Kevin B. Walsh

Endothelial cells are central orchestrators of cytokine amplification during influenza virus infection

Summary Cytokine storm during viral infection is a prospective predictor of morbidity and mortality, yet the cellular sources remain undefined. Here, using genetic and chemical tools to probe functions of the S1P1 receptor, we elucidate cellular and signaling mechanisms that are important in initiating cytokine storm. Whereas S1P1 receptor is expressed on endothelial cells and lymphocytes within lung tissue, S1P1 agonism suppresses cytokines and innate immune cell recruitment in wild-type and lymphocyte-deficient mice, identifying endothelial cells as central regulators of cytokine storm. Furthermore, our data reveal immune cell infiltration and cytokine production as distinct events that are both orchestrated by endothelial cells. Moreover, we demonstrate that suppression of early innate immune responses through S1P1 signaling results in reduced mortality during infection with a human pathogenic strain of influenza virus. Modulation of endothelium with a specific agonist suggests that diseases in which amplification of cytokine storm is a significant pathological component could be chemically tractable.

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

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 hosts 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.

IL-10 directly suppresses CD4 but not CD8 T cell effector and memory responses following acute viral infection

Mounting effective T cell responses is critical for eliciting long-lasting immunity following viral infection and vaccination. A multitude of inhibitory and stimulatory factors are induced following infection, and it is the compilation of these signals that quantitatively and qualitatively program the ensuing effector and memory T cell response. In response to lymphocytic choriomeningitis virus (LCMV) infection, the immunosuppressive cytokine IL-10 is rapidly up-regulated; however, how IL-10 is regulating what is often considered an “optimal” immune response is unclear. We demonstrate that IL-10 directly inhibits effector and memory CD4 T cell responses following an acutely resolved viral infection. Blockade of IL-10 enhanced the magnitude and the functional capacity of effector CD4 T cells that translated into increased and more effective memory responses. On the other hand, lack of IL-10 signaling did not impact memory CD8 T cell development. We propose that blockade of IL-10 may be an effective adjuvant to specifically enhance CD4 T cell immunity and protection following vaccination.

A critical role for the sphingosine analog AAL-R in dampening the cytokine response during influenza virus infection

Pulmonary tissue damage resulting from influenza virus infection is caused by both the cytolytic activity of the virus and the host immune response. Immune-mediated injury results from T cell-mediated destruction of virus-infected cells and by release of cytokines and chemokines that attract polymorphonuclear leukocytes (PML) and macrophages to the infected site. The cytokines/chemokines potentiate dendritic cell (DC) activation and T cell expansion, which further enhances local damage. Here we report that immune modulation by local administration to the respiratory tract of sphingosine analog AAL-R significantly dampens the release of cytokines and chemokines while maintaining protective neutralizing antibody and cytotoxic T cell responses. As a result there was a marked reduction of infiltrating PML and macrophages into the lung and resultant pulmonary tissue injury. DC maturation was suppressed, which limited proliferation of specific antiviral T cells in the lung and draining lymph nodes. Further, AAL-R was effective in controlling CD8+ T cell accumulation in the lungs even when given 4 days after initiation of influenza virus infection. These data indicate that sphingosine analogs display useful potential for controlling the immunopathology caused by influenza virus.

Local not systemic modulation of dendritic cell S1P receptors in lung blunts virus-specific immune responses to influenza

The mechanism by which locally delivered sphingosine analogs regulate host response to localized viral infection has never been addressed. In this report, we show that intratracheal delivery of the chiral sphingosine analog (R)-2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol (AAL-R) or its phosphate ester inhibits the T-cell response to influenza virus infection. In contrast, neither intraperitoneal delivery of AAL-R nor intratracheal instillation of the non-phosphorylatable stereoisomer AAL-S suppressed virus-specific T-cell response, indicating that in vivo phosphorylation of AAL-R and sphingosine 1-phosphate (S1P) receptor modulation in lungs is essential for immunomodulation. Intratracheal delivery of water-soluble S1P1 receptor agonist at doses sufficient to induce systemic lymphopenia did not inhibit virus-specific T-cell response, indicating that S1P1 is not involved in the immunosuppressive activities of AAL-R and that immunosuppression acts independently of naive lymphocyte recirculation. Accumulation of dendritic cells (DCs) in draining lymph nodes was inhibited by intratracheal but not intraperitoneal delivery of AAL-R. Direct modulation of DCs is demonstrated by the impaired ability of virus-infected bone marrow-derived DCs treated in vitro with AAL-R to trigger in vivo T-cell response after adoptive transfer to the airways. Thus, our results suggest that locally delivered sphingosine analogs induce immunosuppression by modulating S1P receptors other than S1P1 or S1P2 on dendritic cells in the lungs after influenza virus infection.

Mapping the innate signaling cascade essential for cytokine storm during influenza virus infection

Significance Cytokine storm plays an essential and commanding role in the clinical outcome and pathogenesis of influenza virus infection. We previously documented that a small molecule that activates sphingosine-1-phosphate-1 receptor (S1P1R) signaling is primarily responsible for blunting cytokine storm to protect the infected host from the consequences of influenza infection. In the present study, we map host innate signaling pathways of cytokine storm and chart where along those pathways the drug is effective. We find that the efficacy of S1P1R agonist in blunting cytokine storm is through global inhibition downstream of myeloid differentiation primary response gene 88 and IFN-β promoter stimulator-1 signaling. During pathogenic influenza virus infection, robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced tissue destruction all contribute to morbidity and mortality. Earlier we reported that modulation of sphingosine-1-phosphate-1 receptor (S1P1R) signaling provided a chemically tractable approach for the effective blunting of cytokine storm, leading to the improvement of clinical and survival outcomes. Here, we show that S1P1R agonist treatment suppresses global cytokine amplification. Importantly, S1P1R agonist treatment was able to blunt cytokine/chemokine production and innate immune cell recruitment in the lung independently of endosomal and cytosolic innate sensing pathways. S1P1R signaling suppression of cytokine amplification was independent of multiple innate signaling adaptor pathways for myeloid differentiation primary response gene 88 (MyD88) and IFN-β promoter stimulator-1 signaling, indicating a common pathway inhibition of cytokine storm. We identify the MyD88 adaptor molecule as responsible for the majority of cytokine amplification observed following influenza virus challenge.

Quelling the storm: utilization of sphingosine-1-phosphate receptor signaling to ameliorate influenza virus-induced cytokine storm

Initial and early tissue injury associated with severe influenza virus infection is the result of both virus-mediated lysis of infected pulmonary cells coupled with an exuberant immune response generated against the virus. The excessive host immune response associated with influenza virus infection has been termed “cytokine storm.” Therapies that target virus replication are available; however, the selective pressure by such antiviral drugs on the virus often results in mutation and the escape of virus progeny now resistant to the antiviral regimen, thereby rendering such treatments ineffective. This event highlights the necessity for developing novel methods to combat morbidity and mortality caused by influenza virus infection. One potential method is restricting the host’s immune response. However, prior treatment regimens employing drugs like corticosteroids that globally suppress the host’s immune response were found unsatisfactory in large part because they disrupted the host’s ability to control virus replication. Here, we discuss a novel therapy that utilizes sphingosine-1-phosphate (S1P) receptor signaling that has the ability to significantly limit immunopathologic injury caused by the host’s innate and adaptive immune response, thereby significantly aborting morbidity and mortality associated with influenza virus infection. Moreover, S1P analog therapy allows for sufficient anti-influenza T cell and antibody formation to control infection. We review the anti-inflammatory effects of S1P signaling pathways and how modulation of these pathways during influenza virus infection restricts immunopathology. Finally, we discuss that combinatorial administration of S1P simultaneously with a current antiviral enhances the treatment efficacy for virulent influenza virus infections above that of either drug treatment alone. Interestingly, the scope of S1P receptor therapy reported here is likely to extend beyond influenza virus infection and could prove useful for the treatment of multiple maladies like other viral infections and autoimmune diseases where the host’s inflammatory response is a major component in the disease process.

Dissecting influenza virus pathogenesis uncovers a novel chemical approach to combat the infection

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.

Type I interferon is a therapeutic target for virus-induced lethal vascular damage

Significance Lassa virus is, after dengue virus, the second most common cause of viral hemorrhagic fever. In susceptible individuals, Lassa virus infection is associated with vascular permeability, leading to tissue edema, organ failure, and death. Hemorrhagic fever viruses efficiently infect vascular endothelial cells, but are generally considered noncytopathic. Thus, the mechanism of virus-induced vascular injury remains unclear. Using the lymphocytic choriomeningitis virus variant clone 13, a prototype of Lassa virus, we show here that lethal vascular leakage in susceptible mice was completely prevented by type I IFN receptor blockade. Therefore, approaches that target type I IFNs or effector molecules induced by these cytokines may be considered for the treatment of Lassa fever and other severe hemorrhagic viral illnesses. The outcome of a viral infection reflects the balance between virus virulence and host susceptibility. The clone 13 (Cl13) variant of lymphocytic choriomeningitis virus—a prototype of Old World arenaviruses closely related to Lassa fever virus—elicits in C57BL/6 and BALB/c mice abundant negative immunoregulatory molecules, associated with T-cell exhaustion, negligible T-cell–mediated injury, and high virus titers that persist. Conversely, here we report that in NZB mice, despite the efficient induction of immunoregulatory molecules and high viremia, Cl13 generated a robust cytotoxic T-cell response, resulting in thrombocytopenia, pulmonary endothelial cell loss, vascular leakage, and death within 6–8 d. These pathogenic events required type I IFN (IFN-I) signaling on nonhematopoietic cells and were completely abrogated by IFN-I receptor blockade. Thus, IFN-I may play a prominent role in hemorrhagic fevers and other acute virus infections associated with severe vascular pathology, and targeting IFN-I or downstream effector molecules may be an effective therapeutic approach.

Animal Model of Respiratory Syncytial Virus: CD8+ T Cells Cause a Cytokine Storm That Is Chemically Tractable by Sphingosine-1-Phosphate 1 Receptor Agonist Therapy

ABSTRACT The cytokine storm is an intensified, dysregulated, tissue-injurious inflammatory response driven by cytokine and immune cell components. The cytokine storm during influenza virus infection, whereby the amplified innate immune response is primarily responsible for pulmonary damage, has been well characterized. Now we describe a novel event where virus-specific T cells induce a cytokine storm. The paramyxovirus pneumonia virus of mice (PVM) is a model of human respiratory syncytial virus (hRSV). Unexpectedly, when C57BL/6 mice were infected with PVM, the innate inflammatory response was undetectable until day 5 postinfection, at which time CD8+ T cells infiltrated into the lung, initiating a cytokine storm by their production of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Administration of an immunomodulatory sphingosine-1-phosphate (S1P) receptor 1 (S1P1R) agonist significantly inhibited PVM-elicited cytokine storm by blunting the PVM-specific CD8+ T cell response, resulting in diminished pulmonary disease and enhanced survival. IMPORTANCE A dysregulated overly exuberant immune response, termed a “cytokine storm,” accompanies virus-induced acute respiratory diseases (VARV), is primarily responsible for the accompanying high morbidity and mortality, and can be controlled therapeutically in influenza virus infection of mice and ferrets by administration of sphingosine-1-phosphate 1 receptor (S1P1R) agonists. Here, two novel findings are recorded. First, in contrast to influenza infection, where the cytokine storm is initiated early by the innate immune system, for pneumonia virus of mice (PVM), a model of RSV, the cytokine storm is initiated late in infection by the adaptive immune response: specifically, by virus-specific CD8 T cells via their release of IFN-γ and TNF-α. Blockading these cytokines with neutralizing antibodies blunts the cytokine storm and protects the host. Second, PVM infection is controlled by administration of an S1P1R agonist.