| dc.description.abstract | Mycobacterium tuberculosis (Mtb), an obligate human pathogen, spreads through aerosols to the lung alveoli, where alveolar macrophages engulf it. These macrophages then migrate to the lung interstitium and trigger an inflammatory response, ultimately forming a granuloma, which is the hallmark of tuberculosis (TB) pathology. TB infections continue to be debilitating to society, and our understanding of the disease is primarily derived from in vitro and animal models. However, animal models either fail to replicate the human TB granuloma or require specialized facilities, and existing in vitro models do not accurately reflect the three-dimensional (3D) cellular environment found in vivo. Since pulmonary Mtb infection primarily occurs in the lung interstitium, where collagen I is the dominant extracellular matrix (ECM) component, my thesis focuses on developing novel biomaterials-based 3D in vitro culture platforms for TB research. We created new 3D human-based infection models using collagen I hydrogels to address these limitations.
1. As an alternative platform to 2D infection studies, we infected THP-monocytes with Mtb in collagen gels, which resulted in a longer duration of infection studies (3 weeks) than corresponding macrophage infection studies in 2D (5 days). The THP-1 cells in the infected collagen gels differentiated into macrophages and recapitulated significant characteristics of human TB infection phenotypically and genotypically. Dual RNA-sequencing of Mtb and THP-1 cells from the infected collagen gels (GSE216503) revealed significant similarities with human caseous TB and lymph node granulomas. We also found pyrazinamide (PZA), a first-line anti-tuberculosis drug, to effectively reduce the bacterial load (by 2 log orders at the clinically relevant concentration range of the drug), proving that the culture system can be a superior alternative to the conventional 2D macrophage infection models. Using this model, we are studying the mechanisms of PZA activity, as its mode of action remains unknown (Gupta VK et al., 2024).
2. TB granulomas are challenging microenvironments in which Mtb resides in vivo. We aimed to generate in vitro tuberculosis granulomas for in-depth characterization. This was achieved by engineering a custom-designed lab-on-a-chip perfusion bioreactor in which collagen I hydrogel matrix containing human peripheral blood mononuclear cells (PBMCs) and Mtb are loaded to recapitulate the in vivo host-pathogen interaction dynamics. Moreover, perfusion of PBMCs at interstitial flow velocities flow (0.1 m/s – 10 m /s) is carried out to mimic the continuous recruitment of immune cells to the lesions in an inflamed TB lung. The cytokine gradients generated in the setup because of flow allow the interactions of immune cells with Mtb to form TB granulomas, recapitulating in vivo conditions. The perfused PBMCs respond to the cytokine gradients in the matrix and accumulate to form large cellular aggregates (≥ 500 m diameter) over two weeks. We are working toward establishing the similarity of these in vitro granulomas with human granulomas by analyzing the spatial organization of cells and their transcriptomic signatures. Our observations indicate that granulomas formed from different donors have significantly different bacterial loads, potentially indicating host summary, using bioengineering strategies, we successfully generated in vitro immune cell aggregates with similar dynamics as human TB granulomas by closely mimicking the in vivo infection conditions. This platform holds great potential as an improvement over the current ones in screening antibiotics against Mtb and host modulators to control bacterial growth.
In conclusion, we have developed two novel in vitro platforms to study host-Mtb infection, which can aid the next generation of antibiotic discoveries and help decipher infection biology better. | en_US |
