TLR4- and TLR2-Mediated B Cell Responses Control the Clearance of the Bacterial Pathogen, Leptospira interrogans This information is current as Cécilia Chassin, Mathieu Picardeau, Jean-Michel Goujon, of September 25, 2021. Pascale Bourhy, Nathalie Quellard, Sylvie Darche, Edgar Badell, Martine Fanton d'Andon, Nathalie Winter, Sonia Lacroix-Lamandé, Dominique Buzoni-Gatel, Alain Vandewalle and Catherine Werts J Immunol 2009; 183:2669-2677; Prepublished online 27 Downloaded from July 2009; doi: 10.4049/jimmunol.0900506 http://www.jimmunol.org/content/183/4/2669 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2009/07/27/jimmunol.090050 Material 6.DC1 References This article cites 31 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/183/4/2669.full#ref-list-1 Why The JI? Submit online. by guest on September 25, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology TLR4- and TLR2-Mediated B Cell Responses Control the Clearance of the Bacterial Pathogen, Leptospira interrogans1 Ce´cilia Chassin,* Mathieu Picardeau,† Jean-Michel Goujon,‡ Pascale Bourhy,† Nathalie Quellard,‡ Sylvie Darche,§ Edgar Badell,¶ Martine Fanton d’Andon,#** Nathalie Winter,¶ Sonia Lacroix-Lamande´,ʈ#** Dominique Buzoni-Gatel,ʈ Alain Vandewalle,* and Catherine Werts2#** Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira interrogans that are transmitted by asymptomatic infected rodents. Leptospiral lipoproteins and LPS have been shown to stimulate murine cells via TLRs 2 and 4. Host defense mechanisms remain obscure, although TLR4 has been shown to be involved in clearing Leptospira. In this study, we show that double (TLR2 and TLR4) knockout (DKO) mice rapidly died from severe hepatic and renal failure following Leptospira inoculation. Strikingly, Downloaded from the severe proinflammatory response detected in the liver and kidney from Leptospira-infected DKO mice appears to be inde- pendent of MyD88, the main adaptor of TLRs. Infection of chimeric mice constructed with wild-type and DKO mice, and infection of several lines of transgenic mice devoid of T and/or B lymphocytes, identified B cells as the crucial lymphocyte subset responsible for the clearance of Leptospira, through the early production of specific TLR4-dependent anti-Leptospira IgMs elicited against the leptospiral LPS. We also found a protective tissue compartmentalized TLR2/TLR4-mediated production of IFN-␥ by B and T lymphocytes, in the liver and kidney, respectively. In contrast, the tissue inflammation observed in Leptospira-infected DKO mice http://www.jimmunol.org/ was further characterized to be mostly due to B lymphocytes in the liver and T cells in the kidney. Altogether these findings demonstrate that TLR2 and TLR4 play a key role in the early control of leptospirosis, but do not directly trigger the inflammation induced by pathogenic Leptospira. The Journal of Immunology, 2009, 183: 2669–2677. eptospirosis is a worldwide zoonosis caused by the highly infectious disease (1). Vaccines with attenuated strains are avail- invasive Leptospira species belonging to the spirochete able in some countries, but the humoral protection is strain specific L phylum. Rodents infected by Leptospira are asymptom- and not long-lasting. Therefore, identification of mechanisms that atic carriers, and shed bacteria in their urine into water or soil, thus control the inflammatory responses triggered by pathogenic Lep- infecting human beings via skin contact. Bacteria disseminate tospira should provide a molecular basis for a better understanding by guest on September 25, 2021 through the systemic circulation to colonize the target organs: of the pathophysiology of leptospirosis. liver, lungs, and kidneys. The clinical symptoms may vary widely TLRs are pattern recognition receptors that recognize conserved from almost asymptomatic or mild manifestations, to very severe microbial components, such as LPS, lipopeptides, or hypomethy- forms with multiorgan failure (Weil’s syndrome). Antibiotic treat- lated bacterial DNA, and trigger an innate response to clear mi- ment is effective in most cases during the early stages of the in- crobes from the infected host. We have shown that leptospiral LPS fection, but untreated infections may lead to tissue damage, such as signals by TLR2 in human cells, but is recognized by both TLR4 tubulo-interstitial nephritis. Occupational or recreational outdoor and TLR2 in murine cells, due to a differential recognition of the activities resulting in close contact with animals or flood water atypical lipid A moiety of the leptospiral LPS, which is recognized increase the risk of leptospirosis in humans. Endemic and epidemic by TLR4 in murine, but not in human cells (2–4). Leptospiral outbreaks of leptospirosis are currently causing significant mor- lipoproteins, in contrast, are recognized by TLR2 in murine kidney bidity and mortality, suggesting that leptospirosis is a re-emerging epithelial cells (5). Young C3H/HeJ mice, carrying a mutation in- activating the trl4 gene, are susceptible to leptospirosis (6), and a recent study has shown that TLR4 is indeed involved in the clear- *INSERM, U773, Centre de Recherche Biome´dicale Bichat-Beaujon (CRB3), BP ance of Leptospira (7). However, the mechanisms involved in the 416, Paris France; Universite´Paris 7-Denis Diderot, Site Bichat, Paris, Paris, France; increased susceptibility to Leptospira associated with TLR4 defi- ‡Centre Hospitalier Universitaire de Poitiers, Unite´de Pathologie Ultrastructurale et ʈ ciency have not been explored. Expe´rimentale, Universite´de Poitiers, Poitiers, France; Institut National de Recher- che Agronomique, U1282, Infectiologie Animale et Sante´Publique, Nouzilly, France; Based on the studies described above, we hypothesized that double Institut Pasteur, †Unite´Postulante Biologie des Spiroche`tes, §Unite´deRe´ponses Pre´- (TLR2 and TLR4) knockout (DKO)3 mice should be more suscepti- coces aux Parasites et Immunopathologie, ¶Unite´Ge´ne´tique Mycobacte´rienne, #G5 Biologie et Ge´ne´tique de la Paroi Bacte´rienne; and **INSERM, Groupe Avenir, Paris, ble to Leptospira infection than their resistant C57BL/6J wild-type France (WT) counterparts. In this study, we investigated the consequence of Received for publication February 13, 2009. Accepted for publication June 17, 2009. leptospiral infection in adult TLR2- and/or TLR4-deficient mice and The costs of publication of this article were defrayed in part by the payment of page WT mice, taking into account several parameters including lethality, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Institut Pasteur and INSERM research grants. C.C. was 3 Abbreviations used in this paper used: DKO, double TLR2 and TLR4 knockout; sponsored by the French Ministe`redelaDe´fense (PhD grant DGA/MRIS). WT, wild type; BUN, blood urea nitrogen; ASAT, aspartate aminotransferase activity; 2 Address correspondence and reprint requests to Dr. C. Werts, Institut Pasteur, G5 iNOS, inducible NO synthase. Biologie et Ge´ne´tique de la Paroi Bacte´rienne, INSERM Groupe Avenir, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900506 2670 TLR4-DEPENDENT B CELL RESPONSES PROTECT AGAINST LEPTOSPIROSIS bacterial load, and the expression of proinflammatory mediators in the sue sections were stained with H&E to evaluate inflammatory changes target organs (liver, kidneys and lungs). We demonstrate that DKO by light microscopy. Immunohistochemical studies, using an Ab raised mice are highly susceptible to pathogenic Leptospira, and die rapidly against outer membrane lipoprotein LipL32 (1/4000), were performed us- ing avidin-biotin coupled to peroxidase substrate kits (Vector Laboratories) from severe renal and hepatic failure, thereby constituting a model of according to the manufacturer’s instructions. Peroxidase activity was re- human leptospirosis in its acute form. Using chimeric mice and trans- vealed with diaminobenzidine (brown reaction) (Dako REAL Detection genic mice, B cells were identified as the crucial lymphocyte cell System). subset involved in the clearance of Leptospira, through TLR4-depen- Transmission electron microscopy dent mechanisms. In contrast, the severe inflammation induced by Leptospira in the organs of susceptible mice is not dependent on TLR Kidney and liver samples were fixed for 30 min in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, embedded in Epon, and processed for transmis- signaling. This implies that TLR4 and TLR2