Victoria H. Freedman

Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α/β

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-γ production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-α (TNF-α), IL-6, IL-12, and IFN-γ mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-α mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-α/β. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

A Combination of Thalidomide plus Antibiotics Protects Rabbits from Mycobacterial Meningitis-Associated Death

Tuberculous meningitis (TBM) is a devastating form of tuberculosis that occurs predominantly in children and in immunocompromised adults. To study the pathogenesis of TBM, a rabbit model of acute mycobacterial central nervous system infection was set up (8-day study). Inoculation of live Mycobacterium bovis Ravenel intracisternally induced leukocytosis (predominantly mononuclear cells), high protein levels, and release of tumor necrosis factor-alpha (TNF-alpha) into the cerebrospinal fluid within 1 day. Histologically, severe meningitis with thickening of the leptomeninges, prominent vasculitis, and encephalitis was apparent, and mortality was 75% by day 8. In animals treated with antituberculous antibiotics only, the inflammation and lesions of the brain persisted despite a decrease in mycobacteria; 50% of the rabbits died. When thalidomide treatment was combined with antibiotics, there was a marked reduction in TNF-alpha levels, leukocytosis, and brain pathology. With this combination treatment, 100% of the infected rabbits survived, suggesting a potential clinical use for thalidomide in TBM.

Mycobacterial Antigens Exacerbate Disease Manifestations in Mycobacterium tuberculosis-Infected Mice

ABSTRACT To control tuberculosis worldwide, the burden of adult pulmonary disease must be reduced. Although widely used, Mycobacterium bovis BCG vaccination given at birth does not protect against adult pulmonary disease. Therefore, postexposure vaccination of adults with mycobacterial antigens is being considered. We examined the effect of various mycobacterial antigens on mice with prior M. tuberculosis infection. Subcutaneous administration of live or heat-treated BCG with or without lipid adjuvants to infected mice induced increased antigen-specific T-cell proliferation but did not reduce the bacterial load in the lungs and caused larger lung granulomas. Similarly, additional mycobacterial antigen delivered directly to the lungs by aerosol infection with viable M. tuberculosis mixed with heat-killed Mycobacterium tuberculosis (1:1) also did not reduce the bacillary load but caused increased expression of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), which was associated with larger granulomas in the lungs. When M. tuberculosis-infected mice were treated with recombinant BCG that secreted cytokines shown to reduce disease in a preinfection vaccine model, the BCG secreting TNF-α, and to a lesser extent, IL-2 and gamma interferon (IFN-γ), caused a significant increase in granuloma size in the lungs. Moreover, treatment of M. tuberculosis-infected mice with recombinant murine TNF-α resulted in increased inflammation in the lungs and accelerated mortality without affecting the bacillary load. Taken together, these studies suggest that administration of mycobacterial antigens to mice with prior M. tuberculosis infection leads to immune activation that may exacerbate lung pathology via TNF-α-induced inflammation without reducing the bacillary load.

Effect of cytokine modulation by thalidomide on the granulomatous response in murine tuberculosis

SETTING Experimental murine tuberculosis. OBJECTIVE To evaluate the effect of cytokine modulation by thalidomide on the progression of the lung granulomatous response following aerosol tuberculosis infection in mice. DESIGN Mice infected by the respiratory route with 200-500 viable Mycobacterium tuberculosis Erdman were treated with daily subcutaneous injections of thalidomide (30 mg/kg) or saline for 4 weeks. The bacillary load, granulomatous response and cytokine production in the lungs were evaluated. RESULTS Aerosol M. tuberculosis infection resulted in a progressive granulomatous response in the lungs. At 28 days after infection, large granulomata with central necrosis and no apoptosis were observed. The infection induced high serum and lung cytokine mRNA levels. Thalidomide treatment resulted in a significant reduction in tumor necrosis factor-alpha, interleukin 6 (IL-6) and IL-10 protein levels (blood) and mRNA expression (lungs). IL-12 and interferon-gamma were unaffected. The lungs of thalidomide-treated mice had smaller granulomata with apoptotic cells and no necrosis. Thalidomide treatment did not change the bacillary load. CONCLUSION Thalidomide immunomodulation reduces inflammatory cytokines and concomitant lung pathology following acute aerosol M. tuberculosis infection, without increasing the bacillary load.

AEROSOL INFECTION OF MICE WITH MYCOBACTERIA USING A NOSE-ONLY EXPOSURE DEVICE

The effectiveness of an aerosol producing system (In-Tox Products, Albuquerque, NM) commonly used for toxicological studies of chemicals and radioisotopes was evaluated for experimental tuberculosis infection of mice. Previously, experimental tuberculosis of rodents has been accomplished by aerosol infection devices which exposed the entire animal to the aerosol within a chamber. We utilized the In-Tox system to infect animals via the respiratory route with Mycobacterium bovis strain Bacille Calmette-Guerin (BCG) as a model for experimental pulmonary tuberculosis. The advantage of the device tested is that exposure to the infectious aerosol is limited to the nose of the animal. The present instrument was found to yield good implantation of mycobacteria into the lungs. The infectious load was reproducible and, with the addition of some filtration, the infectious agent was well contained within the device. Our results showed that this device is easy to operate, produces an effective experimental aerosol infection and is safe for the investigator.

Tuberculosis research comes of age

Abstract Keystone Symposium on Tuberculosis: Molecular Mechanisms and Immunologic Aspects Keystone, CO, USA, April 2–8, 1998

Human α-1-acid Glycoprotein Inhibits TNF Production in the CNS of Rabbits with Meningitis: A Report of Preliminary Observations

Bacterial meningitis is accompanied by an acute inflammatory response which may be exacerbated by antibiotic treatment and subsequent killing of bacteria. Bacterial cell products induce the release of cytokines including TNFα, which contribute to the inflammatory process. Alpha-1-acid glycoprotein (AAG), an acute phase reactant, is elevated during inflammation. To test whether AAG has anti-inflammatory activity we examined its effect on lipopolysaccharide-stimulated human peripheral blood mononuclear cells. Treatment of the cells with AAG in vitro resulted in reduced TNFα production. To test the effects of the molecule in vivo, AAG was administered intrathecally to rabbits with Haemophilus influenzae B lysate induced meningitis. Human AAG reduced TNFα production and leukocytosis in the cerebrospinal fluid. Histopathology of the leptomeninges showed markedly attenuated inflammation. These results indicate that AAG can reduce inflammation in rabbits with experimental meningitis and that the effect may be directly on TNFα production by stimulated mononuclear leukocytes.