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

Effect of antibiotic treatments at the chronic phase of leptospirosis.

<p>C57BL/6J mice chronically infected for 25 days (D25) with 10⁷ MFlum1 were (A) IP injected daily for 7 days with penicillin G (Pen) (brown) or Ciprofloxacin (Cipr) (blue) from 25 until 31 dpi (T1 to T7), (B) injected daily for 7 days with azithromycin (Azi) from 25 until 31 dpi, and from 112 until 118 dpi (T1 to T7). The arrows indicate the duration of the different treatments. All the bioluminescence analyses were done after IP administration of D-luciferin. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² in n = 4 infected treated or untreated mice. <i>p</i> values (+<i>p</i><0.05, ++<i>p</i><0.01, +++<i>p</i><0.001) are indicated in corresponding colors for each group <i>versus</i> the uninfected group. Below are shown images of the tracking of one untreated, infected (MFlum1) mouse and an infected treated (Azi) mouse photographed at different crucial time points. The corresponding <i>p</i> values (*<i>p</i><0.05) were calculated between infected untreated group and treated group (Azi). Images depict photographs overlaid with color representations of luminescence intensity, measured in photons/second/cm² and indicated on the scales, where red is most intense and purple is least intense. PT, days post treatment.</p

Kinetics of dissemination of bioluminescent MFlum1 in mice.

<p>All the bioluminescence analyses were performed after IP administration or addition of D-luciferin. (A) Live imaging tracking of 10⁷ MFlum1 IP injected to albino C57BL/6J mice. Images below the graph show the tracking of one infected mouse, photographed at different crucial time points. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² in n = 4 infected mice, imaged in the dorsal view, except for 30 min post-infection for which only imaging in the ventral view allows the visualization of the leptospiral dissemination in the peritoneal cavity. <i>p</i> values (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001) between infected and uninfected groups. Images depict photographs overlaid with color representations of luminescence intensity, measured in photons/second/cm² as indicated on the scales, where red is the most intense and purple the least intense. (B) Kinetics of leptospiral quantification by q-PCR in blood and urine of albino C57BL/6J mice infected with 10⁷ MFlum1. Below are shown corresponding images of the <i>ex vivo</i> live imaging of MFLum1 in blood, in the presence or absence of ATP. (C) Monitoring expressed as the percentage of weight loss of mice infected or not with 10⁷ MFlum1. Panels B and C are representative of 2 independent experiments with a total of n = 8 mice for each group. <i>p</i> values (*<i>p</i><0.05, **<i>p</i><0.01) between infected and uninfected groups.</p

Primer list for cloning.

<p>Boldface indicates restriction sites.</p><p>Primer list for cloning.</p

Threshold of infective dose of MFlum1 to obtain renal colonization.

<p>(A) Comparison of the dissemination kinetics for MFlum1 injected to albino C57BL/6J at different doses. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² in n = 4 infected mice, imaged in the dorsal view, except for 30 min post-infection for which only imaging in the ventral view allows the visualization of the leptospiral dissemination in the peritoneal cavity. On the right side are shown images of one representative mouse at 30 min post infection as a control of infection and on the left side, images of one representative mouse at the end of the kinetics. <i>p</i> values (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001) are indicated in corresponding colors for each group <i>versus</i> the uninfected group. <i>In vivo</i> (B) and <i>ex vivo</i> (C) live imaging and quantification of albino (W) and black (BL) C57BL/6J mice one month post infection with (10⁷) or without (NI) 10⁷ MFlum1. Bioluminescence imaging in dorsal view was carried out after dorsal shaving of the black mice. Below are shown corresponding images of live mice gated on the kidneys (B) and <i>ex vivo</i> (C) tracking of half-kidneys after sacrifice and addition of D-luciferin. <i>p</i> value (*<i>p</i><0.05) between infected and uninfected groups n = 4 mice <i>per</i> group. (D) Correlation between renal imaging and q-PCR. Bioluminescence imaging at 25 dpi in dorsal view of live chronically infected C57BL/6J mice (from 10⁶ or 10⁷ experiments), gated on the kidneys. Subsequently, mice were euthanized and kidneys were harvested and further processed for determination of the leptospiral load by q-PCR. Each cross represents an individual mouse. (E) <i>Ex vivo</i> imaging in presence or absence of ATP of half-kidneys from 10⁷ MFlum1 infected mice one month post infection. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² for 6 half-kidneys for each group. Below are shown corresponding images of the tracking of half-kidney after sacrifice and addition of D-luciferin. On the left is shown the schematic representation of an infected kidney in longitudinal cross section. (F) <i>Ex vivo</i> live imaging (Left Y axis) and the corresponding number of leptospires measured by q-PCR (Right Y axis) of different organs from 10⁷ MFlum1 infected mice, one month post infection. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm². This panel represents 2 experiments with a total of n = 6 mice infected with MFlum1, and n = 5 naïve mice. Below are shown corresponding images of the live imaging of one representative organ or fluid and a mouse before the sacrifice. For imaging, urine was pooled from several mice, to obtain a minimum volume of 50 µL.</p

Azithromycin prophylactic treatment against leptospirosis.

<p>8 C57BL/6J mice were infected with 2×10⁸ MFlum1 and 4 of them were injected IP two days before infection (T-2) with azithromycin (Azi). As controls, 4 uninfected mice were treated with azithromycin. All bioluminescence analyses were performed after IP administration of D-luciferin. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² in n = 4 mice imaged in the dorsal view, except for 30 min post-infection (D0) for which only imaging in the ventral view allows the visualization of the leptospiral dissemination in the peritoneal cavity. <i>p</i> values (+<i>p</i><0.05, ++<i>p</i><0.01, +++<i>p</i><0.001) are indicated in corresponding colors for each group <i>versus</i> the uninfected treated group. The cross indicates that the mice died or were sacrificed because of acute leptospirosis. On the right side are shown images of the last tracking point (24 dpi) of one treated-infected mouse compared to an uninfected treated mouse.</p

Characterization of bioluminescent <i>Leptospira</i> transformants.

<p>(A) Growth curves of <i>L. interrogans</i> Manilae wild-type and MFlum1 strains in EMJH medium at 28°C (Left Y axis) and corresponding bioluminescence of MFlum1 (Right Y axis). (B) Comparison of bioluminescence according to known numbers of MFlum1, grown to mid-log phase or to old stationary phase (four months). Measures were done with the IVIS Spectrum machine after addition of D-luciferin, in the presence or absence of ATP. Panels A and B are representative of 6 and 2 experiments, respectively. (C) Live imaging tracking over time of 2×10⁸ MFlum1 or bioluminescent <i>L. biflexa</i> Patoc PFlum7 injected intra-peritoneally (IP) into albino C57BL/6J mice (Left Y axis) and corresponding weight losses (Right Y axis) of MFlum1 infected mice. All bioluminescence analyses were carried out after the IP administration of D-luciferin. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² in mice and imaged in the ventral view. This panel represents 3 experiments with a total of n = 12 mice infected with MFlum1, n = 4 mice infected with PFlum7, and n = 8 naïve mice. <i>p</i> values (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001) between infected and uninfected group. The cross indicates that the mice died or were sacrificed because of acute leptospirosis. Below are shown images of the tracking of one mouse photographed at different time post infection. (D) Live imaging tracking over time of 50 µl of blood collected from the 2×10⁸ MFlum1 infected mice at different time points from the experiment shown in panel 1C (Left Y axis) and corresponding number of leptospires measured by q-PCR (Right Y axis). Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² in 50 µL of blood. <i>p</i> values (*<i>p</i><0.05), between groups. This panel represents 2 experiments with a total of n = 6 mice. Below are shown the corresponding images at different dpi in the presence or the absence of ATP. Below D0 are shown images of the MFLum1 in PBS imaged just before IP injection.</p

List of bioluminescent leptospires transformants obtained in this study.

a<p>as annotated in MicroScope (<a href="http://www.genoscope.cns.fr/agc/microscope/home/index.php" target="_blank">http://www.genoscope.cns.fr/agc/microscope/home/index.php</a>).</p><p>italics: insertion in an intergenic region.</p><p>List of bioluminescent leptospires transformants obtained in this study.</p

Model of acute and chronic leptospirosis in mice.

<p>This figure depicts the course of leptospirosis in mice following an IP infection with a lethal dose of bioluminescent MFlum1, leading to a septicemia or with a sub-lethal dose leading to a chronic leptospirosis, and the effects of different antibiotics administered at the acute (upper part of the figure) or chronic phase (lower part of the figure) of the leptospiral infection. Mice depicted without kidneys represent mice at the acute phase of infection. Mice depicted with kidneys represent mice at the chronic phase. Inside the kidneys schemed in longitudinal cross-section, the niches colonized by leptospires, presumably the proximal part of renal tubules (proximal tubules) are depicted by small circles. A grey color scale indicates the degree of leptospiral infection, where white means free of leptospires and black means a maximum of infection or colonization. The cross indicates that the mice died or were sacrificed because of acute leptospirosis. D1 to D15: days post-infection. T1-T7 duration of antibiotic treatments. PT15: 15 days post treatment. T-2 prophylaxis treatment 2 days prior to infection.</p

Survival test of C57BL/6J mice to the infection with <i>L. interrogans</i> Manilae and its mutant MFlum1.

<p>Survival test of C57BL/6J mice to the infection with <i>L. interrogans</i> Manilae and its mutant MFlum1.</p