Organ-specific roles for transcription factor NF-kappaB in reovirus-induced apoptosis and disease

Abstract

Reovirus induces apoptosis in cultured cells and in vivo. In cell culture models, apoptosis is contingent upon a mechanism involving reovirus-induced activation of transcription factor NF-kappaB complexes containing p50 and p65/RelA subunits. To explore the in vivo role of NF-kappaB in this process, we tested the capacity of reovirus to induce apoptosis in mice lacking a functional nfkb1/p50 gene. The genetic defect had no apparent effect on reovirus replication in the intestine or dissemination to secondary sites of infection. In comparison to what was observed in wild-type controls, apoptosis was significantly diminished in the CNS of p50-null mice following reovirus infection. In sharp contrast, the loss of p50 was associated with massive reovirus-induced apoptosis and uncontrolled reovirus replication in the heart. Levels of IFN-beta mRNA were markedly increased in the hearts of wild-type animals but not p50-null animals infected with reovirus. Treatment of p50-null mice with IFN-beta substantially diminished reovirus replication and apoptosis, which suggests that IFN-beta induction by NF-kappaB protects against reovirus-induced myocarditis. These findings reveal an organ-specific role for NF-kappaB in the regulation of reovirus-induced apoptosis, which modulates encephalitis and myocarditis associated with reovirus infection.

Figure 1

NF-κB activation following reovirus infection of HLL mice. Newborn HLL mice were inoculated perorally with PBS (A) or 10⁴ PFU reovirus T3SA⁺ (B). Mice were inoculated intraperitoneally with luciferin 7 days after infection and imaged for luciferase activity as a marker for NF-κB activation. Bioluminescence indicates areas of NF-κB activation.

Figure 2

Reovirus-induced NF-κB gel-shift activity following infection of p50^+/+ and p50^–/– mice. (A and D) Newborn p50^+/+ and p50^–/– mice were inoculated perorally with either 10⁴ PFU reovirus T3SA⁺ or PBS. Brains (A) and hearts (D) were resected 12 days after inoculation, and cell extracts were prepared. Extracts were incubated with a ³²P-labeled NF-κB consensus oligonucleotide and resolved by nondenaturing PAGE. Activated NF-κB complexes are indicated. Shown is a representative experiment of 4 performed. (B and E) Extracts were prepared from either brains (B) or hearts (E) of reovirus-infected p50^+/+ mice and incubated with ³²P-labeled NF-κB consensus oligonucleotide in the presence of unlabeled NF-κB consensus probe (cold competitor) at the molar concentrations shown. Extracts prepared from either brains or hearts of uninfected p50^+/+ mice were incubated with ³²P-labeled NF-κB consensus oligonucleotide and electrophoresed in the lane labeled “Mock.” NF-κB complexes are indicated. (C and F) Extracts were prepared from either brains (C) or hearts (F) of reovirus-infected p50^+/+ mice, and prior to the addition of the ³²P-labeled oligonucleotide probe, extracts were incubated with either a control antibody specific to reovirus protein ς3 (α-Reo) or an antibody specific to NF-κB subunit p65 (α-p65). Supershifted complexes containing p65 are indicated.

Figure 3

Growth of reovirus in p50^+/+ and p50^–/– mice. (A) Titers of reovirus in brain after intracranial inoculation of p50^+/+ and p50^–/– mice. Newborn mice were inoculated with 10⁴ PFU reovirus T3SA⁺. At days 2, 4, and 6 after inoculation, mice were euthanized, brains were harvested, and viral titers were determined by plaque assay. (B) Titers of reovirus in intestine, liver, brain, and heart after peroral inoculation of p50^+/+ and p50^–/– mice. Newborn mice were inoculated with 10⁴ PFU T3SA⁺. At days 4, 6, 8, 10, and 12 after inoculation, mice were euthanized, organs were harvested, and viral titers were determined by plaque assay. The results are expressed as the mean viral titers for 2–4 (A) or 4–8 (B) animals for each time point. Error bars indicate SDs. *P < 0.05 by Student’s t test.

Figure 4

Inflammation, reovirus protein expression, TUNEL staining, and immunohistochemical detection of activated caspase-3 in the brain of reovirus-infected p50^+/+ (A) and p50^–/– (B) mice. Newborn mice were inoculated perorally with 10⁴ PFU reovirus T3SA⁺. At 12 days after inoculation, brains were harvested, paraffin embedded, sectioned, and stained with H&E, polyclonal reovirus-specific antiserum (Reo), TUNEL, or activated caspase-3–specific antiserum as indicated. Shown are consecutive sections of diencephalon. Original magnification, ×100 (top panels) and ×400 (bottom panels). (C) Newborn mice were inoculated intracranially with 10⁴ PFU T3SA⁺ or gelatin saline (Mock). At 6 days after inoculation, mice were euthanized, and brain sections were stained using a TUNEL assay. Shown are sections of the upper brain stem. Original magnification, ×200. Brown staining indicates reovirus protein, fragmented DNA, or activated caspase-3.

Figure 5

Quantitation of TUNEL staining in cortex and hippocampus (A), basal ganglia and diencephalon (B), and brain stem (C) of reovirus-infected p50^+/+ and p50^–/– mice. TUNEL staining was performed using tissue sections prepared 2, 4, and 6 days following intracranial inoculation of p50^+/+ and p50^–/– mice with 10⁴ PFU reovirus T3SA⁺. For each brain region, all positive cells in a single parasagittal section were counted for 4–8 animals. The results are expressed as the mean number of apoptotic cells per region. Error bars indicate SDs. *P < 0.05 by Student’s t test.

Figure 6

Heart pathology following reovirus infection of p50^+/+ and p50^–/– mice. (A and B) Newborn p50^+/+ and p50^–/– mice were inoculated perorally with either PBS (mock) (A) or 10⁴ PFU reovirus T3SA⁺ (B), and heart size was monitored at 2-day intervals. Percent heart weight was calculated as heart weight divided by body weight. The results are expressed as the mean heart weights of at least 4 animals for each time point. Error bars indicate SDs. *P < 0.05 by Student’s t test. (C) Hearts from mice euthanized 12 days following peroral inoculation with reovirus T3SA⁺ or gelatin saline (Mock). (D, E, and F) Electrocardiography and echocardiography of reovirus-infected p50^–/– (D), mock-infected p50^–/– (E), and reovirus-infected (F) p50^+/+ mice. Newborn mice were inoculated perorally with 10⁴ PFU T3SA⁺, and tests were performed 10 days after inoculation. A P-wave/QRS ECG complex is displayed above the corresponding echocardiographic image. Systolic and diastolic LV cavity dimensions are indicated by bars superimposed on the M-mode images.

Figure 7

Inflammation, reovirus protein expression, TUNEL staining, and immunohistochemical detection of activated caspase-3 in the heart of reovirus-infected p50^+/+ (A) and p50^–/– mice (B). Newborn mice were inoculated perorally with 10⁴ PFU reovirus T3SA⁺. At 12 days after inoculation, hearts were harvested, paraffin embedded, sectioned, and stained with H&E, polyclonal reovirus-specific antiserum, TUNEL, or activated caspase-3–specific antiserum as indicated. Original magnification, ×25 (top panels) and ×400 (bottom panels). Brown staining indicates reovirus protein, fragmented DNA, or activated caspase-3.

Figure 8

Levels of IFN-β mRNA in brain and heart of p50^+/+ and p50^–/– mice. Newborn mice were inoculated perorally with either PBS (Mock) or 10⁴ PFU reovirus T3SA⁺. At 12 days after inoculation, brains and hearts were resected, and whole-organ RNA was isolated and used as a template to generate cDNA. Levels of IFN-β and GAPDH cDNA were assessed by real-time PCR. The results are expressed as the mean ratio of IFN-β cDNA to that of GAPDH for 2 animals. Error bars indicate SDs.

Figure 9

Reovirus replication and apoptosis in infected p50^–/– mice following treatment with IFN-β. Newborn mice were inoculated intraperitoneally with either IFN-β or PBS 1 day prior to peroral inoculation with 10⁴ PFU reovirus T3SA⁺. Animals were treated with either IFN-β or PBS for an additional 9 days, and brains and hearts were resected. (A) Viral titers in the brain and heart. Organs were homogenized, and viral titers were determined by plaque assay. The results are expressed as the mean viral titers for 3 animals. Error bars indicate SDs. *P < 0.05 by Student’s t test. (B) Histopathology of reovirus infection in the heart. Hearts of the reovirus-infected p50^–/– animals represented in A were paraffin embedded, sectioned, and stained with polyclonal reovirus-specific antiserum or processed for TUNEL analysis. Original magnification, ×100. Brown staining indicates reovirus protein or fragmented DNA.