Innate immune memory through TLR2 and NOD2 contributes to the control of Leptospira interrogans infection

Leptospira interrogans are pathogenic spirochetes responsible for leptospirosis, a worldwide reemerging zoonosis. Many Leptospira 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 Lactobacillus plantarum 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 Lactobacillus, alleviated acute leptospiral infection. The CL429 treatment was characterized as a training effect since i.) it was linked to peritoneal macrophages that produced ex vivo more pro-inflammatory cytokines and chemokines against 3 different pathogenic serovars of Leptospira, 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 ex vivo 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.
Author summary Leptospirosis is a worldwide zoonotic neglected disease caused by pathogenic Leptospira. In addition to cattle and pets, it affects one million people per year and causes 60000 deaths. Antibiotics are efficient when given early after infection, but because the symptoms are not specific, leptospirosis is difficult to diagnose in a timely fashion. There are ~300 serovars of pathogenic Leptospira, which complicates the development of a universal vaccine against leptospirosis. Therefore, prophylactic treatments would be welcome. Recently, a new kind of immune memory known as “innate immune memory” or “trained immunity” has been described. It is not linked to adaptive immunity but only to innate immune cells, such as macrophages. Initial stimulation by a bacterial or fungal component can “train” macrophages to respond better but nonspecifically to a second encounter with a microbe. In this work, we show that trained immunity can be induced in mice to alleviate the symptoms of experimental acute leptospirosis. Cells from treated mice stimulated ex vivo by different serovars of pathogenic leptospires secreted more antimicrobial compounds and showed enhanced leptospire killing. Moreover, this treatment was able to boost human monocytes. This work presents a host-directed therapeutic approach that could be used as a novel prophylactic strategy against leptospirosis.