Disruption of nuclear factor-interleukin-6, a transcription factor, results in severe mycobacterial infection

Abstract

Nuclear factor-interleukin-6 (NF-IL-6) is one of several nuclear transcription factors (NF-IL-6, NF-kappaB, PU.1, interferon-regulatory factor 1, Egr-1, and Stat-1). NF-IL-6 and NF-kappaB are expressed in macrophages and is induced by bacterial lipopolysaccharides. To evaluate whether NF-IL-6 is required for the inflammatory immune response to mycobacterial infection, in which epithelioid macrophages comprise the leading cell population, we generated NF-IL-6 knockout (KO) mutant mice. Airborne infection of these mice with Mycobacterium tuberculosis strains induced disseminated tuberculosis lacking granuloma formation, although interferon-gamma, tumor necrosis factor-alpha, and interleukin-12 mRNA expression levels were within the normal range compared with those of wild-type mice. Generation of O2- and mycobacterial killing by neutrophils from these mice were impaired severely compared with wild-type mice. We conclude that NF-IL-6 is a critical transcription factor in mycobacterial control as well as in granulocyte-colony stimulating factor induction resulting in neutrophil activation.

Figure 1.

Survival of mice infected with M. tuberculosis Kurono strain. WT and NF-IL-6 KO mice were infected with 10 CFU of the Kurono strain by an airborne route. Percentages of surviving WT (solid circles) and NF-IL-6 KO mice (open squares) are shown.

Figure 2.

CFU in lung and spleen tissues of NF-IL-6 KO and WT mice exposed to 10 CFU of H37Rv strain by the airborne route. At the indicated weeks after infection, four mice from each group were sacrificed and homogenates of lung and spleen tissues were plated. Error bars indicate standard errors of the means.

Figure 3.

Macroscopic appearance of lungs from NF-IL-6 KO (a) and WT (b) mice infected with the Kurono strain by an airborne route. Whitish necrotic nodules are noted in the lungs of NK-IL-6 mice, but nodular lesions are noted in the lungs of WT mice.

Figure 4.

Histological analysis of lung sections after infection with M. tuberculosis (Kurono strain). Lungs were removed from NF-IL-6 KO (a) and WT mice (b) 38 days and 50 days, respectively, after infection with Kurono strain (10 CFU) by an airborne route. Hematoxylin and eosin stain; original magnification, ×200. c: NF-IL-6 KO mice infected with Kurono strain were injected subcutaneously three times at day 0 and at weekly intervals with recombinant murine G-CSF (2 μg/mouse). Thirty-eight days later, the mice were sacrificed and the issues were fixed with 10% buffered formalin. Hematoxylin and eosin stain; original magnification, ×200. d: Stain for acid-fast bacilli in the lung of NF-IL-6 infected with Kurono strain. Original magnification, ×600.

Figure 5.

RT-PCR analysis of cytokine mRNA expression. Total RNA was isolated from lung tissues of NF-IL-6 KO and WT mice infected with M. tuberculosis and subjected to RT-PCR using cytokine-specific primers. G-CSF mRNA expression is lower in the lung of NF-IL-6 mice than that of WT mice. Lung fibroblasts from NF-IL-6 KO mice G-CSF-mRNA significantly.

Figure 6.

CFU assay of peritoneal neutrophils from NF-IL-6 mice. The peritoneal neutrophis (10⁵) were cultured in the presence of H37Rv (5 × 10⁶) in 96-well flat-bottomed microculture plates 2 hours after infection with H37Rv or overnight. The neutrophils were also cultured with H37Rv in the presence of recombinant murine G-CSF (2 × 10⁻ μg) overnight. Thereafter, the suspension containing peritoneal neutrophils were cultured for 4 weeks in Lowenstein-Jensen media and the resulting colonies were counted.