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.

Prostaglandin E₂ Production and T Cell Function in Mouse Adenovirus Type 1 Infection following Allogeneic Bone Marrow Transplantation

<div><p>Adenovirus infections are important complications of bone marrow transplantation (BMT). We demonstrate delayed clearance of mouse adenovirus type 1 (MAV-1) from lungs of mice following allogeneic BMT. Virus-induced prostaglandin E₂ (PGE₂) production was greater in BMT mice than in untransplanted controls, but BMT using PGE₂-deficient donors or recipients failed to improve viral clearance, and treatment of untransplanted mice with the PGE₂ analog misoprostol did not affect virus clearance. Lymphocyte recruitment to the lungs was not significantly affected by BMT. Intracellular cytokine staining of lung lymphocytes demonstrated impaired production of INF-γ and granzyme B by cells from BMT mice, and production of IFN-γ, IL-2, IL-4, and IL-17 following ex vivo stimulation was impaired in lymphocytes obtained from lungs of BMT mice. Viral clearance was not delayed in untransplanted INF-γ-deficient mice, suggesting that delayed viral clearance in BMT mice was not a direct consequence of impaired IFN-γ production. However, lung viral loads were higher in untransplanted CD8-deficient mice than in controls, suggesting that delayed MAV-1 clearance in BMT mice is due to defective CD8 T cell function. We did not detect significant induction of IFN-β expression in lungs of BMT mice or untransplanted controls, and viral clearance was not delayed in untransplanted type I IFN-unresponsive mice. We conclude that PGE₂ overproduction in BMT mice is not directly responsible for delayed viral clearance. PGE₂-independent effects on CD8 T cell function likely contribute to the inability of BMT mice to clear MAV-1 from the lungs.</p></div

Type I IFN production in BMT mice.

<p>A) BMT mice (BALB/c donor, C57BL/6 recipient) and untransplanted BALB/c and C57BL/6 controls were infected i.n. with MAV-1, and lungs were harvested at the indicated time points. RT-qPCR was used to quantify IFN-β mRNA levels. Combined data from n = 7–8 mice per group (n = 3 per group at day 0) are presented as means ± S.E.M. Statistical comparisons were made using two-way ANOVA followed by Bonferroni’s multiple comparison tests. B) Untransplanted C57BL/6 and IFNAR^-/- mice were infected i.n. with MAV-1. DNA was extracted from lungs harvested at the indicated time points. qPCR was used to quantify MAV-1 genome copies in lung DNA. DNA viral loads are expressed as copies of MAV-1 genome per 100 ng of input DNA. Individual circles represent values for individual mice and horizontal bars represent means for each group. Statistical comparisons were made using two-way ANOVA followed by Bonferroni’s multiple comparison tests.</p

PGE₂ is overproduced in BMT mice.

<p>BMT mice (BALB/c donor, C57BL/6 recipient) and untransplanted BALB/c and C57BL/6 controls were infected i.n. with MAV-1 or mock infected with conditioned media. ELISA was used to quantify PGE₂ concentrations in BALF at the indicated time points. Combined data from n = 3–4 mice per group are presented as means ± S.E.M. Statistical comparisons were made using two-way ANOVA followed by Bonferroni’s multiple comparison tests. ***<i>P</i><0.001.</p

Impaired IFN-γ production in BMT mice.

<p>A) BMT mice (BALB/c donor, C57BL/6 recipient) and untransplanted BALB/c controls were infected i.n. with MAV-1 or mock infected with conditioned media, and lung leukocytes were isolated at 7 dpi. Lung leukocytes were stimulated overnight with anti-CD3 antibody and ELISA was used to measure IFN-γ concentrations in supernatants. Combined data from n = 3–8 mice per group are presented as means ± S.E.M. Statistical comparisons were made using one-way ANOVA followed by Tukey’s multiple comparison tests. **<i>P</i><0.01 and *<i>P</i><0.05 comparing mock to MAV-1. ††<i>P</i><0.01 and †<i>P</i><0.05 comparing BALB/c to BMT mice. B) A) IFN-γ^+/+ and IFN-γ^-/- mice were infected i.n. with MAV-1. DNA was extracted from lungs harvested at 14 dpi. qPCR was used to quantify MAV-1 genome copies in lung DNA. DNA viral loads are expressed as copies of MAV-1 genome per 100 ng of input DNA. Individual circles represent values for individual mice and horizontal bars represent means for each group. Statistical comparisons were made using the Mann-Whitney test.</p

T cells in lungs of BMT mice.

<p>BMT mice (BALB/c donor, C57BL/6 recipient) and untransplanted BALB/c and C57BL/6 controls were infected i.n. with MAV-1 or mock infected with conditioned media. Lung leukocytes isolated at 7 dpi were stimulated with PMA/ionomycin and stained to quantify the number of A) TCRβ⁺CD4⁺ T cells, B) TCRβ⁺CD8⁺ T cells, C) IFN-γ⁺ T cells, and D) GzmB⁺ T cells per lung. Combined data from n = 3–4 mice per group are presented as means ± S.E.M. Statistical comparisons were made using one-way ANOVA followed by Bonferroni’s multiple comparison tests. **<i>P</i><0.01 and *<i>P</i><0.05 comparing mock to MAV-1. ††<i>P</i><0.01 and †<i>P</i><0.05 comparing BALB/c to BMT mice.</p

Primers and probes used for real-time PCR analysis.

<p>Primers and probes used for real-time PCR analysis.</p

Virus-induced pulmonary inflammation in BMT mice.

<p>Allogeneic BMT mice (BALB/c donor, C57BL/6 recipient) and untransplanted BALB/c and C57BL/6 mice were infected i.n. with MAV-1 or mock infected at 5 weeks post BMT. Lungs were harvested and hematoxylin-and-eosin-stained sections were prepared from paraffin-embedded specimens. Representative images are shown from mice before infection (A-C) and from infected mice at the indicated time points (D-L). Scale bars, 100 μm. M) Pathology index scores were generated to quantify cellular inflammation in the lungs of mock-infected and infected mice. Data from 2 to 3 mock-infected mice and 3 to 6 infected mice per group are presented as means and standard errors of the means at each time point. Statistical comparisons were made using two-way ANOVA followed by Bonferroni’s multiple comparison tests. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001.</p

Prostaglandin E2 induction during mouse adenovirus type 1 respiratory infection regulates inflammatory mediator generation but does not affect viral pathogenesis.

Respiratory viruses cause substantial disease and are a significant healthcare burden. Virus-induced inflammation can be detrimental to the host, causing symptoms during acute infection and leading to damage that contributes to long-term residual lung disease. Prostaglandin E2 (PGE2) is a lipid mediator that is increased in response to many viral infections, and inhibition of PGE2 production during respiratory viral infection often leads to a decreased inflammatory response. We tested the hypothesis that PGE2 promotes inflammatory responses to mouse adenovirus type 1 (MAV-1) respiratory infection. Acute MAV-1 infection increased COX-2 expression and PGE2 production in wild type mice. Deficiency of the E prostanoid 2 receptor had no apparent effect on MAV-1 pathogenesis. Virus-induced induction of PGE2, IFN-γ, CXCL1, and CCL5 was reduced in mice deficient in microsomal PGE synthase-1 (mPGES-1(-/-) mice). However, there were no differences between mPGES-1(+/+) and mPGES-1(-/-) mice in viral replication, recruitment of leukocytes to airways or lung inflammation. Infection of both mPGES‑1(+/+) and mPGES-1(-/-) mice led to protection against reinfection. Thus, while PGE2 promotes the expression of a variety of cytokines in response to acute MAV-1 infection, PGE2 synthesis does not appear to be essential for generating pulmonary immunity