Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: possible role for limiting pathology

Abstract

Reactivation of latent tuberculosis contributes significantly to the incidence of disease caused by Mycobacterium tuberculosis. The mechanisms involved in the containment of latent tuberculosis are poorly understood. Using the low-dose model of persistent murine tuberculosis in conjunction with MP6-XT22, a monoclonal antibody that functionally neutralizes tumor necrosis factor alpha (TNF-alpha), we examined the effects of TNF-alpha on the immunological response of the host in both persistent and reactivated tuberculous infections. The results confirm an essential role for TNF-alpha in the containment of persistent tuberculosis. TNF-alpha neutralization resulted in fatal reactivation of persistent tuberculosis characterized by a moderately increased tissue bacillary burden and severe pulmonic histopathological deterioration that was associated with changes indicative of squamous metaplasia and fluid accumulation in the alveolar space. Analysis of pulmonic gene and protein expression of mice in the low-dose model revealed that nitric oxide synthase was attenuated during MP6-XT22-induced reactivation, but was not totally suppressed. Interleukin-12p40 and gamma interferon gene expression in TNF-alpha-neutralized mice was similar to that in control mice. In contrast, interleukin-10 expression was augmented in the TNF-alpha-neutralized mice. In summary, results of this study suggest that TNF-alpha plays an essential role in preventing reactivation of persistent tuberculosis, modulates the pulmonic expression of specific immunologic factors, and limits the pathological response of the host.

FIG. 1

TNF-α neutralization with the monoclonal antibody MP6-XT22 resulted in disease reactivation in strain C57BL/6 mice persistently infected with M. tuberculosis strain Erdman. C57BL/6 mice were infected with M. tuberculosis Erdman 6 months prior the initiation of TNF-α neutralization (open squares); controls received rat IgG (closed diamonds). The reactivation was associated with increased (A) bacillary burden in lungs, liver, and spleen, respectively (three to five animals were studied per group per time point; bars represent standard error) and (B) mortality rate (six mice were monitored in the rat IgG-treated group [open squares]). There were 12 mice in the MP6-XT22-treated group (open circles). Asterisks, P ≤ 0.05. This experiment was performed three times with similar results.

FIG. 2

Histopathologic studies (H&E stain) of lung tissues from MP6-XT22-treated C57BL/6 mice persistently infected with M. tuberculosis Erdman. Compared to IgG-treated controls (panels A and C), TNF-α-neutralized mice (panels B and D) exhibited marked histopathological deterioration associated with disorganization of granulomata, diffuse infiltration of inflammatory cells, and changes suggestive of fluid accumulation in the alveolar space. Original magnification: ×100 (panels A and B); ×400 (panels C and D).

FIG. 3

Keratin immunoreactivity in the lungs of mice with MP6-XT22-induced reactivation tuberculosis. In mice with reactivation tuberculosis following TNF-α neutralization, areas of tightly bridged squamous cells and/or multinucleated giant cells were observed in the lungs (panel C). These areas contained eosinophilic material that reacted immunohistochemically to a keratin-specific antibody, a finding indicative of squamous metaplasia (panel D). The sections shown are representative of tissues from mice with reactivating tuberculosis after receiving 40 days of MP6-XT22 treatment beginning at 8.5 months postinfection. Keratin immunoreactivity was also observed in areas of lungs with similar histopathologic changes in mice with disease recrudescence following TNF-α neutralization (duration of treatment, 56 days) initiated at 6 months postinfection (B). Immunohistochemical studies revealed the absence of keratin in the lungs of nonreactivating control mice treated with normal rat IgG (duration of treatment, 56 days) beginning 6 months postinfection (A). Tissue sections presented in C were H&E stained. Sections shown in A, B, and D were counterstained with hematoxylin.

FIG. 4

Immunohistochemical and TUNEL analysis of lungs after TNF-α neutralization in mice with persistent tuberculosis. Mice were treated with rat IgG (left panels) or MP6-XT22 (right panels) beginning at 6 months postinfection. Immunohistochemical staining using anti-NOS2 antibodies demonstrated decreased but not absent expression of NOS2 in the lungs of MP6-XT22-treated mice compared to control animals receiving rat IgG. TUNEL staining of lung tissue showed that TNF-α neutralization increased apoptotic activity (arrows) compared with rat IgG treatment. Sections are representative of tissues obtained from mice 59 days after initiation of antibody treatment. Original magnification, ×400.

FIG. 5

Analysis of NOS2 gene expression by RPA using total lung RNA of TNF-α-neutralized mice with persistent tuberculosis. NOS2 gene expression is reported as a ratio to GAPDH expression. Compared to control mice treated with rat IgG (closed diamonds), the expression of NOS2 mRNA in TNF-α-neutralized animals (open squares) was attenuated. Each point is the mean of the results obtained from three to five mice, and bars represent the standard error. Asterisks, P ≤ 0.05. Similar results were obtained in two additional TNF-α-neutralization experiments.

FIG. 6

RPA analysis of cytokine gene expression in lungs of mice with chronic persistent M. tuberculosis infection. Six months after infection with M. tuberculosis, mice were injected with either rat IgG (closed diamonds) or MP6-XT22 monoclonal anti-TNF-α antibody (open squares). Total RNA from lungs was prepared at initiation of antibody treatment (day 0) and at the indicated times therafter. Three to five mice were analyzed at each time point. The autoradiographs were digitized on a flatbed scanner, and gene expression is reported as a ratio to L32 housekeeping genes; combined data for all mice at each time point are shown for the genes for IFN-γ, IL-12, and IL-10. Bars represent the standard error; asterisks, P ≤ 0.05. Similar results were obtained in two additional TNF-α-neutralization experiments.