Leptospirosis is a neglected tropical zoonosis caused by pathogenic spirochetes of the genus Leptospira. Infected reservoir animals, typically mice and rats, are asymptomatic, carry the pathogen in their renal tubules, and shed pathogenic spirochetes in their urine, contaminating the environment. Humans are accidental hosts of pathogenic Leptospira. Most human infections are mild or asymptomatic. However, 10% of human leptospirosis cases develop into severe forms, including high leptospiremia, multi-organ injuries, and a dramatically increased mortality rate, which can relate to a sepsis-like phenotype. During infection, the triggering of the inflammatory response, especially through the production of cytokines, is essential for the early elimination of pathogens. However, uncontrolled cytokine production can result in a cytokine storm process, followed by a state of immunoparalysis, which can lead to sepsis and associated organ failures. In this review, the involvement of cytokine storm and subsequent immunoparalysis in the development of severe leptospirosis in susceptible hosts will be discussed. The potential contribution of major pro-inflammatory cytokines in the development of tissue lesions and systemic inflammatory response, as well as the role of anti-inflammatory cytokines in contributing to the onset of a deleterious immunosuppressive cascade will also be examined. Data from studies comparing susceptible and resistant mouse models will be included. Lastly, a concise discussion on the use of cytokines for therapeutic purposes or as biomarkers of leptospirosis severity will be provided.

Holzapfel et al. TLR5 Evasion by Leptospires the FlaB, but not the FlaA subunits. Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 recognition, our study suggests that the peculiar central localization and stability of the FlaB monomers in the periplasmic endoflagellae, associated with the downregulation of FlaB subunits in hosts, constitute an efficient strategy of leptospires to escape the TLR5 recognition and the induced immune response.

Leptospira interrogans is a bacterial species responsible for leptospirosis, a neglected worldwide zoonosis. Mice and rats are resistant and can become asymptomatic carriers, whereas humans and some other mammals may develop severe forms of leptospirosis. Uncommon among spirochetes, leptospires contain lipopolysaccharide (LPS) in their outer membrane. LPS is highly immunogenic and forms the basis for a large number of serovars. Vaccination with inactivated leptospires elicits a protective immunity, restricted to serovars with related LPS. This protection that lasts in mice, is not long lasting in humans and requires annual boosts. Leptospires are stealth pathogens that evade the complement system and some pattern recognition receptors from the Toll-like (TLR) and Nod-Like families, therefore limiting antibacterial defense. In macrophages, leptospires totally escape recognition by human TLR4, and escape the TRIF arm of the mouse TLR4 pathway. However, very little is known about the recognition and processing of leptospires by dendritic cells (DCs), although they are crucial cells linking innate and adaptive immunity. Here we tested the activation of primary DCs derived from human monocytes (MO-DCs) and mouse bone marrow (BM-DCs) 24h after stimulation with saprophytic or different pathogenic virulent or avirulent L. interrogans. We measured by flow cytometry the expression of DC-SIGN, a lectin involved in T-cell activation, co-stimulation molecules and MHC-II markers, and pro- and anti-inflammatory cytokines by ELISA. We found that exposure to leptospires, live or heat-killed, activated dendritic cells. However, pathogenic L. interrogans, especially from the Icterohaemorraghiae Verdun strain, triggered less marker upregulation and less cytokine production than the saprophytic Leptospira biflexa. In addition, we showed a better activation with avirulent leptospires, when compared to the virulent parental strains in murine BM-DCs. We did not observe this difference in human MO-DCs, suggesting a role for TLR4 in DC stimulation. Accordingly, using BM-DCs from transgenic deficient mice, we showed that virulent Icterohaemorraghiae and Manilae serovars dampened DC activation, at least partly, through the TLR4 and TRIF pathways. This work shows a novel bacterial immune evasion mechanism to limit DC activation and further illustrates the role of the leptospiral LPS as a virulence factor.

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26Leptospira interrogans are invasive bacteria responsible for leptospirosis, a worldwide zoonosis. They 27 possess two periplasmic endoflagella that allow their motility. L. interrogans are stealth pathogens that 28 escape the innate immune responses of the NOD-like receptors NOD1/2, and the human Toll-like 29 receptor (TLR)4, sensing peptidoglycan and lipopolysaccharide (LPS), respectively. TLR5 is another 30 receptor of bacterial cell wall components, recognizing flagellin subunits. 31To study the contribution of TLR5 in the host defense against leptospires, we infected WT and TLR5 32 deficient mice with pathogenic L. interrogans and tracked the infection by in vivo live imaging of 33 bioluminescent bacteria or by q-PCR. We did not identify any protective or inflammatory role of 34 murine TLR5 to control pathogenic Leptospira. Likewise, subsequent in vitro experiments showed 35 that infections with different live strains of L. interrogans and L. biflexa did not trigger TLR5. 36However, unexpectedly, heat-killed bacteria stimulated human and bovine TLR5, although barely 37 2 mouse TLR5. Abolition of TLR5 recognition required extensive boiling time of the bacteria or 38 proteinase K treatment, showing an unusual high stability of the leptospiral flagellins. Interestingly, 39 using antimicrobial peptides to destabilize live leptospires, we detected some TLR5 activity, 40 suggesting that TLR5 could participate in the fight against leptospires in humans or cattle. Using 41 different Leptospira strains with mutations in flagellin proteins, we further showed that neither FlaAs 42 nor Fcps participated in the recognition by TLR5, suggesting a role for the FlaBs. These have 43 structural homology to Salmonella FliC, and conserved residues important for TLR5 activation, as 44 shown by in silico analyses. Accordingly, we found that leptospires regulate the expression of FlaB 45 mRNA according to the growth phase in vitro, and that infection with L. interrogans in hamsters and 46 in mice downregulated the expression of the FlaBs but not the FlaAs subunits. 47Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 48 recognition, our study suggests that the peculiar central localization and stability of the FlaB 49 monomers in the periplasmic endoflagella, associated with the downregulation of FlaB subunits in 50 hosts, constitute an efficient strategy of leptospires to escape TLR5 recognition and the immune 51 response. 53 55Among the Leptospira genus, which includes more than 60 species and 300 different serovars, 56Leptospira interrogans gathers the most pathogenic strains [2]. Rodents and other animals can carry 57 leptospires asymptomatically in the lumen of proximal renal tubules, excrete the bacteria in their urine 58 and contaminate the environment. Vertebrates get infected through skin or mucosa. In most cases, 59 humans have no symptoms or suffer from a flu-like mild disease, but may also show acute severe, 60 potentially fatal, leptospirosis. Antibiotic treatments are e...

In the original article, there was a mistake in Figure 8A, as published. We showed that we did not get expression of the FLaB1 subunit in Manilae L495 strain. In fact, the forward primer (designed according to the Fiocruz sequence) used to amplify the FlaB1 subunit has two mismatches within the Manilae sequence. We did the RT-PCR with the good primer and found an amplification, showing an enhanced expression at the stationary phase compared to the exponential phase, likewise the other FlaB subunits. The corrected Figure 8 appears below.A correction has been made to Results, "FlaB mRNA Are Upregulated in Stationary Phase".