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Mice Lacking Both TNF and IL-1 Receptors Exhibit Reduced Lung Inflammation and Delay in Onset of Death following Infection with a Highly Virulent H5N1 Virus

  1. Lucy A. Perrone1,
  2. Kristy J. Szretter1,a,
  3. Jacqueline M. Katz1,
  4. Joseph P. Mizgerd2,3,a and
  5. Terrence M. Tumpey1,1
  1. 1Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
  2. 2The Pulmonary Center, Boston University School of Medicine
  3. 3Molecular and Integrative Physiological Sciences Program, Harvard School of Public Health, Boston, Massachusetts
  1. Reprints or correspondence: Dr Terrence M. Tumpey, Influenza Division, NCIRD/ CDC, 1600 Clifton Rd NE, MS G-16, Atlanta, GA 30033 (tft9{at}cdc.gov).

  • ↵a Present affiliations: Department of Internal Medicine, Washington University, St Louis, Missouri (K.J.S.), and The Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts (J.P.M.).

 
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Abstract

Background. Highly pathogenic avian influenza viruses of the H5N1 subtype continue to cross the species barrier to infect humans and cause severe disease. It has been suggested that an exaggerated immune response contributes to the pathogenesis of H5N1 virus infection in mammals. In particular, H5N1 virus infections are associated with a high expression of the proinflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α).

Methods. We investigated the compounding affects of both cytokines on the outcome of H5N1 virus disease by using triple mutant mice deficient in 3 signaling receptors, TNF-R1, TNF-R2, and IL-1-RI.

Results. Triple mutant mice exhibited reduced morbidity and a significant delay in mortality following lethal challenge with a lethal H5N1 virus, whereas no such differences were observed with the less virulent A/PR/8/34 (H1N1) virus. H5N1-infected triple mutant mice displayed diminished cytokine production in lung tissue and a quantifiable decrease of macrophages and neutrophils in the lungs postinfection. Moreover, morphometric analysis of airway sections revealed less extensive inflammation in H5N1-infected triple mutant mice, compared with infected wild-type mice. Conclusions. The combined signaling from the TNF or IL-1 receptors promotes maximal lung inflammation that may contribute to the severity of disease caused by H5N1 virus infection.

The emergence of the highly pathogenic H5N1 avian influenza virus and its associated high lethality still remains a public health concern. Since the reemergence of the H5N1 subtype in 2003, there have been >495 human cases worldwide, of which ∼60% have been fatal [1]. H5N1 virus-infected patients initially present with fever, coughing, dyspnea, and in some cases, radiological evidence of bronchopneumonia [2, 3]. In severe cases, bilateral pneumonia and the development of acute respiratory distress syndrome (ARDS) is followed by progressive respiratory failure [4, 5]. ARDS is a clinical syndrome usually secondary to an intense host cytokine and inflammatory response that includes increased levels of tumor necrosis factor a (TNF-α) [5– 9]. Thus, it has been suggested that elevated levels of cytokines in H5N1-infected patients may contribute to the overall pathogenesis of these viral infections, further complicating recovery [10–13].

The hypothesis that an aberrant cytokine and subsequent cellular immune responses to H5N1 virus detrimentally affect severity and patient outcome has been understudied. In comparison to seasonal influenza strains, H5N1 viruses are potent inducers of TNF and interleukin-1 (IL-1) cytokines both in vivo and in vitro studies [3, 10–15]. In mice, studies demonstrated that no individual proinflammatory cytokine was responsible for the pathogenesis associated with virus or bacterial infection [16–19]. Thus, the lack of these individual cytokine receptors ultimately did not lead to an improvement in host survival following infection with H5N1 virus. However, because of the functional redundancy of some cytokine products, the effect of deleting only 1 cytokine might be masked by compensatory host responses.

To determine the combined contributions of TNF and IL-1 signaling to the inflammatory responses to H5N1 virus infection, we utilized triple mutant mice deficient in TNF (R1 and R2 receptors) and IL-1 (type I receptor) and measured their host responses to a virulent H5N1 virus. We found that triple mutant mice survived longer, exhibited less morbidity, and displayed a reduction in lung inflammation and cytokine response, compared with wild-type mice. The specific immune cell subpopulations contributing to the differences in inflammation observed between infected triple mutant and wild-type mice are also presented here. This study shows an important role of the combined effects of TNF and IL-1 in contributing to the pathogenic outcome of H5N1 virus infection.

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Methods

Virus. Influenza A virus, A/Hong Kong/483/97 (HK/483), isolated from a fatal case [20–22], and A/PR/8/34 (PR8; H1N1) were grown and titered in embryonating hen's eggs, and 50% egg infectious dose (EID50) was calculated as described elsewhere [23, 24]. All experiments were performed in biosafety level 3 laboratories with enhancements as outlined in the Biomedical Microbiological and Biomedical Laboratory [25].

Mice. Mouse strains (aged 6–8 weeks) used in these studies included TNF-R1, TNF-R2, and IL-1-RI triple mutant C57BL/ 6J × 129S background [18, 19, 26, 27]. IL-1-RI single cytokine receptor knock-out mice (IL-1-R knock-out [KO]), C57BL/ 6J × 129Sv -Il1r1tm1Roml/J), and single cytokine receptor TNFR1 KO mice (TNF-R KO, C57BL/6J × 129Sv-Tnfrsf1atm1Imx Tnfrsf1btm1Imx/J) were purchased from The Jackson Laboratory (Bar Harbor, Maine). Age-matched C57B6J × 129 wildtype mice were used as genetic controls and were used for 50% mouse infectious dose (MID50) determination [23].

Inoculations. Mice were anesthetized with Avertin (Sigma- Aldrich) and infected intranasally (50 εL) with 1000 MID50 of HK/483 or PR8 virus [23]. Mice were weighed daily and euthanized when total weight loss exceeded 25% of starting body weight. Survival data and mean days to death were analyzed using SPSS software and Kaplan-Meier survival analysis.

Virus titration and cytokine analysis. Mouse lung tissues were removed on days 3 and 5 postinfection and homogenized in 1 mL of phosphate-buffered saline (PBS). Clarified homogenates were titrated for virus in hens' eggs, as described elsewhere [23], and were expressed as the mean log10 EID50/mL. Statistical differences in virus titers between infection groups were measured by Student's t test. Cytokines were measured in homogenates in duplicate using the Bioplex Protein Array system (Bio-Rad), according to manufacturer's instructions [14]. The mean cytokine values from 6 mock-infected wildtype and triple mutant mice were determined.

Pulmonary histopathology. Lungs were collected from wild-type and triple mutant mice 3 days after intranasal infection with HK/483 virus and were processed for routine histology. Dimensions from a stage micrometer and square grid coherent test systems were reflected onto histology slides with use of a drawing arm microscope. All airway sections with an aspect ratio <2 (ie, cut perpendicular to the longitudinal dimension of the airway) and a diameter of >50 εm from all lung lobes from all mice were included in analyses, for a total of 31–81 distinct airway sections per mouse. The fraction of the airway surface area that was inflamed (defined by the presence of extravasated leukocytes) or denuded (defined by exposed basement membrane) was calculated using standard stereological approaches [19].

Flow cytometry and lung immune cell quantification. Wild-type, IL-1-R KO, TNFR KO and triple mutant mice were infected intranasally with HK/483 virus as described. On days 3 and 5 postinfection, mice were euthanized and exsanguinated, and lungs were removed from individual mice without PBS perfusion. Individual whole lung cell suspensions were prepared as described elsewhere [14], and total viable lung cell numbers were determined by trypan blue exclusion. Lung cell suspensions were incubated with Fc blocking antibody (CD16/32) followed by antibodies to the following cell subpopulations: CD11b- APC (pan-macrophage), CD11c- PE (pan-dendritic), Ly6G/C- FITC (neutrophil), CD3-FITC (pan- T cell), CD4- PE (T cell), and CD8b- APC (T cell) (BD Biosciences). Cells were incubated with antibodies, fixed with 2% paraformaldehyde, and quantified on a LSR II flow cytometer (BD Biosciences). Percentages of cell types were determined by appropriate gating on positively labeled cells. The numbers of cells in each subpopulation were calculated using the percent populations determined by the cytometer statistics and the total lung cell counts. Statistically significant differences in immune cell numbers between experimental groups were determined by analysis of variance.

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Results

Triple mutant mice exhibit a delay in mortality following lethal H5N1 virus challenge. Triple mutant mice lacking the receptors for TNF and IL-1 were inoculated with 1000 MID50 of HK/483 or PR8 virus and mortality was compared with infected wild-type (C57B6 × 129) mice. As shown in Figure 1A, all infected triple mutant and wild-type mice eventually succumbed to H5N1 virus infection; however, the triple mutant mice showed a significant delay in mean time to death, compared with wild-type mice. HK/483-infected triple mutant mice lived an average of 2 days longer (mean days until death, 8), compared with virus-infected wild-type mice (mean days until death, 6) (P < .05). Moreover, infected triple mutant mice lost significantly less weight at earlier time points than infected wildtype mice (Figure 1B), whereas mock-infected controls that received PBS in place of virus gained ∼6% body weight over the observation period (data not shown). In contrast, differences in morbidity and mortality between triple mutant and wild-type mouse strains were not observed among PR8-inoculated mice. These results suggest that the lack of both IL-1- R and TNF-R signaling affects the outcome of H5N1 virus disease in mice.

Figure 1
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Figure 1

Morbidity and mortality in triple mutant (TM) mice. Wild-type (WT) C57B6 × 129 (n = 11 ) and TM mice (n = 7 ) were inoculated intranasally n p 11 n p 7 with 1000 50% mouse infectious doses of A/Hong Kong/483/97 (A and B) or A/PR/8/34 (C and D) virus and monitored daily for survival (A and C) and weight loss (B and D). Kaplan-Meier survival analysis was performed, and significant differences in mean time to death was observed between infected WT and TM mice. Lines represent mean percent weight loss over time for each group, and error bars represent standard errors of the means. * P <.05

Triple mutant mice exhibited similar lung virus titers but displayed increased systemic spread of virus to brain tissue. In an effort to identify biological mechanisms contributing to the enhanced survival of H5N1 virus-infected triple mutant mice, we first assessed virus replication in the lungs of infected mice on days 3 and 5 postinfection, time points that correlated with reductions in weight loss as shown in Figure 1. Triple mutant and wild-type inoculated mice exhibited similar lung virus titers at both time periods (Figure 2). Although virus titers in brain tissue were similar between the infected triple mutant and wild-type groups on day 3 postinfection, virus titers were significantly higher in triple mutant mice, compared with wild-type mice, on day 5 postinfection (P < .05), suggesting a role for TNF and/or IL-1 in controlling extrapulmonary spread of virus.

Figure 2
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Figure 2

Infectious virus in lung and brain tissues. Wild-type (WT) and triple mutant (TM) mice were inoculated intranasally with 1000 50% mouse infectious doses of HK/483 virus, and whole lungs were harvested (4 mice per time point) for virus titration on days 3 (A)and 5 (B) post-infection. The limit of detection was 1 × 101.5 50% egg infectious doses (EID50)/mL. A significant increase in titers was observed from day 3 to 5 in the brains of both WT and TM infected mice. * < .05 , by Student's t test.

Diminished cytokine production in the lungs of H5N1 infected triple mutant mice. We next determined whether cytokine responses were altered in the lungs of triple mutant mice. A panel of cytokines critical for the recruitment and activation of immune cells, shown elsewhere to be secreted at high concentrations in lungs of H5N1-infected mice [14, 17], were measured. Beginning on day 3 postinfection, compared with wildtype mice, the triple mutant infected mice exhibited significantly lower levels of MCP-1 and KC in the lungs, which have been shown to be important for recruitment of monocytes [28] and neutrophils [29], respectively (Figure 3). Conversely, the lung levels of MIP-1α and β, IL-3, IL-12 (p40), IL-12 (p70), and RANTES were generally similar or only moderately higher on day 3 postinfection among triple mutant mice, compared with wild-type mice. However, by day 5 postinfection, all cytokines measured had lower levels in the lungs of infected triple mutant mice, compared with wild-type mice, with significant differences (P < .05) observed in the levels of KC, MIP-1-β, MIP-1-α, IL-3, IFN-γ, G-CSF, and IL-12 (p40).

Figure 3
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Figure 3

Lung cytokine levels from HK/483-infected mice (days 3 and 5 postinfection [p.i.]) were measured individually (4 mice per group) by Bioplex Protein Array, in duplicate. Bars represent means from each infection group ± standard deviation (SD). The mean lung cytokine values among mock-infected wild-type (WT) mice are 10.8 pg/mL for tumor necrosis factor a (TNF-α) and 15.6 pg/mL for RANTES, and the mean cytokine values among mock-infected triple mutant (TM) mice are 11.8 pg/mL for TNF-α and 18.2 pg/mL for RANTES. The following cytokines were not detectable in the mock-infected lungs of either mouse strain: G-CSF, IL-1-α , IL-3, IL-6, IL-12 (p70), IL-12 (p40), MIP-1-α , MCP-1, IL-17, KC, MIP-1-β , and IFN-γ. The minimum cytokine detection was <10 pg/mL. *P < .05, between TM and WT mouse groups for each day p.i., by Student's t test).

Reduced inflammation in the lungs of triple mutant mice. To examine the contribution of TNF and IL-1 signaling to lung inflammation, we performed histopathological analyses of H5N1-infected mouse lung sections (10 wild-type and 7 triple mutant mice). Airway inflammation and injury was examined using morphometry, including all conducting airway sections perpendicular to the longitudinal axis (>30 airway sections per mouse, ranging from 60–620 microns in diameter). There was no detectable inflammation or necrosis among uninfected triple mutant or wild-type mice that received PBS in place in virus (data not shown). The overall type of inflammation, necrotizing bronchitis, and bronchiolitis was qualitatively similar in the 2 genotypes (Figure 4A). However, when the fraction of total airway surface area that was inflamed was quantified, the extent of airway inflammation was lower in the lungs of triple mutant mice, compared with wild-type mice (Figure 4B). The localization of inflammation was similar in both genotypes, with the larger and more central airways exhibiting more inflammation than the smaller and more distal airways (Figure 4C), but triple mutant mice showed less inflammation (across airway sizes) throughout the infected lungs (Figure 4C). Similar fractions of the inflamed regions were necrotic or denuded of epithelial cells regardless of mouse genotype (Figure 4D), suggesting that these cytokine receptors were not essential for progression to epithelial necrosis during airway infection with this H5N1 virus.

Figure 4
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Figure 4

Airway inflammation is reduced in mice infected with H5N1 virus. Triple mutant (TM) and wild-type (WT) mice were infected with HK/483 virus, and 3 days later, lungs were filled with fixative and sections were prepared for histopathological analysis. A, Inflamed airways in TM and WT mice. B, Inflammation throughout the conducting airways was quantified, and percent surface area was calculated as a measure of airway inflammation (7–10 mice per group). C, Pulmonary inflammation was most prominent in larger and more central airways but was less prominent in TM mice throughout the range of airway sizes. Airway diameter was measured throughout the lung sections, and airway sizes were binned within each genotype. Epithelial necrosis is shown in panel D. Inflamed airway sections were screened for areas of cellular necrosis and exposed basement membranes, and the fraction of inflamed airways that was denuded was calculated.

TNF receptor-deficient mice exhibit diminished numbers of macrophages and neutrophils following virus challenge. To discriminate cellular contributions to the differences in inflammation observed between triple mutant and wild-type mice, we measured key immune cell subpopulations in lungs following H5N1 virus or mock (PBS) infection by flow cytometry. For such studies, we included single cytokine KO mice, deficient in the IL-1 receptor (IL-1-R KO) or TNF receptor 1 (TNF-R KO), in attempts to determine whether changes to specific cell populations are attributable to the lack of IL-1R or TNF-R signaling [17]. First, a comprehensive view of the total lung cell profile for each of the KO mice is shown in Figure 5A. The mock wild-type and both single cytokine KO mice exhibited similar total lung cell numbers (1.5×108 mean cells per lung). However, mock triple mutant mice had significantly lower (P < .05) total lung cell numbers (5.0×107 mean cells per lung) at either time point examined. Following H5N1 virus inoculation, all mice exhibited a marked increase (P < .05) in total lung cellularity, compared with their respective uninfected controls, regardless of genetic background. However, triple mutant mice failed to demonstrate an increase in total lung cellularity from days 3 to 5 postinfection (Figure 5A). Thus, infected TNF-R KO, IL-1-R KO, and wild-type mice exhibited an increase of 3.1×107 to 1.1×108 total cells from days 3 to 5 postinfection, whereas a decrease in total lung cell number (by 1.0×107 cells) was measured among H5N1-infected triple mutant mice during the same time period. On day 5 postinfection, infected triple mutant mice had significantly fewer (P = .001) total lung cells than infected single cytokine KO mice (Figure 5A, right panel).

Figure 5
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Figure 5

Effect of cytokine deficiency on lung immune cell populations following H5N1 virus infection. Mice were infected intranasally with 1000 50% mouse infectious doses of HK/483 virus (wild-type [WT], n = 8; triple mutant [TM], n = 7 ; IL-1-R KO, n = 5; TNF-R KO,n = 4) or were given phosphate-buffered saline (PBS) (WT, n = 5; TM, n = 8; IL-1-R KO, n = 3; TNF-R KO, n = 4). Lungs were removed on days 3 and 5 postinfection, and single cell suspensions were prepared from individual lungs. A, Total viable cells from individual lungs were counted on a hemocytometer by trypan blue exclusion prior to antibody labeling and cytometric analysis . Bars represent the average total number of live lung cells (×107 cells) per treatment group ± standard deviation. Statistical differences between treatment groups were measured using the analysis of variance test. *P < .05, between PBS-inoculated mice and H5N1-infected mice in the same genetic group. P < .05, between WT H5N1-infected mice and cytokine-deficient .05 P !.05 H5N1-infected mice. †<.05, between WT PBS-inoculated mice and cytokine-deficient PBS-inoculated mice. ‡<.05, between TM H5N1-infected mice and single cytokine-deficient H5N1-infected mice. B, Lung suspensions from individual mice were stained with fluorescent antibodies and analyzed by flow cytometry to determine immune cell composition following infection. Numbers of macrophages (CD11b+, CD11c−, Ly6G/C+, CD3−, CD4−, CD8−)(a), neutrophils (CD11b+, CD11c+, Ly6G/C+, CD3−, CD4−, CD8−)(b), dendritic cells (CD11b−, CD11c+, Ly6G/C−, CD3−, CD4−, CD8−)(c), and T cell numbers (d and e) (CD11b+ , CD11c−, Ly6G/C−, CD3+ and CD4+ or CD8+) were determined by appropriate gating within the total lung cell population. Bars represent the average number of immune cells (×106 cells) per treatment group ± standard deviation.

Flow cytometry was used to determine the specific immune cell composition in the infected lungs, targeting macrophages, neutrophils, dendritic cells, and T cells. With respect to macrophages, the baseline numbers of these cells were >1×106 cells per lung (range, 1.5×106 to 3.0×106 mean cells/lung) among all PBS-inoculated mouse strains except for PBS-control triple mutant mice, which exhibited 7.2×105 to 8.2×105 mean macrophages per lung on days 3 and 5 postinfection (Figure 5B, panel a). Following H5N1 virus inoculation, the numbers of macrophages increased in the lung tissue in all mouse strains, compared with their respective PBS controls; however, the intensity of macrophage infiltration differed between mouse strains. In IL-1-R KO and wild-type mice on day 3 postinfection, the number of macrophages was ∼20-fold higher among infected mice, compared with PBS-inoculated mice. Infected triple mutant mice exhibited a macrophage increase of 15-fold, compared with PBS-inoculated triple mutant mice; however, no increase in the number of macrophages was observed among infected TNF-R KO mice on day 3 postinfection, and only a 1.8-fold increase of lung macrophages was observed on day 5 postinfection. Importantly, infected wild-type and IL-1-R KO mice exhibited a significantly (P = .002) greater infiltration of macrophages, compared with the other mouse strains lacking TNF receptors.

Differences in the neutrophil response between wild-type and triple mutant or TNF-R KO mice also became apparent following H5N1 infection (Figure 5B, panel b). In general, wildtype and IL-1-R KO infected mice exhibited greater infiltration of neutrophils, compared with TNF-R KO and triple mutant mice. On day 3 postinfection, infected wild-type and IL-1-R KO mice had significantly more neutrophils in their lungs, compared with PBS controls, whereas infected triple mutant and TNF-R KO mice exhibited a more modest increase. Differences in neutrophil numbers between infected wild-type and the other PBS-inoculated groups was most apparent on day 5 postinfection, where infected wild-type mice had 1.5×107 (P = .006) and 6.7×106 more neutrophils than H5N1-infected triple mutant and IL-1-R KO mice, respectively. As was observed with the macrophage dynamics following H5N1 infection, both wild-type and IL-1-R KO mice had more lung neutrophils on both days than either mouse strain lacking TNF receptors.

Dendritic cells were included in this analysis because of their importance in regulating the immune response by bridging innate and adaptive immunity [30]. In general, infection caused an increase in the numbers of dendritic cells, compared with their respective PBS controls; however, the increase among IL- 1-R and TNF-R KO mice was only observed on day 5 post infection (Figure 5B, panel c). In contrast to what was observed for macrophages and neutrophils, dendritic cells were not significantly reduced among infected triple mutant mice and actually exhibited slightly higher numbers in their lungs than all other infected mouse strains on both days examined.

No significant change in total numbers of lung-associated CD4+ T cells was observed among infected wild-type mice on either day, compared with PBS controls, with the exception of IL-1-R KO (P = .014) mice on day 3 postinfection (Figure 5B, panel d). With respect to lung-associated CD8+ T cells, the baseline numbers of this T cell subset was significantly lower among lungs of naive triple mutant mice at both time periods examined (Figure 5B, panel e). In general, CD8+ T cells did not dramatically increase after infection, although infected wildtype mice exhibited slightly higher numbers of CD8+ T cells on day 3 postinfection than any of the cytokine receptor-deficient mice.

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Discussion

ARDS is characterized by diffuse alveolar capillary damage, usually following an intense inflammatory response to infection. Fatal outcome in patients with H5N1 virus infection may be attributed to ARDS characterized by elevated levels of inflammatory mediators [5, 8, 31]. This has prompted the hypothesis that an exacerbated proinflammatory response may contribute to the severity of H5N1 disease [10–15]. TNF and IL-1 are of particular interest in ARDS because they are early response cytokines produced by a variety of pulmonary cells and can instigate a cascade of physiological changes including, recruitment of neutrophils, leading to alveolar capillary damage [5–8]. In a bacterial pneumonia model, inhibiting the actions of TNF or IL-1 individually results in little improvement in lung inflammation, compared with blocking both proinflammatory pathways simultaneously [18, 19, 32], suggesting functional redundancy among these cytokines. Thus, studying the effect of deleting individual cytokines might be of limited significance. In this study, we investigated the effects of inhibiting the actions of both TNF (R1 and R2 receptors) and IL-1 (type I receptor) in mice challenged with a lethal H5N1 virus. We used triple mutant mice to address the hypercytokinemia hypothesis of host pathogenesis and to measure affects of combined cytokine depletion on overall lung inflammation. We observed that H5N1-infected but not H1N1-infected triple mutant mice exhibited reduced morbidity and survived longer, compared with infected wild-type mice. The substantial reduction in lung inflammation and significant delay in mortality were not observed in other H5N1 infection studies testing cytokine KO mice deficient in either TNFR-1 or IL-1 signaling alone [16, 17].

Histologically, the extent of airway inflammation was lower in the lungs of triple mutant mice, compared with wild-type mice, suggesting that TNF and IL-1 signaling pathways contribute to H5N1-induced inflammation of lung tissue. Within the inflamed areas, similar proportions of necrotic or denuded tissue were observed, regardless of mouse genotype, indicating that these cytokine receptors are not essential for progression to epithelial necrosis during H5N1 virus infection. These histopathological observations were supported further by cytometric quantification of immune cell populations in the lung following H5N1 infection. The reduced inflammation observed in H5N1 virus-infected triple mutant mice was largely attributable to the significantly reduced numbers of macrophages and neutrophils observed in lung tissue, compared with wildtype mice. Macrophages and neutrophils accounted for the majority of increased cellularity in the lungs of infected wildtype mice (Figure 5) and in the lungs of H5N1-infected BALB/c mice [14]. Macrophage and neutrophil cell function was not addressed in this study, but there is evidence that TNF-a and IL-1 can activate macrophages and neutrophils [33] and that interrupting TNF-α and IL-1 signaling affects bacterial burdens in pneumonia models [34].

Unlike H5N1, PR8 virus is not a potent inducer of TNF-α and IL-1 cytokines [35], which may partially explain the lack of reduced morbidity and mortality observed among H1N1-infected triple mutant mice. Conversely, the virulent HK/483 virus induced relatively high levels of IL-1α and TNF-α in lungs that persisted up until the death of wild-type (C57B6 × 129) mice, which has been observed in BALB/c mice [17, 35]. We found here that among the 10 cytokines measured in the lungs on day 5 postinfection, levels of 7 were significantly lower in triple mutant mice, compared with wild-type mice. A few cytokines, such as IL-12 (p70) and RANTES, were not reduced in triple mutant mice, suggesting that these proteins are can be produced independent of IL-1 and TNF receptor signaling. Triple mutant mice had lower amounts of 2 key chemokines, MCP-1 and KC. The potent chemoattractant properties of MCP-1 for monocytes and KC for neutrophils are well documented [8, 28]. The question is how these chemokines contribute to H5N1-induced pathogenesis. Normally, IL-1 and TNF receptors are present on the pulmonary epithelium and on neutrophils passing through the interstitium to the alveolar airways [8]. Overall, the inhibition of the TNF receptors has been shown to decrease vascular permeability through the destabilization of endothelial cell cytoskeleton [36, 37] and affects subsequent immune cell transmigration across the endothelium [38, 39]. It has been suggested that alveolar macrophage-produced TNF-α further activates alveolar type II pneumocytes to release KC and MCP-1, which in turn contributes to a proinflammatory cascade and neutrophil sequestration during pulmonary ischemia-reperfusion injury [40–41]. It is interesting to note that type II pneumocytes, not columnar tracheal epithelial cells, are the major site of H5N1 viral replication in patients with fatal H5N1 disease [42–44]. Conceivably, infection of type II pneumocytes and subsequent release of cytokines/chemokines could result in excessive accumulation of neutrophils and macrophages and contribute to tissue damage by causing vascular injury and destruction of the parenchymal cells [45, 46]. The resulting loss of functional alveolar surface area could result in inadequate gas exchange, lower respiration, and ultimately death. Deterioration of pulmonary function has been indicated by the need for hospital ventilator support in H5N1- infected patients [47].

We found that triple mutant mice had no detectable defects in virus clearance in their lungs; however, systemic spread of HK/483, which is a common feature of this H5N1 strain [17], was demonstrated as higher titers were isolated in brain tissues of triple mutant mice. This suggests that, together, TNF and IL-1 are important in controlling extrapulmonary viral spread and should be considered when designing potential prophylactic approaches targeting these cytokines for the treatment of inflammatory conditions induced by H5N1 infection. Treatment with anti-inflammatory drugs has been proposed as a therapeutic option for patients infected with H5N1 viruses; however, preclinical testing in H5N1 virus infection models shows little benefit of these treatments [16, 48, 49]. Moreover, treatments with anti-inflammatory drugs, such as corticosteroids, to combat an exaggerated immune response are far too broad in their effects on the immune system. The development of new, more targeted therapies for H5N1 disease, along with combination antiviral drug treatment, could be effective in reducing acute lung injury and mortality caused by H5N1 virus.

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Acknowledgments

We thank the Hong Kong SAR Ministry of Health for providing the H5N1 virus.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: The American Society for Microbiology/CDC Coordinating Center for Infectious Diseases and the Oak Ridge Institute for Science and Education (fellowships to L.A.P), the National Institutes of Health (R01 HL-068153 to J.P.M), and the Centers for Disease Control and Prevention (J.P.M). Presented in part: IX International Symposium on Respiratory Viral Infections, Bangkok, Thailand. February 2009.

  • Received January 27, 2010.
  • Accepted May 5, 2010.
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