module for classically vs alternatively activated macrophages confirmed previous results that loss of BHLHE40 results in an enrichment for alternatively activated macrophages. The single-cell transcriptomic data revealed that this enrichment for alternative polarization is associated with decreased HIF-1�� expression. This study demonstrated the strength of scRNAseq as a tool to understand the relationship between developing cells in a complex mixture and identified the critical role for BHLHE40 in DC maturation and macrophage polarization. Autophagy, the pathway that allows cells to target unwanted intracellular content to the lysosome for nutrient recycling, has long been implicated in macrophages during infection. It was previously thought that the primary role for autophagy in control of mycobacterial infections was through targeting the pathogen to the lysosome for degradation. We previously showed that Atg5����/����-Lysm- Cre mice, which lack ATG5 and autophagy in LysM-expressing cells, succumb to Mtb infection early via an ATG5-dependent but autophagy-independent mechanism, suggesting that autophagy machinery plays an important role in lung innate immune cells outside of the previously reported role in direct pathogen killing. The susceptibility follows a hyper-neutrophilic inflammatory response, and neutrophil depletion rescues the mice. I determined that there are fewer CD4+ and CD8+ T cells in the lungs of these mice at 19 dpi, when the adaptive immune response is initiating in Atg5����/���� mice. Because these mice have markedly elevated neutrophils in the lung, I tested whether the delay in T cell response could be rescued by depletion of neutrophils. Depletion of neutrophils resulted in a rescue of T cell response to the level of undepleted Atg5����/���� mice. However, there was an additional increase in T cells in the neutrophil depleted Atg5����/���� mice despite similar neutrophil levels after depletion, suggesting that there may be an additional neutrophil-independent role for ATG5 in the initiation of adaptive immunity. Preliminary results suggest that this phenotype is common to mice that lack essential genes of the autophagy pathway in innate immune cells. This study determined that autophagy is required in lung innate immune cells for the efficient initiation of adaptive immunity to Mtb infection. Together, these findings advance our understanding of lung macrophage, monocyte, and dendritic cell pathways required during Mtb infection.

Committee Chair

Christina Stallings

Committee Members

David Hunstad; Jen Philips; Jon Brestoff; Max Artyomov; Tim Peterson

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

5-6-2025

Language

English (en)

Author's ORCID

https://orcid.org/https://orcid.org/0000-0003-2038-1288

Download

Available for download on Wednesday, May 05, 2027

Included in

Biology Commons

Share

COinS