Role of Host Cytokine Responses in the Pathogenesis of Avian H5N1 Influenza Viruses in Mice

Highly pathogenic avian H5N1 influenza viruses are now widespreadin poultry in Asia and have recently spread to some Africanand European countries. Interspecies transmission of these virusesto humans poses a major threat to public health. To better understandthe basis of pathogenesis of H5N1 viruses, we have investigatedthe role of proinflammatory cytokines in transgenic mice deficientin interleukin-6 (IL-6), macrophage inflammatory protein 1 alpha(MIP-1 ${alpha}$ ), IL-1 receptor (IL-1R), or tumor necrosis factor receptor1 (TNFR1) by the use of two avian influenza A viruses isolatedfrom 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 andspread in IL-6- and MIP-1 ${alpha}$ -deficient mice were not differentfrom those observed in wild-type mice during acute infectionwith 1,000 50% mouse infective doses of either H5N1 virus. However,with HK/486 virus, IL-1R-deficient mice exhibited heightenedmorbidity and mortality due to infection, whereas no such differenceswere observed with the more virulent HK/483 virus. Furthermore,TNFR1-deficient mice exhibited significantly reduced morbidityfollowing challenge with either H5N1 virus but no differencein viral replication and spread or ultimate disease outcomecompared 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 innonlethal H5N1 disease.

INTRODUCTION

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Introduction

Highly pathogenic avian (HPAI) influenza A H5N1 viruses werefirst recognized to be capable of infecting humans in 1997,when wholly avian influenza viruses were transmitted directlyfrom birds to humans in Hong Kong, causing 18 cases of respiratorydisease, including six deaths (1, 8, 24, 48, 51, 52). Two additionalhuman cases of H5N1 virus infection were identified in HongKong in early 2003 (37). Since late 2003, HPAI H5N1 viruseshave spread across Asia to the Middle East and into Europe andAfrica, causing outbreaks of disease and death in domestic poultryand wild birds. As of September 2006, approximately 240 laboratory-confirmedhuman cases of H5N1 virus infection have been reported by theWorld Health Organization. Over 50% of the human infectionshave been fatal (3, 58; Cumulative number of confirmed humancases of avian influenza A/(H5N1) reported to WHO, World HealthOrganization 2006, http://www.who.int/csr/disease/avian_influenza/country/cases_table_2006_09_19/en/index.html).

As in the 1997 outbreak, the recent human H5N1 infections haveoccurred primarily among previously healthy children and youngeradult patients, who typically developed fever, respiratory symptoms,leukopenia, and lymphopenia before progressing to primary viralpneumonia complicated by acute respiratory distress syndrome(3, 6, 58, 63). Limited reports of viremia and detection ofviral RNA in extrapulmonary tissues and fluids suggest thatH5N1 viruses can disseminate systemically in humans, at leastin some individuals (7, 11, 60). Elevated levels of serum cytokinesand chemokines accompanied these clinical manifestations. Theoccurrence of this "cytokine storm" has been proposed to contributeto the increased severity of the disease caused by these viruses(10, 37, 56, 63). In support of this hypothesis, H5N1 viruseswere found to elicit substantially higher expression of proinflammatorychemokines and cytokines, in particular tumor necrosis factoralpha (TNF- ${alpha}$ ), in human primary macrophages compared with someH1N1 or H3N2 human influenza viruses (5). To assess the contributionof the proinflammatory cytokine responses to severe diseasein vivo, we used a mouse model which generally reflects theseverity of human H5N1 virus disease (30, 32, 59), althoughthe central nervous system involvement typical of highly virulentH5N1 strains in mice may only be a rare complication in H5N1virus-infected humans (11). Although mouse and human respiratorytissues differ in their overall distribution and type of sialicacid receptor expression, ${alpha}$ -2,3-linked sialic acid, preferentiallyrecognized by avian influenza viruses, is present in the lowerrespiratory tract of both species (21).

In BALB/c mice, H5N1 viruses isolated from humans in 1997 werefound to replicate efficiently without the need for adaptationand could be characterized as being of high or low pathogenicity(14, 18, 24, 25, 32, 59). Systemic spread of virus, cytokinedysregulation, lymphopenia, severe tissue pathology, and deathwere characteristic of the infection of mice with the high-pathogenicityphenotype, represented by A/Hong Kong/483/97 (HK/483) virus,whereas viruses of the low-pathogenicity phenotype, representedby A/Hong Kong/486/97 (HK/486) virus, did not to replicate systemicallyand were generally not lethal for mice. Expression of proinflammatorycytokines and chemokines, including interleukin-1ß(IL-1ß), TNF- ${alpha}$ , IL-6, and macrophage inflammatory protein1 alpha (MIP-1 ${alpha}$ ), in the brain of HK483 virus-infected mice wasassociated with the lethal phenotype of this virus (2, 36, 53,59). These findings suggest that the mouse is suitable as amodel to investigate the role of the host's proinflammatoryresponse in the exceptional virulence of the avian H5N1 influenzaviruses in mammals.

Here we use transgenic C57BL/6 (B6) or B6/129 mice deficientin TNF receptor 1 (TNFR1), IL-1 receptor (IL-1R), IL-6, andMIP-1 ${alpha}$ to better understand the role that these cytokines andchemokines play during H5N1 influenza virus infection. We foundthat a lack of IL-6 or MIP-1 ${alpha}$ had no effect on the kinetics ofthe disease process or virus replication in H5N1 virus-infectedmice, whereas a lack of IL-1R signaling delayed viral clearanceand enhanced the lethality of the low-pathogenicity virus. Incontrast, absence of TNFR1 signaling significantly delayed morbiditybut had no effect on virus replication or on the ultimate outcomeof H5N1 disease in mice. These results provide insight intothe mammalian host immune response during avian H5N1 influenzaA virus infection which is important for the design of immunomodulatorytherapies for H5N1 infection.

MATERIALS AND METHODS

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Materials and Methods

Viruses. The influenza viruses used in this study were the H5N1 virusesA/Hong Kong/483/97 (HK/483) and A/Hong Kong/486/97 (HK/486).Virus stocks were propagated in the allantoic cavity of 10-day-oldembryonated chicken eggs at 37°C for 24 h. The allantoicfluids were harvested, aliquoted, and stored at –70°Cuntil use.

Laboratory facility. Because of the potential risk to humans and poultry, all experimentsusing infectious pathogenic avian H5N1 viruses, including workwith animals, were conducted under conditions of biosafety level3 containment that included enhancements required by the U.S.Department of Agriculture and the Select Agent Program (42).Laboratory workers were required to wear appropriate respiratorequipment (RACAL Health and Safety Inc., Frederick, MD).

Mice. IL-1R (B6; 129S1-Il1r1^tm1Roml)-, IL-6 (B6; 129S2-Il6^tm1Kopf)-,MIP-1 ${alpha}$ (B6; 129P2-Ccl3^tm1Unc/J)-, and TNFR1 (B6; 129S-Tnfrsf1a^tm1ImxTnfrsf1b^tm1Imx)-deficientmice aged 6 to 8 weeks and age-matched C57BL/6J (B6) or B6129SF2/J(B6/129) control mice (Jackson Laboratories, Bar Harbor, ME)were used for these studies. Animal research was conducted underthe guidance of the CDC's Institutional Animal Care and UseCommittee in an Association for Assessment and Accreditationof Laboratory Animal Care International-accredited animal facility.

Viral challenge. Mice were lightly anesthetized with CO₂ and infected intranasallywith 1,000 50% mouse infectious doses (MID₅₀) (10^3.75 egg infectiousdoses [EID₅₀] of HK/483 and 10^5.25 EID₅₀ of HK/486) of virusstocks diluted in phosphate-buffered saline (PBS) in a 50-µlvolume. The MID₅₀ was determined for each mouse strain as previouslydescribed (30). Mice were monitored daily for morbidity andmeasured for weight loss and mortality for up to 14 days postinfection(p.i.). Any mouse that lost more than 25% of its body weightwas euthanized. Due to the limited availability of the transgenicmouse strains a separate experiment was conducted to evaluatevirus replication. Mice were infected with 1,000 MID₅₀, andlung, brain, spleen, and thymus samples were collected fromfour mice each on days 3 and 6 p.i. The samples were immediatelyfrozen and stored at –70°C for subsequent determinationof infectious virus and cytokine protein content.

Viral titration. Tissues were homogenized in 1 ml of cold PBS. For analysis ofinfectivity, the titers of the virus were determined using clarifiedtissue homogenates in eggs from initial dilutions of 1:10. Thelimit of virus detection was 10^1.5 EID₅₀/ml. Virus titers werecalculated by the method of Reed and Muench and are expressedas the mean log₁₀ EID₅₀ per milliliter ± standard errors(SE) of the means (41). Tissues in which no virus was detectedwere given a value of 10^1.5 EID₅₀/ml for calculation of themean titer.

Quantification of cytokines and chemokines. Tissue homogenates from days 3 and 6 p.i. were analyzed by useof an enzyme-linked immunosorbent assay (ELISA) kit (R&DSystems, Minneapolis, MN) according to the manufacturer's protocol.Tissue homogenates were analyzed for levels of IL-1ß,IL-6, MIP-1 ${alpha}$ , and TNF- ${alpha}$ .

TNF- ${alpha}$ neutralization in mice. C57Bl/6 mice were given three doses of either 0.2 ml of rabbitanti-mouse TNF- ${alpha}$ polyclonal sera (representing 250,000 neutralizingunits) (kindly provided by Michael Luster, National Institutefor Occupational Safety and Health, CDC) or 200 µg ofrat neutralizing antisera raised against recombinant mouse TNF- ${alpha}$ (Upstate Biotechnology, Chicago, IL) by the intraperitonealroute (31, 33). Control mice were injected with normal rabbitor rat sera or PBS. Mice received treatment 1 day before infectionand on the day of infection and 1 and 4 days p.i. Mice werebled from the orbital plexus at different intervals to ensurethat saturating levels of antibody were achieved.

Histopathologic and immunohistochemical analysis. Four mice were euthanized on days 3 and 6 p.i. Brain, lung,spleen, and thymus tissues from each time point and each experimentalgroup were removed and fixed in formalin, routinely processed,and embedded in paraffin. Histopathologic examination was performedby using hematoxylin- and eosin (H&E)-stained sections.For antigen staining, a colorimetric indirect immunoalkalinephosphatase immunohistochemistry method was developed as previouslydescribed (64, 65). The primary antibody used in the immunohistochemistryassay was a goat antiserum to A/Tern/South Africa/61 (H5N3)(National Institute of Allergy and Infectious Diseases, Bethesda,MD) that recognizes both surface glycoproteins and internalproteins of the virus.

Peripheral blood leukocyte counts. On days 0, 3, and 6, blood samples were collected from the orbitalplexus of four mice per group. Absolute leukocyte counts wereperformed with heparinized blood diluted 1:10 with Turk's solution(2% acetic acid, 0.01% crystal violet [vol/vol], double-distilledwater). For differential counts, peripheral blood smears werestained with Hema-3 stain (Fisher Diagnostics, Orangeburg, NY),and the numbers of monocytes, neutrophils, and lymphocytes weredetermined.

Statistical analysis. The statistical significance of viral titer and morbidity datawas determined by using a two-tailed, paired Student's t test.The statistical significance of mortality data was determinedby using the null model likelihood ratio test and the Mann-Whitneytest.

RESULTS

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Results

Characterization of H5N1 virus in B6/129 wild-type mice. Although H5N1 viruses are known to replicate efficiently inBALB/c mice (12, 30, 32), replication and lethality of H5N1viruses in B6 or B6/129 mouse strains, typically used to derivecytokine- or chemokine-deficient mice, have not been reportedpreviously. In preliminary experiments, we determined that B6mice exhibited weight loss and lethal disease following infectionwith H5N1 viruses comparable to that observed with the establishedBALB/c mouse model. Titers (50% lethal doses) for the highlylethal HK/483 virus were identical in the two mouse strains(10^0.5 EID₅₀ per 50 µl). We next characterized the relativevirulence of HK/483 and HK/486 viruses for B6/129 mice. Micewere inoculated intranasally with 1,000 MID₅₀ of either of theH5N1 influenza A viruses, a dose that was determined previouslyto consistently produce the two distinct pathogenicity phenotypesin mice, and the kinetics of virus replication, morbidity, andmortality were determined. Lung virus titers measured on days3 and 6 p.i. were high (10^5.25 to 10^6.5 EID₅₀/ml) for both virusesand were not significantly different from each other (Fig. 1A).However, by day 9 p.i. all HK/483 virus-infected mice had succumbed,while 90% of HK/486 virus-infected mice survived (Fig. 1C) andexhibited a mean 4 log₁₀ reduction in lung virus titers comparedwith day 6 titer results (Fig. 1A), indicating that these micewere effectively clearing the viral infection. Furthermore,HK/486 virus-infected mice began to regain weight by day 9 p.i.and had almost returned to preinfection weights by the end ofthe experiment (Fig. 1B). Therefore, both B6 and B6/129 micereproduce the pathogenicity phenotypes for H5N1 viruses previouslyestablished for the BALB/c mouse.

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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. ${dagger}$ , 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.

Expression of cytokines in lungs of H5N1-infected B6/129 mice. To determine the proinflammatory cytokine and chemokine levelsfollowing H5N1 virus infection in B6/129 wild-type mice, lungsfrom mice infected with 1,000 MID₅₀ of HK/483 or HK/486 H5N1influenza A viruses were collected on days 3 and 6 p.i., andhomogenates were subsequently assayed for IL-1ß, IL-6,TNF- ${alpha}$ , and MIP-1 ${alpha}$ levels by ELISA. The levels of all cytokineswere substantially greater than constitutive levels in the lungsof B6/129 mice by day 3 p.i. (Fig. 2). However, in HK/483 virus-infectedmice, IL-1ß levels were significantly reduced (P $≤$ 0.02) (Fig. 2A), whereas IL-6 (P $≤$ 0.02) and TNF- ${alpha}$ levels wereelevated, although the latter result was not significant comparedwith levels found in HK/486 virus-infected mice (Fig. 2B and2C). The levels of MIP-1 ${alpha}$ induced by either H5N1 virus in thelungs on day 6 p.i. were also significantly elevated comparedwith levels in normal mice (Fig. 2D). The cytokine and chemokinepatterns detected in the brain were similar to those previouslyobserved in BALB/c mice in that they were detected in HK/483-infectedmice but not in HK/486-infected mice (data not shown) (2, 56).Therefore, the data from the B6/129 mouse model of H5N1 virusinfection are consistent with our previous findings for theBALB/c mouse and suggest that differential induction of proinflammatorycytokines by HK/483 and HK/486 viruses may contribute to theobserved differences in severity of disease caused by theseH5N1 viruses (59). Thus, B6 or B6/129 cytokine- or chemokine-deficientmice can be used to investigate the role of individual cytokinesor chemokines in the H5N1 virus disease process.

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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-1ß (A), TNF- ${alpha}$ (B), IL-6 (C), and MIP-1 ${alpha}$ (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.

IL-6 and MIP-1 ${alpha}$ have little effect on the outcome of H5N1 influenza virus disease. Because of the elevated levels of IL-6 and MIP-1 ${alpha}$ found duringH5N1 virus infection, we first examined the role of these immuneproducts in mice deficient in either IL-6 cytokine or MIP-1 ${alpha}$ chemokine. IL-6- or MIP-1 ${alpha}$ -deficient mice or B6 wild-type controlmice were infected with 1,000 MID₅₀ of HK/483 or HK/486 H5N1viruses and were monitored for 14 days. IL-6- and MIP-1 ${alpha}$ -deficientmice and wild-type mice infected with HK/483 or HK/486 virusexhibited similar kinetics of weight loss and mortality (datanot shown). Furthermore, viral titers in lung, brain, spleen,or thymus tissue on days 3 and 6 p.i. for either H5N1 virusin IL-6- or MIP-1 ${alpha}$ -deficient mice were comparable to wild-typecontrol titers (data not shown). These results suggest thata lack of IL-6 or MIP-1 ${alpha}$ does not affect the outcome of H5N1virus disease in mice.

IL-1 promotes clearance and protects against virus-induced mortality. Because of the reduced levels of IL-1ß in B6/129 miceduring HK/483 virus infection, we next determined what roleIL-1 may have during H5N1 disease in IL-1R-deficient mice infectedwith 1,000 MID₅₀ of HK/483 or HK/486 viruses. There was no differencein weight loss, survival, or viral titers on days 3 or 6 p.i.observed between HK/483 virus-infected IL-1R-deficient and controlmice. In contrast, IL-1R-deficient mice infected with HK/486virus lost significantly more weight (P $≤$ 0.02) and a greaterpercentage of HK/486 virus-infected IL-1R-deficient mice succumbedto infection after day 8 p.i. compared with B6/129 wild-typemouse results, although this difference did not reach statisticalsignificance (Fig. 3B). There was no difference in viral titersbetween the IL-1R-deficient and control mice on day 3 or 6 p.i.(Fig. 3C and D). However, because the differences observed inweight loss and lethal outcome between IL-1R-deficient miceand wild-type controls infected with HK/486 virus occurred latein the time course of infection, we also assessed viral titerson day 9 p.i. At this time point, HK/486 lung viral titers inIL-1R-deficient mice were 1,000-fold higher than those in wild-typemice (Fig. 3E; P $≤$ 0.0001). The lungs from HK/486 virus-infectedmice of either strain, collected on days 6 and 9 p.i., werealso examined histologically. On day 6 p.i., the lungs fromwild-type control mice exhibited more inflammatory infiltratethan those from the IL-1R-deficient mice, although differenceswere modest (Fig. 4C and D), and immunohistochemical analysesrevealed no visible differences in antigen amounts or distributionbetween the two groups of mice (data not shown). Conversely,by day 9 p.i. the lungs from IL-1R-deficient mice displayedmore-extensive inflammation than those from control mice (Fig.4E and F). Furthermore, immunohistochemical analyses revealedmore-abundant viral antigen in the bronchiolar epithelial cellsin IL-1R-deficient mice, which is consistent with the increasedviral titers detected in these mice versus control mice at day9 p.i. (Fig. 4E, inset; Fig. 4F, inset; and Fig. 3E). Thesedata suggest that the IL-1 response may be important for effectiveclearance of the less-virulent H5N1 virus in mice.

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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.

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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.

TNF- ${alpha}$ contributes to H5N1 virus disease. TNF- ${alpha}$ , a key regulator of inflammation (58), is produced in mouseorgans in response to H5N1 virus replication. To assess thecontribution of TNF- ${alpha}$ production in H5N1 virus pathogenesis,TNFR1 knockout mice were infected with 1,000 MID₅₀ of HK/483or HK/486 virus and the levels of virus replication, morbidity,and mortality were evaluated as described above. Infection ofTNFR1-deficient mice with the highly virulent HK/483 virus resultedin a significant delay in weight loss compared with wild-typemouse infection results from day 3 to 7 p.i. (P $≤$ 0.001 to 0.02)(Fig. 5A), although the data with respect to mean maximum weightlost by the two group of mice were not significantly differentand HK/483 virus infection was ultimately lethal for eithergroup (Fig. 5B). A similar delay in weight loss was observedfor TNFR1-deficient mice infected with HK/486 virus (Fig. 5A),but the majority of animals survived infection with this strain(Fig. 5B). Despite these differences in clinical illness, therewas no significant difference in viral titers in lung, brain,spleen, and thymus tissue for either H5N1 virus between TNFR1-deficientmice and wild-type controls on day 3 or 6 p.i. (Fig. 5C and D).Results similar to those obtained with the TNFR1-deficient micewere obtained with wild-type B6 mice infected with HK/483 virusand administered neutralizing anti-mouse TNF- ${alpha}$ polyclonal ormonoclonal sera (31, 33). In two separate experiments, micereceiving neutralizing anti-TNF- ${alpha}$ sera lost significantly lessweight by day 5 p.i. (P $≤$ 0.01) but showed no significant differencesin mortality or viral titers in lung, brain, spleen, or thymustissue compared with mice that received normal rabbit sera (datanot shown). Similarly, there was no substantial difference inthe inflammation observed in lungs from either strain of miceon day 6 p.i. (Fig. 6C and D). These results suggest that TNF- ${alpha}$ may contribute to symptoms of severe disease in H5N1 virus-infectedmice but does not directly influence viral replication or theultimate outcome of disease in this model.

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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.

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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.

It was of interest to find in the present study that TNFR1-deficientmice did not exhibit the same extent of thymic involution observedin wild-type B6/129 mice 6 days p.i. with HK/483 virus. Previouswork from our group determined that infection of BALB/c micewith the more virulent H5N1 virus, HK/483, led to peripherallymphopenia as well as a decrease in overall cellularity ofthe thymus and, in particular, a significant decrease in thepercentage of double-positive (DP) (CD4⁺ CD8⁺) lymphocytes relativeto the results obtained for uninfected or HK/486-infected mice(59). This observation led us to hypothesize that increasedproduction of TNF- ${alpha}$ during H5N1 virus infection contributed tothe peripheral lymphopenia, of which thymic involution was anindicator. To test this hypothesis, we first performed histologicalanalysis of thymic tissues from HK/483 virus-infected mice.The thymuses of wild-type B6/129 mice exhibited decreased cellularityin the cortex (Fig. 6G) compared with thymuses from TNFR1-deficientmice (Fig. 6H). Consistent with the histological analyses, totalthymocyte counts in HK/483 virus-infected B6/129 mice were eightfoldlower (P $≤$ 0.05) than those seen with uninfected mice in comparisonwith a less than twofold reduction in thymocyte numbers in HK483virus-infected TNFR1-deficient mice (Table 1). Flow cytometricanalysis of thymocytes indicated that HK/483 virus infectionresulted in a decrease in frequency of the DP thymocyte populationrelative to the results seen for uninfected animals in eitherB6/129 wild-type or TNFR1-deficient mice, although the overalleffect with respect to a decrease in total DP cell numbers wasmore pronounced in the wild-type mice (Table 1). However, nodifferences were detected in total blood leukocyte counts betweenthe B6/129 control animals and their TNFR1-deficient counterparts,as both groups exhibited substantial leukopenia and lymphopenia6 days after infection with HK/483 virus (Table 1). Taken together,these results suggest that the TNF- ${alpha}$ response induced in H5N1virus-infected animals contributes to thymic involution butdoes not affect leukocyte depletion in peripheral blood.

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TABLE 1. Analysis of lymphocyte populations in thymus and blood of TNFR1-deficient and wild-type control mice infected with HK/483 virus^a

DISCUSSION

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Discussion

The widespread distribution of highly pathogenic avian H5N1influenza A viruses in wild birds and, in particular, in domesticpoultry populations continues to pose a threat to public health.Severe respiratory disease and a high case-fatality rate havebecome a hallmark of H5N1 infection in humans as well as inother mammalian species (19, 40, 54, 55). Detection of increasedlevels of cytokine expression, also referred to as a "cytokinestorm," in blood or tissues of H5N1 virus-infected patientshas prompted the hypothesis that an exacerbated proinflammatoryresponse may contribute to the severity of H5N1 disease in humans(5, 37, 56, 60, 63). Clinical features that are prominent inhuman H5N1 infections such as acute respiratory distress syndromeand multiple organ dysfunction have previously been associatedwith cytokine dysregulation (4, 6, 37, 56, 58). In this study,we investigated the contribution of individual cytokines orchemokines to the H5N1 viral disease process in mice. We firstestablished morbidity, lethality, viral replication and spread,and cytokine and chemokine expression in the B6/129 mouse, astrain for which numerous cytokine-deficient mice have beenbred. We also determined that there were no substantial differencesin cytokine and chemokine expression between wild-type B6/129and the knockout mice used in this study during H5N1 virus infectionexcept for the expected lack of IL-6 or MIP-1 ${alpha}$ in the respectivecytokine-deficient mouse strains (data not shown). The kineticsand outcome of infection with HK/483 and HK/486, two H5N1 virusesthat show different virulence phenotypes in wild-type mice,were found to be essentially unaffected in mice deficient inIL-6 or MIP-1 ${alpha}$ expression. In contrast, mice deficient in IL-1Rdisplayed delayed clearance of the less virulent HK/486 virus,while mice deficient in TNFR1 exhibited delayed weight lossand death following infection with the more virulent HK/483virus. These results suggest that TNF- ${alpha}$ may contribute to earlydisease severity, whereas IL-1 may play a role in viral clearancelate in H5N1 virus infection.

In contrast to the noted effects of IL-1ß and TNF- ${alpha}$ response and the fact that both H5N1 viruses elicited strongIL-6 production, lack of IL-6 expression did not appreciablyaffect the outcome of H5N1 infection. This may be due, in part,to the redundancy of the biological effects of IL-6 in mammaliansystems. Our findings were consistent with those of Kozak etal., who observed no significant difference in weight loss resultsbetween IL-6-deficient and wild-type mice infected with mouse-adaptedA/Puerto Rico/8/34 (PR/8; H1N1) influenza A virus (27). We alsofailed to detect any significant differences between MIP-1 ${alpha}$ -deficientmice and wild-type mice following infection with either of theH5N1 viruses despite the strong induction of this chemokinein lungs of wild-type mice. Cook et al. had previously demonstratedthat MIP-1 ${alpha}$ -deficient mice infected with a sublethal dose ofPR/8 virus exhibited less pulmonary inflammation and a modestdelay in clearance of virus from the lungs compared with wild-typecontrols, suggesting that MIP-1 ${alpha}$ -mediated recruitment of inflammatorymediators contributed to viral clearance (9). Although MIP-1 ${alpha}$ does not appear to be critical for the induction and functionof influenza virus nucleoprotein-specific CD8⁺ T cells duringprimary influenza virus infection, it may influence CD8⁺ T-cellrecruitment to the site of infection in the lungs (23). Thefact that we observed no difference in the kinetics of weightloss, recovery rate, or viral replication in MIP-1 ${alpha}$ -deficientmice infected with HK/483 or HK/486 virus suggests either thatMIP-1 ${alpha}$ does not contribute directly to the control of viral clearanceduring the primary immune response to H5N1 virus infection orthat other chemokines with overlapping functions can compensatefor its loss.

IL-1 acts locally and systemically during acute phase responsesto bacterial and viral infection or other inflammatory stimuli,causing fever, anorexia, and lethargy in the host (reviewedin reference 12). We observed that IL-1R-deficient mice didnot differ from wild-type mice in the outcome of infection withthe highly lethal H5N1 virus HK/483. In contrast, followinginfection with the less virulent HK/486 virus, IL-1R-deficientmice lost significantly more weight, exhibited delayed clearanceof virus, and had a greater proportion of animals succumb toinfection than their immunocompetent counterparts. In otherstudies, mice deficient in IL-1R or IL-1ß and infectedwith PR/8 virus were reported to exhibit enhanced mortalitywhich was not associated with enhanced weight loss, and in thecase of IL-1R-deficient mice, a modest delay in viral clearance(28, 44). The significant weight loss observed for HK/486 virus-infectedIL-1R-deficient mice in the present study occurred late in infection,when relatively high virus titers were still present in thelungs of IL-1R-deficient mice but not in those of wild-typemice. Others have reported that IL-1R-deficient mice mount diminishedor altered antigen-specific T-cell responses but similar humoralresponses compared with wild-type mice (29, 43, 44). Similarly,we observed no differences in H5 hemagglutinin-specific serumimmunoglobulin M (IgM), IgG, IgA, or hemagglutination inhibitionantibody titers between IL-1R-deficient mice and B6/129 controlmice on either day 10 or 21 p.i. (data not shown). In contrast,Schmitz et al. detected reduced serum and bronchoalveolar lavagevirus-specific IgM responses, but not bronchoalveolar lavagecytotoxic T-cell effector function, on day 10 p.i. in IL-1R-deficientmice infected with PR/8 virus (44). The differences in IgM responsesbetween the results of the two influenza studies may be dueto differences in viral growth kinetics of H5N1 versus H1N1viruses or in the specificity of IgM antibody response measured.Nevertheless, our results suggest that IL-1 is important forthe induction of effective adaptive immune responses againstH5N1 viruses that aid in viral clearance and that the reducedIL-1ß production observed for HK/483 virus-infectedwild-type (Fig. 2A) mice may contribute to the rapid and lethaldisease outcome.

Both H5N1 viruses used in this study, like other influenza Aviruses, induce elevated amounts of TNF- ${alpha}$ following productivereplication of the virus in the lungs of mice. The early delayin weight loss observed in H5N1 virus-infected TNFR1-deficientmice is consistent with a role for TNF- ${alpha}$ in induction of cachexiaand may explain the modest delay in time to death for HK/483virus-infected mice, since no effect on viral replication orspread was observed (57). These results are consistent withthose of other studies using mouse-adapted influenza A virusesin which treatment with TNF- ${alpha}$ neutralizing antibodies delayedmouse weight loss and/or the time to death by approximately1 day but did not alter viral replication in infected lungs(20, 38). In contrast, TNF- ${alpha}$ was reported to have potent antiviralproperties for human influenza A viruses in porcine lung epithelialcells, whereas H5N1 viruses were resistant to the antiviraleffects of TNF- ${alpha}$ as well as of alpha interferon (IFN- ${alpha}$ ), IFN-ß,and IFN- ${gamma}$ (45, 46). The resistance of H5N1 viruses to the antiviraleffects of these cytokines was proposed to contribute to thevirulence of H5N1 viruses in vivo. However, in our mouse H5N1model, TNF- ${alpha}$ does not appear to have affected virus replicationor spread directly but rather promoted the clinical severityof the disease. The antiviral activity of TNF- ${alpha}$ in epithelialcell cultures may be due to its ability to activate the IFNresponse pathway by the direct induction of IFN ßexpression and/or indirectly through the upregulation of Toll-likereceptor 3 and RIG-I expression (22, 34). Thus, the lack ofan antiviral effect of TNF- ${alpha}$ in influenza virus-infected inbredmice may in part be due to the lack of a functional Mx gene,an interferon-inducible gene that is known to play a role inthe host's antiviral response to influenza virus infection (13,50). The role of type I interferon in the pathogenesis of H5N1viruses in this murine model is currently under investigation.

TNFR1-deficient mice infected with HK/483 virus did not exhibitthe same extent of thymic involution or depletion of DP thymocytesas wild-type B6/129 or BALB/c mice (59), suggesting that TNF- ${alpha}$ signaling may contribute to the loss of thymocytes in H5N1 virus-infectedmice. Thymic involution and alteration in the frequency of thymocytepopulations have been observed previously in mice during viralinfection (17, 26, 35). Uninfected transgenic mice that overexpressTNF- ${alpha}$ in thymocytes and T cells also exhibit reduced thymic massand deceased populations of DP cells (15, 16, 39). TNF- ${alpha}$ andrelated TNF-superfamily members, including TNF-related apoptosis-inducingligand, are known to mediate apoptosis of T cells and, in particular,thymocytes (49, 61). This fact, together with our observationsin this study, suggests that the thymic depletion observed inimmunocompetent H5N1 virus-infected mice is primarily due tothe production of increased levels of TNF- ${alpha}$ produced in responseto H5N1 infection rather than to a direct cytolytic effect ofthe virus itself.

The demonstration that H5N1 viruses are potent inducers of certaincytokines and chemokines in primary human alveolar and bronchialepithelial cells and monocyte-derived macrophages has providedsupport for the hypothesis that host innate immune responsesmay contribute, at least in part, to the severity of diseaseinduced by H5N1 virus infection in humans (4, 5). The resultsof the present study confirm that TNF- ${alpha}$ and a lack of IL-1ßsignaling can influence the course of H5N1 disease in vivo,although intrinsic properties of H5N1 viral replication, includinga greater efficiency of replication in mouse cells associatedwith the E627K substitution in the PB2 gene found in the highlypathogenic HK/483 virus, may influence the overall outcome ofinfection (47). Because of the functional redundancy of somecytokine and chemokine products, it is possible that depletionof more than one cytokine and/or chemokine may have even moresubstantial effects on H5N1 disease than those described here.In this respect the mouse offers a suitable preclinical modelfor the evaluation of therapies using antibodies or receptoranalogs that may modulate the inflammatory response inducedby H5N1 viruses, either alone or in combination with antiviraltherapy that would promote viral clearance (5, 37, 62).

ACKNOWLEDGMENTS

Kristy J. Szretter received financial support for this workfrom the Oak Ridge Institute of Science and Education, Oak Ridge,TN. Shivaprakash Gangappa received financial support throughan American Society of Microbiology postdoctoral fellowship.

We thank Michael Luster and Joanna Matheson for their kind contributionof the rabbit anti-mouse TNF- ${alpha}$ polyclonal sera and normal rabbitsera and Xiaohong Mao Davis for her help with the statisticalanalysis.

The findings and conclusions in this report are those of theauthors and do not necessarily represent the views of the fundingagency.

FOOTNOTES

* Corresponding author. Mailing address: Influenza Branch MS G-16, 1600 Clifton Rd. NE, Atlanta, GA 30333. Phone: (404) 639-4966. Fax: (404) 639-2334. E-mail: JKatz{at}cdc.gov.

Published ahead of print on 20 December 2006.

Present address: Emory University School of Medicine, Atlanta,GA 30322.

REFERENCES

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