Role of hypercytokinemia in NF-kappaB p50-deficient mice after H5N1 influenza A virus infection

Abstract

During H5N1 influenza virus infection, proinflammatory cytokines are markedly elevated in the lungs of infected hosts. The significance of this dysregulated cytokine response in H5N1-mediated pathogenesis remains to be determined. To investigate the influence of hypercytokinemia, or "cytokine storm," a transgenic mouse technology was used. The classical NF-kappaB pathway regulates the induction of most proinflammatory cytokines. Deletion of the p50 subunit leads to a markedly reduced expression of the NF-kappaB-regulated cytokines and chemokines. Here we show that H5N1 influenza virus infection of this transgenic mouse model resulted in a lack of hypercytokinemia but not in altered pathogenesis.

FIG. 1.

mRNA levels of cytokines and chemokines at 6 days p.i. with 5 × 10³ PFU H5N1 in NF-κB p50^+/+ (black bars), NF-κB p50^+/− (gray bars), and NF-κB p50^−/− (white bars) mice after reverse transcriptase real-time PCR performed according to the manufacturer's protocol (Qiagen). (A) IL-2; (B) IL-4; (C) IL-10; (D) IL-6; (E) RANTES; (F) TNF-α; (G) IFN-α; (H) IFN-β; (I) IFN-γ; (J) MIG; (K) MIP-1β; (L) IP-10. The bars represent the mRNA levels for three infected mice compared to those for uninfected controls according to Boeuf et al. (3). This experiment was performed twice with similar results. *, Student's t test revealed no significant difference between the three NF-κB mouse strains (P > 0.1). The amounts of cytokines were also analyzed at the protein level by use of Bio-Plex arrays, except for MIG, IP-10, and IFN-β, revealing similar increases of expression (data not shown).

FIG. 2.

Disease indexes (A), weights (B), and survival rates (C) after H5N1 infection. Five female NF-κB p50^+/+ (black squares), p50^+/− (gray triangles), or p50^−/− (open circles) mice at the age of 6 to 8 weeks, obtained from the animal breeding facilities at the Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Tübingen, Germany, were infected intranasally with 5 × 10³ PFU H5N1 (A/mallard/Bavaria/2006) virus. For the estimation of the disease index, animals were scored with different points depending on their health status. Healthy animals scored 0, animals with one symptom (ruffled fur, reduction of normal activity rate, unnatural posture, or fast breathing rate) scored 1, animals with two symptoms scored 2, animals with three or more symptoms scored 3, and dead animals scored 4 for the rest of the 14-day observation period. The graphs represent the mean values for five animals. This experiment was performed twice with comparable results.

FIG. 3.

mRNA levels of TRAIL (A) and FasL (B) in lungs of NF-κB p50^+/+ (black bars), p50^+/− (gray bars) and p50^−/− (white bars) mice at 6 days p.i. with 5 × 10³ PFU H5N1 virus relative to those in uninfected mice after a reverse transcriptase real-time PCR performed according to the manufacturer's protocol (Qiagen). (C) Western blot analysis for the detection of cleaved caspase-3 (anti-cleaved caspase-3 antibody [Cell Signaling]) in lungs of uninfected (left) and MB1 (H5N1)-infected (right) NF-κB p50^+/+, p50^+/−, and p50^−/− mice at 6 days p.i. Detection of ERK (anti-ERK; Santa Cruz Biotechnology) was used as a loading control. (D) Lymphopenia after H5N1 infection. Whole blood samples were stained with fluorescent antibodies against CD3, CD4, CD8, CD19, and CD11b according to the manufacturer's protocol (BD Pharmingen). Reductions of CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, and CD11b⁺ cells in the blood of infected NF-κB p50^+/+ (black bars), p50^+/− (gray bars), and p50^−/− (white bars) mice at 6 days p.i. with 5 × 10³ PFU H5N1 virus are shown relative to the levels in uninfected mice. Numbers of lymphocytes in the blood of uninfected mice were set at 100%. The bars represent the mean values for four animals. There were no significant differences in the reduction of lymphocytes between the NF-κB p50-deficient mouse strains and C57BL/6 controls (P > 0.1). This experiment was performed twice with similar results.