Respiratory syncytial virus (RSV) is a common cause of infection that is associated with a range of respiratory illnesses, from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children <1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected to contribute to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs, including TLR2, TLR6, TLR3, TLR4, and TLR7, that can interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting tumor necrosis factor alpha, interleukin-6, CCL2 (monocyte chemoattractant protein 1), and CCL5 (RANTES). As previously noted, TLR4 also contributes to cytokine activation (

Type 5 adenovirus (Ad5) is a human pathogen that has been widely developed for therapeutic uses, with only limited success to date. We report here the novel finding that human erythrocytes present Coxsackie virus-adenovirus receptor (CAR) providing an Ad5 sequestration mechanism that protects against systemic infection. Interestingly, erythrocytes from neither mice nor rhesus macaques present CAR. Excess Ad5 fiber protein or anti-CAR antibody inhibits the binding of Ad5 to human erythrocytes and cryo-electron microscopy shows attachment via the fiber protein of Ad5, leading to close juxtaposition with the erythrocyte membrane. Human, but not murine, erythrocytes also present complement receptor (CR1), which binds Ad5 in the presence of antibodies and complement. Transplantation of human erythrocytes into nonobese diabetic/severe combined immunodeficiency mice extends blood circulation of intravenous Ad5 but decreases its extravasation into human xenograft tumors. Ad5 also shows extended circulation in transgenic mice presenting CAR on their erythrocytes, although it clears rapidly in transgenic mice presenting erythrocyte CR1. Hepatic infection is inhibited in both transgenic models. Erythrocytes may therefore restrict Ad5 infection (natural and therapeutic) in humans, independent of antibody status, presenting a formidable challenge to Ad5 therapeutics. “Stealthing” of Ad5 using hydrophilic polymers may enable circumvention of these natural virus traps.

We investigated the roles of Toll-like receptor 2 (TLR2) and myeloid differentiation factor 88 (MyD88) in the course of a lymphocytic choriomeningitis virus (LCMV) infection and revealed the following: (i) studies of transfected cells and murine peritoneal macrophages demonstrated that TLR2 and MyD88 are essential for the initial pro-inflammatory cytokine response (human IL-8, mouse IL-6) to LCMV; (ii) TLR2 knockout (KO) mice and MyD88 KO mice challenged with LCMV produced less IL-6 and monocyte chemotactic protein-1 in the serum than wild-type mice; (iii) in contrast to inflammatory cytokines, the production of type 1 IFN (IFN-a) in response to LCMV was MyD88 independent; (iv) MyD88 plays an essential role in antiviral CD8 + T cell responses, CD8 + T cells in MyD88 KO mice were defective in their expression of intracellular antiviral cytokines; and (v) the failure of MyD88 KO mice to activate CD8 + T cells was accompanied by persistent viral infection in MyD88 KO mice. We demonstrate that TLR-mediated responses are important in the innate immune response to LCMV and that MyD88 is essential for the control of the LCMV infection and the maturation/activation of virus-specific CD8 + T cells.

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Coxsackie B viruses (CVB) are enteroviruses that have been associated with a variety of human diseases, including myocarditis. In the present study, we found that MDA5 and its adaptor molecule MAVS are critical for type I interferon responses to CVB, since the absence of either MAVS or MDA5 leads to deficient type I interferon production and early mortality in mice infected with CVB. Pancreatic and hepatic necrosis were observed on histopathological examination of MAVS and MDA5 knockout mice infected with CVB. Inflammatory cytokine production in response to systemic CVB infection was independent of MAVS. Surprisingly, virus titers were not elevated in MAVS-deficient mice, despite significant reductions in type I interferon levels. These data highlight the importance of type I interferon in host defense and provide insight on the mechanisms of innate immune responses following coxsackievirus infection.

Herpes simplex virus 1 (HSV-1) causes a spectrum of disease, including herpes labialis, herpes keratitis, and herpes encephalitis, which can be lethal. Viral recognition by pattern recognition receptors plays a central role in cytokine production and in the generation of antiviral immunity. The relative contributions of different Toll-like receptors (TLRs) in the innate immune response during central nervous system infection with HSV-1 have not been fully characterized. In this study, we investigate the roles of TLR2, TLR9, UNC93B1, and the type I interferon (IFN) receptor in a murine model of HSV-1 encephalitis. TLR2 is responsible for detrimental inflammatory cytokine production following intracranial infection with HSV-1, and the absence of TLR2 expression leads to increased survival in mice. We prove that inflammatory cytokine production by microglial cells, astrocytes, neutrophils, and monocytes is mediated predominantly by TLR2. We also demonstrate that type I IFNs are absolutely required for survival following intracranial HSV-1 infection, as mice lacking the type I IFN receptor succumb rapidly following infection and have high levels of HSV in the brain. However, the absence of TLR9 does not impact survival, type I IFN levels, or viral replication in the brain following infection. The absence of UNC93B1 leads to a survival disadvantage but does not impact viral replication or type I IFN levels in the brain in HSV-1-infected mice. These results illustrate the complex but important roles that innate immune receptors play in host responses to HSV-1 during infection of the central nervous system.

Influenza virus infection of the respiratory tract is characterized by a neutrophil infiltrate accompanied by inflammatory cytokine and chemokine production. We and others have reported that Toll-like receptor (TLR) proteins are present on human neutrophils and that granulocytemacrophage colony-stimulating factor (GM-CSF) treatment enhances IL-8 (CXCL8) secretion in response to stimulation with TLR ligands. We demonstrate that influenza virus can induce IL-8 and other inflammatory cytokines from GM-CSF-primed human neutrophils. Using heat inactivation of influenza virus, we show that viral entry but not replication is required for cytokine induction. Furthermore, endosomal acidification and viral uncoating are necessary. Finally, using single-cell analysis of intracellular cytokine accumulation in neutrophils from knockout mice, we prove that TLR7 is essential for influenza viral recognition and inflammatory cytokine production by murine neutrophils. These studies demonstrate neutrophil activation by influenza virus and highlight the importance of TLR7 and TLR8 in that response. (Blood.

Summary Inflammasome activation is associated with numerous diseases. However, in vivo detection of the activated inflammasome complex has been limited by a dearth of tools. We developed transgenic mice that ectopically express the fluorescent adaptor protein, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), and characterized the formation of assembled inflammasome complexes (“specks”) in primary cells and tissues. In addition to hematopoietic cells, we found that a stromal population in the lung tissues forms specks during the early phase of influenza infection whereas myeloid cells showed speck formation after two days. In a peritonitis and Group B streptococcus infection models, a higher percentage of neutrophils formed specks at early phases of infection, while dendritic cells formed specks at later time points. Furthermore, speck-forming cells underwent pyroptosis, and extensive release of specks to the extracellular milieu in vivo. These data underscore the importance of free specks during inflammatory processes in vivo.