Lung Microbiota Contribute to Pulmonary Inflammation and Disease Progression in Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) causes considerable global morbidity and mortality, and its mechanisms of disease progression are poorly understood. Recent observational studies have reported associations between lung dysbiosis, mortality, and altered host defense gene expression, supporting a role for lung microbiota in IPF. However, the causal significance of altered lung microbiota in disease progression is undetermined. Objectives: To examine the effect of microbiota on local alveolar inflammation and disease progression using both animal models and human subjects with IPF. Methods: For human studies, we characterized lung microbiota in BAL fluid from 68 patients with IPF. For animal modeling, we used a murine model of pulmonary fibrosis in conventional and germ-free mice. Lung bacteria were characterized using 16S rRNA gene sequencing with novel techniques optimized for low-biomass sample load. Microbiota were correlated with alveolar inflammation, measures of pulmonary fibrosis, and disease progression. Measurements and Main Results: Disruption of the lung microbiome predicts disease progression, correlates with local host inflammation, and participates in disease progression. In patients with IPF, lung bacterial burden predicts fibrosis progression, and microbiota diversity and composition correlate with increased alveolar profibrotic cytokines. In murine models of fibrosis, lung dysbiosis precedes peak lung injury and is persistent. In germ-free animals, the absence of a microbiome protects against mortality. Conclusions: Our results demonstrate that lung microbiota contribute to the progression of IPF. We provide biological plausibility for the hypothesis that lung dysbiosis promotes alveolar inflammation and aberrant repair. Manipulation of lung microbiota may represent a novel target for the treatment of IPF.

The Lung Microbiota of Healthy Mice Are Highly Variable, Cluster by Environment, and Reflect Variation in Baseline Lung Innate Immunity.

RATIONALE: The "gut-lung axis" is commonly invoked to explain the microbiome's influence on lung inflammation. Yet the lungs harbor their own microbiome, which is altered in respiratory disease. The relative influence of gut and lung bacteria on lung inflammation is unknown. OBJECTIVES: To determine whether baseline lung immune tone reflects local (lung-lung) or remote (gut-lung) microbe-host interactions. METHODS: We compared lung, tongue, and cecal bacteria in 40 healthy, genetically identical, 10-week-old mice, using 16S ribosomal RNA gene quantification and sequencing. We measured inflammatory cytokines, using a multiplex assay of homogenized lung tissue. We compared lung bacteria in healthy mice treated with varied durations of systemic antibiotics. MEASUREMENTS AND MAIN RESULTS: Lung bacterial communities are highly variable among mice, cluster strongly by cage, shipment, and vendor, and are altered by antibiotics in a microbiologically predictable manner. Baseline lung concentrations of two key inflammatory cytokines (IL-1α and IL-4) are correlated with the diversity and community composition of lung bacterial communities. Lung concentrations of these inflammatory cytokines correlate more strongly with variation in lung bacterial communities than with that of the gut or mouth. CONCLUSIONS: In the lungs of healthy mice, baseline innate immune tone more strongly reflects local (lung-lung) microbe-host interactions than remote (gut-lung) microbe-host interactions. Our results independently confirm the existence and immunologic significance of the murine lung microbiome, even in health. Variation in lung microbiota is likely an important, underappreciated source of experimental and clinical variability. The lung microbiome is an unexplored therapeutic target for the prevention and treatment of inflammatory lung disease.

Matrix Metalloproteinase Activity in Infections by an Encephalitic Virus, Mouse Adenovirus Type 1.

Mouse adenovirus type 1 (MAV-1) infection causes encephalitis in susceptible strains of mice and alters the permeability of infected brains to small molecules, which indicates disruption of the blood-brain barrier (BBB). Under pathological conditions, matrix metalloproteinases (MMPs) can disrupt the BBB through their proteolytic activity on basement membrane and tight junction proteins. We examined whether MAV-1 infection alters MMP activity in vivo and in vitro Infected MAV-1-susceptible SJL mice had higher MMP2 and MMP9 activity in brains, measured by gelatin zymography, than mock-infected mice. Infected MAV-1-resistant BALB/c mice had MMP activity levels equivalent to those in mock infection. Primary SJL mouse brain endothelial cells (a target of MAV-1 in vivo) infected ex vivo with MAV-1 had no difference in activities of secreted MMP2 and MMP9 from mock cells. We show for the first time that astrocytes and microglia are also infected in vivo by MAV-1. Infected mixed primary cultures of astrocytes and microglia had higher levels of MMP2 and MMP9 activity than mock-infected cells. These results indicate that increased MMP activity in the brains of MAV-1-infected susceptible mice may be due to MMP activity produced by endothelial cells, astrocytes, and microglia, which in turn may contribute to BBB disruption and encephalitis in susceptible mice.IMPORTANCE RNA and DNA viruses can cause encephalitis; in some cases, this is accompanied by MMP-mediated disruption of the BBB. Activated MMPs degrade extracellular matrix and cleave tight-junction proteins and cytokines, modulating their functions. MAV-1 infection of susceptible mice is a tractable small-animal model for encephalitis, and the virus causes disruption of the BBB. We showed that MAV-1 infection increases enzymatic activity of two key MMPs known to be secreted and activated in neuroinflammation, MMP2 and MMP9, in brains of susceptible mice. MAV-1 infects endothelial cells, astrocytes, and microglia, cell types in the neurovascular unit that can secrete MMPs. Ex vivo MAV-1 infection of these cell types caused higher MMP activity than mock infection, suggesting that they may contribute to the higher MMP activity seen in vivo To our knowledge, this provides the first evidence of an encephalitic DNA virus in its natural host causing increased MMP activity in brains.

Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli.

BACKGROUND: Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-ß1 and substrate stiffness affect fibroblast Fas expression are not well understood. METHODS: Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young's moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-ß1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. RESULTS: Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-ß1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-ß1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-ß1. CONCLUSIONS: Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.

Targeting Inhibitor of Apoptosis Proteins Protects from Bleomycin-Induced Lung Fibrosis.

Accumulation of apoptosis-resistant fibroblasts is a hallmark of pulmonary fibrosis. We hypothesized that disruption of inhibitor of apoptosis protein (IAP) family proteins would limit lung fibrosis. We first show that transforming growth factor-ß1 and bleomycin increase X-linked IAP (XIAP) and cellular IAP (cIAP)-1 and -2 in murine lungs and mesenchymal cells. Functional blockade of XIAP and the cIAPs with AT-406, an orally bioavailable second mitochondria-derived activator of caspases (Smac) mimetic, abrogated bleomycin-induced lung fibrosis when given both prophylactically and therapeutically. To determine whether the reduction in fibrosis was predominantly due to AT-406-mediated inhibition of XIAP, we compared the fibrotic response of XIAP-deficient mice (XIAP(-/y)) with littermate controls and found no difference. We found no alterations in total inflammatory cells of either wild-type mice treated with AT-406 or XIAP(-/y) mice. AT-406 treatment limited CCL12 and IFN-γ production, whereas XIAP(-/y) mice exhibited increased IL-1ß expression. Surprisingly, XIAP(-/y) mesenchymal cells had increased resistance to Fas-mediated apoptosis. Functional blockade of cIAPs with AT-406 restored sensitivity to Fas-mediated apoptosis in XIAP(-/y) mesenchymal cells in vitro and increased apoptosis of mesenchymal cells in vivo, indicating that the increased apoptosis resistance in XIAP(-/y) mesenchymal cells was the result of increased cIAP expression. Collectively, these results indicate that: (1) IAPs have a role in the pathogenesis of lung fibrosis; (2) a congenital deficiency of XIAP may be overcome by compensatory mechanisms of other IAPs; and (3) broad functional inhibition of IAPs may be an effective strategy for the treatment of lung fibrosis by promoting mesenchymal cell apoptosis.

Influences of innate immunity, autophagy, and fibroblast activation in the pathogenesis of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by accumulation of extracellular matrix (ECM) and impaired gas exchange. The pathobiological mechanisms that account for disease progression are poorly understood but likely involve alterations in innate inflammatory cells, epithelial cells, and fibroblasts. Thus we seek to review the most recent literature highlighting the complex roles of neutrophils and macrophages as both promoters of fibrosis and defenders against infection. With respect to epithelial cells and fibroblasts, we review the data suggesting that defective autophagy promotes the fibrogenic potential of both cell types and discuss new evidence related to matrix metalloproteinases, growth factors, and cellular metabolism in the form of lactic acid generation that may have consequences for promoting fibrogenesis. We discuss potential cross talk between innate and structural cell types and also highlight literature that may help explain the limitations of current IPF therapies.

Identifying Mechanisms of Homeostatic Signaling in Fibroblast Differentiation.

Fibroblasts play an important role in the wound-healing process by generating extracellular matrix (ECM) and undergoing differentiation into myofibroblasts, but these cells can also be involved in pathologic remodeling of tissue. Nascent ECM provides a substrate for re-epithelialization to occur, restoring damaged tissue to a functional state. Dysregulation of this process can result in fibrosis--stiffening and scarring of the tissue. Current treatments cannot halt or reverse this process. The molecular mechanisms underlying fibrotic dysregulation are poorly understood, providing an untapped pool of potential therapeutic targets. Transforming growth factor-ß (TGF-ß) and adhesion signaling are involved in inducing fibroblast differentiation into α-smooth muscle actin (αSMA) expressing myofibroblasts, while prostaglandin E2 (PGE2) has been shown to antagonize TGF-ß signaling; however, the temporal and mechanistic details of this relationship have not yet been fully characterized. We measured αSMA, a marker of fibroblast to myofibroblast differentiation, as a function of: TGF-ß1 receptor-ligand complex internalization, PGE2 binding, and adhesion signaling and developed a mathematical model capturing the molecular mechanisms of fibroblast differentiation. Using our model, we predict the following: Periodic dosing with PGE2 temporarily renders fibroblasts incapable of differentiation and refractory to additional TGF-ß1 stimulation; conversely, periodic dosing with TGF-ß1 in the presence of PGE2 induces a reduced signal response that can be further inhibited by the addition of more PGE2. Controlled fibroblast differentiation is necessary for effective wound healing; however, excessive accumulation of αSMA-expressing myofibroblasts can result in fibrosis. Homeostasis of αSMA in our model requires a balance of positive and negative regulatory signals. Sensitivity analysis predicts that PGE2 availability, TGF-ß1 availability, and the rate of TGF-ß1 receptor recycling each highly influence the rates of αSMA production. With this model, we are able to demonstrate that regulation of both TGF-ß1 and PGE2 signaling levels is essential for preventing fibroblast dysregulation.

γ-Herpes virus-68, but not Pseudomonas aeruginosa or influenza A (H1N1), exacerbates established murine lung fibrosis.

Patients with idiopathic pulmonary fibrosis (IPF) often do worse following infection, but the cause of the decline is not fully understood. We previously demonstrated that infection with a murine gamma herpes virus (γHV-68) could exacerbate established lung fibrosis following administration of fluorescein isothiocyanate (McMillan et al. Am J Respir Crit Care Med 177: 771-780, 2008). In the present study, we anesthetized mice and injected saline or bleomycin intratracheally on day 0. On day 14, mice were anesthetized again and infected with either a Gram-negative bacteria (Pseudomonas aeruginosa), or with H1N1 or γHV-68 viruses. Measurements were then made on days 15, 21, or 35. We demonstrate that infection with P. aeruginosa does not exacerbate extracellular matrix deposition post-bleomycin. Furthermore, fibrotic mice are effectively able to clear P. aeruginosa infection. In contrast, bleomycin-treated mice develop worse lung fibrosis when infected with γHV-68, but not when infected with H1N1. The differential ability of γHV-68 to cause increased collagen deposition could not be explained by differences in inflammatory cell recruitment or whole lung chemokine and cytokine responses. Alveolar epithelial cells from γHV-68-infected mice displayed increased expression of TGFß receptor 1, increased SMAD3 phosphorylation, and evidence of apoptosis measured by cleaved poly-ADP ribose polymerase (PARP). The ability of γHV-68 to augment fibrosis required the ability of the virus to reactivate from latency. This property appears unique to γHV-68, as the ß-herpes virus, cytomegalovirus, did not have the same effect.

Usage of integrin and heparan sulfate as receptors for mouse adenovirus type 1.

Adenovirus fiber knobs are the capsid components that interact with binding receptors on cells, while an Arg-Gly-Asp (RGD) sequence usually found in the penton base protein is important for the interaction of most adenoviruses with integrin entry receptors. Mouse adenovirus type 1 (MAV-1) lacks an RGD sequence in the virion penton base protein. We tested whether an RGD sequence found in the MAV-1 fiber knob plays a role in infection. Treatment of cells with a competitor RGD peptide or a purified recombinant RGD-containing fiber knob prior to infection resulted in reduced virus yields compared to those of controls, indicating the importance of the RGD sequence for infection. An investigation of the role of integrins as possible receptors showed that MAV-1 yields were reduced in the presence of EDTA, an inhibitor of integrin binding, and in the presence of anti-alpha(v) integrin antibody. Moreover, mouse embryo fibroblasts that were genetically deficient in alpha(v) integrin yielded less virus, supporting the hypothesis that alpha(v) integrin is a likely receptor for MAV-1. We also investigated whether glycosaminoglycans play a role in MAV-1 infection. Preincubation of MAV-1 with heparin, a heparan sulfate glycosaminoglycan analog, resulted in a decrease in MAV-1 virus yields. Reduced MAV-1 infectivity was also found with cells that genetically lack heparan sulfate or cells that were treated with heparinase I. Cumulatively, our data demonstrate that the RGD sequence in the MAV-1 fiber knob plays a role in infection by MAV-1, alpha(v) integrin acts as a receptor for the virus, and cell surface heparin sulfate glycosaminoglycans are important in MAV-1 infection.

Mouse adenovirus type 1-induced breakdown of the blood-brain barrier.

Infection with mouse adenovirus type 1 (MAV-1) results in fatal acute encephalomyelitis in susceptible mouse strains via infection of brain endothelial cells. Wild-type (wt) MAV-1 causes less brain inflammation than an early region 3 (E3) null virus in C57BL/6 mice. A mouse brain microvascular endothelial cell line infected with wt MAV-1 had higher expression of mRNAs for the proinflammatory chemokines CCL2 and CCL5 than mock- and E3 null virus-infected cells. Primary mouse brain endothelial cells infected with wt virus had elevated levels of CCL2 compared to mock- or E3 null virus-infected cells. Infection of C57BL/6 mice with wt MAV-1 or the E3 null virus caused a dose-dependent breakdown of the blood-brain barrier, primarily due to direct effects of virus infection rather than inflammation. The tight junction proteins claudin-5 and occludin showed reduced surface expression on primary mouse brain endothelial cells following infection with either wt MAV-1 or the E3 null virus. mRNAs and protein for claudin-5, occludin, and zona occludens 2 were also reduced in infected cells. MAV-1 infection caused a loss of transendothelial electrical resistance in primary mouse brain endothelial cells that was not dependent on E3 or on MAV-1-induced CCL2 expression. Taken together, these results demonstrate that MAV-1 infection caused breakdown of the blood-brain barrier accompanied by decreased surface expression of tight junction proteins. Furthermore, while the MAV-1-induced pathogenesis and inflammation were dependent on E3, MAV-1-induced breakdown of the blood-brain barrier and alteration of endothelial cell function were not dependent on E3 or CCL2.

Lung and gut microbiota are altered by hyperoxia and contribute to oxygen-induced lung injury in mice.

Inhaled oxygen, although commonly administered to patients with respiratory disease, causes severe lung injury in animals and is associated with poor clinical outcomes in humans. The relationship between hyperoxia, lung and gut microbiota, and lung injury is unknown. Here, we show that hyperoxia conferred a selective relative growth advantage on oxygen-tolerant respiratory microbial species (e.g., Staphylococcus aureus) as demonstrated by an observational study of critically ill patients receiving mechanical ventilation and experiments using neonatal and adult mouse models. During exposure of mice to hyperoxia, both lung and gut bacterial communities were altered, and these communities contributed to oxygen-induced lung injury. Disruption of lung and gut microbiota preceded lung injury, and variation in microbial communities correlated with variation in lung inflammation. Germ-free mice were protected from oxygen-induced lung injury, and systemic antibiotic treatment selectively modulated the severity of oxygen-induced lung injury in conventionally housed animals. These results suggest that inhaled oxygen may alter lung and gut microbial communities and that these communities could contribute to lung injury.

Retinoic acid decreases adherence of murine myeloid dendritic cells and increases production of matrix metalloproteinase-9.

Myeloid dendritic cells (DC) are professional antigen presenting cells (APC) that migrate to secondary lymphoid tissues upon antigen stimulation, where they activate naïve T cells. Vitamin A is essential for normal immune function. We investigated the ability of all-trans retinoic acid (atRA), a bioactive metabolite of vitamin A, to modulate DC adhesion in culture. Male BALB/cJ mouse bone marrow cells cultured with granulocyte-macrophage colony-stimulating factor in the presence of retinoic acid receptor (RAR) alpha-specific antagonist showed an increase in the percentage of developing DC that remained adherent compared with cells rescued with atRA treatment from d 8 to 10 of culture (P < 0.05). Replacement of the RARalpha antagonist with atRA on d 8 of the culture period decreased DC surface expression of the adhesion molecule CD11a (P < 0.0001) but not the gene expression. Rescue with atRA also dramatically increased gene and protein expression of pro-matrix metalloproteinase (MMP)-9 (P < 0.05). However, gene expression and protein production of tissue inhibitor of metalloproteinase (TIMP)-1 was unaffected by atRA rescue, altering the molar ratio of secreted pro-MMP-9:TIMP-1, resulting in a fold excess of pro-MMP-9 to its primary inhibitor (P < 0.05). These data suggest that atRA is essential to augment MMP-9 expression in myeloid DC and can alter their surface expression of adhesion molecules.

Erratum: The peripheral blood proteome signature of idiopathic pulmonary fibrosis is distinct from normal and is associated with novel immunological processes.

This corrects the article DOI: 10.1038/srep46560.

The peripheral blood proteome signature of idiopathic pulmonary fibrosis is distinct from normal and is associated with novel immunological processes.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial pneumonia. The disease pathophysiology is poorly understood and the etiology remains unclear. Recent advances have generated new therapies and improved knowledge of the natural history of IPF. These gains have been brokered by advances in technology and improved insight into the role of various genes in mediating disease, but gene expression and protein levels do not always correlate. Thus, in this paper we apply a novel large scale high throughput aptamer approach to identify more than 1100 proteins in the peripheral blood of well-characterized IPF patients and normal volunteers. We use systems biology approaches to identify a unique IPF proteome signature and give insight into biological processes driving IPF. We found IPF plasma to be altered and enriched for proteins involved in defense response, wound healing and protein phosphorylation when compared to normal human plasma. Analysis also revealed a minimal protein signature that differentiated IPF patients from normal controls, which may allow for accurate diagnosis of IPF based on easily-accessible peripheral blood. This report introduces large scale unbiased protein discovery analysis to IPF and describes distinct biological processes that further inform disease biology.

Six-SOMAmer Index Relating to Immune, Protease and Angiogenic Functions Predicts Progression in IPF.

RATIONALE: Biomarkers in easily accessible compartments like peripheral blood that can predict disease progression in idiopathic pulmonary fibrosis (IPF) would be clinically useful regarding clinical trial participation or treatment decisions for patients. In this study, we used unbiased proteomics to identify relevant disease progression biomarkers in IPF. METHODS: Plasma from IPF patients was measured using an 1129 analyte slow off-rate modified aptamer (SOMAmer) array, and patient outcomes were followed over the next 80 weeks. Receiver operating characteristic (ROC) curves evaluated sensitivity and specificity for levels of each biomarker and estimated area under the curve (AUC) when prognostic biomarker thresholds were used to predict disease progression. Both logistic and Cox regression models advised biomarker selection for a composite disease progression index; index biomarkers were weighted via expected progression-free days lost during follow-up with a biomarker on the unfavorable side of the threshold. RESULTS: A six-analyte index, scaled 0 to 11, composed of markers of immune function, proteolysis and angiogenesis [high levels of ficolin-2 (FCN2), cathepsin-S (Cath-S), legumain (LGMN) and soluble vascular endothelial growth factor receptor 2 (VEGFsR2), but low levels of inducible T cell costimulator (ICOS) or trypsin 3 (TRY3)] predicted better progression-free survival in IPF with a ROC AUC of 0.91. An index score ≥ 3 (group ≥ 2) was strongly associated with IPF progression after adjustment for age, gender, smoking status, immunomodulation, forced vital capacity % predicted and diffusing capacity for carbon monoxide % predicted (HR 16.8, 95% CI 2.2-126.7, P = 0.006). CONCLUSION: This index, derived from the largest proteomic analysis of IPF plasma samples to date, could be useful for clinical decision making in IPF, and the identified analytes suggest biological processes that may promote disease progression.

Limited effects of Muc1 deficiency on mouse adenovirus type 1 respiratory infection.

Muc1 (MUC1 in humans) is a membrane-tethered mucin that exerts anti-inflammatory effects in the lung during bacterial infection. Muc1 and other mucins are also likely to form a protective barrier in the lung. We used mouse adenovirus type 1 (MAV-1, also known as MAdV-1) to determine the role of Muc1 in the pathogenesis of an adenovirus in its natural host. Following intranasal inoculation of wild type mice, we detected increased TNF-α, a cytokine linked to Muc1 production, but no consistent changes in the production of lung Muc1, Muc5ac or overall lung mucus production. Viral loads were modestly higher in the lungs of Muc1(-/-) mice compared to Muc1(+/+) mice at several early time points but decreased to similar levels by 14 days post infection in both groups. However, cellular inflammation and the expression of CXCL1, CCL5, and CCL2 did not significantly differ between Muc1(-/-) and Muc1(+/+) mice. Our data therefore suggest that Muc1 may contribute to a physical barrier that protects against MAV-1 respiratory infection. However, our data do not reveal an anti-inflammatory effect of Muc1 that contributes to MAV-1 pathogenesis.

Host genetic variation in susceptibility to Punta Toro virus.

Infection of small laboratory animals by Punta Toro virus (PTV), family Bunyaviridae, genus Phlebovirus, is a model for the study of the human pathogen Rift Valley fever virus (RVFV). We have identified inbred mouse strains with significant differences in host response to the Adames strain of PTV. Nine inbred strains of mice representing major branches in the Mus musculus phylogeny were inoculated subcutaneously with a high dose of PTV in survival experiments. Two inbred strains of mice, NZW/LacJ and 129S1/SvImJ, died ~4 days after PTV infection, whereas 7 other strains survived the challenge and showed no clinical signs of disease. Histologically, 129S1/SvImJ mice showed massive hepatocellular necrosis and had additional lesions in lung, brain, and spleen, whereas NZW/LacJ mice had mild piecemeal hepatocellular necrosis. PTV viral loads in the livers of infected mice were determined by reverse transcriptase quantitative PCR. Inbred mice from strains that showed clinical signs and succumbed to PTV infection had higher liver viral loads than did mice of resistant strains. Hybrid F1 mice were generated by crossing susceptible 129S1 and resistant FVB/N mice and tested for susceptibility. The hybrid F1 mice showed significantly higher viral loads in the liver than the resistant parental FVB/N mice, suggesting that susceptibility is dominant. These findings will enable an unbiased genetic approach to identify host genes mediating susceptibility to PTV.

Mouse adenovirus type 1 infection of macrophages.

Mouse adenovirus type 1 (MAV-1) causes acute and persistent infections in mice, with high levels of virus found in the brain, spinal cord and spleen in acute infections. MAV-1 infects endothelial cells throughout the mouse, and monocytes/macrophages have also been implicated as targets of the virus. Here we determined the extent and functional importance of macrophage infection by MAV-1. Bone marrow-derived macrophages expressed MAV-1 mRNAs and proteins upon ex vivo infection. Adherent peritoneal macrophages from infected mice expressed viral mRNAs and produced infectious virus. Infected chemokine (C-C motif) receptor 2 (CCR2) knockout mice, which are defective for macrophage recruitment, did not show differences in survival or MAV-1 load compared to controls. In contrast, macrophage depletion using clodronate-loaded liposomes resulted in increased virus replication in spleens of a MAV-1-resistant mouse strain, BALB/cJ. Thus macrophages serve both as targets of infection and as effectors of the host response.