Novel therapeutics, associated adverse effects, and changes in immune responses during pulmonary infection with Mycobacterium tuberculosis
Date
2024
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Abstract
Patients diagnosed with multidrug resistant (MDR) or extensively drug resistant (XDR) tuberculosis (TB) have limited treatment options. The Nix-TB clinical trial evaluated a new 6-month regimen containing three-oral-drugs; bedaquiline (B), pretomanid (Pa) and linezolid (L) collectively termed as BPaL for the treatment of TB. This regimen achieved remarkable results as almost 90% of the participants suffering from MDR- or XDR-TB had favorable outcomes. Despite the extraordinary outcomes, many patients also developed severe adverse effects (AEs) which were associated with the long-term administration of the oxazolidinone protein synthesis inhibitor linezolid. Spectinamide 1599 (S) is also a potent protein synthesis inhibitor of Mycobacterium tuberculosis (Mtb) with an excellent safety profile, but which lacks oral bioavailability. In chapter 2, we hypothesized that inhaled spectinamide 1599, combined with BPa ––BPaS regimen ––, has similar efficacy to that of BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the BALB/c (permissive resistant) and C3HeB/FeJ (permissive susceptible) murine chronic TB efficacy models. Both regimens promoted similar bactericidal effects in lung and spleen of both models after 4 weeks. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL treatment also decreased myeloid to erythroid ratio and increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. During therapy both regimens improved the lung lesion burden, reduced neutrophil and cytotoxic T cell counts while increased the number of B and helper and regulatory T cells. This combined data suggests that inhaled spectinamide 1599 combined with BPa is an effective TB therapy that avoids L-associated AEs. The granuloma formation is the pathological hallmark of TB, and several studies suggest that there are temporal and spatial changes in their distinct immune responses. These changes differ not only from one granuloma to another in a single individual but also depend on the severity of the disease. In chapter 3, we attempted to understand longitudinal changes in immune cells, their relationships, and their spatial distribution in granulomas of Mtb infected BALB/c and C3HeB/FeJ mouse models using a novel technique of multispectral imaging microscopy. Multiplex fluorescence immunohistochemistry (mfIHC) is unique in its ability to provide both expression and location of several immune cells along with their co-localization in a single tissue section while preserving tissue architecture and spatial context. The results showed that as the infection progresses, there are also dynamic changes in the immune phenotypes forming the granulomas and those located within the parenchymal tissue. Moreover, the histologically similar granulomas manifested complexity in their immune cell composition mainly due to the presence of adaptive immune responses. The advanced cellular granulomas in BALB/c TB model were mainly predominated by CD4 and CD8 T cells, Ly6G stained neutrophils, B220 B cells and all these were surrounded by F4/80 macrophages. With time post infection, there was an increased uniform recruitment of CD4 and Foxp3 T cells, F4/80 macrophages and Ly6G neutrophils within granulomas compared to parenchymal tissue where IFNγ and IL-10 secreting cells were in abundance. Moreover, B220 B cells and CD8 T cells also showed increased but heterogeneous distribution among the advancing granulomas especially B220 B cells formed clusters. The spatial analysis showed an increased median distance for Ly6G neutrophils, whereas this distance was decreased for B220 B cells when measured from CD4 and CD8 cells. In summary, combining the spatial and temporal data in addition to the mere cell counts helps to uncover interactions and relationships between different immune cells within the granuloma.
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Subject
C3HeB/FeJ
immune responses
tuberculosis
drug therarpy
BALB/c
spectinamide 1599