ORCID
https://orcid.org/0000-0002-0644-6519
Date of Award
12-14-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Infections with Mycobacterium tuberculosis (Mtb), the causative agent of the disease tuberculosis (TB), remain a prominent global health threat that affects millions of people annually. While antibiotic treatments for Mtb infections are available, the increased prevalence of drug-resistant Mtb cases has highlighted the urgent need to develop additional therapeutic options such as host-directed therapies. Development of host-directed therapies, however, first requires a better understanding of the protective immune responses required to control Mtb infection. Such responses must strike a precise balance between pro-inflammatory and anti-inflammatory responses that promote bacterial clearance while also minimizing tissue damage. Here we show that the transcription factor BHLHE40 plays a key role in regulating pro-inflammatory responses during Mtb infection. Previous work conducted in our lab has shown that expression of the transcription factor BHLHE40 is required in mice during Mtb infection. Mice lacking Bhlhe40 expression are highly susceptible to Mtb infection compared to WT mice, and have significantly higher lung bacterial burdens, increased neutrophil inflammation in the lung, and decreased production of the protective cytokines IFN-γ and IL-12. We have shown that BHLHE40 binds the Il10 locus to repress its transcription, and deletion of Il10 in Bhlhe40-/- mice largely rescues the susceptibility of Bhlhe40-/- mice, indicating that BHLHE40 is required in mice during Mtb infection to negatively regulate IL-10 production. We have shown that Bhlhe40 expression is specifically required in CD11c+ lung macrophages and dendritic cells (DCs) in addition to T cells during Mtb infection, though the role of BHLHE40 in these cell types during infection is unknown. To determine whether the role of BHLHE40 in CD11c+ and T cells during Mtb infection was to negatively regulate IL-10 production, I infected mice lacking Il10 and Bhlhe40 expression specifically in these cell types. I found that a primary role for BHLHE40 in these cells is to negatively regulate IL-10 production, as mice lacking BHLHE40 and IL-10 in CD11c+ or T cells had decreased susceptibility to infection compared to mice lacking Bhlhe40 alone in those cell types. To determine if deleting Il10 production in CD11c+ or T cells specifically was sufficient to rescue the susceptibility of Bhlhe40-/- mice, I infected Bhlhe40-/- mice lacking IL-10 production CD11c+ and T cells. I found that while deleting Il10 production from CD11c+ and other myeloid cell types did not rescue the survival of Bhlhe40-/- mice, loss of IL-10 signaling from T cells specifically extended the survival of Bhlhe40-/- mice, indicating IL-10 from T cells contributes to the susceptibility phenotypes Bhlhe40-/- mice. To further investigate the role of BHLHE40 in CD11c+ cells, we utilized a GM-CSF culture system, which has previously been used to study myeloid cell functions. Culturing murine bone marrow in the presence of GM-CSF, which is an abundant cytokine within the lung, results in the development of CD11c+ granulocytes, macrophages, and DCs, all of which we have shown express Bhlhe40. Using this system, I determined that BHLHE40 is required for the induction of multiple GM-CSF-mediated pro-inflammatory responses in both IL-10-dependent and IL-10-independent fashions, suggesting that BHLHE40 plays additional roles in the immune response outside of its role in repressing IL-10 production. Additionally, I discovered that loss of Bhlhe40 expression leads to increased granulocyte frequency and decreased proportions of macrophages and DCs compared to WT mice in an IL-10-independent manner, indicating a novel role for BHLHE40 in the development of myeloid cells cultured in the presence of GM-CSF. To determine the role BHLHE40 plays in myeloid cell development, I utilized single-cell RNA-sequencing (scRNAseq) in collaboration with lab mates Michael McNehlan and Yassin Mreyoud. We first used the increased resolution of this method to characterize the development of WT bone marrow cultured in GM-CSF-containing media and found that the resulting cell populations were more heterogenous than what had been canonically described in the literature. In fact, WT bone marrow differentiated in GM-CSF-containing media resulted in nine transcriptionally distinct cell populations, as opposed to the three populations (granulocytes, macrophages, and DCs) that had classically been reported using flow cytometry and microscopy-based methods. We were then able to determine that loss of Bhlhe40 expression lead to an upregulation of Cebpb expression in macrophage/DC progenitor populations and verified that BHLHE40 binds directly to the Cebpb locus in myeloid cells using previously published ChIP-sequencing data. Expression of the transcription factor C/EBP must be downregulated to facilitate DC development, indicating that BHLHE40 may directly repress the expression of Cebpb to facilitate DC development. Additionally, when we investigated the transcriptional profiles of macrophage/DC progenitor populations, we found that loss of Bhlhe40 expression led to a more neutrophil-like transcriptional profile, suggesting that macrophage/DC progenitors lacking BHLHE40 are skewed towards granulocytic development, which could account for the increased granulocyte frequencies that develop when bone marrow from Bhlhe40-/- mice is differentiated in GM-CSF-containing media. We also found that loss of BHLHE40 led to macrophage populations that were transcriptionally skewed towards anti-inflammatory polarization. I then characterized how loss of BHLHE40 in macrophages led to decreased pro-inflammatory responses in vitro following treatment with heat-killed Mtb (HKTB), and in vivo at 21 days post Mtb infection. Bhlhe40-/- mice infected with Mtb produce significantly higher levels of IL-10 from both innate and adaptive immune cells. What cell type(s) respond to the increase in IL-10 production to contribute to the susceptibility of Bhlhe40-/- mice, however, is unknown. To determine what cell type(s) respond to higher IL-10 in the absence of BHLHE40, I infected Bhlhe40-/- mice lacking the IL-10 receptor, IL-10R, in CD11c+, LysM+, MRP8+, and T cells. I found that while deletion of Il10ra in MRP8+ and T cells did not rescue the susceptibility of Bhlhe40-/- mice, loss of Il10ra expression in CD11c+ and LysM+ cells resulted in partial rescue of survival, indicating responses by these cell types both contribute to the susceptibility of Bhlhe40-/- mice to Mtb infection. Determining whether the partial rescue from Bhlhe40-/- mice lacking IL-10R in CD11c+ and LysM+ cells is additive, or if a non-hematopoietic cell type responds to higher IL-10 in the absence of BHLHE40 to contribute to susceptibility will require subsequent studies. Additionally, further insight into the differential responses to IL-10 signaling in the aforementioned genotypes will require further studies and could shed additional insight into how loss of BHLHE40 leads to the susceptibility of mice to Mtb infection. While there are many remaining gaps in knowledge about the precise immune responses that dictate protection versus progression to active TB disease, IL-10 has been shown to limit protective responses to Mtb infection, and patients with active TB disease have been shown to have higher serum IL-10 levels and lower BHLHE40 expression. The findings herein have provided additional insight into how the essential transcription factor BHLHE40 functions during Mtb infection to facilitate pro-inflammatory responses in murine cells exposed to GM-CSF in IL-10-dependent and IL-10-independent manners, in addition to negatively regulating IL-10 produced from T cells. Together, these findings have contributed to the overall knowledge of key mechanisms that are needed to mount a protective immune response to Mtb infection.
Language
English (en)
Chair and Committee
Christina Stallings
Recommended Citation
Hendrix, Skyler, "Investigating the Role of BHLHE40 in Host Responses to Mycobacterium tuberculosis Infection and Myeloid Cell Development" (2023). Arts & Sciences Electronic Theses and Dissertations. 3203.
https://openscholarship.wustl.edu/art_sci_etds/3203