Isabella Forasteiro Tavares, Rafaela de Souza Andrade, Natasha Cabral, Roberta Olmo Pinheiro, Veronica Schmitz
We found that at least 8 h of M. leprae incubation was necessary to trigger primary and secondary granule degranulation, while tertiary granules began to degranulate within 4 h. At this 4-h mark, LDNs were observed in cell cultures. By 24 h, we confirmed neutrophil degranulation and LDN generation. We observed a correlation between the release of pro-inflammatory cytokines and degranulation markers. The secretion of IL-8 and IL-6 was associated with secondary and tertiary granule degranulation. Selective inhibition of p38 MAPK affected the release of tertiary and secondary granules as well as IL-6 secretion. Moreover, mito-TEMPO treatment showed that mitochondrial ROS production seems to be crucial for M. leprae-induced primary and secondary granules degranulation. Finally, the inhibition of TGF-β receptor 1 indicates that TGF-β modulates M. leprae-induced neutrophil degranulation and LDN generation through binding to the receptor.
INTRODUCTION: Erythema Nodosum Leprosum (ENL) is an inflammatory complication of leprosy that is associated with the progression of peripheral nerve damage and the resultant physical disabilities. Neutrophil-mediated inflammation plays a crucial role in the emergence of ENL. Recent evidence suggests that Mycobacterium leprae triggers neutrophil degranulation and the generation of Low-Density Neutrophils (LDN). This study aimed to investigate whether inhibition of critical regulators of human neutrophil activation can regulate M. leprae-induced neutrophil degranulation.
METHODS: To explore the mechanisms underlying inflammation in vitro, whole-blood cultures were incubated with M. leprae. The effects of selective inhibitors in M. leprae co-cultures were subsequently evaluated by measuring surface expression of granule markers, levels of inflammatory cytokines, and release of granule-related proteins, including MMP-9, PTX3, and MPO.
RESULTS: We found that at least 8 h of M. leprae incubation was necessary to trigger primary and secondary granule degranulation, while tertiary granules began to degranulate within 4 h. At this 4-h mark, LDNs were observed in cell cultures. By 24 h, we confirmed neutrophil degranulation and LDN generation. We observed a correlation between the release of pro-inflammatory cytokines and degranulation markers. The secretion of IL-8 and IL-6 was associated with secondary and tertiary granule degranulation. Selective inhibition of p38 MAPK affected the release of tertiary and secondary granules as well as IL-6 secretion. Moreover, mito-TEMPO treatment showed that mitochondrial ROS production seems to be crucial for M. leprae-induced primary and secondary granules degranulation. Finally, the inhibition of TGF-β receptor 1 indicates that TGF-β modulates M. leprae-induced neutrophil degranulation and LDN generation through binding to the receptor.
DISCUSSION: These results indicate that p38 MAPK pathway and mitochondrial ROS production are important contributors to M. leprae-induced neutrophil degranulation, while TGF-β/TGF-βR1 signaling exerts an important regulatory effect. These findings advance our knowledge of the molecular mechanisms underlying M. leprae-induced degranulation and raise the possibility of new potential therapeutic strategies for targeting ENL-related degranulation.