A rat model of picornavirus-induced airway infection and inflammation

<p>Abstract</p> <p>Background</p> <p>Infection of the lower airways by rhinovirus, a member of the picornavirus family, is an important cause of wheezing illnesses in infants, and plays an important role in the pathogenesis of rhinovirus-induced asthma exacerbations. Given the absence of natural rhinovirus infections in rodents, we investigated whether an attenuated form of mengovirus, a picornavirus whose wild-type form causes systemic rather than respiratory infections in its natural rodent hosts, could induce airway infections in rats with inflammatory responses similar to those in human rhinovirus infections.</p> <p>Results</p> <p>After inoculation with 10⁷plaque-forming units of attenuated mengovirus through an inhalation route, infectious mengovirus was consistently recovered on days 1 and 3 postinoculation from left lung homogenates (median Log₁₀plaque-forming units = 6.0 and 4.8, respectively) and right lung bronchoalveolar lavage fluid (median Log₁₀plaque-forming units = 5.8 and 4.0, respectively). Insufflation of attenuated mengovirus, but not vehicle or UV-inactivated virus, into the lungs of BN rats caused significant increases <it>(P </it>< 0.05) in lower airway neutrophils and lymphocytes in the bronchoalveolar lavage fluid and patchy peribronchiolar, perivascular, and alveolar cellular infiltrates in lung tissue sections. In addition, infection with attenuated mengovirus significantly increased (<it>P </it>< 0.05) lower airway levels of neutrophil chemoattractant CXCR2 ligands [cytokine-induced neutrophil chemoattractant-1 (CINC-1; CXCL1) and macrophage inflammatory protein-2 (MIP-2; CXCL2)] and monocyte chemoattractant protein-1 (MCP-1; CCL2) in comparison to inoculation with vehicle or UV-inactivated virus.</p> <p>Conclusion</p> <p>Attenuated mengovirus caused a respiratory infection in rats with several days of viral shedding accompanied by a lower airway inflammatory response consisting of neutrophils and lymphocytes. These features suggest that mengovirus-induced airway infection in rodents could be a useful model to define mechanisms of rhinovirus-induced airway inflammation in humans.</p

Protective Intestinal Effects of Pituitary Adenylate Cyclase Activating Polypeptide

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide widely distributed throughout the body, including the gastrointestinal tract. Several effects have been described in human and animal intestines. Among others, PACAP infl uences secretion of intestinal glands, blood fl ow, and smooth muscle contraction. PACAP is a well-known cytoprotective peptide with strong anti-apoptotic, anti-infl ammatory, and antioxidant effects. The present review gives an overview of the intestinal protective actions of this neuropeptide. Exogenous PACAP treatment was protective in a rat model of small bowel autotransplantation. Radioimmunoassay (RIA) analysis of the intestinal tissue showed that endogenous PACAP levels gradually decreased with longer-lasting ischemic periods, prevented by PACAP addition. PACAP counteracted deleterious effects of ischemia on oxidative stress markers and cytokines. Another series of experiments investigated the role of endogenous PACAP in intestines in PACAP knockout (KO) mice. Warm ischemia–reperfusion injury and cold preservation models showed that the lack of PACAP caused a higher vulnerability against ischemic periods. Changes were more severe in PACAP KO mice at all examined time points. This fi nding was supported by increased levels of oxidative stress markers and decreased expression of antioxidant molecules. PACAP was proven to be protective not only in ischemic but also in infl ammatory bowel diseases. A recent study showed that PACAP treatment prolonged survival of Toxoplasma gondii infected mice suffering from acute ileitis and was able to reduce the ileal expression of proinfl ammatory cytokines. We completed the present review with recent clinical results obtained in patients suffering from infl ammatory bowel diseases. It was found that PACAP levels were altered depending on the activity, type of the disease, and antibiotic therapy, suggesting its probable role in infl ammatory events of the intestine

Lower Respiratory Tract Infection Induced by a Genetically Modified Picornavirus in Its Natural Murine Host

Infections with the picornavirus, human rhinovirus (HRV), are a major cause of wheezing illnesses and asthma exacerbations. In developing a murine model of picornaviral airway infection, we noted the absence of murine rhinoviruses and that mice are not natural hosts for HRV. The picornavirus, mengovirus, induces lethal systemic infections in its natural murine hosts, but small genetic differences can profoundly affect picornaviral tropism and virulence. We demonstrate that inhalation of a genetically attenuated mengovirus, vMC0, induces lower respiratory tract infections in mice. After intranasal vMC0 inoculation, lung viral titers increased, peaking at 24 h postinoculation with viral shedding persisting for 5 days, whereas HRV-A01a lung viral titers decreased and were undetectable 24 h after intranasal inoculation. Inhalation of vMC0, but not vehicle or UV-inactivated vMC0, induced an acute respiratory illness, with body weight loss and lower airway inflammation, characterized by increased numbers of airway neutrophils and lymphocytes and elevated pulmonary expression of neutrophil chemoattractant CXCR2 ligands (CXCL1, CXCL2, CXCL5) and interleukin-17A. Mice inoculated with vMC0, compared with those inoculated with vehicle or UV-inactivated vMC0, exhibited increased pulmonary expression of interferon (IFN-α, IFN-β, IFN-λ), viral RNA sensors [toll-like receptor (TLR)3, TLR7, nucleotide-binding oligomerization domain containing 2 (NOD2)], and chemokines associated with HRV infection in humans (CXCL10, CCL2). Inhalation of vMC0, but not vehicle or UV-inactivated vMC0, was accompanied by increased airway fluid myeloperoxidase levels, an indicator of neutrophil activation, increased MUC5B gene expression, and lung edema, a sign of infection-related lung injury. Consistent with experimental HRV inoculations of nonallergic, nonasthmatic human subjects, there were no effects on airway hyperresponsiveness after inhalation of vMC0 by healthy mice. This novel murine model of picornaviral airway infection and inflammation should be useful for defining mechanisms of HRV pathogenesis in humans

PACAP is an endogenous protective factor - insights from PACAP-deficient mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widespread neuropeptide with a diverse array of biological functions. Not surprisingly, the lack of endogenous PACAP therefore results in a variety of abnormalities. One of the important effects of PACAP is its neuroprotective and general cytoprotective role. PACAP protects neurons and other tissues against ischemic, toxic, and traumatic lesions. Data obtained from PACAP-deficient mice provide evidence that endogenous PACAP also has protective functions. Mice lacking PACAP are more vulnerable to different in vitro and in vivo insults. The present review summarizes data on the increased sensitivity of PACAP-deficient mice against harmful stimuli. Mice lacking PACAP respond with a higher degree of injury in cerebral ischemia, autoimmune encephalomyelitis, and axonal lesion. Retinal ischemic and excitotoxic injuries also produce increased cell loss in PACAP-deficient mice. In peripheral organs, kidney cell cultures from PACAP-deficient mice are more sensitive to oxidative stress and in vitro hypoxia. In vivo, PACAP-deficient mice have a negative histological outcome and altered cytokine response in kidney and small intestine ischemia/reperfusion injury. Large intestinal inflammation, toxic lesion of the pancreas, and doxorubicin-induced cardiomyopathy are also more severe with a lack of endogenous PACAP. Finally, an increased inflammatory response has been described in subacute endotoxin- induced airway inflammation and in an oxazolone-induced allergic contact dermatitis model. In summary, lack of endogenous PACAP leads to higher vulnerability in a number of injuries in the nervous system and peripheral organs, supporting the hypothesis that PACAP is part of the endogenous cytoprotective machinery

Lung edema in response to inhalation of attenuated mengovirus.

<p>Wet:dry lung weight ratios were measured for lungs harvested on day 2 after intranasal inoculation with 10⁶ PFU of attenuated mengovirus, vMC₀, an equivalent amount of UV-inactivated vMC₀, or vehicle (n = 9 mice per group). Data are presented as box plots; whiskers indicate the 10th and 90th percentiles. * <i>P</i><0.01 (vMC₀ vs. vehicle or UV-inactivated vMC₀).</p

Differences between attenuated mengovirus and HRV in the kinetics of lung viral titers.

<p>Mice received intranasal inoculations of 10⁶ PFU of attenuated mengovirus, vMC₀ [A (n = 7 mice per group), B (n = 6 mice per group)], or 5×10⁶ PFU of HRV-A01a (C; n = 4 mice per group). Lungs were harvested at the indicated times, and viral titers in lung homogenates were determined by plaque assays. Data are the total amount of virus present in the lung homogenates (virus concentrations were multiplied by lung homogenate volumes). No virus was detected in lungs from vehicle-inoculated mice. Data are presented as box plots. For one HRV-A01a-inoculated mouse at 3 h postinoculation, a value of 1 PFU was assigned for graphing purposes because virus was undetectable. ND, not detectable.</p

Recruitment of inflammatory cell infiltrates to the lungs in response to inhalation of attenuated mengovirus.

<p>Giemsa-stained sections of the lungs from mice intransasally inoculated with (A) vehicle, (B) vMC₀ (10⁶ PFU) or (C) an equivalent amount of UV-inactivated vMC₀. Lungs were harvested on day 2 postinoculation. Magnification, 20X.</p

Neutrophilic inflammation in the lungs in response to inhalation of attenuated mengovirus.

<p>Mice received intranasal inoculations of 10⁶ PFU of attenuated mengovirus, vMC₀, an equivalent amount of UV-inactivated vMC₀, or vehicle. (A) Lung-associated MPO levels on day 1 postinoculation. MPO levels in lung tissue homogenates from mice inoculated with vehicle or vMC₀ were determined by ELISA and normalized to total protein levels (n = 4 mice per group). (B) MPO release into airway fluids. BAL fluid was harvested on the indicated days, and MPO levels were determined by ELISA (n = 6 mice per group). Data are the total amount of MPO recovered (ELISA values were multiplied by the BAL fluid volume). MPO levels below the limit of detection were assigned a value of 1 ng for graphing purposes. (C) CXCR2 and (D) IL-17A expression in the lungs on day 1 postinoculation. Levels of CXCR2 and IL-17A mRNA were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels (n = 6 mice per group). Data are presented as box plots. * <i>P</i><0.05 (vMC₀ vs. vehicle). ** <i>P</i><0.01 (vMC₀ vs. vehicle or UV-inactivated vMC₀).</p

Mucin expression in the lungs in response to inhalation of attenuated mengovirus.

<p>Mice received intranasal inoculations of 10⁶ PFU of attenuated mengovirus, vMC₀, or vehicle (n = 5 mice per group). Levels of (A) MUC5B and (B) MUC5AC mRNA in lungs on day 1 postinoculation were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels. * <i>P</i><0.01 (vMC₀ vs. vehicle).</p