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Combined exposure to cigarette smoke and nontypeable Haemophilus influenzae drives development of a COPD phenotype in mice.

Ganesan S, Comstock AT, Kinker B, Mancuso P, Beck JM, Sajjan US - Respir. Res. (2014)

Bottom Line: CS/HK-NTHi-exposed mice also expressed increased levels of mucin genes and cytokines compared to mice in other groups.CS/HK-NTHi-exposed mice infected with RV demonstrated increased viral persistence, sustained neutrophilia, and further increments in mucin gene and chemokine expression compared to other groups.These findings indicate that in addition to CS, bacteria may also contribute to development of COPD, particularly changes in airways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics and Communicable Diseases, University of Michigan, 1150 W, Medical Center Dr,, Ann Arbor, MI 48109-5688, USA. usajjan@umich.edu.

ABSTRACT

Background: Cigarette smoke (CS) is the major etiologic factor of chronic obstructive pulmonary disease (COPD). CS-exposed mice develop emphysema and mild pulmonary inflammation but no airway obstruction, which is also a prominent feature of COPD. Therefore, CS may interact with other factors, particularly respiratory infections, in the pathogenesis of airway remodeling in COPD.

Methods: C57BL/6 mice were exposed to CS for 2 h a day, 5 days a week for 8 weeks. Mice were also exposed to heat-killed non-typeable H. influenzae (HK-NTHi) on days 7 and 21. One day after the last exposure to CS, mice were sacrificed and lung inflammation and mechanics, emphysematous changes, and goblet cell metaplasia were assessed. Mice exposed to CS or HK-NTHi alone or room air served as controls. To determine the susceptibility to viral infections, we also challenged these mice with rhinovirus (RV).

Results: Unlike mice exposed to CS or HK-NTHi alone, animals exposed to CS/HK-NTHi developed emphysema, lung inflammation and goblet cell metaplasia in both large and small airways. CS/HK-NTHi-exposed mice also expressed increased levels of mucin genes and cytokines compared to mice in other groups. CS/HK-NTHi-exposed mice infected with RV demonstrated increased viral persistence, sustained neutrophilia, and further increments in mucin gene and chemokine expression compared to other groups.

Conclusions: These findings indicate that in addition to CS, bacteria may also contribute to development of COPD, particularly changes in airways. Mice exposed to CS/HK-NTHi are also more susceptible to subsequent viral infection than mice exposed to either CS or HK-NTHi alone.

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Assessment of susceptibility of RA, CS, HK-NTHi or CS/HK-NTHi-exposed mice to RV infection. RA, CS, HK-NTHi or CS/HK-NTHi-exposed mice were either infected with RV or equal volume of sham and mice were sacrificed 4 days post-infection. (A) Total RNA was isolated from whole lungs and viral RNA (vRNA) copy number was determined by quantitative qPCR and expressed vRNA copy number per 10 μg of total RNA. (B) Lung homogenates were used to determine infectious viral load by assessing number of plaque forming units (PFU) per lung. (C and D). Cells from bronchoalveolar lavage were used to determine number of neutrophils and lymphocytes and expressed as cells/ml of BAL. Data in A to D represent median with range calculated from 5 to 6 mice per group (* different from RA-exposed mice, p ≤ 0.05, ANOVA on ranks; # different from CS-exposed mice, p ≤ 0.05, ANOVA on ranks; † different from HK-NTHi-exposed mice, p ≤ 0.05, ANOVA on ranks). (E and F) Supernatants from bronchoalveolar lavage were used to determine KC and MIP2 levels. (G) Total lung RNA was reverse transcribed and subjected to qPCR to determine mRNA levels of IP-10 and expressed as fold change over house-keeping gene, G3PDH. Data in E to G represents mean ± SD calculated from 5–6 mice per group (* different from respective sham controls, p ≤ 0.05, ANOVA; # different from CS-exposed RV-infected mice, p ≤ 0.05, ANOVA; † different from HK-NTHi-exposed RV-infected mice, p ≤ 0.05, ANOVA).
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Figure 6: Assessment of susceptibility of RA, CS, HK-NTHi or CS/HK-NTHi-exposed mice to RV infection. RA, CS, HK-NTHi or CS/HK-NTHi-exposed mice were either infected with RV or equal volume of sham and mice were sacrificed 4 days post-infection. (A) Total RNA was isolated from whole lungs and viral RNA (vRNA) copy number was determined by quantitative qPCR and expressed vRNA copy number per 10 μg of total RNA. (B) Lung homogenates were used to determine infectious viral load by assessing number of plaque forming units (PFU) per lung. (C and D). Cells from bronchoalveolar lavage were used to determine number of neutrophils and lymphocytes and expressed as cells/ml of BAL. Data in A to D represent median with range calculated from 5 to 6 mice per group (* different from RA-exposed mice, p ≤ 0.05, ANOVA on ranks; # different from CS-exposed mice, p ≤ 0.05, ANOVA on ranks; † different from HK-NTHi-exposed mice, p ≤ 0.05, ANOVA on ranks). (E and F) Supernatants from bronchoalveolar lavage were used to determine KC and MIP2 levels. (G) Total lung RNA was reverse transcribed and subjected to qPCR to determine mRNA levels of IP-10 and expressed as fold change over house-keeping gene, G3PDH. Data in E to G represents mean ± SD calculated from 5–6 mice per group (* different from respective sham controls, p ≤ 0.05, ANOVA; # different from CS-exposed RV-infected mice, p ≤ 0.05, ANOVA; † different from HK-NTHi-exposed RV-infected mice, p ≤ 0.05, ANOVA).

Mentions: At the end of 8 weeks of exposure to CS, mice were infected with RV or sham, sacrificed after 4 days and examined for viral RNA, chemokine expression, neutrophil infiltration and mucin gene expression. CS/HK-NTHi-exposed mice infected with RV showed one log higher vRNA than similarly infected animals in other groups (p = 0.024) (Figure 6A). CS/HK-NTHi mice also showed higher infectious viral load than mice in other groups (p = 0.007), but it was only 1 to 2 × 102 PFU/lung (Figure 6B). Despite having low levels of infectious viral load, the CS/HK-NTHi group showed remarkably higher neutrophil (p = 0.001) and lymphocyte (p = 0.009) infiltration compared to the sham infected group (Figure 6C and6D). Consistent with the cellular infiltration, CS/HK-NTHi mice infected with RV also showed persistent increases in KC (p = <0.001), MIP-2 (p = 0.001) and IP-10 (0.022), compared to sham infected mice (Figure 6E-6G). RV infected mice in other groups showed very small increases in infiltrated neutorphils, lymphocytes and cytokine levels over their respective sham-infected controls (p values ranged between 0.039 to 0.05 in these animals).


Combined exposure to cigarette smoke and nontypeable Haemophilus influenzae drives development of a COPD phenotype in mice.

Ganesan S, Comstock AT, Kinker B, Mancuso P, Beck JM, Sajjan US - Respir. Res. (2014)

Assessment of susceptibility of RA, CS, HK-NTHi or CS/HK-NTHi-exposed mice to RV infection. RA, CS, HK-NTHi or CS/HK-NTHi-exposed mice were either infected with RV or equal volume of sham and mice were sacrificed 4 days post-infection. (A) Total RNA was isolated from whole lungs and viral RNA (vRNA) copy number was determined by quantitative qPCR and expressed vRNA copy number per 10 μg of total RNA. (B) Lung homogenates were used to determine infectious viral load by assessing number of plaque forming units (PFU) per lung. (C and D). Cells from bronchoalveolar lavage were used to determine number of neutrophils and lymphocytes and expressed as cells/ml of BAL. Data in A to D represent median with range calculated from 5 to 6 mice per group (* different from RA-exposed mice, p ≤ 0.05, ANOVA on ranks; # different from CS-exposed mice, p ≤ 0.05, ANOVA on ranks; † different from HK-NTHi-exposed mice, p ≤ 0.05, ANOVA on ranks). (E and F) Supernatants from bronchoalveolar lavage were used to determine KC and MIP2 levels. (G) Total lung RNA was reverse transcribed and subjected to qPCR to determine mRNA levels of IP-10 and expressed as fold change over house-keeping gene, G3PDH. Data in E to G represents mean ± SD calculated from 5–6 mice per group (* different from respective sham controls, p ≤ 0.05, ANOVA; # different from CS-exposed RV-infected mice, p ≤ 0.05, ANOVA; † different from HK-NTHi-exposed RV-infected mice, p ≤ 0.05, ANOVA).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Assessment of susceptibility of RA, CS, HK-NTHi or CS/HK-NTHi-exposed mice to RV infection. RA, CS, HK-NTHi or CS/HK-NTHi-exposed mice were either infected with RV or equal volume of sham and mice were sacrificed 4 days post-infection. (A) Total RNA was isolated from whole lungs and viral RNA (vRNA) copy number was determined by quantitative qPCR and expressed vRNA copy number per 10 μg of total RNA. (B) Lung homogenates were used to determine infectious viral load by assessing number of plaque forming units (PFU) per lung. (C and D). Cells from bronchoalveolar lavage were used to determine number of neutrophils and lymphocytes and expressed as cells/ml of BAL. Data in A to D represent median with range calculated from 5 to 6 mice per group (* different from RA-exposed mice, p ≤ 0.05, ANOVA on ranks; # different from CS-exposed mice, p ≤ 0.05, ANOVA on ranks; † different from HK-NTHi-exposed mice, p ≤ 0.05, ANOVA on ranks). (E and F) Supernatants from bronchoalveolar lavage were used to determine KC and MIP2 levels. (G) Total lung RNA was reverse transcribed and subjected to qPCR to determine mRNA levels of IP-10 and expressed as fold change over house-keeping gene, G3PDH. Data in E to G represents mean ± SD calculated from 5–6 mice per group (* different from respective sham controls, p ≤ 0.05, ANOVA; # different from CS-exposed RV-infected mice, p ≤ 0.05, ANOVA; † different from HK-NTHi-exposed RV-infected mice, p ≤ 0.05, ANOVA).
Mentions: At the end of 8 weeks of exposure to CS, mice were infected with RV or sham, sacrificed after 4 days and examined for viral RNA, chemokine expression, neutrophil infiltration and mucin gene expression. CS/HK-NTHi-exposed mice infected with RV showed one log higher vRNA than similarly infected animals in other groups (p = 0.024) (Figure 6A). CS/HK-NTHi mice also showed higher infectious viral load than mice in other groups (p = 0.007), but it was only 1 to 2 × 102 PFU/lung (Figure 6B). Despite having low levels of infectious viral load, the CS/HK-NTHi group showed remarkably higher neutrophil (p = 0.001) and lymphocyte (p = 0.009) infiltration compared to the sham infected group (Figure 6C and6D). Consistent with the cellular infiltration, CS/HK-NTHi mice infected with RV also showed persistent increases in KC (p = <0.001), MIP-2 (p = 0.001) and IP-10 (0.022), compared to sham infected mice (Figure 6E-6G). RV infected mice in other groups showed very small increases in infiltrated neutorphils, lymphocytes and cytokine levels over their respective sham-infected controls (p values ranged between 0.039 to 0.05 in these animals).

Bottom Line: CS/HK-NTHi-exposed mice also expressed increased levels of mucin genes and cytokines compared to mice in other groups.CS/HK-NTHi-exposed mice infected with RV demonstrated increased viral persistence, sustained neutrophilia, and further increments in mucin gene and chemokine expression compared to other groups.These findings indicate that in addition to CS, bacteria may also contribute to development of COPD, particularly changes in airways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics and Communicable Diseases, University of Michigan, 1150 W, Medical Center Dr,, Ann Arbor, MI 48109-5688, USA. usajjan@umich.edu.

ABSTRACT

Background: Cigarette smoke (CS) is the major etiologic factor of chronic obstructive pulmonary disease (COPD). CS-exposed mice develop emphysema and mild pulmonary inflammation but no airway obstruction, which is also a prominent feature of COPD. Therefore, CS may interact with other factors, particularly respiratory infections, in the pathogenesis of airway remodeling in COPD.

Methods: C57BL/6 mice were exposed to CS for 2 h a day, 5 days a week for 8 weeks. Mice were also exposed to heat-killed non-typeable H. influenzae (HK-NTHi) on days 7 and 21. One day after the last exposure to CS, mice were sacrificed and lung inflammation and mechanics, emphysematous changes, and goblet cell metaplasia were assessed. Mice exposed to CS or HK-NTHi alone or room air served as controls. To determine the susceptibility to viral infections, we also challenged these mice with rhinovirus (RV).

Results: Unlike mice exposed to CS or HK-NTHi alone, animals exposed to CS/HK-NTHi developed emphysema, lung inflammation and goblet cell metaplasia in both large and small airways. CS/HK-NTHi-exposed mice also expressed increased levels of mucin genes and cytokines compared to mice in other groups. CS/HK-NTHi-exposed mice infected with RV demonstrated increased viral persistence, sustained neutrophilia, and further increments in mucin gene and chemokine expression compared to other groups.

Conclusions: These findings indicate that in addition to CS, bacteria may also contribute to development of COPD, particularly changes in airways. Mice exposed to CS/HK-NTHi are also more susceptible to subsequent viral infection than mice exposed to either CS or HK-NTHi alone.

Show MeSH
Related in: MedlinePlus