Murine myeloid-derived suppressor cells are a source of elevated levels of interleukin-27 in early life and compromise control of bacterial infection.

Microbial infections early in life remain a major cause of infant mortality worldwide. This is consistent with immune deficiencies in this population. Interleukin (IL)-27 is suppressive toward a variety of immune cell types, and we have shown that the production of IL-27 is elevated in humans and mice early in life. We hypothesize that elevated levels of IL-27 oppose protective responses to infection during the neonatal period. In this study, we extended previous findings in neonatal mice to identify a population of IL-27 producers that express Gr-1 and were further identified as myeloid-derived suppressor cells (MDSCs) based on the expression of surface markers and functional studies. In neonates, MDSCs are more abundant and contribute to the elevated pool of IL-27 in this population. Although the ability of MDSCs to regulate T lymphocyte activation has been well-studied, sparingly few studies have investigated the influence of MDSCs on innate immune function during bacterial infection. We demonstrate that macrophages are impaired in their ability to control growth of Escherichia coli when cocultured with MDSCs. This bacterium is a significant concern for neonates as a common cause of bacterial sepsis and meningitis. The suppressive effect of MDSCs on macrophage function is mediated by IL-27; inclusion of a reagent to neutralize IL-27 promotes improved control of bacterial growth. Taken together, these results suggest that the increased abundance of MDSCs may contribute to early life susceptibility to infection and further highlight production of IL-27 as a novel MDSC mechanism to suppress immunity.

Elevated interleukin-27 levels in human neonatal macrophages regulate indoleamine dioxygenase in a STAT-1 and STAT-3-dependent manner.

Microbial infections are a major cause of infant mortality as a result of limitations in immune defences. Interleukin-27 (IL-27) is a heterodimeric cytokine produced primarily by leucocytes and is immunosuppressive toward lymphocytes and leucocytes. Our laboratory demonstrated that human neonatal macrophages express IL-27 more abundantly than adult macrophages. Similarly in mice, IL-27 expression is elevated early in life and maintained through infancy. To determine IL-27-regulated mechanisms that may limit immunity, we evaluated the expression of a number of genes in response to this cytokine in primary human neonatal macrophages. Indoleamine 2,3-dioxygenase (IDO) gene expression was increased dose-responsively by IL-27. We have previously demonstrated inhibition of T-cell proliferation and cytokine production by neonatal macrophage-generated IL-27, and IDO is often implicated in this negative regulation. An increase in IDO protein was demonstrated by immunofluorescence microscopy and was consistent with increased enzyme activity following treatment with IL-27. Inclusion of a soluble receptor to neutralize endogenous IL-27, decreased IDO expression and activity compared with untreated macrophages. In response to IL-27, neonatal macrophages phosphorylate signal transdcuer and activator of transcription 1 (STAT-1) and STAT-3. Both transcription factors are recruited to the IDO regulatory region. STAT-3 dominates during steady-state regulation by lower levels of endogenous IL-27 production. A shift to enhanced STAT-1 recruitment occurs during increased levels of exogenously supplied IL-27. These data suggest an interesting interplay of STAT-1 and STAT-3 to regulate IDO activity and immunosuppression in response to different levels of IL-27 in the microenvironment of the immune response that may further our understanding of this interesting cytokine.

Genetic engineering of Francisella tularensis LVS for use as a novel live vaccine platform against Pseudomonas aeruginosa infections.

Francisella tularensis LVS (Live Vaccine Strain) is an attenuated bacterium that has been used as a live vaccine. Patients immunized with this organism show a very long-term memory response (over 30 years post vaccination) evidenced by the presence of indicators of robust cell-mediated immunity. Because F. tularensis LVS is such a potent vaccine, we hypothesized that this organism would be an effective vaccine platform. First, we sought to determine if we could genetically modify this strain to produce protective antigens of a heterologous pathogen. Currently, there is not a licensed vaccine against the important opportunistic bacterial pathogen, Pseudomonas aeruginosa. Because many P. aeruginosa strains are also drug resistant, the need for effective vaccines is magnified. Here, F. tularensis LVS was genetically modified to express surface proteins PilAPa, OprFPa, and FliCPa of P. aeruginosa. Immunization of mice with LVS expressing the recombinant FliCPa led to a significant production of antibodies specific for P. aeruginosa. However, mice that had been immunized with LVS expressing PilAPa or OprFPa did not produce high levels of antibodies specific for P. aerugionsa. Therefore, the recombinant LVS strain engineered to produce FliCPa may be able to provide immune protection against a P. aeruginosa challenge. However for future use of this vaccine platform, selection of the appropriate recombinant antigen is critical as not all recombinant antigens expressed in this strain were immunogenic.

The presence of interleukin-27 during monocyte-derived dendritic cell differentiation promotes improved antigen processing and stimulation of T cells.

Dendritic cells (DCs) are potent antigen-presenting cells necessary to establish effective adaptive immune responses. The cytokine environment that exists at the time of DC differentiation may be an important but often ignored determinant in the phenotypic and functional properties of DCs. Interleukin-27 (IL-27) is a unique cytokine that has both inflammatory and immune suppressive activities. Although it can both promote and oppose activity of different T-cell subsets, mostly anti-inflammatory activity has been described toward macrophages and DCs. However, the specific effect of IL-27 during DC differentiation and how that may change the nature of the antigen-presenting cell has not been investigated. In this report, we show that IL-27 treatment during monocyte-derived DC differentiation enhanced the ability to process antigens and stimulate T-cell activity. DCs differentiated in the presence of IL-27 showed enhanced acidification of latex bead-containing phagosomes that was consistent with elevated expression of vacuolar-ATPases. This resulted in inhibition of intracellular growth of Staphylococcus aureus. In addition, the levels of MHC class II surface expression were higher in DCs differentiated in the presence of IL-27. Production of IL-12 was also significantly increased during S. aureus infection of IL-27-differentiated DCs. The net effect of these activities was enhanced CD4(+) T-cell proliferation and T helper type 1 cytokine production. These findings are important to a wide number of immunological contexts and should be considered in the development of future vaccines.

IL-12 and IL-27 regulate the phagolysosomal pathway in mycobacteria-infected human macrophages.

BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: Our 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.

The intracellular environment of human macrophages that produce nitric oxide promotes growth of mycobacteria.

Nitric oxide (NO) is a diffusible radical gas produced from the activity of nitric oxide synthase (NOS). NOS activity in murine macrophages has a protective role against mycobacteria through generation of reactive nitrogen intermediates (RNIs). However, the production of NO by human macrophages has remained unclear due to the lack of sensitive reagents to detect NO directly. The purpose of this study was to investigate NO production and the consequence to mycobacteria in primary human macrophages. We found that Mycobacterium bovis BCG or Mycobacterium tuberculosis infection of human macrophages induced expression of NOS2 and NOS3 that resulted in detectable production of NO. Treatment with gamma interferon (IFN-γ), l-arginine, and tetrahydrobiopterin enhanced expression of NOS2 and NOS3 isoforms, as well as NO production. Both of these enzymes were shown to contribute to NO production. The maximal level of NO produced by human macrophages was not bactericidal or bacteriostatic to M. tuberculosis or BCG. The number of viable mycobacteria was increased in macrophages that produced NO, and this requires expression of nitrate reductase. An narG mutant of M. tuberculosis persisted but was unable to grow in human macrophages. Taken together, these data (i) enhance our understanding of primary human macrophage potential to produce NO, (ii) demonstrate that the level of RNIs produced in response to IFN-γ in vitro is not sufficient to limit intracellular mycobacterial growth, and (iii) suggest that mycobacteria may use RNIs to enhance their survival in human macrophages.

Interleukin-27 inhibits phagosomal acidification by blocking vacuolar ATPases.

Interleukin (IL)-27 is a unique cytokine that has a dual role in immune responses. It was originally described to promote Th1 differentiation but also suppresses inflammation by inhibiting these and other inflammatory T cell subsets. Inhibition of inflammatory activity in macrophages has also been reported. These reports have largely focused on cytokine profiles or signaling mechanisms. To date, there have been no reports of how IL-27 may directly influence cellular mechanisms that operate to control microbial growth. Formation of a phagolysosome that acquires antimicrobial properties is an essential step for destruction of pathogens or pathogen-derived materials that are internalized by macrophages. Here we report that IL-27 has a profound influence on this critical innate immunity pathway. Treatment of human macrophages with IL-27 interferes with the acidification of phagosomes by reducing protein levels of V-ATPase and impairs control of bacterial pathogens.

Neonatal macrophages express elevated levels of interleukin-27 that oppose immune responses.

Microbial 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 antigen-presenting cells and is immunosuppressive toward a variety of immune cell types. We show that IL-27 gene expression is elevated in cord blood-derived 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-Guérin vaccine augmented the level of interferon-γ 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.

Interferon-γ, tumor necrosis factor, and interleukin-18 cooperate to control growth of Mycobacterium tuberculosis in human macrophages.

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.

Multiple host proteins that function in phosphatidylinositol-4-phosphate metabolism are recruited to the chlamydial inclusion.

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 alpha, a major producer of Golgi complex-localized PI4P, also localize to chlamydial inclusions. Depletion of OCRL1, Arf1, or PI4KII alpha 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.

Up-regulation of gamma interferon receptor expression due to Chlamydia-toll-like receptor interaction does not enhance signal transducer and activator of transcription 1 signaling.

Gamma interferon (IFN-gamma)-induced indoleamine dioxygenase (IDO), which inhibits chlamydial replication by reducing the availability of tryptophan, is up-regulated by interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha). The mechanisms by which this occurs include an increase in the synthesis of interferon regulatory factor-1 as well as a nuclear factor-kappaB (NF-kappaB)-dependent increase in the expression of IFN-gamma receptors (IFN-gammaR). Although Chlamydia is susceptible to IDO, it up-regulates IFN-gammaR expression to a greater degree than either IL-1beta or TNF-alpha, perhaps through interaction with Toll-like receptors (TLR). The purpose of this study was to determine the mechanism by which Chlamydia psittaci up-regulates IFN-gammaR expression and evaluate this effect on IDO induction. Infection of HEK 293 cells with C. psittaci increased IFN-gammaR expression only in cells expressing either TLR2 or TLR4 and the adaptor protein MD-2. In addition, up-regulation of IFN-gammaR expression in Chlamydia-infected HeLa cells could be blocked either by neutralizing TLRs with anti-TLR2 and/or anti-TLR4 or by inhibiting NF-kappaB transactivation with a proteasome inhibitor. Although the newly expressed IFN-gammaR in Chlamydia-infected cells were capable of binding IFN-gamma, they did not enhance IFN-gamma-induced IDO activity in a manner similar to those observed for IL-1beta and TNF-alpha. Instead, IDO activation in Chlamydia-infected cells was no different than that induced in uninfected cells, despite the increase in IFN-gammaR expression. Furthermore, the amount of IFN-gamma-induced signal transducer and activator of transcription 1 (STAT-1) activation in infected cells paralleled that observed in uninfected cells, suggesting that STAT-1 activation by these newly expressed receptors was impaired.

Upregulation of IFN-gamma receptor expression by proinflammatory cytokines influences IDO activation in epithelial cells.

Interferon-gamma (IFN-gamma) induces the enzyme indoleamine dioxygenase (IDO) in a variety of human cell types. Furthermore, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) synergistically increase IFN-induced IDO activity. Inasmuch as cytokines can upregulate cytokine receptor expression, one mechanism of cytokine synergy may be at the level of receptor expression. To test the hypothesis that this mechanism of IDO regulation is active in epithelial cells, HeLa cells were treated with IFN-gamma, TNF-alpha, or IL-1beta to determine optimal cytokine concentrations and time for maximal cytokine receptor expression. Flow cytometric analysis with antibodies to receptors for IFN-gamma, TNF-alpha, or IL-1beta indicated that each cytokine upregulated expression of the other cytokine receptors by 4 h, with maximal expression observed between 16 and 20 h after cytokine treatment. Furthermore, increases in IFN-gamma receptors (IFNGR) induced by IL-1beta were found to be dependent on NF-kappaB transactivation. To determine if increases in IFNGR expression alone contributes to synergistic IDO induction, cells were stimulated with IL-1beta to upregulate receptor expression, and the NF-kappaB concentration was allowed to return to basal levels. When treated with IFN-gamma, enhanced Stat1 signaling and IDO induction were still observed, indicating that increased cytokine receptor expression contributes to synergistic increases in IDO activity.

Novel lavendamycin analogues as potent HIV-reverse transcriptase inhibitors: synthesis and evaluation of anti-reverse transcriptase activity of amide and ester analogues of lavendamycin.

Novel lavendamycins including two water soluble derivatives were synthesized via short and efficient methods. Pictet-Spengler condensation of 7-N-acylamino-2-formylquinoline-5,8-diones with tryptophans produced lavendamycin esters or amides 11-17. Lavendamycins 18-21 were obtained, respectively, by further transformations of 13-15 and 17. Several lavendamycins were found to be potent HIV reverse transcriptase inhibitors with very low toxicity in vitro and in vivo. Several compounds also acted either additively or synergistically to inhibit enzyme activity together with AZT-triphosphate.