Cytokine Storm Plays a Direct Role in the Morbidity and Mortality from Influenza Virus Infection and is Chemically Treatable with a Single Sphingosine-1-Phosphate Agonist Molecule
Cytokine storm defines a dysregulation of and an excessively exaggerated immune response most often accompanying selected viral infections and several autoimmune diseases. Newly emerging and re-emerging infections of the respiratory tract, especially influenza, SARS, and hantavirus post considerable medical problems. Their morbidities and mortalities are often a direct result of cytokine storm. This chapter visits primarily influenza virus infection and resultant cytokine storm. It provides the compelling evidence that illuminates cytokine storm in influenza pathogenesis and the clear findings that cytokine storm is chemically tractable by therapy directed toward sphingosine-1-phosphate receptor (S1PR) modulation, specifically S1P1R agonist therapy. The mechanism(s) of how S1P1R signaling works and the pathways involved are subjects of this review.
KeywordsInfluenza Virus Alveolar Macrophage Severe Acute Respiratory Syndrome Influenza Infection Influenza Virus Infection
Newly emerging and re-emerging infections of the respiratory tract pose considerable medical and public health concerns as well as economic hardships to humans and countries. The last century witnessed at least five pandemics: 1918/1919, H (viral hemagglutinin) 1 N (viral neuraminidase) 1 Spanish influenza; 1957, H2N2 Asia influenza; 1968, H3N2 Hong Kong influenza; 1977, H1N1 Russian influenza; and 1997, H5N1 bird influenza (reviewed Wright et al. 2007). In twenty-first century alone, two viral pandemics have already occurred—the first was in 2002 when the new viral pandemic, severe acute respiratory syndrome (SARS), appeared (reviewed Oldstone 2010), followed by the first influenza virus pandemic in 2009, H1N1 swine influenza (Dawood et al. 2012). Moreover, Hantaviruses have infected humans in the past and recently in an outbreak at Yellowstone National Park. These viral infections loom as important zoonotic human diseases with the threat of human to human transmission and excessively high mortality rates. For example, 1918/1919 H1N1 influenza infections caused the greatest loss of life from any infectious disease or medical condition known, visiting roughly 5 % of the world’s population and killing 2 % or 40–50 million persons (Ahmed et al. 2007; Johnson and Mueller 2002). The most recent influenza pandemic, 2009 H1N1 swine influenza, rapidly infected millions worldwide with estimates exceeding 290,000 deaths of which more than 201,000 resulted from respiratory failure and over 83,000 from cardiovascular complications (Dawood et al. 2012). All of the above diseases in humans (Arankalle et al. 2010; Cheng et al. 2010; Lee et al. 2011) and experimental animals (Baskin et al. 2009; Kobasa et al. 2007; Marcelin et al. 2011; Zhang et al. 2012) are accompanied by early exacerbation and dysregulation of innate immune responses, a combination of events called “cytokine storm.” Severe disease and death following infection correlated strongly with the cytokine storm.
Susceptibility or resistance to any viral infection is determined by the balance between the virulence of the infecting agent and the resistance of the host including the aggressiveness of the latter’s immune response against the virus infection. When the immune response is limited due to either host genetics, acquired defects like lymphoid diseases, immaturity of the immune system in fetuses, newborns, or young children, or loss of immune vigor in the aged, the advantage is firmly in the virus’s court. However, usually when the infection occurs in individuals with a developed and competent immune system, the advantage is the host’s, unless the infecting virus overwhelms the individual’s immune system or the immune response becomes hyperactive resulting in an excessive innate and adoptive immune reaction, the “cytokine storm” phenomenon. Cytokine storm leads to immune-mediated injury (immunopathology).
When available, vaccination is useful in protecting groups of previously uninfected (naive) individuals from acute viral respiratory diseases. By this means, the spread of infection is diminished. Additionally antiviral drugs, which were developed as effective therapies to diminish or in some instances prevent ongoing infections, are reasonably effective, nevertheless come with two marked limitations. First, antiviral drugs exert selective pressure on viral progeny, promoting their mutation and selection thereby creating a new generation that is more fit and resistant to the drug (Nguyen et al. 2012; Orozovic et al. 2011). Second, the injury associated with these acute viral respiratory diseases, including influenza, results from a combination of the virus’s intrinsic virulence in lysing cells it infects and the intensity of the immune response which can damage tissues and promote a cytokine storm. Antiviral drugs are effective against the virus but not against cytokine storm or immune-mediated injury.
Recently, while studying human H1N1 2009 influenza virus infection in mice (Walsh et al. 2011; Teijaro et al. 2011) and ferrets (Teijaro et al. 2013), we uncovered the first direct and definitive experimental evidence that cytokine storm, per se, was a major factor in the causation of morbidity and mortality from influenza virus and some other acute, severe respiratory infections rather than just the accompanying phenomena. Further, we documented that cytokine storm was chemically treatable using an immunomodulatory small molecule, sphingosine-1-phosphate agonist, which dramatically inhibited the production of cytokines/chemokines and the innate cellular response, thereby blunting both the innate as well as the adoptive antiviral T cell response (Marsolais et al. 2009; Walsh et al. 2011; Teijaro et al. 2011). These events successfully limited immunopathologic injury. Nevertheless, a sufficient host T cell response remained and coupled with the antiviral antibody response curtailed the acute infection while providing recall immunologic memory to any renewed insult by the virus. This review focuses primarily on our experimental work that provided these conclusions.
2 Influenza Virus Infection
2.1 Epidemiologic and Experimental Evidence for Cytokine Storm
An overly aggressive innate immune response, the early recruitment of inflammatory leukocytes to the lung and dysregulated immune gene expression were key contributors to morbidity from the 1918/1919 influenza virus onslaught, as suggested by experimental infection of macaques with the 1918 H1N1 virus strain (Kobasa et al. 2007; Cilloniz et al. 2009). Clinical studies of humans infected by H5N1 bird influenza virus revealed a significant association between excessive early cytokine responses and immune cell recruitment as strongly predictive of poor medical outcomes (de Jong et al. 2006). Recently, similar results for influenza virus infections were reported for experimental animal models (Baskin et al. 2009; Marcelin et al. 2011, Zhang et al. 2012) and for humans (Arankalle et al. 2010; Cheng et al. 2010; Lee et al. 2011). Among reports of H1N1 2009 pandemic influenza infections in humans, that of Arankalle et al. (2010) is illuminating. Analyzing viral events and cytokine storms in critically ill-hospitalized patients, the investigators showed that those who died had no difference in influenza viral load from those who recovered. However, the patients who recovered and left the hospital had significantly lower cytokine storm profiles than the population who succumbed from the infection. My colleague, Hugh Rosen, and I reasoned that calming the host’s aggressive and exaggerated cytokine storm response might provide the opportunity to shift the balance from severe morbidity and mortality to survival. Our laboratories started jointly about 7 years ago to test this hypothesis (Marsolais et al. 2008). We selected the molecule sphingosine 1-phosphate (S1P) and sought to determine if harmful immunologic processes accompanying H1N1 2009 influenza infection could be modulated by S1P receptors in the lung. We selected S1P agonists because of their documented history of modulating lymphoid trafficking by inducing sequestration of lymphocytes in secondary lymphoid regions. By that means, S1P agonists limit the migration of effector lymphocytes to areas where such cells mediate immunologic injury (Rosen et al. 2007, 2009, 2013; see Chaps. 1 (Rosen) and 6 (Cyster) in this volume)). S1P is a signaling lipid present at a concentration of 1–3 nM in plasma and approximately 100 nM in lymph. Physiologically, S1P levels are under tight homeostatic control, and S1P signals through specific S1P receptors of which there are five (S1P receptors 1–5). These five specific S1P receptors are coupled to different G proteins for the purpose of regulating a variety of downstream pathways specific for many cells, tissues, and organs (Rosen et al. 2007, 2009, 2013).
2.2 Tracking and Kinetics of Influenza Virus-Specific CD8 and CD4 T Cells in the Lung and their Modulation by S1P Agonist
2.3 Pulmonary Injury and Disease Associated with Influenza and Resultant Cytokine Storm are Treatable with a Single S1P1 Receptor Agonist Molecule
Thus, severe pulmonary injury and disease associated with influenza infection and resultant cytokine storm were treatable with a preparation composed of only S1P1 receptor agonist molecules, thereby avoiding signaling through S1P2, S1P3, S1P4, and S1P5 receptors. Pharmaceutically this may be of importance if/when individually S1P2, S1P3, S1P4, or S1P5 signaling might lead to unwanted harmful biologic effects.
2.4 S1P1 Receptors are Located on Pulmonary Endothelial Cells, Which Serve as the Gatekeepers for Cytokine Storm
T and B lymphocytes as well as pulmonary endothelial cells were the only cells within the lung that expressed measurable amounts of S1P1-eGFP protein (Fig. 3 Panel a). We therefore determined whether lymphocytes expressing S1P1 receptors were involved in S1P1 agonist inhibition of cytokine storm or were merely bystander cells accompanying the innate immune response to influenza virus infection. Since Rag2-/- mice are deficient in lymphocytes, we reasoned that if such mice, when infected with influenza virus, generated a cytokine storm that could be blocked by S1P1 agonist, then lymphocytes were ruled out as initiators of cytokine storms. Our experiments documented that cytokine storm occurred in Rag2-/- mice infected with influenza virus. Importantly, treatment of infected Rag2-/- mice with the S1P1 agonist CYM-5442 significantly reduced cytokines and chemokines in the bronchial lavage fluids as well as minimalizing the infiltration of innate cells (macrophages/monocytes and NK cells). Recently, John Teijaro (2013), utilizing cell sorting and a biochemical approach, found S1P1 receptor on plasmacytoid dendritic cells (pDC) whose expression was undetectable in the S1P1-γ GFP transgenic mouse model.
2.5 Type I Interferon Signaling is Essential for the Cytokine/Chemokine Response of Cytokine Storm but is not Involved in Recruitment of Innate Inflammatory Cells into the Lung
Influenza virus infection induces a robust interferon type I response, despite the early induction of the viral NS1 protein that suppresses the cellular induction of and response to interferon I (Fernandez-Sesma 2007). The predominant type I interferon produced early following influenza virus infection is alpha, not beta (Fig. 4a). However, the cellular sources of interferon type I-α produced and amounts made by various cell populations have not been clear. The two major pulmonary cell populations known to make type I interferon in vivo following respiratory viral infections are pDCs and alveolar macrophages (Kumagai et al. 2007). To judge the contribution of pDCs to interferon-α production in the lung, we utilized a novel mouse model recently developed at Scripps by Bruce Beutler and termed “feeble.” Feeble mice have a specific genetic defect that prevents their pDCs from producing type I interferon and pro-inflammatory cytokines upon activation of TLR7 and TLR9 ligands by influenza virus stimulation (Blasius et al. 2010). Importantly, there is no disruption of the numbers or vitality of pDCs, and the feeble mouse defect is specifically restricted to pDCs. As displayed in Fig. 4e, when wild type or feeble mice received H1N1 human 2009 swine influenza with or without S1P1 agonist CYM5442 treatment, 75–85 % of total interferon-α was produced by pDCs. Further, interferon-α release was significantly inhibited by the S1P1 agonist CYM-5442. These results were confirmed using a pDC depletion antibody (anti-PDCS-1 clone 120.68), which again resulted in significant depletion of pDCs in the lung and corresponding reductions in interferon-α, CCL2, CCL5, and IL-6. Thus, pDCs are the essential and major producers of interferon-α (75–85 %) and involved in amplification of cytokine/chemokine volumes following influenza infection. Other depletion studies indicate that most of the remaining interferon-α production (~15–25 %) was by alveolar macrophages.
Since S1P1 agonist therapy diminished interferon-α production, and the majority of interferon-α produced was by pDCs, it was important to learn whether or not pDCs expressed S1P1 receptors on their surfaces. We know that alveolar macrophages, the other main albeit minority producers of interferon-α do not express S1P1 receptors (Fig. 2a). Plasmacytoid dendritic cells were recently found to express S1P1 receptors (Teijaro 2013). However, using the S1P1 eGFP receptor knock-in mice and pDCs of more than 98.5 % purity failed to show that these cells expressed S1P1 receptors (Fig. 4e). Thus, the S1P1-specific receptor is found primarily on pulmonary endothelial cells with lesser amounts on pulmonary pDCs. S1P1 receptor agonist acts directly on pulmonary endothelial and pDCs and likely indirectly on alveolar macrophages. We have, as yet, been unable to detect S1P1 receptor on alveolar macrophages.
2.6 Working Model for the Initial Production of Cytokine Storm and its Chemical Tractability by Single S1P1 Molecules
After that initial response, a second stage occurs by day 4–8 (see 2.2) via a mechanism described in our publication (Marsolais et al. 2009). Here influenza virus-specific T cells are activated and expand numerically in the mediastinal lymph nodes and pulmonary tissues. These T cells of the adoptive immune system produce additional inflammatory molecules and lyse influenza virus-infected epithelial cells thereby augmenting cytokine storm and immune-mediated injury. This second phase of tissue injury is primarily influenza virus–specific T cell-mediated and signals through S1P1 but also likely progresses via S1P3 and 4 receptors, as by our preliminary results. However, additional data are required to ensure these observations. What is clear is that treatment with S1P permissive-AAL-R agonists signaling via S1P1,3,4,5 receptors affect adoptive immune T cell-mediated immunopathology by downregulating MHC and co-stimulatory molecules of DCs located in the mediastinal lymph nodes and the lung parenchyma thereby blunting the arming, expansion, and migration of virus-specific CD4 and CD8 T cells into the lung (Marsolais et al. 2009).
The cytokine pathways blunted by S1P1 agonist signaling are displayed in Fig. 5 by the symbol ⊢.
3 Conclusions and Future Studies
Cytokine storm plays an essential role in the pathogenesis and clinical outcome of influenza virus infection. Blockade of cytokine storm provides greater protection than does antiviral therapy, like that with a neuraminidase inhibitor, and does so without compromising the control and clearance of viruses. Moreover, optimal therapy is achieved by combining S1P agonists with anti-neuraminidase treatment. For the foregoing observations, human pathogenic H1N1 09 influenza virus and mouse adapted H1N1 influenza virus were used.
Sphingosine-1-phosphate (S1P) receptor agonists blunt cytokine storm. Importantly, cytokine storm is chemically disarmed by administering one of the five S1P receptors: S1P1.
The molecular mechanism of this event involves S1P1 receptor signaling on pulmonary endothelial cells and pulmonary pDCs but not virally infected epithelial cells or alveolar macrophages. Pulmonary endothelial cells are the major gateway combined with pulmonary pDCs to precipitate a cytokine storm. S1P1 agonism suppresses the recruitment of both cytokines, innate and adoptive immune cells. Blunting of innate immune cell function and virus-specific T cell activity lessens morbidity and prevents mortality associated with experimental models of influenza virus infection in mice and ferrets. In both species, there is a sufficient antiviral response remaining to terminate the virus infection and provide immune memory upon rechallenge.
Immune cell infiltration and cytokine production are distinct events, but both are orchestrated by endothelial and pDC cells. Pro-inflammatory cytokine responses depend on type I interferon signaling; IFN-α is the predominant interferon made. The predominant pulmonary cell making type I interferon is the pDC (over 75–85 %); alveolar macrophages make most of the rest.
3.2 Future Studies
Investigate interferon I as to the cellular source and signaling pathway(s) in the influenza system.
Dissect crosstalk and signaling between pulmonary endothelial cells, infected epithelial cells, and interferon-producing pDCs. Identify the molecules involved. See if these molecules provide potential therapeutic targets.
Determine generalities for other acute respiratory infections, e.g., Hantavirus, respiratory syncytial virus, SARS, pneumococcal pneumonia, in which cytokine storm plays a prominent role.
Study an animal model (subhuman primates) more reflective of influenza in humans. Results from our studies in ferrets (Teijaro et al. 2013) mirror the protection supplied by S1P1 agonist therapy in defending against influenza virus infection in the murine model.
Define the S1P pathway and design a genetic screen to identify humans who are the most susceptible to cytokine storm.
Develop specific S1P receptor agonists and antagonists for human therapeutics, focusing initially on S1P1 molecules.
The experimental work described in this chapter was initiated with and carried out as a collaboration between my laboratory and that of Hugh Rosen. Graduate student (Stuart Cahalan) and former postdoctoral fellows (David Marsolais, Kevin Walsh, and John Teijaro) were instrumental in the findings presented. John Teijaro continues his independent work in this area as a faculty member at Scripps. Yoshi Kawaoka and colleagues in his laboratories have been valued collaborators. This work was funded by NIH grants AI009484 (MBAO), AI074564 (MBAO, HR), MH084512 (HR), and was also supported by the NIH/NIAID under Award Number U54 AI057160 to the Midwest Regional Center of Excellence (MRCE) for Biodefense and Emerging Infectious Diseases Research (MBAO).
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