Evaluation of small-molecule compounds and immunomodulatory agents as potential host-directed therapy for tuberculosis in in vitro models of Mycobacterium tuberculosis infection.

Abstract

Background: Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), is a global health concern that currently affects millions of individuals and causes significant mortality annually. The problem is aggravated by the emergence of drug-resistant Mtb strains and TB-associated tissue pathology, which may result in post-TB lung disease and lung function challenges. New therapeutic strategies are required to treat TB-associated lung pathology. Host-directed therapies (HDTs) offer a novel approach to treating TB and improving outcomes by enhancing the host’s immune response, alleviating tissue damage caused by extreme inflammation, and shortening treatment duration. This study aims to screen small-molecule compounds as potential HDTs to target inflammation, reduce tissue damage, and alleviate pulmonary impairment associated with TB. Methods: High-throughput screening of small-molecule compounds was performed on PBMCs infected with Mtb H37Rv. ELISA was used to quantify the release of TNF-α, IL-1β, and IL-6 to assess the anti-inflammatory effects of the compounds. A promising candidate, Tam-X1, which inhibits IL-5 from binding to its receptor IL-5R, was identified for its capacity to reduce pro-inflammatory cytokine production. Tam-X1 was further evaluated in a 3D biomimetic collagen-alginate model mimicking TB granulomas, using PBMCs infected with luminescent Mtb H37Rv LUX strain. ELISA and luminometry were employed to measure the anti-inflammatory and antimycobacterial effects of Tam-X1 in the 3D model, respectively. H&E staining and immunofluorescence staining were performed on resected TB-diseased lung tissue to examine the spatial association of proteins in the Tam-X1 target pathway, including IL-5 and IL-5Ra, with TB-induced inflammation and granuloma development. Results: Tam-X1 significantly reduced the production of pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6, during Mtb infection and inhibited intracellular Mtb growth within the 3D biomimetic model. Histopathological assays revealed that TB-driven inflammation is mediated by immune cells expressing IL-5Ra, including eosinophils, and that Tam-X1 targets a pathway spatially associated with pathology in the TB-diseased lung. Conclusion: This study highlights the potential of Tam-X1 as a promising HDT for TB. By targeting the IL-5/IL-5R signalling pathway, Tam-X1 reduced the production of TNF-α, IL-1β, and IL-6 during Mtb infection and inhibited Mtb growth. This suggests that treatment with Tam-X1 may mitigate TB-associated tissue pathology by modulating the host's immune response. ABSTRACT: PART B Background: Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (Mtb), remains a global health threat due to drug resistance, poor treatment adherence, and high mortality rates. Host-directed therapies (HDTs) offer a novel approach to improve TB treatment outcomes by enhancing the host’s immune defence, controlling inflammation and reducing post-TB complications and mortality. This study aimed to assess the potential of immunomodulatory compounds targeting Toll-like receptors (TLRs) 7 and 8, as adjunctive HDTs for TB. Methods: Screening of the effects of immunomodulatory agents in THP-1 cells infected with the luminescent Mtb strain, H37Rv LUX, was performed. The effects on the production of the pro-inflammatory cytokines, TNF-α, IL-1β and IL-6 were quantified using ELISA. Luminometry was used to analyse the effects of TLR modulation on bacterial clearance. A promising compound, TLR M, a TLR7/8 agonist, was identified as an activator of macrophage pro-inflammatory responses and underwent further evaluation using an in vitro 3D biomimetic TB granuloma model, analysing the effects on inflammation using ELISA and antimycobacterial effects using luminometry. H&E-stained and immunofluorescent labelled slides were imaged using confocal microscopy to spatially analyse the association of TLR M’s protein target, TLR7/8, with TB-induced inflammation and granuloma formation in resected TB-diseased lung tissue. Results: This study identified TLR7/8 as potential targets for adjunctive HDTs, revealing their potential to enhance the inflammatory response during Mtb infection. TLR7/8 activation with TLR M improved antimycobacterial properties. Using immunofluorescence staining on lung tissue from TB diseased participants, we observed that alveolar macrophages contribute to TLR7/8 mediated signalling, potentially amplifying effector functions and aiding in Mtb clearance. Conclusion: Taken together, TLR M represents a potential adjuvant HDT for TB treatment. The ability of this TLR7/8 agonist to modulate the inflammatory immune response and enhance antimycobacterial activity suggests that it can improve treatment outcomes and mitigate TB-related morbidity and mortality. Further research into TLR7/8-targeted therapies may therefore lead to enhanced strategies for combating TB by enhancing the hosts immune response and addressing the challenges associated with current treatment regimens.