28 research outputs found
Pathophysiologic Effects of Influenza Infection on the Murine Lung and Evaluation of Novel Therapeutic Targets
Respiratory syncytial virus reverses airway hyperresponsiveness to methacholine in ovalbumin-sensitized mice.
Each year, approximately 20% of asthmatics in the United States experience acute symptom exacerbations, which commonly result from pulmonary viral infections. The majority of asthma exacerbations in very young children follow infection with respiratory syncytial virus (RSV). However, pathogenic mechanisms underlying induction of asthma exacerbations by RSV are not well understood. We therefore investigated the effect of post-sensitization RSV infection on lung function in ovalbumin (OVA)-sensitized BALB/c mice as a model of RSV asthma exacerbations. OVA sensitization of uninfected female BALB/c mice increased bronchoalveolar lavage fluid (BALF) eosinophil levels and induced airway hyperresponsiveness to the muscarinic agonist methacholine, as measured by the forced-oscillation technique. In contrast, intranasal infection with replication-competent RSV strain A2 for 2-8 days reduced BALF eosinophil counts and reversed airway hyperresponsiveness in a pertussis toxin-sensitive manner. BALF levels of the chemokine keratinocyte cytokine (KC; a murine homolog of interleukin-8) were elevated in OVA-sensitized, RSV-infected mice and reversal of methacholine hyperresponsiveness in these animals was rapidly inhibited by KC neutralization. Hyporesponsiveness could be induced in OVA-sensitized, uninfected mice by recombinant KC or the GΞ±i agonist melittin. These data suggest that respiratory syncytial virus induces KC-mediated activation of GΞ±i, resulting in cross-inhibition of GΞ±q-mediated M(3)-muscarinic receptor signaling and reversal of airway hyperresponsiveness. As in unsensitized mice, KC therefore appears to play a significant role in induction of airway dysfunction by respiratory syncytial virus. Hence, interleukin-8 may be a promising therapeutic target to normalize lung function in both asthmatics and non-asthmatics with bronchiolitis. However, the OVA-sensitized, RSV-infected mouse may not be an appropriate model for investigating the pathogenesis of viral asthma exacerbations
Keratinocyte cytokine released in response to RSV infection reverses hyperresponsiveness to methacholine in OVA-sensitized mice.
<p>(<b>A</b>) Bronchoalveolar lavage fluid keratinocyte cytokine (KC; ng/ml) levels in unsensitized, uninfected mice (UNSENS/UNINF; <i>n</i>β=β5), OVA-sensitized, uninfected mice (OVA/UNINF; <i>n</i>β=β11), OVA-sensitized, uninfected mice treated with 50 Β΅g/ml heat-inactivated recombinant murine KC (OVA/UNINF + HI-KC; <i>n</i>β=β5), OVA-sensitized, uninfected mice treated with 50 Β΅g/ml recombinant murine KC (OVA/UNINF + KC; <i>n</i>β=β7), OVA-sensitized mice infected with RSV (10<sup>6</sup> pfu/mouse) for 2 days (OVA/DAY 2; <i>n</i>β=β6), OVA-sensitized mice βinfectedβ with UV-inactivated RSV for 2 days (OVA/UVx DAY 2; <i>n</i>β=β4), and OVA-sensitized mice infected with RSV for 8 days (OVA/DAY 8; <i>n</i>β=β6). *<i>P</i><0.05, ***<i>P</i><0.0005, <i>vs</i>. UNSENS/UNINF mice. (<b>B</b>) Bronchoconstrictive responses to increasing doses of nebulized methacholine (MCH) in OVA-sensitized, RSV-infected mice following nebulization of normal rat IgG (50 Β΅g/ml; <i>n</i>β=β5), KC-neutralizing monoclonal antibody (ANTI-KC, 50 Β΅g/ml; <i>n</i>β=β5), pretreatment with pertussis toxin and IgG (PTX + IgG; <i>n</i>β=β6), or pretreatment with pertussis toxin and KC neutralizing antibody (PTX + ANTI-KC; <i>n</i>β=β8). ***MCH dose-response curve differs significantly (<i>P</i><0.0005) from UNSENS/UNINF mice (<i>n</i>β=β16).</p
Keratinocyte cytokine exposure and GΞ±i activation are both sufficient to reverse methacholine hyperresponsiveness in OVA-sensitized, uninfected mice.
<p>Bronchoconstrictive responses to increasing doses of nebulized methacholine (MCH) following nebulization of: (<b>A</b>) heat-inactivated recombinant murine keratinocyte cytokine (HI-rmKC, 50 Β΅g/ml; <i>n</i>β=β6) or recombinant murine keratinocyte cytokine (rmKC, 50 Β΅g/ml; <i>n</i>β=β7); and (<b>B</b>) Melittin (100 Β΅M; <i>n</i>β=β7). ***MCH dose-response curve differs significantly (<i>P</i><0.0005) from OVA/UNINF mice (<i>n</i>β=β8).</p
Schematic timeline of the OVA sensitization/challenge and RSV infection protocol.
<p>Mice were sensitized by intraperitoneal (i.p.) injection of OVA in alum at days β28 and β14. From β7 to β3 days, mice were challenged daily by intranasal (i.n.) OVA instillation. Animals were infected with RSV 3 days after the last OVA challenge (day 0). Airway responsiveness to methacholine (MCH) was measured at 2β8 days post-infection (d.p.i.).</p
RSV infection reverses hyperresponsiveness to methacholine in OVA-sensitized mice via a pertussis toxin-sensitive pathway.
<p>Bronchoconstrictive response to increasing doses of nebulized methacholine (MCH) following pretreatment with saline (100 Β΅l i.p.; <i>n</i>β=β4) or pertussis toxin (PTX, 100 Β΅g/kg in 100 Β΅l saline i.p.; <i>n</i>β=β9). ***MCH dose-response curve differs significantly (<i>P</i><0.0005) from OVA/DAY 2 mice (OVA-sensitized mice infected with 10<sup>6</sup> pfu/mouse RSV A2 for 2 days; <i>n</i>β=β16).</p
RSV infection reduces bronchoalveolar lavage fluid cell counts in OVA-sensitized mice.
<p>Effect of OVA sensitization on (<b>A</b>) Alveolar macrophage (AM), eosinophil (EO) and small lymphocyte (SL) counts in uninfected mice (OVA/UNINF; <i>n</i>β=β8) and unsensitized, uninfected controls (UNSENS/UNINF; <i>n</i>β=β8); (<b>B</b>) Total cell counts in OVA-sensitized mice after mock infection for 2 days (M2; <i>n</i>β=β5), 4 days (M4; <i>n</i>β=β4), or 8 days (M8; <i>n</i>β=β8), and infection with RSV (10<sup>6</sup> pfu/mouse) for 2 days (<i>n</i>β=β16), 4 days (<i>n</i>β=β10), or 8 days (<i>n</i>β=β6); (<b>C</b>) AM, EO, and SL counts after mock infection for 2, 4, or 8 days and infection with RSV for 2β8 days; and (<b>D</b>) Neutrophil (PMN) counts after infection with RSV for 2β8 days. No PMNs were detected in bronchoalveolar lavage fluid from uninfected or mock-infected mice at any timepoint (not shown). *<i>P</i><0.05, **<i>P</i><0.005, ***<i>P</i><0.001. N.D.: None detected.</p