AbstractLeptospirosis is a worldwide re-emerging zoonosis caused by pathogenicLeptospiraspp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human TLR4, whereas it signals through murine TLR4, which mediates mouse resistance to acute leptospirosis. However, leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, using confocal microscopy and flow cytometry, we showed that the LPS ofL. interrogansdid not induce internalization of TLR4 in mouse macrophages, unlike the LPS ofEscherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter O antigen LPS and repurified leptospiral LPS with reporter HEK cells, we further found this TLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel immune escape mechanism linked to leptospiral LPS. We hypothesize that the LPS, already known as a virulence factor, plays a major role in the innate immune evasion of the leptospires, thereby contributing to their stealthiness and chronicity in mice.Author summaryLeptospira interrogansis a bacterial pathogen, responsible for leptospirosis, a worldwide neglected reemerging disease.L. interrogansmay cause an acute severe disease in humans, whereas rodents and other animals asymptomatically carry the leptospires in their kidneys. They can therefore excrete live bacteria in urine and contaminate the environment. Leptospires are stealth pathogens known to escape the innate immune defenses of their hosts. They are covered in lipopolysaccharide (LPS), a bacterial motif recognized in mammals through the Toll-like receptor 4 (TLR4), which triggers two different signaling pathways. We showed previously that pathogenic leptospires escape TLR4 recognition in humans. Here we show in mice that the leptospiral LPS triggers only one arm of the TLR4 pathway and escapes the other, hence avoiding production of antimicrobial compounds. Removing the lipoproteins that always co-purify with the leptospiral LPS, or using shorter LPS, restores the stimulation of both pathways. This suggests a novel escape mechanism linked to the LPS and involving lipoproteins that could be instrumental for leptospires to escape the mouse defense and allows for their chronic renal colonization.