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In Situ IL-12/23p40 Production during Mycobacterial Infection Is Sustained by CD11b^high Dendritic Cells Localized in Tissue Sites Distinct from Those Harboring Bacilli¹

Antonio Gigliotti Rothfuchs^2,*, Jackson G. Egen, Carl G. Feng^*, Lis R. V. Antonelli^*, Andre Bafica^3,*, Nathalie Winter, Richard M. Locksley and Alan Sher^*

^* Immunobiology Section, Laboratory of Parasitic Diseases and Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; Institut Pasteur, Mycobacterial Genetics Unit, Paris, Cedex, France; and Howard Hughes Medical Institute and Department of Medicine and Department of Microbiology and Immunology, University of California, San Francisco, CA 94143

Although IL-12/23p40 is known to play a major role in host resistance to Mycobacterium spp, the cellular source, tissue localization, and regulation of p40 production during mycobacterial infection in vivo has been unclear. In this study, we used IL-12/23p40eYFP (yet40) reporter mice to track expression of the cytokine following Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. We found that in spleens of these mice, p40 production is initiated by a transient burst from CD11b^lowCD11c⁺ dendritic cells (DC) which are later replaced at the onset of granuloma formation by CD11b^highCD11c⁺ DC as the major source of the cytokine. The latter subset was also found to be the key producer of DC-derived p40 in nonlymphoid tissue and in both spleen and liver optimal production of the cytokine was regulated by endogenous TNF-. Although BCG and p40-expressing DC were both observed in splenic white pulp, p40⁺ DC rarely colocalized with bacilli. Indeed, in vitro flow cytometry and confocal microscopy indicated that the presence of intracellular bacteria is not required for p40 production by DC and Transwell experiments confirmed that soluble mycobacterial components are sufficient for inducing cytokine expression by these cells. Moreover, when stimulated with LPS, DC directly infected with BCG showed impaired IL-12p40 production in vitro. Together, our findings establish CD11b^high DC as a major source of IL-12/23p40 during mycobacterial infection in situ and implicate both soluble mycobacterial products and TNF- in stimulating sustained production of p40 by these cells.

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 the Intramural Research Program of the National Institute of Allergy and Infectious Diseases and Grant AI30663 from the National Institute of Allergy and Infectious Diseases (to R.M.L.).

² Address correspondence and reprint requests to Dr. Antonio Gigliotti Rothfuchs, Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Room 6148, Building 50, 50 South Drive, Bethesda, MD 20892-8003. E-mail address: rothfuchsa{at}niaid.nih.gov

³ Current address: Department of Microbiology and Parasitology, Federal University of Santa Catarina, Florianopolis 88.040900, Brazil.

⁴ Abbreviations used in this paper: MTB, Mycobacterium tuberculosis; DC, dendritic cell; eYFP, enhanced yellow fluorescent protein; LysM, lysozyme M; BCG, bacillus Calmette-Guérin; RFP, red fluorescent protein; STAg, soluble tachyzoite from Toxoplasma gondii; SP, splenic; SpDC, splenic DC; MHC, MHC class II; BMDC, bone marrow-derived DC; MOI, multiplicity of infection.