SummaryMicrobial infections are a major cause of infant mortality worldwide because of impaired immune defences in this population. The nature of this work was to further understand the mechanistic limitations of the neonatal and infant immune response. Interleukin-27 (IL-27) is a heterodimeric cytokine of the IL-12 family that is produced primarily by antigenpresenting cells and is immunosuppressive toward a variety of immune cell types. We show that IL-27 gene expression is elevated in cord bloodderived macrophages relative to macrophages originating from healthy adults. We also evaluated the duration over which elevated IL-27 gene expression may impact immune responses in mice. Age-dependent analysis of IL-27 gene expression indicated that levels of IL-27 remained significantly elevated throughout infancy and then declined in adult mice. Flow cytometric analysis of intracellular cytokine-stained splenocytes further confirmed these results. Interleukin-27 may be induced during pregnancy to contribute to the immunosuppressive environment at the fetal-maternal interface because we demonstrate dose-responsive gene expression to progesterone in macrophages. Neutralization of IL-27 in neonatal macrophages improved the ability of these cells to limit bacterial replication. Moreover, neutralization of IL-27 during incubation with the Mycobacterium bovis bacillus Calmette-Gu erin vaccine augmented the level of interferon-c elicited from allogeneic CD4 + T lymphocytes. This suggests that blocking IL-27 during vaccination and infection may improve immune responses in newborn and infant populations. Furthermore, mice will be a suitable model system to further address these possibilities.

Chlamydiae replicate within a nonacidified vacuole, termed an inclusion. As obligate intracellular bacteria, chlamydiae actively modify their vacuole to exploit host signaling and trafficking pathways. Recently, we demonstrated that several Rab GTPases are actively targeted to the inclusion. To define the biological roles of inclusion localized Rab GTPases, we have begun to identify inclusion-localized Rab effectors. Here we demonstrate that oculocerebrorenal syndrome of Lowe protein 1 (OCRL1), a Golgi complex-localized phosphatidylinositol (PI)-5-phosphatase that binds to multiple Rab GTPases, localizes to chlamydial inclusions. By examining the intracellular localization of green fluorescent protein (GFP) fusion proteins that bind to unique phosphoinositide species, we also demonstrate that phosphatidylinositol-4-phosphate (PI4P), the product of OCRL1, is present at the inclusion membrane. Furthermore, two additional host proteins, Arf1, which together with PI4P mediates the recruitment of PI4P-binding proteins to the Golgi complex, and PI4KII␣, a major producer of Golgi complex-localized PI4P, also localize to chlamydial inclusions. Depletion of OCRL1, Arf1, or PI4KII␣ by small interfering RNA (siRNA) decreases inclusion formation and the production of infectious progeny. Infectivity is further decreased in cells simultaneously depleted for all three host proteins, suggesting partially overlapping functions in infected cells. Collectively, these data demonstrate that Chlamydia species create a unique replication-competent vacuolar environment by modulating both the Rab GTPase and the PI composition of the chlamydial inclusion.

BackgroundThe cytokine environment at the site of infection is important to the control of mycobacteria by host macrophages. During chronic infection immunosuppressive cytokines are likely to favor mycobacterial growth, persistence, and an avoidance of proper antigen processing and presentation. The activity of interleukin (IL)-27 toward macrophages is anti-inflammatory and this compromises control of mycobacteria. Modulation of the cytokine environment may enhance both protective and vaccine-induced responses.ResultsIn this study we showed that supplying IL-12 and neutralizing IL-27 enhanced acidification and fusion of mycobacterial-containing phagosomes with lysosomes. This was achieved by phagosomal acquisition of vacuolar ATPase (V-ATPase) and CD63. Both V-ATPase and CD63 protein levels were increased by the addition of IL-12 and neutralization of IL-27. In addition, cathepsin D associated with the bacteria and matured to the active form when IL-12 was supplied and IL-27 was neutralized. Lysosomal acidification and cathepsin D activity were associated with control of mycobacteria. The acidification of lysosomes, association with mycobacteria, and maturation of cathepsin D required macrophage production of IFN-γ and signaling through signal transducer and activator of transcription (STAT)-1. In contrast, STAT-3 signaling opposed these events.ConclusionsOur results have identified novel influences of IL-12, IL-27, and STAT-3 on lysosomal activity and further demonstrate that modulating the cytokine environment promotes enhanced trafficking of mycobacteria to lysosomes in human macrophages. This has important implications in approaches to control infection and improve vaccination. Overcoming bacterial resistance to lysosomal fusion may expand the repertoire of antigens presented to the adaptive arm of the immune response.

Mycobacterium tuberculosis (MTB) remains a leading infectious threat to human health. Macrophages are the cells targeted for infection by the bacterium as well as key effector cells for clearance of the pathogen. Interleukin (IL)-27 opposes macrophage-mediated control of MTB because supplying IL-12 and blocking the activity of IL-27 limits bacterial growth in primary human macrophages. The purpose of this study was to determine the immunological regulators of this macrophage mechanism to restrict MTB growth. Interferon (IFN)-γ, TNF-α, and IL-18 were all demonstrated to be important to the environment that limits bacterial growth when IL-12 is supplied and IL-27 is neutralized. We find IL-18 works in conjunction with IL-12 to achieve optimal IFN-γ production in this system. We also demonstrate novel interactions between these cytokines to influence the expression or responsiveness to one another. Quantitative assays show that IFN-γ enhances expression of the IL-18 receptor signaling chain, as well as TNF expression and secretion. In turn, TNF-α augments expression of the receptor for IFN-γ the amount at the cell surface, and the extent of IFN-γ-induced signaling. We further define how the cytokine environment supports an enhanced state of classical macrophage activation. Collectively, these results describe novel immunological mechanisms that provide additional insights into the effects of IL-12 and IL-27 on macrophage regulation during MTB infection.