Linda B. Adams

IFN-γ and NO in mycobacterial disease: new jobs for old hands

Abstract Granulomatous disease following exposure to Mycobacterium tuberculosis , Mycobacterium leprae or Mycobacterium avium is correlated with strong inflammatory and protective responses. The mouse model of mycobacterial infection provides an excellent tool with which to examine the inter-relationship between protective cell-mediated immunity and tissue-damaging hypersensitivity. It is well established that T cells and interferon (IFN)-γ are necessary components of anti-bacterial protection. We propose that IFN-γ also modulates the local cellular response by downregulating lymphocyte activation and by driving T cells into apoptosis, and that the events that limit excessive inflammation are largely mediated by IFN-γ-induced nitric oxide (NO). In several murine models of mycobacterial infection, the absence of IFN-γ and/or NO results in dysregulated granuloma formation and increased lymphocytic responses, which, in the case of M. avium infection, even leads to reduced bacterial growth.

Mycobacterium leprae-Specific, HLA Class II-Restricted Killing of Human Schwann Cells by CD4+ Th1 Cells: A Novel Immunopathogenic Mechanism of Nerve Damage in Leprosy

Peripheral nerve damage is a major complication of reversal (or type-1) reactions in leprosy. The pathogenesis of nerve damage remains largely unresolved, but detailed in situ analyses suggest that type-1 T cells play an important role. Mycobacterium leprae is known to have a remarkable tropism for Schwann cells of the peripheral nerve. Reversal reactions in leprosy are often accompanied by severe and irreversible nerve destruction and are associated with increased cellular immune reactivity against M. leprae. Thus, a likely immunopathogenic mechanism of Schwann cell and nerve damage in leprosy is that infected Schwann cells process and present Ags of M. leprae to Ag-specific, inflammatory type-1 T cells and that these T cells subsequently damage and lyse infected Schwann cells. Thus far it has been difficult to study this directly because of the inability to grow large numbers of human Schwann cells. We now have established long-term human Schwann cell cultures from sural nerves and show that human Schwann cells express MHC class I and II, ICAM-1, and CD80 surface molecules involved in Ag presentation. Human Schwann cells process and present M. leprae, as well as recombinant proteins and peptides to MHC class II-restricted CD4+ T cells, and are efficiently killed by these activated T cells. These findings elucidate a novel mechanism that is likely involved in the immunopathogenesis of nerve damage in leprosy.

Enumeration of Mycobacterium leprae Using Real-Time PCR

Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae–infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.

An indirect fluoresent antibody staining procedure for detection of Vibrio cholerae serovar 01 cells in aquatic environmental samples

Abstract An immunofluorescence method for detection of Vibrio cholerae serovar 01 in aquatic environmental samples and enrichment broths is described. Antiserum specific for the 01 somatic antigen was produced in rabbits and used in an indirect fluorescent antibody method incorporating fluoresceinisothiocyanate conjugated anti-rabbit globulin goat serum, and rhodamine isothiocynate conjugated bovine serum albumin as background stain. Comparisons of the immunofluorescent procedure and conventional culture methods for isolation of V. cholerae 01 showed that detection occurred significantly more frequently with the fluorescent antibody system.

The Study of Mycobacterium leprae Infection in Interferon-γ Gene—Disrupted Mice as a Model to Explore the Immunopathologic Spectrum of Leprosy

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.

Rapid Microbiologic and Pharmacologic Evaluation of Experimental Compounds against Mycobacterium tuberculosis

ABSTRACT The assessment of physiochemical and pharmacological properties at early stages of drug discovery can accelerate the conversion of hits and leads into candidates for further development. A strategy for streamlined evaluation of compounds against Mycobacterium tuberculosis in the early preclinical stage is presented in this report. As a primary assay to rapidly select experimental compounds with sufficient in vitro activity, the growth inhibition microtiter plate assay was devised as an alternative to current methods. This microdilution plate assay is a liquid culture method based on spectrophotometric readings of the bacillary growth. The performance of this method was compared to the performance of two established susceptibility methods using clinical available tuberculosis (TB) drugs. Data generated from all three assays were similar for all of the tested compounds. A second simple bioassay was devised to assess the oral bioavailability of compounds prior to extensive in vivo efficacy testing. The bioassay estimates drug concentrations in collected serum samples by a microdilution MIC plate method using M. tuberculosis. In the same assay, the MIC of the compound is also determined in the presence of 10% mouse serum as an indication of protein binding. The method was validated using different clinically available TB drugs, and results are discussed in this report. With these methodological advances, screening of compounds against tuberculosis in the preclinical phase will be rapid, can be adapted to semi-high-throughput screening, and will add relevant physicochemical and basic pharmacological criteria to the decision process of drug discovery.

Role of inducible nitric oxide synthase in resistance to Mycobacterium leprae in mice.

ABSTRACT The manifestation of leprosy in humans is largely determined by host immunity to Mycobacterium leprae and is a model for immunoregulation in a human disease. However, animal models available for exploration of the leprosy spectrum are inadequate. This study explored M. leprae infection in mice deficient in inducible nitric oxide synthase, and this report describes elements resembling borderline tuberculoid leprosy in humans.

PARK2 mediates interleukin 6 and monocyte chemoattractant protein 1 production by human macrophages.

Leprosy is a persistent infectious disease caused by Mycobacterium leprae that still affects over 200,000 new patients annually. The host genetic background is an important risk factor for leprosy susceptibility and the PARK2 gene is a replicated leprosy susceptibility candidate gene. The protein product of PARK2, Parkin, is an E3 ubiquitin ligase that is involved in the development of various forms of Parkinsonism. The human macrophage is both a natural host cell of M. leprae as well as a primary mediator of natural immune defenses, in part by secreting important pro-inflammatory cytokines and chemokines. Here, we report that down-regulation of Parkin in THP-1 macrophages, human monocyte-derived macrophages and human Schwann cells resulted in a consistent and specific decrease in interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1/CCL2) production in response to mycobacteria or LPS. Interestingly, production of IL-6 at 6 hours by THP-1 cells stimulated with live M. leprae and M. bovis BCG was dependent on pretreatment with 1,25-dihydroxyvitamin D3 (VD). Parkin knockdown in VD-treated cells blocked IL-6 induction by mycobacteria. However, IκB-α phosphorylation and levels of IκB-ξ, a nuclear protein required for IL-6 expression, were not affected by Parkin silencing. Phosphorylation of MAPK ERK1/2 and p38 was unaffected by Parkin silencing while JNK activation was promoted but did not explain the altered cytokine production. In a final set of experiments we found that genetic risk factors of leprosy located in the PARK2 promoter region were significantly correlated with M. leprae sonicate triggered CCL2 and IL6 transcript levels in whole blood assays. These results associated genetically controlled changes in the production of MCP-1/CCL2 and IL-6 with known leprosy susceptibility factors.

Lymphotoxin-α and TNF Have Essential but Independent Roles in the Evolution of the Granulomatous Response in Experimental Leprosy

Recent studies identified an association between genetic variants in the lymphotoxin-alpha (LTalpha) gene and leprosy. To study the influence of LTalpha on the control of experimental leprosy, both low- and high-dose Mycobacterium leprae foot pad (FP) infections were evaluated in LTalpha-deficient chimeric (cLTalpha(-/-)) and control chimeric (cB6) mice. Cellular responses to low-dose infection in cLTalpha(-/-) mice were dramatically different, with reduced accumulation of CD4(+) and CD8(+) lymphocytes and macrophages and failure to form granulomas. Growth of M. leprae was contained for 6 months, but augmented late in infection. In contrast, tumor necrosis factor knockout and tumor necrosis factor receptor 1 knockout FPs exhibited extensive inflammatory infiltration with an increase in M. leprae growth throughout infection. Following high-dose infection, cB6 FP induration peaked at 4 weeks and was maintained for 12 weeks. Induration was not sustained in cLTalpha(-/-) FPs that contained few lymphocytes and no granulomas. There was a reduction in the expression levels of inflammatory cytokines, chemokines, and chemokine receptors, including nitric oxide synthase 2, vascular cell adhesion molecule, and intercellular cell adhesion molecule. Furthermore, cLTalpha(-/-) popliteal lymph nodes contained a higher proportion of naïve CD44(lo)CD62L(hi) T cells than cB6 mice, suggestive of reduced T cell activation. Therefore, both LTalpha and tumor necrosis factor are essential for the regulation of the granuloma, but they have distinctive roles in the recruitment of lymphocytes and maintenance of the granulomatous response during chronic M. leprae infection.

An In Vitro Model for the Lepromatous Leprosy Granuloma: Fate of Mycobacterium leprae from Target Macrophages after Interaction with Normal and Activated Effector Macrophages

The lepromatous leprosy granuloma is a dynamic entity requiring a steady influx of macrophages (Mφ) for its maintenance. We have developed an in vitro model to study the fate of Mycobacterium leprae in a LL lesion, with and without immunotherapeutic intervention. Target cells, consisting of granuloma Mφ harvested from the footpads of M. leprae-infected athymic nu/nu mice, were cocultured with normal or IFN-γ-activated (ACT) effector Mφ. The bacilli were recovered and assessed for viability by radiorespirometry. M. leprae recovered from target Mφ possessed high metabolic activity, indicating a viable state in this uncultivable organism. M. leprae recovered from target Mφ incubated with normal effector Mφ exhibited significantly higher metabolism. In contrast, bacilli recovered from target Mφ cocultured with ACT effector Μφ displayed a markedly decreased metabolic activity. Inhibition by ACT Mφ required an E:T ratio of at least 5:1, a coculture incubation period of 3–5 days, and the production of reactive nitrogen intermediates, but not reactive oxygen intermediates. Neither IFN-γ nor TNF-α were required during the cocultivation period. However, cell-to-cell contact between the target and effector Mφ was necessary for augmentation of M. leprae metabolism by normal effector Mφ as well as for inhibition of M. leprae by ACT effector Mφ. Conventional fluorescence microscopy and confocal fluorescence microscopy revealed that the bacilli from the target Mφ were acquired by the effector Mφ. Thus, the state of Mφ infiltrating the granuloma may markedly affect the viability of M. leprae residing in Mφ in the lepromatous lesion.