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The cytokine interleukin-12 (IL-12) was thought to have a central role in T cell-mediated responses in inflammation for more than a decade after it was first identified. Discovery of the cytokine IL-23, which shares a common p40 subunit with IL-12, prompted efforts to clarify the relative contribution of these two cytokines in immune regulation. Ustekinumab, a therapeutic agent targeting both cytokines, was recently approved to treat psoriasis and psoriatic arthritis, and related agents are in clinical testing for a variety of inflammatory disorders. Here we discuss the therapeutic rationale for targeting these cytokines, the unintended consequences for host defense and tumor surveillance and potential ways in which these therapies can be applied to treat additional immune disorders.

It is unclear how CD4 T cell memory formation is regulated following pathogen challenge, and when critical mechanisms act to determine effector T cell fate. Here, we report that following influenza infection most effectors require signals from major histocompatibility complex class II molecules and CD70 during a late window well after initial priming to become memory. During this timeframe, effector cells must produce IL-2 or be exposed to high levels of paracrine or exogenously added IL-2 to survive an otherwise rapid default contraction phase. Late IL-2 promotes survival through acute down regulation of apoptotic pathways in effector T cells and by permanently upregulating their IL-7 receptor expression, enabling IL-7 to sustain them as memory T cells. This new paradigm defines a late checkpoint during the effector phase at which cognate interactions direct CD4 T cell memory generation.

Testing of drugs or vaccines in animal models prior to studies in the human population are essential to avoid safety problems in the field. In this chapter, the mouse, guinea pig, rabbit, and nonhuman primate models are discussed. Each model has its strengths and weaknesses, and the choice of the most appropriate model for a study depends on a variety of factors, including cost, available housing, and the question being addressed. The mouse model provides an economical and easily manipulated tool with which to determine the role of specific host or bacterial components in the pathogenesis of tuberculosis. The development of a chronic infection is common to most mammals infected with Mycobacterium tuberculosis. One important factor in using the aerosol model to assess the pathogenesis of M. tuberculosis is the use of bacterial cultures with high viability. Caseous necrosis in the form of small, spherical tubercles is a hallmark of human tuberculosis; indeed, it is the pathologic entity from which the disease derives its name. Nonhuman primates have been used in tuberculosis research for many decades, although cost and containment requirements have reduced the use of this model substantially in the last 30 years. The increasing sophistication of the animal models will lead the way to new findings of great importance in tuberculosis.

Fibrinogen-like protein 2 (Fgl2, fibroleukin) is a leukocyte product that exhibits significant homology to secreted proteins of diverse function, including growth factors, lectins, and components of extracellular matrix. Prior studies found that Fgl2 is IFNγ-inducible, possesses direct coagulant activity, and inhibits T cell proliferation and dendritic cell maturationin vitro. Here, we demonstrate that Fgl2 expression is up-regulated during type 1 immunityin vivoand establish that such up-regulation is IFNγ-, signal transducer and activation of transcription protein 1-, and IFN response factor 1-dependent. To investigate functional roles for Fgl2 during type 1 immunity, we generated Fgl2-deficient mice. Those animals are born at predicted Mendelian frequencies, appear overtly healthy, and contain normal numbers and frequencies of lymphoid cells. Although Fgl2 is IFNγ-inducible and putatively regulates T cell activation/proliferation, we demonstrate that Fgl2-deficient and control mice exhibit similar degrees of T cell expansion, immunopathology, and/or pathogen burdens during protozoan (Toxoplasma gondii), bacterial (Yersinia enterocolitica, Listeria monocytogenes, andMycobacterium tuberculosis), and viral (murine γ-herpesvirus-68 and Sendai) infections. Fgl2-deficient mice also reject allografts with similar kinetics as control mice. Moreover, despite prior reports that Fgl2 functions as a procoagulant enzyme, we demonstrate that Fgl2-deficient and control mice produce similar levels of fibrin, a product of the coagulation cascade, duringT. gondiiinfection and allograft rejection. Together, our findings suggest that Fgl2, although highly conserved and IFNγ-inducible, is not a critical mediator of either type 1 immunity or immune-associated coagulant activity.

Setting : Members of the Mycobacterium avium complex (MAC) are responsible for mycobacterial disease in children, the aged and in immunocompromised individuals. The complex consists of different species, serovars and morphologic forms that vary in virulence. One isolate of the MAC is currently being sequenced (MAC 104) and was chosen based on its derivation from an AIDS patient and the fact that it could be genetically manipulated. Objective : MAC 104 was therefore analyzed for virulence, colony morphotype and expression of the glycopeptidolipid (GPL) responsible for serotying differences and the rough to smooth morphological switch. Results : The isolate was found to be virulent in the murine model of low-dose aerosol infection in that it could colonize the lung, proliferate within the tissue and disseminate to other organs. MAC 104 expressed a variety of colony morphotypes, the most prevalent of which were smooth opaque, smooth transparent and rough. All three morphotypes could persist in the lung; however, the transparent and rough morphotypes grew more rapidly in vivo . The rough morphotype was unusual in that it expressed an atypical form of the GPL usually absent from rough morphotypes. Conclusion : This characterization complements the genome data and confirms that MAC 104 behaves similarly to other MAC isolates.

Mycobacterium leprae infection was evaluated in interferon-gamma knockout (GKO) mice. At 4 months, growth of the bacilli in the footpads of GKO mice plateaued a log(10) higher than that in control mice. Control mice exhibited mild lymphocytic and histiocytic infiltrates, whereas GKO mice developed large, unorganized infiltrates of epithelioid macrophages and scattered CD4 and CD8 T cells. Flow cytometric analysis of popliteal lymph node cells demonstrated similar profiles of T cells; however, GKO cells exhibited an elevated proliferative response to M. leprae antigen. Expression of inducible nitric oxide synthase mRNA was decreased in GKO mice, whereas macrophage inflammatory protein-1alpha and interleukin-4 and -10 mRNA expression were augmented. Control and GKO activated macrophages inhibited bacterial metabolism and produced nitrite. Thus, although deficient in an important Th1 cytokine, GKO mice possess compensatory mechanisms to control M. leprae growth and feature elements resembling mid-borderline leprosy in humans.

Recent evidence suggests that absence of the IL-12p40 subunit is more detrimental to the generation of protective responses than is the absence of the p35 subunit. To determine whether this is the case in tuberculosis, both p35 and p40 knockout mice were infected with Mycobacterium tuberculosis. Mice lacking the p40 subunit were highly susceptible to increased bacterial growth, exhibited reduced production of IFN-γ, and had increased mortality. In contrast, mice lacking the p35 subunit exhibited a moderate ability to control bacterial growth, were able to generate Ag-specific IFN-γ responses, and survived infection longer. The superior Ag-specific responses of the p35 gene-disrupted mice, when compared with the p40 gene-disrupted mice, suggest that the p40 subunit may act other than as a component of IL-12. A candidate molecule capable of driving the protective responses in the p35 gene-disrupted mice is the novel cytokine IL-23. This cytokine is composed of the IL-12 p40 subunit and a p19 subunit. In support of a role for this cytokine in protective responses to M. tuberculosis, we determined that the p19 subunit is induced in the lungs of infected mice.

Of the two common morphotypes of Mycobacterium avium, designated smooth transparent (SmT) or smooth opaque (SmO), the SmO morphotype is avirulent, whereas the SmT morphotype is virulent. The role of the host macrophage in determining these different virulence phenotypes was analyzed using an in vitro model of macrophage infection. Initial studies confirmed previous reports of the increased ability of the SmT bacteria to grow in macrophages; this increased virulence correlated with reduced induction of inflammatory cytokines. Examination of the response of the mitogen-activated protein kinase (MAPK) pathway following infection with either morphotype revealed that all three members of the MAPK pathway were activated. Pharmacologic inhibition of either the extracellular signal-regulated kinase (ERK) or p38MAPK pathways resulted in distinct consequences for the growth of the two morphotypes. In particular, inhibition of the p38MAPK resulted in attenuated growth of the SmT morphotype, which correlated with reduced PGE2 production. Inhibition of cyclooxygenase 2 by indomethacin also inhibited growth of SmT, substantiating the role for PGE2 in promoting the growth of SmT. In contrast, SmO induction of the ERK pathway was increased compared with the SmT morphotype, and inhibition of ERK resulted in decreased TNF-α synthesis and enhanced SmO growth. Pharmacologic inhibitors of the MAPK pathway were present for only the first 4 h of infection and yet had consequences for bacterial growth at 7 days. Therefore, the data suggest that induction of the MAPK pathway during uptake of bacteria is instrumental in determining the eventual fate of the bacteria.

Loss of IL-27R on T cells results in increased protection from Mycobacterium tuberculosis. Torrado et al. demonstrate that IL-27R−/− T cells show improved fitness that is associated with decreased expression of cell death molecules, maintenance of IL-2 production, and preferential accumulation in the lung parenchyma and around infected macrophages., CD4+ T cells mediate protection against Mycobacterium tuberculosis (Mtb); however, the phenotype of protective T cells is undefined, thereby confounding vaccination efforts. IL-27 is highly expressed during human tuberculosis (TB), and absence of IL-27R (Il27ra) specifically on T cells results in increased protection. IL-27R deficiency during chronic Mtb infection does not impact antigen-specific CD4+ T cell number but maintains programmed death-1 (PD-1), CD69, and CD127 expression while reducing T-bet and killer cell lectin-like receptor G1 (KLRG1) expression. Furthermore, T-bet haploinsufficiency results in failure to generate KLRG1+, antigen-specific CD4+ T cells, and in improved protection. T cells in Il27ra−/− mice accumulate preferentially in the lung parenchyma within close proximity to Mtb, and antigen-specific CD4+ T cells lacking IL-27R are intrinsically more fit than intact T cells and maintain IL-2 production. Improved fitness of IL-27R–deficient T cells is not associated with increased proliferation but with decreased expression of cell death–associated markers. Therefore, during Mtb infection, IL-27R acts intrinsically on T cells to limit protection and reduce fitness, whereas the IL-27R–deficient environment alters the phenotype and location of T cells. The significant expression of IL-27 in TB and the negative influence of IL-27R on T cell function demonstrate the pathway by which this cytokine/receptor pair is detrimental in TB.

Mycobacterium bovis Bacille Calmette-Guerin (BCG) is the only vaccine in use to prevent Mycobacterium tuberculosis (Mtb) infection. Here we analyzed the protective efficacy of BCG against Mtb challenges 21 or 120 days after vaccination. Only after 120 days post-vaccination were mice able to efficiently induce early Mtb growth arrest and maintain long-lasting control of Mtb. This protection correlated with the accumulation of CD4(+) T cells expressing IL-17(+)TNF(+)IL-2(+). In contrast, mice challenged with Mtb 21 days after BCG vaccination exhibited only a mild and transient protection, associated with the accumulation of CD4(+) T cells that were mostly IFN-gamma(+)TNF(+) and to a lesser extent IFN-gamma(+)TNF(+)IL-2(+). These data suggest that the memory response generated by BCG vaccination is functionally distinct depending upon the temporal proximity to BCG vaccination. Understanding how these responses are generated and maintained is critical for the development of novel vaccination strategies against tuberculosis. Copyright 2014 Elsevier Ltd. All rights reserved., We thank the ICVS animal facility personnel for excellent animal husbandry. This work was supported by Fundacao para a Ciencia e Tecnologia, Portugal and cofunded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), Quadro de Referencia Estrategico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER) project grants PTDC/SAU-MII/101977/2008, PTDC/BIA-BCM/102776/2008, and from Projeto Estrategico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013). AMC was funded by the National Institute of Allergy and Infectious Diseases at the National Institutes of Health grant AI46530. A.C. received a personal FCT grant SFRH/BPD/3306/2007 and M.S. is an FCT Investigator fellow.

This chapter discusses what happens when the immune response is capable of controlling a bacterial pathogen but not eliminating it. In this case, acquired immunity is defined not as the response capable of eliminating the pathogen but the response that allows survival of the host. The presence of ongoing infection and inflammation is likely to severely interfere with the generation of acquired specific memory. In this chapter the authors have consider the acquired cellular response to bacterial challenge with a focus on members of the genus Mycobacterium. The chapter focuses on the elements of the acquired immune response that mediate both immunity as well as the immunopathologic consequences that are often concomitant with expression of immunity to chronic bacterial pathogens. More recent studies, made possible by the development of tools capable of dissecting the nature of the T-cell response during an infection, have highlighted the truly complex nature of acquired immunity. The nature of the inflammatory response to Mycobacterium tuberculosis in the absence of acquired immune response suggests that the pathogen makes use of the acquired response to generate the damage required for transmission. The relative importance of cytokine cross-regulation, regulatory T-cell control, and the active cytotoxic activity of the bacteria in regulating the acquired response to Mycobacterium ulcerans has yet to be fully defined.

The immune mechanisms regulating epithelial cell repair after injury remain poorly defined. We demonstrate here that lymphotoxin beta receptor (LTβR) signaling in intestinal epithelial cells promotes self-repair after mucosal damage. Using a conditional gene-targeted approach, we demonstrate that LTβR signaling in intestinal epithelial cells is essential for epithelial interleukin-23 (IL-23) production and protection against epithelial injury. We further show that epithelial-derived IL-23 promotes mucosal wound healing by inducing the IL-22-mediated proliferation and survival of epithelial cells and mucus production. Additionally, we identified CD4(-)CCR6(+)T-bet(-) RAR-related orphan receptor gamma t (RORγt)(+) lymphoid tissue inducer cells as the main producers of protective IL-22 after epithelial damage. Thus, our results reveal a novel role for LTβR signaling in epithelial cells in the regulation of intestinal epithelial cell homeostasis to limit mucosal damage.

Inflammation is an essential protective part of the body’s response to infection, yet many diseases are the product of inflammation. For example, inflammation can lead to autoimmune disease and tissue damage, and is a key element in chronic health conditions such as heart disease, diabetes, rheumatoid arthritis, and also drives changes associated with aging. Animal models of infectious and chronic disease are important tools with which to dissect the pathways whereby inflammatory responses are initiated and controlled. Animal models therefore provide a prism through which the role of inflammation in health and disease can be viewed, and are important means by which to dissect mechanisms and identify potential therapies to be tested in the clinic. A meeting, “The Yin and Yang of Inflammation” was organized by Trudeau Institute and was held between April 4-6, 2014. The main goal was to bring together experts from biotechnology and academic organizations to examine and describe critical pathways in inflammation and place these pathways within the context of human disease. A group of ~80 scientists met for three days of intense formal and informal exchanges. A key focus was to stimulate interactions between basic research and industry.

We continue to investigate the nature of the host response to mycobacterial infection to be able to intervene with vaccination and immunotherapy and thereby, improve immunity. Animal models provide a tool to define pathways by which the vertebrate response to mycobacteria occurs, however making these observations relevant to human disease requires examination of these pathways in humans. In this edition of the Journal of Leukocyte Biology [1], the pathway by which Mtb induces an early IL-17 response from naive human peripheral blood cells is shown to involve the following: serine protease activity, the p38MAPK and Erk pathways, and IL-1R. In this model, peripheral blood cells from naive individuals with no known exposure to BCG or a history of positive, purified protein-derivative reaction were cultured with heat-killed Mtb for 7 days, and these cells produced IL-17A by Day 5. During exposure, the frequency of IFN-γ and IL-17 double-positive cells was found to increase tenfold, and although CD45RO-positive CD4 T cells made IL-17, and CD45RA-positive CD4 T cells did not; there was also a tenfold increase in the frequency of γδ T cells producing IL-17. By introducing inhibitors or gene deficiency into this model, the mannose receptor, NOD2, IL-6R, and Jnk pathway were excluded from having a role in this response. In contrast, strong inhibition of the IL-17 response was seen with an IL-1R antagonist as well as chemical inhibitors of the p38 MAPK and Erk pathways. In further support of a role for the activation of IL-1, inhibition of serine protease activity reduced the IL-17 response. Finally, dectin-1 and TLR4 were implicated in the IL-17A response by the ability of laminarin (which blocks dectin-1-dependent pathways [2]) and LPS from Bartonella quintana (which is a natural antagonist for TLR4 [3]) to reduce IL-17A production. So why are we interested in the response of naive people to Mtb exposure? IL-17 has been seen to be a component of the human response to Mtb, but what does this mean? Is it useful? Is it detrimental? Is it always good/bad to have it (Fig. 1)? As we are still asking these questions about IFN-γ [4], a cytokine we have been studying for far longer, it is premature to expect definitive answers to these questions for IL-17. What the data reported in this issue [1] do tell us, however, is that the earliest interaction between Mtb and a naive human host results in an environment that allows for IL-17 production and that specific pathways are required. Figure 1. The role of IL-17 in protective immunity to Mtb is still being determined. (A) We know that IL-17 can be produced by human peripheral blood cells with no known exposure to mycobacterial antigens in response to Mtb. This IL-17 response is dependent on ... How then does this paper help us in our goal of being able to intervene by vaccination or immunotherapy? A major area that is aided by this paper is not linked directly to Mtb but rather, to our ability to induce cellular responses effectively by vaccination. Although this ability lags behind our ability to induce antibody-mediated immunity, we are currently in a growth phase of adjuvant development and use. As we have come to understand the complexity of the immune response, we realize that effective and specific induction of cellular immunity requires defined adjuvants that are targeted exquisitely to induce specific subsets of cells. We know that complete Freund’s adjuvent (CFA) induces a cell-mediated inflammatory response but the pathways by which CFA induces this response are not fully defined. It has not been used in humans as a result of its broadly inflammatory nature, and surely, understanding how specific elements of CFA interact with human cells will improve adjuvant design. As the stimulant used in the paper under discussion here is heat-killed Mtb, and this is the major inflammatory ingredient in CFA, then the paper provides us with understanding of why and how CFA is inflammatory in humans. Combining the work of van de Veerdonks et al.[1] and some excellent recent work about defining the pathway by which one of the major components of Mtb—TDM—binds and stimulates cells, we are finally beginning to define more specifically the activity of CFA. In particular, although recent papers have demonstrated that TDM acts via Mincle [5, 6] and the Fcγ/Syk/CARD9 pathway [7] to induce inflammatory responses and Th1/Th17 acquired immunity, we now also know that IL-1R, dectin-1, and TLR4 are important in the induction of the IL-17 response in humans. With regard to immunotherapy, it is important to know where IL-17 is coming from in the response to Mtb infection. Once we know this, we can induce or inhibit this response from specific cell types and determine the role of these cell types in the protective and the inflammatory response. van de Veerdonk et al. [1] identify IL-17-positive CD4 memory T cells and γδ T cells as potential sources of IL-17, but we know many cells can produce IL-17. However, what should we do with this information? Should we induce a memory response that augments IL-17 production upon infection? We might say this is a good thing, as we have shown that IL-17 is essential for the accelerated recall of memory IFN-γ-producing cells into the lung [8], however IL-17 responses may be detrimental in chronic infection as a result of increased neutrophil responses that can compromise immunity [9, 10]. A recent study using a prime-boost strategy to enhance cellular responses in BCG-vaccinated individuals found that multifunctional cells expressing IL-2, TNF, and IFN were expanded, but they did not see an increase in IL-17; they were not overly concerned with this, as they did not know whether induction of Th17 cells would result in more or less optimal immunity [11]. So what good would IL-17 do in the protective response to mycobacterial infection? To begin to answer this, we need to go to the animal models for definitive data. Although studies with BCG implicated IL-17 recruitment of neutrophils as important in the early inflammatory response and maintenance of Th1 responses [12], later work has implicated IL-17 in maintenance of the granuloma [13]. Although this work was interesting, one is always concerned when BCG is used as a surrogate for Mtb, as it lacks the early secretory antigenic target system 1/region-of-difference 1 virulence locus that has been implicated in the earliest interaction between vertebrate host phagocytes and the invading mycobacteria [14]. This being said, when IL-17-deficient mice are infected with Mtb in this model, they fail to develop significant inflammation and do not control bacterial growth [13]. The source of IL-17 in these models is largely γδ T cells with some CD4 T cell production, consistent with the data in the paper under discussion. Thus, we can hypothesize that early cellular responses to Mtb induce IL-17 and that this response aids in granuloma formation and ability to control bacterial growth. A second animal model using IL-1β-deficient mice has shown recently that the IL-1R pathway is essential for control of Mtb growth and survival [15], however in this model, in contrast to the work by van de Veerdonk et al. [1], caspase-1 activity was not required. Whether a deficiency of IL-17 occurs in the IL-1β-deficient animal model and is linked to this susceptibility was not addressed, but if this were the case, then the paper describing an essential role for IL-1R in the human IL-17 response to Mtb will provide important support for further investigation of this pathway in humans with TB. TB can be a chronic disease, and although the early response may define later events, it is likely that the nature of the acquired cellular response to Mtb will have a great impact on the development and maintenance of immunity. In this regard, the cells studied in the accompanying paper were not assessed for antigen specificity, and it is possible that the memory CD4 T cells and the γδ T cells producing IL-17 were being stimulated nonspecifically (possibly by TDM stimulation of Mincle, as the authors suggest). Although we have made great strides in characterizing the acquired cellular response occurring in TB patients, we still do not know what constitutes a protective response at the site of infection. We know that patients with active TB have high levels of circulating γδ T cells producing IL-17 [16], however when a patient has active disease, there is more antigen and greater levels of inflammation that can drive more effector cell generation but equally, during active disease effector cells are more likely to be sequestered at the active site of disease. Interpreting the importance of specific cell types by their frequency in the peripheral blood is, therefore, a task fraught with complexity. Although IL-17 production may be required for early granuloma formation, it may also be detrimental in the long run, as it promotes neutrophil accumulation, and this may not be good for the generation of a stable mononuclear granuloma [17]. In conclusion, the accompanying paper provides an excellent mechanistic base for our understanding of how Mtb induces an early IL-17 response in naive cells. This is helpful for our development of adjuvants as well as our understanding of the early response to Mtb in humans. What we still do not know, however, is whether an acquired specific and long-term IL-17 response is a good or a bad thing for human immunity to Mtb (Fig. 1).

Isoxyl (ISO), a thiourea (thiocarlide; 4,4′-diisoamyloxythiocarbanilide), demonstrated potent activity against Mycobacterium tuberculosis H37Rv (MIC, 2.5 μg/ml), Mycobacterium bovis BCG (MIC, 0.5 μg/ml), Mycobacterium avium (MIC, 2.0 μg/ml), and Mycobacterium aurum A+ (MIC, 2.0 μg/ml), resulting in complete inhibition of mycobacteria grown on solid media. Importantly, a panel of clinical isolates of M. tuberculosis from different geographical areas with various drug resistance patterns were all sensitive to ISO in the range of 1 to 10 μg/ml. In a murine macrophage model, ISO exhibited bactericidal killing of viable intracellular M. tuberculosis in a dose-dependent manner (0.05 to 2.50 μg/ml). The selective action of ISO on mycolic acid synthesis was studied through the use of [1,2- 14 C]acetate labeling of M. tuberculosis H37Rv, M. bovis BCG, and M. aurum A+. At its MIC for M. tuberculosis , ISO inhibited the synthesis of both fatty acids and mycolic acids (α-mycolates by 91.6%, methoxymycolates by 94.3%, and ketomycolates by 91.1%); at its MIC in M. bovis BCG, ISO inhibited the synthesis of α-mycolates by 87.2% and that of ketomycolates by 88.5%; and the corresponding inhibitions for M. aurum A+ were 87.1% for α-mycolates, 87.2% for ketomycolates, and 86.5% for the wax-ester mycolates. A comparison with isoniazid (INH) and ethionamide (ETH) demonstrated marked similarity in action, i.e., inhibition of the synthesis of all kinds of mycolic acids. However, unlike INH and ETH, ISO also inhibited the synthesis of shorter-chain fatty acids. ISO showed no acute toxicity against primary macrophage cell cultures as demonstrated by diminution of redox activity. A homologous series of ISO derivatives were synthesized. Most derivatives were as effective or more effective than the parent compound in the agar proportion assay. Thus, these thioureas, like INH and ETH, specifically inhibit mycolic acid synthesis and show promise in counteracting a wide variety of drug-sensitive and -resistant strains of M. tuberculosis .

It remains unknown whether the expression of cell-mediated protective immunity and the capacity to mount a delayed-type hypersensitivity (DTH) reaction in tuberculosis infection represent two manifestations of a basic response or are dissociable events. In this study, we present data in favor of the latter hypothesis, by showing that tuberculosis infection in the lungs of mice possessing only a truncated form of intracellular adhesion molecule 1 due to gene disruption was still adequately controlled by the expression of protective immunity in the absence of any sustained influx of macrophages and the lack of formation of appreciable granulomas. These animals also had no detectable DTH response to mycobacterial proteins in the footpad assay, indicating that the accumulation of blood-borne macrophages at sites of mycobacterial infection or antigen deposition is not essential to control of the infection. These data support the hypothesis that the DTH component of the cellular response is not protective but contributes by walling off the sites of infection to prevent dissemination and reactivation disease.

Immunity to Mycobacterium tuberculosis infection is associated with the emergence of protective CD4 T cells that secrete cytokines, resulting in activation of macrophages and the recruitment of monocytes to initiate granuloma formation. The cytokine-mediating macrophage activation is interferon-gamma (IFN-gamma), which is largely dependent on interleukin-12 (IL-12) for its induction. To address the role of IL-12 in immunity to tuberculosis, IL-12 p40(-/-) mice were infected with M. tuberculosis and their capacity to control bacterial growth and other characteristics of their immune response were determined. The IL-12 p40(-/-) mice were unable to control bacterial growth and this appeared to be linked to the absence of both innate and acquired sources of IFN-gamma. T cell activation as measured by delayed type hypersensitivity and lymphocyte accumulation at the site of infection were both markedly reduced in the IL-12 p40(-/-) mice. Therefore, IL-12 is essential to the generation of a protective immune response to M. tuberculosis, with its main functions being the induction of the expression of IFN-gamma and the activation of antigen-specific lymphocytes capable of creating a protective granuloma.

CD8 T cells have been shown to be protective against Mycobacterium tuberculosis infections in the mouse. These cells have been shown to be cytolytic toward M. tuberculosis-infected cells and have also been shown to release the protective cytokine gamma interferon in response to mycobacterial antigen. It has therefore been unclear how these cells mediate their protective response. To dissect this problem, we compared the courses of M. tuberculosis infections in control, perforin gene-knockout, and granzyme gene-knockout mice exposed by the realistic pulmonary route. The inability to express either of these molecules limits the expression of the major lytic pathway but does not appear to influence the course of the infection or result in any discernible histologic differences. These data seem to rule against a lytic role for CD8 T cells in the lungs and hence tend to suggest instead that another type of mechanism, such as cytokine secretion by these cells, is their primary mode of action.

Mice which have disrupted genes for important components of the immune system have been used to study the role of these components in the immune response to infection with Mycobacterium tuberculosis . This has resulted in the identification of interleukin-12 (IL12), interferon-γ (IFNγ), tumour necrosis factor-α (TNFα) and inducible nitric oxide synthase (iNOS) as being essential to the protective response. Less crucial but perhaps more intriguing roles for other molecules such as intercellular adhesion molecule (ICAM) and IL6 have also been suggested by this kind of analysis.

Objective: Use gene disrupted mice to examine the possible role of secretory antibody on resistance to re-exposure to pulmonary tuberculosis. Design: Mice deficient in B cells due to targeted gene disruption were infected by aerosol exposure with Mycobacterium tuberculosis. A further set were identically exposed then given isoniazid to clear the infection and establish a state of memory immunity. Results: Control of the aerosol infection and generation of gamma interferon proceeded in a similar manner in both naive and memory immune mice, regardless of B cell deficiency. Conclusions: The absence of antibody responses did not affect the course of infection, thus confirming the classical literature that antibody plays no significant protective role.

Cell-mediated immune responses are known to be critical for control of mycobacterial infections whereas the role of B cells and humoral immunity is unclear. B cells can modulate immune responses by secretion of immunoglobulin, production of cytokines and antigen-presentation. To define the impact of B cells in the absence of secreted immunoglobulin, we analyzed the progression of Mycobacterium tuberculosis (Mtb) infection in mice that have B cells but which lack secretory immunoglobulin (AID(-/-)µS(-/-)mice). AID(-/-)µS(-/-) mice accumulated a population of activated B cells in the lungs when infected and were more susceptible to aerosol Mtb when compared to wild type (C57BL/6) mice or indeed mice that totally lack B cells. The enhanced susceptibility of AID(-/-)µS(-/-) mice was not associated with defective T cell activation or expression of a type 1 immune response. While delivery of normal serum to AID(-/-)µS(-/-) mice did not reverse susceptibility, susceptibility in the spleen was dependent upon the presence of B cells and susceptibility in the lungs of AID(-/-)µS(-/-)mice was associated with elevated expression of the cytokines IL-6, GM-CSF, IL-10 and molecules made by alternatively activated macrophages. Blocking of IL-10 signaling resulted in reversal of susceptibility in the spleens and lungs of AID(-/-)µS(-/-) mice. These data support the hypothesis that B cells can modulate immunity to Mtb in an organ specific manner via the modulation of cytokine production and macrophage activation.

The outcome of natural infections with pathogenic mycobacteria can range from early asymptomatic clearance through latent infection to clinical disease. Different host and pathogen-specific factors have been implicated in determining the outcome of these infections; however, it is clear that the interaction of mycobacteria with the innate and acquired components of the immune system plays a central role. Specifically, the recognition of mycobacterial components by innate immune cells through different pathogen recognition receptors (PPRs) induces a cytokine response that can promote early control of the infection. In fact, in the majority of individuals that come into contact with mycobacteria, this response is enough to control the infection. Among PRRs, Toll-like receptors (TLRs), Nucleotide Oligomerization Domain (NOD)-like receptors, and C-type lectins have all been implicated in recognition of mycobacteria and in the initiation of the cytokine response. Defining the mechanisms by which distinct mycobacterial components and their receptors stimulate the immune response is an area of intense research.

S UBMITRAL aneurysms occur in the left ventricle adjacent to the mltral annulus. They are the most common subannular aneurysms of the left ventricle. The aneurysms are often large and multiloculated and contain thrombi. They tend to grow into the pericardial space, behind the left atrium, bulging through its floor) A significant segment of the posterior mitral annulus is involved that leads to loss of support for the posterior leaflet with prolapse and consequent regurgitation 2 It may also compress the circumflex coronary artery, leading to angina pectoris or myocardial infarction. Submitral aneurysms are not uncommon in the black population of Africa, and occasional cases have been described m the white population outside of Africa. 2-6 They have also been reported in the Indmn population. 7 The incidence of this condmon is 1% to 2% of isolated mitral valve disease in the African population as reported by Antunes. I The cause is unknown but appears to be related to a defect or developmental weakness between the muscular ventricular wall and the fibrous annulus. The hemodynamlc impairment is related to the systolic expansion of the aneurysm, to valve regurgitation, or to both. The symptoms are those of congestive heart failure and angina pectorls. Occasionally they rupture, s Reported is an experience with two such patients and highlighted are the assocmted problems that might influence the successful anesthetic management of such patients. The authors believe this to be the first report describing the anesthetic management of such cases. A case of grant submitral aneurysm was reported about 6 years ago. 7 The patient died of low cardiac output syndrome after surgery However, the patient is not included in this report due to the unavailability of the anesthetic details.

We show here that infection of murine macrophages with various strains of Mycobacterium tuberculosis induces the rapid in vitro expression of genes encoding chemokines macrophage inflammatory protein 1 alpha and macrophage inflammatory protein 2, which recruit neutrophils to sites of infection, and macrophage-recruiting chemokines 10-kDa, interferon-inducible protein (IP-10) and macrophage chemotactic protein 1. Three strains of M. tuberculosis, Erdman and the clinical isolates CSU 22 and CSU 46, induced similar levels of secretion of macrophage chemotactic protein 1 from infected macrophage monolayers; however, the Erdman strain failed to induce levels of secretion of tumor necrosis factor alpha similar to those induced by either CSU 22 or CSU 46. Using a low-dose aerosol infection model, we also found that while the Erdman strain induced negligible increases in chemokine mRNA levels in the lungs, infection with either CSU 22 or CSU 46 resulted in greater levels of mRNA production for all four chemokines tested. The growth of these strains in the lungs was, however, equally well contained by acquired host immunity. These data allow us to hypothesize that the chemokine response in the lungs probably does not control the protective granulomatous response and that perhaps other T-cell- or macrophage-associated cytokines such as tumor necrosis factor alpha or interleukin 12 may be involved in this process.

Recent advances have characterized the protective immune response against Mycobacterium tuberculosis. These include identification of the phenotype of some protective cells, the antigens to which these cells respond and the cytokines produced in response to infection which modulate disease. Progress has also been made in inducing this response by vaccination.

We review the state of knowledge regarding the role of IL-17 in tuberculosis (TB). IL-17 is clearly induced following exposure to mycobacteria in mice and humans and therefore its role in both protection and the immunopathological consequences of infection must be fully defined. IL-17-producing T cells can be seen in both mice and humans and these cytokine-producing cells are dependent to a large degree upon IL-23. Based on what we know of the function of IL-17 and the nature of TB, it would be surprising if this were a disease where IL-17 would have a dramatic impact; indeed the experimental data suggests that it is not required for control of bacterial growth. However, while it is clear that IL-17 is present during TB, its function(s) is not yet known. Key questions that will help elucidate function include - the role the mycobacteria plays in induction and regulation of the IL-17 response and the role IL-17 plays in modulating the inflammatory response during chronic disease.

Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4+ T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-γ without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4+ effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4+ effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-γ production. During the early phase of infection, ∼10% of P25TCRTh1 cells produced IFN-γ in vivo; this declined to, Author Summary Mycobacterium tuberculosis causes persistent infection even in human or animal hosts that develop antigen-specific CD4+ and CD8+ T cell responses. To understand this phenomenon, we tested the hypothesis that the CD4+ effector T cells that are generated in response to M. tuberculosis infection fail to encounter their antigens at the site of infection in the lungs. Using mice infected with M. tuberculosis, and an assay of in vivo antigen-dependent activation of CD4+ T cells, we found that both polyclonal CD4+ and T cell receptor transgenic CD4+ T cells specific for antigen 85B peptide 25 are activated at low frequencies in the lungs. We found that this is due in part to downregulation of antigen gene expression by M. tuberculosis, as forced expression of the antigen gene resulted in higher frequency activation of CD4+ T cells, as well as CD4+ T cell-dependent reduction in bacterial burdens and prolonged survival of infected mice. We also found that administration of antigen 85B peptide 25, which is recognized by a high proportion of M. tuberculosis-specific CD4+ T cells, reduced the bacterial burden in the lungs, indicating that stimulation of existing antigen-specific CD4+ T cells may be a promising approach to therapy of TB.

There has been a recent increase in our understanding of T cell responses during mycobacterial infection; however we have not yet identified the protective mechanisms capable of mediating vaccine-induced protection in the lung. Novel approaches have allowed the determination of the kinetics and location of naïve T cell activation, the factors impacting expansion of antigen-specific T cell responses, and the factors impacting the balance between protective and immunopathological consequences during the chronic stages of infection. With new and more efficient vaccination strategies urgently needed, the integration of these data will result in improved vaccine strategies.

The expression of protective immunity to Mycobacterium tuberculosis in mice is mediated by T lymphocytes that secrete cytokines. These molecules then mediate a variety of roles, including the activation of parasitized host macrophages, and the recruitment of other mononuclear phagocytes to the site of the infection in order to initiate granuloma formation. Among these cytokines, interferon gamma (IFN-gamma) is believed to play a key role is these events. In confirmation of this hypothesis, we show in this study that mice in which the IFN-gamma gene has been disrupted were unable to contain or control a normally sublethal dose of M. tuberculosis, delivered either intravenously or aerogenically. In such mice, a progressive and widespread tissue destruction and necrosis, associated with very high numbers of acid-fast bacilli, was observed. In contrast, despite the lack of protective immunity, some DTH-like reactivity could still be elicited. These data, therefore, indicate that although IFN-gamma may not be needed for DTH expression, it plays a pivotal and essential role in protective cellular immunity to tuberculosis infection.

RNA splicing is an increasingly recognized regulator of immunity. Here, we demonstrate that after Mycobacterium tuberculosis infection (mRNA) il12rb1 is spliced by dendritic cells (DCs) to form an alternative (mRNA) il12rb1Deltatm that encodes the protein IL-12Rbeta1DeltaTM. Compared with IL-12Rbeta1, IL-12Rbeta1DeltaTM contains an altered C-terminal sequence and lacks a transmembrane domain. Expression of IL-12Rbeta1DeltaTM occurs in CD11c(+) cells in the lungs during M. tuberculosis infection. Selective reconstitution of il12rb1(-/-) DCs with (mRNA) il12rb1 and/or (mRNA) il12rb1Deltatm demonstrates that IL-12Rbeta1DeltaTM augments IL-12Rbeta1-dependent DC migration and activation of M. tuberculosis-specific T cells. It cannot mediate these activities independently of IL12Rbeta1. We hypothesize that M. tuberculosis-exposed DCs express IL-12Rbeta1DeltaTM to enhance IL-12Rbeta1-dependent migration and promote M. tuberculosis-specific T cell activation. IL-12Rbeta1DeltaTM thus represents a novel positive-regulator of IL12Rbeta1-dependent DC function and of the immune response to M. tuberculosis., This work was supported by the Trudeau Institute and the National Institutes of Health (AI067723 to A. M. Cooper; AI49823 to D. L. Woodland [trainee: R. T. Robinson] and AI084397 to R. T. Robinson).

Infection usually leads to the development of acquired immune responses associated with clearance or control of the infecting organism. However, if not adequately regulated, immune-mediated pathology can result. Tuberculosis is a worldwide threat, and development of an effective vaccine requires that the protective immune response to Mycobacterium tuberculosis (Mtb) be dissected from the pathological immune response. This distinction is particularly important if new vaccines are to be delivered to Mtb-exposed individuals, as repeated antigenic exposure can lead to pathological complications. Using a model wherein mice are vaccinated with bacille Calmette-Guérin after Mtb infection, we show that repeated vaccination results in increased IL-17, tumor necrosis factor, IL-6, and MIP-2 expression, influx of granulocytes/neutrophils, and lung tissue damage. This pathological response is abrogated in mice deficient in the gene encoding IL-23p19 or in the presence of IL-17-blocking antibody. This finding that repeated exposure to mycobacterial antigen promotes enhanced IL-17-dependent pathological consequences has important implications for the design of effective vaccines against Mtb., A. Cruz and A.G. Fraga were supported by the Fundacao para a Ciencia e Tecnologia (grants SFRH/BPD/33036/2007 and SFRH/BD/15911/2005). Other funding came from the Health Service of Fundacao Calouste Gulbenkian and Fundacao para a Ciencia e Tecnologia (grant PTDC/SAU-MII/70895/2006), the National Institutes of Health (grants HL69409, AI072689, and AI61511 to T. D. Randall; and grants AI46535, AI69121, and AI67723), the American Lung Association DeSouza Award, and Trudeau Institute funding to A.M. Cooper.

There has been an increase in our understanding of the complexity of the T cell response to mycobacterial infection recently. Improved tools have allowed the determination of the location and kinetics of naive T cell activation in the mouse as well the variety of function of mycobacteria-specific cells in humans. There is also an increased appreciation of the balance required during mycobacterial infection between anti-bacterial activity and control of the immunopathologic response. The integration of the T cell functional data with the consequences of infection should improve rational vaccine design.

IL-17 can impact health in a variety of ways. It is protective for some pathogens but it is also associated with tissue damaging inflammation. By examining the role of IL-17 in a variety of bacterial infections the mechanisms by which this cytokine mediates both protection and damage can be dissected. A key element in understanding the role of this cytokine is determining where and when it is acting. Dissecting its essential protective role from its immunopathologic role will allow for improved intervention in both acute and chronic disease.

We show here that IL-17-secreting CD4 T (Th)17 and CD8 T (Tc)17 effector cells are found in the lung following primary challenge with influenza A and that blocking Ab to IL-17 increases weight loss and reduces survival. Tc17 effectors can be generated in vitro using naive CD8 T cells from OT-I TCR-transgenic mice. T cell numbers expand 20-fold and a majority secretes IL-17, but little IFN-γ. Many of the IL-17-secreting cells also secrete TNF and some secrete IL-2. Tc17 are negative for granzyme B, perforin message, and cytolytic activity, in contrast to Tc1 effectors. Tc17 populations express message for orphan nuclear receptor γt and FoxP3, but are negative for T-bet and GATA-3 transcription factors. The FoxP3-positive, IL-17-secreting and IFN-γ-secreting cells represent three separate populations. The IFN-γ-, granzyme B-, FoxP3-positive cells and cells positive for IL-22 come mainly from memory cells and decrease in number when generated from CD44low rather than unselected CD8 T cells. Cells of this unique subset of CD8 effector T cells expand greatly after transfer to naive recipients following challenge and can protect them against lethal influenza infection. Tc17 protection is accompanied by greater neutrophil influx into the lung than in Tc1-injected mice, and the protection afforded by Tc17 effectors is less perforin but more IFN-γ dependent, implying that different mechanisms are involved.

Tuberculosis (TB) results from an interaction between a potent immune response and a chronically persistent pathogen. The ability of Mycobacterium tuberculosis (Mtb) to induce a strong immune response while being able to resist the ability of the host to clear bacteria provides an excellent tool with which to investigate the role of specific cytokine pathways on the induction, expansion, and control of the effector T-cell response. In this review, the role of interleukin-12p40 (IL-12p40), IL-12p70, IL-23, and IL-27 in the immune response to Mtb are described. We show that IL-12(p40)(2) acts to mediate the activation of dendritic cells to become responsive to homeostatic chemokines. We also show that IL-12p70 is required for the optimal interferon-gamma (IFN-gamma) T-cell response, which is required for control of Mtb growth. IL-23 can induce IFN-gamma responses in the lung if IL-12 is not present, but its major role is in supporting the IL-17 response within the lung. Neither IL-23 nor IL-17 is required for early control of Mtb in the lung. IL-23 and IL-17, however, can be instrumental in vaccine-induced protection. Finally, IL-27 limits protective immunity in the lung, but it is also required for long-term survival. These cytokines are therefore key players in the immune response to TB.

CD4+T cell responses to aerosolMycobacterium tuberculosis(Mtb) infection are characterized by the relatively delayed appearance of effector T cells in the lungs. This delay in the adaptive response is likely critical in allowing the bacteria to establish persistent infection. Because of limitations associated with the detection of low frequencies of naïve T cells, it had not been possible to precisely determine when and where naïve antigen-specific T cells are first activated. We have addressed this problem by using early secreted antigenic target 6 (ESAT-6)-specific transgenic CD4 T cells to monitor early T cell activationin vivo. By using an adoptive transfer approach, we directly show that T cell priming to ESAT-6 occurs only after 10 days of infection, is initially restricted to the mediastinal lymph nodes, and does not involve other lymph nodes or the lungs. Primed CD4 T cells rapidly differentiated into proliferating effector cells and ultimately acquired the ability to produce IFN-γ and TNF-αex vivo. Initiation of T cell priming was enhanced by two full days depending on the magnitude of the challenge inoculum, which suggests that antigen availability is a factor limiting the early CD4 T cell response. These data define a key period in the adaptive immune response to Mtb infection.

SUMMARYDevelopment and forward migration ofLeishmaniaparasites in the sandfly gut is accompanied by morphological transformation to highly motile, non-dividing ‘metacyclic’ forms. Previous studiesin vitrohave demonstrated that this metacyclogenesis is associated with developmentally regulated changes in expression of two major surface glycoconjugates ofLeishmania, the lipophosphoglycan (LPG) and the glycoprotein protease GP63. Studies presented here are the first to examinein situthe changes in expression of these two important surface molecules which occur during amastigote-initiated development ofL. majorin its natural vectorPhlebotomus papatasi. Immunocytochemical analysis using a GP63-specific monoclonal (3.8). and others recognizing metacyclic-specific (3F12) and common (WIC79.3) epitopes of LPG on logarithmic and metacyclic promastigotes, demonstrates: (1) clear expression of LPG and GP63 from 2 and 7 days post-bloodfeeding, respectively; (2) developmental modification of the LPG molecule as parasites undergo forward migration and morphological changes associated with metacyclogenesis; and (3) striking deposition of large amounts of parasite-free excreted LPG on/in the epithelial cells of the gut wall.

The role of IL-23 and IL-17 in the response to fungal infection has been the focus of recent reports. In this issue of the European Journal of Immunology there is an article that reports an important role for IL-23 and IL-17 in limiting fungal control, promoting neutrophillic inflammation and regulating the killing activity of neutrophils. In the fungal model it appears that IL-23 and IL-17 are counter-productive for protection. See accompanying article: http://dx.doi.org/10.1002/eji200737409

Tuberculosis is a chronic disease requiring the constant expression of cellular immunity to limit bacterial growth. The constant expression of immunity also results in chronic inflammation, which requires regulation. While IFN-gamma-producing CD4+ T helper cells (Th1) are required for control of bacterial growth they also initiate and maintain a mononuclear inflammatory response. Other T cell subsets are induced by Mycobacterium tuberculosis (Mtb) infection including those able to produce IL-17 (Th17). IL-17 is a potent inflammatory cytokine capable of inducing chemokine expression and recruitment of cells to parenchymal tissue. Both the IL-17 and the Th17 response to Mtb are largely dependent upon IL-23. Although both Th17 and Th1 cells are induced following primary infection with Mtb, the protective response is significantly altered in the absence of Th1 cells but not in the absence of Th17. In contrast, in vaccinated animals the absence of memory Th17 cells results in loss of both the accelerated memory Th1 response and protection. Th1 and Th17 responses cross-regulate each other during mycobacterial infection and this may be important for immunopathologic consequences not only in tuberculosis but also other mycobacterial infections.

Our understanding of the role of interleukin (IL)-12 in controlling tuberculosis has expanded because of increased interest in other members of the IL-12 family of cytokines. Recent data show that IL-12, IL-23 and IL-27 have specific roles in the initiation, expansion and control of the cellular response to tuberculosis. Specifically, IL-12, and to a lesser degree IL-23, generates protective cellular responses and promotes survival, whereas IL-27 moderates the inflammatory response and is required for long-term survival. Paradoxically, IL-27 also limits bacterial control, suggesting that a balance between bacterial killing and tissue damage is required for survival. Understanding the balance between IL-12, IL-23 and IL-27 is crucial to the development of immune intervention in tuberculosis.

Interferon-gamma is key in limiting Mycobacterium tuberculosis infection. Here we show that vaccination triggered an accelerated interferon-gamma response by CD4(+) T cells in the lung during subsequent M. tuberculosis infection. Interleukin 23 (IL-23) was essential for the accelerated response, for early cessation of bacterial growth and for establishment of an IL-17-producing CD4(+) T cell population in the lung. The recall response of the IL-17-producing CD4(+) T cell population occurred concurrently with expression of the chemokines CXCL9, CXCL10 and CXCL11. Depletion of IL-17 during challenge reduced the chemokine expression and accumulation of CD4(+) T cells producing interferon-gamma in the lung. We propose that vaccination induces IL-17-producing CD4(+) T cells that populate the lung and, after challenge, trigger the production of chemokines that recruit CD4(+) T cells producing interferon-gamma, which ultimately restrict bacterial growth.

T cell responses are important to the control of infection but are deleterious if not regulated. IFN-γ-deficient mice infected with mycobacteria exhibit enhanced accumulation of activated effector T cells and neutrophils within granulomatous lesions. These cells do not control bacterial growth and compromise the integrity of the infected tissue. We show that IFN-γ-deficient mice have increased numbers of IL-17-producing T cells following infection with Mycobacterium bovis bacille Calmette Gue´rin. Furthermore, exogenous IFN-γ increases IL-12 and decreases IL-23 production by bacille Calmette Gue´rin-infected bone marrow-derived dendritic cells and reduces the frequency of IL-17-producing T cells induced by these bone marrow-derived dendritic cells. These data support the hypothesis that, during mycobacterial infection, both IFN-γ- and IL-17-producing T cells are induced, but that IFN-γ serves to limit the IL-17-producing T cell population. This counterregulation pathway may be an important factor in limiting mycobacterially associated immune- mediated pathology., Fundacão para a Ciência e Tecnologia (FCT) - grant SFRH/BD/9624/2002

IL-12p70 induced IFN-γ is required to control Mycobacterium tuberculosis growth; however, in the absence of IL-12p70, an IL-12p40-dependent pathway mediates induction of IFN-γ and initial bacteriostatic activity. IL-23 is an IL-12p40-dependent cytokine containing an IL-12p40 subunit covalently bound to a p19 subunit that is implicated in the induction of CD4 T cells associated with autoimmunity and inflammation. We show that in IL-23 p19-deficient mice, mycobacterial growth is controlled, and there is no diminution in either the number of IFN-γ-producing Ag-specific CD4 T cells or local IFN-γ mRNA expression. Conversely, there is an almost total loss of both IL-17-producing Ag-specific CD4 T cells and local production of IL-17 mRNA in these mice. The absence of IL-17 does not alter expression of the antimycobacterial genes, NO synthase 2 and LRG-47, and the absence of IL-23 or IL-17, both of which are implicated in mediating inflammation, fails to substantially affect the granulomatous response to M. tuberculosis infection of the lung. Despite this redundancy, IL-23 is required to provide a moderate level of protection in the absence of IL-12p70, and this protection correlates with a requirement for IL-23 in the IL-12p70-independent induction of Ag-specific, IFN-γ-producing CD4 T cells. We also show that IL-23 is required for the induction of an IL-17-producing Ag-specific phenotype in naive CD4 T cells in vitro and that absence of IL-12p70 promotes an increase in the number of IL-17-producing Ag-specific CD4 T cells both in vitro and in vivo.

Th1 type cytokine responses are critical in the control of Mycobacterium tuberculosis infection. Recent findings indicate that 5-lipoxygenase-dependent (5-LO-dependent) lipoxins regulate host IL-12 production in vivo. Here, we establish lipoxins as key chemical mediators in resistance to M. tuberculosis infection. High levels of lipoxin A4 (LXA4) were detected in sera from infected WT but not infected 5-LO-deficient mice. Moreover, lungs from M. tuberculosis-infected 5-lo-/- animals showed increased IL-12, IFN-gamma, and NO synthase 2 (NOS2) mRNA levels compared with the same tissues in WT mice. Similarly, splenocyte recall responses were enhanced in mycobacteria-infected 5-lo-/- versus WT mice. Importantly, bacterial burdens in 5-lo-/- lungs were significantly lower than those from WT mice, and this enhancement in the resistance of the 5-lo-/- animals to M. tuberculosis was completely prevented by administration of a stable LXA4 analog. Together our results demonstrate that lipoxins negatively regulate protective Th1 responses against mycobacterial infection in vivo and suggest that the inhibition of lipoxin biosynthesis could serve as a strategy for enhancing host resistance to M. tuberculosis.

Mice lacking expression of granulocyte macrophage-colony stimulating factor (GM-CSF KO) are unable to contain Mycobacterium tuberculosis (M. tuberculosis) growth and succumb to infection by 35 days following pulmonary challenge. GM-CSF KO mice do not express normal levels of the inflammatory cytokine tumor necrosis factor α (TNF-α) nor the chemokines, regulated on activation, normal T expressed and secreted (RANTES), macrophage-inflammatory protein-1β (MIP-1β), MIP-1α, and lymphotactin, which are required for recruitment of lymphocytes and expression of a T helper cell type 1 (TH1) response within the lungs. In contrast, transgenic mice overexpressing GM-CSF in the lungs but with a lack of GM-CSF in other organs (GM+) are able to recruit lymphocytes and to express a TH1 response with production of TNF-α and interferon-γ in the lungs. However, GM+ mice succumb to infection between 60 and 90 days post-challenge, as they are unable to develop a normal granulomatous response. Although GM+ mice are able to express the chemokine RANTES, they lack the ability to express other inflammatory chemokines such as lymphotactin and MIP-1β. We conclude that GM-CSF is essential to the recruitment of lymphocytes and expression of a TH1 response in the lung, to the generation of a normal mononuclear granuloma, and most importantly, to the containment of M. tuberculosis bacterial growth.

Infection by virulentMycobacterium aviumcaused progressive severe lymphopenia in C57BL/6 mice due to increased apoptosis rates. T-cell depletion did not occur in gamma interferon (IFN-γ)-deficient mice which showed increased T-cell numbers and proliferation; in contrast, deficiency in nitric oxide synthase 2 did not prevent T-cell loss. Although T-cell loss was IFN-γ dependent, expression of the IFN-γ receptor on T cells was not required for depletion. Similarly, while T-cell loss was optimal if the T cells expressed IFN-γ, CD8+T-cell depletion could occur in the absence of T-cell-derived IFN-γ. Depletion did not require that the T cells be specific for mycobacterial antigen and was not affected by deficiencies in the tumor necrosis factor receptors p55 or p75, the Fas receptor (CD95), or the respiratory burst enzymes or by forced expression ofbcl-2in hematopoietic cells.

Resistance to tuberculosis (TB) is dependent on the induction of Ag-specific CD4 Th1 T cells capable of expressing IFN-γ. Generation of these T cells is dependent upon IL-12p70, yet other cytokines have also been implicated in this process. One such cytokine, IL-27, augments differentiation of naive T cells toward an IFN-γ-producing phenotype by up-regulating the transcription factor T-bet and promoting expression of the IL-12Rβ2 chain allowing T cells to respond to IL-12p70. We show that the components of IL-27 are induced during TB and that the absence of IL-27 signaling results in an altered disease profile. In the absence of the IL-27R, there is reduced bacterial burden and an increased lymphocytic character to the TB granuloma. Although the number of Ag-specific CD4 IFN-γ-producing cells is unaffected by the absence of the IL-27R, there is a significant decrease in the level of mRNA for IFN-γ and T-bet within the lungs of infected IL-27R−/− mice. Ag-specific CD4 T cells in the lungs of IL-27R−/− also produce less IFN-γ protein per cell. The data show that expression of IL-27 during TB is detrimental to the control of bacteria and that although it does not affect the number of cells capable of producing IFN-γ it does reduce the ability of CD4 T cells to produce large amounts of IFN-γ. Because IFN-γ is detrimental to the survival of effector T cells, we hypothesize that the reduced IFN-γ within the IL-27R−/− lung is responsible for the increased accumulation of lymphocytes within the mycobacterial granuloma.