Protective effect of Toll-like receptor 4 in pulmonary vaccinia infection.

Innate immune responses are essential for controlling poxvirus infection. The threat of a bioterrorist attack using Variola major, the smallpox virus, or zoonotic transmission of other poxviruses has renewed interest in understanding interactions between these viruses and their hosts. We recently determined that TLR3 regulates a detrimental innate immune response that enhances replication, morbidity, and mortality in mice in response to vaccinia virus, a model pathogen for studies of poxviruses. To further investigate Toll-like receptor signaling in vaccinia infection, we first focused on TRIF, the only known adapter protein for TLR3. Unexpectedly, bioluminescence imaging showed that mice lacking TRIF are more susceptible to vaccinia infection than wild-type mice. We then focused on TLR4, the other Toll-like receptor that signals through TRIF. Following respiratory infection with vaccinia, mice lacking TLR4 signaling had greater viral replication, hypothermia, and mortality than control animals. The mechanism of TLR4-mediated protection was not due to increased release of proinflammatory cytokines or changes in total numbers of immune cells recruited to the lung. Challenge of primary bone marrow macrophages isolated from TLR4 mutant and control mice suggested that TLR4 recognizes a viral ligand rather than an endogenous ligand. These data establish that TLR4 mediates a protective innate immune response against vaccinia virus, which informs development of new vaccines and therapeutic agents targeted against poxviruses.

The "genomic storm" induced by bacterial endotoxin is calmed by a nuclear transport modifier that attenuates localized and systemic inflammation.

Lipopolysaccharide (LPS) is a potent microbial virulence factor that can trigger production of proinflammatory mediators involved in the pathogenesis of localized and systemic inflammation. Importantly, the role of nuclear transport of stress responsive transcription factors in this LPS-generated "genomic storm" remains largely undefined. We developed a new nuclear transport modifier (NTM) peptide, cell-penetrating cSN50.1, which targets nuclear transport shuttles importin α5 and importin ß1, to analyze its effect in LPS-induced localized (acute lung injury) and systemic (lethal endotoxic shock) murine inflammation models. We analyzed a human genome database to match 46 genes that encode cytokines, chemokines and their receptors with transcription factors whose nuclear transport is known to be modulated by NTM. We then tested the effect of cSN50.1 peptide on proinflammatory gene expression in murine bone marrow-derived macrophages stimulated with LPS. This NTM suppressed a proinflammatory transcriptome of 37 out of 84 genes analyzed, without altering expression of housekeeping genes or being cytotoxic. Consistent with gene expression analysis in primary macrophages, plasma levels of 23 out of 26 LPS-induced proinflammatory cytokines, chemokines, and growth factors were significantly attenuated in a murine model of LPS-induced systemic inflammation (lethal endotoxic shock) while the anti-inflammatory cytokine, interleukin 10, was enhanced. This anti-inflammatory reprogramming of the endotoxin-induced genomic response was accompanied by complete protection against lethal endotoxic shock with prophylactic NTM treatment, and 75% protection when NTM was first administered after LPS exposure. In a murine model of localized lung inflammation caused by direct airway exposure to LPS, expression of cytokines and chemokines in the bronchoalveolar space was suppressed with a concomitant reduction of neutrophil trafficking. Thus, calming the LPS-triggered "genomic storm" by modulating nuclear transport with cSN50.1 peptide attenuates the systemic inflammatory response associated with lethal shock as well as localized lung inflammation.

TLR3 increases disease morbidity and mortality from vaccinia infection.

Innate immunity is required for effective control of poxvirus infections, but cellular receptors that initiate the host response to these DNA viruses remain poorly defined. Given this information and the fact that functions of TLRs in immunity to DNA viruses remain controversial, we investigated effects of TLR3 on pathogenesis of vaccinia virus, a prototype poxvirus. We used a recombinant strain Western Reserve vaccinia virus that expresses firefly luciferase to infect wild-type C57BL/6 and TLR3-/- mice through intranasal inoculation. Bioluminescence imaging showed that TLR3-/- mice had substantially lower viral replication in the respiratory tract and diminished dissemination of virus to abdominal organs. Mice lacking TLR3 had reduced disease morbidity, as measured by decreased weight loss and hypothermia after infection. Importantly, TLR3-/- mice also had improved survival relative to wild-type mice. Infected TLR3-/- mice had significantly reduced lung inflammation and recruitment of leukocytes to the lung. Mice lacking TLR3 also had lower levels of inflammatory cytokines, including IL-6, MCP-1, and TNF-alpha in serum and/or bronchoalveolar lavage fluid, but levels of IFN-beta did not differ between genotypes of mice. To our knowledge, our findings show for the first time that interactions between TLR3 and vaccinia increase viral replication and contribute to detrimental effects of the host immune response to poxviruses.

Applications of bioluminescence imaging to the study of infectious diseases.

Bioluminescence imaging (BLI) has emerged as a powerful new method to analyse infectious diseases in animal models. BLI offers real-time monitoring of spatial and temporal progression of infection in the same animal, as opposed to euthanizing a cohort of animals and quantifying colony or plaque forming units at multiple time points. Pathogens or mice are engineered to express genetically encoded luciferase enzymes from bacteria, insects, or the sea pansy. The seminal study showing the feasibility of detecting microbially generated luminescence within a living mouse was published by Contag and colleagues in 1995, using Salmonella typhimurium transformed with the lux operon from Photorhabdus luminescens. Following this, they and others performed many studies of infection by bioluminescent Gram-negative and Gram-positive bacteria. Viruses can also be engineered to encode luciferase. Our laboratory has used bioluminescent reporter viruses to follow HSV and vaccinia pathogenesis; others have used an alphavirus or novirhabdovirus. Recently, even eukaryotic parasites Plasmodium, Leishmania and Toxoplasma have been transformed with luciferase and yielded unique insights into their in vivo behaviour. We expect that both the range of organisms and the molecular events able to be studied by BLI will continue to expand, yielding important insights into mechanisms of pathogenesis.

Murine alveolar macrophages limit replication of vaccinia virus.

Because of concerns about zoonotic transmission of monkeypox to humans and the bioterrorism threat posed by orthopoxviruses, there is renewed interest in probing cellular and molecular mechanisms of host defense to these pathogens. In particular, it is essential to understand viral-host interactions in the respiratory tract, which is the route of infection for smallpox and a likely route of transmission for monkeypox. In this study, we analyze functions of alveolar macrophages in poxvirus infection, using a recombinant vaccinia virus expressing firefly luciferase to quantify infection in mice and cell culture. Depletion of alveolar macrophages with liposomal clodronate worsens the overall severity of infection in mice, including greater replication and systemic dissemination of vaccinia as determined by bioluminescence imaging. Absence of alveolar macrophages increases total numbers of granulocytes and granulocytes/monocyte progenitor cells in the lungs during vaccinia infection, indicating that protective effects of alveolar macrophages may be mediated in part by reducing the host inflammation. Alveolar macrophages also limit vaccinia infection in respiratory epithelium, as shown by a co-culture model of cell lines derived from alveolar macrophages and lung epithelium. Collectively, these data demonstrate that alveolar macrophages are key determinants of host defense against local and systemic infection with poxviruses.

Bioluminescence imaging of vaccinia virus: effects of interferon on viral replication and spread.

Whole animal imaging allows viral replication and localization to be monitored in intact animals, which provides significant advantages for determining viral and host factors that determine pathogenesis. To investigate effects of interferons on spatial and temporal progression of vaccinia infection, we generated recombinant viruses that express firefly luciferase or a monomeric orange fluorescent protein. These viruses allow vaccinia infection to be monitored with bioluminescence or fluorescence imaging, respectively. The recombinant viruses were not attenuated in vitro or in vivo relative to a control WR virus. In cell culture, reporters could be detected readily by 4 h post-infection, showing that these viruses can be used as early markers of infection. The magnitude of firefly luciferase activity measured with bioluminescence imaging in vitro and in vivo correlated directly with increasing titers of vaccinia virus, validating imaging data as a marker of viral infection. Replication of vaccinia was significantly greater in mice lacking receptors for type I interferons (IFN I R-/-) compared with wild-type mice, although both genotypes of mice developed focal infections in lungs and brain after intranasal inoculation. IFN I R-/- mice had greater dissemination of virus to liver and spleen than wild-type animals even when mortality occurred at the same time point after infection. Protective effects of type I interferons were mediated primarily through parenchymal cells rather than hematopoietic cells as analyzed by bone marrow transplant experiments. Collectively, our data define a new function for type I interferon signaling in systemic dissemination of vaccinia and validate these reporter viruses for studies of pathogenesis.