Mechanisms of control in Mycobacterium tuberculosis infection

Mycobacterium tuberculosis is an obligate pathogen that has evolved to survive exclusively in the human population and remains a leading cause of mortality due to bacterial infections. The immune responses against M. tuberculosis are diverse, with some people controlling the infection for their lifetime without showing symptoms, whereas others develop progressive disease leading to the spread of the bacterium among people. The underlying causes for the obvious differences in susceptibility to infection are not well understood. Some genes have been associated with severe disease, including genes involved in the interferon-γ (IFN-γ) and interleukin-12 (IL-12) signaling pathways. However, many questions remain about the genetic factors that contribute to susceptibility and resistance to infection. In chapter 1 we highlight our current knowledge about the innate immune responses that are crucial for the recognition of infection and activation of adaptive immune responses. A successful pro-inflammatory response to infection is well-balanced, accomplishing control of infection while limiting excessive pro-inflammatory responses that damage the host. Cytokines such as TNF-α and IL-1 are important for protection, yet excessive or insufficient cytokine production results in progressive disease. Macrophages phagocytose M. tuberculosis and are capable of cell-intrinsic antimicrobial control. Furthermore, they are important for initiating and maintaining inflammation. Neutrophils are highly abundant during infection and are normally associated with control of bacterial infections. However, they can be drivers of hyperinflammatory responses that benefit M. tuberculosis growth and result in host mortality. The role of other innate cells, including natural killer cells and innate T cells, remains enigmatic. Chapter 2 describes the use of a genetically diverse mouse cohort to identify underlying mechanisms of susceptibility to infection. The diverse mouse lines showed a range of susceptibility to infection with several lines harboring 100-fold more bacterial in the lungs compared to the classical C57BL/6 mice. In addition, the abundance of neutrophils in the lung seems to correlate with outcome of infection, showing that high neutrophilic influx associates with high bacterial burden in the lung. Furthermore, we identified a mouse line that controls M. tuberculosis infection in the absence of high numbers of CD4+IFNg+ T cells, which have been shown to drive control of infection in the classic B6 model. Restriction of M. tuberculosis growth by macrophages occurs at the onset of the adaptive immune responses with CD4+ T cells producing IFN-γ that activates macrophages to exert their antimicrobial programs. A master regulator of IFN-γ is the transcription factor HIF-1α, which induces the upregulation of several pathways involved in maintenance of the macrophage’s glycolytic metabolism and bactericidal responses against M. tuberculosis. Many questions remain on how HIF-1α mediates these effects and how HIF-1α is regulated by IFN-γ signaling. In chapter 3 we investigate the factors involved in the constitutive degradation of HIF-1α in resting macrophages. HIF-1α seems to be degraded through a non-canonical pathway that requires the activity of ubiquitin ligase VHL. Although VHL is known for targeting proteins to proteasome-dependent degradation, HIF-1α is degraded by the lysosome, suggesting the involvement of additional proteins that contribute to the activation of the correct degradation pathway. Inhibition of HIF-1α degradation, including the deletion of the gene encoding VHL, results in enhanced cell-intrinsic control in macrophages. This effect is mediated through production of nitric oxide (NO). Surprisingly, very little is known about the pathways downstream of NO and HIF-1α signaling that result in microbicidal responses. Our data shows that expression of a family of solute carrier (SLC) transporter proteins is highly dependent of IFN-γ and HIF-1α expression. The requirement for SLC metabolite transporters in macrophage glycolytic metabolism makes this protein family essential for maintaining the antimicrobial function of macrophages. In Chapter 3 we describe the development of a targeted forward genetics CRISPR approach to identify which SLC transporters are required for cell-intrinsic control of M. tuberculosis infection in macrophages. Taken together, this project aims to elucidate the antimicrobial mechanisms in the host that contribute to cell-intrinsic and -extrinsic control of infection.

Advisor: Stanley, Sarah A