Activation of Canine, Mouse and Human TLR2 and TLR4 by Inactivated Leptospira Vaccine Strains

Abstract

Canine Leptospira vaccines contain inactivated strains of pathogenic Leptospira, the causative agents of leptospirosis. For an effective response to vaccination, activation of the innate immune system via pattern recognition receptors such as TLRs is crucial. However, it is not known which TLRs are activated by Leptospira in dogs. To investigate the involvement of canine TLR2, TLR4, and TLR5 in the recognition of Leptospira, we stimulated canine moDC and reporter cells expressing canine TLR2 with either whole-inactivated bacteria or purified LPS of Leptospira strains, representing the serogroups generally used in canine leptospirosis vaccines. Using the endotoxin neutralizing reagent polymyxin B and TLR4 antagonist RS-LPS, we demonstrate that Leptospira LPS and canine TLR4 are involved in IL-1β production as well as in the uptake of inactivated Leptospira in canine moDC. Furthermore, polymyxin B only partially inhibited IL-1β production induced by inactivated Leptospira, suggesting that next to TLR4, also other TLRs may be involved. The observed activation of canine TLR2-expressing reporter cells by inactivated Leptospira strains indicates that TLR2 could be one of these TLRs. Next, we analyzed TLR2 and TLR4 activating capabilities by the same Leptospira strains using human and mouse TLR-expressing reporter cells. Inactivated Leptospira and leptospiral LPS activated not only mouse, but also human TLR4 and this activation was shown to be LPS dependent in both cases. Additionally, inactivated Leptospira activated mouse and human TLR2-expressing reporter cell lines. In our study, we could not identify significant species differences in the recognition of Leptospira by TLR2 and TLR4 between dog, human and mouse. Lastly, we show that these inactivated Leptospira strains are recognized by both mouse and human TLR5 reporter cells only after exposure to additional heat-treatment. Unfortunately, we were not able to confirm this in the canine system. Our data show that TLR2 and TLR4 are involved in the recognition of Leptospira strains used in the production of canine Leptospira vaccines. This study contributes to the understanding of Leptospira-induced innate immune responses in dogs, humans, and mice. Future studies are needed to further explore the role of canine TLR2, TLR4 and TLR5 in the induction of vaccine-mediated immunity against Leptospira.

Host and Species-Specificities of Pattern Recognition Receptors Upon Infection With Leptospira interrogans

“…Interestingly, it was shown that heat treatment allowed triggering of TLR5 signaling by degraded leptospires (Holzapfel et al, 2020) (Figure 5). Such findings were very recently confirmed by another group (Novak et al, 2022). Leptospiral flagellin monomers, supposedly TLR5 agonists, were unexpectedly released only after boiling for 30 min and have been shown to be unusually thermoresistant in comparison to Salmonella FliC monomers (Holzapfel et al, 2020).…”

“…Finally, unlike E. coli LPS, the leptospiral LPS is lacking a 4′-phosphate group and the 1phosphate group is methylated (Figure 2) and therefore does not present the usual negative charges. The peculiarities of the L. interrogans lipid A are conserved in several pathogenic serovars (Lai, Icterohaemorrhagiae, and Manilae) (Que-Gewirth et al, 2004;Eshghi et al, 2015;Novak et al, 2022).…”

“…Leptospires are potent agonists of human, mouse, and canine TLR2 (Werts et al, 2001;Nahori et al, 2005;Hsu et al, 2010;Novak et al, 2022). Furthermore, in mice, TLR2 activation is dependent on TLR2/1 heterodimer formation (Nahori et al, 2005), leading to the recognition of tri-acylated lipoproteins.…”

“…However, it remains to be confirmed since this study was recently brought into question (Li et al, 2021). In addition, other studies reported IL-1b release upon stimulation of canine whole blood and monocyte-derived dendritic cells (moDCs) in response to Leptospira (Rajeev et al, 2020;Novak et al, 2022), suggesting that functional inflammasomes could also be triggered in dogs, although the mechanism remains uncharacterized. Taken together, these data suggest that leptospires could trigger NLRP3 inflammasome and induce IL-1b secretion in different hosts.…”

Alive Pathogenic and Saprophytic Leptospires Enter and Exit Human and Mouse Macrophages With No Intracellular Replication

“…In addition to the numerous leptospiral species described ( Guglielmini et al., 2019 ; Vincent et al., 2019 ), hundreds of different serovars of leptospires have been characterized according to their different O antigens ( Adler, 2015 ; Caimi and Ruybal, 2020 ; Bertasio et al., 2020 ). Nevertheless, the lipid A part of LPS is conserved among L. interrogans ( Que-Gewirth et al., 2004 ; Eshghi et al., 2015 ; Novak et al., 2022 ). Leptospiral LPS possesses peculiar structural features (a methylated phosphate group and four amide bonds in the acyl chains) ( Que-Gewirth et al., 2004 ), which most probably account for its escape from recognition by human TLR4 ( Werts et al., 2001 ).…”