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 β1, 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

Increased viral replication in TLR4 mutant mice is more pronounced at higher viral dose.

<p>C3HeB/FeJ and C3H/HeJ mice were infected with 5×10⁵ pfu Vac-GFL intranasally. (A) Head luminescence. (B) Chest luminescence. (C) Representative chest images. C3HeB/FeJ (left) and C3H/HeJ (right), 30 s exposure, f-stop 1. Purple denotes lower luminescence intensity; red, higher luminescence intensity.*<i>p</i><0.05. Error bars denote SEM.</p

Lack of TLR4 does not impair IFN-β production.

<p>IFN-β concentrations in lung homogenate supernatants were measured by ELISA. Points represent individual mice. Dashed line represents lower limit of reliable detection on standard curve. Solid lines represent mean IFN-β concentration.</p

TLR4 alters the inflammatory response to vaccinia infection: representative photomicrographs.

<p>Mice were infected intranasally with 5×10⁵ pfu Vac-GFL. Lungs were harvested on days 3 and 5 post-infection, preserved in 10% formalin, paraffin-embedded, and stained with H&E. (A, B) representative sections of C3H/HeJ (A) and C3HeB/FeJ (B) lung tissue obtained on day 3 post-infection. B = bronchiole; V = blood vessel; arrows denote inflammatory foci.</p

TLR4 mutant mice are more susceptible to vaccinia than controls.

<p>C3HeB/FeJ and C3H/HeJ mice were infected with 1×10⁴ pfu Vac-GFL. (A) Body temperature. (B) Weight loss, expressed as percent of initial weight; (C) Chest luminescence. (D) Abdominal luminescence, expressed as photon flux. *<i>p</i><0.05. Error bars denote SEM.</p

Lack of TLR4 does not impair proinflammatory cytokine production.

<p>IL-6 levels in the lung homogenate supernatants measured by ELISA. Error bars denote SEM. *<i>p</i><0.05.</p

TLR4 alters the inflammatory response to vaccinia infection: quantification of foci of inflammation.

<p>Data are expressed as number of foci per section. *<i>p</i><0.01. Error bars denote SEM.</p

Increased viral replication in TLR4 mutant abdomens is more pronounced at higher viral dose.

<p>C3HeB/FeJ and C3H/HeJ mice were infected with 5×10⁵ pfu Vac-GFL intranasally. (A) Abdominal luminescence. (B) Representative images of splenic luminescence C3HeB/FeJ (left) and C3H/HeJ (right) mice, 30 s exposure, f-stop 1. Purple denotes lower luminescence intensity; red, higher luminescence intensity.*<i>p</i><0.05. Error bars denote SEM.</p