*3.1. Activation of Bcl10-Mediated NF-κB in Severe Asthmatic Fibroblasts*

Persistent activation of the NF-κB signaling pathway characterized the peripheral blood mononuclear cells isolated from patients with severe asthma [3]. We therefore first examined the expression of various components of this pathway in bronchial fibroblasts from severe asthmatics (S-As) and healthy subjects. To compare the basal expression levels, normal human bronchial fibroblasts (NHBF) and asthmatic diseased human bronchial fibroblasts (DHBF) in culture were pelleted and lysed for qRT-PCR analysis. The mRNA expression of TLR4 (*p* = 0.0056), MALT1 (*p* = 0.0006) and CARMA3 (*p* = 0.0067) was significantly upregulated in DHBF relative to NHBF (Figure 1A). IKBα gene expression was significantly downregulated in DHBF (*p* = 0.0116). Furthermore, A20 deubiquitinase, another negative regulator of NF-κB that terminates downstream signaling events [25], was also lowered in DHBF compared to NHBF (Figure 1A). Increased mRNA levels of RELA subunit were also found in DHBF compared to NHBF, albeit without any statistical significance (*p* = 0.09). Since increased expression of the intermediates of the NF-κB pathway was noted, we next measured the expression of NF-κB-target genes, including IL-6 and IL-8. While a significant increase in IL-8 expression was observed in DHBF (*p* = 0.0231), IL-6 transcript levels showed an increased trend in DHBF when compared to NHBF. Bcl10 being a critical mediator of NF-κB signaling, we next investigated the protein expression of Bcl10 in these fibroblasts at basal levels. It was interesting to note a trend of 3-fold increase in the relative protein expression of Bcl10 in DHBF in comparison to NHBF (*p* = 0.0524) (Figure 1B).

The elevated expression of Bcl10 in bronchial fibroblasts from severe asthmatic subjects and the increased signature of key NF-κB genes at baseline suggest activation of the Bcl10 mediated NF-κB pathway in S-As fibroblasts.
