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. Author manuscript; available in PMC: 2014 Jan 5.
Published in final edited form as: Virology. 2013 Jan 5;435(1):92–101. doi: 10.1016/j.virol.2012.09.039

Dissecting Influenza Virus Pathogenesis Uncovers a Novel Chemical Approach to Combat the Infection

Michael BA Oldstone a,*, John R Teijaro a, Kevin B Walsh a,+, Hugh Rosen b
PMCID: PMC3523270  NIHMSID: NIHMS412385  PMID: 23217619

Abstract

The cytokine storm is an aggressive immune response characterized by the recruitment of inflammatory leukocytes and exaggerated levels of cytokines and chemokines at the site of infection. Here we review evidence that cytokine storm directly contributes to the morbidity and mortality resulting from influenza virus infection and that sphingosine-1-phosphate (S1P) receptor agonists can abort cytokine storms providing significant protection against pathogenic human influenza viral infections. In experiments using murine models and the human pathogenic 2009 influenza viruses, S1P1 receptor agonist alone reduced deaths from influenza virus by over 80% as compared to lesser protection (50%) offered by the antiviral neuraminidase inhibitor oseltamivir. Optimal protection of 96% was achieved by combined therapy with the S1P1 receptor agonist and oseltamivir. The functional mechanism of S1P receptor agonist(s) action and the predominant role played by pulmonary endothelial cells as amplifiers of cytokine storm during influenza infection are described.

Keywords: cytokine storm, influenza, pulmonary endothelial cells, sphingosine-1-phosphate (S1P) receptor (rec) signaling, S1P receptor agonist therapy

Introduction

Influenza virus infections were responsible for nearly 100 million human deaths in the last century. Further, during the two-year period of 1918–1919, influenza caused the greatest loss of life of any infectious disease or medical condition known (Ahmed et al., 2007; Johnson and Mueller, 2002). During that period influenza visited roughly 5% of the world’s population, killing 2%. Since that most virulent episode, several influenza pandemics have raged, the most recent being the 2009 attack of swine flu. The 2009 H1N1 influenza viruses rapidly infected millions of humans worldwide with an estimated 293,500 deaths of which 201,200 resulted from respiratory failures and 83,300 cardiovascular insults (Dawood et al., 2012).

Susceptibility or resistance to any viral infection is determined by a balance between the virulence of the microbe, the resistance of the host including the aggressiveness of its immune response against the infecting agent. When the immune response is limited either because of host genetic or acquired defects, or temporarily due to lack of normal differentiation of the immune system of newborns and young children; or decreasing immune responses of the elderly, the advantage goes to the virus. When infection occurs in those with a fully developed and competent immune system, the advantage goes to the host unless the infecting virus overwhelms the individuals immune system or when the mechanism that modulates the immune response fails resulting in an over abundant excessive innate and adaptive immune response termed “cytokine storm.”

Vaccination is employed to protect uninfected reservoirs of individuals and thereby diminishing the spread of infection. Antiviral drugs are the primary effective therapy used to diminish ongoing disease. Antiviral drugs are effective, nevertheless, there are two compelling limitations to their total efficiency and effectiveness. First, antiviral drugs exert selective pressure on the virus, resulting in the generation and selection of more fit viral progeny that per se become resistant to the drug (Nguyen et al., 2012; Orozovic et al., 2011; Wang et al., 2010). Second, the pathogenic injury associated with influenza virus infection results both from the intrinsic virulence of the virus which is attacked by anti-influenza viral drug therapy and the intensity of the immune response (cytokine storm and viral-induced immunopathologic tissue damage) which antiviral drugs do not engage. Here, using a small animal model, we review the evidence from our laboratories that cytokine storm alone plays an important and essential role in causing significant tissue injury and mortality following human pathogenic H1N1 2009 influenza virus infection. We document that dampening the host’s immune response against influenza virus using specific immunomodulatory sphingosine-1-phosphate (S1P) receptor (rec) agonists provide significant protection from mortality over that observed by the neuraminidase inhibitor oseltamivir. Further, we demonstrate that specific agonists against S1P1 receptor inhibits innate cellular and cytokine/chemokine responses that limit virus-induced immunopathologic injury yet still maintain host control and termination of virus replication by anti-influenza virus cytotoxic T cells and neutralizing antibodies. Utilizing genetic, molecular, and chemical tools we locate S1P1 receptor on pulmonary endothelial cells, identify endothelial cells as the central regulators of cytokine storm and show a mandatory role for interferon type I signaling in this process. Due to space limitations we do not discuss the role played by the virus and its genes as virulence factors in H1N1 influenza virus infection but point the reader to several publications in this area (Ahmed et al., 2007; Chou et al., 2011; Fukuyama and Kawaoka, 2011; Hai et al., 2010; Song et al., 2011; Watanabe et al., 2012).

Epidemiologic and experimental evidence for cytokine storm

When accompanied by manifestations of cytokine storm or acute respiratory distress syndrome, infected individuals display high mortality with elevated cytokines/chemokines, leukocyte inflammation and edematous lungs during H1N1 1918–1919 and H1N1 2009 pandemic influenza virus infections in experimental animal models (Baskin et al., 2009; Kobasa et al., 2007; Marcelin et al., 2011; Zhang et al., 2012) and for 2009 infection in humans (Arankalle et al., 2010; Cheng et al., 2011; Lee et al., 2011). Among the reports of H1N1 2009 infection in humans, that of Arankalle and colleagues (Arankalle et al., 2010) is illuminating. These investigators analyzed viral load in lungs of critically ill patients who died and those who recovered. Both groups showed roughly equivalent titers of virus. In contrast, mortality correlated directly with cytokine storm. Thus, the patients who died had higher cytokine/chemokine levels but equivalent viral titers in pulmonary samples when compared to patients having a milder infection course and recovering.

In summary, both experimental studies and human clinical observations suggested to us that: 1) cytokine storm is associated with poor outcome in influenza virus infection, and 2) calming the host’s aggressive and exaggerated cytokine storm response might provide the opportunity to decrease morbidity and enhanced survival to influenza and likely other acute respiratory diseases like SARS, Hantavirus infection, and pneumococcal pneumonia that manifest severe cytokine storm. To test this possibility we turned our attention to the molecule sphingosine-1-phosphate (S1P) and determined if the harmful immunologic processes accompanying H1N1 influenza virus infection could be modulated by S1P receptors in the lung. If so, influenza could be chemically tractable and successfully treated pharmacologically with therapy directed against the host’s over-aggressive immune response. Further, this anti-host immune therapy would be unlikely to generate viral escape variants that is an issue with antiviral therapy. In addition, such an approach would also provide new insights into pathogenesis of influenza viral infections and may uncover surrogate markers useful for identifying those most susceptible to influenza virus infection.

Sphingosine-1-phosphate (S1P) properties

S1P is a signaling lipid present at a concentration of 1–3 μM in plasma and at roughly 100 nM in lymph. The vast majority of S1P in plasma is bound to high density lipoprotein, leaving a free concentration between 15–45 nM in blood. The metabolism of S1P is displayed in Figure 1A. S1P is generated by phosphorylation of sphingosine by the actions of two intracellular sphingosine kinases. S1P is degraded either reversible by dephosphorylation or irreversible by cleavage (reviewed in (Rosen et al., 2009; Rosen et al., 2007; Scott, 2011)). Physiologically, S1P levels are under tight homeostatic control and S1P signals through specific S1P receptors of which there are five: S1P receptor 1–5. These five specific S1P receptors are coupled to different G proteins in order to regulate a variety of downstream signaling pathways that are specific for many cells, tissues, and organs (Rosen et al., 2009; Rosen et al., 2007; Scott, 2011). S1P and its analogs have been used clinically to induce sequestration of lymphocytes in secondary lymphoid organs (Figure 1B) and by that means limit the migration of effector lymphocytes to areas where such cells might mediate immunopathologic injury leading to diseases (Rosen et al., 2009; Rosen et al., 2007; Scott, 2011). Indeed, S1P agonist therapy is currently being prescribed for treatment of multiple sclerosis (MS) and being considered for treatment of other inflammatory disorders.

Figure 1.

Figure 1

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SIP biochemistry and activity in lymphoid organs. Panel A: Synthesis and regulation of S1P. Panel B: Cartoon of systemic activity of S1P in secondary lymphoid tissues (see ref. Rosen et al., 2007, 2009 for details). Figure adapted from Rosen et al., 2009.

Immune virus-specific T cell trafficking in vivo during influenza virus infection

Infiltration of lymphoid cells in pulmonary tissues is one of the signatures of influenza virus infection. To study the kinetics of influenza virus-specific CD8 and CD4 T cell entry into the lung and their anatomic distribution during influenza infection we designed an in vivo model taking advantage of the wealth of reagents we and others have generated to lymphocytic choriomeningitis virus (LCMV). First, a recombinant influenza virus A/WSN/33 (H1N1 WSN) was engineered using reverse genetics by our collaborator, Yoshihiro Kawaoka, to express the immunodominant H-2Db (H-2b MHC class I background) restricted CD8 T (glycoprotein [GP] aa 33-41) and IAb (H-2b MHC class II background) CD4 (GP aa 65-77) T cell epitopes for LCMV into the influenza neuraminidase (NA) stalk (flu/LCMV) (Marsolais et al., 2009; Neumann et al., 1999). This maneuver still allowed NA function and influenza replication (Marsolais et al., 2009). GP 33-41 and GP 65-77 incorporated into H1N1 influenza, when expressed in infected lung cells and bound to H-2Db or H-2IAb, served as recognition epitopes for lymphocytes from H-2b transgenic mice created to express the T cells bearing receptors (TCR) specific for these two LCMV immunodominant epitopes. By crossing such TCR mice with H-2b transgenic mice expressing either GFP or RFP under transcriptional control of beta actin gene, GFP- or RFP-labeled GP 33 CD8 T cells and GP 65 CD4 T cells allows the isolation of >98% of GFP- or RFP-labeled LCMV-specific T cells (Marsolais et al., 2009; McGavern et al., 2002). 2.5 × 104 of these GFP or RFP labeled LCMV-specific CD8 or CD4 T cells were then adoptively transferred into H-2b (C57Bl/6) naïve adult 8-week-old mice. One day later 1 × 105 PFU of flu/LCMV recombinant was administered intranasally (Marsolais et al., 2009). This protocol allows visualization of influenza virus replication in the lung by use of immunocytochemistry with specific fluoroprobe-labeled antibody to influenza, in vivo trafficking and quantitation of influenza virus-specific CD8 and CD4 T cells in the lung and visualization of virus-specific T cell/influenza virus-infected cell interaction (Marsolais et al., 2009; McGavern et al., 2002). Quantitation of number of CD8 and CD4 virus-specific T cells is also measured by FACS providing a complimentary assay. Quantitative differences between in vivo labeling of T cells and FACS identification assays is less than 10% (McGavern et al., 2002).

Both in vivo trafficking studies and FACS analysis six days after flu/LCMV recombinant virus infection, accumulation of GFP virus-specific CD8 T cells and CD4 T cells occurred. The plateau reached in trafficking virus-specific T cells occurred between day 6 and 8 post-infection and then dramatically declined at day 10. When analyzed at day 7 post-infection viral antigen was found throughout the lung parenchyma. High resolution light microscopy identified influenza antigens in epithelial cells lining bronchioles and alveolar cells. Infiltration of virus-specific RFP-GP 33 CD8 T and GFP-GP 65 CD4 T cells was evaluated at day 7 following intranasal inoculation of flu/LCMV recombinant. Numbers of trafficking CD8 T cells into the lung was always greater than the trafficking of CD4 T cells. The ratio of virus-specific CD8 T cells to CD4 T cells was 2.6 to 1. In addition, this model also provided the opportunity to determine if infiltrating virus-specific T cells could be modulated by chemical probes that would limit the resultant pulmonary injury mediated by the adoptive immune response. Since sphingosine-1-phosphate (S1P) receptor agonist have been reported earlier to sequester lymphocytes in secondary lymphoid organs and retard their migration to sites of tissue injury (Rosen et al., 2009; Rosen et al., 2007), we turned our attention to beneficial use of such probes in terms of altering the infiltration of virus-specific T cells and the outcome of influenza virus infection.

S1P receptor signaling system and effect of S1P receptor agonists in modulating the adoptive immune response and clinical course of influenza virus infection

There are five specific S1P receptors that couple to different G proteins that regulate multiple downstream signaling pathways (Rosen et al., 2009; Rosen et al., 2007). The biologic functions of S1P are dependent on the cell/tissue location of these receptors and their relative expression. We began our study for the in vivo influenza model using a non-selective S1P receptor pro-drug agonist AAL-R that signals on receptors S1P1, S1P3, S1P4, and S1P5, but not S1P2 (Fig. 2A) following phosphorylation by sphingosine kinase 2. Local administration of a single 0.1 mg/kg dose of AAL-R by intratracheal (i.t.) administration when given two hours (data shown, Fig. 2B, middle row) or three to four days (data not shown) after intranasal (i.n.) infection with 1 × 105 PFU of H1N1 WSN/LCMV significantly down-modulated virus-specific CD8 T cell accumulation in the lung whereas administration of vehicle alone (Fig. 2B, top row) or the chiral enantiomer molecule, AAL-S, that cannot be phosphorylated efficiently in vivo was unable to restrict T cell infiltration (Fig. 2B, bottom row) into the lung. This assay was performed by adoptively transferring a pure population (>99%) of 5 × 104 GFP-labeled GP 33 LCMV-specific T cells 24 hours before infection, sacrificing mice 7 days post-infection, isolating T cells from the lung, gating on GFP and quantitative analysis by FACS. We then assessed, in other groups of mice treated the same way, the ability of the remaining virus-specific T cells to generate a protective anti-influenza viral immune response that controlled and terminated the infection. Administration of AAL-R, which significantly retarded numbers of virus-specific T cells entering influenza virus-infected lungs, did not alter the immune response sufficiently to alter or raise the viral burden in the lung when compared to infected mice receiving vehicle or AAL-S. Ten days after influenza infection AAL-R, AAL-S and vehicle treated mice all cleared virus from their lungs. Indeed, AAL-R mice sacrificed at 7 days post-influenza virus challenge showed a robust virus-specific CTL response (51Cr release assay) and a vigorous specific memory T cell response upon virus stimulation occurred at 40 days post-influenza virus challenge. Further, the kinetics, titers, and immunoglobulin subtypes of neutralizing antibodies in sera of influenza virus-infected mice treated with AAL-R, AAL-S or vehicle were equivalent. Taken together these results document that AAL-R therapy given locally into the respiratory tract down-modulated the migration of virus-specific CD8 T cells in the lung. However, neither influenza virus replication nor the generation of protective neutralizing antibodies was adversely effected. Despite the reduction in numbers of virus-specific CD8 T cells by AAL-R activity, influenza viral infection was still controlled.

Figure 2.

Figure 2

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The non-selective AAL-R S1P receptor agonist retarded influenza virus specific CD8 T cell expansion in influenza virus infected lungs resulting in significant protection from pulmonary tissue injury and related mortality. Panel A: AAL-R is a non-selective S1P agonist and signals on S1P1, S1P3, S1P4 and S1P5 receptors but not the S1P2 receptor. Biologic functions of signaling on S1P1, S1P3, S1P4 and S1P5 receptors and their different G protein couplings are displayed. Panel B: AAL-R therapy significantly decreased numbers of influenza virus specific CD8 T cells in influenza virus infected lungs (middle row) compared to controls given vehicle (top row) or the chiral enantiomer of AAL-R, AAL-S (bottom row). Panel C: significant protection from influenza virus induced death of C57Bl/6 mice treated with AAL-R (yellow color), compared to vehicle (magenta) treatment. Significant protection also followed oseltamivir (blue) therapy when compared to vehicle (magenta). Although AAL-R alone provided significantly greater protection than oseltamivir, the best protection from influenza infection produced by a lethal challenge with non-mouse passed human H1N1 2009 (shown) or mouse adapted WSN virus (not shown) came from combined AAL-R with oseltamivir (green) therapy. Panel D complements the mortality graft in Panel C with histopathologic analysis following treatment with vehicle, AAL-R, oseltamivir or AAL-R combined with oseltamivir. The black bar indicates similar magnification for each tissue sample. Greatest degree of hemorrhage and loss of alveolar air space were from vehicle < oseltamivir < AAL-R < AAL-R+oseltamivir. Representative tissue samples came from over 10 pulmonary sections from 4 mice in each group. The graph on the right displays outcomes detected in bronchial washes (BALF) of influenza virus-infected lung samples after various treatments. Figure adapted from Walsh et al., 2011.

Associated with the decreased accumulation of virus-specific T lymphocytes in the lung was enhanced survival of mice receiving a lethal challenge with H1N1 human pandemic 2009 isolate A/Wisconsin/WSLH3439/09 2 × 105 PFU i.n. (data shown, Fig. 2C), A/California/04/2009 (data not shown), or WSN mouse adopted virus (data not shown). Neither human H1N1 viruses isolated from humans had been passaged before in mice. Thus, while 6 of 28 mice (21%) receiving only vehicle survived i.n. infection with 2 × 105 PFU of virus, 23 of 28 mice (82%) receiving AAL-R were protected (P < 0.001). Susceptibility and death correlated directly with massive lung consolidation, hemorrhage, and exudate in lungs of mice receiving vehicle or AAL-S. Significantly less lung pathology occurred in influenza virus infected mice receiving AAL-R (Fig. 2D, left panel). Tissue histochemistry and biochemical analysis of pulmonary exudate (Fig. 2) was done 10 days after initiating the influenza infection. Quantitation of lung exudate was by measuring LDH and total protein in the bronchial lavage fluid (BALF) (Fig. 2D, right panel).

AAL-R also significantly down-regulated cytokine/chemokine synthesis in vivo. Upon influenza infection there was significant up-regulation of IL-1α, IL-1β, IL-6, IL-10, IL-12, MIP-1, TNF-α, MIP-1α, GM-CSF, and RANTES in the BALF. IL-6 and MCP-1 levels were strikingly enhanced similar to reports in both H5N1 infected humans (de Jong et al., 2006) and 1918-1919 H1N1 infected macaques (Kobasa et al., 2007) and mice (Kash et al., 2006). Treatment with AAL-R significantly inhibited synthesis of IL-1α, IL-1β, IL-6, IL-10, MCP-1, TNF-α, and GM-CSF in BALF two days post-infection when compared to mice treated with AAL-S or vehicle. Cytokine/chemokine suppression was associated with a decrease of GR-1+ polymorphic leukocytes and F4/80+ macrophages in the lung which directly correlated with the diminished lung parenchyma pathology, infiltration, and amount of exudate.

Oseltamivir is a potent anti-influenza drug by its action of inhibiting the viral neuraminidase. We then tested the therapeutic potential of an optimal dose of oseltamivir, 5 mg/kg, dissolved in water administered by gavage for five consecutive days starting on post-infection day 4 alone or in combination with AAL-R to protect against a lethal challenge of 2 × 105 PFU i.n. of A/Wisconsin/WSLH34939/09. Vehicle or 0.2 mg/kg of AAL-R was given i.t. one hour after virus infection. As shown in Figure 2C, oseltamivir given alone protected 14 of 28 mice (50%), a result significantly improved over vehicle. However, AAL-R therapy alone provided significantly greater protection than that of oseltamivir (82% vs. 50%). Optimal therapy occurred when both AAL-R and oseltamivir therapy were combined as 27 of 28 mice (96%) survived the influenza infection (Fig. 2C) (Walsh et al., 2011). Superior survival observed in treatment of combined AAL-R and oseltamivir, and of AAL-R over oseltamivir when administered independently closely mirrored the degree of histologic evidence of pulmonary injury and degree of exudate in BALF (Fig. 2D).

The last series in this category investigated the mechanism(s) of how AAL-R impaired the number of virus-specific CD8 T cells that trafficked to and deposited in the lung parenchyma. Upon pulmonary infection, influenza virus-specific T cells are induced and proliferate in mediastinal lymph nodes (MLNs), then migrate to infected sites in the lung (Baumgarth and Kelso, 1996; Belz et al., 2004). AAL-R significantly reduced the numbers of virus-specific CD8 and CD4 T cells in MLNs at days 5 and 6 post-infection (Marsolais et al., 2009) and in the lung days 6–8 post-infection indicating that this S1P receptor agonist inhibited clonal expansion of T cells. Analysis for dead cells (annexin V-positive virus-specific T cells) in MLNs and lungs indicated that these cells were not killed by local respiratory tract therapy with AAL-R. This result suggested that AAL-R likely altered T cell stimulation by influenza virus antigen presenting dendritic cells (DC) (Allan et al., 2006) rather than by T cell deletion. Study of AAL-R effect on DCs found that AAL-R did not reduce the numbers of DCs or their specific subsets (Marsolais et al., 2009). However, AAL-R suppressed influenza virus-induced DC activation in the lungs and MLNs as measured by reduction in surface expression of MHC-I, MHC-II, and B7.2 molecules on the DC surfaces. AAL-R treatment also impaired the stimulatory capacity of DCs as confirmed by inefficient induction of virus-specific CD8 T cell proliferation in vitro (Marsolais et al., 2009). Thus, AAL-R locally administered in the respiratory tract during influenza infection disrupts the antigen-presenting DC network (Steinman and Hemmi, 2006) by blocking DC-mediated signal transmission from the infected site to MLNs, leading to a dramatic decrease in T cell expansion in MLNs and in lungs.

Specific S1P1 receptor agonists blunt the exaggerated innate immune host reaction “cytokine storm” by modulating S1P1 signaling of pulmonary endothelial cells

The non-selective agonist AAL-R reacts by modulating S1P1, 3, 4, 5, and not S1P2 receptors (Rosen et al., 2009; Rosen et al., 2007). To determine if a single or multiple receptors were involved in the initial cytokine storm and the later immunopathologic adoptive immune response, discovery and optimization of S1P receptor subtype selective agonists and antagonists were begun by Hugh Rosen and colleagues. Further, a variety of genetically engineered S1P1 receptor knock-out and S1P1 receptor eGFP knock-in mice were utilized that were physiologically and pharmacologically normal when compared to wild-type controls. Initially, a series of well-characterized S1P1 selective agonists were administered i.t. (CYM-5442: 2 mg/kg; RP-002: 3 mg/kg), or orally (RP-002: 6 mg/kg) (Teijaro et al., 2011). All S1P1 selective agonists provide protection against a lethal i.n. challenge with human H1N1 A/Wisconsin/WSLH34939 or A/California/04/209 (Fig. 3A) and blunted cytokine storm (Fig. 3B,C) to a degree equivalent to that observed earlier with non-selective S1P agonist AAL-R (Fig. 3D,E). The S1P1 receptor agonists significantly inhibited secretion of cytokines and chemokines associated with influenza infection including IFN-α, CCL-2, IL-6, TNF-α, CCL-3, CCL-5, CXCL-2, IL-1α, and IFN-γ. While most of these cytokines/chemokines were inhibited to a similar degree as with AAL-R therapy, suppression of CXCL-2, TNF-α, and IFN-γ was not as effective, suggesting, perhaps, a role for other S1P receptor subtypes in modulating these cytokine/chemokines. In addition, the S1P1 selective agonists significantly blunted the accumulation of innate infiltration of macrophages/monocytes (CD11b+, F480+, LyG6−), neutrophils (CD11b+, LyG6+, F480−), and natural killer (NK) cells (NK1.1+, CD3−). Further, the expression of the activation marker CD69 was significantly reduced following S1P1 agonist treatment. As with earlier results with selective S1P receptor agonist AAL-R, there was no increase in viral titers following chemical treatment. Further, viral infection was effectively terminated and both humoral (antibody) and cell-mediated (CD8 T cell) arms of the immune response were generated during S1P1rec agonist therapy. Since cytokine storm, pathologic injury to the lung parenchyma, and survival of influenza virus infection were all achieved with S1P1 agonist therapy, our results implicated S1P1 receptor signaling as the essential player in the initiation of cytokine storm and resultant immune-mediated injury. Importantly, our results also indicated that a severe pulmonary disease associated with cytokine storm was chemically tractable with a single chemical molecule, S1P1 agonist that avoided signaling through S1P2, 3, 4, 5 receptors.

Figure 3.

Figure 3

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Severe influenza respiratory disease is blunted by S1P1 receptor agonist. Protection by S1P1 specific receptor agonist is comparable to protection provided by AAL-R S1P1, S1P3, S1P4, S1P5 receptor agonist. Panels A, B, C: Significant protection and ablation of cytokine storm by S1P1 receptor agonist RP-002. Panels D and E: Significant protection by S1P1 receptor agonist CYM-5442 and its comparison to the protection provided by AAL-R agonist to S1P1, S1P3, S1P4 and S1P5 receptor signaling. Figure adapted from Teijaro et al., 2011, with permission from Elsevier.

Having identified S1P1rec signaling as the primary pathway for the initiation of cytokine storm we sought to identify the cell or cells in the lung that expressed the S1P1 receptor. Since epithelial cells are the primary cell infected by influenza virus we suspected that S1P1 receptor was located on that cell. To determine the pulmonary cell(s) bearing the S1P1 receptor, we took advantage of eGFP-S1P1 receptor knock-in mice made by Stuart Cahalan in the Rosen laboratory (Cahalan et al., 2011). In this mouse, the native S1P1 receptor was homologously replaced with a functional fused eGFP-tagged S1P1 receptor (Cahalan et al., 2011). Utilizing this mouse model we could directly detect eGFP-S1P1 receptor protein expression of pulmonary cells which we could then identify by antibodies to specific pulmonary cell markers and flow cytometry. Additional conformation was achieved by biochemical analysis of purified pulmonary cells. High levels of S1P1-eGFP receptor expression was found on lung lymphatic (CD45−, CD31+, GP38+) and vascular (CD45−, CD31+, GP38−) endothelial cells but surprisingly was absent on pulmonary epithelial cells (CD45−, CD31−, EpCAM+) (Fig. 4A, top panel). These results were confirmed by doing Western blots on >98.5% pure populations of pulmonary endothelial and epithelial cells (Fig. 4C). As expected and previously reported (19,20), CD4 T cells (CD4+, CD3+), CD8 T cells (CD8+, CD3+), and B cells (B200+, CD19+) also expressed S1P1-eGFP receptor (Fig. 4A). In contrast, pulmonary leukocytes, including macrophages/monocytes (CD11c+, CD11b−, F480+), DCs (CD11c+, IA-IE+, CD205+, F480−), neutrophils, NK cells (NK1.1+, CD3−) (Fig. 4B), and immature lymphoid cells (LIN−, SCA-1+) failed to express significant levels of eGFP-S1P1 receptor protein. S1P1-eGFP receptor expression was similar whether cells were harvested from mice that were uninfected or infected with influenza virus. Other experiments in infected mice (Fig. 4C) indicated that S1P1-eGFP receptor expression is not altered during influenza virus infection. Importantly, S1P1 agonist treatment of infected eGFP-S1P1 receptor knock-in mice did not lessen expression of S1P1-eGFP receptor indicating that administration of specific S1P1 agonist does not degrade the endothelial S1P1 receptor. These results indicated that functional agonism of S1P1 and not antagonism effect of receptor degradation is the mechanism by which S1P1 receptor blocking molecules CYM-5442 and RP-002 suppressed cytokine storm.

Figure 4.

Figure 4

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S1P1 receptor is present on pulmonary endothelial cells but not on pulmonary epithelial cells. Panel A identifies S1P1 receptor primarily on pulmonary endothelial cells, and modestly on CD4 T cells as well as CD8 T cells but not on pulmonary epithelial cells. Panel B displays absence of S1P1 receptor on pulmonary macrophages, monocytes, dendritic cells, neutrophils or NK cells. Data represented in Panels A and B used eGFP-S1P1 receptor knock in mice, antibodies to specific markers of various cell populations and FACS. Cell populations were >98.5% pure. Panel C: Western blotting shows S1P1 receptor primarily on pulmonary endothelial cells but none on pulmonary epithelial cells. Panel D: Rag2−/− knock-out mice do not contain T cells. S1P1 specific agonist C M5442 blocked cytokine storm (chemokines/cytokines in bronchial wash: left panel; and migration of innate inflammatory cells: right panel) indicating that the S1P1 receptor agonist acted on pulmonary endothelial cells, not lymphoid cells to dampen the cytokine storm. Figure adapted from Teijaro et al., 2011, with permission from Elsevier.

T and B lymphocytes as well as pulmonary endothelial cells are the only ones within the lung that express measurable amounts of S1P1-eGFP protein (Fig. 4). We therefore determined whether lymphocytes expressing S1P1 receptor were involved in S1P1 agonist inhibition of cytokine storm or were merely bystander cells accompanying the innate immune response to influenza virus infection. Rag2−/− mice are deficient in lymphocytes and we reasoned that if such influenza virus infected Rag2−/− mice generated a cytokine storm that could be blocked by S1P1 agonist then lymphocytes did not contribute to initiation of cytokine storm and could be excluded as key regulators of influenza virus-induced cytokine storm. Figure 4D documents that cytokine storm occured in Rag2−/− mice infected with influenza virus and that treatment with S1P1 agonist CYM-5442 significantly reduced cytokines/chemokines (shown IFN-α, CCL-2, IL-6, TNF-α, IFN-γ) and innate infiltration of macrophages/monocytes and NK cells. A further series of experiments conclusively documented (Teijaro et al., 2011) that cytokine/chemokine production and innate inflammatory cell recruitment were independent events that occurred after influenza virus infection. However, both cytokine/chemokine production and innate inflammatory cell recruitment were inhibited by S1P1 receptor agonism of pulmonary endothelial cells. An important observation is that initial inflammatory cell infiltration into the lungs was not dependent or required for cytokine/chemokine production (Teijaro et al., 2011).

Type I interferon signaling is essential for cytokine/chemokine response of cytokine storm but is independent of innate inflammatory cell recruitment into the lung

Our observations with influenza and those of others have repeatedly documented that type I interferon, predominantly the IFN-α species, are elevated early after acute respiratory viral infection. The release and action of type I interferon is believed crucial for the production of proinflammatory cytokines/chemokines. We found that S1P1 receptor agonists inhibited the production of IFN-α in the pulmonary parenchyma early after influenza infection (Figs. 3, 4) (Teijaro et al., 2011). To prove that blunting of IFN-α production was a mechanism by which S1P1 receptor agonist inhibited cytokine storm, IFNα-βrec knock-out and IFNα-βrec sufficient mice infected with 1 × 105 PFU i.n. of H1N1 virus were treated with S1P1 receptor agonist CYM-5442 or vehicle and both cytokine/chemokine proteins and innate inflammatory cell recruitment measured in the bronchial lavage fluid at 48 hours post-infection. Cytokines/chemokines IFN-α, CCL-2, IL-6, IFN-γ (shown), CCL-5, and CXCL-10 in the bronchial lavage fluid were significantly reduced in IFNα-βrec sufficient and knock-out mice (Fig. 5A) but inflammatory cell infiltration of macrophages/monocytes, neutrophils and NK cells following S1P1 receptor agonist therapy were only down-regulated in IFNα-β sufficient and not IFNα-β knock-out mice (Fig. 5B). Thus, regulation of innate inflammatory cell recruitment into lung was mediated by endothelial cells and was independent of type I interferon signaling. Cytokine/chemokine production in the lung was also mediated by endothelial cells but, in contrast, S1P1 receptor agonism of endothelial cells inhibited IFN-α production leading to dampening of production of inflammatory cytokine/chemokine responses.

Figure 5.

Figure 5

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Interferon production is essential to yield the cytokine/chemokine component of cytokine storm. Regulation of innate inflammatory cell recruitment into the lung is also mediated by endothelial cells but is independent of type 1 interferon signaling. Eliminating interferon occurred by use of influenza virus infected interferon - receptor knock-out mice. Panel A shows that chemokines/cytokines were significantly reduced when interferon -sufficient or interferon - deficient mice were treated with S1P1 receptor agonist. In contrast, inflammatory cell infiltration of macrophages/monocytes, neutrophils and NK cells following S1P1 receptor agonist therapy were down-regulated only in interferon - sufficient mice but not their interferon - receptor knock-out counterparts. Figure adapted from Teijaro et al., 2011, with permission from Elsevier.

Crystal structure of S1P1 receptor complexed with S1P1 receptor antagonist

Recently, Hugh Rosen and Ray Stevens’ laboratories solved the crystal structure of the lyso-phospholipid sphingosine-1-phosphate-1 receptor fused to T4 lysozyme in complex with an antagonist sphingolipid analogue ML-056 (W-146) (Hanson et al., 2012) (Fig. 6). Unique features of the S1P1 receptor were not predictable from the extensive and rigorous analysis by mutagenesis previously performed (Fujiwara et al., 2007; Fujiwara et al., 2005; Inagaki et al., 2005; Jo et al., 2005; Parrill et al., 2000a; Parrill et al., 2000b; Schurer et al., 2008; Wang et al., 2001). The extracellular face of S1P1 receptor is tightly structured (Hanson et al., 2012) where the N-terminus a-helix folds over the top of the receptor to block access to the binding pocket from the aqueous phase. This static view of the receptor suggests that S1P might require an alternate route into the binding pocket.

Figure 6.

Figure 6

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The alpha-carbon tracing of the S1P1 receptor at 2.8 Angstrom resolution. The orthosteric antagonist ML-056 is fully visualized within the binding pocket immediately above the rhodopsin GPCR family conserved W269, that serves as a rotamer toggle switch in rhodopsin, but in S1P1 serves as the binding point of contact for alternative binding mode agonists like CYM-5442. Extracellular loops (ECL) 1, 2 and 3 are shown. There are 7™ domains and N-term, I, 1st ICL, II, 1st ECL, III, 2nd ICL, IV, 2 ECL, V, 3rd ICL, VI, 3rd ECL, VII, C-term are shown. V and VI are not marked in this pose. See Figures 3, 4, and 5 for biologic action of the S1P1 agonist CYM-5442 and reference Hanson et al., 2012, for additional structural details and amino acid alignments. Figure adapted from Hanson et al., 2012, reprinted with permission from AAAS.

Shown in Figure 6 is the alpha-carbon tracing of S1P1 receptor at 2.8 Angstrom resolution. The orthosteric antagonist ML-056 is fully visualized within the binding pocket immediately above the rhodopsin GPCR family conserved W269, which serves as a rotamer toggle switch in rhodopsin, but in S1P1 serves as the binding point of contact for alternate binding mode agonists like CYM-5442. By virtue of these unique interactions with the receptor, these molecules no longer require the zwitterionic amino-phosphate headgroup and thus have enhanced physico-chemical properties that allow delivery to the lung for the influenza studies. The crystal structure provides the detailed insight into molecular interaction that may rationally enhance the properties of S1P1 agonists for the relief of cytokine storm.

Conclusions and future directions

Conclusions reached indicate the following four major points displayed in tabular form at

  1. Cytokine storm plays an essential role in the pathogenesis and clinical outcome of influenza virus infection.

    1. Blockade of cytokine storm provides greater protection than antiviral therapy to inhibit virus replication and does so without compromising the host’s ability to control and terminate influenza virus infection.

    2. Observations made with human pathogenic H1N1 Swine 09 influenza virus isolates and mouse adapted H1N1 influenza virus.

  2. Sphingosine-1-phosphate (S1P) receptor agonists blunts cytokine storm thus cytokine storm is chemical tractable.

    1. Blunting of cytokine storm is mediated by just one of the five S1P receptors: S1P1.

  3. Molecular mechanism: S1P1 receptor signaling occurs on pulmonary endothelial cells and not influenza virus infected epithelial cells.

    1. S1P1 receptor is located on pulmonary endothelial cells and not on pulmonary epithelial cells.

    2. S1P1 agonism suppresses cytokine and innate immune cell recruitment.

  4. Immune cell infiltration and cytokine production are distinct events both orchestrated by pulmonary endothelial cells.

    1. Proinflammatory cytokine responses depend on type I interferon signaling.

A kinetic outline of events of molecular pathogenesis of influenza virus infection in the lung leading to cytokine storm and its resultant morbidity and mortality is sketched below

Cytokine storm has an early and late stage. The early stage is reflective of a pulmonary endothelial amplification network taking place within the first few days of infection. This phase is mediated by S1P1 signaling on pulmonary endothelial cells. The second phase of immunopathologic injury and activity occurs later during influenza virus infection, days 6–8 post-infection, is T cell-mediated and signals likely through S1P3, 4, 5rec signaling, with S1P3rec and S1P4rec the most likely culprits.

  • Step 1

    Influenza virus enters the upper respiratory tract and infects primary epithelial cells in the lung parenchyma.

  • Step 2

    Infected epithelial cells produce and release a signaling molecule(s), not yet defined, that cross-talk with and activate endothelial cells.

  • Step 3

    Pulmonary endothelial cells augment influenza virus-induced cytokine storm by two distinct mechanisms. First, such non-influenza virus-infected endothelial cells release a currently unidentified molecule(s) that activate primarily plasmacytoid1 DCs to produce IFN-α.

  • Step 4

    IFN-α production stimulates the expression of proinflammatory molecules leading to the initiation of cytokine storm.

  • Step 5

    The second mechanism by which pulmonary endothelial cells augment influenza virus-induced cytokine storm is by attracting the recruitment of innate inflammatory cells into the lung. Such innate inflammation infiltrates exacerbate cytokine storm by producing additional proinflammatory molecules.

  • Step 6

    The late stage occurs by day 6–8. Influenza virus-specific T cells activated and expanded in MLN and in pulmonary tissues produce additional inflammatory molecules, lyse virus-infected epithelial cells and thereby augmenting cytokine storm and immune-mediated injury.

Future directions include investigating IFN-I as to its cellular source, species and signaling pathways while dissecting molecular cross-talk and signaling between pulmonary endothelial cells and other pulmonary cells, especially influenza virus infected epithelial cells and plasmacytoid dendritic cells.

Expansion and generalities of our findings for other acute respiratory infections, infectious and autoimmune disorders in which cytokine storm is a major component. Indeed, preliminary investigations with Matthew Friedman at the University of Maryland Medical School suggest a similar scenario for SARS. Studies by Kevin Walsh from our laboratory recently found that S1P agonist successfully aborts pneumonia virus of mice, a murine model of human respiratory syncytial virus (RSV) disease2.

Additionally, in collaboration with the NIH and Battelle group, and with Yoshi Kawaoka, we have been studying human pathogenic H1N1 2009 virus in ferrets, a host whose infection with influenza virus is more akin to humans than the mouse. Samples from infected ferrets not treated; or treated with S1P agonists only, oseltamivir alone, or S1P agonist combined with oseltamivir are currently under analysis. Lastly, it is likely that genetic aberrations in the S1P pathway being uncovered may prove to be of use for screening and identifying those humans who would be most susceptible to severe cytokine storm during an influenza viral infection. We are currently exploring such genetic profiling.

HIGHLIGHTS.

  • Cytokine storm plays a direct role in causation of injury due to influenza.

  • Signaling via S1P1 receptor on pulmonary endothelial cells initiates lung pathology.

  • S1P1 receptor agonist blocks the influenza-induced cytokine storm.

  • S1P1 receptor agonist does not block host immune control over influenza.

  • Pulmonary endothelial cells are the central regulators of cytokine storm.

Acknowledgments

Parts of the work were done with the active collaboration and consultation with Yoshihiro Kawaoka, Stuart Cahalan, David Marsolais, Ray Stevens, Fiona Scott, Edward Roberts, and Robert Peach. The research was supported by NIH grants AI074564 (MBAO, HR), AI009484 (MBAO), AI005509 (HR), MH084512 (HR), and NIH training grants NS041219 and AI007244 (K. Walsh), NIH training grant AI007364 and American Heart Association Fellowship 11POST7430106 (J. Teijaro). We thank Marcus Boehm, Liming Huang, and Bryan Clemons (Receptos, Inc.) for helping provide RP-002 as a chemical tool. HR is a founder of Receptos, Inc.

Footnotes

1

Teijaro, J., H. Rosen, and M.B.A. Oldstone. Unpublished observations, 2012.

2

Walsh, K.B., J.R. Teijaro, M. Welch, and M.B.A. Oldstone. Manuscript submitted, 2012.

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Contributor Information

Michael B.A. Oldstone, Email: mbaobo@scripps.edu.

John R. Teijaro, Email: teijaro@scripps.edu.

Kevin B. Walsh, Email: walsh.kevin@gene.com.

Hugh Rosen, Email: hrosen@scripps.edu.

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