Innate Invariant NKT Cells Recognize Mycobacterium tuberculosis–Infected Macrophages, Produce Interferon-γ, and Kill Intracellular Bacteria

and Kill Intracellular Bacteria. PLoS Pathog 4(12): e1000239. doi:10.1371/journal.ppat.1000239 Innate Invariant NKT Cells Recognize Mycobacterium tuberculosis- Infected Macrophages, Produce Interferon-c, and Kill Intracellular Bacteria Isabel Sada-Ovalle 0 1 Asako Chiba 0 1 Adaena Gonzales 0 1 Michael B. Brenner 0 1 Samuel M. Behar 0 1 JoAnne L. Flynn, University of Pittsburgh School of Medicine, United States of America 0 Funding: This work was supported by National Institutes of Health grants R01 HL80312 and HL080330 to SMB 1 1 Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard Medical School , Boston , Massachusetts, United States of America, 2 Immunochemistry Department, National Institute of Respiratory Diseases , Mexico City , Mexico Cellular immunity to Mycobacterium tuberculosis (Mtb) requires a coordinated response between the innate and adaptive arms of the immune system, resulting in a type 1 cytokine response, which is associated with control of infection. The contribution of innate lymphocytes to immunity against Mtb remains controversial. We established an in vitro system to study this question. Interferon-c is produced when splenocytes from uninfected mice are cultured with Mtb-infected macrophages, and, under these conditions, bacterial replication is suppressed. This innate control of bacterial replication is dependent on CD1d-restricted invariant NKT (iNKT) cells, and their activation requires CD1d expression by infected macrophages as well as IL-12 and IL-18. We show that iNKT cells, even in limiting quantities, are sufficient to restrict Mtb replication. To determine whether iNKT cells contribute to host defense against tuberculosis in vivo, we adoptively transferred iNKT cells into mice. Primary splenic iNKT cells obtained from uninfected mice significantly reduce the bacterial burden in the lungs of mice infected with virulent Mtb by the aerosol route. Thus, iNKT cells have a direct bactericidal effect, even in the absence of synthetic ligands such as a-galactosylceramide. Our finding that iNKT cells protect mice against aerosol Mtb infection is the first evidence that CD1d-restricted NKT cells mediate protection against Mtb in vivo. - Cells of the innate immune system use several receptor systems to recognize pathogens and act as the first line of defense against infection. In contrast, the expression of clonal antigen receptors and the capacity to differentiate into memory cells distinguish B and T lymphocytes as the central components of the adaptive immune system. Certain T subsets, such as cd T cells and NKT cells, have features of innate immune cells including a partially activated phenotype, a rapid response following detection of infected cells, and the modulation of other cell types [1]. Together with NK cells, these cell subsets are functionally defined as innate lymphocytes. While innate lymphocytes serve important roles in host resistance to different infections, it remains controversial whether these cells contribute to immunity against Mycobacterium tuberculosis (Mtb) infection. Following Mtb infection, NK cells become activated and are early and rapid producers of interferon-c (IFN-c), a cytokine critical for the activation of macrophages (MQ) [2,3]. However, mouse models in which NK cells are defective or are depleted in vivo have failed to show that NK cells are essential for immunity to tuberculosis [3]. Similarly, cd T cells are frequently activated by a variety of pathogens including Mtb [4]. Mice lacking cd T cells succumb more rapidly than control mice following intravenous challenge with virulent Mtb; however, such a difference has not been observed following infection by the aerosol route [5,6]. Although cd T cells may not be required for optimum control of bacterial replication following pulmonary infection, cd T cell deficient mice form disorganized granulomas dominated by foamy MQ and granulocytes instead of lymphocytes [6]. Similarly, while CD1d-restricted NKT cells rapidly produce large amounts of IFNc when activated and play a role in granuloma formation under certain conditions, there is little evidence to support their requirement for optimum immunity against Mtb infection, although their pharmacological activation confers a significant survival advantage to susceptible mouse strains [714]. The mouse model of tuberculosis has been useful in delineating how different cell types contribute to immunity against Mtb. Many important components of the human immune response to Mtb were first identified or have been successfully modeled in the mouse including the critical role of IL-12, IFN-c, TNF, and CD4+ T cells. CD4+ T cells have unambiguously been identified as the most important lymphocyte subset in the mouse for mediating protection. However, the dominant role of CD4+ T cells may obscure the contribution of other immune mechanisms. Factors such as inoculum size, Mtb strain virulence, and experimental variability limit the dynamic range of the end points measured and reduce the capacity to detect subtle defects in immunity. We established an in vitro model to address whether innate lymphocytes have a role in immunity against Mtb. In our model, splenocytes obtained from uninfected mice are cultured with primary MQ infected with virulent Mtb. Under these conditions, splenocytes secrete IFN-c, Host resistance to Mycobacterium tuberculosis (Mtb) requires a coordinated response by the different components of the immune system. We established an in vitro model to study the contribution of innate lymphocytes to immunity against Mtb. When co-cultured with Mtbinfected macrophages, splenocytes from uninfected mice become activated and suppress bacterial replication. By fractionating the different splenocyte cell populations, we discovered that the invariant NKT (iNKT) cell is essential for suppressing intracellular bacterial replication. iNKT cells, which are conserved in rodents and humans, recognize lipids presented by the antigen-presenting molecule CD1d. While we had previously shown that iNKT celldeficient mice are not more susceptible to tuberculosis, a potential contribution of iNKT cells during the early phase of immunity may have been masked. To address this issue, we showed that highly purified iNKT cells were sufficient to reduce the lung bacterial burden of mice infected with virulent Mtb. This is the first evidence that CD1d-restricted iNKT cells play a physiological role in mediating protection against aerosol Mtb infection in vivo. Thus, by being an early producer of interferon-g and suppressing intracellular bacterial growth, iNKT cells function as an important part of the early immune response against Mtb. stimulate NOS2 upregulation and NO production, and suppress intracellular Mtb replication. In this report, we identify the cellular mechanism that mediates this innate effector function against the intracellular human pathogen Mtb. Splenocytes from Nave Mice (...truncated)