Leptospira interrogans are pathogenic bacteria responsible for leptospirosis, a zoonosis impacting 1 million people per year worldwide. Leptospires can infect all vertebrates, but not all hosts develop similar symptoms. Human and cattle may suffer from mild to acute illnesses and are therefore considered as sensitive to leptospirosis. In contrast, mice and rats remain asymptomatic upon infection, although they get chronically colonized in their kidneys. Upon infection, leptospires are stealth pathogens that partially escape the recognition by the host innate immune system. Although leptospires are mainly extracellular bacteria, it was suggested that they could also replicate within macrophages. However, contradictory data in the current literature led us to reevaluate these findings. Using a gentamicin–protection assay coupled to high-content (HC) microscopy, we observed that leptospires were internalized in vivo upon peritoneal infection of C57BL/6J mice. Additionally, three different serotypes of pathogenic L. interrogans and the saprophytic L. biflexa actively infected both human (PMA differentiated) THP1 and mouse RAW264.7 macrophage cell lines. Next, we assessed the intracellular fate of leptospires using bioluminescent strains, and we observed a drastic reduction in the leptospiral intracellular load between 3 h and 6 h post-infection, suggesting that leptospires do not replicate within these cells. Surprisingly, the classical macrophage microbicidal mechanisms (phagocytosis, autophagy, TLR–mediated ROS, and RNS production) were not responsible for the observed decrease. Finally, we demonstrated that the reduction in the intracellular load was associated with an increase of the bacteria in the supernatant, suggesting that leptospires exit both human and murine macrophages. Overall, our study reevaluated the intracellular fate of leptospires and favors an active entrance followed by a rapid exit, suggesting that leptospires do not have an intracellular lifestyle in macrophages.

Leptospira interrogans are bacteria that can infect all vertebrates and are responsible for leptospirosis, a neglected zoonosis. Some hosts, such as humans, are susceptible to the disease, whereas mice are resistant and get chronically colonized. Although leptospires escape recognition by some immune receptors, they activate the NOD-like receptor pyrin 3–inflammasome and trigger IL-1β secretion. Classically, IL-1β secretion is associated with lytic inflammatory cell death called pyroptosis, resulting from cytosolic LPS binding to inflammatory caspases, such as caspase 11. Interestingly, we showed that L. interrogans and Leptospira biflexa do not trigger cell death in either murine, human, hamster, or bovine macrophages, escaping both pyroptosis and apoptosis. We showed, in murine cells, that the mild IL-1β secretion induced by leptospires occurred through nonlytic caspase 8–dependent gasdermin D pore formation and not through activation of caspase 11/noncanonical inflammasome. Strikingly, we demonstrated a potent antagonistic effect of pathogenic L. interrogans and their atypical LPS on spontaneous and Escherichia coli LPS-induced cell death. Indeed, LPS of L. interrogans efficiently prevents caspase 11 dimerization and subsequent massive gasdermin D cleavage. Finally, we showed that pyroptosis escape by leptospires prevents massive IL-1β release, and we consistently found no major role of IL-1R in controlling experimental leptospirosis in vivo. Overall, to our knowledge, our findings described a novel mechanism by which leptospires dampen inflammation, thus potentially contributing to their stealthiness.

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