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Interleukin-12p40 modulates human metapneumovirus-induced pulmonary disease in an acute mouse model of infection.

Chakraborty K, Zhou Z, Wakamatsu N, Guerrero-Plata A - PLoS ONE (2012)

Bottom Line: IL-12p40, a known important mediator in limiting lung inflammation, is induced by hMPV and its production is sustained after the resolution phase of infection suggesting that this cytokine plays a role in the immune response against hMPV.However, hMPV infection in these mice does not have an effect on viral replication.These results identify an important regulatory role of IL-12p40 in hMPV infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiological Sciences, Louisiana State University, Baton Rouge, Louisiana, United States of America.

ABSTRACT
The mechanisms that regulate the host immune response induced by human metapneumovirus (hMPV), a newly-recognized member of the Paramyxoviridae family, are largely unknown. Cytokines play an important role in modulating inflammatory responses during viral infections. IL-12p40, a known important mediator in limiting lung inflammation, is induced by hMPV and its production is sustained after the resolution phase of infection suggesting that this cytokine plays a role in the immune response against hMPV. In this work, we demonstrated that in mice deficient in IL-12p40, hMPV infection induced an exacerbated pulmonary inflammatory response and mucus production, altered cytokine response, and decreased lung function. However, hMPV infection in these mice does not have an effect on viral replication. These results identify an important regulatory role of IL-12p40 in hMPV infection.

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Mucus production after hMPV infection in IL-12p40 deficient mice.C57BL/6 and IL-12p40-KO mice were infected i.n. with 5×106 PFU of hMPV or mock-infected and lungs were harvested at day 7 after hMPV infection. (A) Lung tissue was fixed for slide preparation, PAS stained and tissue sections were digitalized. Representative stained lung tissue sections from the indicated treatment. Mucus-producing cells in the airways were identified by positive PAS staining. Scale bar = 100 µm. (B) Each area of airway epithelium was measured to calculate the Mucus index (as described in Materials and Methods). An average of 52 individual airways were measured per mouse. n = 3 mice/group. (C) RNA was isolated from the lungs of mock- and hMPV-infected mice (WT and KO) and transcribed into cDNA. Samples were assessed for expression of MUC5ac and MUC5b using quantitative RT-PCR by SYBR green. Each sample was normalized using GAPDH control and the bar graphs represent average fold increase to RNA obtained before infection. n = 3 mice/group. *P<0.05.
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pone-0037173-g004: Mucus production after hMPV infection in IL-12p40 deficient mice.C57BL/6 and IL-12p40-KO mice were infected i.n. with 5×106 PFU of hMPV or mock-infected and lungs were harvested at day 7 after hMPV infection. (A) Lung tissue was fixed for slide preparation, PAS stained and tissue sections were digitalized. Representative stained lung tissue sections from the indicated treatment. Mucus-producing cells in the airways were identified by positive PAS staining. Scale bar = 100 µm. (B) Each area of airway epithelium was measured to calculate the Mucus index (as described in Materials and Methods). An average of 52 individual airways were measured per mouse. n = 3 mice/group. (C) RNA was isolated from the lungs of mock- and hMPV-infected mice (WT and KO) and transcribed into cDNA. Samples were assessed for expression of MUC5ac and MUC5b using quantitative RT-PCR by SYBR green. Each sample was normalized using GAPDH control and the bar graphs represent average fold increase to RNA obtained before infection. n = 3 mice/group. *P<0.05.

Mentions: Increased production of mucus in the airways is usually correlated with exacerbated lung inflammation. In order to determine whether the absence of IL-12p40 would also alter the mucus production, the presence of mucin-expressing goblet cells in the airways as well as the expression of mucins in lung, were investigated. Lung sections from mice infected i.n. with 5×106 PFU of hMPV, were collected at day 7 after infection [same time point as in the determination of pulmonary inflammation, (Fig. 2)]. Periodic acid-Schiff (PAS) staining was used to detect mucin-expressing cells in the airway epithelium. In mock-infected WT and IL-12p40-KO mice, the presence of goblet cells was almost absent (Fig. 4A, upper panels). However, after hMPV infection, the number of goblet cells was increased in the airways of WT mice (Fig. 4A, left lower panel) and further exacerbated in those mice lacking the expression of IL-12p40 (Fig. 4A, right lower panel). Quantification of mucus producing cells in the airways was determined by assessing the mucus index (as described in Methods). As shown in Fig. 4B, the mucus index was increased after hMPV infection in WT mice. Furthermore, we observed an increased mucus index in the absence of IL-12p40 by more than two times when compared to WT mice infected with hMPV. In order to confirm the exacerbated mucus production as indicated by the mucus index, in a separate set of experiments, the expression of mucin 5 subtype AC (Muc5ac) and Muc5b was investigated. Muc5ac and Muc5b have been implicated as markers of goblet cell metaplasia in lung pathologies based upon expression studies in humans, in animal models, and in cell cultures [34], [35], [36]. Therefore, lung tissue was collected at day 7 after hMPV infection to quantify the expression of the mucins by quantitative RT-PCR. As shown in Fig. 4C, the expression of Muc5ac was modestly induced after hMPV infection in WT mice when compared to WT mock- infected mice. However, following hMPV infection, the absence of IL-12p40 exacerbated Muc5ac expression over 8 times when compared to WT mice. On the other hand, Muc5b was not induced after hMPV infection in WT mice, as similar expression was observed in mock-infected mice. However, it was increased in IL-12p40-KO mice after hMPV infection, although in less magnitude than Muc5ac. Thus, the absence of IL-12p40 resulted in significantly increased mucin expression in the airways of hMPV-infected mice.


Interleukin-12p40 modulates human metapneumovirus-induced pulmonary disease in an acute mouse model of infection.

Chakraborty K, Zhou Z, Wakamatsu N, Guerrero-Plata A - PLoS ONE (2012)

Mucus production after hMPV infection in IL-12p40 deficient mice.C57BL/6 and IL-12p40-KO mice were infected i.n. with 5×106 PFU of hMPV or mock-infected and lungs were harvested at day 7 after hMPV infection. (A) Lung tissue was fixed for slide preparation, PAS stained and tissue sections were digitalized. Representative stained lung tissue sections from the indicated treatment. Mucus-producing cells in the airways were identified by positive PAS staining. Scale bar = 100 µm. (B) Each area of airway epithelium was measured to calculate the Mucus index (as described in Materials and Methods). An average of 52 individual airways were measured per mouse. n = 3 mice/group. (C) RNA was isolated from the lungs of mock- and hMPV-infected mice (WT and KO) and transcribed into cDNA. Samples were assessed for expression of MUC5ac and MUC5b using quantitative RT-PCR by SYBR green. Each sample was normalized using GAPDH control and the bar graphs represent average fold increase to RNA obtained before infection. n = 3 mice/group. *P<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351396&req=5

pone-0037173-g004: Mucus production after hMPV infection in IL-12p40 deficient mice.C57BL/6 and IL-12p40-KO mice were infected i.n. with 5×106 PFU of hMPV or mock-infected and lungs were harvested at day 7 after hMPV infection. (A) Lung tissue was fixed for slide preparation, PAS stained and tissue sections were digitalized. Representative stained lung tissue sections from the indicated treatment. Mucus-producing cells in the airways were identified by positive PAS staining. Scale bar = 100 µm. (B) Each area of airway epithelium was measured to calculate the Mucus index (as described in Materials and Methods). An average of 52 individual airways were measured per mouse. n = 3 mice/group. (C) RNA was isolated from the lungs of mock- and hMPV-infected mice (WT and KO) and transcribed into cDNA. Samples were assessed for expression of MUC5ac and MUC5b using quantitative RT-PCR by SYBR green. Each sample was normalized using GAPDH control and the bar graphs represent average fold increase to RNA obtained before infection. n = 3 mice/group. *P<0.05.
Mentions: Increased production of mucus in the airways is usually correlated with exacerbated lung inflammation. In order to determine whether the absence of IL-12p40 would also alter the mucus production, the presence of mucin-expressing goblet cells in the airways as well as the expression of mucins in lung, were investigated. Lung sections from mice infected i.n. with 5×106 PFU of hMPV, were collected at day 7 after infection [same time point as in the determination of pulmonary inflammation, (Fig. 2)]. Periodic acid-Schiff (PAS) staining was used to detect mucin-expressing cells in the airway epithelium. In mock-infected WT and IL-12p40-KO mice, the presence of goblet cells was almost absent (Fig. 4A, upper panels). However, after hMPV infection, the number of goblet cells was increased in the airways of WT mice (Fig. 4A, left lower panel) and further exacerbated in those mice lacking the expression of IL-12p40 (Fig. 4A, right lower panel). Quantification of mucus producing cells in the airways was determined by assessing the mucus index (as described in Methods). As shown in Fig. 4B, the mucus index was increased after hMPV infection in WT mice. Furthermore, we observed an increased mucus index in the absence of IL-12p40 by more than two times when compared to WT mice infected with hMPV. In order to confirm the exacerbated mucus production as indicated by the mucus index, in a separate set of experiments, the expression of mucin 5 subtype AC (Muc5ac) and Muc5b was investigated. Muc5ac and Muc5b have been implicated as markers of goblet cell metaplasia in lung pathologies based upon expression studies in humans, in animal models, and in cell cultures [34], [35], [36]. Therefore, lung tissue was collected at day 7 after hMPV infection to quantify the expression of the mucins by quantitative RT-PCR. As shown in Fig. 4C, the expression of Muc5ac was modestly induced after hMPV infection in WT mice when compared to WT mock- infected mice. However, following hMPV infection, the absence of IL-12p40 exacerbated Muc5ac expression over 8 times when compared to WT mice. On the other hand, Muc5b was not induced after hMPV infection in WT mice, as similar expression was observed in mock-infected mice. However, it was increased in IL-12p40-KO mice after hMPV infection, although in less magnitude than Muc5ac. Thus, the absence of IL-12p40 resulted in significantly increased mucin expression in the airways of hMPV-infected mice.

Bottom Line: IL-12p40, a known important mediator in limiting lung inflammation, is induced by hMPV and its production is sustained after the resolution phase of infection suggesting that this cytokine plays a role in the immune response against hMPV.However, hMPV infection in these mice does not have an effect on viral replication.These results identify an important regulatory role of IL-12p40 in hMPV infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiological Sciences, Louisiana State University, Baton Rouge, Louisiana, United States of America.

ABSTRACT
The mechanisms that regulate the host immune response induced by human metapneumovirus (hMPV), a newly-recognized member of the Paramyxoviridae family, are largely unknown. Cytokines play an important role in modulating inflammatory responses during viral infections. IL-12p40, a known important mediator in limiting lung inflammation, is induced by hMPV and its production is sustained after the resolution phase of infection suggesting that this cytokine plays a role in the immune response against hMPV. In this work, we demonstrated that in mice deficient in IL-12p40, hMPV infection induced an exacerbated pulmonary inflammatory response and mucus production, altered cytokine response, and decreased lung function. However, hMPV infection in these mice does not have an effect on viral replication. These results identify an important regulatory role of IL-12p40 in hMPV infection.

Show MeSH
Related in: MedlinePlus