Andrew G. D. Bean

TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection.

Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF−/−) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b+ macrophages and CD4+ T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF−/− mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF−/− mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF−/− mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.

Up-Regulation of VCAM-1 and Differential Expansion of β Integrin-Expressing T Lymphocytes Are Associated with Immunity to Pulmonary Mycobacterium tuberculosis Infection

Immune responses rely on an intricate system of adhesion molecules to coordinate the homing and retention of lymphocytes in both secondary lymphoid tissues and at sites of infection. To define the events associated with pulmonary immune responses, the expression of endothelial addressins and integrins on T cells was analyzed during Mycobacterium tuberculosis infection. In infected lung, expression of endothelial VCAM-1, but not mucosal addressin cell adhesion molecule-1, was up-regulated from 4 wk postinfection and persisted to at least 12 wk. Subsequent analysis of the corresponding integrins expressed on lung CD4+ and CD8+ T cells revealed an accumulation of β1high/β7−/low, and to a lesser extent β7high, integrin-expressing T cells during infection. Examination of integrin heterodimers showed that while α4 integrin was predominantly expressed on β1high/β7−/low cells, αE integrin was primarily associated with β7high. The majority of activated/memory T cells recruited during infection expressed high levels of β1 integrin and undetectable or low levels of β7 integrin. These T cells were capable of producing IFN-γ, a cytokine crucial for controlling M. tuberculosis infection. Rapid expansion of β1high, β7−, and β7high T cell populations in the lung upon secondary mycobacterial infection indicates the participation of these populations in the acquired immune response to the infection. Furthermore, treatment of infected mice with mAb to α4 or α4β7 integrin led to a reduction in lymphocytes and increase in granulocytes in the pulmonary infiltrate. These results reveal a crucial role for adhesion molecules in the generation of an effective pulmonary immune response to M. tuberculosis infection.

Prebiotics Modulate Immune Responses in the Gut-Associated Lymphoid Tissue of Chickens

The recent European Union ban on the prophylactic use of in-feed antibiotics has escalated the search for alternatives for use within the poultry industry. When evaluating the efficacy of potential antibiotic alternatives on bird health and productivity, it is important to analyze the competence of the immune cells in the gut-associated lymphoid tissue (GALT), because it is routinely involved in the surveillance of colonizing microbes as well as in interacting with the ingested feed antigens. Therefore, we studied the effect of the prebiotics mannan-oligosaccharide (MOS) and fructo-oligosaccharide (FOS) on the phenotypic and functional competence of immune cells in cecal tonsil (CT), which is a major GALT. Day-old Cobb 500 male broilers were randomized to 4 groups. Control chickens were fed the basal diet only. Chickens in experimental groups received 0.05 g/kg zinc bacitracin or 5 g/kg of either FOS or MOS in addition to basal diet. At the end of 25 d, our comparison of the experimental groups with controls revealed that the addition of prebiotics to diet resulted in a significant reduction in the proportion of B cells and in mitogen responsiveness of lymphocytes in CT. Furthermore, FOS treatment significantly enhanced the IgM and IgG antibody titers in plasma. These findings emphasize the need for the analyses of the gut immune function following treatment with novel feed additives. The knowledge obtained from such analyses may aid in understanding the mechanisms underlying the immune competence of the birds, which needs consideration when selecting and optimizing new feed additives instead of antibiotics for poultry production.

Avian cytokines - the natural approach to therapeutics.

While the effective use of antibiotics for the control of human disease has saved countless lives and has increased life expectancy over the past few decades, there are concerns arising from their usage in livestock. The use of antibiotic feed additives in food production animals has been linked to the emergence in the food chain of multiple drug-resistant bacteria that appear impervious to even the most powerful antimicrobial agents. Furthermore, the use of chemical antimicrobials has led to concerns involving environmental contamination and unwanted residues in food products. The imminent banning of antibiotic usage in livestock feed has intensified the search for environmentally-friendly alternative methods to control disease. Cytokines, as natural mediators and regulators of the immune response, offer exciting new alternatives to conventional chemical-based therapeutics. The utilisation of cytokines is becoming more feasible, particularly in poultry, with the recent cloning of a number of avian cytokine genes. Chickens offer an attractive small animal model system with which to study the effectiveness of cytokine therapy in the control of disease in intensive livestock. In this report we will review the status of avian cytokines and focus on our recent studies involving the therapeutic potential of chicken interferon gamma (ChIFN-gamma) as a vaccine adjuvant and a growth promoter.

Stimulation of Dendritic Cells via CD40 Enhances Immune Responses to Mycobacterium tuberculosis Infection

ABSTRACT The resolution of pulmonary tuberculosis (TB) critically depends on the development of the Th1 type of immune responses, as exemplified by the exacerbation of TB in IL-12-deficient mice. Therefore, vaccination strategies optimizing IL-12 production by antigen-presenting cells (APC) in response to mycobacteria may have enhanced protective efficacy. Since dendritic cells (DC) are the critical APC for activation of CD4+ and CD8+ T cells, we examined whether stimulation of Mycobacterium bovis bacillus Calmette Guérin (BCG)-infected DC via CD40 increased their ability to generate Th1-oriented cellular immune responses. Incubation of DC with an agonistic anti-CD40 antibody activated CD40 signaling in DC, as shown by increased expression of major histocompatibility complex class II and costimulatory molecules, mRNA production for proinflammatory cytokines and interleukin 12 (IL-12) p40. This activation pattern was maintained when DC were stimulated with anti-CD40 antibody and infected with BCG. Importantly, CD40-stimulated BCG-infected DC displayed increased capacity to release bioactive IL-12 and to activate gamma interferon (IFN-γ) producing T cells in vitro. Moreover, when C57BL/6 mice were immunized with these DC and challenged with aerosol Mycobacterium tuberculosis, increased levels of mRNA for IL-12 p40, IL-18, and IFN-γ were present in the draining mediastinal lymph nodes. However, the mycobacterial burden in the lungs was not reduced compared to that in mice immunized with BCG-infected non-CD40-stimulated DC. Therefore, although the manipulation of DC via CD40 is effective for enhancing immune responses to mycobacteria in vivo, additional strategies are required to increase protection against virulent M. tuberculosis infection.

Lymphocyte Recruitment and Protective Efficacy against Pulmonary Mycobacterial Infection Are Independent of the Route of Prior Mycobacterium bovis BCG Immunization

ABSTRACT Mycobacterium tuberculosis infects humans through the lung, and immunity to this chronic infection is mediated primarily by CD4+ T lymphocytes. Recently we have demonstrated that the recruitment of lymphocytes to the lung during primary aerosol M. tuberculosis infection in mice occurs predominantly through the interaction of α4β1 integrin on CD4+ T cells and vascular cell adhesion molecule-1 on the pulmonary endothelium. To investigate the effect of route of immunization with Mycobacterium bovis BCG on the pattern of T-cell recruitment to the lung, we have analyzed the differences in expression of integrins on activated memory CD4+ T cells infiltrating the lung following primary BCG immunization by aerosol, intravenous, and subcutaneous routes and after subsequent aerosol challenge with M. tuberculosis. There were marked differences in the patterns of recruitment of activated CD4+ T cells to the lung following primary immunization by the three routes. Expansion of CD44hi CD62Llow CD4+ T cells in the lung occurred following aerosol and intravenous BCG immunizations, and the lymphocyte recruitment was proportional to the pulmonary bacterial load. The majority of infiltrating CD4+ T cells expressed α4β1 integrin. On subsequent exposure to aerosol BCG rapid expansion of gamma interferon-secreting α4β1+ CD4+ T cells occurred to the same extent in all immunized mice, regardless of the route of immunization. Similar expansion of α4β1+ CD4+ memory T cells occurred following M. tuberculosis challenge. The three routes of BCG immunization resulted in the same level of protection against aerosol M. tuberculosis or BCG challenge in both the lungs and spleen. Therefore, recruitment of effector T lymphocytes and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior BCG immunization.

Endogenous inhibition of antimycobacterial immunity by IL-10 varies between mycobacterial species.

Interleukin (IL)‐10 is an immunoregulatory cytokine that inhibits both Th1‐like T cell responses and macrophage activation. Deficiency of IL‐10 has been associated with increased Th1‐like CD4+ T‐cell responses and increased clearance of some intracellular pathogens, however, its role in mycobacterial infections is controversial. In order to examine the effects of mycobacterial virulence on the outcome of infection we compared infection with Mycobacterium avium and virulent Mycobacterium tuberculosis in C57Bl/6 IL‐10−/– mice. M. avium infection in IL‐10−/– mice resulted in sustained increases in interferon (IFN)‐γ‐secreting T‐cell responses and was associated with the increased clearance of M. avium from the liver and lung. By contrast, M. tuberculosis infection in IL‐10−/– mice led to a transient increase in IFN‐γ T‐cell responses at 4u2003weeks postinfection, with reduced bacterial burden in the lungs. This was not sustained so that by 8u2003weeks there was no difference to wild‐type (WT) mice. In vitro infection of IL‐10−/– macrophages with M. avium, but not M. tuberculosis, led to an increased IL‐12 production. Therefore, endogenous IL‐10 exerts a significant inhibition on specific IFN‐γ T‐cell responses to M. avium infection, however, this effect is short lived during the M. tuberculosis infection, and fails to influence the long‐term course of infection.

Molecular Cloning, Expression, and Characterization of Chicken IFN -λ

Interferons (IFN) provide a critical first line of defense against viral infection in vertebrates. Moreover, IFN-lambda, a recently identified group of mammalian IFN, has demonstrated antiviral potential in the treatment of mammalian viruses. With the growing concern over such diseases as avian influenza (AI), there is a pressing need for new antiviral strategies to manage problem viruses in poultry. Furthermore, the use of immune molecules, such as IFN-lambda, provides an attractive option for treating poultry by augmenting the host response to virus. With this in mind, we report here the first cloning, expression, and analysis of biologic activity of chicken IFN-lambda (ChIFN-lambda). We compared the similarity of ChIFN-lambda to those identified in other species and demonstrate that ChIFN-lambda has antiviral properties similar to those of human IFN-lambda (HuIFN-lambda). Our results demonstrate that in the chicken, as in human, the antiviral activity demonstrated by ChIFN-lambda supports its inclusion in therapeutic strategies directed against viral infections.

Protective effect of DNA immunization against mycobacterial infection is associated with the early emergence of interferon-gamma (IFN-γ)-secreting lymphocytes

The development of more effective anti‐tuberculosis (TB) vaccines would contribute to the global control of TB. Understanding the activated/memory T cell response to mycobacterial infection and identifying immunological correlates of protective immunity will facilitate the design and assessment of new candidate vaccines. Therefore, we investigated the kinetics of the CD4+ T cell response and IFN‐γ production in an intravenous challenge model of Mycobacterium bovis bacille Calmette–Guérin (BCG) before and after DNA immunization. Activated/memory CD4+ T cells, defined as CD44hiCD45RBlo, expanded following infection, peaking at 3–4u2003weeks, and decreased as the bacterial load fell. Activated/memory CD4+ T cells were the major source of IFN‐γ and the level of antigen‐specific IFN‐γ‐secreting lymphocytes, detected by ELISPOT, paralleled the changes in bacterial load. To examine the effects of a DNA vaccine, we immunized mice with a plasmid expressing the mycobacterial secreted antigen 85B (Ag85B). This led to a significant reduction in mycobacteria in the liver, spleen and lung. This protective effect was associated with the rapid emergence of antigen‐specific IFN‐γ‐secreting lymphocytes which were detected earlier, at day 4, and at higher levels than in infected animals immunized with a control vector. This early and increased response of IFN‐γ‐secreting T cells may serve as a correlate of protective immunity for anti‐TB vaccines.

Studying immunity to zoonotic diseases in the natural host — keeping it real

Zoonotic viruses that emerge from wildlife and domesticated animals pose a serious threat to human and animal health. In many instances, mouse models have improved our understanding of the human immune response to infection; however, when dealing with emerging zoonotic diseases, they may be of limited use. This is particularly the case when the model fails to reproduce the disease status that is seen in the natural reservoir, transmission species or human host. In this Review, we discuss how researchers are placing more emphasis on the study of the immune response to zoonotic infections in the natural reservoir hosts and spillover species. Such studies will not only lead to a greater understanding of how these infections induce variable disease and immune responses in distinct species but also offer important insights into the evolution of mammalian immune systems.