Sahar Aly

Location of Persisting Mycobacteria in a Guinea Pig Model of Tuberculosis Revealed by R207910

ABSTRACT The lengthy chemotherapy of tuberculosis reflects the ability of a small subpopulation of Mycobacterium tuberculosis bacteria to persist in infected individuals. To date, the exact location of these persisting bacteria is not known. Lung lesions in guinea pigs infected with M. tuberculosis have striking similarities, such as necrosis, mineralization, and hypoxia, to natural infections in humans. Guinea pigs develop necrotic primary lesions after aerosol infection that differ in their morphology compared to secondary lesions resulting from hematogenous dissemination. In infected guinea pigs conventional therapy for tuberculosis during 6 weeks reduced the bacterial load by 1.7 logs in the lungs and, although this completely reversed lung inflammation associated with secondary lesions, the primary granulomas remained largely unaffected. Treatment of animals with the experimental drug R207910 (TMC207) for 6 weeks was highly effective with almost complete eradication of the bacteria throughout both the primary and the secondary lesions. Most importantly, the few remnants of acid-fast bacilli remaining after R207910 treatment were to be found extracellular, in a microenvironment of residual primary lesion necrosis with incomplete dystrophic calcification. This zone of the primary granuloma is hypoxic and is morphologically similar to what has been described for human lung lesions. These results show that this acellular rim may, therefore, be a primary location of persisting bacilli withstanding drug treatment.

Interferon-gamma-dependent mechanisms of mycobacteria-induced pulmonary immunopathology: the role of angiostasis and CXCR3-targeted chemokines for granuloma necrosis†

The mechanisms leading to granuloma caseation, a hallmark of tuberculosis (TB) in humans, are poorly understood. Lung histopathology of C57BL/6 (WT) mice 16 weeks after aerosol infection with Mycobacterium avium strain TMC724 is uniquely characterized by centrally necrotizing granulomas, strongly resembling human TB lesions. However, IFN‐γ‐deficient (GKO) and IFN‐γ‐receptor‐deficient (GRKO) mice did not develop granuloma necrosis following M. avium infection. Comparison of differentially expressed genes in infected WT and GKO lungs by DNA microarray and RNase protection assays revealed that the angiostatic chemokines CXCL9–11 were significantly reduced in GKO mice. In contrast, angiogenic mediators such as angiopoietin and vascular endothelial growth factor, and angiogenic chemokines such as CXCL2, CCL3, and CCL4, remained unchanged or were expressed at higher levels than in infected WT mice, suggesting impaired neovascularization of the granuloma as a possible mechanism for caseation in WT mice. Granuloma vascularization was significantly decreased in central, but not peripheral, areas of granulomas of infected WT compared to GKO mice. In contrast to GRKO mice, WT mice showed signs of severe hypoxia in cells immediately surrounding the necrotic core of granulomas as measured immunohistochemically with a reagent detecting pimonidazole adducts. To test the hypothesis that CXCR3, the common receptor for the angiostatic chemokines CXCL9–11, is involved in granuloma caseation, histomorphology was assessed in M. avium‐infected mice deficient for CXCR3 (CXCR3‐KO). 16 weeks after infection, these mice developed caseating granulomas similar to WT mice. We conclude that IFN‐γ causes a dysbalance between angiostatic and angiogenic mediators and a concomitant reduction in granuloma vascularization, but that CXCR3‐targeted chemokines are not sufficient to induce granuloma necrosis in a mouse model of mycobacteria‐induced immunopathology. Copyright

Selectin Ligand-Independent Priming and Maintenance of T Cell Immunity during Airborne Tuberculosis

Immunity to Mycobacterium tuberculosis infection is critically dependent on the timely priming of T effector lymphocytes and their efficient recruitment to the site of mycobacterial implantation in the lung. E-, P-, and L-selectin counterreceptors control lymphocyte homing to lymph nodes and leukocyte trafficking to peripheral sites of acute inflammation, their adhesive function depending on fucosylation by fucosyltransferases (FucT) IV and VII. To address the relative importance of differentially glycosylated selectin counterreceptors for priming of T cell effector functions in a model of mycobacteria-induced granulomatous pulmonary inflammation, we used aerosol-borne M. tuberculosis to infect FucT-IV−/−, FucT-VII−/−, FucT-IV−/−/FucT-VII−/−, or wild-type control mice. In lymph nodes, infected FucT-IV−/−/FucT-VII−/− and, to a lesser extent, FucT-VII−/− mice had severely reduced numbers of T cells and reduced Ag-specific effector responses. By contrast, recruitment of activated T cells into the lungs was similar in all four groups of mice during infection and expression of T cell, and macrophage effector functions were only delayed in lungs of FucT-IV−/−/FucT-VII−/− mice. Importantly, lungs from all groups expressed CXCL13, CCL21, and CCL19 and displayed organized follicular neolymphoid structures after infection with M. tuberculosis, which suggests that the lung served as a selectin ligand-independent priming site for immune responses to mycobacterial infection. All FucT-deficient strains were fully capable of restricting M. tuberculosis growth in infected organs until at least 150 days postinfection. Our observations indicate that leukocyte recruitment functions dictated by FucT-IV and FucT-VII-dependent selectin ligand activities are not critical for inducing or maintaining T cell effector responses at levels necessary to control pulmonary tuberculosis.

Mycobacterium tuberculosis embB Codon 306 Mutations Confer Moderately Increased Resistance to Ethambutol In Vitro and In Vivo

ABSTRACT Ethambutol (EMB) is a major component of the first-line therapy of tuberculosis. Mutations in codon 306 of embB (embB306) were suggested as a major resistance mechanism in clinical isolates. To directly analyze the impact of individual embB306 mutations on EMB resistance, we used allelic exchange experiments to generate embB306 mutants of M. tuberculosis H37Rv. The level of EMB resistance conferred by particular mutations was measured in vitro and in vivo after EMB therapy by daily gavage in a mouse model of aerogenic tuberculosis. The wild-type embB306 ATG codon was replaced by embB306 ATC, ATA, or GTG, respectively. All of the obtained embB306 mutants exhibited a 2- to 4-fold increase in EMB MIC compared to the wild-type H37Rv. In vivo, the one selected embB306 GTG mutant required a higher dose of ethambutol to restrict its growth in the lung compared to wild-type H37Rv. These experiments demonstrate that embB306 point mutations enhance the EMB MIC in vitro to a moderate, but significant extent, and reduce the efficacy of EMB treatment in the animal model. We propose that conventional EMB susceptibility testing, in combination with embB306 genotyping, may guide dose adjustment to avoid clinical treatment failure in these low-level resistant strains.

Mycobacteria-induced granuloma necrosis depends on IRF-1.

In a mouse model of mycobacteria‐induced immunopathology, wild‐type C57BL/6 (WT), IL‐18‐knockout (KO) and IFN‐αβ receptor‐KO mice developed circumscript, centrally necrotizing granulomatous lesions in response to aerosol infection with M. avium, whereas mice deficient in the IFN‐γ receptor, STAT‐1 or IRF‐1 did not exhibit granuloma necrosis. Comparative, microarray‐based gene expression analysis in the lungs of infected WT and IRF‐1‐KO mice identified a set of genes whose differential regulation was closely associated with granuloma necrosis, among them cathepsin K, cystatin F and matrix metalloprotease 10. Further microarray‐based comparison of gene expression in the lungs of infected WT, IFN‐γ‐KO and IRF‐1‐KO mice revealed four distinct clusters of genes with variable dependence on the presence of IFN‐γ, IRF‐1 or both. In particular, IRF‐1 appeared to be directly involved in the differentiation of a type I immune response to mycobacterial infection. In summary, IRF‐1, rather than being a mere transcription factor downstream of IFN‐γ, may be a master regulator of mycobacteria‐induced immunopathology.

Control of Mycobacterial Infections in Mice Expressing Human Tumor Necrosis Factor (TNF) but Not Mouse TNF

ABSTRACT Tumor necrosis factor (TNF) is an important cytokine for host defense against pathogens but is also associated with the development of human immunopathologies. TNF blockade effectively ameliorates many chronic inflammatory conditions but compromises host immunity to tuberculosis. The search for novel, more specific human TNF blockers requires the development of a reliable animal model. We used a novel mouse model with complete replacement of the mouse TNF gene by its human ortholog (human TNF [huTNF] knock-in [KI] mice) to determine resistance to Mycobacterium bovis BCG and M. tuberculosis infections and to investigate whether TNF inhibitors in clinical use reduce host immunity. Our results show that macrophages from huTNF KI mice responded to BCG and lipopolysaccharide similarly to wild-type macrophages by NF-κB activation and cytokine production. While TNF-deficient mice rapidly succumbed to mycobacterial infection, huTNF KI mice survived, controlling the bacterial burden and activating bactericidal mechanisms. Administration of TNF-neutralizing biologics disrupted the control of mycobacterial infection in huTNF KI mice, leading to an increased bacterial burden and hyperinflammation. Thus, our findings demonstrate that human TNF can functionally replace murine TNF in vivo, providing mycobacterial resistance that could be compromised by TNF neutralization. This new animal model will be helpful for the testing of specific biologics neutralizing human TNF.

Cathepsin G in Experimental Tuberculosis: Relevance for Antibacterial Protection and Potential for Immunotherapy

Neutrophil serine proteases, such as cathepsin G (CG) and neutrophil elastase (NE), have been implicated in the protective response against infections, including experimental mycobacterial infections. The goal of this study was to explore the role of CG in immunocompetent mice challenged aerogenically with Mycobacterium tuberculosis. We used genetically CG- or CG/NE-deficient mice to define the importance of these neutrophil serine proteases for antibacterial protection, granulomatous response, and survival. In addition, we explored the effect of intratracheally delivered liposomally encapsulated CG/NE as a therapeutic approach early during M. tuberculosis infection. Our data show that the presence of CG or CG/NE prolongs survival in M. tuberculosis–infected mice. However, CG is not directly involved in antibacterial defenses, and exogenous intratracheal administration of CG combined with NE does not reduce bacterial loads in the lungs of M. tuberculosis–infected mice.

11 - Measuring Immune Responses In Vivo

Publisher Summary This chapter measures immune responses In Vivo. When analyzing the immune response during infection in human patients or in experimentally infected mice, the two major goals are (a) to detect differences in the kinetics and magnitude of, for instance, mediator or receptor expression in individual tissues or experimental groups and (b) to define the cellular localization of this response. The most sensitive methods involve amplification strategies of reverse-transcribed mRNAs, for example cytokines and chemokines, in tissue biopsies. Intracellular or surface fluorescence staining and flow cytometric analysis of single cell suspensions isolated from infected tissues may be used to obtain that information but is often not sensitive enough and requires in vitro polyclonal or antigen-specific restimulation of ex vivo isolated cell populations to boost protein expression. The standard procedure to allow detection of molecules associated with individual cells within structurally intact tissue is immunohistochemistry, which may also be employed to gather information on the physiology of the tissue environment. The chapter further explains ex Vivo mRNA analysis by real-time PCR.