SUMMARY NLR genes mediate host immunity to various pathogenic stimuli. However, in vivo evidence for NLR involvement in viral sensing has not been widely investigated and remains controversial. As an ultimate test of the physiologic role of NLRP3 during RNA viral infection, this work explores the in vivo role of NLRP3 inflammasome components during influenza virus infection. Mice lacking Nlrp3, ASC, or Caspase-1, but not Nlrc4, exhibit dramatically increased mortality but reduced immune response following influenza virus exposure. Utilizing analogs of dsRNA (poly(I:C)) and ssRNA (ssRNA40), we demonstrate that NLRP3-mediated response can be activated by RNA species. Mechanistically, NLRP3 inflammasome activation by influenza virus is dependent upon lysosomal maturation and reactive oxygen species. Inhibition of ROS induction eliminated IL-1β production in animals during influenza infection. Together, these data place the NLRP3 inflammasome as an essential component in host defense against influenza infection through the sensing of viral RNA.

SUMMARY The nucleotide-binding domain and leucine-rich repeat containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed Nlrx1−/− mice exhibited increased expression of antiviral signaling molecules IFN-β, STAT2, OAS1 and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1−/− mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS-activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.

Airway mucus forms the structural basis of the local innate immune defense mechanism. It is an integrated, active, viscoelastic gel matrix evolved to protect the exposed lung from physical, chemical, and pathological erosion. Exosomes are biologically active vesicles secreted by different cell types including epithelial, hematopoietic, and some tumor cells. They are also present in some biological fluids such as serum, urine, breast milk, and bronchoalveolar lavage fluid. In this study, we demonstrate for the first time that exosome-like vesicles with antiviral properties are present in human tracheobronchial epithelial (HTBE) cell culture secretions. These vesicles have been isolated by differential centrifugation and are characterized further by mass spectrometry, flow cytometry, immunoblotting, electron microscopy, and light-scattering methods. HTBE vesicles exhibited characteristic exosomal size (30-100 nm) and morphology (cup-shaped) with a buoyant density in sucrose of 1.12-1.18 g/ml. Biochemical characterization further revealed typical surface, cytoskeletal, and cytoplasmic proteins characteristic of exosomes, including the multivesicular and late endosomal membrane markers Tsg101 and CD63. The presence of RNA was also observed. The epithelial mucins MUC1, MUC4, and MUC16 also contributed to the vesicles' structure. Notably, alpha-2,6-linked sialic acid was associated with these mucin molecules and subsequent functional analysis showed that these vesicles have a neutralizing effect on human influenza virus, which is known to bind sialic acid. Taken together, these findings suggest that airway epithelial cells release exosome-like vesicles and that these structures may be involved in diverse physiological processes in airway biology, including innate mucosal defense.

SummaryInterferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene SERPINE1, encoding plasminogen activator inhibitor 1 (PAI-1). By targeting extracellular airway proteases, PAI-1 inhibits IAV glycoprotein cleavage, thereby reducing infectivity of progeny viruses. This was biologically relevant for IAV restriction in vivo. Further, partial PAI-1 deficiency, attributable to a polymorphism in human SERPINE1, conferred increased susceptibility to IAV in vitro. Together, our findings reveal that manipulating the extracellular environment to inhibit the last step in a virus life cycle is an important mechanism of the antiviral response.

Studying human hepatotropic pathogens such as hepatitis B and C viruses and malaria will be necessary for understanding host-pathogen interactions, and developing therapy and prophylaxis. Unfortunately, existing in vitro liver models typically employ either cell lines that exhibit aberrant physiology, or primary human hepatocytes in culture configurations wherein they rapidly lose their hepatic functional phenotype. Stable, robust, and reliable in vitro primary human hepatocyte models are needed as platforms for infectious disease applications. For this purpose, we describe the application of micropatterned co-cultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive cells. Using this system, we demonstrate how to recapitulate in vitro liver infection by the hepatitis B and C viruses and Plasmodium pathogens. In turn, the MPCC platform can be used to uncover aspects of host-pathogen interactions, and has the potential to be used for medium-throughput drug screening and vaccine development.

In spite of the vast collective experience in tissue engineering, control of both tissue architecture and scale are fundamental translational roadblocks. An experimental framework that enables investigation into how architecture and scaling may be coupled is needed. Here, we introduce an approach called ‘SEEDs’ (‘in Situ Expansion of Engineered Devices’), in which we fabricate a structurally organized engineered tissue unit that expands in response to regenerative cues after implantation. We find that tissues containing pre-patterned human primary hepatocytes, endothelial cells, and stromal cells in degradable hydrogel expand over 50-fold over the course of 11 weeks in animals with liver injury, with concomitant increased function as characterized by the production of multiple human liver proteins. Histologically, we observe the emergence of stereotypical microstructure via coordinated growth of hepatocytes in close juxtaposition with a perfused, chimeric vasculature. Importantly, we demonstrate the utility of this platform for probing the impact of multicellular geometric architecture on tissue expansion in response to regenerative cues. This approach represents a hybrid strategy that harnesses both biology and engineering to deploy a limited cell mass more efficiently than either approach could do in isolation, and thus offers a new convergent paradigm for tissue engineering.

Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37°C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32°C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40°C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32°C and 37°C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32°C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32°C may represent a critical evolutionary step enabling human-to-human transmission.

The emergence in 2009 of a swine-origin H1N1 influenza virus as the first pandemic of the 21st Century is a timely reminder of the international public health impact of influenza viruses, even those associated with mild disease. The widespread distribution of highly pathogenic H5N1 influenza virus in the avian population has spawned concern that it may give rise to a human influenza pandemic. The mortality rate associated with occasional human infection by H5N1 virus approximates 60%, suggesting that an H5N1 pandemic would be devastating to global health and economy. To date, the H5N1 virus has not acquired the propensity to transmit efficiently between humans. The reasons behind this are unclear, especially given the high mutation rate associated with influenza virus replication. Here we used a panel of recombinant H5 hemagglutinin (HA) variants to demonstrate the potential for H5 HA to bind human airway epithelium, the predominant target tissue for influenza virus infection and spread. While parental H5 HA exhibited limited binding to human tracheal epithelium, introduction of selected mutations converted the binding profile to that of a current human influenza strain HA. Strikingly, these amino-acid changes required multiple simultaneous mutations in the genomes of naturally occurring H5 isolates. Moreover, H5 HAs bearing intermediate sequences failed to bind airway tissues and likely represent mutations that are an evolutionary “dead end.” We conclude that, although genetic changes that adapt H5 to human airways can be demonstrated, they may not readily arise during natural virus replication. This genetic barrier limits the likelihood that current H5 viruses will originate a human pandemic.