Relative normalized gene expression levels and <i>Leptospira</i> burdens of the hamsters at day 3 post-infections and p-values according to outcome in the LD50 experiments.

<p>Relative normalized gene expression levels and <i>Leptospira</i> burdens of the hamsters at day 3 post-infections and p-values according to outcome in the LD50 experiments.</p

IFNγ, TGFβ and IL-6 gene expression levels at day 3 post <i>Leptospira</i> infection (relative normalized expression, see text).

<p>Error bars indicate 95% confidence intervals. The insert shows the high variability observed in IL-6 gene expression levels.</p

Mean expression levels of differentially expressed genes (relative normalized expression, see text).

<p>Expression levels are shown at day 3 post-infection in the LD50 <i>Leptospira</i> infection (according to the outcome) and after the injection of a high dose of a virulent or the high-passage variant (A) and <i>Leptospira</i> burdens (B) in infected hamsters. Error bars indicate 95% confidence intervals.</p

Primers used in this study, amplicon size and elongation time.

<p>Primers used in this study, amplicon size and elongation time.</p

Preliminary experiments results leading to the selection of day 3 post-infection (as shown by the red lines) as an appropriate study time point.

<p>A. Time course of mortalities in the LD50 infection model (n = 70, green line) and in the animals sampled for the gene expression studies (n = 36, blue line). B. Relative normalized gene expression levels (see text) time courses (y-axes use different scales for different gene targets).</p

Innate immune memory through TLR2 and NOD2 contributes to the control of <i>Leptospira interrogans</i> infection

<div><p><i>Leptospira interrogans</i> are pathogenic spirochetes responsible for leptospirosis, a worldwide reemerging zoonosis. Many <i>Leptospira</i> serovars have been described, and prophylaxis using inactivated bacteria provides only short-term serovar-specific protection. Therefore, alternative approaches to limit severe leptospirosis in humans and morbidity in cattle would be welcome. Innate immune cells, including macrophages, play a key role in fighting infection and pathogen clearance. Recently, it has been shown that functional reprograming of innate immune cells through the activation of pattern recognition receptors leads to enhanced nonspecific antimicrobial responses upon a subsequent microbial encounter. This mechanism is known as trained immunity or innate immune memory. We have previously shown that oral treatment with <i>Lactobacillus plantarum</i> confers a beneficial effect against acute leptospirosis. Here, using a macrophage depletion protocol and live imaging in mice, we established the role of peritoneal macrophages in limiting the initial dissemination of leptospires. We further showed that intraperitoneal priming of mice with CL429, a TLR2 and NOD2 agonist known to mimic the modulatory effect of <i>Lactobacillus</i>, alleviated acute leptospiral infection. The CL429 treatment was characterized as a training effect since i.) it was linked to peritoneal macrophages that produced <i>ex vivo</i> more pro-inflammatory cytokines and chemokines against 3 different pathogenic serovars of <i>Leptospira</i>, independently of the presence of B and T cells, ii.) it had systemic effects on splenic cells and bone marrow derived macrophages, and iii.) it was sustained for 3 months. Importantly, trained macrophages produced more nitric oxide, a potent antimicrobial compound, which has not been previously linked to trained immunity. Accordingly, trained macrophages better restrict leptospiral survival. Finally, we could use CL429 to train <i>ex vivo</i> human monocytes that produced more cytokines upon leptospiral stimulation. In conclusion, host-directed treatment using a TLR2/NOD2 agonist could be envisioned as a novel prophylactic strategy against acute leptospirosis.</p></div

Expression of LipL21 impairs <i>E</i>. <i>coli</i> peptidoglycan digestion into muropeptides and recognition by NOD receptors.

<p>(A) Color phenotypes of strains expressing alkaline phosphatase (phoA) derivatives and grown as colonies on agar with chloramphenicol and 5-bromo-4-chloro-3-indoyl-phosphate (XP). FL-<i>phoA</i>, Δ(2–22)phoA, FL-<i>lipl21-phoA</i> and ΔN-<i>lipl21-phoA</i> correspond respectively to <i>E</i>. <i>coli</i> (BTH₁₀₁) expressing the full length phoA (positive control), <i>E</i>. <i>coli</i> expressing phoA without signal peptide (negative control), <i>E</i>. <i>coli</i> expressing the full length LipL21 fused with Δ(2–22)<i>phoA</i> and <i>E</i>. <i>coli</i> expressing the LipL21 without signal peptide fused with Δ(2–22)<i>phoA</i>. (B) Growth curves of BL-21 Rosetta-2 <i>E</i>. <i>coli</i> expressing the LipL21 lipoprotein (pLipL21) or not (pEmpty) in the pRSFDuet-1 vector, without IPTG induction. (C) Crude bacterial extracts of BL-21 Rosetta-2 <i>E</i>. <i>coli</i> with empty pASK-IBA6 vector (pEmpty) or LipL21 expressing vector (pLipL21), induced by anhydrous-Tetracyclin or not (NI) and migrated on 12% acrylamide gel. Stain free coloration (left) and immunodetection (right) using a LipL21 antiserum. The Rainbow marker ladder gives an indication for the LipL21size.(D) HPLC separation profiles of muropeptides after digestion with mutanolysin. Each peptidoglycan was extracted with both the 0.5 h and 4 h protocols from BL-21 Rosetta-2 <i>E</i>. <i>coli</i> expressing the LipL21 lipoprotein (pLipL21) or not (pEmpty), after induction with anhydrous-Tetracyclin. (E) and (F) <i>E</i>. <i>coli</i> PGs, extracted from the 0.5 h protocol, were used to stimulate HEK 293T cells expressing human NOD1 (E) or NOD2 (F). HEK 293T cells were co-transfected with 6 μg of PG or 100 nM of muropeptides controls (MTP for NOD1 and MDP for NOD2, along with the reporter constructions and NOD1 or NOD2 expression plasmids. Luciferase activity was measured 24 h after transfection. Cells left untreated with muropeptides were used as negative control (water). Data are expressed as the mean of triplicates ± SEM of relative light units representing luciferase activity normalized with respect to β-galactosidase activity. The graph shown is representative of 3 equivalent experiments. The unpaired <i>t</i> test was used to compare the recognition of PGs prepared from <i>E</i>. <i>coli</i> with the empty vector (pEmpty) to the PG prepared from <i>E</i>. <i>coli</i> expressing LipL21 (pLipL21). A <i>p</i> value < 0.05 was considered significant. <i>p</i> values: *** <i>p</i> < 0.001. For clarity, statistics comparing each PG or muropeptide control to the water treated cells have not been indicated but are all significant with at least p<0,05.</p

Missense non synonymous SNP mutations found by DNA sequencing of genome of M58 compared to the parental strain L495.

<p>Missense non synonymous SNP mutations found by DNA sequencing of genome of M58 compared to the parental strain L495.</p

NOD1 and NOD2 receptors do not affect <i>L</i>. <i>interrogans</i> dissemination.

<p>(A) Survival curves (left panel) and weight variation (right panel) of C57BL6/J (WT) and NOD1 and NOD2 deficient (NOD1/2DKO) mice after intraperitoneal infection with 4 different doses (from 5.10⁸ to 4.10⁶ leptospires/mouse) of MFLum1, a bioluminescent strain of <i>L</i>. <i>interrogans</i> serovar Manilae L495 (weekly passage 14). Survival curves and weight loss were established within the 5 days following the infection, corresponding to the acute phase of the disease. Percentage of weight loss is represented as the mean ± SEM of individual mice compared to their initial weights before infection at day 0 (D0). For each dose, n = 5 WT and n = 5 NOD1/2 DKO mice. (B) Bacterial loads in WT and NOD1/2DKO mice IP infected with a sub-lethal dose of 10⁷ bioluminescent <i>L</i>. <i>interrogans</i> (MFLum1). Bacterial loads were measured by qPCR in blood at 8, 24, 48 and 72 hours post infection, and in urine at 5 and 7 days post infection, and by live imaging (IVIS), 1 month post infection. The graphs represent a compilation of 3 independent experiments with n = 3 to 6 mice for the blood panel, 1 experiment for the urine panel and 1 experiment representative of 3 with n = 4 mice for the IVIS panel. (A),(B). Statistics are not indicated as no significant differences were found at any time points between WT and NOD1/2DKO mice.</p

M58 is not virulent and the complementation does not restore virulence.

<p>(A)(B) M58 is not virulent in mice and the loss of virulence is not due to PG sensing. (A) C57BL6/J (WT) mice and mice deficient for both NOD1 and NOD2 receptors (NOD1/2DKO) and (B) FVB (WT) mice and mice deficient for lysozyme expressed in macrophages (LysMKO) were IP infected with 2.10⁷ M58 <i>lipl21</i>- mutant. Bacterial loads were measured by qPCR in blood at 8, 24 and 72 hours post infection. These experiments are representative of 2 equivalent experiments with n = 4 to 7 mice in each group. Statistics are not indicated as no significant differences were found at any time points between WT and NOD1/2DKO mice (A), or WT and LysMKO mice (B). (C) <i>L</i>. <i>interrogans</i> are not susceptible to lysozyme <i>in vitro</i>; leptospires were incubated at 28°C in presence of lysozyme and EDTA for the indicated times, then washed and further cultured for 5 days in EMJH medium. The Alamar blue dye was added and further incubated for 2 days. A pink color means that the bacteria are viable whereas the blue color indicates dead bacteria, reflecting the killing by lysozyme in presence of EDTA. Each plate included a positive control (bacteria with EDTA (+) and without lysozyme (-)), and a negative control (medium only). (D) The LipL21 complementation does not restore the virulence of M58 (<i>lipl21</i>-) in mice (left panel) nor in gerbils (right panel). C57BL6/J (WT) mice were IP infected with a sub-lethal dose of 10⁷ L495 strain or with 2.10⁷ of M58, the <i>lipl21</i>- mutant or C5M58, the complemented strain of M58 expressing LipL21 (<i>lipl21-/+</i>). Bacterial loads were measured by qPCR in blood at 8, 24, 48 and 72 hours post infection. This experiment is representative of 2 equivalent experiments with n = 4 to 7 mice in each group. Statistics are not indicated as no significant differences were found at any time points between M58 and C5M58 complemented strain. Survival curves of gerbils IP infected with 10⁶ bacteria/ ml in EMJH. n = 4 gerbils /group.</p