Kari Ann Shirey

The TLR4 Antagonist, Eritoran, Protects Mice from Lethal Influenza Infection

There is a pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury caused by chemical or microbial insults is secondary to the generation of host-derived, oxidized phospholipid that potently stimulates Toll-like receptor 4 (TLR4)-dependent inflammation. Subsequently, we reported that Tlr4−/− mice are highly refractory to influenza-induced lethality, and proposed that therapeutic antagonism of TLR4 signalling would protect against influenza-induced acute lung injury. Here we report that therapeutic administration of Eritoran (also known as E5564)—a potent, well-tolerated, synthetic TLR4 antagonist—blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titres. CD14 and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2. Thus, Eritoran blockade of TLR signalling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other infections.

Control of RSV-induced lung injury by alternatively activated macrophages is IL-4R|[alpha]|-, TLR4-, and IFN-|[beta]|-dependent

Severe respiratory syncytial virus (RSV)-induced bronchiolitis has been associated with a mixed “Th1” and “Th2” cytokine storm. We hypothesized that differentiation of “alternatively activated” macrophages (AA-Mφ) would mediate the resolution of RSV-induced lung injury. RSV induced interleukin (IL)-4 and IL-13 by murine lung and peritoneal macrophages, IL-4Rα/STAT6-dependent AA-Mφ differentiation, and significantly enhanced inflammation in the lungs of IL-4Rα−/− mice. Adoptive transfer of wildtype macrophages to IL-4Rα−/− mice restored RSV-inducible AA-Mφ phenotype and diminished lung pathology. RSV-infected Toll-like receptor (TLR)4−/− and interferon (IFN)-β−/− macrophages and mice also failed to express AA-Mφ markers, but exhibited sustained proinflammatory cytokine production (e.g., IL-12) in vitro and in vivo and epithelial damage in vivo. TLR4 signaling is required for peroxisome proliferator-activated receptorγ expression, a DNA-binding protein that induces AA-Mφ genes, whereas IFN-β regulates IL-4, IL-13, IL-4Rα, and IL-10 expression in response to RSV. RSV-infected cotton rats treated with a cyclooxygenase-2 inhibitor increased expression of lung AA-Mφ. These data suggest new treatment strategies for RSV that promote AA-Mφ differentiation.

Francisella tularensis live vaccine strain induces macrophage alternative activation as a survival mechanism.

Francisella tularensis (Ft), the causative agent of tularemia, elicits a potent inflammatory response early in infection, yet persists within host macrophages and can be lethal if left unchecked. We report in this study that Ft live vaccine strain (LVS) infection of murine macrophages induced TLR2-dependent expression of alternative activation markers that followed the appearance of classically activated markers. Intraperitoneal infection with Ft LVS also resulted in induction of alternatively activated macrophages (AA-Mφ). Induction of AA-Mφ by treatment of cells with rIL-4 or by infection with Ft LVS promoted replication of intracellular Ftn, in contrast to classically activated (IFN-γ plus LPS) macrophages that promoted intracellular killing of Ft LVS. Ft LVS failed to induce alternative activation in IL-4Rα−/− or STAT6−/− macrophages and prolonged the classical inflammatory response in these cells, resulting in intracellular killing of Ft. Treatment of macrophages with anti-IL-4 and anti-IL-13 Ab blunted Ft-induced AA-Mφ differentiation and resulted in increased expression of IL-12 p70 and decreased bacterial replication. In vivo, Ft-infected IL-4Rα−/− mice exhibited increased survival compared with wild-type mice. Thus, redirection of macrophage differentiation by Ft LVS from a classical to an alternative activation state enables the organism to survive at the expense of the host.

Toll-Like Receptor 2-Mediated Signaling Requirements for Francisella tularensis Live Vaccine Strain Infection of Murine Macrophages

ABSTRACT Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously that F. tularensis live vaccine strain (LVS) elicited strong, dose-dependent NF-κB reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report that F. tularensis LVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2−/− macrophages. F. tularensis LVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers from F. tularensis LVS-infected mice. Colocalization of intracellular F. tularensis LVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if the F. tularensis LVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis. F. tularensis LVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVSΔguaA, an F. tularensis LVS guanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-κB in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-type F. tularensis LVS. Collectively, these data indicate that the primary macrophage response to F. tularensis LVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellular F. tularensis replication.

Type I IL-4Rs Selectively Activate IRS-2 to Induce Target Gene Expression in Macrophages

The ability of IL-4, but not IL-13, to efficiently activate IRS-2 leads to the expression of a subset of genes in alternatively activated macrophages. Selective Activation The T cell–derived cytokines interleukin-4 (IL-4) and IL-13 are closely related and are associated with allergic inflammatory responses, such as those that occur in asthma. IL-4 binds to the IL-4 receptor α (IL-4Rα) subunit, which heterodimerizes with either the γC subunit or the IL-13Rα1 subunit to form the type I or type II IL-4 receptors, respectively. Thus, whereas IL-4 activates both type I and type II receptors, IL-13 activates type II receptors. In allergic asthma, IL-4 is important in driving the differentiation of T helper type 2 (TH2) cells, whereas IL-13 plays a role in mucus secretion and airway hyperresponsiveness. Because of their overlap in receptor usage, it has been difficult to tease apart the different contributions of IL-4 and IL-13 (see the Perspective by Wills-Karp and Finkelman). By comparing the responses to each cytokine of a human cell line expressing type I and type II receptors to that of cells expressing only type II receptors, Heller et al. have found a critical difference in the signaling pathways activated by IL-4 and IL-13. Although both cytokines activated signal transducer and activator of transcription 6 to a similar extent, IL-4–stimulated phosphorylation and activation of insulin receptor substrate 2 (IRS-2) was much more efficient than that of IL-13 and was dependent on the presence of the γC subunit. Moreover, type I IL-4 receptor–mediated activation of IRS-2 resulted in the expression of a subset of genes associated with alternatively activated macrophages. By increasing our understanding of the differences between IL-4– and IL-13–dependent signaling pathways, these data may help in the development of more selective therapies against allergic asthma. Although interleukin-4 (IL-4) and IL-13 participate in allergic inflammation and share a receptor subunit (IL-4Rα), they have different functions. We compared cells expressing type I and II IL-4Rs with cells expressing only type II receptors for their responsiveness to these cytokines. IL-4 induced highly efficient, γC-dependent tyrosine phosphorylation of insulin receptor substrate 2 (IRS-2), whereas IL-13 was less effective, even when phosphorylation of signal transducer and activator of transcription 6 (STAT6) was maximal. Only type I receptor, γC-dependent signaling induced efficient association of IRS-2 with the p85 subunit of phosphoinositide 3-kinase or the adaptor protein growth factor receptor–bound protein 2. In addition, IL-4 signaling through type I IL-4Rs induced more robust expression of a subset of genes associated with alternatively activated macrophages than did IL-13. Thus, IL-4 activates signaling pathways through type I IL-4Rs qualitatively differently from IL-13, which cooperate to induce optimal gene expression.

Macrophage Proinflammatory Response to Francisella tularensis Live Vaccine Strain Requires Coordination of Multiple Signaling Pathways

The macrophage proinflammatory response to Francisella tularensis (Ft) live vaccine strain (LVS) was shown previously to be TLR2 dependent. The observation that intracellular Ft LVS colocalizes with TLR2 and MyD88 inside macrophages suggested that Ft LVS might signal from within the phagosome. Macrophages infected with LVSΔiglC, a Ft LVS mutant that fails to escape from the phagosome, displayed greatly increased expression of a subset of TLR2-dependent, proinflammatory genes (e.g., Tnf) but decreased expression of others (e.g., Ifnb1). This latter subset was similarly mitigated in IFN-β−/− macrophages indicating that while Ft LVS-induced TLR2 signaling is necessary, cytosolic sensing of Ft to induce IFN-β is required for full induction of the macrophage proinflammatory response. Although LVSΔiglC greatly increased IL-1β mRNA in wild-type macrophages, protein secretion was not observed. IL-1β secretion was also diminished in Ft LVS-infected IFN-β−/− macrophages. rIFN-β failed to restore IL-1β secretion in LVSΔiglC-infected macrophages, suggesting that signals in addition to IFN-β are required for assembly of the inflammasome and activation of caspase-1. IFN-β plays a central role in controlling the macrophage bacterial burden: bacterial recovery was greater in IFN-β−/− than in wild-type macrophages and treatment of Ft LVS-infected macrophages with rIFN-β or 5,6-dimethylxanthenone-4-acetic acid, a potent IFN-β inducer, greatly decreased the intracellular Ft LVS burden. In toto, these observations support the hypothesis that the host inflammatory response to Ft LVS is complex and requires engagement of multiple signaling pathways downstream of TLR2 including production of IFN-β via an unknown cytosolic sensor and activation of the inflammasome.

Transcriptional Regulation of Murine IL-33 by TLR and Non-TLR Agonists

IL-33, a member of the IL-1 family of cytokines, is produced by many cell types, including macrophages, yet its regulation is largely unknown. Treatment of primary murine macrophages with a panel of TLR (e.g., TLR2, TLR3, TLR4, and TLR9) agonists and non-TLR (e.g., MDA5, RIG-I) agonists revealed a pattern of gene and protein expression consistent with a role for IFN regulatory factor-3 (IRF-3) in the expression of IL-33. Accordingly, induction of IL-33 mRNA was attenuated in IRF-3−/− macrophages and TBK-1−/− mouse embryonic fibroblasts. Despite the fact that all IL-33 agonists were IRF-3 dependent, LPS-induced IL-33 mRNA was fully inducible in IFN-β−/− macrophages, indicating that IL-33 is not dependent on IFN-β as an intermediate. Epinephrine and Bordetella pertussis adenylate cyclase toxin (ACT), cAMP-activating agents, activate CREB and greatly synergize with LPS to induce IL-33 mRNA in macrophages. Both LPS-induced and ACT/LPS-enhanced expression of IL-33 mRNA was partially, but significantly, inhibited by the protein kinase A inhibitor H-89 but not by tyrosine kinase or protein kinase C inhibitors. Two IL-33 mRNA species derived from two alternative promoters encode full-length IL-33; however, the shorter “A” species is preferentially induced by all IL-33–inducing agonists except Newcastle disease virus, a RIG-I agonist that induced expression of both “A” and “B” transcripts. Together, these studies greatly extend what is currently known about the regulation of IL-33 induction in macrophages stimulated by bacterial and viral agonists that engage distinct innate immune signaling pathways.

Sustained generation of nitric oxide and control of mycobacterial infection requires argininosuccinate synthase 1

Nitric oxide (NO) defends against intracellular pathogens, but its synthesis must be regulated due to cell and tissue toxicity. During infection, macrophages import extracellular arginine to synthesize NO, generating the byproduct citrulline. Accumulated intracellular citrulline is thought to fuel arginine synthesis catalyzed by argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl), which would lead to abundant NO production. Instead, we find that citrulline is exported from macrophages during early stages of NO production with <2% retained for recycling via the Ass1-Asl pathway. Later, extracellular arginine is depleted, and Ass1 expression allows macrophages to synthesize arginine from imported citrulline to sustain NO output. Ass1-deficient macrophages fail to salvage citrulline in arginine-scarce conditions, leading to their inability to control mycobacteria infection. Thus, extracellular arginine fuels rapid NO production in activated macrophages, and citrulline recycling via Ass1 and Asl is a fail-safe system that sustains optimum NO production.

Respiratory Syncytial Virus Fusion Protein-Induced Toll-Like Receptor 4 (TLR4) Signaling Is Inhibited by the TLR4 Antagonists Rhodobacter sphaeroides Lipopolysaccharide and Eritoran (E5564) and Requires Direct Interaction with MD-2

ABSTRACT Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1β (IL-1β) mRNA in WT versus MD-2−/− macrophages, where MD-2−/− macrophages failed to show IL-1β expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4–CD14–MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology. IMPORTANCE This study shows for the first time that the fusion (F) protein of respiratory syncytial virus (RSV), a major cause of bronchiolitis and death, particularly in infants and young children, physically interacts with the Toll-like receptor 4 (TLR4) coreceptor, MD-2, through its N-terminal domain. We show that F protein-induced TLR4 activation can be blocked by lipid A analog antagonists. This observation provides a strong experimental rationale for testing such antagonists in animal models of RSV infection for potential use in people. This study shows for the first time that the fusion (F) protein of respiratory syncytial virus (RSV), a major cause of bronchiolitis and death, particularly in infants and young children, physically interacts with the Toll-like receptor 4 (TLR4) coreceptor, MD-2, through its N-terminal domain. We show that F protein-induced TLR4 activation can be blocked by lipid A analog antagonists. This observation provides a strong experimental rationale for testing such antagonists in animal models of RSV infection for potential use in people.

New insights for development of a safe and protective RSV vaccine.

Respiratory Syncytial Virus (RSV) is the leading cause of pneumonia and bronchiolitis in infants and children