Poly(I:C)-Induced Protection of Neonatal Mice Against Intestinal Cryptosporidium parvum Infection Requires an Additional TLR5 Signal Provided by the Gut Flora

MAJOR ARTICLE Poly(I:C)-Induced Protection of Neonatal Mice Against Intestinal Cryptosporidium parvum Infection Requires an Additional TLR5 Signal Provided by the Gut Flora Louis Lantier,1,2 Françoise Drouet,1,2 William Guesdon,1,2 Roselyne Mancassola,1,2 Coralie Metton,1,2 Richard Lo-Man,3 Catherine Werts,4 Fabrice Laurent,1,2,a and Sonia Lacroix-Lamandé1,2,a 1 INRA Val de Loire, UMR1282 Infectiologie et Santé Publique, Nouzilly; 2Université François Rabelais, UMR1282 Infectiologie et Santé Publique, Tours; Institut Pasteur, Unité de Régulation Immunitaire et Vaccinologie, Paris; and 4Institut Pasteur, Unité de Biologie et Génétique de la Paroi Bactérienne, Paris, France 3 Keywords. poly(I:C); TLR3; neonatal mice; Cryptosporidium parvum; intestine; microbiota; dendritic cells. At the time of birth, the immune system of neonates is still undergoing development. The neonatal immune response has been shown to be quantitatively and qualitatively distinct from that of adults, rendering neonates more susceptible to infections [1, 2]. Lacking a fully developed adaptive immune system, newborns must rely on innate immune responses and maternally transmitted immunity for early protection against pathogens [3]. The development of effective strategies to protect against neonatal diseases is challenging. After delivery, the intestine of neonates is rapidly colonized with a vast diversity of microbes. Bacterial Received 25 February 2013; accepted 17 July 2013; electronically published 6 September 2013. a F. L. and S. L.-L. contributed equally to this study. Correspondence: Sonia Lacroix-Lamandé, PhD, Laboratoire Contrôle et Immunologie des Maladies Entériques du Nouveau-né, UMR1282 Infectiologie et Santé Publique, INRA Val de Loire, 37380 Nouzilly, France (). The Journal of Infectious Diseases 2014;209:457–67 © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: . DOI: 10.1093/infdis/jit432 recognition of the intestinal microbial flora by pathogen recognition receptor has been shown to contribute to developmental programming of epithelial barrier function and gut homeostasis, as well as the innate and adaptive host immune function [4]. In the gut mucosa, commensal-related bacteria are constantly in contact with epithelial cells and mononuclear phagocytes that extend dendrites across the epithelial barrier [5]. Moreover, the loss of gut epithelium integrity when mice are infected or after chemical exposure favors interaction between bacteria and lamina propria resident dendritic cells (DCs). Cryptosporidiosis is a zoonotic disease caused by the protozoan Cryptosporidium parvum. This intracellular parasite infects intestinal epithelial cells and causes debilitating diarrhea [6]. The severity of the disease is related to the immune status of its host, with young and immunocompromised individuals being the primary targets of this infection. In the neonatal mouse model of infection, cytokines such as interleukin 12 (IL-12) and interferon (IFN) γ have been shown to be involved in controlling the infection within 2–3 Poly(I:C) for Neonatal Gut Defense • JID 2014:209 (1 February) • 457 The neonatal intestinal immune system is still undergoing development at birth, leading to a higher susceptibility to mucosal infections. In this study, we investigated the effect of poly(I:C) on controlling enteric infection by the protozoan Cryptosporidium parvum in neonatal mice. After poly(I:C) administration, a rapid reduction in parasite burden was observed and proved to be dependent on CD11c+ cells and TLR3/TRIF signaling. Protection against C. parvum required additional signals provided by the gut flora through TLR5 and MyD88 signaling. This cooperation gave rise to higher levels of expression of critical mutually dependent cytokines such as interleukin 12p40 and type 1 and type 2 interferons, the last 2 being known to play a key role in the elimination of infected enterocytes. Our findings demonstrate in neonatal mice how gut flora synergizes with poly(I:C) to elicit protective intestinal immunity against an intracellular pathogen. METHODS Mouse Models IFN-γ−/−, IL-12p40−/−, Toll-like receptor (TLR) 2/4−/−, Nod1/ 2−/−, IFN-αR−/−, MyD88−/−, TLR5−/−, Lps2−/− (TRIF), TLR9−/−, and CD11c-DTR mice, all with a C57BL/6 genetic background, were raised and maintained in animal facilities of the Plateforme d’Infectiologie Expérimentale (INRA-Tours) or Institut Pasteur in accordance with European guidelines. Parasite Preparation and Mouse Infections Oocysts of the C. parvum CpINRA isolate were initially purified from the feces of an infected child and were maintained by regular passages in newborn calves. Purification of C. parvum oocysts was performed as previously described [7]. Threeday-old neonatal mice were infected orally with 5 × 105 C. parvum oocysts. The level of infection in individual neonatal mice was assessed by counting the number of oocysts in the intestinal content, as previously described [13]. Collection of Mesenteric Lymph Node Cells, Flow Cytometry, and Cell Sorting After dissociation, cells of mesenteric lymph nodes (MLNs) were first incubated with anti-CD16/CD32 antibody and then with the antibodies against cell surface molecules for 30 minutes. All antibodies were purchased from BD Pharmingen: APC-antiCD11, PE-anti-CD40, PE-anti-CD86, PE-anti-CD103, APCH7-anti-CD8α and FITC-anti-I-A/I-E antibodies. Cells were analyzed on a Becton-Dickinson fluorescence-activated cell sorter with FSC Express3 software. To isolate CD103+ CD8α+ CD11c+ and CD103+ CD8α− CD11c+ subsets from the MLNs, pools were constituted from 10–40 infected neonatal mice. Cells were first gated for CD11c+ MHCII+ and further analyzed for CD8α expression among the CD103+ population. Sorting was performed on a MoFlo highspeed cell sorter. Analysis of mRNA Levels by Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Array and Quantitative RT-PCR RNA was extracted from purified cells and tissue using Picopure RNA isolation kit and TRIZOL solution, respectively. Gene expression for inflammatory cytokines, chemokines, and their receptors was measured by real-time RT-PCR using commercially available reagents (RT2 profiler system), according to the manufacturer’s protocol. Raw data were acquired and processed with the Chromo4 software to calculate the threshold cycle (Ct) value and relative gene expression values, subsequently determined according to the standard ΔΔCt method. For quantitative RT-PCR, samples were normalized internally using the average cycle quantification (Cq) of the 3 most suitable reference genes—hypoxanthine phosphoribosyltransferase (HPRT), TATAA-box binding protein (TBP), and peptidylprolyl isomerase A (PPia)—selected using geNorm application among 5 commonly used genes. Primers used for mRNA quantification were described in Supplementary Table 1. Expression data are expressed as relative va (...truncated)