Guinea Pig Model of Pulmonary Tuberculosis
In the past 30 years, a modest literature has accumulated regarding the link between diet, antimyco-bacterial immunity, and disease resistance in TB. The vast majority of this work has been conducted in a highly relevant guinea pig model of low-dose pulmonary TB. The pathogenesis of TB in this model mimics essentially all of the important aspects of TB in humans.
Early studies established that moderate, chronic deficiencies of protein and other nutrients (e.g., zinc) could be induced in guinea pigs, and that the resulting nutritional states had many of the metabolic hallmarks of human dietary deficiencies. In general, the design of these experiments called for giving BCG vaccine to one group of guinea pigs among a larger group receiving different diet treatments and measuring a number of antigen-specific immune responses in vitro and in vivo several weeks later. Groups of vaccinated and nonvaccinated animals from each diet group were then challenged with an aerosol containing a low dose of virulent M. tuberculosis. The ability of the guinea pigs to control the infection was assessed quantitatively by culture of viable mycobacteria from the lungs and spleens.
Most of the work involving this model has been carried out with moderate, chronic protein deficiency.
Feeding a 10% ovalbumin-based diet over several weeks resulted in a dramatic loss of T cell functions in BCG-vaccinated guinea pigs. Thus, protein-deprived animals had much smaller PPD skin tests and their lymphocytes proliferated poorly to mito-genic and antigenic stimuli in vitro. PPD-stimulated T cells from low-protein animals produced significantly less interleukin (IL)-2 and interferon (IFN), and macrophage-lymphocyte cocultures from malnourished animals produced less tumor necrosis factor-a (TNF-a) in response to infection of the macrophages with virulent M. tuberculosis. Following virulent pulmonary challenge, protein-deficient guinea pigs were unable to form mature, well-circumscribed granulomas in the lung, and they expressed significantly less BCG-induced resistance in the lung and spleen. Not only was BCG-induced protection diminished by protein deficiency but also the response to exogenous reinfection was impaired. Furthermore, although immune cells from normally nourished guinea pigs adoptively protected syngeneic, protein-deficient guinea pigs against aerosol infection, the reverse was not true. That is, immune lymphocytes from low-protein animals did not protect naive, normally nourished recipients.
Protein malnutrition altered the absolute and relative numbers of total T lymphocytes and various subpopulations, including CD2+, CD4+, CD8+, and Fc receptor-bearing T cell subsets in the circulation and lymphoid organs (e.g., spleen and broncho-tracheal lymph nodes draining the infected lung). Taken together, these results imply that protein deficiency is accompanied by alterations in the ability of guinea pigs to regulate the normal recirculation and trafficking of T lymphocytes that would be required for the formation of protective granulomas. These phenomena could be explained by diet-induced changes in the production or function of chemo-kines, which have been observed to be altered in TB, or by perturbations in the expression of adhesion molecules on T cells or endothelial cells.
Finally, macrophages from TB patients are known to produce suppressive factors for T cells, including transforming growth factor-^ (TGF-/3). Alveolar macrophages are particularly effective at downregu-lating T cell proliferation in many species, including humans. Although protein deficiency was not associated with loss of some macrophage functions in the guinea pig model, alveolar macrophages suppress the mitogen-induced proliferation of autologous splenic lymphocytes at macrophage-to-lymphocyte ratios of 1:4 or greater. More important, the intrinsic suppression of alveolar macrophages was enhanced 10-fold in this system when the cells were derived from protein-deficient guinea pigs. In a separate series of experiments, it was demonstrated that TGF-,3 was produced in higher amounts by cells from protein-deprived guinea pigs, and that recombinant human TGF-,3 injected daily into guinea pigs infected with virulent M. tuberculosis suppressed T cell functions and impaired bacillary control in the lungs and spleens of treated animals. Thus, macrophages from protein-deprived guinea pigs appear to be more suppressive for T lymphocyte functions, and this suppression may be mediated, in part, by overproduction of TGF-A
It should be noted that the profound loss of T cell-mediated resistance that accompanies chronic dietary protein deprivation in this model is substantially and rapidly reversible. BCG-vaccinated guinea pigs maintained on a low-protein diet during the entire 6-week period postvaccination, but given a normal diet beginning on the day of virulent pulmonary challenge, displayed PPD skin test reactivity and vaccine-induced control of bacillary loads in the lungs and spleens 2-4 weeks later. These reactions were indistinguishable from those in BCG-vaccinated animals that had never been protein deficient. One possible interpretation of these observations is that protein deficiency interferes with the expression, but not the development, of T cell-mediated protective mechanisms in TB.
These basic observations were confirmed and extended by studies performed in protein-malnourished mice. Using a high-dose, intravenous challenge model, Chan and colleagues observed many of the same T cell defects that have been reported in low-protein guinea pigs, including loss of control of the virulent infection and impaired granuloma formation and also recovery of resistance following refeeding with an adequate diet. They concluded that loss of resistance to TB in their model was a result of diminished nitric oxide (NO) production by activated macrophages, which occurred secondary to an IFN-7 defect in malnourished animals. These are important studies because they confirm the fundamental nature of the effects of protein deprivation in TB even when such crucial variables as host species and infection dose and route are altered.
Zinc Chronic dietary zinc deficiency was found to exert a profound suppressive effect on T lymphocyte functions in BCG-vaccinated guinea pigs. Thus, there was significant anergy in response to PPD skin tests in zinc-deficient animals and dramatic loss of PPD-induced lymphoproliferation in vitro. The activity of a cytokine, macrophage migration inhibitory factor, was also impaired by zinc deficiency. Taken together, these data implied that zinc deficiency interfered with the ability of BCG vaccine to induce protection against virulent, pulmonary challenge. However, no differences were observed between the bacillary loads of zinc-deficient and normally nourished guinea pigs 4 weeks following aerosol infection, and BCG exerted the same protective effect regardless of zinc status in this model.
Vitamin D Calcitriol [1,25(OH)2 vitamin D3] is a potent coactivator of macrophages. Several in vitro studies demonstrated that the addition of calcitriol to cultured human macrophages enhanced the ability of the cells to control the intracellular replication of virulent M. tuberculosis over several days in culture. The role of dietary vitamin D deficiency was examined in the guinea pig model of pulmonary TB. Feeding a diet completely devoid of vitamin D for several weeks resulted in marked depletion of serum levels of the calcitriol precursor, 25(OH) vitamin D3, and resulted in significant loss of some T cell functions in BCG-vaccinated guinea pigs. However, vitamin D deficiency did not alter the course of TB disease in nonvaccinated guinea pigs, nor did it impair the protective efficacy of BCG vaccination in this model.
The previously discussed studies confirm that protein deficiency, in particular, can have devastating consequences on both innate and vaccine-induced resistance against TB in animal models. Certain micronutrient deficiencies, although not as well studied, also appear to affect the immune response to M. tuberculosis, but the effect on the course of the disease is less clear. The precise mechanisms by which diet exerts these effects remain to be elucidated. However, the results of the experiments summarized previously point to defects in T cell trafficking and antigen-induced proliferation, the inability to form mature granulomas, diminished production of 'protective' cytokines (e.g., IL-2, IFN-7, and TNF-a) and antimycobacterial effector molecules (e.g., NO in mice), and increased suppression by adherent cells, perhaps secondary to increased TGF-,3 production.
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