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Delivery of Biologically Active Anti-Inflammatory Cytokines IL-10 and IL-1ra In Vivo by the Shigella Type III Secretion Apparatus¹

Mustapha Chamekh^2,*, Armelle Phalipon, Renaud Quertainmont^*, Isabelle Salmon, Philippe Sansonetti and Abdelmounaaïm Allaoui^*

^* Laboratoire de Bactériologie Moléculaire and Laboratoire de Pathologie, Faculté de Médecine, Université Libre de Bruxelles, Brussels, Belgium; and Unité de Pathogénie Microbienne Moléculaire, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 786, Institut Pasteur, Paris, France

Pathogenicity of many Gram-negative bacteria relies on a type III secretion (T3S) apparatus, which is used for delivery of bacterial effectors into the host cell cytoplasm allowing the bacteria to manipulate host cell cytoskeleton network as well as to interfere with intracellular signaling pathways. In this study, we investigated the potential of the Shigella flexneri T3SA as an in vivo delivery system for biologically active molecules such as cytokines. The anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist (IL-1ra) were genetically fused to the first 30 or 60 residues of the Shigella T3S effector IpaH9.8 or to the first 50 residues of the Yersinia enterocolitica effector YopE and the recombinant fusion proteins were expressed in S. flexneri. YopE₅₀-IL-10, IpaH₆₀-IL-10, and IpaH₆₀-IL-1ra were efficiently secreted via the T3S apparatus of Shigella. Moreover, these recombinant proteins did not impair the invasive ability of the bacteria in vitro. In a murine model, Shigella strains expressing YopE₅₀-IL-10, IpaH₆₀-IL-10, and IpaH₆₀-IL-1ra induced a lower mortality in mice that was associated with reduced inflammation and a restricted localization of bacteria within the lung tissues as compared with wild-type Shigella. Moreover, the level of TNF- and IL-1β mRNA were reduced in the lungs following infection by IL-10- and IL-1ra-secreting Shigella, respectively. These findings demonstrate that the Shigella T3S apparatus can deliver biologically active cytokines in vivo, thus opening new avenues for the use of attenuated bacteria to deliver proteins for immunomodulation or gene therapy purposes.

The costs of publication of this article were defrayed in part by the payment of page 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 grant from the European Union (QLK2-C1999-00938), the Actions de Recherche concertées (convention 98/03-224) de la Communauté Française de Belgique, and by Fonds national de la Recherche Scientifique Médicale, convention 3.4623.06. P.S. is a Howard Hughes Medical Institute Scholar.

² Address correspondence and reprint requests to Dr. Mustapha Chamekh, Institute for Medical Immunology, Université Libre de Bruxelles, Gosselies, Belgium. E-mail address: mchamekh{at}ulb.ac.be

³ Abbreviations used in this paper: PMN, polymorphonuclear leukocyte(s); T3S, type III secretion; IL-1ra, IL-1 receptor antagonist; MOI, multiplicity of infection; i.n., intranasal(ly); Ipa, invasion plasmid Ag.