Obesity has been epidemiologically and empirically linked with more severe diseases upon influenza infection. To ameliorate severe disease, treatment with antivirals, such as the neuraminidase inhibitor oseltamivir, is suggested to begin within days of infection especially in high-risk hosts. However, this treatment can be poorly effective and may generate resistance variants within the treated host. Here, we hypothesized that obesity would reduce oseltamivir treatment effectiveness in the genetically obese mouse model. We demonstrated that oseltamivir treatment does not improve viral clearance in obese mice. While no traditional variants associated with oseltamivir resistance emerged, we did note that drug treatment failed to quench the viral population and did lead to phenotypic drug resistance in vitro. Together, these studies suggest that the unique pathogenesis and immune responses in obese mice could have implications for pharmaceutical interventions and the within-host dynamics of the influenza virus population. IMPORTANCE Influenza virus infections, while typically resolving within days to weeks, can turn critical, especially in high-risk populations. Prompt antiviral administration is crucial to mitigating these severe sequalae, yet concerns remain if antiviral treatment is effective in hosts with obesity. Here, we show that oseltamivir does not improve viral clearance in genetically obese or type I interferon receptor-deficient mice. This suggests a blunted immune response may impair oseltamivir efficacy and render a host more susceptible to severe disease. This study furthers our understanding of oseltamivir treatment dynamics both systemically and in the lungs of obese mice, as well as the consequences of oseltamivir treatment for the within-host emergence of drug-resistant variants.

Norepinephrine is a key sympathetic neurotransmitter, which acts to suppress CD8 + T cell cytokine secretion and lytic activity by signaling through the β2-adrenergic receptor (ADRB2). Although ADRB2 signaling is considered generally immunosuppressive, its role in regulating the differentiation of effector T cells in response to infection has not been investigated. Using an adoptive transfer approach, we compared the expansion and differentiation of wild type (WT) to Adrb2-/- CD8 + T cells throughout the primary response to vesicular stomatitis virus (VSV) infection in vivo. We measured the dynamic changes in transcriptome profiles of antigen-specific CD8 + T cells as they responded to VSV. Within the first 7 days of infection, WT cells out-paced the expansion of Adrb2-/- cells, which correlated with reduced expression of IL-2 and the IL-2Rα in the absence of ADRB2. RNASeq analysis identified over 300 differentially expressed genes that were both temporally regulated following infection and selectively regulated in WT vs Adrb2-/- cells. These genes contributed to major transcriptional pathways including cytokine receptor activation, signaling in cancer, immune deficiency, and neurotransmitter pathways. By parsing genes within groups that were either induced or repressed over time in response to infection, we identified three main branches of genes that were differentially regulated by the ADRB2. These gene sets were predicted to be regulated by specific transcription factors involved in effector T cell development, such as Tbx21 and Eomes. Collectively, these data demonstrate a significant role for ADRB2 signaling in regulating key transcriptional pathways during CD8 + T cells responses to infection that may dramatically impact their functional capabilities and downstream memory cell development.

Obesity is associated with increased disease severity, elevated viral titers in exhaled breath, and significantly prolonged viral shed during influenza A virus infection. Due to the mutable nature of RNA viruses, we questioned whether obesity could also influence influenza virus population diversity. Here, we show that minor variants rapidly emerge in obese mice. The variants exhibit increased viral replication, resulting in enhanced virulence in wild-type mice. The increased diversity of the viral population correlated with decreased type I interferon responses, and treatment of obese mice with recombinant interferon reduced viral diversity, suggesting that the delayed antiviral response exhibited in obesity permits the emergence of a more virulent influenza virus population. This is not unique to obese mice. Obesity-derived normal human bronchial epithelial (NHBE) cells also showed decreased interferon responses and increased viral replication, suggesting that viral diversity also was impacted in this increasing population. IMPORTANCE Currently, 50% of the adult population worldwide is overweight or obese. In these studies, we demonstrate that obesity not only enhances the severity of influenza infection but also impacts viral diversity. The altered microenvironment associated with obesity supports a more diverse viral quasispecies and affords the emergence of potentially pathogenic variants capable of inducing greater disease severity in lean hosts. This is likely due to the impaired interferon response, which is seen in both obese mice and obesity-derived human bronchial epithelial cells, suggesting that obesity, aside from its impact on influenza virus pathogenesis, permits the stochastic accumulation of potentially pathogenic viral variants, raising concerns about its public health impact as the prevalence of obesity continues to rise.

Since their discovery in the United States in 1963, outbreaks of infection with equine influenza virus (H3N8) have been associated with serious respiratory disease in horses worldwide. Genomic analysis suggests that equine H3 viruses are of an avian lineage, likely originating in wild birds. Equine-like internal genes have been identified in avian influenza viruses isolated from wild birds in the Southern Cone of South America. However, an equine-like H3 hemagglutinin has not been identified. We isolated 6 distinct H3 viruses from wild birds in Chile that have hemagglutinin, nucleoprotein, nonstructural protein 1, and polymerase acidic genes with high nucleotide homology to the 1963 H3N8 equine influenza virus lineage. Despite the nucleotide similarity, viruses from Chile were antigenically more closely related to avian viruses and transmitted effectively in chickens, suggesting adaptation to the avian host. These studies provide the initial demonstration that equine-like H3 hemagglutinin continues to circulate in a wild bird reservoir.

Fifty percent of the United States population is predicted to be obese (body mass index > 30) by 2030. Obese individuals are considered a high-risk group for developing severe influenza disease. To compound this, studies have shown that inactivated influenza vaccination has decreased efficacy in this high-risk population. However, the efficacy of live-attenuated vaccines has not been investigated to date. Therefore, we sought to determine if a cold-adapted A/California/04/2009 (caCA/09) H1N1 influenza virus vaccine protected obese mice from lethal influenza virus challenge. In these studies, we demonstrate that intranasal administration of caCA/09 vaccine protected genetically and diet-induced obese (DIO) mice from a lethal challenge with wild-type influenza virus (CA/09) 4 weeks post-vaccination. Protection was not due to route of administration, as intranasal vaccination with inactivated caCA/09 virus was not protective. Unlike non-vaccinated obese mice, caCA/09 vaccinated obese mice cleared the virus and controlled viral spread, as determined by lung TCID50 and immunohistochemistry, respectively. Protection of obese mice lasts at least 17 weeks post-vaccination. Mechanistically, we found that effector memory CD4 and CD8 T cells were increased in lungs of vaccinated mice that may provide heterosubtypic protection. Indeed, the vaccinated obese mice were protected from a lethal challenge with an H5 deselected influenza virus. In conclusion, live-attenuated virus vaccination is a viable option to protect obese hosts against increased disease severity and mortality.

The severity of the 2017-18 influenza season, combined with the low efficacy for some vaccine components, highlights the need to improve our current seasonal influenza vaccine. Thus, the National Institute of Allergy and Infectious Diseases recently announced a strategic plan to improve current influenza vaccines and eventually develop a "universal" influenza vaccine. This review will highlight the many different strategies being undertaken in pursuit of this goal and the exciting advances made by the influenza community. There is no doubt that an improved influenza vaccine is on the horizon.

Immune memory is critical for protection from repeated infections by pathogenic organisms and is the basis for the success of vaccines. Memory CD8+ T cell development is guided by important signals that alter their function and transcriptional program, enabling them to persist as pools of primed memory cells capable of rapid response. Lymphoid organs are highly innervated by sympathetic neurons that secrete norepinephrine as the primary neurotransmitter, which places the nervous and immune systems in direct communication. CD8+ T cells express the β2-adrenergic receptor (ADRB2), and we found that it was required for the development of long-lived memory cells in response to virus infection. The failure of Adrb2−/− T cells to form memory populations correlated with profound differences in gene expression and predicted upstream regulators during the early stages of T cell priming. Thus, the sympathetic nervous system controls memory CD8+ T cell development through ADRB2 signaling.

The mammalian nervous system communicates important information about the environment to the immune system, but the underlying mechanisms are largely unknown. Secondary lymphoid organs are highly innervated by sympathetic neurons that secrete norepinephrine (NE) as the primary neurotransmitter. Immune cells express adrenergic receptors, enabling the sympathetic nervous system to directly control immune function. NE is a potent immunosuppressive factor and markedly inhibits TNF-α secretion from innate cells in response to lipopolysaccharide (LPS). In this study, we demonstrate that NE blocks the secretion of a variety of proinflammatory cytokines by rapidly inducing IL-10 secretion from innate cells in response to multiple Toll-like receptor (TLR) signals. NE mediated these effects exclusively through the β2-adrenergic receptor (ADRB2). Consequently, Adrb2-/- animals were more susceptible to L. monocytogenes infection and to intestinal inflammation in a dextran sodium sulfate (DSS) model of colitis. Further, Adrb2-/- animals rapidly succumbed to endotoxemia in response to a sub-lethal LPS challenge and exhibited elevated serum levels of TNF-α and reduced IL-10. LPS-mediated lethality in WT animals was rescued by administering a β 2-specific agonist and in Adrb2-/- animals by exogenous IL-10. These findings reveal a critical role for ADRB2 signaling in controlling inflammation through the rapid induction of IL-10. Our findings provide a fundamental insight into how the sympathetic nervous system controls a critical facet of immune function through ADRB2 signaling.

The nervous and immune systems reciprocally regulate their functions through the release of chemical messengers. Norepinephrine (NE), a neurotransmitter released by catecholaminergic nerve endings, allows the sympathetic nervous system to communicate with immune cells through adrenergic receptors (ADR). We identified a novel role for the β2-ADR (ADRB2) in controlling inflammation. NE suppresses pro-inflammatory cytokine secretion from primary macrophages in response to multiple TLR agonists, and ADRB2 signaling enhances early induction of IL-10. We assessed the in vivo role of this pathway using a LPS endotoxemia model. ADRB2 −/− animals rapidly succumbed to a sub-lethal LPS challenge, which correlated with elevated serum levels of TNFα and reduced IL-10. The lethality in the ADRB2 −/− animals was rescued by administering exogenous IL-10. Additionally, the ADRB2-specific agonist salmeterol rescued wild-type animals from a lethal LPS challenge, which was reversed by neutralizing anti-IL-10 antibody. Additionally, ADRB2 −/− mice are unable to control inflammation models of infection and colitis. These observations suggest that ADRB2 signaling is critical for controlling inflammation through the rapid induction of IL-10. Through transcriptome analysis, the NR4A nuclear orphan family members were induced by NE, and the presence of several putative NR4A binding sites within the IL-10 promoter suggests that these factors may directly regulate IL-10 expression in response to ADRB2 signaling. Further studies will test the control of IL-10 expression by the NR4A family of transcription factors. Understanding this pathway will provide new insights into how the nervous and immune systems communicate through ADRB2 signaling.

Innervation of the lymphoid tissues by catecholaminergic nerve endings allow the sympathetic nervous system to communicate with immune cells through the release of norepinephrine (NE). NE signals through adrenergic receptors, which are widely expressed by immune cells. We have identified a novel pathway where NE can suppress pro-inflammatory cytokine secretion from bone marrow-derived macrophages in response to multiple TLR agonists. This pathway operates through the beta2-adrenergic receptor (ADRB2), and the suppressive effect of NE depends on the early induction of the anti-inflammatory cytokine IL-10. We assessed the in vivo role of this pathway using a LPS endotoxemia model and found that ADRB2 knockout (KO) animals rapidly succumbed to a sub-lethal LPS challenge. This sensitivity is correlated with elevated serum levels of TNFα and suppressed IL-10 in ADRB2 KO animals. The lethality in the ADRB2 KO animals was rescued by administering exogenous IL-10. Additionally, the ADRB2-specific agonist salmeterol rescued wild-type animals from a lethal LPS challenge, which was reversed by neutralizing anti-IL-10 antibody. These observations suggest that ADRB2 signaling is critical for controlling inflammation through the rapid induction of IL-10. Through transcriptome analysis, the NR4A nuclear orphan family members were induced by NE, and the presence of several putative NR4A binding sites within the IL-10 promoter suggests that these factors may directly regulate IL-10 expression in response to ADRB2 signaling. Further studies will directly test the control of IL-10 expression by NR4A factors. Understanding this pathway will provide new insights into how the nervous and immune systems communicate through ADRB2 signaling.

Cytotoxic T lymphocytes (CTLs) play a critical role in immune responses to intracellular pathogens. CTL responses are initiated by T cell receptor signaling, and they can be modulated by secreted factors, such as cytokines and other extracellular signals. Previous research from our lab demonstrated that the acute effector functions of CTLs are regulated by the neurotransmitter norepinephrine (NE) via beta2-adrenergic receptor (ADRB2) signaling in vitro. We now demonstrate that ADRB2 signaling intrinsically modulates in vivo memory development in response to viral infection using an adoptive transfer model. Expansion of ADRB2-deficient CTLs is unaffected during the initial priming phase in response to the model pathogen, vesicular stomatitis virus. However, ADRB2-deficient CTLs contract rapidly and do not yield any detectable pools of memory cells when compared to wild-type counterparts. Transcriptome analysis revealed a variety of differentially expressed genes, both expressed in resting naïve cells as well as in cells obtained during the primary expansion phase of infection. Among them, we found that the expression of the high affinity IL-2Ra (CD25) was reduced during the activation phase in ADRB2-deficient cells, which was confirmed at the protein level by flow cytometry. While IL-2 responsiveness is a key determinant in programming memory cell development, treatment with IL-2/anti-IL-2 complexes during early phases of infection failed to rescue memory formation of ADRB2-deficient cells, despite a restoration of CD25 expression on day 5 post-infection. Future experiments will dissect the role of ADRB2 signaling during CTL development on the capacity to form memory upon antigen exposure.

Type I interferon (IFN‐α/β) plays a critical role in suppressing viral replication by driving the transcription of hundreds of interferon‐sensitive genes (ISGs). While many ISGs are transcriptionally activated by the ISGF3 complex, the significance of other signaling intermediates in IFN‐α/β‐mediated gene regulation remains elusive, particularly in rare cases of gene silencing. In human Th2 cells, IFN‐α/β signaling suppressed IL5 and IL13 mRNA expression during recall responses to T‐cell receptor (TCR) activation. This suppression occurred through a rapid reduction in the rate of nascent transcription, independent of de novo expression of ISGs. Further, IFN‐α/β‐mediated STAT4 activation was required for repressing the human IL5 gene, and disrupting STAT4 dimerization reversed this effect. This is the first demonstration of STAT4 acting as a transcriptional repressor in response to IFN‐α/β signaling and highlights the unique activity of this cytokine to acutely block the expression of an inflammatory cytokine in human T cells.

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2‐adrenergic receptor (ADRB2) on primary human CD8+ effector memory T cells. Treatment of both human and murine CD8+ T cells with NE decreased IFN‐γ and TNF‐α secretion and suppressed their cytolytic capacity in response to T‐cell receptor (TCR) activation. The effects of NE were specifically reversed by β2‐specific antagonists. Adrb2−/− CD8+ T cells were completely resistant to the effects of NE. Further, the ADRB2‐specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8+ T cells. While both TCR activation and stimulation with IL‐12 + IL‐18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN‐γ secretion in response to IL‐12 + IL18. Finally, the long‐acting ADRB2‐specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8+ T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8+ T‐cell function and underscores the novel role this pathway plays in adaptive T‐cell responses to infection.

There is growing evidence to suggest that various aspects of immune function are controlled by neural networks. Both cholinergic and adrenergic receptors are expressed by various major immune cell types. Specifically, expression of the β2-adrenergic receptor (ADRB2) has been characterized in different cells of the immune system; however, our lab has demonstrated for the first time a differential expression of this receptor in subsets of effector and memory CD8+ T cells. We found that the ADRB2 is upregulated in human effector memory CD8+ T cells compared to central memory/naïve counterparts. Further, human CD8+ T cells that are acutely activated in the presence of the ADRB2 natural ligand, nor-epinephrine (NE), secrete significantly less IFN-γ and TNF-α. Similarly, the cytolytic capacity of these cells is reduced when NE is present. This downregulation by NE was reversed in the presence of a ADRB2-specific antagonist, but not a pan α-adrenergic or β1-adrenergic receptor specific antagonists. Additionally, mouse CD8+ T cells behaved similarly to human cells to these antagonists, suggesting species conservation of this neural-immune network. Moreover, ADRB2-KO CD8+ T cells were completely resistant to the effects of NE. Current experiments using an adoptive transfer model will elucidate how ADRB2 signaling modulates CD8+ T cell function in vivo. These experiments will provide insight into how the nervous system controls CD8+ T cell function during the course of an infection.

CD8+ cytotoxic T lymphocytes (CTLs) play a major role in defense against intracellular pathogens. During development, antigen-presenting cells secrete innate cytokines such as IL-12 and IFN-α, which drive CTL differentiation into diverse populations of effector and long-lived memory cells. Using whole transcriptome analyses, the serine/threonine protein kinase Tpl2/MAP3K8 was found to be induced by IL-12 and selectively expressed by effector memory (TEM) CTLs. Tpl2 regulates various inflammatory pathways by activating the ERK mediated MAP kinase pathway in innate immune cells such as macrophages and dendritic cells. In this study, we found that a specific small molecule Tpl2 inhibitor blocked IFN-γ and TNF-α secretion as well as cytolytic activity of human CTLs. This pathway was specific for human effector CTLs, as the Tpl2 inhibitor did not block IFN-γ and TNF-α secretion from murine effector CTLs. Further, IL-12 failed to induce expression of Tpl2 in murine CTLs, and Tpl2 deficient murine CTLs did not exhibit any functional deficiency either in vitro or in vivo in response to L. monocytogenes infection. In summary, we identified a species-specific role for Tpl2 in effector function of human CTLs, which plays a major role in adaptive immune responses to intracellular pathogens and tumors.

Abstract The nervous system controls a variety of physiological responses through the release of several chemical messengers. Although the immune system has been thought to be a self-regulated system, there is growing evidence that the immune system and the nervous system are capable of reciprocally regulating their function. We recently identified a unique role for adrenergic signaling in modulating the innate response to pathogen-associated molecular patterns. First, we found that norepinephrine (NE) can suppress pro-inflammatory cytokine secretion from bone marrow-derived macrophages and dendritic cells in response to TLR signaling in vitro. This suppressive effect depends on the early induction of the anti-inflammatory cytokine IL-10, where neutralizing the autocrine IL-10 signaling restores the cytokine secretion. NE exerts its effects by signaling through the beta2-adrenergic receptor (ADRB2). Second, upon challenging mice with a sub-lethal dose of Listeria monocytogenes, ADRB2 knockout mice present with splenomegaly and enhanced bacterial clearance. Additionally, treatment of whole human blood with TLR ligands in the presence of the ADRB2 agonist albuterol suppresses TNFα secretion, demonstrating the presence of this pathway in humans. This supports the hypothesis that ADRB2 signaling is a negative regulator of TLR signaling and suggests that this pathway plays an important role in immune activation and homeostasis.

Albuterol, which signals through the ADRB2, is a widely used rescue treatment for asthma attacks. Our data demonstrates that signaling through ADRB2 down-regulates the function of human CD8+T cells. We hypothesize that chronic use of albuterol suppresses the anti-viral immune response, therefore making patients more susceptible to viral infections. We collected peripheral blood from adult healthy human donors, between the ages of 20-30. CD8+ and CD8+CCR7lo T cells were isolated through magnetic bead separation or fluorescence-assisted cell sorting (FACS), respectively. Cells were stimulated using plate-bound anti-CD3 and anti-CD28 or anti-CD3 alone in the presence or absence of nor-epinephrine or albuterol. Cytokine secretion was assayed 24 hours post-stimulation by ELISA. Additionally, the effect of nor-epinephrine on the ability of CD8+T cells to specifically lyse target cells was tested using a chromium release assay. To link the specificity of ADRB2 signaling, the adrenergic receptor antagonists phentolamine (pan α), atenolol (β1) and ICI-118,551 (β2) were used. Bulk CD8+ T cells and CD8+CCR7loT cells acutely activated in the presence of nor-epinephrine or albuterol secrete less IFN-γ and TNF-α. Similarly, these cells lower their ability to kill target cells when nor-epinephrine is present. This inhibition by nor-epinephrine/albuterol was reversed in the presence of the β2 antagonist but not the pan α or β1 antagonists. ADRB2 signaling significantly suppresses human CD8+T cell function.