Role of airway epithelial cells for GC-therapy of asthma in inducible GR SPCcreERT2

mice

The possibility to treat mice lacking the GR in T and B, DCs and myeloid cells, the experiments with mast cell deficient mice and bone marrow chimeras suggested that non-immune cells (like epithelial cells of the lung) play an important role in the suppression of inflammatory reactions by GCs during asthma therapy.

To investigate the role of the epithelial cells as targets of GC-therapy inducible GR^SPCcreERT2 mice were used in this work. Because of the efficiently diminished expression of the GR in AT2 epithelial cells and the normal lung physiology of these mutant mice they could be used to investigate this issue without limitations. While the pathogenesis of the asthmatic reaction was similar compared to wild type animals, the analysis of the therapeutic efficiency showed that the treatability of the mutant mice was impaired. The cellular infiltration of the mutant lungs was only partially reduced after GC therapy. The analysis of the BAL cell composition

demonstrated almost the same level of eosinophilia in samples of GRSPCcreERT2

with or without treatment. Based on these data, the AT2 cells play a crucial role during asthma therapy with GCs.

Gene expression analysis of sorted AT2 cells was done to identify regulatory mechanisms of GCs in the treatment of asthma.

Figure 66: Effects of GC therapy on gene expression in AT2 cells isolated from Dexamethasone treated AAI mice of wild type and GR^SPCcreERT2 genotype. The data represent an overview of the results obtained by RT-qPCR analysis.

Current publications reported an important role of pulmonary epithelial cells during lung infection and a critical role of epithelial cells in the initiation of an asthmatic reaction. An important role in this process was assigned to the TLR-mediated pathway. After contact with microbes or microbial products, TLR-mediated signals result in the production of TNF-α, IL-1β, TSLP and IL-33 by epithelial cells. These cytokines stimulate the expression of adhesions molecules on vascular endothelial cells, activate DCs and promote a T_H2 response, initiating an asthmatic reaction. Several publications point towards TLR as a possible target for a more specific asthma therapy. The data from the initial analysis of GR^SPCcreERT2 mice revealed a strong upregulation of mediators that are correlated with TLR-signals and diminished effects of GC-therapy on their downregulation compared to the wild type samples.

Although these results represent only a first data set a correlation between the treatability of the mice and the expression pattern of AT2 cells could be seen. The regulation of all analysed genes by GC treatment was different in mutant mice compared to wild type samples. While

GCs in wild type animals successfully downregulated the mRNA expression of critical inflammatory mediators like IL-1β, IL-33 and iNOS these genes showed increased expression in samples of GR^SPCcreERT2 mice. The expression of TSLP was increased in AT2 samples of GC-treated GR^SPCcreERT2 mice while the expression of other important mediator like RANTES, Eotaxin-2 and TNF-α reminded unaltered independent on GC therapy. The barrier function of epithelial cells was impaired in both genotypes after induction of AAI but only in wild type mice the GC treatment led to an increased expression of ZO-1. The upregulation of ZO-1 expression is reported to be a sign of an improvement of the epithelial barrier function during the therapy and is important to closer contact of DCs with the environment.

Based on the initial observations, the following genes could be key players for the therapeutic efficiency of GCs: ZO-1 (important for the barrier function); TNFα, IL-1β and IL-33 (initiator and mediators of inflammatory response), Eotaxin-2 and RANTES (mediators of cellular infiltration) and TSLP (activation of DCs and initiation of asthmatic reaction). A possible explanation of the observed effects could be the regulation of TLR signalling by GCs, which could lead to a strong inhibition of DCs maturation in the lungs. The fact that the recruitment, activation and intraepithelial migration of DCs in response to microbial components as inhaled endotoxin requires only triggering of epithelial TLR-4 (Hammad H. et al., 2009) demonstrates how important the interaction between the immune system and epithelial cells and effect of GC treatment on the epithelial cells activation is. Finally, mast cell activation and subsequent secretion of proinflammatory mediators is directly induced by TLR ligands as well and can be regulated by GCs via effects on TLR expression.

4.3.3. Conclusion

The experimental data obtained with cell type specific GR knockout mutant mice and GR^dim bone marrow chimeras suggest a crucial role of non-immune cells of the lungs during the therapy of asthmatic reactions with GCs. The loss of GR expression in T cells, myeloid cells, DCs and B cells did not result in an impaired therapeutic efficiency of GCs. Also when the immune system did not respond to GCs, inflammation could still be suppressed by GCs in bone marrow chimera with wild type mice as recipients. The data on GR^SPCcreERT2 mice further support the hypothesis that AT2 epithelial cells are important targets of GCs during asthma treatment. Based on the gene expression analysis and current publications in field, AT2 cells seems to play a key role in the development of asthma and its pathogenesis and might be critical for the therapeutic efficiency of GCs.