Multi-species transcriptome analysis of the monocyte and macrophage immune response against Mycobacterium tuberculosis

Background: Despite extensive research and vaccine development, tuberculosis remains one of the top causes of death related to a single pathogen. The central dogma of protective Tuber-culosis (TB) immunity remains highly controversial and previous findings suggest differences between species in the host response to Mycobacterium tuberculosis (Mtb) infection. Compar-ing activated anti-mycobacterial mechanisms of macrophages and monocytes between relevant host species, might reveal novel targets against Mtb infection, important for the development of novel intervention strategies.
Methods: This thesis used a two-way approach to investigate and compare anti-mycobacterial defense mechanisms between macrophages or monocytes from mouse, human and cattle. The Smart Seq2 protocol was used as a basis for an agnostic bulk RNA sequencing approach to systemically compare Interferon-γ activated immune responses to Mtb infection in murine bone marrow derived macrophages and human and bovine monocyte derived macrophages. Addi-tionally human and bovine monocyte immune responses to Mtb whole cell lysate were investi-gated by analyzing relative expression and expression fold changes (EFC) of genes of interest, determined by a previous single cell RNA sequencing study.
Results: Using bulk RNA sequencing, a strong pro-inflammatory response was shown for both murine and human macrophages. These responses were closely related, sharing many similar enriched gene ontology associations as well as signaling pathways. However, human macro-phages lacked expression of NOS2 and genes of the guanylate-binding protein (GBP) family. Contrasting the other investigated species, bovine macrophages were shown to respond with a lower number of differentially expressed genes, decreased fold changes and less associations to an inflammatory response. This low inflammatory phenotype was shared by bovine mono-cytes which only significantly increased PTGS2, IL1B and IL6 expression after stimulation with Mtb whole cell lysate or LPS. Human monocytes in turn reflected the strong inflammatory response of human macrophages, increasing CXCL10, TNF, PTGS2, IL1B as well as IL6 ex-pression after in vivo stimulation.
Conclusions: The agnostic approach of bulk RNA sequencing revealed absent NOS2 and GBP expression in human macrophages which might contribute to the inability of interferon-γ (IFNγ) activated human macrophages fail to control Mtb infection even though their response is similar to murine macrophages that are known to control Mtb in an IFNγ dependent manner. Although this thesis could not show a bovine specific anti-mycobacterial defense mechanism which could explain the attenuation of Mtb in cattle, a unique low responding phenotype for both bovine macrophages and monocytes was observed. All together this phenotype might be relevant for protective TB immunity, since infected cells do not promote excessive chronic inflammation.



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