Role of host cytokine responses in the pathogenesis of avian H5N1 influenza viruses in mice

Abstract

Highly pathogenic avian H5N1 influenza viruses are now widespread in poultry in Asia and have recently spread to some African and European countries. Interspecies transmission of these viruses to humans poses a major threat to public health. To better understand the basis of pathogenesis of H5N1 viruses, we have investigated the role of proinflammatory cytokines in transgenic mice deficient in interleukin-6 (IL-6), macrophage inflammatory protein 1 alpha (MIP-1alpha), IL-1 receptor (IL-1R), or tumor necrosis factor receptor 1 (TNFR1) by the use of two avian influenza A viruses isolated from humans, A/Hong Kong/483/97 (HK/483) and A/Hong Kong/486/97 (HK/486), which exhibit high and low lethality in mice, respectively. The course of disease and the extent of virus replication and spread in IL-6- and MIP-1alpha-deficient mice were not different from those observed in wild-type mice during acute infection with 1,000 50% mouse infective doses of either H5N1 virus. However, with HK/486 virus, IL-1R-deficient mice exhibited heightened morbidity and mortality due to infection, whereas no such differences were observed with the more virulent HK/483 virus. Furthermore, TNFR1-deficient mice exhibited significantly reduced morbidity following challenge with either H5N1 virus but no difference in viral replication and spread or ultimate disease outcome compared with wild-type mice. These results suggest that TNF-alpha may contribute to morbidity during H5N1 influenza virus infection, while IL-1 may be important for effective virus clearance in nonlethal H5N1 disease.

FIG. 1.

Characterization of H5N1 pathogenicity in B6/129 mice. (A) B6/129 mice were infected with 1,000 MID₅₀ of HK/483 or HK/486 virus. Four mice from each virus-infected group were euthanized on day 3, 6, or 9 p.i., and viral titers of individual tissues were determined. Data are expressed as log₁₀ of mean titers ± SE. The limit of virus detection was 10^1.5 EID₅₀/ml. †, HK/483 virus-infected mice did not survive to day 9 postinfection. (B and C) Ten mice were observed for weight loss (B) and survival (C) for 14 days p.i. Data are expressed as a percentage of mean starting weight or total survival.

FIG. 2.

Determination of proinflammatory cytokine levels in lungs of B6/129 mice. B6/129 mice were infected with 1,000 MID₅₀ of HK/483 or HK/486 virus. Four mice from each virus-infected group were euthanized on either day 3 or day 6 p.i., and tissue homogenates were generated for analysis. IL-1β (A), TNF-α (B), IL-6 (C), and MIP-1α (D) levels were assayed by ELISA as described in Materials and Methods. Bars represent mean cytokine concentrations (in picograms per milliliter) ± SE. The horizontal lines denote constitutive levels present in uninfected B6/129 mice. *, P ≤ 0.02.

FIG. 3.

Effect of IL-1R deficiency on H5N1 disease outcome. B6/129 and IL-1R-deficient mice were infected with 1,000 MID₅₀ of HK/483 or HK/486 virus. (A and B) Ten to 15 mice were observed for weight loss (A) and survival (B) for 14 days p.i. Data are expressed as a percentage of mean starting weight or total survival. (C to E) Four mice from each virus-infected group were euthanized on day 3 (C), day 6 (D), or day 9 (E) p.i., and viral titers of individual tissues were determined. Data are expressed as log₁₀ values of mean viral titers ± SE. *, P ≤ 0.02; **, P ≤ 0.0001.

FIG. 4.

Representative images of the histopathologic effect of IL-1R deficiency in mice infected with HK/486 virus. B6/129 and IL-1R-deficient mice were infected with 1,000 MID₅₀ of HK/483 or HK/486 virus. Tissues were removed on the indicated day p.i. and routinely processed for histopathologic and immunohistochemical analyses. (A and B) Representative H&E images showing similar histologic features in lungs of naïve B6/129 (A) and IL-1R-deficient (B) mice. (C to F) Representative H&E images showing inflammatory infiltrates in the lungs of B6/129 (C and E) and IL-1R-deficient (D and F) mice on day 6 and 9 p.i. with HK/486 virus. Immunostaining of influenza virus antigen was observed in bronchiolar epithelial cells of IL-1R-deficient mice on day 9 p.i. (F, inset), whereas B6/129 mice showed only scattered antigen staining in alveolar cells (E, inset). Bars, 100 μm.

FIG. 5.

Effect of TNFR1 deficiency on H5N1 virus disease outcome. B6/129 and TNFR1-deficient mice were infected with 1,000 MID₅₀ of HK/483 or HK/486 virus. (A and B) Ten mice were observed for weight loss (A) and survival (B) for 14 days p.i. Data are expressed as a percentage of mean starting weight or total survival. (C and D) Four mice from each virus-infected group were euthanized on either day 3 (C) or day 6 (D) p.i., and viral titers of individual tissues were determined. Data are expressed as log₁₀ of mean viral titers ± SE. *, P ≤ 0.001; **, P ≤ 0.02.

FIG. 6.

Effect of TNFR1 deficiency on HK/483 virus pathology in lung and thymus. B6/129 and TNFR1-deficient mice were infected with 1,000 MID₅₀ of HK/483 or HK/486 virus. Tissues were removed on the indicated day p.i. and routinely processed for histochemical analyses. Representative H&E images show similar histologic features in naïve lungs and thymuses of B6/129 (A and E) and TNFR1-deficient (B and F) mice. Representative H&E images show similar amounts of inflammatory infiltrates in the lungs of B6/129 (C) and TNFR1-deficient (D) mice on day 6 p.i. with HK/483 virus. Representative H&E images show a higher density of thymocytes in the thymic cortex of TNFR1-deficient mice (H) compared with B6/129 mouse results (G) on day 6 p.i. with HK/483 virus. Bars, 100 μm. C, cortex; M, medulla.