Corrigendum: Escape of TLR5 recognition by Leptospira spp.: A rationale for atypical endoflagella.

[This corrects the article DOI: 10.3389/fimmu.2020.02007.].

Pathogenic Leptospires Limit Dendritic Cell Activation Through Avoidance of TLR4 and TRIF Signaling.

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.

Escape of TLR5 Recognition by Leptospira spp.: A Rationale for Atypical Endoflagella.

Leptospira (L.) interrogans are invasive bacteria responsible for leptospirosis, a worldwide zoonosis. They possess two periplasmic endoflagellae that allow their motility. L. interrogans are stealth pathogens that escape the innate immune recognition of the NOD-like receptors NOD1/2, and the human Toll-like receptor (TLR)4, which senses peptidoglycan and lipopolysaccharide (LPS), respectively. TLR5 is another receptor of bacterial cell wall components, recognizing flagellin subunits. To study the contribution of TLR5 in the host defense against leptospires, we infected WT and TLR5 deficient mice with pathogenic L. interrogans and tracked the infection by in vivo live imaging of bioluminescent bacteria or by qPCR. We did not identify any protective or inflammatory role of murine TLR5 for controlling pathogenic Leptospira. Likewise, subsequent in vitro experiments showed that infections with different live strains of L. interrogans and L. biflexa did not trigger TLR5 signaling. However, unexpectedly, heat-killed bacteria stimulated human and bovine TLR5, but did not, or barely induced stimulation via murine TLR5. Abolition of TLR5 recognition required extensive boiling time of the bacteria or proteinase K treatment, showing an unusual high stability of the leptospiral flagellins. Interestingly, after using antimicrobial peptides to destabilize live leptospires, we detected TLR5 activity, suggesting that TLR5 could participate in the fight against leptospires in humans or cattle. Using different Leptospira strains with mutations in the flagellin proteins, we further showed that neither FlaA nor Fcp participated in the recognition by TLR5, suggesting a role for the FlaB. FlaB have structural homology to Salmonella FliC, and possess conserved residues important for TLR5 activation, as shown by in silico analyses. Accordingly, we found that leptospires regulate the expression of FlaB mRNA according to the growth phase in vitro, and that infection with L. interrogans in hamsters and in mice downregulated the expression of 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.

Use of Golden Syrian Hamster as an Animal Model to Study Leptospirosis-Associated Immune Responses.

Experimental infections greatly contribute to further deepen our knowledge of infectious diseases. In the case of leptospirosis, hamsters as well as gerbils and guinea pigs have been used as animal models of acute leptospirosis in studying the pathophysiology of the disease. Here we describe a typical Leptospira infection using golden Syrian hamsters. We will also present techniques we use to study the resulting bacterial burden and gene expression patterns in the host in order to decipher the innate immune response to leptospirosis.

Leptospirosis Pathophysiology: Into the Storm of Cytokines.

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.

The Hippo kinase promotes Scalloped cytoplasmic localization independently of Warts in a CRM1/Exportin1-dependent manner in Drosophila.

Scalloped (SD) is a transcription factor characterized by a TEA/ATTS DNA binding domain. To activate transcription, SD must interact with its coactivators, including Yorkie (YKI) or Vestigial (VG). YKI is the downstream effector of the Hippo signaling pathway that plays a key role in the control of tissue growth. The core components of this pathway are two kinases, Hippo (HPO) and Warts (WTS), which negatively regulate the activity of the SD/YKI complex, retaining YKI in the cytoplasm. We previously showed that HPO kinase can also reduce SD/VG transcriptional activity in Drosophila S2 cells. We further investigated the relationship between the SD/VG complex and the Hippo pathway. We show here that HPO overexpression suppresses overgrowth induced by SD/VG in vivo during Drosophila development. Using S2 cells, we show that HPO promotes the translocation of SD to the cytoplasm in a CRM1-dependent manner, thereby inhibiting the induction of SD/VG target genes. Using RNAi-mediated depletion of yki and a mutant SD protein unable to interact with YKI, we demonstrate that HPO regulates SD localization independently of YKI. This function requires HPO kinase activity, yet surprisingly, not its downstream effector kinase WTS. Taken together, these observations reveal a new and unexpected role of HPO kinase in the regulation of a transcription factor independently of YKI.

Alteration of TEAD1 expression levels confers apoptotic resistance through the transcriptional up-regulation of Livin.

BACKGROUND: TEA domain (TEAD) proteins are highly conserved transcription factors involved in embryonic development and differentiation of various tissues. More recently, emerging evidences for a contribution of these proteins towards apoptosis and cell proliferation regulation have also been proposed. These effects appear to be mediated by the interaction between TEAD and its co-activator Yes-Associated Protein (YAP), the downstream effector of the Hippo tumour suppressor pathway. METHODOLOGY/PRINCIPAL FINDINGS: We further investigated the mechanisms underlying TEAD-mediated apoptosis regulation and showed that overexpression or RNAi-mediated silencing of the TEAD1 protein is sufficient to protect mammalian cell lines from induced apoptosis, suggesting a proapoptotic function for TEAD1 and a non physiological cytoprotective effect for overexpressed TEAD1. Moreover we show that the apoptotic resistance conferred by altered TEAD1 expression is mediated by the transcriptional up-regulation of Livin, a member of the Inhibitor of Apoptosis Protein (IAP) family. In addition, we show that overexpression of a repressive form of TEAD1 can induce Livin up-regulation, indicating that the effect of TEAD1 on Livin expression is indirect and favoring a model in which TEAD1 activates a repressor of Livin by interacting with a limiting cofactor that gets titrated upon TEAD1 up-regulation. Interestingly, we show that overexpression of a mutated form of TEAD1 (Y421H) implicated in Sveinsson's chorioretinal atrophy that strongly reduces its interaction with YAP as well as its activation, can induce Livin expression and protect cells from induced apoptosis, suggesting that YAP is not the cofactor involved in this process. CONCLUSIONS/SIGNIFICANCE: Taken together our data reveal a new, Livin-dependent, apoptotic role for TEAD1 in mammals and provide mechanistic insight downstream of TEAD1 deregulation in cancers.