6.1.1 RAGE on type 1 alveolar epithelium vs. RAGE on vessel endothelium
It seems that RAGE in pulmonary parenchymal, not hematopoietic, cells is important for development of AAI.162 RAGE is expressed in AT1 epithelial cells and pulmonary endothelial cells (Figure 34, Figure 35).82, 88 It is unclear if RAGE is important on epithelial cells, endothelial cells, or perhaps both for development of a type 2 response.
To differentiate the importance of RAGE on these two cell types, tissue-specific inducible RAGE KO mice could be used. Allergen treatments can be administered to wild-type mice, global RAGE KO mice, mice with RAGE knocked out only in AT1 epithelial cells (via an aquaporin-5 (Aqp5) AT1-epithelial-cell-specific promoter),320 and mice with RAGE knocked out
only in endothelial cells (via a Tie2 endothelial-specific promoter).321 Type 2 cytokine levels, eosinophilic inflammation, IL-33 levels, and ILC2 numbers in the lung can be measured to assess differences in AAI in these mice. It is expected that if RAGE is necessary for development of AAI in the specific cell type of interest, then tissue-specific knockout of RAGE in those cells will result in no development of AAI in response to allergen. If RAGE is not necessary in those cells for AAI, then AAI should develop normally in mice lacking RAGE in those specific cells. The answer may be more complicated than expected, however. For example, loss of RAGE in the alveolar epithelium may attenuate IL-33 production/release, but alternative mechanisms may be in place to activate ILC2s, which may still be able to migrate into the lung via endothelial RAGE. The possibilities are many, but use of tissue-specific RAGE KO mice will be an excellent tool for uncovering some of these complicated interactions.
Additionally, in vitro signaling studies using primary AT1 and AT2 epithelial cells will be helpful to better understand how RAGE promotes AAI. Since RAGE is known to be expressed on AT1 epithelial cells and IL-33 is known to be highly expressed in AT2 epithelial cells, it would be interesting to investigate if there are paracrine signals between the two cell types through which RAGE signaling on AT1 cells promotes IL-33 release from AT2 cells. AT1 cell cultures322, 323 can be treated with RAGE ligands (i.e. HMGB1, AGES, S100 proteins), and changes in gene expression and signaling proteins (i.e. NF-κB-related proteins, ERK 1/2, etc.)
can be assessed via microarrays, qRT-PCR, Western blot, and ELISA. Media from the RAGE-stimulated AT1 epithelial cells can also be collected and placed onto cultures of AT2 epithelial cells324 to see if release of IL-33 can be induced from AT2 cells. Further studies would then be needed to identify what specific molecule(s) within the AT1 cell media induces IL-33 release from AT2 cells. This could be done by comparing media from unstimulated AT1 cells with
media from RAGE-stimulated AT1 cells using mass spectrometry. Proteins that are increased in the RAGE-stimulated media would be an excellent starting point and may provide some insight into if and how RAGE on AT1 cells can induce IL-33 release from AT2 epithelial cells. Uric acid and ATP are two molecules that have previously been shown to promote IL-33 release from cells and may also be worth investigating more closely in these studies.176, 177
6.1.2 RAGE on ILC2s
While ILC2 qRT-PCR (Figure 24) and studies of ILC2 reactions in the gut of RAGE KO mice (Figure 18, Figure 19, Figure 20) suggest that RAGE directly on ILC2s is not important for their development or function, more precise examination of RAGE expression on ILC2s is important.
First, since ILC2s have now been shown to be expressed in the spleen of RAGE KO mice at high levels (Figure 20, Figure 21, Figure 22), RAGE KO ILC2s can be more easily isolated. Prior to these studies, very low ILC2 numbers were seen in the lung and thus RAGE KO ILC2s could not be successfully isolated. RAGE KO ILC2s will be a much-needed proper negative control to assess RAGE expression in these cells. Once enough wild-type and RAGE KO ILC2s can be isolated from mouse spleens, qRT-PCR and Western blots can be carried out to look at both RAGE mRNA and protein expression. Development of a specific RAGE antibody for flow cytometry studies on ILC2s would also be extremely helpful in future studies.
As discussed previously, an absence of ILC2s accumulating in the lungs of RAGE KO mice in response to allergen could be due to decreased recruitment or proliferation of ILC2s.
Another way to test if RAGE expression on ILC2s directly is important for their proliferation is to activate the isolated ILC2s in vitro as depicted in Figure 22. If wild-type ILC2s proliferate normally and RAGE KO ILC2s do not, then RAGE may be important for ILC2 proliferation in
vivo as well. Wild-type ILC2s can also be cultured in the presence of a RAGE-blocking antibody
or a small molecule inhibitor or RAGE to see if this can block normal ILC2 proliferation. qRT-PCR studies on activated wild-type ILC2s in culture may also reveal changes in RAGE expression over time in these cells. For example, RAGE may not be expressed on ILC2s until they are activated.
Examination of RAGE on specific cell types using tissue-specific knockout mice and in vitro studies will help to focus future studies on the cell types that are important for RAGE-driven AAI.