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 important in initiating cytokine storm. While 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 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 where amplification of cytokine storm is a significant pathological component could be chemically tractable.

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.

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 (S1P 1 R) 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 S1P 1 R agonist treatment suppresses global cytokine amplification. Importantly, S1P 1 R 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. S1P 1 R 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.pathology | pulmonary O verabundant innate immune responses correlate with increased morbidity and mortality during multiple pathogenic respiratory viral infections (1-5). When studying human pandemic H1N1/2009 influenza virus in mice and ferrets, we found direct evidence that cytokine storm was chemically tractable using a sphingosine-1-phosphate receptor-1 (S1P 1 R) selective agonists. S1P 1 R agonist therapy suppressed innate immune cell recruitment, cytokine-chemokine production, and improved survival without altering viral clearance, indicating that cytokine storm was causative to disease pathogenesis and that S1P therapy could suppress detrimental innate immune responses without hindering virus control (6, 7). The identification that S1P 1 R agonists suppress detrimental innate immune responses without hindering virus control indicates that S1P 1 R probes may serve as both viable drug leads to curb influenza virus morbidity and mortality, and as research tools to identify additional cellular signaling pathways that can be targeted to improve clinical outcomes during respiratory viral infection.To generate a molecular understanding how S1P 1 R agonist therapy effectively blunts pathological innate inflammatory responses, we systematically assessed the role various innate signaling pathways play in S1P 1 R-mediated suppression of inflammation following influenza virus infection. Using an S1P 1 R selective agonist synergistically with genetic and biochemical tools, we reveal that S1P 1 R signaling effectively suppressed global cytokine amplification at a point that converged downstream of multiple innate signaling pathways. We reveal that S1P 1 R signaling can suppress innate cellular recruitment and cytokine amplification downstream of both endosome and cytosolic innate sensing pathways. Moreover, we identify myeloid differentiation primary response gene 88 (MyD88) as the ...

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 cellmediated 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.immunopathology ͉ dendritic cells ͉ cytokine storm ͉ T cells T he antiviral host response evolved to limit the spread of infection at the cost of causing tissue injury. There is a balance between the protective and injurious responses that leads either to the purging of infectious virus and host recovery or to severe disease and even death. Thus, strategies to balance the antiviral immune response in favor of host outcome need to be developed.T cell response elicited early in the course of infection recognizes, attacks and lyses virus-infected cells to eliminate potential factories of progeny viruses (1-5). The protective influenza antibody response is elicited later and plays a role in controlling re-infection (6, 7). The innate and adaptive immune systems release wide varieties of cytokines and chemokines that activate and attract inflammatory cells to the site of infection (2,8,9). However, these molecules can also cause the host harm by a phenomenon known as cytokine storm. Cytokine storm has been convincingly documented both in experimental animals infected with the 1918/1919 and H5N1 influenza viruses (2, 10-13) as well as in humans (14-18) succumbing to H5N1 infection. Although antiviral drugs can be used to treat the virus, a strategy to balance the resultant cytokine release and lung injury while maintaining benefits of the antiviral protective immune response is needed (19). Influenza virus replication is most often limited primarily to the respiratory tract but the systemic signs and symptoms of disease, e.g., fever, muscle pain and shakes, intestinal tract involvement, are related to cytokine effects (20, 21).We reported that intratracheal (i.t.) delivery of the sphingosine analog AAL-R or its phosphate ester inhibited virus-specifi...

Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.

Mounting effective T cell responses is critical for eliciting longlasting 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. Following infection, antigen-presenting cells (APC) present viral antigens to T cells and, in the context of multiple cell-based and soluble factors, instruct virus-specific T cells to proliferate and acquire multiple antiviral and immune-stimulatory effector functions. Virus-specific CD8 T cells acquire the ability to lyse virally infected cells both directly through cell to cell killing and indirectly through the secretion of cytokines, including TNFα and IFNγ (reviewed in ref. 1). Simultaneously, virus-specific CD4 T cells proliferate and produce immunostimulatory cytokines (such as IL-2) that help to orchestrate the direction of the immune response, sustain and program memory CD8 T cell differentiation, and provide help to B cells for antibody production (2). Following clearance of the infection, effector virus-specific CD4 and CD8 T cells contract and, through a complex differentiation program, generate a stable memory T cell population able to rapidly respond and clear future infections with the same or similar virus (3). Early IL-2 signals are important for secondary CD8 T cell responses, and, once formed, T cell memory is sustained via homeostatic signals including the cytokines IL-7 and IL-15 (4, 5). Yet it is still poorly understood how different factors interact to initially instruct distinct immune responses and how the factors and signals that a T cell initially encounters are integrated to program long-term memory differentiation. Thus, the interplay between positive and negative regulatory signals toward memory development is largely unclear.Viral replication triggers the production of multiple immune stimulatory factors that activate and program T cell responses. As a counterbalance, negative regulatory factors are also produced to control the magnitude of the antiviral immune response and attenuate T cell responses following viral clearance to prevent excessive immunopathology. Many immune and nonimmune cell types...

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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 nonphosphorylatable 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 S1P 1 receptor agonist at doses sufficient to induce systemic lymphopenia did not inhibit virus-specific T-cell response, indicating that S1P 1 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 S1P 1 or S1P 2 on dendritic cells in the lungs after influenza virus infection.