*3.2. Increased Cytoplasmic Bcl10 Expression in Subepithelial Fibroblasts from Severe Asthma Patients*

In order to validate the increased protein expression of Bcl10 in severe asthma, we evaluated its immunohistochemical expression and distribution in bronchial biopsies obtained from normal individuals and asthma patients of varying severities. Bcl10 protein was variably expressed in the airways of both normal and asthma patients.

Expression of Bcl10 was strong and more pronounced in the subepithelial fibroblasts in severe asthmatic patients compared to weak to moderate expression noted in mild and moderate asthmatics, respectively (Figure 2C–F). The non-asthmatic healthy individuals showed almost no expression of Bcl10 in subepithelial fibroblasts (Figure 2A,B). On the other hand, the mucosal bronchial epithelial cells showed variable nuclear and cytoplasmic staining for Bcl10 in both healthy and asthmatic patients.

**Figure 1.** Activation of NF-κB in severe asthmatic fibroblasts. NHBF and DHBF were cultured in DMEM complete medium post serum-starvation. (**A**) Under basal conditions, mRNA expression of NF-κB pathway members, TLR4, BCL10, MALT1, CARMA3, IκBα, A20, RELA, IL-6 and IL-8, in NHBF and DHBF was analyzed by qRT-PCR and expressed as fold expression change relative to NHBF post normalization to housekeeping gene 18s rRNA. (**B**) Whole cell lysates were subjected to immunoblot analysis of Bcl10 protein levels. β-actin was used as loading control. Data are represented as mean ± SEM from at least 3 unique donors in each group. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 determined by unpaired two-tailed Student *t*-test.

In the control biopsies, the surface epithelium showed positive Bcl10 expression in both the cytoplasm and the nuclei whereas the subepithelial fibroblasts were mostly negative for Bcl10 (Figure 2A,B). Biopsies from mild and moderate cases of asthma displayed moderate Bcl10 expression in both the epithelium and subepithelium with very few subepithelial fibroblasts staining positively for Bcl10 (Figure 2C,D). Biopsies from severe asthmatic subjects showed an increased number of fibroblasts in the subepithelium with intense Bcl10 staining (Figure 2E,F). These findings suggest that the intensity of positive Bcl10 staining as well as the distribution of Bcl10-positive cells increased with increasing severity of asthma.

**Figure 2.** Increased cytoplasmic Bcl10 expression in subepithelial fibroblasts of bronchial biopsies from asthma patients with increasing severity. Representative images taken at 400X magnification showing Bcl10 expression. Representative bronchial biopsy sections from (**A**,**B**) healthy control showing positive Bcl10 staining in surface epithelium and no Bcl10 staining in subepithelial fibroblasts, (**C**,**D**) mild and moderate asthma patients showing minimal and weak Bcl10 cytoplasmic expression in subepithelial fibroblasts, (**E**,**F**) severe asthmatic patients showing numerous subepithelial fibroblasts with strong Bcl10 expression. (Red arrows indicate Bcl10-positive cells).
