Suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus

Abstract

Human pandemic H1N1 2009 influenza virus rapidly infected millions worldwide and was associated with significant mortality. Antiviral drugs that inhibit influenza virus replication are the primary therapy used to diminish disease; however, there are two significant limitations to their effective use: (i) antiviral drugs exert selective pressure on the virus, resulting in the generation of more fit viral progeny that are resistant to treatment; and (ii) antiviral drugs do not directly inhibit immune-mediated pulmonary injury that is a significant component of disease. Here we show that dampening the host's immune response against influenza virus using an immunomodulatory drug, AAL-R, provides significant protection from mortality (82%) over that of the neuraminidase inhibitor oseltamivir alone (50%). AAL-R combined with oseltamivir provided maximum protection against a lethal challenge of influenza virus (96%). Mechanistically, AAL-R inhibits cellular and cytokine/chemokine responses to limit immunopathologic damage, while maintaining host control of virus replication. With cytokine storm playing a role in the pathogenesis of a wide assortment of viral, bacterial, and immunologic diseases, a therapeutic approach using sphingosine analogs is of particular interest.

Factors implicated in high morbidity and mortality from influenza virus infection include robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced tissue destruction (1). Immune-mediated pulmonary injury has been observed in avian influenza virus infection (H5N1) as well as pandemic influenza of 1918–1919 that resulted in as many as 50 million deaths (2, 3). More recently, the pandemic H1N1 2009 influenza virus resulted in more than 18,000 confirmed deaths, with associated pulmonary damage (4–6). This new pandemic strain was of particular concern because person-to-person transmission of this virus was efficient (7), isolates resistant to available antivirals emerged (8), and reassortments of potentially increased virulence were identified (9, 10). Hence, it is necessary to develop drugs for the treatment of influenza virus-induced disease that are less susceptible to virus selection, mutation, and resistance. Corticosteroids and COX-2 inhibitors, drugs that are less susceptible to virus resistance because they inhibit inflammation, have been tested as inhibitors of influenza virus-induced immunopathology, but their effectiveness has been limited (11–13). We hypothesized that targeting of sphingosine-1-phosphate (S1P) receptors known to modulate human inflammatory responses (14) would provide protection from cytokine storm accompanying pandemic H1N1 2009 influenza virus infection. AAL-R is phosphorylated in vivo by sphingosine kinase 2 to AFD-R, an agonist for the S1P receptors S1P1 and S1P3-5 (15). AAL-R administration modulates the innate and adaptive immune responses in mice infected with mouse-adapted A/WSN/33 (WSN; H1N1) influenza virus and limits immune-mediated tissue damage (15, 16). Here we test the impact of the sphingosine analog AAL-R, a chiral sphingosine analog of the clinical drug FTY720 (finglimod), on host outcome during infection with human pathogenic H1N1 2009 influenza virus of swine origin. We find that AAL-R is significantly more effective than the antiviral drug oseltamivir in promoting survival and limiting inflammation by dampening immune-mediated pulmonary injury (immunopathology).

Results

AAL-R Administration Significantly Improved Survival by Limiting Pulmonary Injury.

For our studies, mice were treated with either vehicle or AAL-R intratracheally (i.t.) 1 h after intranasal (i.n.) infection with the human pathogenic pandemic A/Wisconsin/WSLH34939/09 (5) influenza virus isolate. Mice then received 5 mg/kg of oseltamivir by gavage for 5 d starting on postinfection day 4. AAL-R administration alone significantly lengthened the survival time of mice (82%; P < 0.0001) compared with those that received vehicle (Fig. 1A). Similarly, oseltamivir treatment alone significantly increased the number of survivors (50%; P = 0.010) compared with vehicle recipients (21%), but protection was significantly less than that from AAL-R treatment (50% vs. 82%; P = 0.005; Fig. 1A). Combined AAL-R and oseltamivir administration resulted in 96% survival (P < 0.0001 compared with vehicle recipients) on postinfection day 12 and provided greater survival for a longer period than either AAL-R (P < 0.08) or oseltamivir (P < 0.0001) alone.

Fig. 1.

AAL-R administration significantly protects against human pathogenic H1N1 2009 influenza virus infection and inhibits immunopathology associated with infection. Vehicle or 0.2 mg/kg AAL-R was administered i.t. to mice 1 h after i.n. infection with 2 × 10⁵ pfu of A/Wisconsin/WSLH34939/09 (5) influenza virus. Mice then received by gavage vehicle or 5 mg/kg oseltamivir for 5 d starting on postinfection day 4. (A) Mice were monitored for survival. For recipients of vehicle alone, only 21% survived by 12 d after infection, whereas 50% of mice that received oseltamivir only survived until 12 d after infection (P = 0.010). AAL-R administration alone also significantly enhanced survival compared with vehicle (82%; P < 0.0001) and was more efficient than oseltamivir alone (82% vs. 50%; P = 0.005). Notably, combined treatment with AAL-R and oseltamivir further enhanced survival compared with vehicle (96% vs. 21%; P < 0.0001) at day 12 after infection. Protection afforded by combined AAL-R and oseltamivir therapy was enhanced compared with AAL-R (96% vs. 82%; P = 0.076) and oseltamivir (96% vs. 50%; P < 0.0001) treatments alone. Data are derived from three separate combined experiments with a total of 28 mice per group. Data on graph below individual survival curves represents number of survivors/total number of mice, percentage of mice that survived. Survival curves shown were ended day 12 after infection because further mortality did not occur at later time points. (B–I) Histopathologic analysis on postinfection day 10 revealed diminished inflammation characterized by reduced mononuclear cell infiltration, alveolitis, bronchiolitis, vascular hemorrhaging, and edema, as well as significantly decreased tissue inflammation scores in AAL-R (C and G), oseltamivir (D and H), and combined AAL-R and oseltamivir-treated mice (E and I) compared with vehicle recipients (B and F). Tissue inflammation, from greatest to least was: vehicle > oseltamivir > AAL-R > combine AAL-R and oseltamivir. ^aTissue inflammation score; numbers are derived from the mean lung involvement of six separate random fields per tissue section ± SEM. Numbers correspond as follows: 0–1 = ≤10% or less lung involvement; 1–2 = 10–30% lung involvement; 2–3 = 30–60% lung involvement; 3–4 = ≥60% lung involvement. Fields shown are a representative section from four mice per group per time point, six tissue sections per mouse. Tissue sections are representative from six separate sections per lung from four mice per group. ***P ≤ 0.0005 compared with vehicle recipients. (Magnification: B–E, 200×; F–I, 400×; scale bars, 100 μm in B–E, 50 μm in F–I.) (J–L) Biochemical analysis of lung exudate in the BALF. Significant reductions of total protein (J) as well as LDH enzymatic activity (L) in AAL-R and combined AAL-R and oseltamivir-treated mice compared with vehicle and oseltamivir recipients. (K) AAL-R, oseltamivir, and combined AAL-R and oseltamivir-treated mice exhibited significantly diminished levels of IgM compared with those that received vehicle. Data represents average ± SEM from four to five mice per group. Data are representative of two independent experiments. (J–L) *P ≤ 0.05; **P ≤ 0.005. The half-life of AAL-R in vivo is ≈24 h (15).

Enhanced survival afforded by AAL-R treatment correlated directly with pulmonary injury. Histopathologic analysis revealed that AAL-R recipients displayed a reduction in inflammatory cells, alveolitis, bronchiolitis, and significantly diminished tissue inflammation scores on days 4, 7, and 10 after infection with the less lethal pandemic A/California/04/2009 (5) influenza virus isolate (Fig. S1 A–F) compared with vehicle-treated mice. Similarly, when using the more lethal pandemic isolate, A/Wisconsin/WSLH34939/09 (5), treatment with AAL-R or oseltamivir alone, or in combination, significantly diminished immune-mediated tissue damage compared with vehicle-treated mice (Fig. 1 B–L). Histopathologic analysis of lungs from such infected mice treated with AAL-R and combined AAL-R and oseltamivir revealed markedly reduced tissue injury, mononuclear cell accumulation, hemorrhage, and pulmonary edema, as well as significantly reduced tissue inflammation scores compared with recipients receiving vehicle on postinfection day 10 (Fig. 1 B–I). Oseltamivir-treated mice (Fig. 1 D and H) had similar inflammation scores as AAL-R–treated as well as combination AAL-R and oseltamivir-treated mice when at least four independent pulmonary areas on two separate levels of pulmonary tissue were studied. However, in the oseltamivir-alone treated group, areas of increased tissue consolidation and vascular hemorrhaging were detectable, as shown in Fig. 1 D and H. In AAL-R (Fig. 1 C and G) alone as well as combination AAL-R and oseltamivir-treated mice (Fig. 1 E and I), areas of tissue consolidation and hemorrhage were less numerous, decreased in size, and less confluent compared with mice that received oseltamivir alone. These histopathologic findings directly mirror the mortality findings in each group, as shown in Fig. 1A. In agreement with the mortality and histology data, AAL-R as well as combined AAL-R and oseltamivir recipients had significantly diminished total protein (Fig. 1J) and lactate dehydrogenase (LDH) enzymatic activity (Fig. 1L) in the bronchoalveolar lavage fluid (BALF) on postinfection day 7 compared with vehicle- and oseltamivir-treated mice. Vehicle-treated recipients displayed significantly increased IgM (Fig. 1K) in the BALF compared with all other treatment groups, indicating more extensive exudates. In summary, AAL-R therapy alone enhanced outcome compared with oseltamivir therapy at these doses. Mice receiving AAL-R alone had reduced lung exudates and enhanced survival after a lethal inoculum of pandemic H1N1 2009 influenza virus compared with oseltamivir alone.

AAL-R Limits Innate Inflammation Induced by Pathogenic H1N1 2009 Influenza Virus Infection.

Robust innate proinflammatory cytokine expression can cause direct tissue insult and recruit potentially tissue destructive inflammatory cells. Analysis of cytokines/chemokines in AAL-R–treated mice revealed significant reductions in IFN-α, IL-6, IFN-γ, chemokine (C-C motif) ligand 2 (CCL2), CCL3, CCL5, chemokine (C-X-C motif) ligand 2 (CXCL2), and CXCL10 2 d after infection compared with recipients receiving vehicle (Fig. 2A). In addition, AAL-R-treated mice produced significantly less IFN-γ, CCL2, and CCL3 on day 4 after infection (Fig. 2B). Immune cell infiltration and activation can be affected by the reductions in cytokine/chemokine production in AAL-R–treated mice. Analysis of macrophages/monocytes revealed a significant reduction in the accumulation of CD69⁺ (Fig. 2C) and MHC class II⁺ (I-A/I-E; Fig. 2E) cells in the lung on postinfection days 2 and 4. At these same time points, mean fluorescence intensity (MFI) of the early activation marker CD69 (Fig. 2D) and MHC class II (Fig. 2F) on macrophages/monocytes was significantly diminished, indicating a reduction in cell activation. AAL-R treatment significantly reduced the accumulation of activated, CD69⁺ natural killer (NK) cells in the lung on postinfection days 2 and 4 (Fig. 2G). In addition, the MFI of CD69 on NK cells was significantly reduced on postinfection day 2 (Fig. 2H). In correlation with diminished inflammation and pulmonary injury early after pandemic H1N1 2009 influenza virus infection (Fig. S1 A and D), AAL-R significantly inhibited cytokine/chemokine production, as well as the accumulation of activated innate inflammatory infiltrates.

Fig. 2.

AAL-R treatment blunts the innate immune response to human pathogenic H1N1 2009 influenza virus. Mice were infected with 1 × 10⁵ pfu of A/Wisconsin/WSLH34939/09 (5) influenza virus i.n. and treated with vehicle or 0.2 mg/kg AAL-R i.t. 1 h after infection. On postinfection days 2 and 4, mice were euthanized, and BALF was collected for cytokine and chemokine analysis by ELISA. AAL-R administration inhibits the production of cytokines/chemokines on postinfection days 2 (A) and 4 (B). Data represent average ± SEM. (C–H) Two and four days after infection, lungs were harvested, and CD69 as well as MHC class II expression was assessed on macrophages/monocytes (CD11b⁺, F480⁺, Ly6G⁻) and NK cells (NK1.1⁺, CD3⁻). AAL-R treatment significantly reduced total numbers of CD69⁺ (C) and MHC class II⁺ (E) macrophages/monocytes on postinfection days 2 and 4. At these same time points, expression (MFI) of CD69 (D) and MHC class II (F) was significantly impaired on the surface of macrophages/monocytes from AAL-R recipients. AAL-R-treated mice displayed significant reductions in the number of CD69⁺ NK cells (G, postinfection days 2 and 4) and surface expression (MFI) of CD69 (F, postinfection day 2). (C–H) Circles represent individual mice, and black bars signify the mean. (D, E, and H) Data are displayed as the average ratio of MFI from individual infected and treated mice over the average MFI of uninfected mice. (A–H) ^#P ≤ 0.1; *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005 compared with vehicle-treated mice. Samples were collected from four to five mice per group. Data are representative of two independent experiments.

AAL-R Treatment Inhibits the Proinflammatory T-Cell Response Without Altering the Generation of Influenza Virus-Neutralizing Antibodies.

Continued elevated expression of cytokines/chemokines as well as T-cell responses during the later phase of influenza virus infection contribute to pulmonary injury (1). Analysis of cytokines and chemokines on postinfection day 7 revealed that AAL-R and combined AAL-R and oseltamivir recipients had significantly reduced levels of CCL3 and TNF-α (Fig. 3 A and B) in the BALF compared with vehicle-treated mice. In addition, AAL-R administration significantly inhibited CCL3 production compared with oseltamivir recipients (Fig. 3A). We next assessed effector T-cell accumulation in the lungs because such cells are sources of CCL3 and TNF-α. T-cell analysis in AAL-R and combined AAL-R and oseltamivir-treated mice revealed significant inhibition of CD44⁺ effector CD4⁺ (Fig. 3C) and CD8⁺ (Fig. 3D) T-cell infiltration on postinfection day 7 compared with vehicle recipients. Combined AAL-R and oseltamivir-treated mice had significantly reduced numbers of effector T cells compared with oseltamivir recipients and significantly reduced effector CD8⁺ T cells compared with AAL-R–treated mice (Fig. 3 C and D). Reduced effector T cells in the lungs of AAL-R recipients is likely the result of reduced T-cell proliferation due to significantly diminished expression of CD80, CD86, MHC class I, and MHC class II on pulmonary dendritic cells (Fig. S2 A–D) (16). Importantly, AAL-R administration, which down-modulates the cellular immune response, did not significantly increase viral burden (Fig. 3E), demonstrating that AAL-R enhances protection without altering the host's ability to control infection. Lung virus burden was below the limit of detection in the plaque assay on days 8 and 11 after infection with A/Wisconsin/WSLH34939/09 influenza virus treated with vehicle, AAL-R, oseltamivir, and combined AAL-R and oseltamivir therapy. Thus, AAL-R does not interfere with the host's ability to clear virus in the presence or absence of oseltamivir. Administration of AAL-R is effective at limiting CCL3 and TNF-α production, as well as T-cell recruitment in the presence or absence of oseltamivir, whereas oseltamivir treatment alone did not significantly inhibit these inflammatory responses. The joint administration of oseltamivir to the AAL-R therapeutic regimen did not abrogate the antiinflammatory effects of AAL-R. Further, this combined treatment was effective at limiting infection and improving survival, demonstrating that both drugs can be used successfully in concert. Analysis of neutralizing antibody titer in serum of mice treated with AAL-R with or without oseltamivir on day 21 after infection with influenza virus isolate A/Wisconsin/WSLH34939/09 revealed levels equivalent to those in vehicle-treated mice, demonstrating that the use of AAL-R and/or oseltamivir does not disrupt the protective neutralizing antibody response against influenza virus (Fig. 3F).

Fig. 3.

AAL-R administration, but not oseltamivir, inhibits the proinflammatory effector T-cell response to human pathogenic H1N1 2009 influenza virus. (A–E) Mice were treated with vehicle or 0.2 mg/kg of AAL-R 1 h after infection with 1 × 10⁵ pfu of H1N1 A/Wisconsin/WSLH34939/09 (5) influenza virus i.n. Mice then received 5 mg/kg of oseltamivir or water control for 5 d starting on postinfection day 4. (A and B) ELISAs were performed on BALF samples collected on postinfection day 7. (A) AAL-R and combined AAL-R and oseltamivir (Osel.) treatment significantly impaired the secretion of CCL3 compared with mice administered vehicle and oseltamivir alone. (B) Significant reductions in TNF-α production were noted in AAL-R and combined AAL-R and oseltamivir recipients compared with mice that received vehicle. Data represent the average ± SEM. (C and D) Assessment of effector T-cell accumulation in the lung. The infiltration of CD44⁺ CD4⁺ (C) and CD8⁺ T cells (D) was significantly impaired in AAL-R and combined AAL-R and oseltamivir recipients compared with those that received vehicle. Combined AAL-R and oseltamivir recipients displayed significantly less effector CD4⁺ (C) and CD8⁺ (D) T cells than mice treated with oseltamivir alone. Combined AAL-R and oseltamivir treatment significantly hampered CD44⁺ CD8⁺ T-cell accumulation in the lung compared with AAL-R administration alone. Circles and diamonds represent individual mice, and black bars signify the mean. (E) AAL-R administration did not alter amounts of virus in the lung at the time points assayed. Solid and shaded circles represent individual mice studied for viral load. Black bars signify the average virus burden of mice with detectable virus within the group. (F) Mice were infected with 5 × 10⁴ pfu of H1N1 A/Wisconsin/WSLH34939/09 influenza virus (5) i.n. and were treated with AAL-R and/or oseltamivir as stated above. Equivalent levels of neutralizing antibodies to influenza virus (A/Wisconsin/WSLH34939/09) occurred in all four treatment groups. Symbols designate individual mice, and black bars represent the average neutralizing antibody concentration required to inhibit 50% of virus plaque formation. (A–F) Four to five mice per group per time point. Data are representative of two independent experiments. (A–D) *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.

Discussion

Aberrant and excessive cytokine production correlates with morbidity and mortality in macaques (17, 18) and humans (19, 20) infected with highly virulent influenza viruses. Mouse models have demonstrated that many cytokines/chemokines are essential for the control of virus replication but also exacerbate morbidity and tissue injury (1). IFN-α and IFN-γ activate inflammatory cells and stimulate expression of multiple cytokines and chemokines (21–23). IL-6 expression is directly linked to host morbidity (24, 25), and TNF-α secretion enhances pulmonary injury. CCL2, CCL3, CCL5, and CXCL10 production during severe influenza virus infections correlates directly with disease severity and mortality (17, 19, 26). CCL2, CCL3, and CXCL2 expression recruits innate inflammatory cells that damage pulmonary tissue and contribute to morbidity and mortality (27–29).

A single administration of an inhibitory, immunomodulating S1P receptor agonist prodrug (AAL-R) directed against the host's early immune response provided significant protection against a lethal challenge of pandemic H1N1 2009 influenza virus infection over antiviral therapy using oseltamivir. The mechanism of AAL-R′s action was blocking cytokines/chemokines, as well as infiltration and activation of inflammatory cells over the course of influenza virus infection (Fig. 4) that resulted in diminished immune-mediated tissue injury and increased survival. Oseltamivir administration also resulted in reduced tissue injury. The mechanism is likely due to reduced viral burden. However, this still occurred in the presence of enhanced lung exudates, proinflammatory cytokine expression, and effector T-cell accumulation. This indicates both the contribution of immunopathologic injury during influenza virus infection and the therapeutic advantage of limiting immune-mediated pulmonary tissue injury. Combined administration of both AAL-R and oseltamivir significantly blunted tissue injury and should theoretically be the best approach for treatment because it inhibited both virus load and immunopathologic injury in the lung. These results again suggest that cytokine storm plays a significant, if not dominant role in influenza-induced lung injury. Importantly, although AAL-R diminished the inflammatory response induced by cytokine storm, thereby enhancing preservation of lung tissue and host survival, it does so while not hampering the host's ability to generate a protective neutralizing antibody response (Fig. 3F) or generate a sufficient antiinfluenza virus T-cell response that terminates the infection.

Fig. 4.

Schematic cartoon of the effects of AAL-R and oseltamivir on the pathogenesis of human pathogenic H1N1 2009 influenza virus infection. (A) Influenza virus infects and replicates primarily in lung epithelial cells. Killed infected cells release viral progeny, which results in direct virus-induced pulmonary injury. Oseltamivir inhibits influenza virus neuraminidase activity, preventing the release of newly replicated virions from the surface of lysed cells. Diminished virus dissemination by oseltamivir ultimately reduces virus burden as well as viral antigen. (B) Infection induces innate inflammation characterized by cytokine/chemokine production as well as the infiltration and activation of inflammatory cells. AAL-R inhibits cytokine/chemokine production of tissue resident cells and infiltrating inflammatory cells. In addition, AAL-R limits the accumulation of activated inflammatory cell infiltrates that contributes to decreased cytokine/chemokine secretion. (C) Dendritic cells are activated, engulf virus antigen, and become infected. AAL-R impairs surface expression of costimulatory and MHC molecules on activated dendritic cells (16). (D) Dendritic cells migrate into the draining lymph nodes and are necessary to stimulate proliferation of virus-specific CD4⁺ and CD8⁺ T cells. Because of blunted dendritic cell activation by AAL-R treatment, the proliferation of T cells is hampered, resulting in a smaller pool of virus-specific T cells (16). (E) T cells migrate from the draining lymph node into the lung and act directly on cells displaying virus antigen, resulting in the control of virus replication. Because of diminished dendritic cell activation and subsequent T-cell proliferation from AAL-R administration, fewer effector T cells migrate into the lung, but the numbers are sufficient to control viral load. (A–E) Control of virus replication and inflammation by combined AAL-R and oseltamivir administration provides protection from pulmonary injury that in conjunction with a sufficient host adaptive immune response diminishes morbidity and enhances host survival to influenza virus infection.

Cytokine storm is believed to play a prominent role in several other infections, such as hantavirus (30), sudden acute respiratory syndrome (31), HIV (32), pneumococcal pneumonia (33, 34), and a number of immunological diseases (35–38). Thus, the potential benefit of using a similar sphingosine analog therapy should be considered in these cases. Indeed the recent US Food and Drug Administration approval of the sphingosine analog FTY720 (fingolimod) for the treatment of remitting-relapsing multiple sclerosis (39) highlights this potential use of S1P receptor agonists. However, both AAL-R and the phosphorylated form of FTY720 (fingolimod) signal through multiple S1P receptors (40). Signaling by some of these receptors, like the S1P3 receptor, may adversely affect the cardiovascular system (41, 42). Thus, an important future direction is the identification of specific S1P receptor(s) involved in protection against cytokine storm and the design of receptor-specific agonists to limit adverse effects induced by signaling of nonessential but injurious S1P receptor(s). In addition, potential therapies using multiple administrations or a single delayed administration at the time when the immune response is most destructive are under investigation. Effective inhibition of inflammation and existing clinical utilization of sphingosine analog therapy highlights the potential use of such drugs not only for respiratory virus infections but all human maladies in which inflammation contributes to disease severity.

Materials and Methods

Mice, Virus, and Compounds.

C57BL/6 male mice, at 6–8 wk of age, were obtained from Jackson Laboratories and The Scripps Research Institutes Rodent Breeding colony. Influenza viruses A/Wisconsin/WSLH34939/09 and A/California/04/2009 (5) were amplified and plaqued on Madin-Darby canine kidney (MDCK) cells. Mice under isoflurane anesthesia were infected i.n. with 1 × 10⁵ or 2 × 10⁵ of influenza virus A/Wisconsin/WSLH34939/09 or 1 × 10⁵ A/California/04/2009. One hour after infection, mice were anesthetized by isoflurane inhalation for i.t. delivery of vehicle (100 μL of water) or AAL-R (0.2 mg/kg dissolved in water). AAL-R was synthesized according to published methods (15). Oseltamivir (5 mg/kg dissolved in 100 μL of water) or water (100 μL) was administered by gavage for 5 d starting on postinfection day 4. Mice monitored for survival were euthanized when 25% of their starting weight was lost.

Cytokine and Chemokine Analysis.

The trachea of euthanized mice were exposed, transected, and intubated with a blunt 18-gauge needle that delivered 1 mL of PBS supplemented with Complete Mini, EDTA-free Protease Inhibitor Mixture (Roche). Infusion of the 1-mL volume was repeated three times, and the fluid was recovered. The recovered BALF was centrifuged at 3,000 × g for 3 min at 4 °C and stored at −80 °C until use. ELISAs were performed on the BALF using CCL2 (MCP-1), CCL5 (RANTES), CXCL10 (IP-10), and IL-6 Duoset kits (R&D Systems), as well as the CCL3 and CXCL2 Quantikine kits (R&D Systems). IFN-α was quantitated using the VeriKine Mouse IFN-Alpha ELISA Kit (R & D Systems).

Cellular Analysis by Flow Cytometry.

Lungs were harvested from PBS-perfused mice and mechanically diced using surgical scissors. Diced lungs were suspended in 4 mL of CDTI buffer [0.5 mg/mL collagenase from Clostridium histolyticum type IV (Sigma), 0.1 mg/mL Dnase I from bovine pancreas grade II (Roche), 1 mg/mL trypsin inhibitor type Ii-s (Sigma) in DMEM] for 1 h at 37 °C. Lungs were then disrupted mechanically through a 100-μm filter, and red blood cells were lysed using red blood cell lysis buffer [0.02 Tris-HCL (pH 7.4), 0.14 NH₄Cl]. Inflammatory cells were purified by centrifugation in 35% PBS-buffered Percoll (GE Healthcare Life Sciences) at 500 × g for 15 min. Cell pellets were resuspended in staining buffer, and Fc receptors were blocked using 25 μg/mL anti-mouse CD16/32 (BD Biosciences). Cells were stained with fluorescently labeled antibodies against the following mouse proteins: CD69, CD11b, CD11c, F480, Ly6G, 7/4, NK1.1, CD3, CD4, CD8a, CD44, CD103, DEC205, H2-Db, I-A/I-E, CD40, CD80, and CD86. Flow cytometry acquisition was performed with a BD FACSDiva-driven BD LSR II flow cytometer. Data were analyzed with FlowJo software (Treestar).

Histopathology and BALF Protein Content.

Tissues were harvested and placed in PBS-buffered formalin. Lungs were then blocked in paraffin, and 10-μm tissue sections were cut, placed on glass slides, and stained with hematoxylin and eosin. Slides were analyzed by three separate pathologists who were blinded to the various experimental treatments. Total protein content in the BALF was assessed using the Pierce protein BCA assay kit (Thermo Scientific). IgM levels in the BALF were quantified using the mouse IgM quantitation kit (Bethyl Laboratories). Determination of LDH enzymatic activity was determined using the Cytotox 96 nonradioactive cytotoxicity assay (Promega).

Virus Neutralization Assay.

Sera harvested from C57BL/6 mice 21 d after infection with 5 × 10⁴ pfu of the A/Wisconsin/WSLH34939/09 influenza virus isolate were heat-inactivated for 45 min at 56 °C. Serial 10-fold dilutions of the sera were incubated with 100 pfu of the A/Wisconsin/WSLH34939/09 influenza virus isolate (1:1) for 60 min at 25 °C. Samples were then used for plaque assays performed on MDCK cells. The serum dilution required for 50% inhibition of plaque formation was calculated using the Reed-Muench method.

Statistical Analysis.

For the survival study, a Gehan-Breslow-Wilcoxon test was performed, and a P value of 0.05 (95% confidence level) was deemed significant. A bidirectional paired Student t test was used for all other analyses, with a 95% confidence level being significant.