Effect of macrophage number on tumor necrosis factor-α levels in a tuberculous granuloma model: an in vitro study

Abstract

Background: Tumor necrosis factor-alpha (TNF-α) is a key cytokine secreted by macrophages in the formation and integrity of tuberculous granulomas.

Objective: This study aimed to analyze the influence of macrophage number variation on TNF-α levels in an in vitro tuberculous granuloma model.

Methods: The granuloma model was constructed by co-culturing peripheral blood mononuclear cells (PBMC) from a healthy donor with autologous macrophages at numbers of 1x10⁵, 2x10⁵, and 3x10⁵ cells/well, followed by infection with Mycobacterium tuberculosis H37Rv. Supernatants were collected up to the 5th day of incubation, and TNF-α levels were measured by ELISA. Data were analyzed using statistical tests.

Results: TNF-α levels varied between the number groups. The lowest mean TNF-α level was found in the 1x10⁵ cells/well macrophage number group (0.013 pg/mL). Results from the ANOVA and post-hoc tests indicated a significant difference within the groups (p=0.009) and between specific groups: between the 1x10⁵ cells/well macrophage group and the control (p=0.026), the 2x10⁵ cells/well group (p=0.035), and the 3x10⁵ cells/well group (p=0.033) at a 0.05 significance level.

Conclusion: Macrophage number affects TNF-α levels in the in vitro tuberculous granuloma model in a non-linear manner, suggesting threshold-dependent immune regulation. These findings are confined to the in vitro context and require further validation.