IL-13 overexpression in mouse lungs triggers systemic genotoxicity in peripheral blood

Abstract

Asthma is a common heterogeneous disease with both genetic and environmental factors that affects millions of individuals worldwide. Activated type 2 helper T cells secrete a panel of cytokines, including IL-13, a central immune regulator of many of the hallmark type 2 disease characteristics found in asthma. IL-13 has been directly implicated as a potent stimulator of asthma induced airway remodeling. Although IL-13 is known to play a major role in the development and persistence of asthma, the complex combination of environmental and genetic origin of the disease obfuscate the solitary role of IL-13 in the disease. We therefore, used a genetically modified mouse model which conditionally overexpresses IL-13 in the lungs to study the independent role of IL-13 in the progression of asthma. Our results demonstrate IL-13 is associated with a systemic induction of genotoxic parameters such as oxidative DNA damage, single and double DNA strand breaks, micronucleus formation, and protein nitration. Furthermore we show that inflammation induced genotoxicity found in asthma extends beyond the primary site of the lung to circulating leukocytes and erythroblasts in the bone marrow eliciting systemic effects driven by IL-13 over-expression.

Fig.1

IgE concentration assessed via sandwhich ELISA. **indicates p<, 0.007 n= 5 in both IL-13 and WT animals. Assay was performed with triplicate blood samples from each WT (n=5) and Il-13 (n=5) mouse. (color)

Fig. 2. IL-13 over-expression induces lung inflammation in asthmatic mice

Representative lung histology Hematoxylin & Eosin (H&E) staining at indicated magnifications. (A) 10x image of Wild type (WT) lung and Interleukin 13 over expressed mice (B), both at one month old. Arrows in (B) 10x image indicate formation of granuloma metafoci surrounding eosinophilic crystals. 40x image of WT (C) and (D) 40x image of IL-13 mice. Arrows in (D) indicate eosinophil migration, goblet cell metaplasia, and eosinophilic crystal formation in bronchial lumen. n=9 for WT and n=10 for IL-13 mice. (color)

Fig.3. Inflammatory cell composition of bronchial alveolar lavage fluid (BALF)

Differential cell analysis were determined by light microscopic evaluation n=9 for WT, and n=10 for IL-13 animals,* indicates p<0.05,*** indicates p<0.0004 respectively analysis were conducted using two tailed Student's unpaired T-test with Mann-Whitney determination.

Fig.4. Assessment of cytokine panel in lung mRNA measured by quantitative real-time PCR

Mean expression divided by Gapdh, the internal control gene. * indicates p<0.01, ** indicates p<0.001, analysis were conducted using two tailed Student's unpaired T-test. n=9 for WT animals and n=10 for IL-13 animals.

Fig.5. Staining of markers of genotoxicity and oxidative protein damage in lung tissue as measured by immunohistochemistry

Markers of double stranded breaks (A-J), reactive oxygen species (B-K), and inflammation (C-L) induced genotoxicity were stained in WT and IL-13 mice. Lung tissue in IL-13 mice (G-L) exhibited increased staining in all genotoxic parameters in comparison to WT mice (A-F). n=9 for WT animals and n=10 for IL-13 animals. (color)

Fig.6. Persistent genotoxicity measured via inflammation induced 8-oxoguanine and double stranded breaks measured via γH2AX in peripheral blood

A.) Percent positive cells for 8-oxoguanine induction in white blood cells. Presence of 8-oxoguanine was confirmed by immunofluorescence. Positive cells stain brightly green compared to no immunofluorescent staining for negative cells. White bars indicate Wild type (WT) animals and black bars indicate IL-13 animals. Data represent mean ± SEM. Statistical analyses were done using ANOVA testing and Tukey's post hoc analysis. n=5 in all groups. ** indicates p<0.001.B Assessment of double strand breaks measured via γH2AX assay, were counted per cell using fluorescent microscopy before doxycycline administration at Day 0 and after doxycycline administration at days 3,9,12,16,18 and day 21 using a linear mixed model to determine genotoxic accumulation over time. * indicates p<0.02, ** indicates p<0.002 n=5 for WT and IL-13 animals. (color)

Fig.7. IL-13 over-expression induced single stranded breaks and micronucleated cells in peripheral blood

A.) Assessment of single strand breaks were measured via comet assay before doxycycline administration at Day 0 and after doxycycline administration at days 6. At least 100 olive tail moments were counted via fluorescent microscopy and assessed using CASP software. White bars indicate Wild type (WT) animals and black bars indicate IL-13 animals. Data represent mean ± SEM. Statistical analyses were done using ANOVA testing and Tukey's post hoc analysis. * indicates p<0.05 n=5 for WT and IL-13 animals. B.) Number of micronucleated cells per 4000 normorchromatic erythrocytes. Presence of micronuclei were confirmed by light microscope at 100X. White bars indicate Wild type (WT) animals and black bars indicate IL-13 animals. Data represent mean ± SEM. Statistical analyses were done using ANOVA testing and Tukey's post hoc analysis. n=9 for WT and n=10 for IL-13.* indicates p<0.05.